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IntroductionIntroductionIntroductionIntroduction

Introduction

1.1 Vegetable oils

Oils are liquid fats. A major portion of oils for human consumption comes

from plants. Plants producing oil either for human consumption or for animal

feed play a major role in the economics of the region producing them. Oils and

fats derived from plants are considered as vegetable oils. A steep hike in the

prices of petroleum products to its prohibitive limits has turned people on to a

cheaper source of vegetable oils with manifold purposes (Gunstone, 2011).

1.1.1 Importance of non edible vegetable oils

Vegetable oils may or may not be edible. Edible oils include various

cooking oils such as coconut oil, mustard oil, soybean oil, rapeseed oil,

cottonseed oil, sunflower oil, groundnut oil, sesame oil etc. Some of the

prominent non edible oils include processed linseed oil, tung oil and castor oil.

These oils are used in the production of biodiesel, lubricants, paints, cosmetics,

pharmaceuticals and other industrial products.

Non-edible oilseed plants are mostly of forest origin where these plants

are growing wild and they don’t need any systematic efforts for their

domestication as they are mostly toxic plants and only used in plant based

treatment of ailments or rituals (Keith Syers et al., 2008). Choice of non edible oil

as fuel can prove as good alternative source to the conventional petroleum based

fuels (Kapilan et al., 2009). Biofuels are gaining importance as potential sources

of energy, particularly in developing countries like India where there are many

plant species whose seeds remain unutilized and underutilized (Padhi and

Singh. 2011).

Introduction

1.2 Castor oil

Castor oil is one of the most versatile vegetable oils obtained from the

castor bean (Ricinus communis). Its unique chemical composition makes it useful

in a large number of applications. It has found usage in many chemical

industries. It is a raw material for paints, coatings, inks, lubricants and a wide

variety of other products (Ogunniyi, 2006). It is a triglyceride in which

approximately ninety percent of fatty acid chains are ricinoleic acid. Castor oil is

fast becoming one of the most sought after plant oils, owing to its rich properties

and variety of end-uses in lubricant, pharmaceutical and cosmetic preparations.

Castor oil was one of the world's first medicinal oil because it naturally

contains a unique and beneficial mixture of triglycerides or fatty acids (Caupin,

1997). The presence of unusual hydroxy fatty acid ricinoleate (ricinoleic acid)

makes this oil very unique by imparting very high density to the oil. Castor oil is

a colourless to very pale yellow liquid with mild or no odour or taste. Various

attributes of castor oil like unsaturated bonds, low melting point (5°C), very low

solidification point (−12°C to −18°C), high boiling point (313°C), high density (961

kg3), with the highest and most stable viscosity (9.5–10.0 Pa s-1) make it

industrially useful than any other vegetable oil (Miller et al., 2009). Ricinoleic

acid (12-hydroxy-9-cis octadecenoic acid) a major component of castor oil is an

unsaturated omega 9-fatty acid that naturally occurs in mature castor seeds.

Ricinoleic acid is abundant in castor oil (90%) but many common vegetable oils

and oil seeds contain lower amounts of this particular fatty acid; its content

Introduction

amounts to 0.27% in cottonseed oil and 0.03% in soybean oil (Yamamoto et al.,

2008). Ricinoleic acid was discovered in 1848 (Saalmuller, 1848). The seed oils

of Jatropha gossypifolia and Hevea brasiliensis were also found to contain high

content of ricinoleic acid (about 18%). Apart from ricinoleic acid, castor oil also

contains saturated fatty acids like palmitic acid and stearic acid. (Wilhelm et al.,

2009).

Castor oil contains distinctive mixture of fatty acids as compare to other

vegetable oils as (Table 1).

Table 1: Average fatty acid composition of castor oil (Rompp, 1974).

Common name Acid name Average Percentage

Range

Ricinoleate Ricinoleic acid 85 to 95%

Oleate Oleic acid 6 to 2%

Linoleate Linoleic acid (LA) 5 to 1%

Linolenate Linolenic acid

(ALA)

1 to 0.5%

Stearate Stearic acid 1 to 0.5%

Palmitate Palmitic acid 1 to 0.5%

Dihydroxystearate Dihydroxystearic

acid

0.5 to 0.3%

Other Fatty acids 0.5 to 0.2%

1.2.1 Industrial and medicinal uses of castor oil

Castor oil finds its application in manufacture of ever expanding products

such as nylon, lubricants, hydraulic fluids, dyes, greases and ointments (Pathak,

Introduction

2003). Because of its hydroxyl functionality, the oil is suitable for use in

isocyanate reactions to make polyurethane elastomers (Quipeng et al., 1990),

polyurethane millables (Kirk-Othmer, 1979; Yeganeh and Mehdizadeh, 2004),

castables (Heiss, 1960; Lyon and Garret, 1973), adhesives and coatings (Yeadon

et al., 1959; Trevino and Trumbo, 2002; Somani et al., 2003), interpenetrating

polymer network from castor oil-based polyurethane (Patel and Suthar, 1988;

Xie and Guo, 2002) and polyurethane foam (Ehrlich et al., 1959; Ogunniyi et al.,

1996). Semi-rigid foam that has potential uses in thermal insulation was

produced when castor oil along with polyether mixture was reacted with toluene

diisocyanate (Ogunniyi et al., 1996). Sebacic acid, a 10-carbon dicarboxylic acid is

manufactured by heating castor oil to high temperatures with alkali. This

treatment results in saponification of the castor oil to ricinoleic acid which is then

cleaved to give capryl alcohol (2-octanol) and sebacic acid. Sebacic acid and

hexamethylene diisocyanate react through condensation polymerization to

produce nylon- 6, 10. The esters of sebacic acid are also used as plasticizers for

vinyl resins and in the manufacture of dioctylsebacate-a jet lubricant and

lubricant in air cooled combustion motors (Vasishtha et al., 1990). The pyrolysis

of castor oil at 700°C under reduced pressure has been used to obtain

heptaldehyde and undecylenic acids which are important intermediates in the

preparation of perfume formulations. When undecylenic acid is mixed with

isobutylamine, an insecticidal synergist is obtained. Heptaldehyde can be further

hydrogenated to produce alcohol for use as a plasticizer. Also, undecylenic acid

Introduction

is used in preparing athlete‘s foot remedy (Das et al., 1989). Hydrogen bonding

of hydroxyl group of castor oil confers high viscosity to the oil and this makes the

oil an important component in blending lubricants (Kirk-Othmer, 1979). After oil

extraction, the leftover detoxified seed cake is an excellent source of nitrogen and

hence used in making fertilizer (Woodend, 1993).

Blown or oxidized castor oil is prepared by blowing air or oxygen in to

castor oil at temperatures of 80–130°C, with or without catalyst to obtain oils of

varying viscosity. The blown oil is used widely as a plasticizer in lacquers,

artificial leathers, hydraulic fluids and adhesives (Weiss, 1971; Kirk-Othmer,

1979). Castor oil can be modified by reduction with hydrogen to produce

hydrogenated castor oil (HCO), which is a wax-like material with melting point

of 86°C. HCO is used in cosmetics, hair dressing, ointments, preparation of

hydrostearic acid and its derivatives and in certain cases as wax substitutes for

polishes. HCO is used as a paint additive, solid lubricant, for making transparent

typewriter printing inks and releasing agent in the manufacture of plastics and

rubber goods (Kirk-Othmer, 1979; Weiss, 1971). Sulphonated castor oil

commonly called as Turkey Red Oil is used in dyeing and finishing of cotton and

linen. Blown castor oil is used in making pestle colours.

For industrial applications, ricinoleic acid is manufactured by

saponification or by fractional distillation of castor oil. Polyglycerolpoly-

ricininoleate, a polymer of ricinoleic acid is used as an emulsifier in chocolate.

Ricinoleic acid, like its derivative undecylenic acid inhibits the growth of many

Introduction

viruses, bacteria, yeasts, and molds. Also, topical application castor oil with the

main component as ricinoleic acid, exerts remarkable analgesic and anti-

inflammatory effects.

Castor oil has a long history as a medicinal agent in folk healing and

mainstream medicine. Castor oil was used in lamps by the Egyptians more than

4,000 years ago and castor seeds have been found in their ancient tombs (Weiss,

1971). In his book The Oil That Heals, McGarey (1993) described Edgar Cayce who

treated his patients with castor oil during early Middle Ages in Europe claimed

that castor oil helped to heal the lymphatic tissue in the small intestines, thus

increasing absorption of fatty acids and allowing for tissue growth and repair.

Herodotus and other Greek travellers noted the use of castor seed oil for lighting,

body ointments, and improving hair growth and texture. Dissidents and regime

opponents of Italian dictator Benito Mussolini were forced to ingest the oil in

large amounts, triggering severe diarrhoea and dehydration, which would

ultimately cause death (Karp, 1986). Castor oil was widely used to induce labour

by US midwives in the past, but now the use has declined (Sicuranza and

Figueroa 2003; Tenore, 2003). The use of castor bean oil in India has been

documented since 2000 BC in lamps and in local medicine as a laxative, purgative

and cathartic in Unani, Ayurvedic and other ethanomedical systems. The oil has

long been used as a laxative and purgative following treatment for intestinal

parasites. The components of castor oil are known to exert a cathartic effect (Audi

et al., 2005; Burdock et al., 2006; Rajshekhar, 2004). The United States Food and

Introduction

Drug Administration (USFDA) has categorized castor oil as "generally

recognized as safe and effective"(GRASE) for over-the-counter use as a laxative,

with its major site of action as small intestine and can be used for constipation. In

ayurveda, castor oil is considered as king of medicines as it is having excellent

purgative and laxative properties (FDA, 2003). Undecylenic acid, a castor oil

derivative, is also FDA approved for over-the-counter use on skin disorders or

skin problems. A small randomized clinical trial evaluating the efficacy of castor

oil eye drops in treating meibomian gland dysfunction resulted in an increase in

tear stability and lubricating effect (Goto et al., 2002). Castor oil is used for its

water-insoluble lipid and surfactant properties in certain oral and injectable

drugs and vitamin preparations, including cyclosporine A, phytonadione,

tacrolimus, and carbemazepine (Riegert-Johnson and Volcheck, 2002; Tayrouz,

et al., 2003; Strickley, 2004). Castor oil is often found in topical analgesics for

wound healing for effective treatment for skin ulcers combined with Peru balsam

and trypsin (Glenn, 2006). Castor oil is used in burns, sunburns, skin disorders,

skin cuts, abrasions and acne-healing. It is one of the important ingredients in

several pharmaceutical preparations (Gray and Jones, 2004). Therapeutically

modern drugs are rarely given in a pure chemical state, so most active

ingredients are combined with excipients or additives. Castor oil, or a castor oil

derivative such as Cremophor EL (polyethoxylated castor oil, a nonionic

surfactant) are added to many modern drugs, including: Miconazole, an

antifungal agent; Paclitaxel, a mitotic inhibitor used in cancer chemotherapy;

Introduction

Sand immune (cyclosporine injection, USP), an immunosuppressant drug widely

used in connection with organ transplant to reduce the activity of the patient's

immune system; Nelfinavirmesylate, an HIV protease inhibitor; Saperconazole, a

triazole antifungal agent (contains Emulphor EL-719P, a castor oil derivative);

Tacrolimus, an immunosuppressive drug (contains HCO-60, polyoxyl 60

hydrogenated castor oil); Aci-Jel (composed of ricinoleic acid from castor oil,

with acetic acid and oxyquinoline) is used to maintain the acidity of the vagina.

1.2.2 Castor oil-an alternative source of fuel

Biodiesel refers to any diesel equivalent fuel made from renewable

biological materials such as vegetable oils or animal fats. The term biodiesel

usually refers to an ester or oxygenate made from oils. It is usually produced by a

transesterification of vegetable oil with a low molecular weight alcohol such as

ethanol and methanol. There are certain parameters like ash content (0.02%),

sulfur (<0.04%), potassium (negligible), heating value (39.5 gigajoules per metric

tons), iodine value (80), cetane number (45 i.e. higher than petrol or diesel) that

determines the suitability of castor oil as biodiesel (Sudhakarababu et al., 2006).

1.3 Castor bean plant

Owing to the profit gained by multipurpose oil derived from the castor

seeds (containing 40% - 60% oil), agriculturists and farmers are now diverting for

cultivation of castor bean plants. Castor plant is well adapted to arid and

semiarid environments and is better suited for harsh growing conditions.

Moreover, it is not foraged by cattle. Technological advances in the castor crop

Introduction

breeding have resulted in exploitation of high level of heterosis through

development of hybrids and large scale production for achieving high oil yields

(Hegde and Sudhakara Babu, 2002). In the search for biodegradable and

environmentally friendly fuels, the use of improved varieties of castor is

cultivated for high yield of castor oil as biofuel for technical and ecological

benefits. Castor oil stands as an opportunity for agricultural development in arid

and impoverished areas (Gressel, 2008).

Growth and habitation

The original home of castor bean plant is considered to be Africa and

India but presently it is cultivated in all warmer parts of the world (Weiss, 1971).

However the tropical regions of the world are contributing to the major oil yields

(Govaerts et al., 2000). The plant is an important oleaginous candidate of

Euphorbiaceae family with more than four thousand species.

Castor is perennial in its natural growing area, while commercially treated

as annual or biennial and exhibits a bushy appearance (Atsmon, 1989; Moshkin,

1980; Weiss, 1971). Castor plants appear in green or red-violet shades with a thin,

waxy coating with average height of 10-13 m in the tropical regions, but usually

behave as an annual in the temperate regions with a height of 1–3 m; with the

succulent and herbaceous shoot 7.5–15 cm in diameter which is variable in all

aspects. The stem bears alternate, orbicular, palmately compound 6–11 toothed

lobed leaves with glabrous texture on long (25-60 cm) leaf stalks; the root system

of the plant consists of a well developed tap root that reaches depths of 3-4 m.

Introduction

The plant possesses long raceme inflorescences located on the ends of branches

with male flowers at the base and female flowers at the tips. Fruits are three-

celled spherical or oblong capsules, with one seed in each cell, usually spiny and

green which turns brown on ripening. Seeds are oval, lustrous, with or without

papilla-shaped caruncle, often appearing in shades of gray to dark red. Castor is

a cross pollinated crop and is usually cultivated as a hybrid in India, as hybrids

give significantly greater yields than pure lines or varieties (Moll et al., 1962;

Birchler et al., 2003; Reif et al., 2007). Seeds of castor bean are allergic and

possibly fatal containing potent neurotoxin called ricin. Commercial varieties of

castor contain 2200 to 3200 seeds per kg (Reed, 1976).

Highest yields of castor seeds are obtained under irrigation on fine or

medium textured soils, where low relative humidity prevails. Castor plant

requires at least 140-days from planting until harvesting to produce satisfactory

yields of castor seed; though a 150 to 160-day season is more advantageous for

the crop (Brigham, 1993).

1.3.1 Cultivation of castor

Castor bean is one of the major oil seed in India whose cultivation earns

good foreign exchange for the nation. The major countries for maximum castor

bean production are India, China and Brazil. India takes up around 62% of the

world average production of castor (Damodaran and Hegde, 2002). The Indian

variety of castor has an oil content of 48%. Out of 48%, about 42% of oil is being

extracted and the cake retains the rest (Castor seed Seasonal Report, 2008). The

Introduction

average productivity of castor bean during 2000-2001 was found to be 805 kg/ha

(Damodaran and Hegde, 2002).

It is extensively cultivated in a few states in India namely Gujarat,

Rajasthan and Andhra Pradesh which have suitable climatic conditions. Gujarat

shares about 43% of its land and produces around 74% of total seed production

of the country (Castor outlook, 2005). But owing to the climate there are some

major and minor diseases prevalent in the castor bean plant that cause a major

effect on the total yield.

1.3.2 Major diseases prevalent in castor bean

Plant diseases are a normal part of nature and they helps in keeping plants

and pathogens to maintain equilibrium within the ecosystem. Plant cells contain

special signaling pathways that enhance their defenses against insects, animals,

and pathogens. Although each plant species is susceptible to some diseases, the

occurrence and prevalence of plant diseases vary from place to place, season to

season, depending on the presence of the pathogen, environmental conditions

and the crops and varieties grown. Some plant varieties are particularly subject to

certain outbreaks of diseases while others are more resistant to them depending

upon the interaction occurring between plant and pathogen.

Castor is attacked by numerous diseases but only few of them cause major

economic loss in the seed production. Many fungi, viruses, bacteria and

nematodes are reported to cause yield loss in castor bean. The major ones are

Alternaria Blight caused by Alternaria ricini that causes defoliation to varying

degrees in susceptible cultivars (Brigham, 1970). This disease is prevalent in

Introduction

Brunei, Egypt, India, and United States (Singh et al., 1955; Stone and Culp, 1959;

Kolte, 1995). Bacterial leaf blight is a bacterial disease caused by Xanthomonas

ricinicola that causes serious damage to susceptible cultivars. Gray mold, caused

by Botryotinia ricini is prominent disease in India where it is observed frequently

in states like Andhra Pradesh and Tamilnadu (Moses and Reddy, 1989).

Charcoal root rot caused by Macrophomina phaseolina has been observed in dry

regions of India, Ceylon and Eastern U.S (Ashby, 1927). In India it is observed in

few states like Andhra Pradesh, Gujarat, Maharashtra, Bihar and Tamilnadu

(Maiti and Raoof, 1984; Das and Prasad, 1989). Castor plants, both wild and

cultivated are prone to Verticillium wilt caused by Verticillium species that causes

major yield loss but some castor cultivars are resistant to the pathogen (Brigham

and Minton, 1969). Seedling Blight of castor bean has been first reported in Bihar

(Dastur, 1913) where the disease outbreak is often favored by prolonged rainy

season.

One of the most important diseases of castor bean plants is fusarium wilt

that was first observed in Morocco (Reuif, 1970). In India this disease was first

observed in Udaipur (Nanda and Prasad, 1970). Fusarium wilt causes major yield

loss in the castor growing states. In state of Gujarat, the disease is very severe and

causes 85% yield loss of cultivars of castor hybrids in North Gujarat (Dange 1997;

Dange et al. 2003).

1.3.2.1 Fusarium wilt

Fusarium wilt of castor is caused by xylem inhabiting fungus Fusarium

oxysporum f. sp. ricini (For). Castor growing states are endemic to fusarium wilt

and the disease destroys around 80-100% crop yield (Anjani et al., 2004).

Introduction

1.4 Fusarium oxysporum

The distribution of pathogen Fusarium oxysporum is known to be

cosmopolitan. Fusarium includes numerous pathogenic strains causing wilt in

many agricultural and ornamental plants.

1.4.1 Physiological specialization

Pathogenicity factors present in fungus are responsible for determination

of plant species as hosts. The whole population will contain certain

morphological and phenotypic variation; there will be many genetic variants that

affect fungal pathogenicity. Strains with same limited host range are grouped

into formae specialies (Armstrong and Armstrong, 1981) although some formae

speciali are further subdivided into pathogenic races. Some strains have

capability to colonize xylem while other strains are incapable of causing diseases

(Katan, 1971). The non pathogenic strains are aggressive colonizers of plant roots

(Gordon et al., 1989).

These formae speciales (f. sp.) can infect a variety of hosts causing various

diseases in potato, sugarcane, garden bean, cowpea and banana (Raabe et al.,

1981). The examples are Fusarium oxysporum f. sp. asparagi (fusarium yellows on

asparagus); f. sp. callistephi (wilt on china aster); f. sp. cubense (Panama

disease/wilt on banana); f. sp. dianthi (wilt on carnation); f. sp. koae (wilt on koa);

f. sp. lycopersici (wilt on tomato); f. sp. melonis (fusarium wilt on muskmelon); f.

sp. niveum (fusarium wilt on watermelon); f. sp. pisi (wilt on edible-podded pea);

f. sp. tracheiphilum (wilt on Soya bean); and f. sp. zingiberi (fusarium yellows on

ginger).

Introduction

Genus Fusarium is known to cause the following symptoms: vascular wilt,

yellows, root rot and damping-off. The most important of these is vascular wilt.

Of the vascular wilt-causing Fusaria, Fusarium oxysporum is the most important

species (Agrios, 1988; Smith et al., 1988). Strains that are rather poorly

specialized may induce yellows, rot and damping-off, rather than the more

severe vascular wilt (Smith et al., 1988).

Fusarium wilts first appear as slight vein clearing on the outer portion of

the younger leaves, followed by epinasty (downward drooping) of the older

leaves. At the seedling stage, plants infected by F. oxysporum may wilt and die

soon after symptoms appear. In older plants, vein clearing and leaf epinasty are

often followed by stunting, yellowing of the lower leaves, formation of

adventitious roots, wilting of leaves and young stems, defoliation, marginal

necrosis of remaining leaves and finally death of the entire plant (Agrios, 1988).

Browning of the vascular tissue is a strong evidence of fusarium wilt. On older

plants, symptoms generally become more apparent during the period between

blossoming and fruit maturation (Jones et al., 1982; Smith et al., 1988).

1.4.2 Disease cycle of Fusarium oxysporum f. sp. ricini

Fusarium oxysporum f. sp. ricini is the causal organism of the wilt in castor

bean. Though the pathogen is host specific, it shows great variation in

pathogenicity.

The pathogen is classified as follows:

Kingdom: Fungi;

Division: Eumycota;

Introduction

Subdivision: Deuteromycotina;

Class: Hyphomycetes;

Order: Moniales;

Family: Tuberculariaceae.

The fungus forms white fluffy colony on PDA plate and turns pinkish at

later stages. It is an imperfecti fungus with no known sexual reproduction.

Spores are produced in the form of microconidia, macro conidia and

chlamydospores. Micro-conidia are single celled and range from 5.25-14 x 3.5-7

μm in size. They are hyaline, round to oval in shape, single celled but rarely

septate. Macroconidia are hyaline, few in number, having 2-6 septa, straight,

spindle as well as sickle shaped and measure 17.5-70 x 3.50-5.25 μm (Desai et al.,

2003). Generally, chlamydo-spores both terminal and intercalary are developed

in later stages of growth after in two weeks old of inoculation (Kolte, 1995).

1.4.3 Role of fungal toxins

The genus Fusarium is known to produce many mycotoxins. These

mycotoxins when play role in plant invasion are called as phytotoxins. Fusaric

acid is a well-known phytotoxin that is produced by several Fusarium species,

particularly pathogenic strains of F. oxysporum causing wilt diseases in a great

variety of plants. This toxin is different from other mycotoxins synthesized by

various Fusarium sp., e.g. moniliformin, deoxynivalenol, and zearalenone, which

are limited to only a few taxonomic entities among a species population. Fusaric

acid is known to play an important role in the plant disease process (Gaumann,

1957) and enhance the toxicity of other mycotoxins (Bacon, 1995). Fusaric acid (5-

Introduction

butylpicolinic acid) was first discovered during the laboratory culture of

Fusarium heterosporum and was one of the first fungal metabolites implicated in

the pathogenesis of wilt symptoms of plants especially under adverse conditions.

Since fusaric acid is considered a wilt toxin, it has been examined for its

production and role in the wilt of field maize (Bacon, 2006). Correlation between

fusaric acid production and virulence of isolates of F. oxysporum species have

been reported in lily (Curir, 2000), date palm (Bouizgarne, 2004) and Arabidopsis

thaliana (Bouizgarne, 2006).

Screening and selection of plant tissue in vitro for resistance to Fusarium

fungal toxin or culture filtrate has been successful for several species (Behnke,

1979; Hartman et al., 1984). Fusaric acid has been characterized as the phytotoxin

and is reported to be significantly involved in the development of fusarium

yellows (Remottiet et al., 1997) using such in vitro studies. The semi-purified

culture filtrate elicits morphological and physiological symptoms like browning

and wilting which is very similar to the symptoms produced by fungal infection

(Prachi et al., 2000) during plant invasion. It is therefore possible to use the

culture filtrate in selection and assessment of disease resistance.

1.4.4 Host Infection

The pathogen is soil borne and survives in soil in the form of macro

conidia, micro conidia and chlamydospores (Smith et al., 1988). F. oxysporum

spores can also survive on non-host plants in the absence of a susceptible host.

When non-host plants become infected they show few symptoms and become a

carrier of the pathogen. Underground rhizomes are often another means of

Introduction

spreading the disease. Healthy plants can also be infected by F. oxysporum if the

soil in which they are growing is contaminated with the fungus. The fungus can

invade a plant either with its sporangial germ tube or mycelium by invading the

plant roots (Figure 1). The roots can be infected directly through the root tips,

through wounds in the roots, or at the formation point of lateral roots (Agrios,

1988). Once inside the plant, the mycelium grows through the root cortex

intercellularly. When the mycelium reaches the xylem, it invades the vessels

through the xylem's pits. At this point, the mycelium remains in the vessels,

where it usually advances upwards toward the stem and crown of the plant. As it

grows the mycelium branches and produces microconidia, which are carried

upward within the vessel by way of the plants sap stream. When the

microconidia germinate, the fungal mycelium can penetrate the upper wall of the

xylem vessel, enabling more microconidia to be produced in the next vessel. The

fungus can also advance laterally as the mycelium penetrates the adjacent xylem

vessels through the xylem pits (Agrios, 1988).

Figure 1: Disease cycle of wilt of castor caused by Fusarium oxysporum f. sp.

ricini (Dange et al., 2003).

Introduction

1.4.5 Epidemiology

The disease appears at all growth stages of the crop but becomes more

prominent and severe at the time of flowering and spike formation. Favorable

temperature for infection is 13-15°C and for symptom expression is 22-25°C

(Andreeva, 1979). Monocropping of castor practiced by the farmers as a result of

high economic return may lead in the endemic development of the castor wilt

which makes field wilt sick. Infected seeds also play an important role in the

perpetuation and spread of the pathogen.

Fungal–Nematode interactions: Reniform nematode Rotylenchulus

reniformis was found to be involved in the wilt of castor. The castor plants

attacked by reniform nematode are predisposed for the infection of wilt pathogen

F. oxysporum f. sp. ricini (Chattopadhyay and Reddy, 1995). Nematodes play vital

role in the breakdown of wilt resistance in castor hybrid and thus, increasing the

severity of the castor wilts (Pathak, 2003).

1.4.6 Disease management

Uses of healthy seeds, crop rotation, ploughing and field sanitation reduce

the incidence of the disease. Since the pathogen is mainly soil-borne and survives

in soil for long period, soil inhabiting nematodes like R. reniformis facilitates the

Fusarium infection and makes the castor crop vulnerable to fusarium wilt. Soil

solarization technique may reduce the wilt incidence and nematode population

considerably (Raoof and Rao, 1997).

Introduction

1.4.6.1 Chemical control

Seed borne infection of F. oxysporum f. sp. ricini from castor seeds can be

eradicated by treating the seeds with Emisan-6 and Thiram at 3 g/kg seed

(Andreeva, 1979, Siddaramaiah et al., 1980).

1.4.6.2 Biological control

Biological control is particularly attractive in respect of soil borne

pathogens. It’s not only economical but also non hazardous. Use of biocontrol

agents against fusarium wilt is well known in many crops. Trichoderma harzianum,

Trichoderma viride, Gliocladium virens, Aspergillus flavus and Aspergillus niger have

been screened for their antagonistic activity against castor wilt pathogen F.

oxysporum f. sp. ricini and promising results have been obtained (Pushpawathi et

al., 1998).

Use of resistant genotypes is least expensive, easiest, and safest and one of

the most effective means of controlling plant diseases. Resistant varieties not only

eliminate losses from spray and other methods but also avoid addition of toxic

chemicals to the environment.

1.5 Plant responses to diseases

Plants have developed means to prevent or tolerate the pathogen attack.

For the reason that plants are unable to escape the challenges, they have

developed some unique strategies to overcome such stresses. Plants hold two

lines of defense to defend against pathogen.

Introduction

The first line of defense is passive defences that provides basal resistance

against all potential pathogens and is based on recognition of conserved

microbial features known as pathogen-associated molecular patterns (PAMPS) by

so called PAMP recognition receptors (PRRs) that activate PAMP triggered

immunity (PTI) and prevent further colonization of the host. One of the best

known microbial PAMPs is chitin, a major structural component of fungal cell

wall for which two LysM-type of receptor like kinases are involved in its

recognition have been characterized in rice (OsCERK1) and Arabidopsis (LysM

RLK1), respectively.

The second line of defense is an active defense that is more specific and

guided by the interaction of R genes and Avr genes. This gene-for-gene

interaction hypothesis states that for every dominant avirulence (Avr) gene in the

pathogen there is a cognate resistance (R) gene in the host and the interaction

between the products of these genes leads to activation of host defense response

such as hypersensitive response (HR) that arrests the growth of pathogen (Flor,

1971).

Resistance mechanisms in plants can be subdivided into two categories:

1.5.1 Passive mechanism

This type of mechanism is constitutive where the host plant either includes

some structural elements for prevention of entry of pathogen or may synthesize

antimicrobial compounds such as phytoanticipins or phytoalexins as chemical

barriers for the entry of pathogen. For example root tips are one type of structural

Introduction

barrier that rapidly elongates and protected by the root border cells that guard

the root tip from the pathogen attack. These passive preformed/induced defence

mechanisms are described below:

1.5.1.1 Modification of cell walls

The Cuticle: Cutin and waxes together comprise the cuticle that prevents

the entry of many pathogens apart from the fungal pathogens that have

pathogenicity factors for direct penetration inside the cell (Kolattukudy, 1980).

The cuticle is a hydrophobic surface and thus prevents water from accumulating

as a film on cell surfaces and it also restricts the flow of nutrients to the cell

surface. This prevents certain microorganisms from becoming established on

plant surfaces.

Components of wax, as well as cutin acids, inhibit germination of certain

pathogens (Wang and Pinckard, 1973), but there are some pathogens that use

cutin as a sole carbon source for growth in artificial media (Kolattukudy, 1980).

Cuticle thickness may vary with levels of resistance to fungi that penetrate

directly into tissues (Bell, 1974; Wang and Pinckard, 1973).

1.5.1.2 Carbohydrate depositions

As fungi begin to penetrate the cell wall, either with infectious hyphae or

haustoria, the resistant host responds by synthesizing particularly callose and

cellulose, which are added to the inside of the cell wall just outside the

plasmalemma. These appositions may continue even after the fungus penetrates

the original wall, until they become dome shaped or elongate and are called

Introduction

papillae (Aist, 1976). Cells adjoining or near to those invaded by pathogens also

may deposit new carbohydrates onto thickening secondary walls. In susceptible

hosts, papillae and secondary wall thickening are often poorly developed (Aist,

1977; Hohl and Stossel, 1976). Callose enlarges with time, covering the lesion

area, and then gradually disappears as necrosis and browning of cells progress.

In some cases, even after complete browning, callose can be seen in walls of live

cells around lesions in plasmodesmata (Stobbs and Manocha, 1979) and sieve

plate pores (Favali et al., 1978), and around membrane-bound bundles of virus

particles in necrotic cells. Cell wall thickening and callose deposition also occur

in healthy cells surrounding necrotic lesions caused by fungi (Garcia and

Sagasta, 1978).

1.5.1.3 Structural proteins

The importance of structural proteins in cell walls has become increasingly

apparent in recent years. Lignin complexes formed with structural proteins are

much more resistant to acid hydrolysis as compared to complexes with

carbohydrates (Lin and Kolattukudy, 1978). Many wall-bound proteins also have

enzymatic activities and probably are important as chemical barriers in cell walls.

1.5.1.4 Lignin and Phenolic Acid Complexes

Lignin is a phenolic polymer formed mostly by the free radical

condensation of hydroxycinnamyl alcohols. Lignin forms covalent bonds with

cellulose, pectates and structural proteins when synthesized in the presence of

these compounds (Lin and Kolattukudy, 1978). It forms ester linkages with fatty

Introduction

acid polyesters to yield suberin and suberized cells are rarely penetrated by

pathogens (Kolattukudy, 1980).

The hydroxycinnamic acids also form complexes with polysaccharides,

proteins, suberin, and cutin by esterification (Kolattukudy, 1980). Thus, both

lignin and cinnamic acids cause modifications of cell walls that may contribute to

disease resistance. Lignifications of cell walls is stimulated in and around viral

local lesions (Appiano et al., 1979) and in necrotic cells formed near nematodes in

resistant cultivars whereas no such stimulation occurs in susceptible cultivars

(Giebel et al., 1970).

Key enzymes in the synthesis of lignin are: phenylalanine ammonia lyase

(PAL) and tyrosine ammonia lyase (TAL) which converts phenylalanine and

tyrosine to cinnamic acid and 4-hydroxycinnamic acid. Increased activity of all of

these enzymes slightly precedes accumulation of lignin and such increases are

correlated with disease resistance (Vance and Sherwood, 1977).

PAL is an important enzyme involved in biosynthesis of dihydroxy-

phenols. Increase in this enzyme is often greater in resistant than in susceptible

cultivars in response to various infectious agents (Yamamoto, 1977), particularly

in plant species that form caffeic acid esters as their major dihydroxy-phenols.

1.5.1.5 Production of tannins and melanins for defence

Necrosis associated with race-specific resistance normally is characterized

by the formation of brown to black pigments (melanin) throughout the cell walls

and the collapsed protoplasts along with walls of adjoining live cells. The

Introduction

intensity of melanin formation often is greatest in highly resistant plants,

suggesting that melanins or their precursors contribute to resistance. Melanins in

plants are formed principally from various ortho-dihydroxyphenolic compounds

(Mace et al., 1972). The enzymes polyphenoloxidase (PPO) and peroxidase (PO)

oxidize the colourless dihydroxyphenols to give the coloured ortho-quinones

(Mayer and Harel, 1979). Certain dihydroxyphenols are conjugated with each

other or with glucose hydroxyl groups to form polydihydroxy phenolic

oligomers and polymers called tannins. The coloured condensation products of

quinones and tannins constitute the plant melanins.

Peroxidases, besides forming quinones, converts dihydroxyphenols to free

radicals that may undergo various reactions with cellular constituents. The

tannins and the ortho-quinones have some toxicity to most microorganisms as

they inactivate extracellular enzymes produced by microorganisms (Beckman,

1974).

To estimate the importance of phenol oxidation and melanin generation, it

is necessary to measure activities of enzymes involved in dihydroxyphenol

synthesis such as PAL that also feed intermediates into lignins, and phytoalexins

and also to measure concentrations of dihydroxyphenols that are transient

intermediates in the synthesis of melanin involved in plant’s defence mechanism.

1.5.2 Active mechanisms

This type of defence includes induced responses in plants that can be

triggered by a number of factors. Induction may be in response to non specific

Introduction

elicitors or may follow gene for gene relationship. One of the most observed

plant defence is hypersensitive response that is localized death of plant cells.

Hypersensitive response limits the growth of biotrophic fungi that limits the

growth of pathogen and minimizes the loss of living cells of host tissues.

1.5.2.1 Gene for gene interaction

Many plant species have developed mechanism to defend against

pathogen based on host specific resistance. In host specific resistance the

recognition of pathogen by plant has been associated with gene for gene

hypothesis (Flor, 1971). This hypothesis is based on observation that both plant

and pathogen synthesize a gene product: plant R protein and pathogen Avr-

protein.

Induced defence mechanism is effective in some plants against some

pathogens controlled by one or few genes. Disease resistance in plants is often

controlled by gene/genes that confer high levels of resistance but only to specific

pathogen genotypes.

1.5.2.2 Resistance genes

Plant disease resistance genes (R genes) encode proteins that detect

pathogens. R genes have been used in resistance breeding programs for decades,

with varying degrees of success. Resistance (R) genes that are employed on a

large scale in agriculture typically lose their effectiveness over time owing to

shifts in the pathogen population to forms that are virulent on cultivars carrying

the gene. Recent molecular research on R proteins and downstream signal

Introduction

transduction networks has provided exciting insights, which will augment the

use of R genes for disease prevention. Definition of conserved structural motifs in

R proteins has facilitated the cloning of useful R genes, including several that are

functional in multiple crop species and/or provide resistance to a relatively wide

range of pathogens. Because R genes confer resistance against specific pathogens

it is possible to transfer an R gene from one plant to another and make a plant

resistant to a particular pathogen (Shoresh et al., 2010).

Genetic analysis of resistance in numerous host species and specific

virulence in the corresponding pathogens has led to the general acceptance of the

gene-for-gene model (Flor, 1971), where specific R genes interact with specific

avirulence (Avr) genes in the pathogen to cause resistance. Over 30 disease

resistance genes have now been isolated from a variety of plant species (Shoresh

et al., 2010). In the simplest models to account for gene-for-gene resistances, the R

gene products somehow recognize the pathogen Avr gene, either by a direct

interaction with its protein product or by an interaction with product made by

the Avr genes (Yedidia, 1999). Once this recognition has occurred, defense

responses are triggered. These are often characterized by a hypersensitive

response, which involves the death of the first cell or cells infected and the local

accumulation of antimicrobial compounds (Staskawicz et al., 1995).

1.5.2.3 Proteins Coded by R Genes

The proteins encoded by most characterized resistance genes carry motifs

found in other receptor and signal transduction proteins (Figure 2).

Introduction

• The majority of R proteins contain tandem leucine- rich repeats (LRRs)

which have a major role in recognition specificity (Jones, 2001).

• The largest group of resistance genes carries leucine-rich repeats and

nucleotide-binding site (NBS) domains. These genes are very abundant in

plant genomes, comprising an estimated of 1% of the genes in the

Arabidopsis genome (Meyers et al., 1999). The NBS-LRR class of R genes

can be further subdivided based on their ability to code for other

recognizable domains.

• One subclass codes for a TIR domain (homology to the Drosophila Toll and

mammalian Interleukin-1 receptors) at the N terminus of the protein.

• NBS-LRR proteins without a TIR domain typically code for a coiled-coil

domain (CC) near the N terminus, sometimes in the form of a leucine

zipper. This type is much more common in cereals, where the members

with a TIR domain have not yet been identified (Meyers et al., 1999; Pan,

et al., 2000).

• Three other classes of resistance genes carry LRRs or kinase domains or

both LRRs and kinase domains.

Introduction

Figure 2: The major domains of NBS LRR proteins (Mchale et al.

2006).

LRR domains are typically thought to be the major determinant of

specificity in R genes that carry them based on their known history in other

proteins and the high levels of polymorphism between alleles in these domains

(Kobe and Deisenhofer, 1994; Kobe and Deisenhofer, 1995). Apparently LRR

region is considered as the “specificity domain”. Domain exchange between

alleles of the L locus demonstrated that sequences at the amino-terminal end of

the protein are also involved in specificity (Ellis et al., 2000). The predicted

cellular location of an R protein reflects where it interacts with its corresponding

elicitor (Hwang et al., 2000).

The LRR-TM (transmembrane domain) and LRRTM-Kinase classes of proteins

are predicted to space the cell membrane, with an extracellular LRR. These

include rice Xa21D for resistance against Xanthomonas and the cf genes of tomato

that confer resistance against Cladosporium fulvum. The tomato Ve1 and Ve2

Introduction

proteins contain putative extracellular LRRs, along with polypeptide sequences

enriched in proline (P), glutamate (E), serine (S), and threonine (T).

In contrast, the NBS-LRR genes are predicted to be cytoplasmic, although

they may be membrane associated also (Boyes et al., 1998). The LRR-kinase

superfamily consists of extracytoplasmic leucine-rich repeats (eLRR) fused to a

cystoplasmic serine-threonine kinase domain (KIN). One resistance protein

(tomato Pto) is a Ser-Thr kinase without LRRs, and another (maize Rpg1)

contains two kinase domains. The Arabidopsis RPW8 protein contains a

membrane anchor, fused to a putative coiled-coil domain (CC).

These genes also confer resistance to an amazing diversity of different

organisms including fungi from three different taxonomic classes with very

different modes of pathogenicity, from biotrophic rusts, powdery mildews, and

downy mildews, to hemibiotrophic fungi like Magnaporthe and vascular wilts like

Fusarium wilt. They also control resistance to nematodes and insects. The

observed interaction with intracellular R gene products has lead the technology

to investigate how these diverse organisms deliver elicitors into plant cells. Along

with R gene products, other genes are also involved in R gene–mediated

resistance that had been identified either by mutagenesis or biochemical

approaches (Zhou, 2000). Some of these other components are involved in

downstream signalling steps, but some may be components of an elicitor

recognition complex.

Briefly, resistance can be developed in plants through a number of

mechanisms including:

Introduction

• The R protein interacting directly with an Avr gene (Avirulence gene)

product of a pathogen.

• The R protein being guarded by another protein that detects degradation

by an Avr gene.

• The R protein may detect a Pathogen- associated molecular pattern-

PAMP (alternatively called MAMP for microbe- associated molecular

pattern).

• The R protein may be an enzyme that degrades toxin produced by a

pathogen.

During fusarium wilt disease, resistance and susceptibility of host towards

fungal pathogen is partly determined by the interaction that occurs between the

pathogen and the host in two possible means:

(1) Incompatible interaction in which the fungus is apparently contained

within the vessel it has invaded,

(2) Compatible interaction where the fungus invades the neighboring

parenchyma tissue and spreads laterally to other vessels, eventually

colonizing the entire vascular system (Gao et al., 1995; Mes et al., 2000).

As discussed earlier, for every R gene there is cognate Avr gene in the

pathogen that plays a major role in plant disease. Comparative genomics of

fungal pathogens will be useful in identification of new effector proteins and

possibly in prediction of their virulence functions.

1.5.2.4 Avirulence genes

Introduction

Avirulence (Avr) genes exist in many fungi that share a gene-for-gene

relationship with their host plant. Interaction between elicitors (primary or

secondary products of Avr genes) and host receptors in resistant plants causes

induction of various defence responses often involving a hypersensitive

response. Avr genes encode effectors that suppress PTI (PAMP triggered

immunity) thus enabling a pathogen to infect its host plant and cause disease.

Avr proteins are diverse in nature and functions. They represent unique genetic

determinants that prevent fungi from causing disease on resistant plants that

possess matching Resistance (R) genes. Once the basal defence system of plants is

overcome by the pathogen, plants respond with the development of more

specialized recognition system based on effectors’ perception by R-proteins and

subsequent activation of effectors triggered immunity (ETI) and leads to rapid

and acute defence response in plants the characteristic of which is Hyper

sensitive Reaction (HR). This triggers a second wave of co evolutionary arms race

between pathogen and plants, during which pathogens respond by mutating or

losing effectors or by developing novel effectors that can avoid or suppress ETI

whereas plants develop novel R proteins mediating recognition of novel

effectors.

Avr genes have been successfully isolated by reverse genetics and

positional cloning. Five cultivar-specific Avr genes (Avr4, Avr9, and Ecp2 from

Cladosporium fulvum; nip1 from Rhynchosporium secalis; and Avr2-YAMO from

Magnaporthe grisea) and three species-specific Avr genes (PWL1 and PWL2 from

Introduction

M. grisea and inf1 from Phytophthora infestans) have been cloned. Isolation of

additional Avr genes from these fungi, as well as from other fungi such as

Uromyces vignae, Melampsora lini, Phytophthora sojae, and Leptosphaeria maculans, is

in also reported (Lauge and De Wit, 1998). Molecular analyses of non functional

Avr gene alleles show that these originate from deletions or mutations in the

open reading frame or the promoter sequence of an Avr gene. Although intrinsic

biological functions of most Avr gene products are still unknown, recent studies

have shown that two Avr genes, nip1 and Ecp2, encode products that are

important pathogenicity factors. All fungal Avr genes cloned so far have been

demonstrated or predicted to encode extracellular proteins (Stergiopoulos and

De Wit, 2009).

Fusarium oxysporum f. sp. lycopersici (Fol) an extracellular pathogen along

with its host Lycopersicon esculentum is one of the best studied models for

unraveling host pathogen relationship. Fol colonizes the xylem cells and four

small proteins designated as Six1 to Six 4 are found to be secreted in xylem

during infection. All these proteins are found to have different roles in

pathogenicity. Six 1 (Avr 3) is a 32 kDa protein required for full virulence. Its

expression is triggered when living plant tissue comes in vicinity of fungus. Six 4

(Avr 1) is a small cysteine rich protein that functions as suppressor of I-2 and I-3

mediated resistance. Six 2 is not found in other strains of Fusarium oxysporum and

also absent in non-pathogenic strains of Fol. Six 3 is present in both virulent and

avirulent strains and is required for full virulence (Rep et al., 2004).

1.6 Molecular markers

Introduction

Currently numerous laboratories are adopting molecular methods for

plant pathogen detection (Lopez et al., 2003; Schaad et al., 2003; Hernandez-

Delgado et al., 2009). With the advent of molecular markers, a new generation of

markers has been introduced over the last two decades, which has revolutionized

the entire scenario of biological sciences (Joshi, 1999). Molecular markers, useful

for plant genome analysis, have now become an important tool in this revolution.

Molecular markers are based on naturally occurring polymorphisms in DNA

sequences (i.e. base pair deletions, substitutions, additions or patterns). There are

various methods to detect and amplify these polymorphisms so that they can be

used for different plant genome analysis. Molecular markers are superior to other

forms of MAS (Marker Assisted Selection) because they are relatively simple to

detect, abundant throughout the genome even in highly bred cultivars,

completely independent of environmental conditions and can be detected at

virtually any stage of plant development.

There are five conditions that characterize a suitable molecular marker:

i. Must be polymorphic

ii. Co-dominant inheritance

iii. Randomly and frequently distributed throughout the genome.

iv. Reproducible

v. Easy and cheap to detect

Introduction

Molecular markers can be used for several applications including

germplasm characterization, genetic diagnostics, characterization of

transformants, study of genome organization and phylogenic analysis.

The development of molecular techniques for genetic analysis has led to a

great increase in our knowledge of plant genetics and understanding structure

and behavior of plant genomics. These molecular techniques, in particular the

use of molecular markers, have been used to monitor DNA sequence variation in

and among the species. Identification of markers linked to useful traits has been

based on complete linkage maps and bulk segregate analysis (BSA). In recent

years, different marker systems such as Randomly Amplified Polymorphic DNA

(RAPD), Restriction Fragment Length Polymorphisms (RFLP), Sequence Tagged

Sites (STS), Amplified Fragment Length Polymorphisms (AFLP), Simple

Sequences Repeats (SSR) or microsatellites, Single Nucleotide Polymorphisms

(SNPs), Sequence Characterized Amplified Regions (SCARs) and many others

have been developed and applied to a range of plant genome analysis..

Among these, SSR a molecular marker based on PCR amplification of

sample of DNA from short oligonucleotide sequences is an easy, convenient and

economical method. The microsatellites are a class of DNA sequences that are

repeated several times at various points in all organisms, both the eukaryotes and

prokaryotes. The term microsatellites was coined by Litt and Luty (1989), also

referred to simple sequence repeats (SSRs), short tandem repeats (STRs), or

simple sequence length polymorphism (SSLPs) (Morgante et al., 2002).

Introduction

Simple Sequence Repeats (also termed microsatellites) are stretches of

DNA, consisting of tandemly repeating mono-, di-, tri-, tetra-, or penta-

nucleotide units that range in size from 1 to 6 base pairs and they are present in

both the coding and non coding regions arranged throughout the genomes of

most eukaryotic species. The PCR reaction for microsatellites is determined by

using specific oligo-nucleotides designed by using information concerning the

repeats of the flanking regions. Hence, the forward and reverse primers already

designed are used to anneal at the 5’ and 3’ end of the template DNA

respectively. The great strength of the microsatellites is that are a Mendelian

inherited co-dominant markers. Their polymorphism, abundance and their

distribution throughout the genome has made the microsatellites one of the most

popular markers (Morgante et al., 2002; Wright and Bentzen, 1994). Moreover,

this technique is PCR-based analysis, so only low quantity of DNA template is

required.

Introduction

1.7 Objectives:

• Isolation of Fusarium oxysporum f. sp. ricini from infected castor plants and

determine the level of toxin produced by the pathogen.

• To categorize selected cultivars of castor bean into fusarium wilt resistant

and susceptible ones by in vivo and in vitro bioassays.

• To investigate various biochemical changes occurring in both cultivars

during infection by Fusarium oxysporum f. sp. ricini.

• To discriminate resistant and susceptible cultivars at molecular level using

SSR markers.

• To examine the presence of R genes in both resistant and susceptible

cultivars using specific primers.

• To analyze the expression of virulence gene in a susceptible cultivar of

castor infected by host specific Fusarium oxysporum f. sp. ricini and to

confirm its identification.

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