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3
GENERAL INTRODUCTION
Tobacco is an important industrial crop of India providing
employment to 38 million people including 6 million tobacco farmers. It
fetches 20,000 crores as internal excise revenue and foreign exchange.
About 750 million kg of tobacco is produced and 260 million kg is
exported annually. Tobacco in India is grown in an area of around 4.5
lakh ha. India is the second largest producer of tobacco in the world next
to Peoples Republic of China. The global tobacco production is 7.1
million kg of dry weight (Anonymous, 2011). Indian tobacco is exported
to more than 80 countries. Tobacco provides livelihood to 38 million
people, both direct and indirect, and to over 70% of those engaged in
agriculture.
Apart from the above aspects, tobacco is a promising crop to be
used in the field of Biotechnology (Ganapathi et al., 2004) for the
production of different products including many life saving ones (Cramer
et al., 2004).
Tobacco remains as the earliest plant to be genetically engineered
and is widely used to test suitability of plant based systems for
bioproduction of recombinant proteins. Some plant based biotech
companies (Crop Tech crop; Bio Source Technologies, Inc. and Plant
Biotechnology) are already targeting tobacco for biopharmaceutical
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production. Tobacco is an excellent biomass and seed producer. Hence
this hastens the time in which a product can be scaled up and brought to
market. Thus it is referred to as a promising protein factory (Daniell,
2005). The beauty of the system is its potential versatility that can be
adapted to fight against other pathogens as well. Gu et al., (2005) have
confirmed the expression of Ure B gene in tobacco plants for the
production of plant based vaccines. Vaccines produced by transgenic
plants have the potential to change the traditional means of production
and injecting, which reduces the cost of vaccine production. Daniell
(2005) said “we can provide enough doses of a safe and effective vaccine
for all Americans from just one acre of tobacco plants”.
In India tobacco cultivation was introduced by Portuguese in 1605.
Tobacco holds an unparalleled position among crop plants in several
ways. It is one of the very few crops entering world market entirely on
leaf basis and is the most widely grown non-food crop. In many
countries, it is an instrument of very high importance in financial and
economic policy matters. Originally it had a religious significance and
subsequently claims are made regarding medicinal benefits (Anonymous,
2005). It was used as an intoxicant and also as cure for all kinds of ills
and for paying homage to deities (Gopalachari, 1984).
At present tobacco is cultivated for the manufacture of cigarettes,
bidi, cigar, cheroot, hookah, snuff, chewing types (Gopalachari, 1984;
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Manickavasagan et al., 2007). Different types of tobacco cultivated are
Bidi, Cheroot, Flue Cured Virginia (FCV), Natu, Hookah, Cigar and
Snuff and percentage of their cultivation is given in Table1 and their
distribution throughout India is shown in Fig. 1.
Table 1. Different varieties of tobacco cultivated in India
Sl. No. Varieties Percentage
of cultivation
1 Bidi 29.5
2 Burley 1.0
3 Cheroot 29.1
4 Cigar 1.5
5 FCV 23.6
6 HDBGG 1.5
7 Hookah 6.6
8 Natu 8.1
9 Snuff 1.5
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Fig. 1. Tobacco map of India
Among the varieties, FCV tobacco is of export quality and is
cultivated in the transitional belt between the eastern slopes of Western
Ghats and the plains in Karnataka under monsoon conditions. This
transitional belt is also Karnataka Light Soil (KLS) region (Fig. 2).
Karnataka is one of the important states where FCV tobacco is produced
next to Andhra Pradesh, Orissa and Maharashtra.
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In Karnataka, presently the crop is grown in about 85,000ha. The
yields per ha are around 1,250kg. West European, some African Middle
East and South East Asian countries mostly prefer this tobacco.
Various soil zones under varied rainfall conditions in Andhra
Pradesh, Orissa and Maharashtra are Northern Black Soils (NBS), Central
Black Soils (CBS), Southern Black Soils (SBS), Southern Light Soils
(SLS) and Northern Light Soils (NLS). In Karnataka the soils are sandy
loams and loamy sand. The light soils existing in Mysore, Hassan,
Shimoga, Davangere, Coorg, Chikkamagalore and Chitradurga districts
constitute the light soils of Karnataka and the zone is known as Karnataka
Light Soils (KLS). The crop in this transitional zone is raised as a
monsoon crop with sufficient rainfall during the crop growth period.
Amount of rain fall in this tract ranges from 800mm to 1000mm. Tobacco
nurseries are raised during March-May (premonsoon period). The
seedlings are transplanted in May-June period during the onset of
southwest monsoon and harvested in August-September, cured tobacco is
marketed by December (Devaki, 1991; Gopalachari, 1984).
The optimum weather conditions during cultivation of tobacco in
the field after transplantation also favours number of diseases. Wilt
disease is one of the important diseases of priority as it causes severe loss
of yield and quality of tobacco.
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Fusarium species occur in all major geographic regions of the
world. Individual species have a cosmopolitan geographic distribution.
Around 80 species are reported from different countries (Leslie and
Summerell, 2006).
In the monograph “Die Fusarein” by Woollenweber and Reinking
(1935), the large genus was divided into 16 sections with few exceptions
and placed all Fusaria causing wilt of plants in Section: Elegans.
However, Snyder and Hansen (1940) suggested that the Elegans section
should contain only one species Fusarium oxysporum (Schlecht) with
various formae specialis to be designated, based on pathogenicity, for
single plant species.
Fig. 2. Flue Cured Virginia tobacco growing areas of Karnataka
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Fusarium species have been important for many years as plant
pathogens causing diseases such as crown rot, head blight, and scab on
cereal grains, vascular wilts on a wide range of horticultural crops, root
rots, cankers, and other diseases such as pokkah-boeng on sugarcane and
bakanae disease of rice (Gilardi et al., 2007; Kausar et al., 2009; Nelson
et al., 1981; Tawfik and Allam, 2004). These are a widespread
cosmopolitan group of fungi and commonly colonise aerial and
subterranean plant parts, either as primary or secondary invaders. Some
species are common in soil and they result in necrosis of roots of many
agricultural crops (Nelson et al., 1983). Of all diseases caused by
Fusarium, probably the most important ones are the vascular wilt
diseases caused by the formae specialis of Fusarium oxysporum.
Symptoms of diseased plants include wilt, chlorosis and necrosis of
leaves and their apices, growth retardation, asymmetric growth, dark
longitudinal streaks on stems and petioles and stem necrosis, vascular
discoloration, root rot, and plant death (Dutky and Wolkow, 1994;
Holcomb and Reed, 1994). The disease may severely reduce crop
productivity and yield. The pathogen may be transmitted via seeds also
(Elmer et al., 1994; Keinath, 1994; Martini and Gullino, 1991).
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Fusarium spp. are long-lived as soil inhabitants that can survive
extended periods in the absence of their host by colonizing crop debris
and producing chlamydospores, dormant resting propagules (Haware et
al., 1978; Nelson, 1981). Chlamydospores can with-stand extreme
environmental conditions (Nash et al., 1961) and readily germinate when
conditions are favorable (Schippers and Van Eck, 1981). Soil carbon
depletion, nutrient requirements (carbon, nitrogen and minerals), nutrient
stress, and light quality are factors in quenching chlamydospore
production (Mondal et al., 1996; Oritsejafor, 1986). Hebbar et al., (1997,
1998) described a one-step liquid fermentation system using a low
utilizable carbon substrate that produced large quantities of
chlamydospores of a F. oxysporum isolate.
Fusarium infection of a host plant is accomplished either by
germinating conidia or by direct hyphal penetration (Nelson, 1981;
Beckman and Roberts, 1995). Various factors including nitrogen
amendments and host root exudates affect the survival of Fusarium
chlamydospores. Flavonoids and other host root exudates stimulate
chlamydospore germination (Mondal et al., 1996; Ruan et al., 1995;
Schroth et al., 1963). The impact of non-host exudation on
phytopathogenic species has received far less attention but might,
nonetheless, play an important role in soil fungistasis (Schroth and
Hendrix, 1962; Schroth and Hildebrand, 1964). Since the fungus can
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survive in the soil for several years, it is not possible to control the
disease through normal crop rotations.
Fusarium wilt is one of the “stem diseases” largely confined to
transplanted crop and is one of the destructive field diseases (Garner,
1951). Many tobacco germ plasm lines have been reported as resistant to
wilt from different countries of the world (Nene et al., 1981). Resistance
to vascular wilt may be expressed before the pathogen gains entry into the
xylem of plants or even after that. Since vascular fungi are facultative
parasites, they obviously find the xylem environment relatively free of
severe host reactions. These peculiar features have to be born in mind to
identify the factors of resistance to wilt disease (Farooq et al., 2005).
Populations of Fusaria in agricultural field soils can be greater than
1,00,000 propagules (Smith and Snyder, 1971). Fusarium species were
frequently isolated from diseased plant roots (Gherbawy and Prillinger,
2000; Zhang et al., 1996). Fusarium oxysporum Schlecht. has one of the
broadest host ranges of many plant pathogenic fungi.
In Karnataka, tobacco is grown as a monocrop every year. This is
ideal for perpetuation and development of pathogens under favourable
environmental conditions. Diseases resulted due to these pathogens which
become epidemic causing severe losses.
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Fusarium wilt is one of the devastating diseases of tobacco and has
become a threat to its cultivation. A view of healthy tobacco (Fig. 3) and
wilt affected crop (Fig. 4) is given below:
Fig. 3. Healthy tobacco crop
Fig. 4. Wilt affected tobacco crop
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Scanty work was available on this disease. Literature review and
consultations with the scientists of CTRI, Research station, Hunsur, have
revealed the necessity for further investigation in the area of wilt of
tobacco.
Hence, investigation on this disease of priority is taken up for the
present study involving the following objectives:
Part A: Biology of the Pathogen
1. Disease survey
2. Identification of the pathogen and pathogenicity studies
3. Studies on wilt complex
4. Molecular studies
5. Cultural studies
6. Biochemical studies
Part B: Management of the Disease
7. In vitro evaluation of chemical fungicides
8. In vitro evaluation of botanicals
9. In vitro evaluation of biocontrol agents
10. Comparative evaluation of chemical fungicides and bioagent
formulations in the field
14
BIOLOGY OF THE PATHOGEN
INTRODUCTION
Fusarium wilt was first observed by Johnson (1921) in Southern
Mary Land in light soil in which tobacco was grown for many years (Fig.
4). It is caused by Fusarium oxysporum f. sp. nicotianae (Johnson) W. C.
Snyder & H. N. Hansen (FON). This fungus is an anamorph and is
classified under Kingdom: Mycetae, Division: Amastigomycota, Class:
Deuteromycetes, Order: Moniliales, Family: Tuberculariaceae, Genus:
Fusarium, Species: oxysporum (Alexopoulos and Mims, 1979). This
pathogen affects the vascular system of the host. The symptoms observed
in wilt affected plants are chlorosis (Fig. 5a), vascular discoloration
(Fig. 5b), blackened roots (Fig. 5c), finally wilting of plant (Fig. 5d). In
many cases wilting is confined to one side of the plant and the top is bent
towards the diseased side i.e., unilateral wilting of tobacco plant. It is
characterized by sudden wilting and drying up of the green parts, as if
they are suffering from drought. At the later stage due to the rapid growth
of the mycelium complete blockage of the xylem vessel is observed. This
results in wilting symptom.
Wilt pathogen produces conidia in short vertically-branched
conidiophores in sporodochia. The conidia produced are of two types viz.,
15
microconidia and macroconidia. Microconidia being single or two celled,
hyaline and ellipsoidal measure 5-12x2.5-3µm.
Fig. 5. Wilt disease affected tobacco plant showing
a. Chlorosis, b.Vascular discoloration
c. Blackened roots and d. Wilting
Macroconidia being 3-4 septate, sickle-shaped, hyaline conidia
measure 40-50x2-3.5µm. The smooth spherical one celled
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chlamydospores formed by the fungus are either terminal or intercalary
and are about 8µm in diameter.
Fusarium wilt fungus is a soil inhabitor and can persist in the soil
for many years. After spore germination the mycelium enters the roots
and quickly invades the water conducting elements. Spores and
chlamydospores are formed within the vessels and when the plant dies
and decomposes the fungus is returned to the soil, where it may persist
indefinitely. Strains of the fungus are known to survive more than 10
years.
Fusarium wilt is known to be associated with root-knot nematode
attack (Lucas, 1975). Nematode feeding punctures the roots and allows
the entry of Fusarium fungus causing infection (Gopalachari, 1984).
Increase in Fusarium wilt incidence occurred in those plants which were
infected with nematodes (Powell et al., 1971).
This fungus is reported to be highly variable both morphologically
and biochemically. The extent of variations of the Fusarium wilt fungus
in KLS region of Karnataka is not known. Hence the present investigation
is carried out to understand the morphological and biochemical
characteristics of different isolates collected from this region. This part of
the thesis is split into 6 chapters. At the beginning of each chapter
separate introduction is given.