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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

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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.,

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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.