variability in macrophomina phaseolina (tassi) goid. causing...
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Variability in Macrophomina phaseolina (Tassi) Goid. causing
charcoal rot of maize and its management
BY
W A Q A S A S H R A F 2004-ag-1541
M.Sc. (Hons.) Agriculture
A thesis submitted in partial fulfillment of the requirements For the degree of
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DOCTOR OF PHILOSOPHY
IN PLANT PATHOLOGY
Department of Plant Pathology
FACULTY OF AGRICULTURE,
UNIVERSITY OF AGRICULTURE,
FAISALABAD, PAKISTAN.
2015
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To,
The Controller of Examinations,
University of Agriculture,
Faisalabad.
‘‘We, the supervisory committee, certify that the contents and form of the
thesis submitted by Waqas Ashraf Regd. No. 2004-ag-1541 have been found
satisfactory and recommend that it be processed for evaluation, by External
Examiner(s) for the award of the degree’’.
SUPERVISORY COMMITTEE:
Chairman: ----------------------------------------
Prof. Dr. Shahbaz Talib Sahi
Member: ----------------------------------------
Dr. Imran ul Haq
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Member: ----------------------------------------
Prof. Dr. Sohail Ahmed
Dedicated to my loving
FATHER,
MOTHER,
Brothers and Sister
Without their efforts and sacrifices
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I could not have reached this position in my life
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C O N T E N T S
Acknowledgements i
List of Tables ii
List of Figures iii
List of plates iv
Abstract v
Chapter I INTRODUCTION 1
Chapter II REVIEW OF LITERATURE 5
2.1 Symptomology and history 5
2.2 Taxonomy and Nomenclature 6
2.3 Disease cycle 7
2.4 Viability 8
2.5 Distribution 9
2.6 Factors affecting the infection and severity of the charcoal rot disease 9
2.7 Macrophomina phaseolina in other hosts 10
2.8 Macrophomina phaseolina diversity 12
2.9 VARIATION IN CULTURAL AND MORPHOLOGICAL CHARACTERS 13
2.9.1 Growth on solid media 13
2.9.2 Growth on liquid media 15
2.9.3 Morphology of sclerotia and time for sclerotial initiation 15
2.10 PHYSIOLOGICAL VARIATION 17
2.10.1 Effect of pH on M. phaseolina 17
2.10.2 Effect of temperature on M. phaseolina 18
2.10.3 Variation in pathogenicity 18
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2.10.4 Sensitivity of M. phaseolina isolates to chemicals 20
2.11 MANAGEMENT 22
2.11.1 Through diverse germplasm 22
2.11.2 Management through fungicides 23
2.11.3 Management through plant extracts 23
Chapter III MATERIALS AND METHODS 30
3.1 GENERAL PROCEDURE 30
3.1.1 Glassware and cleaning 30
3.1.2 Sterilization 30
3.2 COLLECTION OF DISEASED SPECIMEN 30
3.3 ISOLATION, IDENTIFICATION AND MAINTENANCE OF
MACROPHOMINA PHASEOLINA
30
3.4 MASS-CULTURING OF M. PHASEOLINA 31
3.5 TESTING VIRULENCE OF THE ISOLATES 34
3.5.1 Detached stem technique 34
3.5.2 Confirmation of pathogencity 34
3.6 STUDIES ON VARIABILITY AMONG THE ISOLATES 35
3.6.1 Morphological characters on PDA and Corn Meal Agar Media
35
3.6.2 Cultural characters on Potato Dextrose and Corn Meal Media 35
3.6.2.1 Growth on solid media 35
3.6.2.2 Growth on liquid media 35
3.7 SENSITIVITY OF ISOLATES TO CHEMICALS 36
3.7.1 Sensitivity of isolates to Copper sulphate and Benomyl 36
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3.8 EFFECT OF DIFFERENT pH LEVELS ON THE ISOLATES OF M. PHASEOLINA 36
3.9 EFFECT OF DIFFERENT TEMPERATURE LEVELS ON THE ISOLATES OF
M.PHASEOLINA
37
3.10 PATHOGENIC VARIABILITY AMONG ISOLATES 37
3.11 EFFECT OF SOWING DATE AND TIME OF INOCULATION ON
DISEASE
37
3.12 EVALUATION OF PLANT EXTRACTS FOR THEIR EFFICTIVENESS AGAINST M.
PHASEOLINA
37
3.12.1 In vitro evaluation of plant extracts 38
3.12.2 Pot culture assay 38
3.13 EVALUATION OF DIFFERENT FUNGICIDES FOR THEIR EFFECTIVENESS AGAINST
M. PHASEOLINA
39
3.13.1 In vitro evaluation of fungicides 39
3.13.2 Pot culture assay 39
3.14 COLLECTION OF MAIZE GERMPLASM FOR ESTABLISHMENT OF DISEASE
SCREENING NURSERY
39
3.15 MANAGEMENT OF CHARCOAL ROT OF MAIZE THROUGH ARTIFICIAL
INOCULATION AND INFESTED FIELD
40
3.16 STATISTICAL ANALYSIS 40
Chapter IV RESULTS 41
4.1 COLLECTION OF INFECTED PLANT SAMPLES AND ISOLATION OF THE FUNGUS 41
4.2 IDENTIFICATION OF THE FUNGUS 41
4.3 PATHOGENICITY 41
4.4 CULTURAL STUDIES FOR VARIABILITY 42
4.4.1 Growth on solid media 42
4.4.1.1 Growth on Potato Dextrose Agar (PDA) 42
4.4.1.2 Growth on Corn meal medium 45
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4.4.2 Growth on liquid media 45
4.4.3 Sensitivity to chemicals 56
4.4.3.1 Sensitivity of isolates to benomyl 56
4.4.4 Effect of pH on isolates 59
4.4.5 Effect of temperature on the growth of isolates 59
4.4.6 Pathogenic variability 60
4.5 INHIBITORY EFFECT OF FUNGICIDES ON THE RADIAL GROWTH OF M.
PHASEOLINA
66
4.5.1 Effect of fungicides on plant survival of maize 66
4.6 EFFECTS OF PLANT EXTRACTS ON THE GROWTH OF M. PHASEOLINA 68
4.6.1 Effects of plant extracts on plant survival of maize 69
4.7 SCREENING OF GERMPLASM LINES AGAINST CHARCOAL ROT OF MAIZE 70
4.8 EFFECT OF INOCULATION TIME AND SOWING DATES ON DISEASE 74
4.9 MANAGEMENT OF CHARCOAL ROT OF MAIZE IN INFESTED PLOT 75
Chapter V DISCUSSION 78
Chapter VI SUMMARY 89
LITERATURE CITED 92
APPENDICES 114
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CHAPTER I INTRODUCTION
Maize or corn (Zea mays L.) belongs to the family of grasses (Poaceae). The
Mesoamerican region, now Mexico and Central America is considered as the center of origin
for Z. mays (Watson & Dallwitz, 1992). Archaeological records suggest that domestication of
maize took place independently in regions of the south western United States, Mexico and
Central America began about 6000 years ago, (Mangelsdorf, 1974). The Portuguese brought
maize to Southeast Asia from America in the 16th century. Maize was introduced into Spain
after the return of Columbus from America and from Spain it went to France, Italy and
Turkey. In India, Portuguese introduced maize during the seventeenth century. From India, it
reached to China and later it was introduced in Philippines and the East Indies. Corn now is
being grown in USA, China, Brazil, Argentina, Mexico, South Africa, Rumania, Yugoslavia,
India and Pakistan.
Maize is a tall, determinate, monoecious, annual plant. It produces large, narrow,
opposite leaves, borne alternatively along the length of stem. All maize varieties follow the
same general pattern of development, although specific time and interval between stages and
total numbers of leaves developed may vary between different hybrids, seasons, time of
planting and location.
It is cultivated globally as one of the most important cereal crops worldwide. Maize is
not only an important food crop for human consumption, but also a basic element of animal
feed and raw material for manufacturing of many industrial products. The products include
corn starch, maltodextrins, corn oil, corn syrup and products of fermentation and distallaries.
It is also being used in the production of bio-fuel.
Maize is a versatile crop grown over a range of agro-climatic zones. In fact the
suitability of maize to diverse environments is unmatched by any other crop plant. It is grown
from 58°N to 40°S, to an altitudes higher than 3000 M, and in areas with 250 mm to 5000
mm of rainfall per year (Shaw, 1988; Dowswell et al., 1996) and with a growing cycle
ranging from 3 to 13 months (CIMMYT, 2000). However, the major maize production areas
are located in temperate regions of the globe. The United States, China, Brazil and Mexico
account for 70% of global production. India has 5% of corn acreage and contributes 2% of
world production (FAO, 2000).
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The use of maize varies in different countries. In USA, EU, Canada and other
developed countries, maize is used mainly to feed animals directly or sold to feed industries
and as raw material for extractive/fermentation industries (Morris, 1998; Galinat, 1988;
Mexico, 1994). In developing countries, use of maize is variable. In Latin America and
Africa the main use of maize is for food while in Asia it is used for food and animal feed. In
fact, in many countries it is the basic staple food and an important dietary ingredient. Maize
grain contains about 72% starch, 10% protein, 4.8% oil, 5.8 % fiber, 3.0% sugar and 1.7%
ash (Gopalan et al., 2007).
Maize is an important crop in the current cropping systems of Pakistan. It ranks third
after wheat and rice in Pakistan for its grain production. Maize is grown in all provinces of
the country but Punjab and Khyber Pakhtunkhwa (KPK) are the main areas of production.
KPK produces around 0.8-0.9 million tons of maize per year. According to an estimate,
Punjab and KPK account for 84 percent of the total maize production from 95 percent of the
maize cultivation area. Yield of the crop in central Punjab has gone up to 4,600 kg/acre
whereas it is generally between 700-1, 200 kg/acre in KPK (GOP, 2013). Average maize
yield is very low (3.5 t/ha) in Pakistan due to non-availability of high yielding varieties.
Extremely expensive hybrid seed of maize is un-affordable for majority of the farmers.
During autumn, more than 50 percent area under maize is sown with local varieties. The
significance of open pollinated maize varieties cannot be denied even in the era of hybrid
maize as these can better withstand the extreme temperatures giving stable yields with
comparatively very low seed rate and inputs cost.
In Pakistan maize crop is sown mainly in two seasons Feb-March and July-August. A
number of fungal diseases attack on the maize crop but charcoal rot caused by
Macrophomina phaseolina (Tassi.) Goid. is globally the most important disease of maize
(Edmunds, 1962).
Maize suffers from about 110 diseases on a global basis caused by fungi, bacteria and
viruses. The disease spectrum varies in different agro-climatic zones. Several diseases such
as seed and seedling blights, foliar disease, downy mildews, stalk rots and leaf blight are
prevalent in various cultivations of maize in Pakistan. It has been estimated that about 13.2%
of the economic production of maize is lost annually due to diseases in India (Dhillon and
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Prasanna, 2001). The information has been collected on distribution and appearance of
diseases in different maize growing regions in India (Sharma and Lal, 1998).
M. phaseolina causes disease on more than 500 plant species worldwide (Salik,
2007). About 67 hosts of this pathogen have been reported from Pakistan (Mirza and
Qureshi, 1982; Shahzad and Ghaffar, 1986; Shahzad et al., 1988). Tropical crop plants are
seriously affected by this pathogen (Malaguti, 1990). M. phaseolina is classified as a
Deuteromycete which shows two asexual sub-phases, a mycelial phase named as Rhizoctonia
bataticola (Taub) and the other a pycnidial phase called M. phaseolina (Dhingra and Sinclair,
1978). The fungus is classified in the Botryosphaeriaceae according to recent phylogenetic
data (Crous et al., 2006). The fungus produces dark brown lesions on the epicotyls and
hypocotyls of seedlings. The seedling dies because of obstruction of xylem vessels. In adult
plants, the pathogen causes red to brown lesions on roots and stems and produces dark
mycelium and black microsclerotia. The stem shows longitudinal dark lesions and the plant
becomes defoliated and wilted (Abawi and Pastor-Corrales, 1990). The black, 0.1-1 mm
sized microsclerotia are formed in soil, infected seeds or host tissues and constitute the
primary inoculum source of the pathogen (Bouhot, 1968). They can survive up to 15 years
depending on environmental conditions, and whether or not the sclerotia are associated with
host residues (Cook et al., 1973; Papavizas, 1977; Short et al., 1980).
M. phaseolina is a heat tolerant pathogen since sclerotia could withstand a
temperature range of 60-65ºC (Bega and Smith, 1962; Mihail and Alcorn, 1984). The
evidence suggests that it is primarily a root inhabiting fungus and produces tuber or cushion
shaped 1-8 mm diameter black sclerotia. These sclerotia serve as a primary means of survival
(Smith, 1969; Kaiser et al., 1980). Macrophomina phaseolina isolates from different hosts,
soils or geographical regions differ in terms of their morphological, physiological and
pathogenecity characteristics (Dhingra and Sinclair, 1978). This variation helps the pathogen
to survive in a diverse range of environments. Various advance techniques like PCR and
others are used for the more precise identification of variability among isolates. An adequate
knowledge of the existence of variability in the pathogen would be the most important and
highly desirable to strengthen breeding attempts in attaining host plant resistance. The
hypothesis of the current studies is to characterize different isolates of M. phaseolina on the
basis of cultural, morphological, physiological characteristics and pathogenicity in the
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current scenario of varying disease severities to devise better management strategies for
charcoal rot of maize.
Among maize crop diseases in Pakistan, stalk rot is considered to be a great threat. At
the National Agricultural Research Centre (NARC), Islamabad, Pakistan, pathogenicity of
Fusarium moniliforme, F. graminearum and Macrophomina phaseolina isolated from stalk
rot affected maize was tested on susceptible maize varieties in different seasons and at
different stages of plant growth. F. moniliforme and M. phaseolina in combination produced
77.9 and 89.0% infection with 21.7 and 25.9% reduction in grain yield during spring and
summer seasons of 1988, respectively; whereas F. graminearum produced 32.8 and 49.4 %
disease severity with 0.9 and 1.4 % reduction in grain yield during the same spring and
summer seasons (Ahmad, 1997).
Efficient management of this disease is important because of the severity of economic
yield loss caused by the pathogen. Now-a-days crop protection is dependent on chemical
pesticides because chemical control is easy, direct and quick but continuous dependence on
pesticides has proved unsuitable and in reality has led to greater problems in pest control but
integrated management can help to avoid such problems, moreover it is environmental
friendly and suitable strategy as it minimize the use of chemicals by placing more reliance on
biological control, resistant varieties and non-chemical methods. The ultimate objective is to
increase the quality and quantity of the produce at minimum cost. Approaches are required
that aim at avoiding the frequent re-occurrences of charcoal rot disease epidemics. The
information regarding the variability and management of this pathogen on maize is lacking in
this part of world, thus the current study was planned with following objectives.
1. To determine the morphological, pathogenic, physiological and cultural variability
among isolates of M. phaseolina prevalent in different maize growing areas.
2. To observe the effect of sowing date and time of inoculation on the appearance and
incidence of charcoal rot disease of maize.
3. To determine the source of resistance against charcoal rot of maize.
4. Evaluation of different plant extracts and fungicides for management of charcoal rot
disease of maize.
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CHAPTER II REVIEW OF LITERATURE
The present investigation included different aspects on the variation of
Macrophomina phaseolina (Tassi) Goid. causing charcoal rot of maize. The literature
pertaining to studies on the variability and management aspects of M. phaseolina is reviewed
below.
2.1 Symptomology and history
The causal organism of charcoal rot is a common soil-borne fungus often known by
its imperfect state Macrophomina phaseolina (Tassi) Goid. It was first reported in India by
Uppal (1931 and 1934). It is the pycnidial stage of Rhizoctonia bataticola (Ashby, 1927)
with the following synonyms.
Botryodiplodia phaseoli (Maubl.) Thirum
Dothiorella cajani Syd,. P. Syd. and E. J. Butl.
Manophoma cajani Syd., P. Syd. adn E. J. Butl.
Macrophomina phaseoli Maubl.
Rhizoctonia lamellifera Small
Sclerotium bataticola Taubenh
Rhizoctonia bataticola (Taubenh) E. J. Butler (anamorph)
Goidanich (1947) examined the original material of M. phaseoli collected by Tassi
and renamed it as Macrophomina phaseolina (Tassi) Goid. Rhizoctonia is predominantly
found in subterranean habitat both as parasite and saprophyte.
Soil-borne pathogens are especially challenging because their infection propagules
often survive for several years in the soil and they reduce yield and quality in numerous
crops. Soil-borne diseases are difficult to detect, predict and manage because the soil matrix
is complex, making it difficult to understand the variables that govern infection processes and
epidemics. M. phaseolina is the causal agent of seedling blight, root rot and charcoal rot of
more than 500 crop and non-crop species, including economically important crops such as
soybean, corn, sorghum and cotton. The causal organism of charcoal rot is a common soil-
borne fungus often known by its imperfect state M. phaseolina. Charcoal rot is a worldwide
disease and has been reported from all the ecologically diverse areas of sorghum cultivation
in the tropics, subtropics and temperate regions (Tarr, 1962). Charcoal rot of sorghum caused
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by M. phaseolina has been identified as a destructive disease in the post rainy season in
Karnataka, Maharashtra, Gujarat and Andhra Pradesh (Anahosur and Patil, 1983).
Disease symptoms are clearly visible from the time of emergence and can be
evaluated at various stages of development of the plant. M. phaseolina can infect roots which
show necrotic lesions (Bouhot, 1967; Adam, 1986). On adult plants, M. phaseolina causes
lesions on stem, spike and seed. On stems, lesions are light brown and appear at the
ramification point of the lateral secondary branches. Colonized tissues become gray and
covered with abundant minute black punctuations. Initially these punctuations are immersed,
becoming gradually more prominent. The most striking symptom is the sudden wilting and
drying of the whole plant while most of the leaves remain green. The stem and branches are
then covered with black bodies and give the charcoal or ashy appearance of dead plants.
Withering can be observed from seedling to mature stage and is the result of necrosis of
roots, stems and mechanical plugging of xylem vessels by microsclerotia, but also by toxin
production, and enzymatic action (Chan and Sackston, 1973; Kuti et al., 1997; Jones and
Wang, 1997).
Charcoal rot is characterized by lodging of plants as they approach maturity. Lodging
is due to the weakened condition of the stalk because of disintegration of the pith and cortex
by the pathogen, leaving the lignified fibro-vascular bundles suspended as separate strands in
the hollow stalk. Hence the disease was first named “hollow stalk of sorghum” by Uppal et
al., (1936). The vascular bundles are profusely covered with tiny black sclerotia of the
pathogen, which give the charcoal appearance to the affected area. Thus, the name“Charcoal
rot” describes the appearance of the disease inside the infected roots and stalks.
2.2 Taxonomy and Nomenclature
Macrophomina phaseolina (Tassi) Goid. [Tiarosporella phaseolina (Tassi) Vander
Aa] is a soil-borne plant pathogenic fungus. It belongs to the anamorphic Ascomycetes and is
characterized by the production of both pycnidia and sclerotia in host tissues and culture
media. The pycnidial state was initially named Macrophoma phaseolina by Tassi in 1901 and
Macrophoma phaseoli by Maublanc in 1905. In 1927, Ashby maintained the name
Macrophomina phaseoli, while Goidanich (1947) proposed Macrophomina phaseolina.
Mihail (1992) indicated that there is an unconfirmed report of a teleomorph named Orbilia
obscura (Ghosh et al., 1964) of M. phaseolina, but since then no further evidence appeared
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for the telemorph state. The sclerotial state was described for the first time by Halsted as
Rhizoctonia bataticola (Taub) by Butler on sweet potato in 1890. According to Dhingra and
Sinclair (1978) the same fungus was isolated from cowpea in India in 1912 by Shaw and was
then named Sclerotium bataticola. Recently, Crous et al., (2006) demonstrated that although
the telemorph is unknown M. phaseolina is a member of the family Botryosphaeriaceae. The
authors pointed out the differences between Tiarosporella and Macrophomina, which
produces percurrently proliferating conidiogenous cells in the pycnidia. The pycnidiospores
are ellipsoid to oblong-ovoid, and measure 16-32 × 6-11 μm. During the sclerotial formation,
50–200 individual hyphal cells aggregate to give multicellular bodies named microsclerotia.
The microsclerotia are black and are variable in size (50-150 μm) depending on the available
nutrients of the substrate on which the propagules are produced (Short and Wyllie, 1978).
2.3 Disease cycle
M. phaseolina causes seedling blight, root rot and stem rot of more than 500
cultivated and wild plant species including economically important crops. There are soybean,
common bean, sorghum, maize, cotton, peanut, cowpea (Gray et al., 1990; Hall, 1991;
Diourte et al., 1995) softwood forest trees, Abies, Pinus, Pseudotsuga (Mc Cain and Scharpf,
1989) fruit trees, Citrus spp, Cocoa nucifera, Coffea spp and weed species (Songa and
Hillocks, 1996). The fungus was reported in North and South America, Asia, Africa and
Europe. However, it is economically more important in subtropical and tropical countries
with a semi-aridclimate (Wrather et al., 1997; 2003).
M. phaseolina produces sclerotia in root and stem tissues of its hosts that enable it to
survive adverse environmental conditions (Cook et al., 1973; Meyer et al., 1974; Short et al.,
1980). In PDA, pycnidia are not produced except under some specific incubation conditions
(Gaetán et al., 2006) and only some times in host crops (Mihail and Taylor, 1995), and their
importance in the epidemiology of the fungus likely depends on the host involved as well as
the fungal isolate (Ahmed and Ahmed, 1969). On cowpea, pycnidia are produced at the end
of the rainy season, but their epidemiological significance seems minor. On the contrary, in
jute crops, pycnidiospores produced on early infected stem and leaf tissues have been
reported to be responsible for secondary spread of the disease.
Microsclerotia in soil, infected seeds or host tissues serve as primary inoculum
(Bouhot, 1968; Dhingra and Sinclair, 1977; Abawi and Pastor-Corrales, 1990). Root
17
exudates induce germination of microsclerotia and root infection of hosts. The infective
hyphae enter into the plant through root epidermal cells or wounds. During the initial stages
of pathogenesis, the mycelium penetrates the root epidermis and is restricted primarily to the
intercellular spaces of the cortex of the primary roots. As a result, adjacent cells collapse and
heavily infected plantlets may die. At flower onset, the fungal hyphae grow intracellularly
through the xylem and form microsclerotia that plug the vessels (Short et al., 1978; Mayek-
Pérez et al., 2002) and disrupt host cells. The infected plants show necrotic lesions on stems,
branches and peduncles. From pod peduncles, the fungus spreads to the pods and invades
grains. Heavily infected plants die prematurely due to the production of fungal toxins like
phaseolinone (Bhattacharya et al., 1994) and production of fungal tissue that plugs the host
vessels. In soybean, formation of microsclerotia is conditioned by flowering and pod setting
(Wyllie and Calvert, 1969) and may be indicative of initiation of death of the host (Short and
Wyllie, 1978). After plant death, colonization by mycelia and formation of sclerotia in host
tissue continues until tissues are dry. The mycelium and microsclerotia produced in infected
plant material, including plant residues, are the means of propagation of the pathogen.
Microsclerotia in soil, host root and stems are the main surviving propagules. After decay of
root and plant debris, microsclerotia are released into the soil. They are distributed generally
in clusters at the soil surface and are localized mainly at a depth of 0-20 cm (Alabouvette,
1976; Mihail, 1989; Campbell and Van der Gaag, 1993). They can survive for 15 years
depending on environmental conditions and whether or not the sclerotia are associated with
host residue (Baird et al., 2003). Factors that adversely affect the survival of these propagules
includes repeated freezing and thawing of soil, low carbon: nitrogen ratios in soil and soil
moisture content (Dhingra and Sinclair, 1974; Dhingra and Sinclair, 1975).
2.4 Viability
M. phaseolina is known to survive as sclerotia in corn and sorghum stalk residues for
18 and 16 months, respectively. Large numbers of viable sclerotia were isolated from corn
and sorghum stalk in final collection. M. phaseolinais capable of living saprophytically on
dead organic tissues particularly on many of its natural hosts producing sclerotial bodies (Sen
and Bandopadhyaya, 1988).
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2.5 Distribution
The charcoal root rot organism, M. phaseolina is one of the most damaging pathogens
in arid region and has a wide host range (Waller, 1976). In India, it has been reported from
Andhra Pradesh, Bihar, Gujarat, Karnataka, Maharashtra, Madhya Pradesh, Rajasthan, Tamil
Nadu, Uttar Pradesh and West Bengal (Uppal, 1931; Likhite and Kulkarni, 1934; Deshpande
et al., 1969 and Philip et al., 1969). Dry root rot caused by R. bataticola has been reported
from Australia, Ethiopia, India, Iran, Lebanon, Syria, Turkey and United States (Nene, 1978).
2.6 Factors affecting the infection and severity of the charcoal rot disease
Root infection is affected by growth stage and environment. High root infection can
occur before reproductive development and is then associated with hot and dry weather early
in the growing season (Cloud and Rupe, 1994). M. phaseolina can infect beans also under
relatively dry conditions (Olaya and Abawi, 1993). However, there are also reports where a
high moisture holding capacity (40-50%) resulted in greater M. phaseolina colonization on
peanut (Husain and Ghaffar, 1995). Agarwal and Goswani (1973) reported a significant
synergistic effect in soybean when the root-knot nematode Meloidogyne incognita preceded
infection by M. phaseolina by three weeks, and suggested that M. incognita predisposes
plants to the fungal infection similar to the vascular pathogens Fusarium oxysporum and
Verticillium dahliae. In white clover M. phaseolina also tends to be associated with higher
final densities of the plant pathogenic nematodes Meloidogyne trifoliophila, Helicotylenchus
dihystera and Heterodera trifolii (Zahid et al., 2002). In contrast, in a pot experiment the
simultaneous addition of M. phsaeolina and Meloidogyne javanica resulted in reduced
nematode galls, which was described to the effect of toxic fungal metabolites on the
nematode (Gupta and Mehta, 1989). Visible symptoms of the disease in the field are most
apparent under conditions that reduce plant vigor suchas; poor soil fertility (Sinclair and
Backman, 1989), high seeding rates (Pearson et al., 1984), low soil water content (Ali and
Ghaffar, 1991; Sheikh and Ghaffar, 1979; Kendig et al., 2000), high temperatures (Odvody
and Dunkle, 1979) and root injury.Timing of host reproduction is another factor that has a
strong influence on charcoal rot development. In Euphorbia lathyris, early flowering plants
are more prone to charcoal rot than later flowering ones (Canaday et al., 1986). In sorghum,
post-flowering water-stressed plants showed more severe charcoal rot symptoms than plants
without water stress (Diourte et al., 1995). Initial population density of sclerotia in soil was
19
directly correlated with the severity of charcoal rot of soybean and was inversely related to
soybean yield (Short et al., 1980). Mihail (1989) found that average symptom expression and
mortality increased with increasing soil temperature and that mortality increased markedly
when the temperature was 28-30°C at 5 cm soil depth.
2.7Macrophomina phaseolina in other hosts
Reichert and Hellinger (1947) listed a wide number of plant species around the world
affected by M. phaseolina. The most economically important are: pepper (Capsicum annum
L.), papaya (Carica papaya L.), sweet orange (Citrus sinensis L. Osbeck), coffee (Coffea
arabica L.), soybean (Glycine max (L.) Merr) sunflower (Helianthus annus L.), sweet potato
(Ipomoea batatas L) Lam.) apple (Malus domestica Borkh.), alfalfa (Medicago sativa L)
plantain (Musa paradisiaca L.), tobacco (Nicotiana tabacum L.), common bean (Phaseolus
vulgaris L.), garden pea (Pisum sativum L.), tomato (Lycopersicon sculentum Mill.), potato
(Solanum tuberosum L.), grain sorghum (Sorghum bicolor L.) Moench), cacao (Theobroma
cacao L.), clover (Trifolium sp.), grape (Vitis vinifera L.), and corn (Zea mays L.). Host
range is wide spread in the tropics and subtropics, and includes cereals, legumes, fruits,
vegetables, herbaceous, and woody plants (Dhingra and Sinclair, 1978; Holliday et al.,
1970).
The pathogen is being reported in new hosts and areas causing charcoal rot in
previously reported hosts. For example, in spring 2006, tan-brown wilted canola plants
(Brassica napus L.) were observed in an experimental plot at Merredin, Western Australia.
Longitudinal streaks along the main stem, wilting of branches, and shriveled pods were the
characteristic symptoms caused by M. phaseolina (Khangura and Aberra, 2009). Charcoal rot
of canola was also reported Argentina in 2006 (Gaetan et al., 2006). Tropical soda apple
(Solanum viarum Dunal), considered one of the most invasive weeds in Florida, was reported
showing symptoms of progressive necrosis from leaves to petiole caused by M. phaseolina in
2006. Production of pycnidia and pycnidiospores was also observed on infected tissues.
Iriarte (2007) suggested that M. phaseolina could be a limiting factor for the spread of this
weed. However, the fungus is a pathogen for desirable crops produced in the area and
tropical soda apple may be a reservoir for the pathogen.
In 2006 in southern Spain (Aviles et al., 2008) confirmed M. phaseolina causing
crown and root rot in several strawberry cultivars. Affected plants presented necrotic roots,
20
dark brown necrotic areas around the crown and along the woody vascular ring. The same
disease and symptoms were also reported in strawberries in Florida (United States) and Israel
in 2005.
M. phaseolina is able to cause different symptoms: hollow stem rot, wilt, and pre-
emergent and post-emergent damping off, depending on the plant tissue colonized. In the
case of melon, M. phaseolina infects plant roots and later colonizes the fruit via the peduncle
through the abscission zone. Once the fruit is colonized, M. phaseolina infects the seed coat
and cotyledons, and these infected seeds give rise to diseased seedlings that can increase the
inoculum potential in the soil. Melon is one of the hosts in which M. phaseolina serves as a
soilborne and seed borne pathogen (Reuveni et al., 1983).
Charcoal rot of sunflower (H. annuus) was first reported in 1927 in Sri Lanka and in
subsequent years in various continents around the world (Wyllie and Scott, 1988); the most
recent report described by (Mahmoud and Budak, 2011) in Turkey. Predominant symptoms
of charcoal rot on sunflower are gray to black discoloration with lesions on the stem above
the soil line, black microsclerotia usually observed in the fibro-vascular system of roots and
lower internodes covering an average of one-third of the plant height (Khan, 2007; Raut,
1985; Yang and Owen, 1982). Like soybean, disease severity is high dependent upon
environmental conditions, specially drought and high air temperatures. Dawar and Ghaffar
(1998) pointed out that there is a significant correlation between the level of inoculum in the
soil and infection and colonization of sunflower roots by M. phaseolina.
M. phaseolina is reported to be soil, seed and stubble born in the specific case of
sunflower. The pathogen has been reported to cause seedling blight, damping off, basal stem
rot and early maturity of sunflower (Khan, 2007). One infected plant can have up to 44
percent infected seeds (Raut, 1985). Yang et al., (1983) reported that M. phaseolina also can
be spread by insects in this crop. A small percentage of Cylindrocopturus adsperus
(sunflower stem weevil) carry M. phaseolina as they emerge after over wintering in roots and
stalks. Later, the insects spread the pathogen while feeding and ovipositioning on other
plants. Presumably, M. phaseolina infests the egg cavity, and grows and spreads through the
stalks via larval tunnels.
21
2.8 Macrophomina phaseolina diversity
It was noted that among the isolates of M. phaseolina, there are cultural
morphological variations such as hyphal pigmentation, microsclerotia size and shape and
presence or absence of pycnidia. In general, colonies are gray or white with short mycelium
inclined towards the growth direction, although aerial mycelium is not often produced. Under
the microscope, hyphae branch at right angles from the main hyphae, but later hyphae bend
and grow nearly parallel to the main hyphae (Hartman et al., 1999). Sometimes hyphae
present gloomy colors from pale brown to grey. Septal width varies from approximately 2 to
11 µm and cells measure at least 46 µm in length. However, the most important
characteristics regarding taxonomy and classification are the production size and composition
of microsclerotia (Reichert and Hellinger, 1947).
Microsclerotia of M. phaseolina are black bodies composed of 50-200 hyphal cells
aggregated by a melanin-like cementing agent that gives its color (Gangopadhyay and
Wyllie, 1974; Short and Wyllie, 1978). Microsclerotia vary in size (60-200 µm) and number
depending upon the nutrient availability in the culture media or a specific host. In addition,
the number of cells and germ tubes are directly related with the size of microsclerotia
(Dhingra and Sinclair, 1977). M. phaseolina microsclerotia are produced in five different
ways during mycelial growth. The most frequent formation is by the spontaneous production
of swollen barrel-shaped dark cells from a single hyphae measured from 4 to 23 µm, that
later are self-divided or segmented forming a microsclerotium. It was also observed that
barrel-shaped cells growing from different hyphae fuse to generate a microsclerotium.
Normal cells from a single or various hyphae can also fuse and intertwine or normal hyphal
cells fuse with swollen-barrel cells to form microsclerotia. These types of microsclerotia
formations were observed among isolates from various hosts, fusion of barrel-shaped cell
being the most common type of formation. Fusion of mature-formed microsclerotia also was
observed. It was noted M. phaseolina is a soil-borne pathogen with a wide range of hosts,
about 500 species of plants, and must have a good genetic variability to be able to not
discriminate in its host selection (Wyllie and Scott, 1988). However, differentiation of
isolates in this species is problematic to the plant pathologists and mycologists because the
morphological characteristics are highly variable (Babu et al., 2010). Isolates from different
hosts, soils or geographical regions can differ in their morphological characteristics,
22
production of microsclerotia, pycnidial size, pycnidiospores and pathogenicity. Due to this
variability between isolates, morphological or phenotypic criteria are often not reliable (Babu
et al., 2007; Saleh et al., 2009).
Wyllie and Brown (1970) described a microsclerotium cross section and pointed out
that all the inner cells have the cellular organelles necessary for germination, such as;
mitochondria, endoplasmic reticulum, lomasomes, lipids, and one to three nuclei per cell.
They also reported the observation of septal pores that connect the sclerotial cells and
hypothesized that they allow the continuity of the cytoplasm and that some microsclerotia
cells serve as a nutrient suppliers to others during the microsclerotia germination process.
A morphological description of germ tubes and hyphae from M. phaseolina
microsclerotia germinated on diluted corn meal agar (CMA) is given by (Pratt, 2006). On this
media an average of four germ tubes are produced per microsclerotium, with a length of a
few micrometers to several hundred after 18 to 24 hours of exposure to this media. Pratt
(2006) also reported that germ tubes were sometimes unbranched and with curly growth
patterns.
M. phaseolina pycnidia are obtained by alternating the light regime by 12 hours dark
and light intervals and growing the fungus on fresh plant tissue. The pycnidial stage has been
reported on different bean tissues such as garden and jute bean and there are reports of the
pycnidial stage on soybean (Mihail, 1992). However, Ma et al., (2010) reported that conidia
and pycnidia are produced in an optimum manner on a special culture media, peanut butter
extract-saturated filter paper over soynut butter extact agar (PESEA).
2.9 VARIATION IN CULTURAL AND MORPHOLOGICAL CHARACTERS
2.9.1 Growth on solid media
Uppal et al., (1936) reported variation in growth rate and colony characteristics for
two sorghum strains of M. phaseolina from two different localities, one from Broach farm
and another from Mohol farm in Maharashtra. Cultures of Broach strain showed concentric
rings on PDA, while that of Mohol strain produced aerial mycelium.
Vasudeva (1937) reported that R. bataticola grew equally well on Richards agar,
cotton root extract and synthetic agar but the sclerotial formation was favoured by the last
two media and was poor on Browns agar. Thomas (1938) observed that the pycinidia of M.
phaseoli developed in cultures on several solid media.
23
Adam and Stokes (1942) observed that mean diameter of sclerotia varied from 63-165
µm on Dox’s agar and 18-50 µm on Potato dextrose agar. Goidanich and Camici (1947)
observed similar cultural and morphological characters among the strains of M. phaseoli
isolated from bean (Phaseolus vulgaris Linn.) and lavender (Lavandula officinalis).
Khare et al.,(1970) reported variability in growth pattern and rate of growth among
six isolates of M. phaseolina from roots, stem, pod, leaf, seeds and soil in case of urdbean
when grown on different media. Further, they observed that the soil isolate had the least
amount of growth in almost all media whereas the pod isolate showed maximum growth.
Dhingra and Sinclair (1973) noticed variation in growth rate among the isolates from
the same plant and between isolates from different soybean plants on different media. The
stem isolates produced fluffy growth; root isolates showed partially fluffy growth and all
other isolates produced appressed growth. Seed and petiole isolates produced concentric
alternating zones of sclerotia and this character was more pronounced for the seed isolate.
They also collected nine isolates of M. phaseoli from various parts of the United States and
reported variation in growth rate and colony characteristics. Further, the growth rate was
correlated with pathogenicity. Contrary to this, Sobthi and Sharma (1992) reported no
correlation between radial growth and virulence and or virulence and pycnidial formation in
isolates of R. bataticola from groundnut in Rajasthan.
Ghosh and Sen (1973) reported that Richards’s agar permitted the best growth and
sclerotial formation of R. bataticola as compared to PDA, Czapeks Dox agar, Sabourauds
agar, and Steinberg’s agar and Jute extract agar.
Jain et al., (1973) isolated M. phaseolina from urdbean plant parts i.e. from root,
stem, leaf, pod and seeds and one isolate from the soil where urdbean crop was grown. In the
study for growth on different media, the isolates differed from each other in the growth
pattern either in the diameter of colony or nature of aerial mycelium or the type of myeclial
growth. Raut and Bhombe (1976) reported that the growth of two isolates of R. bataticola
from sorghum was maximum on Elliott’s medium at 30°C. Waseer et al., 1990 isolated M.
phaseolina from soybean and reported that the pathogen grew better on potato dextrose agar
or potato dextrose juice at 35ºC and little growth occurred at either 20°C or 40°C.
Chowdary and Govindaiah (2007) reported that the radial growth was maximum after
24 hours of incubation on PDA (40.3 mm), followed by potato sucrose agar (39.0 mm),
24
Czapeks dox agar (36.0 mm) and Oatmeal agar (35.5 mm). Likewise after 36 hrs of
incubation period, the radial growth was found maximum on PDA (81.3 mm) followed by
Czapeks dox agar (78.0 mm). After 48 hours of incubation, maximum radial growth of
pathogen was recorded on Potato dextrose agar (90.0 mm) and minimum on Oat meal agar
(80.0 mm).
2.9.2 Growth on liquid media
Uppal et al., (1936) had grown Broach farm and Mohol farm isolates of Rhizoctonia
on seven different liquid media and found that in Meyer’s solution, both the strains grew well
and produced abundant sclerotia, while in Fermis and Coons solution; both the strains had
very poor growth with less number of sclerotia. It was concluded that vigorous mycelial
growth induced abundant sclerotial formation. Rhizoctonia follows thegrowth curve in liquid
medium, having an initial period of accelerating growth followed by a phase of very rapid
growth and then a decrease in weight due to autolysis (Ikeno, 1933; Townsend, 1957; Israel
and Ali, 1964).
Shanmugam and Govindswamy (1973) reported that Richards’ medium gave the
maximum growth of M. phaseolina of groundnut. Satishchandra (1977) reported that the
fungus attained maximum growth after 11 days of incubation in Richard’s medium.
Chowdary and Govindaiah (2007) reported that maximum mycelial dry weight of M.
phaseolina in potato dextrose broth (998.3 mg) and minimum in Oatmeal broth (439.3 mg),
whereas 823.3, 811.6 and 806.0 mg of mycelial dry weight was recorded in Czapeks dox,
Richards’ and Potato sucrose broth, respectively. Byadgi and Hegde (1985) reported
variation in dry mycelial weight of six isolates of R. bataticola. Bean isolate produced
maximum growth with mean mycelial weight of 190 mg followed by Bengal gram, cowpea,
sorghum, soybean and glyricidia isolates.
2.9.3 Morphology of sclerotia and time for sclerotial initiation
Haigh (1928) classified 27 strains of M. phaseolina into three groups A, B and C,
where C group included those strains having a mean sclerotial diameter of 120 µm or less,
the B group included those with about 200 µm, while group A comprised of sclerotia that
could conveniently be measured in milli meters. On the basis of differences in size and
number of sclerotia, Hildebrand et al., (1945) differentiated two strains of M. phaseolina
isolated from soybean, the Ontario strain having mean sclerotial diameter of 90.4 x 75.8 µm
25
on host plant and 99.9 x 89.4 µm in culture and from cotton, the Texas strain whose sclerotia
in culture averaged 85.4 x 73.0 µm and were produced in greater number. Goidanich and
Camici (1947) recorded sclerotial diameter of 65 to 75 µm on host and 100 to 115 µm in
cultures with bean isolate and 115 to 135 µm on culture with broad bean (Vicia fabia Linn.)
isolates. Bean, eggplant, potato, chilli, tomato, Bengalgram and pumpkin isolates produced
smooth and round sclerotia, whereas those of cotton, tobacco and sesame isolates were more
or less irregular. Similarly, studies with eight isolates (B-4, B-6, B-43, B-45, B-46, B-50, B-
61 and B-68) revealed that the sclerotia of B-4 and B-43 were smaller, less compact and of
irregular shape as compared with the other strains (Reichert and Hellinger, 1947).
Dhingra and Sinclair (1973) observed irregular shaped sclerotia with the root isolates
and round to elliptical sclerotia in other isolates involved in their study. Raut and Bhombe
(1976) reported that stem isolate of sorghum produced more sclerotial bodies than that of the
leaf isolate on Elliotts’ medium at 30°C. Anilkumar and Sastry (1980) reported that the
ability of the isolates to produce sclerotia changed with the growth media. Sunflower and
Horsegram isolates produced maximum number of sclerotia on Rose Bengal Agar (RBA) and
Nutrient Agar (NA) respectively. The brinjal isolate produced least sclerotia on NA and no
sclerotia on RBA, while, the sesamum and bean isolates did not develop sclerotia on NA.
They also reported that the sclerotial initiation was quick in sunflower, on all the media tried,
while the brinjal and cowpea isolates took the maximum time for sclerotial initiation (ten
days).
Smits and Noguera (1988) used SEM and light microscopy and reported that the
sclerotial formation began with branching and intertwining of adjacent hyphal filaments, an
increase in size of associated cells and compaction of the sclerotial mass. They also observed
that the external cells of peripheral hyphae collapsed, but internally, sclerotia were uniformly
reticulate. Raut and Ingle (1989) reported differences in sclerotial size of isolates of R.
bataticola (M. phaseolina) from 15 crops including sorghum, soybean and safflower. Waseer
et al., (1990) isolated M. phaseolina from soybean and reported that, sclerotial size on PDA
differed from other media. However, it was within the range described for M. phaseolina.
Dhar and Sarbhoy (1993) isolated the pod and seed isolates of soybean causing ashy stem
blight and reported the smallest known sclerotium in the world (mean 32 x 24 µm). Lokesh
and Benagi (2004) reported maximum sclerotial production on PDA i.e. 123.3/microscopic
26
field (10x). The ICRISAT isolate recorded maximum production (71.2 sclerotia/microscopic
field), followed by Bidar isolate (68.68 sclerotia /microscopic field).
Shekhar et al.,(2006a) recorded that Hyderabad isolate produced highest number of
sclerotia (180.3 sclerotia/ 9 mm disc and 52.0 sclerotia/microscopic field (10x) of bigger size
(95.7 µm), whereas Coimbatore isolate produced minimum number of sclerotia (169) with
smaller size (66.9 µm). They also observed that on the basis of sclerotial morphology, two
groups of isolates could be formed, the one with oblong shape having irregular edges and the
other being round with regular edges. Sub species of M. phaseolina were classified based on
differences in microsclerotial size, cultural characteristics, chlorate sensitivity, pycnidia
formation and pathogenicity (Cloud and Rupe, 1988 and 1991; Mihail, 1992; Karunanithi et
al., 1999 and Suriachandraselvan and Seetharam, 2000).
2.10 PHYSIOLOGICAL VARIATION
2.10.1 Effect of pH on M. phaseolina
Uppal et al.,(1936) while studying variation in M. phaseolina, revealed that both
Mohol and Broach strain can tolerate wide range of pH, while the optimum range lies
between pH 3.4 and 6.4, indicating that acidic mediumfavours the growth of the isolates.
Sclerotial formation was abundant in acidic condition compared to alkaline condition. Khare
et al., (1970) while studying the variation among Rhizoctonia bataticola from urdbean plant
parts in vitro found differences in growth pattern and sclerotial size at pH 6.5. All the six
isolates varied in their growth pattern and growth rate at different pH levels.
Shanmugam and Govindswamy (1973) studied the physiologic aspects of M.
phaseolina causing groundnut root rot and found that M. phaseolina grew best on Richard’s
medium at optimum of pH 5.0. Chowdary and Govindaiah (2007) observed that the growth
of M. phaseolina (causal agent of root rot of mulberry) was maximum (90.0 mm) at pH 7.0,
whereas it recorded 59.0 mm at pH 8.0. At different levels of pH (6.0, 6.5 and 7.5), growth of
pathogen varied 62.3, 84.3 and 87.6 mm respectively. Similarly, maximum number (120) of
sclerotia /9 mm mycelia disc was recorded at pH 7.0 and the same was found minimum
(16.3) at pH 6.0. The growth rate of pathogen and number of sclerotia varied at different
levels of pH.
27
Ghosh and Sen (1973) conducted physiologic studies on four isolates of
Macrophomina phaseolina. According to them, the optimum pH for the growth of these
isolates was 6.8. Bainade et al., (2006) studied various pH levels for the growth of M.
phaseolina causal agent of leaf spot of mung bean. They observed that the pH levels of 7.0
(neutral) recorded 89.66 mm growth followed by pH 8.0 (61.33 mm growth).
2.10.2 Effect of temperature on M. phaseolina
Uppal et al., (1936) studied the optimum temperature of two strains of M. phaseolina
and revealed that Broach strain grew best between 30 and 35°C and Mohol strain between 30
and 37°C. At maximum temperatures studied, the radial growth of the two strains fell
sharply; they also noted very slow growth at lower temperature. Khare et al., (1970) while
studying variation among the isolates of R. bataticola from urdbean plants found differences
in growth pattern and sclerotial size at 5 and 25°C. All the six isolates differed from their
growth pattern and rate of growth at different temperature levels.
Waseer et al., (1990) isolated M. phaseolina from soybean and reported that pathogen
grew best on PD agar and PD juice at 35°C. Least growth was recorded at 20°C or 40°C.
Bansal and Gupta (2000) while investigating cultural variability among seven isolates of
Rhizoctonia reported 30-35°C temperature as optimum for mycelial growth. Bainade et al.,
(2006) studied various levels of temperature for culturing M. phaseolina causing leaf spot of
mungbean and observed that 38°C temperature favoured the maximum growth and sclerotial
production.
Chowdary and Govindaiah (2007) studied the growth and sclerotial production of M.
phaseolina (inciting root rot of mulberry) at different temperature in vitro and found that
growth was found maximum (90 mm) at 30 and 35°C and minimum (40 mm) at 20°C. The
number of sclerotia/9 mm disc was found maximum (162.3 sclerotia) at 40°C and gradually
reduced with the decrease in temperature to 35°C (150.3 sclerotia), 30°C (120.0 sclerotia)
and 20°C (8.66 sclerotia). It failed to produce any sclerotia below 20°C.
2.10.3 Variation in pathogenicity
Mukherjee (1956) suggested the possibility of existence of pathogenic races of the
fungus as indicated by the differential response of same variety under different conditions.
During pathogenicity test on seedlings of JRD-632 (corchorus), B-43 was the most virulent
28
of the eight forms followed by B-46 and B-50, while B-45 and B-35 were least severe in their
action.
Sulaiman and Patil (1966) reported that out of 19 isolates of M. phaseolina on cotton,
race-1 caused 100 per cent mortality of host plants, while race-2 caused 59.4 per cent
mortality. Dhingra and Sinclair (1973) reported 80 to 100, 60 to 100, 30 to 60 and 10 to 20%
mortality of wound inoculated soybean plants with root, petiole, stem and pod isolates,
respectively.
Than et al.,(1991) working on relationship among R. bataticola isolates in rice based
cropping systems, based on colony fusion types, concluded that the isolates from soybean
and bean were related to the chickpea isolates. Subramanian (1994) studied the variation
among M. phaseolina isolates of sorghum from Andhra Pradesh, Karnataka and Maharashtra
and reported that all the isolates were pathogenic on sorghum (CSH-5) and to their respective
hosts. He also made studies for the first time on ‘detached stem technique’ which revealed a
higher length of spread among the sorghum isolates in comparison with non-sorghum
isolates.
According to Jana et al., (2003) design and use of a RAPD primer (OPA-13) was able
to distinguish M. phaseolina isolates from soybean, sesame, ground nut, chickpea, cotton,
common bean, and other hosts, however the study was confined to a particular area of the
United States including states like Arkansas, Texas and Alabama. Through AFLP analysis,
Vandemark et al., (2000) concluded that it was impossible to obtain a correlation at the DNA
polymorphism level with geographic location or host. Even with these molecular approaches,
there is not sufficient evidence to suggest a formae specialis or subspecies within the M.
phaseolina. It was noted that genetic variation has always been evident among M. phaseolina
isolates. Even isolates taken from a single host have different levels of pathogenicity. Using
114 isolates representing four host families and two continents, Mihail and Taylor (1995)
were able to obtain hyphal fusions between M. phaseolina isolates from Somalia and
Arizona, a geographical scale in which geographical isolation would appear reasonable.
Their study suggested that M. phaseolina does not have genetic barriers for non-sexual
genetic interchange. A plausible explanation of the variability in M. phaseolina is presented
in studies conducted by Punithalingam (1983). This author reported that conidiogenous cells
and hyphal cells are initially uninucleate, but a single nucleus can undergo various mitotic
29
divisions and some conidia (pycnidiospores) possess up to 36 nuclei each. As the conidia
produce germ tubes the nuclei move into the developing hyphae dividing mitotically during
migration. The chromosome number most frequently observed is six, however, aneuploid,
haploid and diploid nuclei have been observed in different M. phaseolina isolates.
2.10.4 Sensitivity of M. phaseolina isolates to chemicals
Owing to less availability of investigation relating to sensitivity of different isolates to
copper sulphate and carbendazim, review of work relating to other chemicals has been
recorded in this section. Some of the fungicides which have been reported to be effective
against M. phaseolina are captan, thiram and agallol (Grewal and Vir, 1958; Clinton, 1960;
Bhargava, 1965; Sahai, 1969 and Masih et al., 1970). Brassicol (Pentachloronitrobenzene) a
soil fungicide that has been reported to be effective against M. phaseolina isolated from
various crops other than sorghum (Geol and Mehrotra, 1973; Shanmugan and Govindswamy,
1973 and Ilyas et al., 1975). Further, it has also been reported that M. phaseolina has
developed resistance against increasing concentrations of brassicol (Grover and Chopra,
1970; Mathur and Singh, 1973) other workers also reported that brassicol did not control the
disease in field trials (conducted between 1976-78). During in vitro evaluation on PDA, it
inhibited the growth upto 86.75 per cent at 5000 ppm, but in soil at the same concentration,
inhibition was only upto15.25 per cent (Anahosur and Patil, 1983). Among the systemic
fungicides, benomyl has been reported to be most effective (AL-Beldawi et al., 1973 and
Ilyas et al., 1975). Rajkule et al., (1979) reported that soil treatment with thiram at sowing,
did not effectively control charcoal rot, but reduced it by 25 per cent. Satishchandra (1977)
observed that, saprophytic activity of R. bataticola was controlled to a large extent with
brassicol and captan seed treatment. Ramamurthy (1982) reported that carbendazim, captan
or thiram when applied as soil drench, effectively reduced the cotton root rot caused by R.
bataticola.
Taneja and Grover (1982) reported complete control of sunflower and sesamum root
rot, by seed treatment with benomyl or thiophanate methyl at two gram product per kg seed.
Anahosur et al., (1984) found that an herbicide called ametryne inhibited the growth of the
pathogen (28.5 to 60.15% at 250 to 2000 ppm). Sekhar (1985) observed that rhizolex
followed by quintozene were significantly superior in suppressing saprophyte activity of M.
30
phaseolina. Hundekar (1987) reported that carbendazim and rhizolex were effective against
M. phaseolina and thiram was effective among non-systemic fungicides.
Pall et al., (1990) opined that seed treatment with MBC (carbendazim) followed by
thiram were the best fungicides for controlling M. phaseolina in urd and green gram. Reddy
et al.,(1991) found that, carbendazim was the most effective fungicide when tested in vitro,
along with five other fungicides for controlling R. bataticola (M. phaseolina) and other seed
borne fungi on groundnut. The next best chemicals were carbendazim + thiram and captan. In
vivo studies relating to seedling vigour, showed that carbendazim + thiram was the most
effective treatment for enhancing seed germination, shoot and root growth and total dry
weight per plant.
Gupta and Roy (1993) opined that S-methyl-S, S-diphenyl phosphorotrithioate was
most active against R. solani, while its P-methyl analogue was more effective against R.
bataticola (M. phaseolina). Ramdoss and Sivaprakasan (1987) reported inhibitory effect of
carbendazim and quintozene, carbendazim at 100 ppm and thiram at 500 ppm were
fungicidal, while quintozene and all insecticides were fungistatic.
Alagarsamy and Sivaprakasan (1988) reported that carbendazim did not show any
adverse effect on T. viride and T. harziamum in vitro and in pot culture studies. Patel and
Patel (1990) reported that fungicides benomyl, thiram and carbendazim were on par and
significantly more effective than rest of the fungicides when tested in vitro against M.
phaseolina which caused charcoal rot of sesamum. Complete growth inhibition was obtained
with benomyl (0.05%) while copper oxychloride gave minimum growth inhibition (57.70%)
while mancozeb showed non-significant difference for growth inhibition when compared
with the control treatment.
Singh and Kaiser (1995) reported that among the eight fungicides tested under in
vitro and field conditions, carbendazim was found most effective against M. phaseolina.
31
2.11 MANAGEMENT
2.11.1 Through diverse germplasm
Sources of resistance to some soil-borne pathogens have been identified but highly
resistant cultivars are often not available for polyphagous and unspecialized pathogens like
M. phaseolina. Pastor-Corrales and Abawi (1988) reported some selected bean lines with
stable resistance to the fungus. Resistance in beans to M. phaseolina has been associated with
drought tolerance. In common bean resistance to M. phaseolina is controlled by two
dominant complementary genes (Olaya et al., 1996). Grezes-Besset et al., (1996) reported
resistance to M. phaseolina in Ricinus persicus (Castor-oil Plant) and incorporated this in
cultivated castor bean. The improved lines showed high seedling resistance to the disease.
Based on seed yield and the levels of lower stem and tap root colonization, Smith and Carvil
(1997) identified four resistant cultivars among 24 soybean cultivars screened for resistance
to M. phaseolina. Research on sources for resistance to M. phaseolina in sorghum has led to
breeding lines and cultivars with stable performance. Resistance in sorghum was associated
with delayed leaf senescence (Ducan, 1984). The durability of this resistance, however, was
influenced by water stress.
Attempts to find out sources of resistance to charcoal rot for breeding programs were
started in USA in the 1940’s. In one of the most comprehensive testing programs, Hoffmaster
and Tullis (1944) screened 232 sorghum lines of diverse genetic background at four locations
for four years. Although, they found differences in the stability of performance of the lines
from year to year, they concluded that it was impossible from the data available to
recommend certain varieties for localities in which Macrophomina dry rot is a limiting
factor. Lack of stability to different levels of drought stress and hence different levels of pre-
disposition to the disease was evident. Patil (1980) tested 180 genotypes and recorded the
resistant genotypes for charcoalrot disease namely SPV-249, BJ-112, SBP-315, SPV-324,
SPV-326, SPV-346, SPV-352, SPV-353. Hiremath and Palakshappa (1991) recorded
resistant genotypes such as SPV-967, SPH-296 and SPH-509. DSV-4 (9-13) is a high
yielding sorghum with built in resistance and released in Karnataka (as Rabivariety) (Singh
et al., 1990).
32
2.11.2 Management through fungicides
Ilyas et al., (1975) studied the efficacy of various fungicides and observed that
carbendazim, quintozene and mancozeb reduced M. phaseolina population under laboratory
conditions.
Alagarsamy and Sivaprakasam (1988) conducted studies on cowpea post emergence
mortality caused by M. phaseolina and they suggested that there was no adverse effect of
seed treatment on carbendazim with biocontrol agents apart from reducing the mortality.
Hooda and Grover (1988) reported inefficacy of five fungicides seed treatments
(Carbendazim, Thiophanate-methyl, Captafol, Thiram, and PMA) for the control of charcoal
rot on five different plant species. Under in vitro conditions, mungbean and cotton root
exudates reduced the efficacy of Carbendazim and Captafol at low concentrations in more
than 50% compared to the non-treated control. Researchers also have investigated the in vitro
sensitivity of different isolates of M. phaseolina to fungicides (Al-beldawi et al., 1973) and
the efficacy of fungicide application to seed and soil to reduce fungal germination and
infection (Kannaiyan et al., 1980; Alice et al., 1996). However, until now, chemical control
of M. phaseolina is difficult and neither profitable nor advisable (Pearson et al., 1986).
Bavistin 50 WP [carbendazim] was the most effective against M. phaseolina, a major
pathogen of mothbean (Rathore and Rathore, 1999). Application of carbendazim in
combination with Thiram resulted in the highest seed germination percentage and lowest root
rot incidence in chickpea (Prajapati et al., 2003).
Kulkarni (2000) screened various chemicals against safflower root rot caused by M
.phaseolina and concluded that carbendazim and propiconazole were the most effective
fungicides.
2.11.3 Management through plant extracts
To avoid the implication of yield losses due to plant diseases, variety of control
measures presently are in use. Chemical control of M. phaseolina is not much effective and
economical because the pathogen is seed borne and difficult to eradicate. This situation
demands an alternative approach for management of seed borne and other saprophytic and
parasitic fungi.
33
Striubaite (1960) reported that Ranunculus lanuginosus extract was more toxic than
that of R. polyanthemos. During the same year, Peturshova (1960) also observed the highest
antifungal activity in the plant species belonging to families: Ranunculaeceae, Crucifereae,
Liliaceae, Rosaceae, Solanaceae and Anarcadiaceae.
Abdullaeva (1962) studied the antifungal activity of Allium sativum and A. cepa
extracts against Verticillium dahliae, Fusarium oxysporum f. sp. vasinfectum and Rhizoctonia
sp. by the inhibition zone method. Out of those, onion extract showed more antifungal
activities to volatile fractions. Mkervali (1963) tested the sap extracts of Rhusveria, Allium
sativum and decoction of Grocus pulps (in vitro) against Colletotrichum lauri and found it
highly toxic in this fungus.
Nene and Thapliyal (1965) studied the antifungal properties of the extracts of
Anagallis arvensis. It was found to be effective against Colletotrichum papayae L. This
extract was effective against the test fungus even at its very low concentration. Nene and
Kumar (1966) for the first time reported the antifungal properties of Erigeron linifolius
against Helminthosporium turicum. During 1968, Kumar and Nene reported that the leaf
extracts of Cleome isocandra checked the growth of the different fungi namely
Helminthosporium maydis, Alternaria solani and Sclerotium rolfsii. Besides leaf extracts,
roots, stem, flowers and seeds extracts also showed inhibitory effects on the growth of these
fungi. Thapliyal and Nene (1970) compared the influence of growth stages of Anagallis
arvensis L. and observed that antifungal properties of A. arvensis was gradually increased
from the stages of seedling to flower initiation and later decreased with the advance in the
age of the plant.
Misra et al., (1974) tested leaf extracts of Allium sativum and Ranunculus clematis on
spore germination of Alternaria alternata, Helmithosporium gramineum and Curuvlaria
lunata and reported that plant extracts showed antifungal activity against all these test fungi.
Dixit and Tripathi (1975) studied that the extracts of Brassica compestris, B. oleraceae, and
Raphanus sativus showed strong fungistatic activity against Cephalosporium sacchari and
Fusarium nivale. While the extracts of B. juncea and B. pekinensis stimulated the
germination of the above fungi. According to (Dixit et al., 1976) rose flowers possessed
antifungal principles against Cephalosporium sacchari, Curvularia pallescens and Fusarium
34
nivale. Gallic acid present in the extract was found to be responsible for their antifungal
activity, even at 3 percent concentration.
Misra and Dixit (1977) found that the leaf extracts of Allium sativum, Clematis
gouriana and Ranunculus scleratus inhibited the spore germination of 30 test fungi. Some of
these were Alternaria alternata, Curvularia lunata, Fusarium nivale and Helminthosporium
gramineum. During the same year, Singh et al., (1977) studied the influence of aqueous
extracts of eight Indian Ferns and three gymnosperms on three soil fungi: Colletotrichum
flacatum, Fusarium oxysporum and Helminthosporium sativum. Out of these, Araucaria
bidwillii and Lindsaya spp. showed maximum antifungal activity.
Pariya and Chakravarti (1977) reported antifungal activity in the plant extracts of
Allium cepa, Euphorbia ligularia, Glycyrriza glabra, Embelia ribes and Tinospora cordifolia
against Helminthosporium oryzae, Alternaria alternata, Sclerotium rolfsii and Aspergillus
Niger. They observed that the growth of Helminthosporium oryzae was completely checked
by the extract of onion bulb, Sclerotium rolfsii by the root and stem extract of T. cardifolia;
A. alternata by the seed extract of E. ribes, G. glabra, root extract and stem and root extract
of T. cordifolia. Egawa et al., (1977) in Japan identified antifungal substances from the
extracts of eleven spp. of Eucalyptus those inhibited the conidial germination of Alternaria
solani and Cochliobolus miyabeanus. Narain and Satapathy (1978) reported that leaf, flower,
stem and root extracts of Vinca rosea showed antifungal activity against Helinthosprium
nodulosum, Sclerotium rolfsii, Fusarium oxysporum, Colletotrichum sp. and Aspergillus
niger. The antifungal activity of leaf extracts was found to be higher than stem and root
extracts.
Misra and Dixit (1978) studied the antifungal properties of Clematis gouriana and
found that the leaf extract of C. gouriana completely inhibited the growth of Alternaria
alternata, Curvularia lunata, Fusarium nivale and Helminthosporium gramineum. The
fungitoxic principle was identified to be protoanemonin and was found to be strongly
fungitoxic even at dilution of 1:10000. Misra and Dixit (1978) also tested the antifungal
properties of leaf extract of Ranunculus scleratus against 26 test fungi including Alternaria
alternata and Fusraium nivale etc. actively acquired the rmostable upto 10°C and retained
activity on autoclaving and for upto 15 days at room temperature. The extract was found to
be lethal to fungal spores at 1:40 dilution.
35
Kemp (1978) reported Falcarindiol, as an antifungal substance, in Aegopodium
podagraria. Falcarindiol was found to be inhibitory to the growth of Helminthosporium
gramineum and Fusarium nivale. Misra (1978) studied the antifungal principles of vapours
of some medicinal plants and reported that the growth of the test fungi namely Alternaria
alternata, Curvularia lunata, Fusarium nivale and Helminthosporium gramineum etc were
inhibited partially and completely. According to Kumar et al., (1979) aqueous extracts of
onion, garlic, kalanchoe, Parthenium histopum, cotton and Phaseolus atropupureus
completely inhibited the spore germination of Alternaria alternata, Fusarium oxysporum,
Drechslera rostrata and Corynespora cassiicola.
During 1979, Chaumont tested the fungistatic properties of the extracts of eight
flowering plants against 51 fungi. Out of these, Fusarium oxysporum, Phytophthora spp.,
Pythium spp., Pyrenchaeta spp., Verticillium sp., were the most resistant fungi. Singh et al.,
(1979) reported that maximum effects were produced by the seed extract of Trachyspermum
ammi against Rhizoctonia solani, Fusarium chlamydosporum and Curvularia lunata.
Blackman and Atkinson (1979) reported that leaf and seed extracts of Chrysanthemum
parthenium in chloroform solvent inhibited the growth of certain filamentous fungi, yeast
and also certain gram positive bacteria.
In 1980, Renu and his associates, studied the fungitoxic properties of leaves of
Cestrum diurnum and observed that the mycelial growth of all the 39 test fungi including
Alternaria alternata, Helminthosporium gramineum and Fusarium nivale was inhibited.
Tripathi et al., (1981) tested the antifungal activities of pollens of various plants against
Alternaria solani, germination of Aegle marmele completely inhibited spore germination of
Alternaria solani but some of the pollens also stimulated the spore germination. Kapoor et
al., (1981) observed that petal extracts of five members of Convolvulaceae almost
completely inhibited the spore germination and mycelial growth of Alternaria brassicicola,
A. brassicae and Fusarium oxysporum. The extracts from Convolvulus pluricaulis and C.
alsinoides were also completely fungicidal against all these test fungi.
Biswas et al., (1981) reported that the leaf extracts from Didymocarpus pedicellata
inhibited several plant pathogens including Alternaria alternata, Sclerotium rolfsii and
Helminthosporium oryzae. They observed that extracts inhibited the growth of these fungi
completely or partially. Bhowmick and Vardhan (1982) studied the leaf extracts of ten
36
medicinal plants. Extracts from Vitex negundo and Cotharanthus roseus completely inhibited
the growth of Drechslera turcica.
Pandey et al., (1982) reported that ehtanolic extracts of seeds of Dodonaea viscose,
soybean, lentil Leonotis nepetaefolia, Paspalum scrobiculatum and Peltophorum
pterocarpum exhibited 100 percent antifungal activity against Alternaria alternata.
Bhowmick and Choudhary (1982) tested the antifungal principles of leaf extracts of some
medicinal plants against Alternaria alternata. The leaf extracts obtained from Acalypha
indica, Vitex negundo and Azadirachta indica completely inhibited the growth of A.
alternata. Chaudhari and Sen in 1982 observed the benzene extract from the Piper nigrum
showed considerable fungitoxic activity against Sclerotium rolfsii, moderately against
Rhizoctonia solani and the least against Sclerotinia sclerotiorum. The extract was
comparatively inhibitorier to mycelial growth than to sclerotial growth.
Annapurna et al., (1983) observed a broad spectrum antimicrobial activity in leaf
extract of Polyalthia longifolia against different fungi including Alternaria alternata and
some bacteria. Agarwal et al., (1983) tested the effects of extracts of different common plants
and found that extract of onion, garlic, neem, holybasil, Leucas sp. and lemon were partially
to completely inhibitory to the spore of Colletotrichum graminicolum and C. capsici. Renu
(1980) screened leaf extract of 30 species of higher plants. Out of these, Aogle marmelos and
Cestrum diurum exhibited better fungitoxicity. The activity was unaffected even by heating
to 100°C.
Jaiswal et al., (1984) observed the antifungal efficiency of root oil of various plants
against some fungi including Fusarium solani, Aspergillus flavus and Cladosporium sp.
According to Strivastava et al., (1984) the antifungal activity of extracts of inflorescence,
leaves and stem of Parthenium hysterophorus showed minimum inhibition against
Aspergillus fuigatus, A. nigerand Microsporum gypseum.
Rafiq et al., (1984) tested the antifungal activity of five wild plants namely Anagallis
arvensis (Billi booti), Ashodelus tenuifolius (Piazi), Chinopodium album (Bathu),
Chinopodium murale (Karund) and Convoluvlus arvensis (Lehli) against Helminthosporium
turcicum, H. oryzae, H. carbonum, H. maydis, Alternaria lini and A. brassicicola. Out of
these, the water extracts of leaf of Anagallis arvensis inhibited the growth of H. trucicum, H.
oryzae, H. maydis and H. carbonum. Whereas, stem extract of A. arvensis showed antifungal
37
activity against H. turcicum and H. carbonum. However, none of plant showed activity
against A. lini and A. brassicae. Akhtar et al., (1986) tested the alcoholic extract of garlic,
pepper, neem, onion, ginger and turmeric against Fusarium coeroleum, the cause of potato
dry root. Among these, maximum antifungal activity was shown by turmeric extract.
Bhatti in 1988 tested the leaf decoctions of 16 phanerogams against leaf rust of wheat
and observed that decoctions of Acacia milotica, Calatropis procera, Datura stramonium,
Dodonaca viscose and Rhazya stricta effectively controlled the rust on detached leaves of
wheat, whereas, Cassia senna enhanced the uredospore germination.
Dwivedi and Dubey (1986) discovered that volatile fractions of two medicinal pants.
Azadirachta indica and Eucalyptus globulus showed pronounced effect on the sclerotial
germination of Macrophomina phaseolina as compared to non-volatile fractions. Rizki et al.,
(1987) studied the antifungal properties of indigenous plants and tested the crude extracts of
50 higher plants belonging to 28 families against Aspergillus Niger, Alternaria spp. and
found that 18% of the plants possessed some antifungal activity.
Eswaramurthy et al., (1988) evaluated the effect of neem leaf water extracts on the
mycelial growth of rice sheath rot pathogen (Sarocladium oryzae) and onion bulb rot and wilt
pathogen (Fusarium oxysporum f. sp. cepae). It was observed that mean mycelial growth
inhibition of Sarocladium oryzae was 34% and that for Fusarium oxysporum f. sp. cepae was
24.7%. According to Saleem (1988) 2400 species of plants possess biologically active
compounds that control various pests. Among these, the neem tree (Azadirachta indica) is
listed as one of the most effective plant known to control various pests including fungi and
nematodes.
Khan (1989) studied the effect of seed and leaf extracts of neem (Azadirachta indica)
against Alternaria radicina and Helminthosporium turcicum, Ascochyta rabiei and
Macrophomina phaseolina. Seed extract showed antifungal activity (in vitro) against all the
test fungi, and leaf extracts showed activity against three out of four test fungi namely
Ascochyta rabiei, Alternaria radicina and Helminthosporium turcicum. Kazmi et al., (1991)
reported that n-Hexane extracted nee mol from samples of Hyderabad checked the growth of
Fusarium moniliforms the best and it proved to be near the Benlate whereas, neem oil from
Karachi significantly checked the growth of Rhizoctonia solani.
38
Scientists are on their way to achieve some plant derived compounds to control
diseases (Singh et al., 2004). Natural products or plant derived compounds contribute to a
great extent in fighting against pathogenic microorganisms (Vyvyan, 2002). Aqueous extract
of many allelopathic plants are known to exhibit antifungal properties. Plant extracts or plant
derived compounds are likely to provide a valuable source of new medicinal agents
(Carvalho and Ferreira, 2001; Kayser and Kiderlen, 2001) and the urgent need for alternative
treatment has led to screen natural products for potential use in the therapy of leishmaniasis
and fungal infections. Bajwa et al., (2001) found inhibitory potential in aqueous extracts of
three asteraceous allelopathic species against growth of Aspergillus Niger. Recently Shafique
et al., (2005) have reported that the aqueous extracts of allelopathic plants have considerable
potential to control seed borne mycoflora. With the objective to contribute to these studies,
the antifungal activity of crude extracts obtained from P. hysterophorus and A. conyzoides
was investigated against M. phaseolina.
Several studies have dealt with the antimycotic effect of plant compounds on M.
phaseolina. The essential oil actinidine isolated from Nepeta clarkei was effective in vitro
against M. phaseolina (Saxena and Matela, 1997). Kazmi et al., (1995) reported that in vitro
neem oil was more or equally effective compared to benomyl and carbendazim. However,
neem seed extracted samples of different locations showed variable suppression of growth of
the pathogen. More effective inhibition of the growth of M. phaseolina was obtained by
aqueous extracts of Cymbogon citratus (Bankole and Adebanjo, 1995). Powder of Datura
fastulosa (Datura) was also reported to be effective against M. phaseolina and Meloidogyne
javanica infection in a pot experiment (Ehteshamul et al., 1996). The aqueous extracts of
Tephrosia candida and Boehmeria nivea could well inhibit the formation of sclerotia of M.
phaseolina (Anuradha et al., 2003). Extracts of pulverized bark of Prosopis africana and
leaves of Nauclea latifolia 100 % inhibited both radial mycelial growth and sclerotial
formation of M. phaseolina (Oluma et al., 2002). Datar (1999) found that aqueous extracts of
Polyalthia longifolia, Allium sativum and Parthenium hysterophorus were found most
effective in reducing mycelial growth of M. phaseolina. Neem leaf extract, Marigold leaf
extract and Garlic bulb extract at 5 % as seed treatments significantly reduced the charcoal
rot incidence and increased yield (Sinha and Sinha, 2004).
39
CHAPTER III MATERIALS AND METHODS
The present field level investigations were carried out during 2011 and 2012 at
research area of Plant Pathology at University of Agricultural Faisalabad, Pakistan, which is
situated on a longitude 73°74ˈ East, latitude 30°31.5ˈ North, with an elevation of 184 metres
(604 ft) above sea level. The average yearly rainfall 300 mm (12 in). The details of the
material used and the methodology followed during the course of the present investigation
are described in this chapter.
3.1 GENERAL PROCEDURE
3.1.1 Glassware and cleaning
Corning and Borosil glassware was used for all the experiments. The glassware was
kept in the cleaning solution containing Poassium dichromate (K2Cr2O7), concentrated
Sulphuric acid (H2SO4) @ 60 g and 60 ml respectively in one litre of water. After immersing
in the cleaning solution, all glassware was washed with detergent powder and rinsed with tap
water followed by a final rinse with distilled water.
3.1.2 Sterilization
All glassware was sterilized in a hot air oven at 160°C for two hours. Both solid and
liquid culture media were sterilized at 1.1 kg/cm2 for 20 minutes. Soil used for experiments
was sterilized for two hours at 1.33 kg/cm2 in an autoclave.
3.2 COLLECTION OF DISEASED SPECIMEN
Charcoal rot affected maize plants were collected from farmer’s fields of major maize
growing districts Kasur, Pakpatan, Okara and Sahiwal.
3.3 ISOLATION, IDENTIFICATION AND MAINTENANCE OF
Macrophomina phaseolina
Potato Dextrose Agar (PDA) medium was used for culturing the fungus during this
study. PDA was prepared according to the following recipe;
Peeled and Sliced potatoes = 200g
Dextrose (C6H12O6 ) = 20g
Agar = 20g
Distilled water to make up the volume = 1000 ml
40
The potatoes were boiled in 400 ml of distilled water and the extract was collected by
filtering through a muslin cloth. Agar was melted separately in 400 ml of distilled water. The
potato extract was mixed in the molten agar and 20 g of dextrose was added to the mixture.
The volume was made 1000 ml with distilled water and sterilized at 1.1 kg/cm2 pressure for
15 min. Infected portion of stalk was cut into small pieces of 5 to 7 mm. These bits were
surface sterilized with Sodium hypochlorite (0.1%) for one minute and rinsed thrice with
sterile water before transferring to sterile PDA in Petriplates. These plates were incubated at
27 ± 1°C for four days to obtain good growth of fungi. Colony morphology and formation of
sclerotia were the principle characteristics used for identification of pure cultures of M.
phaseolina. These features were compared according to the descriptions of Ashby (1927) and
Goidanich (1947). Further purification of cultures was done after 15 days of fungal growth
on PDA slants and cultures were preserved in refrigerator at 4°C for further experimental
use.
3.4 MASS-CULTURING OF M. phaseolina
Seeds of rice were moistened (1 g rice seeds: 1 ml water) and placed inconical flasks.
Each flask was closed with cotton plug, wrapped in aluminum foil and autoclaved at
kg/cm2at 121°C for 3 h. After cooling for 12 h, the flasks were again autoclaved at 121°C for
another 3 h. Upon cooling, the seeds were inoculated with a 5 mm diameter mycelial plug
from a 7-day old culture of M. phaseolina and incubated at 27±1°C for 15 days. The flasks
were shaken at alternate days for uniform colonization of the grains. The inoculum was
stored at 4°C until used in the field. For confirmation of the fungus, the colonized rice seeds
were plated on PDA plates and incubated at 27±1°C for 5 days. The plates were examined
under stereoscope for mycelial growth.
41
Table 3.1 Locations for the collection of M. phaseolina isolates
SR.NO District Location
1
KASUR
Bheala
2 Garewala
3 Talwandi
4 Atari
5 Noor Pur
6 Khudian Khas
7
OKARA
Aktharaabad
8 Ahmadabad
9 Basir Pur
10 Havali Lakha
11 Hujra Shah Muqeem
12 Renala Khurd
13
SAHIWAL
Kassowal
14 Chak 42/12 L
15 Chak 21/11 L
16 Chak 44/12 L
17 Adde Pur
18 Bashera
19
PAKPATAN
Chak 17 SP
20 Jaman Bodla
21 Bunga Hayat
22 Malka Hans
23 Chak 50 SP
24 Chak 30 SP
42
(6 Isolates) (6 Isolates) (6 Isolates) (6 Isolates)
Plate.1. Map of Pakistan and Punjab
43
3.5 TEST OF VIRULENCE OF THE ISOLATES
For inoculation, toothpicks infested with M. phaseolina were used. To prepare
infested toothpicks M. phaseolina culture in potato dextrose broth was used. Potato dextrose
broth was prepared in water with 200 g potatoes, 20 g dextrose per litre (Tuite, 1969). The
potato broth was sterilized at 1.1 kg/cm² for 15 minutes in 100 ml flasks. After cooling,
respective M. phaseolina isolates were inoculated to two 100 ml flasks to obtain a rich
suspension. Tooth picks were boiled in water for two hours and rinsed with sterilized water
to remove toxic substances that may inhibit the growth of fungi. When tooth picks became
dry, ten of them were placed in 100 ml flasks and were sterilized at 1.1 kg/cm² for 20
minutes. The suspension, prepared earlier, was poured into two tooth pick flasks to cover
lower one third of the tooth picks. Thus, for each isolate, two separate sets of tooth picks
were prepared. The flasks were inoculated for seven days at 30°C, by which time, tooth
picks, were covered with the fungal growth and were ready for inoculation. Maize plants of
Sahiwal 2002 (highly susceptible variety) were inoculated 15 days after flowering on second
internode at 45˚ angle by piercing a sterilized needle (1-2 mm diameter) followed by
insertion of the infested tooth picks.
3.5.1 Detached stem technique
Thirty centimeter basal stalk region, excluding the root portion of 70 days old Maize
plants raised in pots were inoculated at the second internode with a 7 day old culture of 24
different isolates. Three replications were maintained for each of the 24 isolates. The stalks
were incubated at 27 ± 1°C and split open on the eighth day. The spread of infection was
recorded and observations were subjected to statistical analysis (Subramaniam, 1994), to
know the variation in the pathogenicity of individual isolate of M. phaseolina to Maize
cultivar (Sahiwal 2002).
3.5.2 Confirmation of pathogencity
Observations for charcoal rot were made after 120 days after sowing on Sahiwal 2002
Maize in pot as well as in field condition. Detached stalks were split open after eight days
and observed for pathogencity. Reisolation of the pathogen from infected stalks was done
and the reisolated cultures of the pathogen were compared with their respective original
cultures.
44
3.6 STUDIES ON VARIABILITY AMONG THE ISOLATES
The virulence variation in 24 isolates of M. phaseolina was studied by employing the
following techniques.
3.6.1 Morphological characters on PDA and Corn Meal Agar Media
Fifteen ml of the medium was poured into each of the 90 mm petridishes. One ml of
100 ppm streptomycin sulphate was added to the medium just before pouring intothe plates.
Inoculation was made by transferring 0.5 cm growth disc of M. phaseolina taken from the
periphery of seven-day-old culture. The separate inoculations were made for each of the 24
isolates. The plates were incubated at 27± 1°C. Differences in morphology, rate of growth
and days to form sclerotia were recorded. For each of the 24 isolates grown after stipulated
period, the growth of each isolate was measured in terms of colony diameter with the help of
verniarcalipers and their means were computed. For measuring sclerotial size, slides from
seven days old pure culture of M. Phaseolina isolates were prepared and examined under
microscope. Sizes of ten randomly selected sclerotia were measured using ocular micrometer
and their means were found.
3.6.2 Cultural characters on Potato Dextrose and Corn Meal Media
3.6.2.1 Growth on solid media
Colony diameter on PDA and corn meal media (Tuite, 1969) was recorded from the
second day, till the growth completely covered the plates (fourth day).
3.6.2.2 Growth on liquid media
Twenty ml of potato dextrose broth (PDB) and corn meal broth (CMB) were taken
separately in each of the 150 ml flasks. These flaks were sterilized at 1.1 kg/cm² for 10
minutes. Inoculum disc of five mm diameter of each of 24 isolates, taken from periphery of
seven-day-old cultures were transferred asceptically to the flasks. Each treatment was
replicated three times. The flasks were incubated at 27 ± 1°C. Since the growth of the fungus
was found to be the maximum on the eleventh day (Satishchandra, 1977 and Ramamurthy,
1982) of incubation, all the cultures were harvested on the eleventh day. Cultures were
filtered through Whatman No. 1 filter paper disc of nine cm diameter and were dried to a
constant weight at 60°C. The mycelial mat on the filter paper was washed with distilled
water to remove any matter likely to be associated with the mycelium. The filter papers,
along with mycelial mat, were dried to a constant weight in an electric oven at 60°C and
45
then, cooled in desicator and weighed immediately on an electric balance and weight of dry
mycelia was recorded. The data were analyzed statistically.
3.7 SENSITIVITY OF ISOLATES TO CHEMICALS
3.7.1 Sensitivity of isolates to Copper sulphate and Benomyl
Sensitivity of all the 24 isolates to copper sulphate and benomyl was tested by
following standard ‘Poisoned Food Technique’ (Schmitz, 1930). In this study, sterilized PDA
medium was prepared and copper sulphate and benomyl were incorporated separately into
the molten medium aseptically so as to get the required concentration of 500, 1500 and 2500
ppm. Twenty ml of molten medium was poured into 90 mm petriplates. After solidification
of medium, plates were inoculated with five mm discs obtained from the periphery of a seven
day old culture for each of the 24 isolates. The plates were incubated at 27 ± 1°C.
Unamended medium served as control. The treatments were replicated thrice and
observations for each of the three concentrations maintained in three replications were
recorded, when the fungal growth reached maximum growth of 90 mm in their respective
controls. The results were expressed as per cent inhibition of the mycelial growth over the
control by using formula given by Vincent (1927).
I = C – T x 100
C
Where,
I = Per cent inhibition
C = Rate of growth in the control T = Rate of growth in the treatment
3.8 EFFECT OF DIFFERENT pH LEVELS ON THE ISOLATES OF M. phaseolina
Isolates of M. phaseolina were grown on the Potato Dextrose Broth in selected pH
levels of 5.0, 6.0, 6.5 and 7.0. The pH levels were adjusted by adding 1N alkali (NaOH) or
acid (HCl). Seven days old, five mm mycelial discs of the isolates were inoculated separately
into conical flasks containing 30 ml medium at different pH levels. Three replications were
maintained. The flasks were incubated at 27± 1°C. After 10 days, the mycelia were
harvested, washed and dried in hot air oven and the dry weights were recorded and analyzed
statistically.
46
3.9 EFFECT OF DIFFERENT TEMPERATURE LEVELS ON THE ISOLATES of M.
phaseolina
Growth of each isolate was tested at 20, 25, 30, 35 and 40°C. Thirty ml of potato
dextrose broth was poured into 150 ml conical flasks and sterilized. Seven days old, five mm
mycelial discs of the isolates were inoculated separately into conical flasks. Three
replications were maintained and incubated at selected temperatures. The cultures were
filtered through Whatman No. 1 filter paper and dry mycelial weight was recorded and
analyzed statistically.
3.10 PATHOGENIC VARIABILITY AMONG ISOLATES
All the 24 isolates were inoculated to a susceptible variety Sahiwal 2002 and to a
popular cultivar MMRI yellow. The Randomized complete block design with three
replications for each of the 24 isolates was adopted. In the field experiment, care was taken to
see that the area chosen was homogenous for soil factors and was kept to the minimum
possible extent, so as to; overcome soil heterogeneity. Each of the isolates was inoculated by
toothpick method at the second internode, 15 days after flowering. Observations were
recorded after 110 days of sowing. The data was collected by measuring the length of spread
with the help of scale.
3.11 EFFECT OF SOWING DATE AND TIME OF INOCULATION ON DISEASE
Influence of various sowing dates (10, 15 and 20 days after optimum sowing date)
and inoculation time (50, 60, 70, 80, 90 days after sowing) was applied to maize. The
treatments were repeated thrice.
3.12 EVALUATION OF PLANT EXTRACTS FOR THEIR EFFECTIVENESS
AGAINST M. phaseolina.
Aqueous extracts of five different plant species were used for their evaluation against
phytopathogenic fungus M. phaseolina in vitro and pot culture assay. For the prepreation of
plant extracts leaves of test plants were surface sterilized for 2 minutes in 70% ethanol.
Samples were then rinsed twice in sterilized distilled water, dried under room temperature for
21 days and ground to powder separately. For preparation of aqueous extracts of crushed dry
sample of each plant was soaked in sterilized distilled water at 1:1 w/v, vigorously stirred and
left for 24 hrs. The suspensions were passed separately through 4 ply muslin cloth, filtered
through Whatman’s filter paper No.41. The filtrates were further passed through Millipore
47
filter of 0.2 μm pore size to avoid the bacterial contamination and stored at 4°C until use. The
extracts thus obtained were arbitrarily termed as ‘S’ (100 %). Further dilutions were prepared
by the addition of requisite amount of distilled water.
Table 3.2 Description of plants
SR-N0 COMMON NAME BOTANICAL NAME PLANT PART USED
1 Neem Azadirachta indica Leaf
2 Shisham Dalbergia sissoo Leaf
3 Datura Datura stramonium Leaf
4 Aak Calotropis procera Leaf
5 Sohanjna Moringa oleifera Leaf
3.12.1 In vitro evaluation of plant extracts
For in vitro evaluation of aqueous plant extracts, poisoned food technique (Nene and
Thapliyal, 1982) was used. Potato dextrose agar (PDA) medium was used in the study.
Twenty five ml of sterile plant extract from each concentration was mixed with 175 ml
potato dextrose agar medium amended with chloramphenicol and carefully agitated to allow
for proper mixing of extract and media. Aliquots of 15 ml of the amended media were
dispensed into 90 mm Petridishes. Once the amended agar had solidified, 5 mm discs from
the actively growing edge of a 7 days old PDA culture of M. phaseolina was placed in the
center of each plate and incubated at (25±1°C) for 6 days. The medium with Inoculum
discbut without any extract served as control. Each treatment was replicated three times. The
inhibition of mycelial growth was determined by the following formula;
Control area – Treatment area
% Mycelial inhibition = ––––––––––––––––––––––––––––––––––––– x 100
Control area
3.12.2 Pot culture assay
Seeds of maize (Sahiwal 2002) were surface sterilized for 10 min in 1% commercial
sodium hypochlorite solution, washed in sterile distilled water and air dried. The seeds were
then soaked in different solutions of botanical extracts for 30 min and then air dried for 3-4
hours. Control seeds treated with sterile distilled water were planted in soils amended with
rice seeds colonized with M. phaseolina @ 2gm/kg soil. Ten seeds were planted in each pot.
Each treatment was replicated three times. Data on percentage germination/plant survival
was recorded after 20 days.
48
3.13 EVALUATION OF DIFFERENT FUNGICIDES FOR THEIR EFFECTIVENESS
AGAINST M.PHASEOLINA.
Five fungicides were tested for their efficiency to reduce the mycelia growth of M.
phaseolina in vitro and through pot culture assay.
Table 3.3 Fungicides description
SR-NO FUNGICIDE CHEMICAL NAME FORMULATION MANUFACTURER
1 Dithane M-45 Mancozeb 80 WP Rohm & Hass Ltd.
2 Derosal Carbendazim 50 WP Bayer (Pvt) Ltd.
3 Captan Captan 50 WP ICI (Pvt) Ltd.
4 Benlate Benomyl 50 WP Du Pont
5 Ridomil Gold Matalaxyl+Mancozeb 68 WP Syngenta
3.13.1 In vitro evaluation of fungicides
The efficacy of the test fungicides was evaluated by using poisoned food technique
(Nene and Thapliyal, 1982). Requisite quantity of active ingredient of each fungicide was
mixed in autoclaved PDA to obtain the required concentrations of 50, 100 and 150 ppm.
Poisoned medium was then poured into each sterilized 90 mm diameter sterilized Petriplate,
allowed to solidify and processed as described earlier.
3.13.2 Pot culture assay
For testing the effectivity of fungicides against M. phaseolina inpots, surface sterilized
seeds each of maize (sahiwal 2002) were treated @ 1, 2 and 3 gm of active ingredient of each
fungicide per kg of seed by slurry method. Control seeds were treated with sterile distilled
water. Ten seeds in three replications were sown in sterile pots containing a mixture of soil
and sand at the rate of 1:1 (v: v) amended with the rice seeds colonized with M. phaseolina
@ 2gm/kg soil. The pots were kept in growth rooms at 30 °C. Data on percent
germination/plant survival was recorded after 20 days.
3.14 COLLECTION OF MAIZE GERMPLASM FOR ESTABLISHMENT OF
DISEASE SCREENING NURSERY
Different hybrids commonly used by the farmers were collected from different seed
providing companies and also some local varieties which are mostly use by the people as
food from local market. Nineteen maize genotypes were screened against charcoal rot during
spring 2012 and spring 2013 at University of Agriculture Faisalabad. Screening was done in
49
infested plots infested @ 2g of rice seeds infected with M. phaseolina per meter of row
followed by artificial inoculation with toothpicks method. The susceptible check Sahiwal
2002 was sown after every two test entries. Observations on percent charcoal rot incidence,
mean length of spread (cm), mean number of nodes crossed and thousand grain weight were
recorded for screening purpose. The genotypes were graded using 0-9 scale (Mayee and
Datar, 1986) and grouped into respective categories as follows.
Table 3.4 Disease rating scale
GRADE PER CENT INFECTION REACTION
0 0 Immune
1 >1 Highly resistant
3 2-10 Resistant
5 11-25 Moderately resistant
7 26-50 Susceptible
9 51-100 Highly susceptible
3.15 MANAGEMENT OF CHARCOAL ROT OF MAIZE THROUGH ARTIFICIAL
INOCULATION AND INFESTED FIELD
In the management experiment treatment of seeds of Sahiwal 2002 was done with
Benomyl @ 1g/kg. 2g/kg and 3g/kg and Datura plant extract (@ 50%, 75% and 100%. Plot
was infested @ 2g of rice seeds infected with M. phaseolina per meter of row followed by
artificial inoculation with tooth pick method. Charcoal rot disease parameters are recorded at
physiological maturity (black layer stage) of the crop including length of spread and number
of nodes crossed.
3.16 STATISTICAL ANALYSIS
Collected data was interoperated by statistical analysis. All statistical tests were
performed by using MINITAB/STAT statistical analysis software (Minitab, 2010). For the
lab experiments CRD and for the field experiments RCBD design was used.
50
CHAPTER IV RESULTS
The present studies were conducted on various aspects of research project. These
aspects were focused on collection of samples, isolation and identification of the fungus,
pathogenicity, cultural behaviour of isolates and morphology of seclerotia, sensitivity
towards chemicals and response of different plant extracts on the growth of Macrophomina
phaseolina. The results are presented here under.
4.1 COLLECTION OF INFECTED PLANT SAMPLES AND
ISOLATION OF THE FUNGUS
Twenty four charcoal rot infected maize samples were collected from major maize
growing districts of Punjab (Kasur, Okara, Sahiwal and Pakpatan). All 24 diseased samples
were collected for variability study. Isolation of the fungus was done following standard
isolation procedure as described earlier.
4.2 IDENTIFICATION OF THE FUNGUS
The growth of the fungal mycelium on Potato Dextrose Agar (PDA) was fast on
comparative basis. Hyphae were branched and sparsely septate, fluffy and brown to black
initially. As the culture grewold, it turned black completely. Abundant production of sclerotia
was observed. The sclerotia were spherical to asymmetrical, dark brown, varying in size and
radiating to mycelium. Pycnidia were not observed in the culture. The morphological and
cultural typeset were the most important characters considered for identification of pure
cultures of M. phaseolina. These characters were compared with those described by Ashby
(1927) and Goidanich (1947) and the present fungus under study was identified as M.
phaseolina.
4.3 PATHOGENICITY
Pathogenicity test of M. phaseolina isolates was carried out on pot sown susceptible
maize variety (Sahiwal 2002) as well as in field sown crop by following tooth pick method,
apartfrom, detached stem technique, conducted under laboratory conditions as descried under
materials and methods. Results of pathogenicity test indicated that all the 24 isolates under
study were pathogenic to maize (25th days after inoculation under field condition and 7th days
after inoculation on detached stem) causing typical charcoal rot symptoms on maize. The
pathogen was reisolated and was found to resemble the original culture of M. phaseolina.
51
4.4 CULTURAL STUDIES FOR VARIABILITY
The cultural studies were conducted on solid medium PDA, corn meal agar as well as
on two liquid medium
4.4.1 Growth on solid media
Cultural and morphological features of 24 isolates of M. phaseolina were observed by
growing the isolates on PDA medium and corn meal medium as described in materials and
methods. The parameters taken into account for assessing the existence of variation were
colony characters, sclerotial size, days taken for sclerotial formation and number of sclerotia
per unit area.
4.4.1.1 Growth on Potato Dextrose Agar (PDA)
Based on the mean colony diameter the 24 isolates ranged from 88.1 to 59.1 (Fig 4.1)
and grouped into four categories. The first group consisted of 12 isolates with a spread of
80.0 to 88.2 mm diameter, second group included four isolates with a mean growth of 71.7 to
79.9 mm, third group included six isolates with an average colony diameter of 63.4 to 71.6
and the fourth group included two isolates with an average colony diameter of 55.1 to 63.3
(Table 4.2.1). Based on the fragmentation of colony characters the cultures were assigned to
four major groups viz., greyish white, medium black, deep black and blackish grey. Among
these, some produced feathery colonies and some produced flat colonies as indicated in
(Table 4.2.2).
The size of sclerotia varied from 29.0 µm to 90 µm (Fig 4.2). It was observed that
Khudian Khas and Atari isolates produced largest sclerotia, while the smallest sclerotia
produced by Chak 44/12 L and Kassowal isolate. Round shaped sclerotia were observed in
K-2, K-3, K-4, O-2, O-5, O-6, P-6, S-1, S-2 and S-6 isolates, while the oblong shaped
sclerotia were observed in K-1, K-5, K-6, O-1, O-3, O-4, P-1, P-2, P-3, P-4, P-5, S-3, S-4 and
S-5 isolates as indicated in (Table 4.3.2).
Based on average sclerotial size, the isolates were categorized into six groups (Table
4.3.1). Maximum numbers of isolates (7) were included in first group where the range of
sclerotial size was 79.9 to 90.1 followed by five isolates in 3rd group with a range of 59.3 to
69.5 mm, group 5th and six include four isolates in the range of (38.7 to 48.9 and 28.4 to 38.6
) respectively. However, minimum number (2) of isolates were found in 2nd and 4th group
52
where the range was (69.6 to 79.8 and 49.0 to 59.2) which indicated the variation in the
sclerotial size among the isolates.
Most of the isolates took 5-6 days for sclerotial formation. Among 24 isolates, 12
isolates took 5 days for sclerotial formation, 12 isolates took 6 days for sclerotial formation.
Maximum number of sclerotia of 56 per microscopic field (10x) was recorded in Chak 50 SP
isolate, while the minimum number was observed in Basir Pur (35) isolate (Fig 4.3).
The number of sclerotia/9 mm disc varied from 56 to 112 on 5th day, 75 to 147 on 7th
day and 99 to 179 on 9th day after inoculation (Fig 4.4). Among the isolates, Chak 17 SP,
Chak 42/12 L and Chak 44/12 L isolates produced comparatively large number of sclerotia/9
mm disc during 5th, 7th and 9th day, while the minimum number was observed in Hujra Shah
Muqeem, Renala Khurd and Chak 30 SP during 5th, 7th and 9th day (Fig 4.4).
53
Table 4.1.1: Collection of isolates of Macrophomina phaseolina
Sr. No Place of collection Code of isolate Part of the plant slected
Kasur
1 Bheala K-1 Infected stem
2 Garewala K-2 Infected stem
3 Talwandi K-3 Infected stem
4 Atari K-4 Infected stem
5 Noor Pur K-5 Infected stem
6 Khudian Khas K-6 Infected stem
Okara
7 Aktharaabad O-1 Infected stem
8 Ahmadabad O-2 Infected stem
9 Basir Pur O-3 Infected stem
10 Havali Lakha O-4 Infected stem
11 Hujra Shah Muqeem O-5 Infected stem
12 Renala Khurd O-6 Infected stem
Pakpatan
13 Chak 17 SP P-1 Infected stem
14 Jaman Bodla P-2 Infected stem
15 Bunga Hayat P-3 Infected stem
16 Malka Hans P-4 Infected stem
17 Chak 50 SP P-5 Infected stem
18 Chak 30 SP P-6 Infected stem
Sahiwal
19 Kassowal S-1 Infected stem
20 Chak 42/12 L S-2 Infected stem
21 Chak 21/11 L S-3 Infected stem
22 Chak 44/12 L S-4 Infected stem
23 Adde Pur S-5 Infected stem
24 Bashera S-6 Infected stem
54
4.4.1.2 Growth on Corn meal medium
Mean colony diameter four days after inoculation (4 DAI) colony diameter and
characters are indicated in (Fig 4.5) and (Table 4.4.2). The study revealed greater variation
among isolates with respect to growth rate. Based on mean colony diameter, the isolates were
grouped into four categories. The first group consisted of 8 isolates (K-1, K-2, K-3, K-4, K-5,
P-4, P-5 and P-6) with a spread of 80.0 to 87.2 mm, second group included six isolates (K-6,
S-2, S-3, S-4, S-5 and S-6 ) with a growth of 72.7 to 79.9 mm, third group included 5 isolates
(0-3, O-4, P-1, P-3 and S-1) with a mean spread of 65.4 to 72.6, fourth group also included
five isolates (0-1, O-2, O-5, 0-6 and P-2) with a mean spread of 58.1 to 65.3 (Table 4.4.1).
The study indicated the existence of considerable variation among the isolates. Based
on the colony characters in Corn meal medium the cultures were included in three group’s
viz., blackish gray, deep black and grayish white colour. Isolates could also be assigned
further into two groups based on the colony texture. Some isolates produced fluffy colonies
and some produced flat colonies (Table 4.2.2).
4.4.2 Growth on liquid media
All the 24 isolates were grown on potato dextrose broth (PDB) and corn meal broth
(CMB) and harvested on eleventh day of incubation and growth was studied as the growth
was maximum on that day (Subramaniam, 1994) as described under materials and methods.
Growth of different isolates on potato dextrose broth and corn meal broth was highly
significant (Fig 4.6) and isolates were categorized into 5 groups each based on their dry
mycelial weight on PDB (4.5.1) and four groups on corn meal broth (Table 4.5.2). Isolates
such as K-1, K-2, P-1 and P-3 produced exceptionally good growth in PDA (Fig 4.6).
Six isolates fallen in group first where in the range of dry mycelia weight was 277 to
319 mg. Five isolates were in group 2nd producing medium growth in the range of 234 to 276
mg and six in group third in the range of 191 to 233. Four isolates fallen in group 4th where in
the range of mycelial dry weight were 148 to 190 mg and three isolates in 5 th group with a
minimum range 105 to 147 mg on PDA as indicated in (Table 4.5.1)
Similarly, there was significant variation among the isolates, when grown in corn
meal broth (Fig 4.6). Group first included seven isolates which fell in the range of 224 to 266
mg dry mycelial weight. Group 2nd included eleven isolates falling in the range of 181 to 223.
55
Group 3rd included two isolate with a range of 138 to 180 and fourth included four isolates
recorded minimum 55 mycelia weight with a range of 95 to 137 (Table 4.5.2)
Fig 4.1: Growth pattern of isolates of M. phaseolina on PDA
Table 4.2.1: Grouping of isolates of Macrophomina phaseolina based on colony diameter
produced on PDA
Group Range
(mm)
Number of
isolates
Location of Isolates
1 80.0-88.2 12
Bheala, Garewala, Talwandi, Atari, Noor Pur,
Khudian Khas, Chak 17 SP, Jaman Bodla,
Bunga Hayat, Malka Hans, Chak 50 SP,
Chak 30 SP
2 71.7-79.9 4 Ahmadabad, Basir Pur, Havali Lakha,
Chak 44/12 L
3 63.4-71.6 6 Aktharaabad, Renala Khurd, Chak 42/12 L,
Chak 21/11 L, Adde Pur, Bashera
4 55.1-63.3 2 Hujra Shah Muqeem, Kassowal
56
Table 4.2.2: Colony characters of different isolates on PDA medium
Isolates codes Colony character
K1 Blackish gray, slight fluffy colony
K2 Medium black, flat colony
K3 Blackish gray, flat colony
K4 Blackish gray, flat colony
K5 Grayish white, fluffy colony
K6 Medium black, flat colony
O1 Medium black, flat colony
O2 Deep black, slight fluffy colony
O3 Blackish gray, flat colony
O4 Blackish gray, slight fluffy colony
O5 Deep black, slight fluffy colony
O6 Blackish gray, flat colony
P1 Medium black, flat colony
P2 Blackish gray, slight fluffy colony
P3 Blackish gray, flat colony
P4 Grayish white, fluffy colony
P5 Blackish gray, flat colony
P6 Deep black, flat colony
S1 Blackish gray, slight fluffy colony
S2 Medium black, flat colony
S3 Medium black, flat colony
S4 Blackish gray, slight fluffy colony
S5 Deep black, slight fluffy colony
S6 Grayish white, fluffy colony
57
Plate.2. Colony characters of isolates of M.phaseolina on potato dextrose agar
medium
58
Fig 4.2: Morphology of sclerotia produced by different isolates of M.
Phaseolina
Table 4.3.1: Grouping of isolates of Macrophomina phaseolina based on sclerotial size
Group Range (µm) Number of isolates Location of Isolates
1 79.9-90.1 7
Atari, Noor Pur, Khudian Khas,
Aktharaabad, Ahmadabad, Basir Pur,
Hujra Shah Muqeem
2 69.6-79.8 2 Havali Lakha, Renala Khurd
3 59.3-69.5 5 Bheala, Garewala, Talwandi,
Chak 17 SP, Jaman Bodla
4 49.0-59.2 2 Adde Pur, Bashera
5 38.7-48.9 4 Bunga Hayat, Malka Hans, Chak 50 SP,
Chak 30 SP
6 28.4-38.6 4 Kassowal, Chak 42/12 L, Chak 21/11 L,
Chak 44/12 L
59
Table 4.3.2: Shape of sclerotia observed in different isolates
Isolates codes Oblong Round
K1 Oblong
K2 Round
K3 Round
K4 Round
K5 Oblong
K6 Oblong
O1 Oblong
O2 Round
O3 Oblong
O4 Oblong
O5 Round
O6 Round
P1 Oblong
P2 Oblong
P3 Oblong
P4 Oblong
P5 Oblong
P6 Round
S1 Round
S2 Round
S3 Oblong
S4 Oblong
S5 Round
S6 Round
60
Fig 4.3: Time for initiation and density of sclerotia produced by different
isolates of M. phaseolina
Fig 4.4: Number of sclerotia in different isolates of M. phaseolina at
different time duration
61
Fig 4.5: Colony diameter (mm) and colony characters of isolates (M.
phaseolina) on corn meal medium
Table 4.4.1: Grouping of isolates of Macrophomina phaseolina based on colony diameter
produced on corn meal medium
Group Range
(mm)
Number of
isolates
Location of Isolates
1 80.0-87.2 8 Bheala, Garewala, Talwandi, Atari, Noor Pur,
Malka Hans, Chak 50 SP, Chak 30 SP
2 72.7-79.9 6 Khudian Khas, Chak 42/12 L, Chak 21/11 L,
Chak 44/12 L, Adde Pur, Bashera
3 65.4-72.6 5 Basir Pur, Havali Lakha, Chak 17 SP, Bunga
Hayat, Kassowal,
4 58.1-65.3 5
Aktharaabad, Ahmadabad,
Hujra Shah Muqeem, Renala Khurd, Jaman
Bodla
62
Table 4.4.2: Colony characters of different isolates on corn meal medium
Isolates codes Colony character
K1 Grayish white, slightly fluffy colony
K2 Deep black, flat colony
K3 Grayish white, slightly fluffy colony
K4 Blackish gray, slightly fluffy colony
K5 Blackish gray, flat colony
K6 Grayish white, slightly fluffy colony
O1 Blackish gray, flat colony
O2 Blackish gray, slightly fluffy colony
O3 Grayish white, slight fluffy colony
O4 Blackish gray, flat colony
O5 Blackish gray, flat colony
O6 Grayish white, slightly fluffy colony
P1 Blackish gray, slight fluffy colony
P2 Deep black, flat colony
P3 Blackish gray, slightly fluffy colony
P4 Deep black, flat colony
P5 Blackish gray, slightly fluffy colony
P6 Blackish gray, flat colony
S1 Deep black, flat colony
S2 Grayish white, slightly fluffy colony
S3 Blackish gray, slightly fluffy colony
S4 Blackish gray, flat colony
S5 Grayish white, slightly fluffy colony
S6 Deep black, flat colony
63
Plate.3. Colony chatracters of isolates of M. phaseolina on corn meal agar medium
64
Fig 4.6: Weight of dry mycelial (mg) of isolates of M. phaseolina on two
different liquid media
Table 4.5.1: Grouping of isolates of Macrophomina phaseolina based on dry mycelia
weight in PDB (on 11th day)
Group Range (mg) Number of
isolates
Location of Isolates
1 277-319 6 Bheala, Garewala, Talwandi,
Chak 17 SP, Jaman Bodla, Bunga Hayat
2 234-276 5 Atari, Noor Pur, Khudian Khas,
Malka Hans, Chak 50 SP
3 191-233 6 Ahmadabad, Basir Pur, Havali Lakha,
Chak 30 SP, Kassowal, Chak 42/12 L
4 148-190 4 Aktharaabad, Chak 21/11 L, Chak 44/12 L,
Adde Pur
5 105-147 3 Hujra Shah Muqeem, Renala Khurd,
Bashera
Table 4.5.2: Grouping of isolates of Macrophomina phaseolina based on dry mycelial
weight in CMB (on 11th day)
Group Range (mg) Number of
isolates
Location of Isolates
1 224-266 7 Garewala, Talwandi, Atari, Noor Pur,
Jaman Bodla, Bunga Hayat, Chak 30 SP
2 181-223 11
Bheala, Khudian Khas, Aktharaabad,
Ahmadabad, Basir Pur, Chak 17 SP,
Malka Hans, Chak 50 SP, Chak 42/12 L,
Chak 21/11 L, Chak 44/12 L
3 138-180 2 Havali Lakha, Kassowal
4 95-137 4 Hujra Shah Muqeem, Renala Khurd, Adde
Pur, Bashera
65
4.4.3 Sensitivity to chemicals
All the 24 isolates were grown on potato dextrose agar medium, incorporated with
500, 1500 and 2500 ppm concentration of copper sulphate. Observations for each of the three
concentrations maintained in three replication were noted, when the fungal growth reached a
maximum growth of 90 mm in their respective controls. The results are presented in (Fig
4.7).
Differences among the isolates, different concentration of copper sulphate and their
interaction were significant. Garewala isolate showed highest per cent inhibition and lowest
per cent inhibition was observed in case of Renala Khurd and Bashera isolate. In general,
maximum per cent inhibition of growth was observed at 2500 ppm and the minimum at 500
ppm (Fig 4.7).
In 500 ppm concentration, highest per cent inhibition of growth (53.1%) was
observed in Bheala isolate followed by Garewala (47.1%) and Bashera (38.1%) isolate. The
lowest per cent inhibition was observed in Kassowal (4.1%) and Chak 42/12 L (8.1%)
followed by Renala Khurd, Chak 21/11 L and Chak 44/12 L isolate (11.1%). In 1500 ppm
concentration highest inhibition of growth (100%). The least inhibition of growth was
observed in Hujra Shah Muqeem isolate (50.1%) followed by Bashera isolate (61.1%) in
1500 ppm. Data (Fig 4.7) indicated that, each isolate, has an ability to resist the chemical at
varying level, revealing the existence of variability among the isolates.
Based on the sensitivity to copper sulphate, all the 24 isolates of M. phaseolina could
be categorized into four groups which show the different degree of sensitivity as depicted in
(Table 4.6.1)
4.4.3.1 Sensitivity of isolates to benomyl
The procedure followed here was the same as for copper sulphate sensitivity. The
study revealed that there was 100 percent inhibition of all 24 isolates at all the three
concentrations tested (500, 1500 and 2500 ppm). The variation was not evident among the
isolates with respect to the sensitivity to benomyl (Table 4.6.2).
66
Fig 4.7: Sensitivity of M. phaseolina isolates to different concentrations of
copper sulphate
Table 4.6.1: Grouping of isolates of Macrophomina phaseolina based on sensitivity to
different concentration of copper sulphate
Group % inhibition Number of
isolates
Location of Isolates
1 76.35-82.07 5 Bheala, Garewala, Khudian Khas, Chak 50
SP, Chak 30 SP
2 70.62-76.34 7 Talwandi, Atari, Aktharaabad, Chak 17 SP,
Jaman Bodla, Bunga Hayat, Malka Hans
3 64.89-70.61 5 Noor Pur, Ahmadabad, Kassowal,
Chak 42/12 L, Chak 21/11 L
4 59.16-64.88 7
Basir Pur, Havali Lakha, Hujra Shah
Muqeem, Renala Khurd, Chak 44/12 L, Adde
Pur, Bashera
67
Table 4.6.2: Sensitivity of M. phaseolina isolates to different concentrations
of Benomyl
Inhibition percentage of Macrophomina phaseolina
Isolates 500 ppm 1500 ppm 2500 ppm Mean
K1 100 100 100 100
K2 100 100 100 100
K3 100 100 100 100
K4 100 100 100 100
K5 100 100 100 100
K6 100 100 100 100
O1 100 100 100 100
O2 100 100 100 100
O3 100 100 100 100
O4 100 100 100 100
O5 100 100 100 100
O6 100 100 100 100
P1 100 100 100 100
P2 100 100 100 100
P3 100 100 100 100
P4 100 100 100 100
P5 100 100 100 100
P6 100 100 100 100
S1 100 100 100 100
S2 100 100 100 100
S3 100 100 100 100
S4 100 100 100 100
S5 100 100 100 100
S6 100 100 100 100
68
4.4.4 Effect of pH on isolates
Variation due to change in hydrogen ion concentration (pH) was also evident in M.
phaseolina (Fig 4.8). Highest mean (321 mg) dry mycelial weight was observed at pH 7.0
followed by 282 mg at pH 6.5. Least growth (83 mg) was observed at pH 5.0 indicating its
inability to support the growth of M. phaseolina isolates. Within the same range of pH also
variation was evident either in the high or low growth of isolates indicating the variation
among the isolates. It is clear from the (Fig4.8) that within given pH of 6.5, Bheala isolate
produced maximum of 282 mg and Havali Lakha isolate least amount of dry mycelial mass
(125 mg).
All the 24 isolates of M. phaseolina were grouped into three groups (Table 4.7). The
first group had highest number of isolates (11) in a range of 207.49 to 244.76 mg, where as
the last group consisted of nine isolates with a range of 132.93 to 170.20 mg.
4.4.5 Effect of temperature on the growth of isolates
Variation also existed in M. phaseolina at different temperature ranges. Isolates from
Bheala, Noor Pur, Khudian Khas, Aktharaabad, Ahmadabad, Basir Pur, Havali Lakha, Hujra
Shah Muqeem and Renala Khurd produced maximum growth in the range of 199.0 to 241.1
mg dry mycelial weight (Table 4.8). Bunga Hayat, Chak 30 SP and Chak 21/11 L isolates
produced the least growth with 162.80, 163.80 and 161.60 mg (dry mycelial weight)
respectively on the mean of all five temperature levels (Fig 4.9).
Among the different temperatures tested at 35ºC was most favourable (331 mg) and it
was closely followed by 40ºC (297 mg) for the growth of M. phaseolina. At 20ºC, 25ºC and
30ºC poor growth was observed indicating isolates’ preference towards higher temperature
for the growth (Fig 4.9). At 35ºC; maximum growth was noticed with Talwandi isolate (331
mg), whereas minimum growth was recorded by Chak 30 SP (192 mg).
Based on mean mycelial weight, isolates were categorized into two groups. Group
first included nine isolates with growth range (199.0 to 241.1 mg) and group 2nd included
fifteen isolates with a growth range 155.9 to 198.0 (Table 4.8).
69
4.4.6 Pathogenic variability
All the 24 isolates were inoculated to a susceptible variety (Sahiwal 2002) and also to
another popular variety (MMRI-yellow). The detailed procedure is given under materials and
methods.
The results of length of spread recorded in field sown crops of Sahiwal 2002 and
MMRI-yellow plants were found to be highly significant (Fig 4.10) indicating the pathogenic
variability among the isolates. Grouping of 24 isolates based on length of spread in field
sown Sahiwal 2002 and MMRI-yellow are furnished in Table 4.9.1 and 4.9.2.
Grouping of isolates based on length of spread in field sown Sahiwal 2002 plants
(Table 4.9.1) revealed five groups. Group first consisted of four isolates (K-5, K-6, P-5 and
P-6) under the range for maximum length of spread (20.4 to 24.4 cm) group 2 nd, 3rd and 4th
had length of spread 16.3 to 20.3, 12.2 to 16.2 and 8.1 to 12.1 respectively. The last group
consisted eight isolates (O-1, 0-2, O-3, O-6, S-1, S-3, S-5 and S-6) showing the least length
of spread (4.0 to 8.0 cm).
All 24 isolates were arranged in three groups based on length of spread in MMRI-
yellow (Table 4.9.2). The maximum number of isolates (14) was included in group 3 rd
showing the range of spread of 3.3 to 7.3 cm followed by five isolates in group 2 nd 7.4 to
11.4 cm. The first group consisted 5 isolates (Bheala, Garewala, Noor Pur, Malka Hans and
Chak 44/12 L) which showed the maximum length of spread (11.5 to 15.5 cm).
The results of length of spread (cm) obtained from “Detached stem
technique“conducted on Sahiwal 2002 was found to be significant indicating the variability
among the isolates (Fig 4.11). In this experiment three groups of the isolates were possible
based on length of spread. Jaman Bodla isolate recorded the highest length of spread (15.6
cm), while Chak 44/12 L and Adde Pur isolate recorded lowest spread of 2.3 and 3.5 cm
respectively. Ten isolates fell under the range of 6.4 to 11.0 cm (Table 4.10).
70
Fig 4.8: Effect of pH on the growth of isolates of Macrophomina phaseolina
Table 4.7: Grouping of isolates of Macrophomina phaseolina based on dry mycelial
weight at different pH levels
Group Range (mg) Number of
isolates
Location of Isolates
1 207.49-244.76 11
Bheala, Talwandi, Atari, Noor Pur,
Khudian Khas, Chak 17 SP, Jaman Bodla,
Bunga Hayat, Malka Hans, Chak 50 SP,
Chak 30 SP
2 170.21-207.48 4 Garewala, Chak 21/11 L, Chak 44/12 L,
Adde Pur
3 132.93-170.20 9
Aktharaabad, Ahmadabad, Basir Pur,
Havali Lakha, Hujra Shah Muqeem,
Renala Khurd, Kassowal, Chak 42/12 L,
Bashera
71
Fig 4.9: Effect of temperature levels on growth of different isolates of M.
phaseolina
Table 4.8: Grouping of isolates of Macrophomina phaseolina based on dry mycelial
weight at different temperatures
Group Range (mg) Number of
isolates
Location of Isolates
1 199.0-241.1 9
Bheala, Noor Pur, Khudian Khas,
Aktharaabad, Ahmadabad, Basir Pur, Havali
Lakha, Hujra Shah Muqeem, Renala Khurd
2 155.9-198.00 15
Garewala, Talwandi, Atari, Chak 17 SP,
Jaman Bodla, Bunga Hayat, Malka Hans,
Chak 50 SP, Chak 30 SP, Kassowal,
Chak 42/12 L, Chak 21/11 L, Chak 44/12 L,
Adde Pur, Bashera
72
Fig 4.10: Spread of infection in Sahiwal 2002 and MMRI-yellow plants
Table 4.9.1: Grouping of isolates of Macrophomina phaseolina based on length of
spread in Sahiwal 2002
Group spread (cm) Number of
isolates
Location of Isolates
1 20.4-24.4 4 Noor Pur, Khudian Khas, Chak 50 SP,
Chak 30 SP
2 16.3-20.3 3 Bheala, Chak 17 SP, Jaman Bodla
3 12.2-16.2 3 Garewala, Talwandi, Atari
4 8.1-12.1 6
Havali Lakha, Hujra Shah Muqeem,
Bunga Hayat, Malka Hans, Chak 42/12 L,
Chak 44/12 L
5 4.0-8.0 8
Aktharaabad, Ahmadabad, Basir Pur,
Renala Khurd, Kassowal, Chak 21/11 L,
Adde Pur, Bashera
73
Table 4.9.2: Grouping of isolates of Macrophomina phaseolina based on length of
spread in MMRI-yellow
Group spread (cm) Number of
isolates
Location of Isolates
1 11.5-15.5 5 Bheala, Garewala, Noor Pur, Malka Hans,
Chak 44/12 L
2 7.3-11.4 5 Atari, Khudian Khas, Jaman Bodla,
Chak 30 SP, Chak 42/12 L
3 3.3-7.2 14
Talwandi, Aktharaabad, Ahmadabad, Basir
Pur, Havali Lakha, Hujra Shah Muqeem,
Renala Khurd, Chak 17 SP, Bunga Hayat,
Chak 50 SP, Kassowal, Chak 21/11 L,
Adde Pur, Bashera
Fig 4.11: Spread of infection in detached stem of Sahiwal 2002 by different
isolates of M. phaseolina
74
Plate.4. Detached stem technique
75
Table 4.10: Grouping of isolates of Macrophomina phaseolina based on detached stem
technique in Sahiwal 2002
Group spread (cm) Number of
isolates
Location of Isolates
1 11.1-15.7 5 Noor Pur, Khudian Khas, Jaman Bodla,
Bunga Hayat, Malka Hans
2 6.4-11.0 10
Garewala, Talwandi, Atari, Aktharaabad,
Ahmadabad, Basir Pur, Havali Lakha,
Hujra Shah Muqeem, Chak 17 SP, Chak 50
SP
3 1.7-6.3 9
Bheala, Renala Khurd, Chak 30 SP,
Kassowal, Chak 42/12 L, Chak 21/11 L,
Chak 44/12 L, Adde Pur, Bashera
4.5 INHIBITORY EFFECT OF FUNGICIDES ON THE RADIAL GROWTH OF M.
phaseolina
Highly significant inhibitory effects of fungicides and their concentrations were
recorded on the growth of M. phaseolina. All the fungicides caused significant inhibition of
the fungus over control.
Maximum individual inhibition of the growth of the fungus was recorded by Benomyl
(86%) followed by Carbendazim (82%) at a concentration of 150 ppm. Captan at a
concentration of 50 ppm gave the minimum inhibition (17%). The individual inhibitions
caused by the fungicides are given in the (Fig 4.12.1). Different concentrations also had
significant inhibitory effects on the growth of the fungus. All the fungicides caused
maximum inhibition of the growth of the fungus at a concentration of 150 ppm.
With the decrease in the concentration, the inhibition in the growth also decreased.
The inhibition of growth was found to be directly proportional to the concentration.
4.5.1 Effect of fungicides on plant survival of maize
Fungicides also affected significantly the plant survival of maize over control.
Maximum plant survival was observed where the seeds were treated with Benomyl followed
by Carbendazim. However, Mancozeb and Captan treated seeds gave the minimum survival
of plants. Doses also had a significant effect on the plant survival. Maximum survival was
recorded where the seeds were treated with a concentration of 150 ppm and minimum was
76
recorded in case of 50 ppm concentration. The plant survivals of maize are shown in (Fig
4.12.2).
It was also observed that higher concentrations of all the fungicides were significantly
better as compared to other concentrations. With the decrease in the concentration the
survival decreased significantly showing a direct relationship between concentrations and
plant survival. Benomyl at 150 ppm concentration showed highest rate of plant survival 79%
whereas Carbendazim and metalexyl+mencozeb on same concentration exhibited 66 and
59% plant survival respectively. Other fungicides also showed enhanced effect on 150 ppm
as compared to other concentrations. All chemicals were significant on all concentrations by
statistical analysis.
Fig 4.12.1: In Vitro radial growth inhibition of M. Phaseolina
77
Fig 4.12.2: Effect of fungicides on plant survival of maize against charcoal
rot (M. phaseolina)
4.6 EFFECTS OF PLANT EXTRACTS ON THE GROWTH OF M. phaseolina
The aqueous extracts of all the tested plants significantly suppressed the growth of M.
phaseolina of all the plants. Datura stramonium proved to be the most effective in
suppressing the growth of the pathogen at all concentrations followed by C. procera and A.
indica. M. oleifera and D. sisso which appeared to be least effective. The individual growth
inhibitions at four concentrations of test plants are given in (Fig 4.13.1). Similarly,
concentrations also had significant inhibitory effect on the growth, being the maximum at
100 % concentration of the extracts. As the concentration of extracts lowered, the magnitude
of inhibition of growth of the fungus also decreased significantly. A direct relationship
between concentrations and growth inhibitions was observed in case of all the test plants as
shown in (Fig 4.13.1).
78
Fig 4.13.1: Effect of plant extracts on the growth of M. phaseolina
4.6.1 Effects of plant extracts on plant survival of maize
All the plant extracts when used as seed treatment significantly enhanced the plant
survival. Of all the test plants, Datura stramonium showed maximum increase in survival of
maize plants over control followed by A.indica and C. procera. On the other hand M. oleifera
and D. sisso appeared to be the least effective in reducing the damage of the pathogen. The
maximum individual survival (84 %) was achieved with 100 % concentration of Datura
stramonium as against the minimum of 31 % obtained with C. procera at 25 %
concentration. The individual percent increases of plant survival atfour concentrations of the
test plants are given in (Fig 4.13.2). Significant effect of concentrations was also observed.
Maximum plant increase in survival was recorded at 100% concentration of extracts. The
effect of the plant extracts diminished as the concentration decreased.
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Fig 4.13.2: Effect of plant extracts on plant survival of maize against
charcoal rot (M. phaseolina)
4.7 SCREENING OF GERMPLASM LINES AGAINST CHARCOAL ROT OF
MAIZE
In 2012, MMRI-yellow recorded minimum charcoal rot incidence (9.70%)
followed by BL-71 (12.10%), Pak-Afgoyee (15.50%), 34N43 (16.80%), M-6142 (18.30%).
The maximum disease incidence was recorded in Sahiwal 2002 (55.70%) and overall rot
incidence ranged from 9.60 to 55.69 percent in various genotypes screened.
Least mean nodes crossed noticed in genotypes MMRI-yellow (1.70). Highest
number of nodes crossed in M-6142 (3.50) followed by Sahiwal 2002 (3.40) genotype.
Least mean length of spread (cm), was noticed in MMRI-yellow (13.40) followed
by 8811 (20.20), 32B33 (23.70), Monsento-6525 (23.20) and 34N43 (24.30). Highest mean
length of spread was noticed Sahiwal 2002 (34.80 cms).
Highest thousand grain weight (g) was recorded in MMRI-yellow (385) followed
by P-1541 (364), Pak-Afgoyee (355) and 34N43 (354) Fig 4.14.1.
During 2013, regarding per cent Charcoal Rot incidence, number of node crossed
and thousand grains weight similar trend was observed as that of 2012.
80
Least mean length of spread (cm), was noticed in MMRI-yellow (13.80) followed
by 34N43 (23.40), BL-71 (23.70), Monsento-6525 (24.30), Pop-corn (26.60). Highest mean
length of spread was noticed Sahiwal 2002 (34.80 cms) Fig 4.14.1.
Out of 19 genotypes screened against charcoal rot incidence in infested plot, one of
the genotypes showed resistant reaction. Nine genotypes showed moderately resistant
reaction. Eight genotypes showed susceptible reaction. One genotype i.e. Sahiwal 2002
showed highly susceptible reaction (Table 4.11).
Fig 4.14.1: Screening of maize germplasm against charcoal rot 2012
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Fig 4.14.2: Screening of maize germplasm against charcoal rot 2013
Table 4.11: Reaction of maize genotypes to charcoal rot
Charcoal rot scale (%) Genotypes response
0 ------ Immune
1 ------ Highly Resistant
3 MM RI-yellow Resistant
5 34N43, 8711, M6142, BL 71, 32B33,
Monsento 6525, S8441, P1541,
Pak Afgoyee
Moderately Resistant
7 Agaiti 85, Desi- Punjab, EV 5098,
EV 6089, Agaiti 2002, Pearl, Pop-
Corn, Neelum
Susceptible
9 Sahiwal 2002 Highly Susceptible
82
Tooth pick method of inoculation
Measuring the lesion length with scale Highly susceptible variety Sahiwal 2002
Plate.5. Screening of varieties
83
4.8 EFFECT OF INOCULATION TIME AND SOWING DATES ON DISEASE
Inoculation done after 50, 60, 70, 80 and 90 days, the maximum number of nodes
crossed in the plants inoculated after 60 days (3.4) and the minimum nodes crossed in plants
inoculated after 90 days (2.3) Fig 4.15.1 and 4.15.2.
In sowing dates the maximum number of nodes crossed when sowing of crop
done after 10 days of optimum sowing date (3.5) and minimum (2.5) in sowing date after 20
days of optimum sowing (Fig 4.15.1 and 4.15.2).
Fig 4.15.1: Effect of inoculation time on disease
84
Fig 4.15.2: Effect of sowing date on disease
4.9 MANAGEMENT OF CHARCOAL ROT OF MAIZE IN INFESTED PLOT
The seed treatment with Benomyl showed superior results over Datura in highly
susceptible variety Sahiwal 2002.
Regarding number of nodes crossed in Benomyl, the least nodes crossed at
concentration 3g/kg is (1.00) as compare to control (3.5) followed by (1.40) and (1.80) at
2g/kg and 1g/kg respectively (Fig 4.16.1).
In D. stramonium extract, the least number of nodes crossed at 100%
concentration (2.10) as compare to control, followed by (2.50) and (2.80) at 75% and 50%
respectively (Fig 4.16.1).
Regarding length of spread least length of spread recorded in Benomyl was
(13.70) as compare to control (35.10) followed by (15.80) and (17.70 cms) at 1g/kg and
2g/kg (Fig 4.16.2).
In D. stramonium extract, least length of spread was recorded at 100%
concentration (24.60) as compare to control followed by (26.50) and (27.80) at 75% and 50%
respectively (Fig 4.16.2).
85
Fig 4.16.1: Effect of different levels of Benomyl and different percentages
of datura on disese under field conditions (nodes crossed)
Fig 4.16.2 Effect of different levels of Benomyl and different percentages
of Datura on disese under field conditions (length of spread)
86
Plate.6. Number of nodes crossed and length of spread
87
CHAPTER V DISCUSSION
Maize is cultivated globally being one of the most important cereal crops worldwide.
Maize is not only an important food crop for human consumption, but also a basic element of
animal feed and raw material for manufacturing of many industrial products. The products
include corn starch, maltodextrins, corn oil, corn syrup and products of fermentation and
distallaries. It is also being recently used in the production of biofuel. Maize is grown in all
the provinces of the country, Punjab and KPK are the main areas of production. Khyber
Pakhtunkhwa produces around 0.8-0.9 million tons of maize per year. According to an
estimate, Punjab and KPK account for 84 per cent of the total maize production of 95 per
cent of the maize cultivation area.
Maize suffers from about 110 diseases on a global basis caused by fungi, bacteria and
viruses. The disease spectrum varies in different agro-climatic zones. Several diseases such
as seed and seedling delights, foliar disease, downy mildews, stalk rots and leaf as sheath
blight occur in various parts of the country. It has been reported that about 13.2% of the
economic product of maize is estimated to be lost annually due to diseases (Dhillon and
Prasanna, 2001). A number of fungal diseases attack on the maize crop but charcoal rot
caused by Macrophomina phaseolina (Tassi) Goid. has the potential on successful crop
production of maize all over the world (Edmunds, 1962).
Inspite of its importance, research on charcoal rot has been largely superficial. Wide
gaps still exist in areas relating to existence of variability in the fungus and disease
management.
Isolation of the fungus was done by following standard isolation process and the
fungus was confirmed with respect to the characters described by Ashby (1927) and
Goidanich (1947) and recognized as M. phaseolina.
The pathogen isolated from each location was considered as an isolate. The names of
maize isolates were assigned after the location from which the samples were obtained.
Pathogenicity of M. phaseolina isolates was carried out on susceptible Sahiwal 2002
variety by toothpick method. Results of pathogenic test indicated that all the 24 isolates
under study were pathogenic to maize. These results are in agreement with findings of
88
Sekhar (1985) and Hundekar (1987) that M. phaseolina isolated from sorghum was
pathogenic to sorghum.
A potential pathogen is often blessed with biodiversity within its population. Mostly,
distinction in pathogen is desirable trait for its survival in nature. This variability among the
pathogens underlined their diverse nature and ability to endure the host environment.
Variability of pathogen studied by cultural and morphological characters, nutritional
prerequisite, enzyme activity and pathogenic behavior focused existence of variation in M.
phaseolina.
Morphological variability
Inherent ability of different isolates to grow on potato dextrose agar and corn meal
medium could vary and association of such trait to any of pathological properties of pathogen
either in M. phaseolina or other fungi seldom exists in literature. However, isolates with
ability to grow faster which have advantage in terms of population built up for causing
disease.
Cultural and morphological characters on potato dextrose agar (PDA) and corn meal
agar (CMA) medium showed variation among the 24 isolates of M. phaseolina. The mean
colony diameter (mm) of most of the 24 isolates was higher on PDA compared to corn meal
agar indicating that, in general, most of isolates under study preferred PDA.
Based on morphological characters of 24 isolates on potato dextrose agar, they could
be grouped into four categories K-2, K-6, O-1, P-1, S-2 and S-3 isolates produced medium
black, flat colonies, K-5, P-4 and S-6 isolates produced grayish white, fluffy colonies. K-1,
K-3, K-4, O-3, O-4, O-6, P-2, P-3, P-5, S-1 and S-4 isolates produced blackish gray, flat and
fluffy colonies and O-2, O-5, P-6 and S-5 isolates produced deep black, flat colonies.
Shekhar et al., (2006a) made similar observations observed in the isolates of M. phaseolina,
where in they produced grayish white, blackish gray, deep black centre with creamish
periphery, cottony white colour colonies. The fast growth of some isolates related with the
virulence K-4, K-5, P-3 and P-4 was the most virulent on Sahiwal 2002 and have more
colony diameter above 83.1 on PDA
Mean colony diameter among 24 isolates varied considerably. A higher mean colony
diameter (81.0-88.1 mm) was observed in 12 isolates (K-1, K-2, K-3, K4, K-5, K-6, P-1, P-2,
P-3, P-4, P-5 and P-6). Significantly lower mean colony diameter (59.1- 69.1 mm) was
89
observed in O-5, S-1, S-2 and S-3 isolates. This property of an isolate indirectly reflects the
inherent ability to grow faster. However, observed property on PDA reflects the natural
variation remains to be elucidated in M. phaseolina. Similar observations among the isolates
were made by Subramaniam (1994) and Kavita (2007).
Regarding the size of sclerotia, it was observed that K-6, K-4 and O-2 isolates
produced larger sized sclerotia (90, 88.3 and 88.3µm respectively), while the smallest sized
sclerotia (29 µm) was recorded in S-4 isolate. Out of 24 isolates 13 was oblong and 11 was
round shaped. Similar observations were reported by Shekhar et al., (2006a), that the size of
sclerotia varied from 95.7 ım to 66.9 µm throughout the study. It was observed that the
District kasur isolates produced large sclerotia followed by Okara, Pakpatan and Sahiwal
isolates. Similar observations were made by Kavita (2007) regaurding the variation in size of
the seclerotia.
Average sclerotial size revealed five groups with K-6 isolate recorded maximum size
(90 µm) and S-4 isolate recorded the lowest (29 µm). Several workers (Goidanich and
Camici, 1947; Dhingra and Sinclair, 1973; Ghosh and Sen, 1973; Raut and Ingle, 1989,
Waseer et al., 1990; Subramaniam, 1994; Shekhar et al., 2006a and Kavita, 2007 have
reported variation in size and shape of sclerotial bodies of different isolates of M. phaseolina.
Haigh (1928) categorized isolates based on average sclerotial size, while Arca and
Yildizin (1989) grouped isolates based on number of sclerotial bodies. Anilkumar and Sastry
(1980) reported that the capability of the isolate to fabricate sclerotia changed with the
growth media. Waseer et al., (1990) reported that the sclerotial size of soybean isolate on
PDA was dissimilar from that of other media, while, it was found to be within the range
described for M. phaseolina. Subramaniam (1994) reported that the average sclerotial size
revealed 18 groups and Kavita (2007) reported that the average sclerotial size revealed eight
groups.
The number of sclerotia/microscopic field 10x was maximum of 56 per microscopic
field in P-5 isolate, while minimum number was observed in O-3 isolate (35 /microscopic
field). Similar observations were made by Shekhar et al.,(2006a) where in maximum
numbers of sclerotia/microscopic field (52.0) in Hyderabad isolate while minimum number
was observed in Coimbatore isolate (44/microscipic field).
90
All of the isolates took five- six days for sclerotial formation. Such variations, in days
required for sclerotial formation has been reported by AnilKumar and Sastry (1980), who
observed that, the sunflower isolates took least time (2-3 days for sclerotial formation), while
the brinjal and cowpea isolates took 4 to 10 days depending on the media used. Subramaniam
(1994) also noticed that most of the isolates took 3-4 days for sclerotial formation, except oil
palm (OP-1, OP-2 and OP-4), groundnut (Gn) and soybean (So) isolates, which took 5-6
days for sclerotial formation. Similar studies were made by Kavita (2007) and the results
support the present study.
Number of sclerotia/9 mm disc at different interval was recorded. Among the isolates,
P-1 and S-4 isolates produced comparatively large number of (112, 147 and 176 and 110,
131 and 179 sclerotia/9 mm disc) during 5th, 7th and 9th day, while the minimum number was
observed in P-6 during 5th, 7th and 9th day. Similar observations were made by Shekhar et al.,
(2006b). They observed that Hyderabad isolate had maximum number of sclerotia/9 mm disc
(180), while the minimum sclerotia number was observed in Coimbatore isolate (169/9 mm
disc).
Grouping of isolates four days of inoculation on corn meal agar also yielded four
groups as in PDA. But having less colony diameter indicating the preference of isolates to
grow better on PDA. The O-6 isolate produced the least colony diameter (62.3 mm) and was
found to differ significantly from the rest of the isolates indicating its slow growing nature on
corn meal medium.
It is obvious that the isolates vary in their colony diameter not only with respect to
each other in the same medium, but also, within the sub-group, in contrast with, its own
growth on two different media. Therefore, it is possible to use a particular medium for the
point of grouping isolates into pathotypes. Differences in cultural and morphological
characters are however important from the point of view of the biology of the organism at the
similar time. It is logically strongly associated with the pathogenicity and endurance of
physiologic races and requires further studies to correlate the same.
Nutrients are essential for the growth and development of micro-organisms.
Capability of isolates to exploit and change these nutrients into bio-mass was studied by
growing them on diverse liquid media.
91
Variations were also observed in growth among isolates in liquid medium. On potato
dextrose broth higher dry mycelial weight was recorded as compared with corn meal broth,
both at the highest and lowest range levels, indicating that potato dextrose medium is more
favorable growth medium than corn meal medium for all the 24 isolates as evidented on the
over all growth attained in the present study.
Groupings made on the basis of dry mycelial weight on PDB and corn meal broth
revealed five and four groups respectively each (each group with a difference of 42 mg. dry
mycelial weight). In PDB, 6 isolates recorded higher mycelial weights (277-319 mg) and 5th
group isolates (O-5, O-6 and S-6) recorded lowest range of mycelial weight (105-147 mg),
clearly indicating variation in growth of different isolates. K-1 isolate recorded highest dry
mycelial weight (318 mg) in PDB while O-6 isolate, recorded the lowest (121.00 mg).
On corn meal broth, O-5 and O-6 isolate produced less dry mycelial weight compared
to potato dextrose broth. This disparity behaviour of isolates on PDB and corn meal medium
clearly indicated the variability in O-5 and O-6 isolates.
The results obtained in the present study are in confirmation with results obtained by
Waseer et al., (1990) while, working with M. phaseolina from soybean and they concluded
that growth was maximum in potato dextrose broth. Similar, studies on the effect of different
media on growth and sporulation has been made by several workers (Subramaniam, 1994 and
Kulkarni, 2000).
An age old traditional chemical, like copper sulphate, still enjoys a good repute for
controlling much fungal growth and yet, it has not been tried with respect to different isolates
of M. phaseolina. In the present study, the sensitivity of different isolates to copper sulphate
at three different levels of concentration (500, 1500 and 2500 ppm), was tried and the isolates
were found to differ in their sensitivity to various concentrations of copper sulphate tested.
Among the 24 isolates, except O-5, S-6, O-3 and O-6 all other isolates were highly sensitive
to 1500 ppm concentration. But at 2500 ppm, all the isolates were inhibited to the maximum.
Similar studies on the effect of copper sulphate at different concentrations have been made
by Subramaniam (1994) against M. phaseolina and support the present study.
Studies on benomyl sensitivity against 24 isolates of M. phaseolina did not reveal any
variation at all the three concentrations tested (500, 1500 and 2500 ppm). In all these three
concentrations, growth of all the 24 isolates was inhibited cent percent highlighting the
92
comparative efficacy of the chemical. The studies are in line with the work done by Ramdoss
and Sivaprakasam (1987) and Singh and Kaiser (1995) where in they reported the higher
efficacy of carbendazim against M. phaseolina both at in vitro and in vivo conditions. Khan
and Khan (2006) found that both benomyl and carbendazim inhibited 100% mycelial growth
of M. phaseolina. We used the minimum concentration of copper sulphate 500 ppm so; we
also used the benomyl minimum concentration 500 ppm to compare the response of most
premitve fungicide with the modern fungicide.
In the present study, variation in M. phaseolina due to change in hydrogen ion
concentration (pH) was recorded. Highest mean growth was observed in 7.0 pH with 321 mg
mean mycelial weight closely followed by 282 mg dry mycelial weight at 6.5 pH indicating
preferential range to be between 6.5 and 7.00 pH. Within the same range of pH also,
variation was observed and it was clearly illustrated by K-1 and O-4 isolates, producing
maximum (321 mg) and minimum (125 mg) amount of dry mycelial mass at 7.0 pH.
The hydrogen ion concentration of the medium and growth of the isolates are
mutually supporting. Every organism requires best possible pH for its optimum growth and
sporulation. Bai et al., (1991) reported 6.7 to 7.5 to be optimum pH for the growth of
Fusarium sp. isolated from maize plants in Northern China.
Uppal et al., (1936) while studying difference among M. phaseolina observed isolates
supporting wide range of pH among 3.4 to 6.4 and concluded that optimum pH range varies
with media composition. In support of the present study, Khare et al., (1970) observed
difference in growth pattern and sclerotial size of R. bataticola isolates from urdbean at 6.5
pH and recognized the variation to changes in biochemical processes in the plant parts from
which they were isolated.
Kulkarni (2000) has studied the effect of different pH levels on 13 isolates M.
phaseolina isolated from maize. He observed that the highest growth was observed at 7.0 pH
and second best was at 6.5 pH. These 13 isolates were categorized into three groups based on
their dry mycelial weight. These results support the observation made in the present study
where-in 24 isolates have been categorized into three groups. The first group had highest
number of 11 isolates in the range of 20.49 - 244.76 mg of dry mycelial weight and the third
group had nine isolates with 132.93-170.20 mg of dry mycelial weight.
93
The metabolic events like transformation of substrate into product are carried out with
the help of biological catalysts (enzymes) and these enzymes require set of pH. Hence,
present investigation will help in long run to study the various characters of these organisms
or isolates by grouping them at optimum pH level. In the current study we used the range of
pH between 5.0-7.0 to check the response of isolates on acidic and basic medium and most of
major maize growing area soils has pH upto 7.0 so, we can not use the more than 7.0 in our
study. Pathogenicity related to pH the most virulent isolate has length of spread P-2 on
Sahiwal 2002 have dry mycelia weight 242.50 mg at all the pH levels
Temperature influences the rate of growth, metabolism and morphological characters
of the fungi. The fungi can sustain under wider range of temperatures and requires optimum
range of temperature for proper growth.
Variation due to change in temperature range was evident in 24 M. phaseolina
isolates. Significant higher growth of all the isolates was observed at 35ºC (mean dry
mycelial weight of 331 mg) and 40ºC (285 mg) and clearly indicates the preference of
isolates towards higher temperature range between 35ºC and 40ºC. Waseer et al., (1990)
while working with M. phaseolina isolates from soybean reported similar results and
obtained best growth on PDA and PD juice at 35ºC. The results of the present study are in
confirmation with the studies made by Bansal and Gupta, 2000; Bainade et al., 2006;
Chowdary and Govindaiah, 2007.
The pathogenic fungus, M. phaseolina, has a broad host range and exits in two
asexual forms which maintain its survival better (Dhingra and Sinclair, 1978; Cloud and
Rupe, 1991; Mihail and Taylor, 1992). Some workers also related variability with the
phenomena of host specialization in M. phaseolina. Su et al., (2001) found host
specialization in maize on the basis of pathogenic, genetic and physiological differences.
Similarly, Cloud and Rupe (1991) analyzed host specialization in soyabean. This mechanism
takes long time to establish with in a specific host. Mihail and Taylor (1995) suggested that
due to heterogenic nature of M. phaseolina, categorization into distinct sub groups based
upon pathogenicity and morphology could not take place. Pathogenesis along with genetic
diversity plays a specific role in host plant resistance. Isolates having morphological
similarity are not necessarily identical genetically, they might have some differences. The
variable genetic pattern contributes for variation in morphology and pathogenesis, which has
94
been confirmed by using different molecular tools (Fuhlbohm, 1997; Mayek-Parez et al.,
2001; Jana et al., 2003; Reyes et al., 2006; Rajkumar et al., 2007 and Allaghebandzadeh et
al., 2008). As the pathogen has no sexual phase, genetic diversity is produced either by
fusion of vegetative cells or by para-sexual recombination between nuclear genes (Carlile,
1996). Greatest genetic variability makes survival of fungus better in nature (Rajkumar et al.,
2007).
It is quite evident that variability in morphology, physiology, genetics, pathogenicity
etc. is imperative for the fungus to have better adaptation in response to diversified
environmental behavior. It also leads to host-plant resistance, development of resistant
varieties of different crops against disease and implementation of new disease controlling
strategies (Mayek-Parez et al., 2001; Purkayastha et al., 2006).
Based on the results obtained from pathogenicity studies, (Length of spread due to
charcoal rot in Sahiwal 2002 and MMRI-yellow) the 24 isolates can be categorized as highly
virulent, moderately virulent and least virulent.
Based on length of spread of infection in Sahiwal 2002, the isolates K-5, K-6, P-5, P-
6, P-1, P-2 and K-1 was grouped as highly virulent (producing 16.4 - 24.3 cm spread).
Isolates K-2, K-3, K-4, O-4, O-5, P-3, P-4, S-2 and S-4 was grouped as moderately virulent
(6.2-15.1 cm spread). While, isolates O-1, O-2, O-3, O-6, S-1, S-3, S-5, and S-6 was grouped
as least virulent (5.4–13.7 cm spread).
Based on length of spread on MMRI yellow, the isolates K-1, K-2, K-5, P-4, and S-4
was grouped as highly virulent (11.5– 15.5 cm spread). Isolates K-4, K-6, P-2, P-6 and S-2
as moderately virulent (7.4-11.4 cm spread) while, K-3, O-1, O-2, O-3, O-4, O-5, O-6, P-1,
P-3, P-5, S-1, S-3, S-5 and S-6 was grouped as least virulent isolates (3.3-7.3 cm spread).
K-5 isolate was found to be more pathogenic to MMRI yellow (with the highest mean
length of spread of 15.4 cm) than to Sahiwal 2002 (with a mean length of spread of 23.3 cm)
clearly emphasizing differential reaction of a single isolate on the same genotype (MMRI-
yellow) as well as with a different genotype (Sahiwal 2002), with respect to length of spread.
Studies on Detached stem technique on Sahiwal 2002 revealed a higher length of
spread in K-5, K-6, P-2, P-3, and P-4 isolates. However, the relative length of spread was
lower in this technique compared to the observation recorded with intact stalks of Sahiwal
2002. Similar results were obtained by Subramaniam (1994) wherein he recorded contrary
95
results in detached stem technique on CSH-5 (sorghum cultivar) compared to observations on
intact stalks. Similar results on differential relation of M. phaseolina on different cultivars
and same cultivar was reported by Shamarao Jahagirdar et al., (2001) against M. phaseolina.
Pathogenicity refers to the ability of an organism to cause disease (harm the host).
This ability represents a genetic component of the pathogen and the overt damage done to the
host is a property of the host-pathogen interactions. Virulence is the degree of pathogenicity
within a group or species of parasites as indicated by case fatality rates and/or the ability of
the organism to invade the tissues of the host.
Benomyl has successfully controlled many diseases of different crops as leaf spot in
sugar beet (Kalaoglanidis et al., 2003), rice blast (Kamelwass-rao, 1976), scab and powdery
mildew of apples, cucurbits and strawberries (Scot et al., 1979). Marley and Genga (2004)
found that benomyl condensed the mycelial growth of Stenocarpella maydis in vitro. It also
repressed the growth of F. oxysporum (El- Tobshy et al., 1981). Mamza et al., (2010)
reported that benomyl beside with thiram and tricyclazole censored growth of F.
pallideroseum isolated from castor. Khan and Khan (2006) observed that benomyl and
carbendazim inhibited 100% mycelia development of M. phaseolina.
A numeral of mechanisms is involved in the control and reticence of pathogens by
fungicides. It was found from the present study that fungicides considerably caused
diminution in development of M. phaseolina and improved germination of maize. Fungicides
act by binding with B-tubulin polymers of pathogens which take part in a key role in nuclear
partition and result in reticence of polymerizing activity of microtubules. These also cause
barrier in diverse dictatorial cellular activities including mitosis, meiosis and cell form
preservation etc. (Nene and Thapliyal, 1982). Similarly, Carbendazim inactivates tubulin role
of pathogen necessary for their maintenance and growth (Butlers et al., 1995).
Aqueous extracts of five test plants significantly inhibited growth of M. phaseolina in
vitro and improved seedling emergence when tested in pots. Fungicidal activities of
antagonistic plants against pathogenic fungi are well documented. A. indica has shown
efficacy against F. solani, C. lunata and R. bataticola on brinjal and sunflower (Hussain et
al., 2000; Joseph et al., 2008).Ahmed et al., (2002) reported the efficacy of A. indica against
Bipolaris oryzae under in vitro conditions. Likewise, aqueous and ethanolic extract of
96
Alchemia cardifolia and Moringa oleifera suppressed the growth of F. verticilliodes and M.
phaseolina (Enikuomehin and Oyedeji, 2010).
In maize, germination increased as a result of treatment with plant extracts. The
enhanced germination might be attributed due to the deposition of chemical compounds
around seed surface and prevented penetration of the pathogen. These chemicals might have
caused lysis of sclerotia and triggered plant growth hormones which resulted in increased
germination and decreased disease incidence. Hasan et al., (2005) reported that seed
treatment with A. indica gave 99.33% seed germination in wheat. Similarly, seed treatment
with garlic extract against Colletolachum corchori, M. phaseolina, Botrydioiploida
theobromae, Fusarium spp, Penicillium spp, A. niger and A. flavus resulted in upto 77.50 %
increase in germination of jute (Islam et al., 2001). Also, seed dressing with caraway and
peppermint before sowing controlled the root rot disease. It was concluded that natural
products have strong fungicidal activity and can be applied for the control of different soil-
borne diseases.
The results of germplasm screening fall in line with the studies made by Padagaonkar
and Mayee (1990). They were of the opinion that genotypes with low stem water depletion
rate will tolerate infection from M. phaseolina. Anahosur and Naik (1985) reported that
quantity of sugar was more in resistant genotypes than susceptible genotype. Nalawade et al.,
2008 reported a higher levels of sugar and phenol in the charcoal rot tolerant varieties. In the
present study, this may be the reason in the nine germplasm lines which showed moderately
resistant reaction and one showed resistant reaction.
The effect of sowing date and inoculation time showed that more disease was
recorded when sowing was done ten days after optimum sowing time and inoculation after 60
days. This was due to the rise in temperature. The Macrophomina phaseolina colonized
maize tissue near the end of growing season so; this fungus is more growth at high
temperature colonized more and more disease recorded.
Studies on management of charcoal rot of maize under field conditions revealed that
the seed treatment method benomyl and Datura extract showed superior results over the
control in Sahiwal 2002 variety. The action of fungicide and plant extract has two-fold action
in the soil either by reducing the population of the inoculant fungus or by changing the
population of the native soil microflora. The present study revealed the importance of
97
fungicide and plant extract affecting the survival of the maize charcoal rot pathogen by
curtailing it to colonize the substrates and thereby reducing the inoculum density for
infection of the subsequent crop (Karunakar et al., 1994).
98
CHAPTER VI SUMMARY
Charcoal rot of maize caused by Macrophomina phaseolina is a root and stalk disease
and it is potentially caustic nature, predominantly in spring season grown crop.
The present investigation included collection, of diseased samples. Study of variation
in pathogen on the basis of morphological, cultural characters, evaluation of fungicides and
plant extracts and management of disease by identification of resistant sources, best
fungicide and plant extract to the disease.
Twenty four infected maize stalk samples were collected from major maize growing
districts of Punjab to assess the variability in the pathogen under in vitro conditions. From the
collected samples, Kasur (K-1, K-2, K-3, K-4, K-5 and K-6), Okara (O-1, O-2, O-3, O-4, O-5
and O-6), Pakpatan (P-1, P-2, P-3, P-4, P-5 and P-6) and Sahiwal (S-1, S-2, S-3, S-4, S-5 and
S-6), M. phaseolina was isolated to study the variability and each samples was considered as
an isolate. Based on colony pigmentation, the cultures were assigned to four major groups on
PDA and corn meal medium.
Grouping of isolates based on colony diameter on PDA revealed four groups; the first
group consisted of twelve isolates with a spread of 80.0 to 88.2 mm diameter, while the last
group included only two isolates with a mean colony diameter of 55.1 to 63.3 indicating the
existence of considerable variation among the isolates.
Most of the isolates took five to six days for sclerotial formation. Maximum number
of sclerotia of 56 per microscopic field (10x) was recorded in P-1 isolate, while the minimum
number was observed in S-6 isolate.
Based on mean colony diameter, the 24 isolates grown on corn meal medium was
grouped into four categories. The first group consisted of 8 isolates with a spread of 80.0 to
87.2 mm diameter.
Growth of different isolates on potato dextrose broth and corn meal broth was also
highly significant and isolates were categorized into groups each based on their dry mycelial
weight on PDB and CMB.
The sensitivity of different isolates to copper sulphate at three different levels of
concentrations (500, 1500 and 2500 ppm) were studied and isolates were found to differ in
99
their sensitivity to various concentrations tested. Except S-6, O-3, O-5and O-6 all other
isolates were highly sensitive at 1500 ppm concentration. But at 2500 ppm, all the isolates
were inhibited to the maximum (100%). Studies on benomyl against 24 isolates of M.
phaseolina did not reveal any variation at all the three concentration tested. In all these three
concentrations, all the isolates were consistently repressed cent per cent, indicating the
utmost efficacy of the chemical. Thus, benomyl could be used as seed dressing fungicide in
the event of non availibility of other suggested fungicides.
The results of study on diversity of the isolates in pH with esteem to mean mycelia
growth revealed that utmost growth was observed in 7.0 pH (321 mg mean mycelia weight)
closely followed by 282 mg mean mycelial weight at 6.5 pH representing privileged range to
be between 6.5 and 7.0 pH. With regard to temperature prerequisite, it was evident that all
the isolates preferred 35-40ºC range of temperature.
Based on the results of pathogencity studies (length of spread in Sahiwal 2002 and
MMRI yellow), the 24 isolates could be categorized as extremely virulent, fairly virulent and
least virulent. From the study, further it became clear that isolates from one geographical
location have shown varying behaviour even in the intra group collection.
Studies on Detached stem technique on Sahiwal 2002 revealed a higher length of
spread in K-5, K-6, P-2, P-3, and P-4 isolates. However, the relative length of spread was
lower in this technique compared to the observation recorded with intact stalks of Sahiwal
2002.
All the fungicides inhibited the growth of M. phaseolina and increased plant survival
significantly. The maximum inhibition was observed with Benomyl (86%) and Carbendazim
(82%) while Captan gave the minimum (44%) inhibition at 150 ppm concentration over
control. Plant survival was found to be the maximum in maize when seeds were treated with
Benomyl and the minimum when seeds were treated with Captan.
Similarly, all the aqueous extracts of all the test plants, significantly inhibited the
growth of M. phaseolina. Maximum inhibition was observed in case of Datura stramonium
(84%) and Calotropis procera (79%) while Dalbergia sissoo gave the minimum (42%)
inhibition at 100% concentration over control. Survival of maize plants were found to be the
maximum when seeds were treated with Datura stramonium and Azadirachta indica at 100%
concentration. On the other hand, M. oleifera showed minimum increase in plant survival.
100
Nineteen germplasm lines of maize screened against M. phaseolina for two seasons in
infested plot conditions. The results indicated that the charcoal rot was least in MMRI-
yellow, followed by BL-71 and Pak Afgoyee. One of the genotype showed resistant reaction,
9 lines showed moderately resistant reaction, 8 lines showed susceptible reaction.Thus, from
the results it was clear that employment of new source of resistance sources in breeding
programme to manage charcoal rot of maize.
Sowing of maize after ten days after sowing date and inoculation after sixty days of
sowing more disease recorded.
Studies on management of charcoal rot of sorghum in field conditions revealed that
the seed treatment with Benomyl and seed treatment with Datura stramonium was showing
superior results over other treatments.
OVER ALL CONCLUSIONS
The isolates collected from different localities varied significantly on the basis of
morphological characters and pathogenecity.
Datura stramonium and Calotropis procera exhibited significant inhibitory effect
against the pathogen and increased plant survival.
Benomyl and Carbendazim significantly inhibited growth of the pathogen and
enhanced plant survival.
One accession of maize was found resistant, nine moderately resistant, eight
susceptible and one is highly susceptible against charcoal rot.
101
LITERATURE CITED
Abawi, G.S and M.A. P. Corrales. 1990. Root rots of beans in Latin America and Africa;
diagnosis, research methodologies and management strategies. CIAT, Colombia. pp-
114.
Abdullaceva, A. 1962.Effects of phytoncides of onion and garlic on some group of
microorganisms. Uizbek. Biol. Zh. 6: 40-5.
Adam, D.B. and J. Stokes. 1942. The association of Rhizoctonia bataticola with rotting flax
in South Australia. Proceedings Linn.Society, N.S.W., 32: 313-317.
Adam, T. 1986. Contribution à la connaissance des maladies du niébé (Vigna unguiculata
L.) Walp.au Niger avec mention spéciale au Macrophomina phaseolina (Tassi) Goïd.
Université de Renne I. Thèse de doctorat. 117p.
Agarwal, D.K. and B.D. Goswani. 1973. Interrelationships between a fungus Macrophomina
phaseoli (Maubl.) Ashby and root-knot nematode Meloidogyne incognita (Kofoid and
White) Chitwood in soybean (Glycine max L.) Merill.). Proc. Ind. Nat. Sci.Acad. B.,
39: 701-4.
Agarwal, M.B., J.S. Gupta and R.B. Dixit. 1983. Effect of distillation and autoclaving on
phytonicidal principles of extracts of some plants. J. Ind. Bot. Soc. 62: 423-25.
Ahmad, S., 1997.Response to selection for grain yield and its componentsin a maize
population.M.Sc. Thesis Department of Plant Breedingand Genetics University of
Agriculture, Faisalabad–Pakistan
Ahmed, M.F., K.M. Khalequzzaman, M.N. Islam, M.K. Anam and M.T. Islam. 2002. Effect
of plant extracts against Bipolaris oryzae of rice under in vitro conditions. Pak. J.
Biol. Sci., 6: 442-45.
Ahmed, N. and Q.A. Ahmed.1969.Physiologic specialization in Macrophomina phaseoli
(Maubl.)Ashby causing stem rot of jute, Corchorus species.Mycopath., 39: 129-38.
Akhtar, T., A. Sattar and I. Khan, 1986. Antifungal activity of some plant extracts against
potato dry rot Fusarium coeruleum. Sarhad J. Agri. 2: 187-91.
Alabouvette, C. 1976. Recherches sur l'écologie des champignons parasites dans le sol.
VIII.Etude écologique de Macrophomina phaseolina grâce à une technique d'analyse
sélective. Ann. Phytopathol., 8:147-157.
102
Alagarsamy, G. and K. Sivaprakasam. 1988. Effect of antagonists in combination with
carbendazim against Macrophomina phaseolina infection in cowpea. Biological
control. J. Biological.Con, 2:123-5.
Al-Beldawi., A.S. Shaikh, H.M. Reddy and Al-Hashimi. 1973. Studies on the control of
charcoal rot of sesame with benomyl. Phytopath. Medit.,12 : 83-6.
Ali, F. and A. Ghaffar. 1991. Effect of water stress on rhizosphere mycoflora and root
infection of soybean. Pak. J. Bot., 23:135-9.
Alice, D., E.G. Ebenezar and K. Siraprakasan. 1996. Biocontrol of Macrophomina
phaseolina causing root rot of jasmine. J. Ecobiol. 8:17-20.
Allaghebandzadeh, N., S. Rezaee, B. Mahmoudi and H.Z. Zadeh. 2008. Pathogenic and
genotypic analysis among Iranian isolates of Macrophomina phaseolina. Phytopath.,
98(6): 11.
Anahosur, K.H. and S.H. Patil. 1983. Assessment of loss in sorghum seed weight due to
charcoal rot. Ind. Phytopath., 36:85-6.
Anahosur, K.H. and S.T. Naik. 1985. Relationship of sugars and phenols of root and stalk of
sorghum with charcoal rot. Ind. Phytopath., 38:131-4.
Anahosur, K.H., Patil, S.H. and R.K Hedge. 1984, Effect of herbicides on Macrophomina
phaseolina causing charcoal rot of sorghum. Pesticides., 18 :11-12.
Anilkumar, T.B. and M.N.L. Sastry. 1980.Nutritional and pathological variation among
isolates of Rhizoctonia bataticola from sunflower. Zbl. Microbiol., 137:228-32.
Annapurna, Y., S. Mitra, D.S. Iyengar, S.N. Rao and U.T. Bhalerao. 1983. Antimicrobial
activity of leaf extracts of Polyalthia longfolia. Phytopath.Zeitschrift., 106: 183-85.
Anuradha, B., S.N. Ghosh and A.K. Das. 2003. In vitro evaluation of some plant extracts for
antimicrobial activity. J. Mycopath. Res., 41:205-9.
Arca, G. and Yildzin. 1989. Investigation on the Physiological variation of Macrophomina
phaseolina in Aegean region. J. Turkish Phytopath., 18:39-45.
Ashby, S.F. 1927. Macrophomina phaseoli (Maubl.) Comb. Nov. the pycnidial stage of
Rhizoctonia bataticola (Taub.) Butl.Trans. Br. Mycol. Soc., 12:141-7.
Aviles, M., S. Castillo, J. Bascon, T. Z.Bonilla, P.M. M.Sanchez and R.M. Perez-Jimenez.
2008. First report of Macrophomina phaseolina causing crown and root rot of
strawberry in Spain. Plant Path.,57:382.
103
Babu, B.K., A.K. Srivastava and D.K. Arora. 2007. Identification and detection of
Macrophomina phaseolina by using species-specific oligonucleotide primers and
probe. Mycologia., 99:797-803.
Babu, B.K., R.Saikia and D.K. Arora. 2010. Molecular characterization and diagnosis of
Macrophomina phaseolina. a charcoal rot fungus. In: molecular identification of
fungi, Y. Gherbawy, and K. Voigt, cds. (Springer Berlin Heidelberg), p.179-193.
Bai, T.K., Z. Yin and HU J.C. 1991.A study on the pathogen of maize stalk rot in Northeast
China. Phytopath., 44:99-111.
Bainade, P.S., B.P. Tripathi, N. Khare and V.K. Yadav. 2006. Growth of Macrophomina
phaseolina on different media, pH and temperature levels. J.Plant .Dis. Sci., 1:70-3.
Baird, R.E., C.E. Watson and M. Scruggs. 2003. Relative longevity of Macrophomina
phaseolina and associated mycobiota on residual soybean roots in soil. Plant Dis., 87:
563-6.
Bajwa, R., N. Akhtar and A. Javaid. 2001. Antifungal activity of Allelopathic plant extracts
effect of allelopathic plant extracts of three allelopathic asteraceous species on growth
of Aspergillii. Pak. J. Biol. Sci., 4:503-7.
Bankole, S.A. and A.A Debanjo. 1995. Inhibition of some plant pathogenic fungi using
extracts from some Nigerian plants. Int. J. Trop. Plant Dis. 13:91-5.
Bansal, R.K and R.K. Gupta. 2000. Evaluation of plant extracts against Rhizoctonia
bataticola. Ind. Phytopath.,53:107-8.
Bega, R.V and R.S. Smith. 1962. Time and temperature relationships in thermal inactivation
of sclerotia of Macrophomina phaseolina. Phytopath., 52:632-5.
Bhargava, S.N. 1965. Studies on charcoal rot of potato. Phytopathol, 53:35- 44.
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. J.
Appl. Bacteriol., 77:129-33.
Bhatti, M.H.R. 1988. Antifungal properties of plant leaf decoctions against leaf rust of wheat.
Pak. J. Bot., 20:259-63.
Bhowmick, B.N. and B.K. Choudhary. 1982. Antifungal activity of leaf extracts of medicinal
plants on Alternaria alternata. Ind. Bot. Rep. 1: 164-5.
104
Bhowmick, B.N.and V. Vardhan. 1982. Antimycotic activity of leaf extracts of some
medicinal plants on Drechslera turcica. Bio. Bull. India 4: 58-60.
Biswas, P., A. Bhattacharyya, P.C. Bose, N. Mukherjee and N. Adityachaudhury,
1981.Further studies on the sensitivity of plant pathogenic microorganisms towards
some naturally occurring chalcones and flavanons. Experientia 37: 397-8.
Blakeman, J.P. and P. Atkinson, 1979.Antimicrobial properties and possible role in host-
pathogen interactions of parthenolide.Phys. Pl. Path. UK 15:183-2.
Bouhot, D. 1967. Étude du Macrophomina phaseoli sur arachide.Agr.Tropic., 22:1165-71.
Bouhot, D. 1968. Le Macrophomina phaseoli sur les plantes cultivées au Sénégal. Agri.
Tropic., 23:1172-81.
Butlers, J. A., S. J. Kendall, I. E. Wheeler and D. W. Hollomon. 1995. Tubulins:Lessons
from Existing Products That Can Be Applied to Target NewAntifungals. In:
Antifungal Agents, Discovery and Mode of Actions. G. K.Dixon, L. G. Copping, D.
W. Howwomon (Eds.), BIOS, Oxford .pp. 173-191.
Byadgi, A.S. and R.K. Hegde. 1985. Variation among the isolates of Rhizoctonia bataticola
from different host plants. Ind. Phytopath., 38:297-301.
Campbell, C.L. and D.J. V.Gaag. 1993. Temporal and spatial dynamics of microsclerotia of
Macrophomina phaseolina in three fields in North Carolina over four to five years.
Phytopath., 83:1434-40.
Canaday, C.H., D.G. Helsel and T.D. Wyllie. 1986. Effects of herbicide induced stress on
root colonization of soybeans by Macrophomina phaseolina. Plant Dis., 70: 863-866.
Carlile, M.J. 1996. Genetic exchange and gene flow: Their promotion and prevention. In:
Evolutionary Biological of the Fungi, ed. by A.D.M. Rayner, C. M. Brasier and D.
Moore. Pp. 203-214. Cambridge University Press, Cambridge, UK.
Carvalho, P.B., E.I. Ferreira. 2001. Leishmaniasis phytotherapy. Nature’s leadership against
an ancient disease-review. Fitoterapia., 72:599-618.
Chan, Y.H. and W.E. Sackston. 1973. Non-specificity of the necrosis inducing toxin of
Sclerotium bataticola. Can. J. Bot., 51:690-2.
Chaudhari, T. and C. Sen. 1982. Effects of some plant extracts on three sclerotia forming
fungal pathogens, Zeitschrift fur Pflanzenkrankheiten and Pflanzenschutz 89.
105
Chaumont, J.P. 1979. Fungistatic proielties of eight phanergams with respect to plant
pathogenic fungi.Bulletin de la societe Botanique de France 126: 537-541.
Chowdary, N.B. and Govindaiah. 2007. Influence of different abiotic conditions on the
growth and sclerotial production of Macrophomina phaseolina. Ind. J. of Sericul.
46:186-188.
CIMMYT. 2000. CGIAR Research, Areas of Research: Maize (Zea mays L.).
<http://www.cgiar.org/areas/maize.htm>.
Clinton, P.K.S. 1960. Some pests and diseases of sorghum and their control in the central
rain lands of the Sudan.Emp. J. Exp.Agri.,90:294-304.
Cloud, G.L. and J.C. Rupe. 1988. Preferential host selection by isolates of Macrophomina
phaseolina (Abstract). Phytopath., 78:1563.
Cloud, G.L. and J.C. Rupe. 1991. Comparison of three media for enumeration of sclerotia of
Macrophomina phaseolina. Plant Dis., 75:771-2.
Cloud, G.L. and J.C. Rupe. 1994. Influence of nitrogen, plant growth stage, and environment
on charcoal rot of grain sorghum caused by Macrophomina phaseolina (Tassi) Goid.
Plant Soil, 158:203-10.
Cook, G.E., M.G. Boosalis, L.D. Dunkle and G.W. Odvondny. 1973. Survival of
Macrophomina phaselina in corn and sorghum stalk residue. Plant Dis., Reptr.,
57:873-5.
Crous, P.W., B. Slippers, M.J. Wingfield, J. Rheeder, W.F.O. Marasas, A.J.L. Philips, A.
Alves, T. Burgess, P. Barber and J. Groenewald. 2006. Phylogenetic lineages in the
Botryosphaeriaceae studies. Mycol., 55:235-53.
Datar.V.V. 1999. Bioefficacy of plant extracts against Macrophominaphaseolina (Tassi.)
Goid.The incitant of charcoal-rot of sorghum. J. Mycol and Pl. Path., 29:251-53.
Dawar, S and A. Ghaffar. 1998. Effect of sclerotial inoculum density of Macrophomina
phaseolina on charcoal rot of sunflower. Pak. J. Bot., 30: 287-90.
Deshpande, A.L., J.P. Agarwal and B.H. Mathur. 1969. Rhizoctonia bataticola causing root
rot of opium in Rajasthan. Ind. Phytopath., 22:510-11.
Dhar, V and A.K. Sarbhoy. 1993. A typical isolate of Rhizoctonia bataticola. Ind.
Phytopathol., 46:245-6.
106
Dhillon, B.S and B.M. Prasanna. 2001. Maize; In "Breeding Food Crops." Ed. Chopra V.L.
pp 147-185. Oxford & IBH, New Delhi.
Dhingra, C.D. and J.B. Sinclair. 1973. Variation among the isolates of Macrophomina
phaseolina (Rhizoctonia bataticola) from different regions. Phytopathology.76:200-4.
Dhingra, O.D and J.B. Sinclair. 1974. Effect of soil moisture and carbon: nitrogen ratio on
survival of Macrophomina phaseolina in soybean stems in soil. Plant Dis.,Rep.,
58:1034-7.
Dhingra, O.D. and J.B. Sinclair. 1978. Biology and pathology of Macrophomina phaseolina
Minas Gerais Brazil, Universidade Federal de Vicosa.
Dhingra, O.D. and J.B. Sinclair. 1975. Survival of Macrophomina phaseolina sclerotia in
soil: Effect of soil moisture, carbon: nitrogen ratio, carbonsources, and nitrogen
concentrations. Phytopath., 65:236-40.
Dhingra, O.D. and J.B. Sinclair. 1977. An annotated bibliography of Macrophomina
phaseolina 1905-1975 Universidade Federal de Vicosa. Urbana, Brazil, University of
Illinois.
Diourte, M., J.L.Starr, M.J. Jeger, J.P. Stack and D.T. Rosenow. 1995. Charcoal rot
(Macrophomina phaseolina) resistance and the effects of stress on disease
development in sorghum. Plant Pathol., 44:196-202.
Dixit, S.N. and S.C. Tripathi, 1975.Fungistatic properties of some seedling extracts.Curr. Sci.
44: 279-80.
Dixit, S.N., S.C. Tripathi and R.R. Upadhyay. 1976. The antifungal substances of rose
flowers (Rosa indica). Econ. Bot., 30:371-4.
Dowswell, C.R., R.L. Paliwal and R.P. Cantrell. 1996. Maize in the Third World. West view
Press, Boulder, USA.
Ducan, R.R. 1984. The association of plant senescence with root and stalk diseases in
sorghum. Pp 99-110.In: Mughogho, L.K. (ed.) Sorghum root and stalk rots: A Critical
Review. Pantacheru, India: Int.crop res.Inst.for the semi-arid tropics.
Dwivedi, R.S. and R.C. Dubey. 1986. Effect of volatile and non-volatile fractions of two
medicinal plants on germination of Macroplomina phaseolina sclerotia. Trans. Brit.
Mycol. Soc., 87:326-8.
107
Edmunds, L.K. 1962. The relation of plant maturity, temperature and soil moisture to
charcoal stalk rot development in grain sorghum. Phytopath., 52:731.
Egawa, H., O. Tsutsui, K. Tatsuyamo and T. Hatta, 1977. Antifungal substances found in
leaves of Eucalyptus spp. Experientia 33.
Enikuomehin, O.A. and E.O. Oyedeji. 2010. Fungitoxic effect of some plant extracts against
tomato fruit rot pathogens. Arch. Phytopath. and Pl. Prot., 43:233-40.
Enteshamul, H.S., M. Abib. V. Sultana, J. Ara and A. Ghaffar. 1996. Use of organic
amendments on the efficacy of bio-control agents in the control of root rot and root
knot disease complex of okra. Nematol. Medit., 24:13-6.
Eswaramurthy, S., M. Muthusamy, V. Mariappan, R. Icyasekar and S. Natarajan. 1988.
Inhibitory effect of neem extracts on Sarcocladium oryzae and Fusarium oxysperum.
Proc. National seminar on control of plant disease Tamil Nadue, India.
FAO.2000a. Species Description.Tripsacum laxum Scrib.and Merr. http://www.fao.
org/WAICENT/faoinfo/agricult/agp/agpc/doc/gbase/data/pf000336.htm.
Gaetan, S.A., L. Fernande and M. Madia. 2006. Occurrence of charcoal rot caused by M.
phaseolina on canola in Argentina. Plant Dis.,90: 524-524.
Galinat, W.C. 1988. The origin of corn.In: Sprague, G.F. and Dudley, J.W. (Eds.) Corn and
corn improvement. Agronomy Monographs No. 18; pp. 1-31. American Society of
Agronomy: Madison, Wisconsin.
Gangopadhyay, S. and T.D. Wyllie. 1974. Melanin like compound in the sclerotia of
Macrophomina phaseolina. Ind. Phytopath., 27: 661-3.
Geol, S.K. and R.S. Mehrotra. 1973. Rhizoctonia root rot and damping off of okra and its
control. Acta Botany Indica.,1 : 45-8.
Ghosh, S.K. and C. Sen. 1973.Comparative physiological study on four isolates of M.
phaseolina.Ind. Phytopath., 35:225-6.
Ghosh, T., N. Mukherji and M. Basak. 1964. On the occurrence of a new species ofOrbilaria
Fr. Jute. Bull., 27: 134–41.
Goidanich, G and Camici. 1947. The prevalence and injuriousness of Macrophomina
phaseolina (Tassi) Goid. existing as a polyphagous parasite in Italy. Ann.
Sper.Agr.,(N.S), 1:485-520.
108
Goidanich, G. 1947. A revision of the genus Macrophomina phaseolina petrak type species:
Macrophomina phaseolina (Tassi.) Goid. Macrophomina phaseolina (Maubl)
Ashby.Ann. Sper. Agr. (NS), 1: 449-61.
GOP, 2013.Economic survey of Pakistan, Economic advisor Wing, Finance Division,
Islamabad. Pakistan.
Gopalan, C., B.V.R. Sastri and S. Balasubramanian. 2007. Nutritive value of Indian foods,
published by national institute of nutrition (NIN), ICMR.
Gray, F.A., B.J. Kolp and M.A. Mohamed. 1990. A disease survey of crops grown in the Bay
egion of Somalia, East Africa. FAO. Pl. Prot. Bult., 38:39-47.
Grewal, J.S. and D. Vir. 1958. Efficacy of different fungicides and seed disinfection in
relation to stem rot of jute and stripe disease of barley. Ind. Phytopathol, 11:175-8.
Grezes, B.B., N. Lucante., V. Kelechian, R. Dargent and H. Muller. 1996. Evaluation of
castor bean resistance to sclerotial wilt disease caused by Macrophomina phaseolina.
Plant Dis., 80:842-6.
Grover, R.K. and B.L. Chopra. 1970. Adaptation of Rhizoctonia species to two oxathin
compounds and manifestation of the adapted isolates. Acta.Phytopath. Acad.Sci,
Hung., 5:113-21.
Gupta, D.C. and N. Mehta. 1989. Interaction studies between different levels of Meloidogyne
javanica and Rhizoctonia spp. on mungbean (Vigna radiate (L) Wilczek.). Ind.J.
Nematol., 19:138-3.
Gupta, R.L. and N.K. Roy. 1993. Fungitoxicity and quantitative structure activity
relationships of S-alkyl, S-diamyl phosphorotrithioates. Pesticide. Res., 5:16-22.
Haigh, J.C. 1928. Macrophomina phaseolina (Maubl.) Ashby, The pycnidial stage of
Rhizoctonia bataticola (Taub) Butl. Tropic.Agric., 70:77-9.
Hall, R. 1991. Compendium of bean diseases.American Phytopathol.Soc., St. Paul, MN.73 p.
Hartman, G.L., J.B. Sinclair and J.C. Rupe. 1999. Compendium of Soybean Diseases, (4 th
ED). St. Paul, Minnesota.
Hasan, M.M., S.P. Chowdhury, S. Aslam, B. Hossain and M.S. Alam. 2005. Antifungal
effects of plant extracts on seed borne fungi of wheat seed regarding seed
germination, Seed health and vigour index. Pak. J. Biol. Sci., 8:1284-89.
109
Hildebrand, A.A., J.J. Miller and L.W. Koch. 1945. Some studies on Macrophomina phaseoli
Ashby in Ontario. Sci. Agric., 25:690-706.
Hiremath, R.V. and M.G. Palakshappa. 1991. Severe incidence of charcoal rot of sorghum at
Dharwad. Current Sci., 33:44.
Hoffmaster, D.E. and E.C. Tullis. 1944. Susceptibility of sorghum varieties to
Macrophomina dry rot (charcoal rot). PD Rep. 28:1175-84.
Holliday, P., E. Punithalingam. 1970. Macrophomina phaseolina. Descriptions of Fungi and
Bacteria (Wallingford UK, CAB International).
Hooda, I. and R.K. Grover. 1988. Studies on different isolates age and quantity of inoculum
of Rhizoctonia bataticola in relation to disease development in mung bean. Ind.
Phytopathol., 35: 619-23.
Hundekar, A. 1987. Studies on some aspects of stalk rot complex of sorghum (Sorghum
bicolor (L.) Moench).M. Sc. (Agri.) Thesis, University of Agricultural
Sciences,Dharwad, p. 146.
Husain, T and A. Ghaffar. 1995. Effect of soil moisture on the colonization of
Macrophomina phaseolina on roots of chickpea. Pak. J. Bot., 27: 221-5.
Hussain, S.Z., R.J. Anandam and A.S. Rao. 2000. Effect of different fungicides and
homeopathic drugs on seed borne fungi of sunflower (Helianthus annus. L). Ind. J. Pl.
Prot., 28: 148-1.
Ikeno, S., 1933, Studies on Sclerotium diseases of the rice plant VII. On the relation of
temperature and period of continuous wetting to the infection of soybean by the
sclerotia of Hypnochnus sasakii and an autolysis of the same fungus.Forsch. Get.
Pflanzenfrankh Kyoto., 2: 238-256.
Ilyas, M.B., M.A. Ellis and J.B. Sinclair. 1975. Evaluation of soil fungicides for the control
of charcoal rot of soybean. Plant Disease., Rep., 59:560-9.
Iriarte, F., E. Rosskopf., M. Hilf., G. McCollum., J. Albano and S. Adkins. 2007. First report
of Macrophomina phaseolina causing leaf and stem blight of tropical soda apple in
Florida. Plant Health Progress: Online (DOI: 10.1094/PHP-2007-1115-01-BR).
Islam, S.M.A., I. Hossain., G.A. Fakir and M. A. U. Ullah. 2001. Effect of physical seed
sorting, seed treatment with garlic extract and vitavax 200 on seed borne fungal flora
and seed yield of jute (Corchorus capsularis L.). Pak. J. Biol. Sci., 4:1509-11.
110
Israel, O.P. and M.S. Ali. 1964. Effect of carbohydrates on the growth of Rhizoctonia solani
Rahuri. Biol., 6:84-7.
Jain, N.K., M.N. Khare and E.C. Sharma. 1973. Variation among Rhizoctonia bataticola
isolates from urd bean plants and soil. Mysore J. Agric, Sci., 7:411-8.
Jaiswal, S., A. Batra and B.K. Mehta. 1984. The antimicrobial efficiency of root oil against
human pathogenic bacteria and phytopathogenic fungi. Phytopathal. Zeitschrift., 109:
90-3.
Jana, T., T.R. Sharma, R.D. Prasad and D.K. Arora. 2003. Molecular characterization of
Macrophomina phaseolina and Fusarium species by a single primer RAPD
technique. Microbiol. Res.,158:249-57.
Jones, R.W. and H.Y. Wang. 1997. Immunolocalization of a beta-1, 4-endoglucanase from
Macrophomina phaseolina expressed in plant. Can. J. Microbiol., 43:491-5.
Joseph, B., M.A. Dar and V. Kumar. 2008. Bio-efficacy of plant extracts to control Fusarium
solani f .sp. Melongenae Incitant of Brinjal Wilt. Global. J. Biotech. Biochem., 3:56
59.
Kaiser. W. J. and G. M. Horner. 1980. Root rot of irrigated lentils in Iran. Can. J. Bot., 58
(24): 2549-56.
Kalaoglanidis, G. S., D. A. Karadimos, P. M. Ioannidis and P. I. Ioannidis. 2003. Sensitivity
of Cercospora beticola populations to Fentinacetate, Benomyl and Flutriatol in
Greece. Crop. prot., 22:735-40.
Kamelwass, R. 1976. Efficacy of organic fungicides for the control of rice blast.Pesticides.,
10: 25-9.
Kannaiyan, J., Y.L. Nene and V.K. Sheila. 1980. Control of mycoflora associated with
pigeon pea seeds. Ind. J. Pl. Protect., 8:93-8.
Kapoor, A., R. Mahor, N. Vaishawpayan and N. Gautam, 1981. Antifungal spectrum of some
petal extracts. Geobios 8: 66-7.
Karunakar, R.K., K. Satyaprasad and S. Pande. 1994. Competitive saprophytic ability of the
sorghum stalk rot pathogens in fungicide amended soils. Ind.J. of Mycol.y and Plant
Pathol., 24:186-9.
111
Karunanithi, K., M. Muthuswamy and K. Seetharaman. 1999. Cultural and pathogenic
variability among the isolates of Macrophomina phaseolina causing root rot of
sesame. Plant Dis. Res., 14:113-7.
Kavita, T.R. 2007. Morphological and genetic variability and host resistance response of
sorghum recombinant inbred lines (RILs) to a virulent isolate of Macrophomina
phaseolina (Tassi) Goid. M. Sc. (Agri.) Thesis, Uni. Agric. Sci. Dharwad, p. 64-6.
Kayser, O. and A.F. Kiderlen. 2001. In vitro leishmanicidal activity of naturally occurring
chalcones. Phytotherapy. Res., 15:148-52.
Kazmi, S., S. Saleem, N. Ishrat, S. Shahzad and I. Niaz. 1995. Effect of neem oil and
benomyl on the growth of the root infecting fungi. Pak. J. Bot., 27: 217-20.
Kazmi, S.A.R., G. Jilani and A.H. Solani. 1991. Phytochemical variations and biological
efficacy of the neem tree. Tech. Report. Trop. Agri. Res. Inst. PARC, Karachi
University.
Kemp, M.S. 1978. Falcarindiol: an antifungal polyacetylene from Aegopodium podagraria.
Phytochemistry 17: 1002.
Kendig, S.R., J.C. Rupe and H.D. Scott. 2000. Effect of irrigation and soil water stress on
densities of Macrophomina phaseolina in soil and roots of two soybean
cultivars.Plant Disease., 84:895-900.
Khan, A.A and R.U. Khan. 2006. Management of Macrophomina leaf spot of Vigna radiata
by fungicides. Ann. Pl. Prot. Sci., 14: 258-9.
Khan, S.N. 2007.Macrophomina phaseolina as a causal agent of charcoal rot of sunflower.
Mycopathol.,5: 111-8.
Khan, T.Z. 1989. Some studies on antifungal properties of certain plant extracts against some
important plant pathogens. M.Sc. Thesis, Dept. of Pl. Path., Univ. of Agri.,
Faisalabad.
Khangura, R., and M. Aberra. 2009. First report of charcoal rot on canola caused by
Macrophomina phaseolina in Western Australia. Plant Dis., 93,666-7.
Khare, M.N., N.K. Jain and H.C. Sharma. 1970. Variation among Rhizoctonia bataticola
isolates from urdbean plant parts and soil (Abstract).Phytopath., 60:1298.
112
Kulkarni, S. 2000. Biology and management of dry stalk rots of maize (Zea mays L.) caused
by Fusarium moniliformae Sheild and Macrophomina phaseolina (Tassi.) Goid.
Ph.D. Thesis, University of Agricultural Sciences, Dharwad, pp.160-4.
Kumar, B.P., M.A.S. Chary and S.M. Reddy. 1979. Screening of plant extracts for antifungal
properties. New Botanist 6: 41-3.
Kumar, K. and Y.L. Nene. 1968. Antifungal properties of Cleome isocandra L. extracts. Ind.
Phytopath., 21: 445-6.
Kuti, J.O., R.L. Schading.G.V. Latigo and J. M. Braford. 1997. Differential responses of
guayule (Parthenium argentatum G.) genotypes to culture filtrate and toxin from
Macrophomina phaseolina (Tassi) Goid. J. Phytopathol., 145:305-311.
Likhite, V.N. and V.G. Kulkarni. 1934. Relative parasitism of cotton root rot organism from
Gujarat Soils. Current Sci., 3:252-4.
Lokesh, N.M. and V.I. Benagi. 2004. Studies on cultural variability of isolates of
Macrophomina phaseolina (Tassi) Goid. Karnataka J.Agric.Sci., 17:721-4.
Ma, J., C.B. Hill and G.L. Hartman. 2010. Production of Macrophomina phaseolina conidia
by multiple soybean isolates in culture. Plant Disease.,94:1088-1092.
Mahmoud, A. and H. Budak. 2011. First report of charcoal rot caused by Macrophomina
phaseolina in sunflower in Turkey. PlantDisease.,95:223.
Malaguti. G. 1990. Half a century of a plant pathologist in a tropical country Venezuela;
Annu. Rev. Phytopath., 28:1-10.
Mamza, W.S., A.B. Zarafi and O. Alabi. 2010. In vitro evaluaion of six fungicides on radial
mycelial growth and regrowth of Fusarium pallidoroseum isolated from castor
(Ricinus communis) in Samaru, Nigeria. Arch. Phytopathol. Pl. Prot., 43: 116-22.
Manglesdorf, P.C. 1974. Corn: Its origin, evolution and improvement. pp 1-262. Harvard
University Press; Cambridge, Massachusetts.
Marley, P.S and O. Gbenga. 2004. Fungicide control of Stenocarpella maydis in the Nigerian
Savanna. Arch. Phytopath. Pl. Prot., 37:19-28.
Masih, B., H.C. Sankhla and R.L. Mathur. 1970. Laboratory evaluation of the fungitoxicity
of PCNB and captan towards three soil fungi. Ind. Phytopathol. 23:136-7.
113
Mathur, R.I. and R.R. Singh. 1973. Control of root rot of cotton caused by Rhizoctonia
bataticola (Taub.) Butl.with soil application of Brassicol.Science and Culture,
39:221-2.
Mayee, C.D and V.V Datar. 1986. Phytopathometry, Technical Bulletin, Published by
Marathwada Agricultural University, Parbhani, Maharashtra, p. 146.
Mayek, P. N., R. G. Espinosa, C. L. Castañeda, J. A. A. Gallegos and J. Simpson. 2002.
Water relations, histopathology, and growth of commonbean (Phaseolus vulgaris L.)
during pathogenesis of Macrophomina phaseolina under drought stress. Physiol. Pl.
Path., 60:185-95.
Mayek-Perez. N, C. Lopez-Castaneda, M. Gonzalez-Chavira, R. Garcia-Espinosa, J. Acosta
Gallegos., O.M. De-la-vega and J. Simpson. 2001. Variability of Mexican isolates of
Macrophomina phaseolina based on pathogenesis and AFLP genotype. Physiol. Mol.
Pl. Pathol., 59 (5): 257-64.
McCain., A.H. and R.F. Scharpf. 1989. Effect of inoculum density of Macrophomina
phaseolina on seedling susceptibility of six conifer species. Eur. J. For. Pathol.,
19:119-23.
Mexico, D.F. 1994. Maize seed industries revisited: emerging roles of the public and private
sectors. World maize facts and trends. 1993/94. CIMMYT.
Meyer. W.A., J.B. Sinclair and M.N. Khare. 1974. Factors affecting charcoal rot of soybean
seedlings. Phytopathol., 64:845-49.
Mihail, D.J. 1989. Macrophomina phaseolina: Spatio-temporal dynamics of inoculum and of
disease in a highly susceptible crop. Phytopath., 79:848-55.
Mihail, J.D. 1992.Methods for research on soilborne phytopathogenic fungi. L.L. Singleton,
J.D. Mihail and C.M. Rush (eds). American Phytopathol. Soc., St. Paul, MN.
Mihail, J.D. and M. Alcorn. 1984. Effects of soil solarization on Macrophomina phaseolina
and Sclerotium rolfsii. Plant Dis., 68:156-9.
Mihail, J.D. and S. J. Taylor. 1995. Interpreting variability among isolates of Macrophomina
phaseolina in pathogenicity, pycnidium production and chlorate utilization. Can J.
Bot. Rev., 73:1596-603.
Minitab 17 Statistical Software (2010). [Computer software]. State College, PA: Minitab,
Inc. (www.minitab.com)
114
Mirza.J.H. and M.S.A. Qureshi. 1982. Fungi of Pakistan. Dept. Plant Pathology, Univ.
Agric., Faisalabad, Pakistan, pp: 311.
Misra, S.B. 1978.Antifungal activity of vapours of some plants. Acta Botanica Indica
6(Suppl.): 118-21.
Misra, S.B. and S.N. Dixit 1978.Screening of some medicinal plants for antifungal activity.
Geobios 4(4):129-32 (Rev. Pl. Path. 57(3):939, 1978).
Misra, S.B. and S.N. Dixit. 1977. Screening of some medicinal plants for antifungal activity.
Geobios 4:129-32.
Misra, S.B., R.R.B. Misra and S.M. Dixit, 1974.Screening of higher plants for antifungal
activity. Nat. Acad. Sci. India., 76:203-5.
Mkervali, V.G. 1963. Korichnevaya yatnistost Blagorodnogo Levrai merybor’y snei.(Brown
spot of Laurus nobilis and its control).Subtrop. Kulture. 1:106-14.
Moriss, M. L. 1998. Overview of the world maize economy. In: Morris M.L. (eds). Maize
Seed Industries in Developing Countries. pp 13-34. Lynne Rienner Publishers, Inc
and CIMMYT, Int.
Mukherjee, D. 1956. Studies on pigeonpea wilt. Ph. D. Thesis, Ind. Agric. Res. Inst., New
Delhi, p. 50.
Nalawade, S.V., G.D. Agarkar and B.B. Chirame. 2008. Biochemical mechanism of host
resistance to Macrophomina phaseolina (Tassi.) Goid.of sorghum.J. Maharashtra
Agric. Univ.,33:193-95.
Narain, A. and J.N. Satapathy, 1978. Antifungal characteristics of Vinca rosea extracts. Ind.
Phytopath., 30:36-40.
Nene .Y.L and P.N. Thapliyal. 1982. Fungicides in plant disease control. Oxford and IBH
Publishing Company, New Delhi., 507 pp.
Nene, Y.L. 1978. A world list of pigeonpea (Cajanus cajanL.) and chickpea (Cicer arietinum
L.) pathogens. ICRISAT Pulse Pathology Progress Report, 8:1-14.
Nene, Y.L. and K. Kumar. 1966. Antifungal properties of Erigeran linifolius L. extracts.
Nature., 53:363.
Nene, Y.L. and P.N. Thapliyal, 1965. Antifungal properties of Anagallis arvensis L. extracts.
Nature 52:89-90.
115
Odvody, G.N. and L.D. Dunkle. 1979. Charcoal stalk rot of sorghum: Effect of environment
on host parasite relation. Phytopath., 69:250-54.
Olaya, G. and Abawi, G.S. 1993. Effect of water potential on germination, growth and
sclerotial production of M. phaseolina.Phytopath., 83:1394.
Olaya, G., G.S. Abawi and N.F. Weeden. 1996. Inheritance of the resistance to
Macrophomina phaseolina and identification of RAPD markers linked to resistance
genes in beans. Phytopath., 86:674-79
Oluma, H.O.A., E.U. Amuta and R. Sha'ato. 2002. Antifungal activity of extracts of some
medicinal plants against Macrophomina phaseolina (Tassi) Goid. J. Agric and
Environ., 96:85-95.
Padagaonkar, S.M and C.D. Mayee. 1990. Stalk water potential in relation to charcoal rot of
sorghum. Ind. Phytopath., 43:192-96.
Pall, B.S., J.P. Lakhani and A.B. Beohar. 1990. Efficacy of fungicides for controlling
Macrophomina phaseolina (Tassi.) Goid in (Vigna mungo L.).Res. Dev. Rep., 7213.
Pandey, D.K., R.N. Tripathi, N.N. Tripathi and R.D. Tripathi. 1982. Antifungal activity in
some seed extracts. Environ. India., 4:83-85.
Papavizas, G.C. 1977. Some factors affecting survival of sclerotia of Macrophomina in soil.
Soil. Biol. Biochem., 9:337-41.
Pariya, S. and D.K. Chakravarti. 1977. Antifungal activity of some Indian medicinal plant
extracts on phytopathogenic fungi. Phytopath.Mediterranea., 16: 33-34
Pastor, C. M.A. and G.S. Abawi. 1988. Reactions of selected bean accessions to infection by
Macrophomina phaseolina. Plant Dis., 72:39-41.
Patel, K.K. and A.J. Patel. 1990. Control of charcoal rot of sesamum. Ind.J.Mycol. and Plant
Path., 20:62-3.
Patil, S.H. 1980. Studies on charcoal rot of sorghum caused by Macrophomina phaseolina
(Tassi.) Goid. M. Sc. (Agri.) Thesis, University of Agricultural Sciences, Bangalore,
p.66-68.
Pearson, C.A.S., F.W. Schwenk, F.J. Crowe and K. Kelly. 1984. Colonization of soybean
roots by Macrophomina phaseolina. Plant Dis., 68:1086-88.
Pearson, C.A.S., J.F. Leslie and F.W. Schwenk. 1986. Variable chlorate resistance in
Macrophomina phaseolina from corn soybean and soil. Phytopath., 76:646-49.
116
Peturshova, N.I. 1960. On the antimicrobial properties of higher plants.All union conference
on immunology from diseases and pest of plants. Kishiner, Minist. Agric. Mold,
U.S.S.R.
Philip, C.T., K.K. Kantha, R.K. Joshi and K.G. Nema. 1969. A Rhizoctonia disease of
Mungo (Phaseolus mungo Roxb.) in Madhya Pradesh. Jawaharlal Nehru Krishi
Vishwa Vidyalaya Res. J., 5:40-45.
Prajapati, R.K., S.S.L. Srivastava and R.G. Chaudhary. 2003. Incidence of chickpea dry root
rot in Uttar Pradesh and Uttar anchal and efficacy of seed dressing fungicides on seed
germination and seedling infection. Farm.Sci.. 12:170-71.
Pratt, R.G. 2006. A direct observation technique for evaluating sclerotium germination by
Macrophomina phaseolina and effects of biocontrol materials on survival of sclerotia
in soil.Mycopath.,162: 121-31.
Punithalingam, E. 1983.The nuclei of Macrophomina phaseolina (Tassi) Goid. Nova
Hedwigia 38:339-67.
Purkayastha, S., B. Kaur. N. Dilbaghi and A. Chaudhury. 2006. Characterization of
Macrophomina phaseolina, the charcoal rot pathogen of cluster bean, using
conventional techniques and PCR- based molecular markers. Pl. Pathol., 55: 106-116.
Rafiq, M., M.A. Nasir and M.A.R. Bhatti. 1984. Antifungal properties of certain common
wild plants against different fungi. Pak. J. Agri. Res., 5: 236-38.
Rajkule, P.N., H.L.Chauhan and K. Desai. 1979. Chemical control of charcoal rot. Sorghum
Newsletter.22:120.
Rajkumar, F. Bashasab and M.S. Kuruvinashetti. 2007. Genetic Variability of Sorghum
Charcoal Rot Pathogen (Macrophomina phaseolina) Assessed by Random DNA
Markers. Pl. Pathol., 23(2): 45-50.
Ramamurthy, R. 1982. Studies on root rot caused by Rhizoctonia bataticola (Taub) Butler.
M. Sc. (Agri.) Thesis, University of Agricultural Sciences, Bangalore, p. 106.
Ramdoss, S. and K. Sivaprakasan. 1987. Effect of fungicides and insecticides on the linear
growth of Macrophomina phaseolina (Tassi.) Goid.Madras Agri.J., 74:29-33.
Rathore, B.S and R.S. Rathore. 1999. Effect of seed dressers on Macrophomina root rot of
mothbean. J. Mycol. Pl. Pathol., 29:389-392.
117
Raut, J.G. 1985. Effect of charcoal rot caused by Macrophomina phaseolina on sunflower
plants. Ind. Path. 38: 345-46.
Raut, J.G. and B.B. Bhombe. 1976. Studies of two isolates of Rhizoctonia bataticola on
sorghum. J. Maharashtra Agri. Uni., 16:264-67.
Raut, J.G. and R.W. Ingle. 1989. Variation in isolates of Rhizoctonia bataticola. Ind.
Phytopath., 42:506-508.
Reddy, G.R., A.G.R. Reddy and K.C. Rao. 1991. Effect of different seed dressing fungicides
against seed borne fungi on groundnut. J. Oil Seed. Res., 8:79-83.
Reichert, I. and E. Hellinger. 1947. On the occurrence, morphology and parasitism of
Sclerotium bataticola. Palestine J. Bot., 6:107-47.
Renu, K., R.D. Tripathi and S.N. Dixit. 1980. Fungitoxic properties of Cestrum diurnum.
Naturwissenschaften 67: 150-51.
Reuveni, R., A. Nachmias and J. Krikun. 1983. The role of seed borne inoculum on the
development of Macrophomina phaseolina on melon. Plant Dis., 67: 280-81.
Reyes, F., M.C., S.H. Delgado, R.B. Fernández, M.M. Fernández, J. Simpson and N.M.
Pérez. 2006. Pathogenic variability within Macrophomina phaseolina from Mexio
and other countries. J. Phytopath., 154: 447-53.
Rizki, Y.M., K. Fatima, A. Askari, S.I. Ahmad and Y. Babar. 1987. Studies on the antifungal
properties of indegenous plant from Karachi region. Pak. J. Sci. Ind. Res., 30: 760-63.
Sahai, D. 1969. Evaluation of certain fungicides against Macrophomina phaseolina from
charcoal rot infected potato. Sci.and Cult., 35:686-87.
Saleem, A. A. 1988. Use of neem and other botanical products for the control of nematodes
and fungi infesting banana and other crop.A review. National Seminar on Banana
Disease Problem in Sindh held on July 11-13, Hyderabad Pakistan.
Saleh, A.A., H.U. Ahmed, T.C. Todd, S. E. Travers, K.A. Zeller, J.F. Leslie. and K.A.
Garrett. 2009. Relatedness of Macrophomina phaseolina isolates from tallgrass
prairie, maize, soybean and sorghum. Mol. Ecol., 19:79-91.
Salik, N.K. 2007.Macrophomina phaseolina as causal agent for Charcoal rot of sunflower. 2:
111-18.
118
Satishchandra, K.M. 1977. Saprophytic activity and survival of Rhizoctonia bataticola
(Taub) Butl.in different soils. M. Sc. (Agri.) Thesis, University of Agricultural
Sciences, Bangalore, pp. 80.
Saxena, J. and C.S. Mathela. 1997. Antifungal activity of new compounds from Nepata
leucophylla and Nepata clarkei. J. Appl. Env. Microbiol., 62:702-704.
Schmitz, H. 1930. Poisoned Food Technique. Ind. Eng. Chem. Analt., pp. 361.
Scot, D.H., A.D. Draper and J.I. Maos. 1979. Benomyl for control of powdery mildew on
strawberry plants in greenhouse. Plant Dis. Rept., 54:362-63.
Sekhar, G. 1985. Studies on the effect of rhizosphere on development of charcoal rot in
sorghum and saprophytic survival of Macrophomina phaseolina (Tassi.) Goid.Ph. D.
Thesis, University of Agricultural Sciences, Bangalore.
Sen, C. and S. Bandyopadhyay. 1988. Some aspects of ecological behaviour, disease
development and biological inoculum of Macrophomina phaseolina. Perspective in
Mycology and Plant Pathology, Ed. Agnihotri, V. P., Sarbhoy, A.K. and Diniesh
Kumar, 1988, Malhotra Publishing House, New Delhi, pp. 418-43.
Shafique, S., S. Shafique and A. Javaid. 2005. Fungitoxicity of aqueous extracts of
allelopathic plants against seed borne mycoflora of maize. Mycopath., 3:23-6.
Shahzad, S. and A. Ghaffar. 1986. Macrophomina phaseolina (Tassi) Goid, on some new
hosts in Pakistan. FAO. Pl. Prot. Bull., 34:163.
Shahzad, S., A. Sattar and A. Ghaffar. 1988. Addition to the hosts of Macrophomina
phaseolina. Pak. J. Bot., 20:151-52.
Shamarao, M.S. Patil and S. Indira. 2001. Biological control of charcoal rot of sorghum
caused by M. phaseolina. Agric.Sci.Digest., 21: 153-56.
Shanmugam, N. and C.V. Govindswamy. 1973. Control of Macrophomina root rot of
groundnut. Madras. Agri. J., 60:500-503.
Sharma, R. C. and S. Lal. 1998. Maize diseases and their management. Indian farming.48:92-
96.
Shaw, R. H. 1988. Climate requirement.In: Sprague G.F., Dudly J.W (eds.) Corn and Corn
638 Improvement, 3rd ed Madism, WI: ASA 609.
Sheikh, A. and A. Ghaffar. 1979. Relation of sclerotial inoculum density and soil moisture to
infection of field crops by Macrophomina phaseolina. Pak. J. Bot., 11: 185–89.
119
Shekhar, M., R.C. Sharma, L. Singh and R. Dutta.2006a. Morphological and pathogenic
variability of Macrophomina phaseolina (Tassi.) Goid incitant of charcoal rot of
maize in India. Ind.Phytopath., 59:453-59.
Shekhar, M., R.C. Sharma, L. Singh and R. Dutta.2006b. Genetic variability of
Macrophomina phaseolina (Tassi) Goid.incitant of charcoal rot of maize in India.
Ind. Phytopathol.,59:294-98.
Short, G. E., T. D. Wyllie, and V. D. Ammon. 1978. Quantitave enumeration
ofMacrophomina phaseolina in soybean tissues. Phytopathol., 68: 736–741.
Short, G.E. and T.D. Wyllie. 1978. Inoculum potential of Macrophominaphaseolina.
Phytopath., 68:742-46.
Short, G.E., T.D. Wyllie and P.R. Bristow. 1980. Survival of Macrophomina phaseolina in
soil and residue of soybean. Phytopathol., 70:13-7.
Sinclair, J. B. and P.A.Backma. 1989. Compendium of soybean diseases.3rd ed. American
Phytopathol.Society, St. Paul, MN.106 p.
Singh, D.B., S.P. Singh and R.C. Gupta, 1979.Effect of volatiles from seeds of some
umbelliferae. Trans. Brit. Mycol. Soc. 73: 349-50.
Singh, H.P., D.R. Batish and R.K. Kohli. 2004. Allelopathic interactions and allelochemicals:
New possibilities for sustainable weed management. Cri. Rev. Plant .Sci. 22:239-311.
Singh, L., M. Sharm and R.P. Singh. 1977. In vitro fungitoxicity of certain Indian ferns and
gymnosperms. Biovigyanum 3: 17-23.
Singh, R.D.N. and S.K.M. Kaiser. 1995. Evaluation of some elite genotypes of maize for
resistance to charcoal rot disease. J. Mycopath. Res., 29:141-7.
Singh, S.K., Y.L. Nene and M.V. Reddy. 1990. Influence of cropping system on
Macrophomina phaseolina in soil. Plant Disease., 74:812-14.
Sinha, R., K.P and B.B.P. Sinha. 2004. Effect of potash, botanicals and fungicides against
wilt disease complex in lentil. Ann. Pl. Prot. Sci.,12 :454-55.
Smith, G.S., and O.N. Carvil. 1997. Field screening of commercial and experimental soybean
cultivars for their reaction to Macrophominaphaseolina. Plant Disease., 81:363-368
Smith, W.H. 1969. Germination of Macrophomina phaseolina sclerotia as affected by Pinus
lambertiana root exudate. Can. J. Microbiol., 15:1387-91.
120
Smits, B.G. and R. Noguera. 1988. The ontogeny and morphogenesis of sclerotia and
pycnidia of Macrophomina phaseolina. Agron. Trop., 38:69-78.
Sobti, A.K. and L.C. Shama. 1992. Cultural and pathogenic variations in isolates of
Rhizoctonia bataticola from groundnut in Rajasthan. Ind. Phytopath., 45:117-19.
Songa, W. and R. J. Hillocks. 1996. Legume hosts of Macrophomina phaseolina in Kenya
and effect of crop species on soil inoculum level. J. Phythopath., 144:387-91.
Striubaite, J. 1960. Verdryniniu fitoncidal, JuPoveikis Irybame ir prakitinio naudojimo
Perspektyvoe, (Phytoconcides of Renunculaceae, their effect on the growth of fungi
and prospects for their practical use). J. Biol. Geog. Geol. 36: 165-173. (Absts. Rev.
Pl. Appl. Mycol. 41: 575, 1962).
Strivastava, J.N., R.K.S. Kushwaha, J.N. Strivastava and J.P. Shukla. 1984. Antifungal
activity of Parthenium hysterophorus. Curr. Sci. 53: 712.
Su, G., S.O. Suh and R.W. Schneider. 2001. Host specialization in the charcoal rot fungus,
Macrophomina phaseolina. Phytopathol, 91:120-6.
Subramaniam, J. 1994. Variability in Macrophomina phaseolina (Tassi) Goid causing
charcoal rot of sorghum. M. Sc. (Agri.) Thesis, University of Agricultural Sciences,
Dharwad.
Sulaiman, M. and B.C. Patil. 1966. Existence of physiological races of Macrophomina
phaseoli causing root rot of cotton. Beitr. Trop. Subtrop. Land-Wirtsch.Tropen.
Veterinarmed.4 :291-98.
Suriachandraselvan, M. and K. Seetharam. 2000. Relationship among pigment synthesis,
culture media, growth and virulence of the geographical isolates of Macrophomina
phaseolina causing charcoal rot of sunflower. J. Myc. and Pl. Pathol.,30:370-4.
Taneja, M. and R.K. Grover. 1982. Efficacy of benzimidazole and related fungicides against
Rhizoctonia solani and Rhizoctonia bataticola. Annals of Appl. Biol., 100:425-32.
Tarr, S.A.J. 1962. Diseases of Sorghum, Sudan Grass and Broom Corn, Kew, Surrey, D.K.
Common wealth Mycol.Inst.p.380.
Than, H., M.M. Thein and S.S. Mvint. 1991. Relationship among Rhizoctonia bataticola
isolates in rice based cropping system based on colony fusion types.Int. Chickpea
Newsletter., 25:29-31.
121
Thapliyal, P.N. and Y.L. Nene. 1970. Influence of growth stages of Anagallis gravensis L. on
its fungitoxicity. Econ. Bot., 24: 283-85.
Thomas, K.M. 1938. Detailed administration report of the Government mycologist, Madras
for the year 1937-38, p. 21.
Townsend, B.B. 1957. Nutritional factors influencing the production of sclerotia by certain
fungi.Ann. Botany(NS)., 21:153-6.
Tripathi, R.N., N.K. Dubey and S.N. Dixit. 1981. Antifungal activity in pollens of Gorakhpur
locality. Nat. Acad. Sci. Lett. 4: 107-109.
Tuite, J., 1969. PlantPathologicalMethods; Fungi and Bacteria, Burgess Publishing
Company, Minneapolis, p. 239.
Uppal, B.N. 1931.Rhizoctonia bataticola on sorghum in Bombay presidency.Internat.Bull.
Pl. Protec., 5:163.
Uppal, B.N. 1934.Summary of work done under the plant pathologist to government,
Bombay Presidency, Poona for the year 1932-33.Annu.Report, Department of
Agriculture, Bombay Presidency. 1932-33, p. 171-175.
Uppal, B.N., K.G. Kolhatkar and M.K. Patel. 1936. Blight and hollow stem of sorghum. Ind.
J. Agri.Sci., 6:1323-34.
Vandemark, G., O. Martinez, V. Pecina and M.D. Alvarado. 2000. Assessment of genetic
relationships among isolates of Macrophomina phaseolina using a simplified AFLP
technique and two different methods of analysis. Mycologia., 92:656-664.
Vasudeva, R.S. 1937. Studies on the root rot disease of cotton in Punjab III. The effect of
some physical and chemical factors on Sclerotia formation.Ind. J. Agri. Sci., 7:259-
70.
Vincent, J.M. 1927. Distortion of fungal hyphae in the presence of certain inhibitors.
Nature.159:850.
Vyvyan, J.R. 2002. Allelochemicals as leads for new herbicides and agrochemicals.
Tetrahedron., 58:1631-46.
Waller, J.M.1976. Plant Disease in arid climate.SPAN, 19:125-26.
Waseer, N.A., M.A. Pathan, Wondiarm and G.R. Solangi. 1990. Studies on charcoal rot of
soybean caused by Macrophomina phaseolina (Tassi) Goid. Rajasthan. J. Phytopath.,
2:22-30.
122
Watson, L. and M.J. Dallwitz. 1992. Grass genera of the world: descriptions, illustrations,
identification, and information retrieval; including synonyms, morphology, anatomy,
physiology, photochemistry, cytology, classification, pathogens, world and local
distribution, and reference. Version: 18thaugust 1999.http://biodiversity.uno.edu/
delta>.
Wrather, J. A., S.R. Koenning and T.R. Anderson. 2003. Effect of diseases on soybean yields
in the United States and Ontario (1999-2002). Online: plant health progress,
http://www.plantmanagmentnetwork.org/pub/php/review/2003/soybean/.
Wrather, J.A., T.R. Anderson, D.M. Arsyad, J. Gai, L.D. Ploper, A.P. Puglia, H.H. Ram and
J.T. Yorinori. 1997. Soybean disease loss estimates for the top 10 soybean producing
countries in 1994. Plant Dis., 81: 107–10.
Wyllie, T.D and D.H. Scott. 1988. Charcoal rot of soybeans - current status. In: Soybean
Diseases of the North Central region (Indianapolis, Ind., APS Press, p.106-113.
Wyllie, T.D. and O. H. Calvert. 1969. Effect of flower removal and pod set on the formation
of sclerotia and infection of Glycine max by Macrophomina phaseoli. Phytopath., 59:
1243-45.
Wyllie, T.D.and M. F. Brown. 1970. Ultra-structural formation of sclerotia of Macrophomina
phaseoli. Phytopathol., 60: 524-28.
Yang, S.M., and D. F. Owen.1982.Symptomatology and detection of Macrophomina
phaseolina in sunflower plants parasitized by Cylindrocopturus adspersus larvae.
Phytopath.,72: 819-21.
Yang, S.M., C. E. Rogers, and N. D. Luciani.1983. Transmission of Macrophomina
phaseolina in sunflower by Cylindrocopoturus adspersus. Phytopathol.,73: 1467-69.
Zahid, M.I., G. M. Gurr, A. Nikandrow, M. Hodda, W. J. Fulkerson and H. I. Nicol. 2002.
Effects of root and stolon infecting fungi on root-colonizing nematodes of white
clover. Pl. Pathol. 51: 242–50.