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IntroductionIntroductionIntroductionIntroduction

Chapter 1

Ph.D. Thesis; BRD School of Biosciences, Sardar Patel University 1

1.1 Historical overview of chickpea wilt

Fusarium wilt of chickpea was first noticed in 1918 (Nene et al., 1980),

and later Prasad and Padwick, (1939) isolated Fusarium from wilted

chickpeas, made hundreds of isolates, and conducted pathogenicity tests

by infesting sterilized soil. They reported that isolated Fusarium species

were classified in the section Elegans and sub-section Orthoceras.

However, Prasad and Padwick (1939) did not name the fungal isolate as a

separate species. A comparison of the isolates with all known species of

the sub-section under identical conditions was necessary. The problems

were elaborately studied by Padwick (1940) who named the pathogen as

Fusarium orthoceras var. ciceri. Later, the causal agent was reported to

be Fusarium lateritum f. sp. ciceri (Erwin, 1958) based on several isolates

collected in 1954, 1955 and 1956 from wilt affected plants in California.

Since the isolates identified as Fusarium orthoceras var. ciceri by Padwick

(1940) were not included in Erwin’s (1958) study, whether the two

isolates were different or similar was questionable. Erwin (1958) also

mentioned the disagreement in nomenclature and the difficulty in

resolving the issue directly.

As reported by Nene et al., (1980), several researchers were not

convinced that Fusarium was the causal agent. According to Nene et al.,

(1980), a study of the wilt complex in Punjab state of India from 1947 to

1954 concluded that wilt was caused by soil and weather factors and not

pathogenic fungi. Virus-induced wilt of chickpea, as reported from Iran

(Kaiser and Danesh, 1971), also contributed to the prevailing view that

pathogenic fungi were not the only cause of wilting in chickpeas. This

prompted a conference on ‘Problems of wilt and breeding for wilt

resistance’ that was held in New Delhi, India in 1973. The conclusion,

according to Jain and Bahl (1974), was that physiological, agronomical,

environmental and pathological factors could contribute to wilt

development. The word “wilt-complex” was coined after a series of

investigations and conferences whereby any dead/dried chickpea plant

was considered wilted due to the “wilt-complex”. The situation was

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clarified when the International Crops Research Institute for Semi-arid

Tropics (ICRISAT) initiated a program to study and understand the “wilt-

complex” (Nene et al., 1980). ICRISAT concluded that the “wilt-complex”

could result from a number of distinct diagnosable diseases.

Characteristic symptoms include sudden drooping of leaves and petioles,

internal (xylem) discoloration, yellowing of leaves, stunting and finally

death of the plant (Nene et al., 1980).

1.2 Facts about chickpea- the host plant

Role of pulses in Indian agriculture needs hardly any emphasis. India is

a premier pulse growing country. Pulses are an integral part of the

cropping systems of the farmers all over the country because these crops

fit in well in the crop rotation and crop mixtures followed by them. Pulses

are important constituents of the Indian diet and supply major part of

the protein requirements. Pulse crops, besides being rich in protein and

some of the essential amino acids, enrich the soil through symbolic

nitrogen fixation from atmosphere.

Gram commonly known as 'chick pea' or Bengal gram is the most

important pulse crop in India. It is used for human consumption as well

as for feeding animals. It is eaten both whole fried or boiled and salted or

more generally in the form of split pulse which is cooked and eaten. Both

husks and bits of the 'dal' are valuable cattle feed. Fresh green leaves are

used as vegetable (sag). Straw of chick pea is an excellent fodder for

cattle. The grains are also used as vegetable (chhole). Chickpea is

considered to have medicinal effects and it is used for blood purification.

Chickpea is a highly nutritious grain legume crop. It is an important

source of energy, protein, minerals, vitamins, fibers and other potentially

health beneficial phyto-chemicals. Mature chickpea grains contain 12-

31% proteins — higher than any other pulse crop; 60-65% carbohydrates

and 6% fat (Geervani, 1991). Chickpea is also a good source of soluble

and insoluble fibres, vitamins (B vitamins), potassium and phosphorus,

hence is increasingly advocated in health-conscious diets. Chickpea is

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one of the cheapest sources of protein (Joshi et al., 2002), hence it can

play an important role in overcoming problems related to nutritional

insecurity of the poor in developing countries where it is grown and

consumed.

1.3 Global chickpea distribution and production

Chickpea is the largest produced food legume in South Asia and the third

largest produced food legume globally, after common bean (Phaseolus

vulgaris L.) and field pea (Pisum sativum L.). India is the largest chickpea

producing country accounting for 64% of the global chickpea production

(Fig. 1). During the triennium 2004-2007, the global chickpea area was

about 11.0 m ha with a production of 8.8 m tons and average yield of

nearly 800 kg ha-1.

Figure 1- Share of different countries in global chickpea production (2004

to 2007) (Gaur et al., 2010)

1.4 Chickpea in Gujarat

Chickpea is one of the most important post-rainy season pulse crops

grown in Gujarat state of India. It occupies an important niche in the

rain fed farming system of resource poor farmers of the state. Since

chickpea is grown on receding residual soil moisture during the post

rainy season, soil moisture is a critical factor from the beginning of plant

establishment to grain development and maturity. This limiting factor is

much more important in a state like Gujarat, where the winters are short

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and comparatively warm, and potential evaporation is far excess of the

annual rainfall. The problem of moisture stress in the post-rainy season

on soils with poor water holding capacity has been tackled to some

extent by selecting early maturing varieties to fit the length of the

growing season. Many biotic and abiotic constraints inflict serious yield

losses and destabilize chickpea production. Scientists and government

agencies face the challenge of finding ways of raising the yield per

hectare in a situation where area expansion is increasingly constrained.

1.5 General classification of chickpea

The Indian grams have been classified into two broader groups:

1. Desi or Brown Gram (Cicer arietinum L.) (Fig. 2A):

In this group the color of the seed ranges from yellow to dark brown.

Seed size is usually small. It is the most widely grown group. Plants are

small with good branching ability. The chromosome number is 2n=16.

2. Kabuli or White Gram (Cicer kabulium) (Fig. 2B):

In this group the colour of the seed is usually white. Grains are bold and

attractive. Yield potential of this group is poor as compared to desi or

brown gram. Plants are generally taller than the desi gram and stand

more or less erect. The chromosome number is 2n=16. The desi type

however, is more prominent– it accounts for close to 80% of the global

chickpea production, and kabuli type chickpea comprises the rest

(Agbola et al., 2002). Desi chickpea can withstand cooler temperatures

and matures quicker than kabuli chickpea.

A B

Figure 2- Figures showing desi or brown gram (A) and Kabuli or white gram (B)

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1.6 Botanical description

Chickpea belongs to family Leguminoseae. It is a small, much branched

herbaceous plant rarely exceeding 60 cm height. The botanical

description of main parts of gram plant is given as below.

1.6.1 Root system

Chickpea has a well developed root system. The roots usually include a

central strong tap root, with numerous lateral branches that spread out

in all directions in the upper layer of soils. There are numerous nodules

on roots. The rhizobium bacteria present in these nodules fix up

atmospheric nitrogen.

1.6.2 Stem

Stem is generally grayish in appearance. It is branched and covered with

glandular hairs. The main branch in gram usually produces not more

than one secondary shoot, but in some types the main branches may

produce numerous lateral branches.

1.6.3 Leaves

The leaves are pinnately compound, usually with one terminal leaflet.

The number as well as the size of the leaflet, however, varies in different

types. There are 9-15 pairs of leaflets. The leaflets of the pinnate leaves

are small, and have serrated edges. The leaves are covered with

glandular hairs. The colour of the leaves also varies; some being light

green while others are green or dark green. Certain types possess leaflets

with red margins.

1.6.4 Flowers

The flowers are typical papilionaceous consisting of five sepals, five petals

comprised of one standard, two wings and two keels, ten stamens, nine

fused to form one staminal column and one free and a carpel with the

style borne laterally on the ovary. The flowers are usually solitary and are

present in the axils of the leaves. They are of various colours from white

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to shades of pink or blue. Self pollination is the rule, but cross

pollination may occur to the extent of about 5-10% due to agency of

insects. The pod is about 2 cm long and usually contains two seeds. A

single plant produces about 50 to 150 pods.

1.6.5 Seed

The seeds are spherical in shape and wrinkled with a pointed beak. They

vary a great deal in size as well as in colour. Colour of seed may vary

from white, light fawn, yellowish-orange, brown, dark brownish and with

a little bluish tinge. The seed coat may be smooth or puckered and

wrinkled. The cotyledons are thick and yellowish in colour.

1.7 Chickpea varieties

Unlike cereals, high yielding photo-insensitive cultivars are not available

in pulses and this appears to be the most important reason for low

productivity of pulse crops in the country. However, several improved

varieties of chickpea have been evolved in different chick pea growing

states (Table 1).

1.8 Production constraints

Singh (1993) described a wide range of constraints to chickpea

production in South Asia. These cover various aspects of crop

management, and many abiotic and biotic constraints. Abiotic factors

include terminal drought, heat, cold, salinity, low pH and soil fertility.

The biotic constraints include “wilt complex” diseases in hot and dry

areas, foliar diseases in cooler areas, various insect pests (especially

Helicoverpa spp.), and parasitic weeds also occur (Singh, 1993).

Johansen et al., (1994) assessed a range of biotic and abiotic stresses for

chickpea in Asia and Africa.

During the last two decades, chickpea cultivation has declined in

India, and almost collapsed in Bangladesh and Nepal. In India, the

largest decline in area occurred in the Indo-Gangetic plains, primarily

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because of its declining competitiveness with respect to competing crops

like wheat and rapeseed-mustard.

Table 1- High yielding varieties of chickpea recommended for general cultivation in

different states (Yadav, 2009)

State Recommended varieties

Andhra Pradesh ICCV-2, ICCV-37, ICCV-4, ICCV-10

Assam KWR-108, BG-256, L-550, KPG-59

Bihar KWR-108, Avrodhi, BG-256, Pant G-114, Pusa-209, L-

550, Pusa-1003

Gujarat Pusa-319, Vijay, ICCV-4, Pusa-240, GG-1, Pusa-1053

Haryana Haryana Chana-1, GNG-469, Pusa-362, Gora Hisari,

Karnal Chana, Gaurav, H-208, H-335, Pusa-1053

Himachal Pradesh BBG-1, Haryana Chana-1, L-550

Jammu & Kashmir GNG-469, L-550, PBG-1, Haryana Chana-1

Karnataka BDN 9-3, ICCV-10, ICCV-2 Annegiri-1

Madhya Pradesh JG-74, JG-315, Vijay, Pusa-256, Phule G-5, Pusa-1053

Maharashtra Vijay, Phule G-5, Vishal, ICCV-10, Pusa-1053

North-Eastern States KWR-108, Avrodhi, KPG-59, BG-256

Orissa Radhey, ICCV-10, L-550, Pusa-372, Pusa-1003

Punjab PBG-1, GNG-469, Haryana Chana-1, Gaurav, L-550, C-

235, G-543, Pusa-1053, GPF-2, PDG-3

Rajasthan GNG-416, GNG-469, GNG-663, PBG-1, L-550, Pusa-256,

RSG-44, Pusa-1053, PDG 84-1

Tamil Nadu ICCV-10, BDN 9-3, CO-3, CO-4

Uttar Pradesh KWR-108, Avrodhi, BG-256, K-850, Pant G-186, Pusa-

372, Radhey, JG-315, Uday (KPG-75), Pusa-1003, Pusa-

1053

West Bengal Pusa-372, KWR-108, KPG-59, BG-256, Pusa-1003

1.9 Fusarium oxysporum f. sp. ciceris- the pathogen

The fungus F. oxysporum f. sp. ciceris (Foc), is both seed and soil borne

and persists in soil in the absence of chickpea for more than five years

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(Kraft et al., 1994). Booth, (1971) described F. oxysporum as a

cosmopolitan soil-borne filamentous fungus. It is an anamorphic species

that includes numerous plant pathogenic strains causing wilt diseases of

a broad range of agricultural and ornamental host plant species (Appel

and Gordon, 1996).

Plant pathogenic forms of F. oxysporum are divided into formae

speciales based on hosts they attack (Armstrong and Armstrong, 1981).

The wide host range of Fusarium oxysporum includes banana (Musa spp.)

(F. oxysporum f. sp. cubense), cabbage (Brassica spp.) (F. oxysporum f. sp.

conglutinans), cotton (Gossypium spp.) (F. oxysporum f. sp. vasinfectum),

flax (Linum spp.) (F. oxysporum f. sp. lini), muskmelon (Cucumis spp.) (F.

oxysporum f. sp. melonis), onion (Allium spp.) (F. oxysporum f. sp. cepae),

pea (Pisum spp.) (F. oxysporum f. sp. pisi), tomato (Lycopersicon spp.) (F.

oxysporum f. sp. lycopersici), watermelon (Citrullus spp.) (F. oxysporum f.

sp. niveum), china aster (Calistephus spp.) (F. oxysporum f. sp.

callistephi), carnation (Dianthus spp.) (F. oxysporum f. sp. dianthi),

chrysanthemum (Chrysanthemum spp.) (F. oxysporum f. sp.

chrysanthemi), gladioli (Gladiolus spp.) (F. oxysporum f. sp. gladioli) and

tulip (Tulipa spp.) (F. oxysporum f. sp. tulipae) (Armstrong and

Armstrong, 1981; MacHardy and Beckman, 1981).

1.9.1 Classsification of Fusarium oxysporum

Based on the structure in or on which conidiogenous hyphae are borne,

Fusarium spp. are classified in Kingdom:-Mycota, Divison:-Eumycota,

Sub divison:- Deuteromycotina, Class:-Hyphomycetes, Order:-Hyphales.

1.9.2 Sporulation

F. oxysporum produces three types of asexual spores: microconidia,

macroconidia and chlamydospores (Nelson et al., 1983). Conidia are

produced on monophialides and in sporodochia. They are scattered

loosely over the surface of a mycelium (Griffin, 1994). Microconidia are

predominantly uninucleate and germinate poorly and variably, with

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germination efficiency ranging from 1-20% (Ebbole and Sachs, 1990).

Macroconidia, produced abundantly, are multinucleate and germinate

rapidly, thereby reproducing fungus efficiently. Chlamydospores are

viable, asexually produced accessory spores resulting from structural

modification of vegetative hyphal segment(s) or conidial cell possessing a

thick wall, mainly consisting of newly synthesized cell wall material. Its

function is primarily survival in soil. Asexual reproduction in F.

oxysporum is accomplished by macroconidia and microconidia, while

sexual state of the fungus has never been observed (Booth, 1971).

1.9.3 Infection and pathogenesis

A common host symptom of Fusarium wilt infection is vascular vessel

colonization. Some pathogens penetrate the roots directly while others

need wounds (MacHardy and Beckmann, 1981). The most common site

of penetration is at or near the root tip. However, in chickpeas,

penetration occurs mainly through the cotyledons and or to a lesser

extent in the zone of elongation and maturation (Jimenez-Diaz et al.,

1989) (Fig. 3).

Figure 3- A chickpea plant showing cotyledonary infection

Fusarium oxysporum commonly penetrates root hairs or epidermal

cells just behind the root tip or within the zone of elongation and

proceeds intercellularly and intracellularly to the primary meristem as

has been shown in other crops (MacHardy and Beckmann, 1981). After

the endodermal and pericycle tissues have been penetrated, the xylem is

colonized and the pathogen is distributed upward (Nelson, 1981). The

upward movement of spores in the vascular system varies with the host

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and the pathogen. In banana, the microconidia of F. oxysporum f. sp.

cubense travel only short distances in the sap stream up to a perforation

plate which permits sap flow but screens out microconidia. The spore

then germinates and the germ tube penetrates the perforation plate.

After penetration, fresh spores are produced beyond the perforation

plate; they are carried a short distance up and germinate to penetrate

the perforation plate again. The pathogen becomes established quickly by

this repeated process (MacHardy and Beckmann, 1981). Because of

proliferation of the pathogen below the perforation plates of the xylem

vessel elements, sap flow is blocked (Mace et al., 1971).

1.10 The wilting phenomenon

Wilting caused by Fusarium oxysporum f. sp. ciceris can be either by

mechanical plugging of water ducts (xylem) by mycelium, wilt toxins or

hydrolytic enzymes (Green, 1981). According to Green (1981), the first

chemically defined wilt toxins were lycomaramasmin and fusaric acid.

“Fusaric acid (5-butylpicolinic acid) increases transpiration and causes

furrowing over the vascular bundles” (Lakshminarayanan and

Subramanian, 1955).

1.11 Vascular wilt diseases

1.11.1 Vascular discolouration

Vascular discolouration or browning is a diagnostic symptom in vascular

wilt diseases. Dark melanin pigment accumulation in host cells results

from an increase in both phenolic and polyphenoloxidase and other

enzyme activities (Green, 1981). The browning originates in xylem

parenchyma adjacent to colonized vessels and, staining is transferred to

the vessels (Pegg, 1981). Vessels could also be blocked by the staining

(Corden and Chambers, 1966). Vascular discolouration can occur in

infected plants prior to the expression of external symptoms as shown in

tomato (Lycopersicon esculentum L.) (Mace et al., 1971). Wilting appears

to be host originated resulting from water shortage and hormonal

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imbalances and the shortage arises in response to the injury of tissues

and not as the cause of injury by the pathogens (Beckman, 1987).

Figure 4- Symptoms of Fusarium wilt in a chickpea field

1.11.2 Factors determining wilt

There are two conditions which must be satisfied before a wilt disease

can develop: the fungus must first gain entry to the vascular system of

the host; it must then continue to colonize the vascular system more or

less extensively and intensively. However, it is sometimes possible for

both of these conditions to be fulfilled without development of disease,

because certain supplementary conditions may also have to be satisfied.

It may be necessary, for example, for the leaves to be in a particular

physiological condition before the characteristic symptoms appear.

Therefore, even if the two primary conditions are satisfied the disease

does not necessarily develop; but if they are not satisfied it cannot

develop. It follows that any mechanism which tends to exclude the

pathogen from the xylem, or to limit its systemic distribution through the

vascular system, will contribute to wilt resistance. Factors which

influence the frequency and intensity of the initial vascular infection will

be termed 'primary determinants' (Talboys, I964) those which influence

the spread and activity of the pathogen after it has entered the vascular

system will be described as 'secondary determinants'.

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1.11.3 Primary determinants

Griffiths (1971) in a series of electron micrographs, demonstrated the

accumulation of vesicular material between the host plasmalemma and

the cell wall within 12 hrs of the entry of the fungus into the adjacent

cell. Penetration hyphae pass through the cell wall and into the newly

formed deposit, but continued accumulation around the hyphae of

vesicular material extruded through the host plasmalemma results in the

formation of lignin tubers. Eventually the hyphal tip enclosed in the

lignin tuber undergoes lysis and no further development occurs. The

mechanism appears to provide an effective barrier in cells which are in

an active metabolic state, as in the younger parts of the root, and a high

proportion of incipient infections of the epidermis and cortex are

excluded at this early stage.

Endodermal cells can respond initially in the same way as

epidermal and cortical cells, but as they mature the cell walls become

more or less heavily suberinized, and become highly resistant to

penetration by fungi (Talboys, 1964). Suberinization is a normal feature

of endodermal development but the process tends to be accelerated by

infection of the cortex or by other forms of damage.

1.11.4 Secondary determinants

The secondary determinants are all those resistance mechanisms which

operate after the initial establishment of vascular infection. Such

infection commonly results in occlusion of vessels by tyloses, gums or

gels (Talboys, 1964). Tyloses result from distension of the pit-closing

membranes between vessels and adjacent xylem parenchyma or ray

cells, forming balloon-like structures which commonly have well-

developed cell walls, sometimes with reticulated lignified thickening,

dense cell contents and often masses of starch grains. The nucleus of the

original cell apparently migrates into one of the several tyloses that may

arise from it. The formation of tyloses involves considerable synthesis of

cell wall and cytoplasmic material. They may fill the vessels so completely

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that they present the appearance of a parenchymatous tissue. Occluding

gums are also formed by living cells adjacent to vessels and are

apparently extruded through the pits into the vessel lumens. The gels

which Beckman (1964) reported in banana vascular tissues invaded by

Fusarium oxysporum f. sp. cubense were largely of pectic materials and

appeared to result from the swelling of perforation plates and end-wall

membranes of vessels. Talboys, (1964) proposed that such swelling

results from 'conditioning' by the accumulation of fungal respiratory

carbon dioxide at night, and subsequent solution of calcium from the

membranes by organic acids.

The effects of vascular occlusions depend upon their rates of

formation and the number of vessels that are affected. If they form in

advance of the pathogen at a rate sufficient to prevent the transport of a

new 'generation' of conidia in the transpiration stream, they will

contribute to resistance (Talboys, 1964). They will in any case reduce the

capacity of the vascular system for water transport. If few vessels are

affected this will not cause damage to the plant, but if many are affected

the plant may collapse as a result of water shortage. According to

circumstances, therefore, occlusion may contribute to pathogenesis or to

resistance. Dixon and Pegg (1969) showed that there may subsequently

be a decrease in the amount of mycelium present in the vessels,

apparently as a result of lysis, and that this effect is most pronounced in

certain wilt-resistant varieties in which as much as 75% of the mycelium

may ultimately disappear.

1.12 Fusarium oxysporum f. sp. ciceris race situation

Fusarium wilt, caused by Fusarium oxysporum f. sp. ciceris (Padwick)

Matuo and Sato, is the most important soilborne disease of chickpea

throughout the world, particularly in the Indian subcontinent, the

Mediterranean region, and California (Jalali and Chand, 1992).

Two pathotypes exhibiting differential symptoms, namely yellowing

and wilting, exist in Foc populations. The yellowing pathotype induces

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progressive foliar yellowing with vascular discolouration, while the wilting

pathotype induces rapid and severe chlorosis, flaccidity, and vascular

discolouration (Trapero-Casas and Jiménez-Díaz, 1985).

In addition to these two pathotypes, there exist eight races (race 0,

1A, 1B/C, 2, 3, 4, 5, and 6) of Foc that can be identified based on the

disease reactions of a set of differential chickpea cultivars (Haware and

Nene, 1982). Races 0 and 1B/C belong to the yellowing pathotype,

whereas the remaining races form the wilting pathotype (Kelly et al.,

1994).

The eight races also have a distinct geographic distribution. Races

2, 3, and 4 have been reported only in India (Haware and Nene, 1982),

whereas races 0, 1B/C, 5, and 6 are found mainly in the Mediterranean

region and California (Kelly et al., 1994). Unlike the other races, race 1A

is more widespread and has been reported in India, California, and the

Mediterranean region (Haware and Nene, 1982; Kelly et al., 1994). An

intraspecific phylogeny of Foc races inferred from DNA fingerprinting

with repetitive sequences indicated that each of the eight races forms a

monophyletic lineage and that they have evolved in a simple stepwise

pattern, with race 0 being hypothesized as ancestor of the wilting races

(Jiménez-Gasco et al., 2001).

1.12.1 Stepwise evolution of races in Foc

For asexually reproducing fungal pathogens such as formae speciales of

F. oxysporum, races capable of overcoming resistance gene are thought to

evolve by successive accumulation of mutations. The evolution of

geographically distinct virulence appears to be correlated to cultivation of

chickpea germplasm lines in these regions. Chickpeas are of two types

desi and kabuli. Between these two, desi genotypes are grown mainly in

India while kabuli in the Mediterranean region and California. Resistance

to wilt occurs mostly in desi genotypes (Haware et al., 1980).

Interestingly, races 1A, 2, 3 and 4 which inhabit India are also the most

virulent ones, whereas, those from the Mediterranean region or the USA

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are less virulent (Jiménez-Gasco et al., 2004). Evidently, there exists a

correlation between evolution to races and cultivation of chickpea lines.

1.13 Major Facilitator Superfamily (MFS)

The Major Facilitator Superfamily (MFS) is a large and diverse group of

secondary transporters that includes uniporters, symporters, and

antiporters. MFS proteins facilitate the transport across cytoplasmic or

internal membranes of a variety of substrates including ions, sugar

phosphates, drugs, neurotransmitters, nucleosides, amino acids, and

peptides (Schüβler et al., 2006). They do so using the electrochemical

potential of the transported substrates. Uniporters transport a single

substrate, while symporters and antiporters transport two substrates in

the same or in opposite directions, respectively, across membranes. MFS

proteins are typically 400 to 600 amino acids in length, and the majority

contain 12 transmembrane alpha helices (TMs) connected by hydrophilic

loops. The N- and C-terminal halves of these proteins display weak

similarity and may be the result of a gene duplication/fusion event.

Based on kinetic studies and the structures of a few bacterial

superfamily members, GlpT (glycerol-3-phosphate transporter), LacY

(lactose permease), and EmrD (multidrug transporter), MFS proteins are

thought to function through a single substrate binding site, alternating-

access mechanism involving a rocker-switch type of movement. Bacterial

members function primarily for nutrient uptake, and as drug-efflux

pumps to confer antibiotic resistance. Some MFS proteins have medical

significance in humans such as the glucose transporter Glut4, which is

impaired in type II diabetes, and glucose-6-phosphate transporter

(G6PT), which causes glycogen storage disease when mutated (Saier et

al., 1999).

1.13.1 Sugar transporters

Sugar efflux is important for the development of plants, feeding

developing seeds and pollen, for example. Despite having important roles

in such basic physiological processes, the mechanisms underlying sugar

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efflux have remained unclear. Furthermore, plant pathogens can use

these plant-derived sugars as nutrients, meaning that plants must

carefully regulate sugar efflux. The transporters, which are of the major

facilitator transporter family, allow fungi to grow on an extremely diverse

set of materials and are one of the reasons why fungi can occupy such a

large number of ecological niches. All transport proteins identified so far

in symbiotic or pathogenic fungus/plant interactions are specific for

monosaccharides (Schüβler et al., 2006, Polidori et al., 2007) and

catalyze the uptake of glucose or fructose and, to a lesser extent, of

hexoses. It was speculated that these hexose transporters act in

combination with fungal and/or plant derived cell wall invertases (Tang

et al., 1996) to supply the pathogen with carbon derived from

extracellular sucrose hydrolysis. The impact of these transporters on the

development of fungal pathogens within the host plant has never been

proven. However, plants have evolved mechanisms to sense extracellular

(apoplastic) changes in glucose concentrations, e.g., produced from

extracellular sucrose hydrolysis, and respond to these changes with the

induction of defense responses (Kocal et al., 2008).

1.14 Resistance in plants to pathogens

1.14.1 Terminology

A diverse range of organisms constantly challenge plants, but not all of

them are able to cause disease. When an organism is able to invade and

multiply within plants, they are referred to as pathogens. Sometimes

pathogens can live on a susceptible host without causing any disease. In

such case they are called as saprophytes. If, however, conditions become

favourable for infection and disease, they are called parasites.

The ability of a pathogen to cause disease to a host plant is often

dependent on how a plant responds. If infection takes place with

subsequent disease development, a plant is considered susceptible to

the pathogen. Susceptibility may be caused by an inability of the plant to

recognize the pathogen and/or produce an effective and rapid defense

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response. Such an interaction between plant and pathogen is termed a

compatible interaction. If, however, plants are able to restrict pathogen

multiplication or movement from the initial site of infection they are

resistant and the interaction is incompatible. The speed and extent of

the defense response often establishes whether a plant is resistant or

susceptible (Nurnberger and Lipka, 2005). Tolerance/ partial

resistance is the ability of a plant to sustain the effects of a disease

without suffering serious yield losses and dying as a result of infection.

Partial resistance is also known as field resistance. Some crops that are

resistant comprise varieties that do not develop any disease, despite

challenge by a known pathogen under favourable environmental

conditions. Such varieties are then considered as immune. When an

entire plant species is resistant to a pathogen, it is called non-host

resistance (Nurnberger and Lipka, 2005). The prerequisite for successful

invasion of a plant by a pathogen, therefore is basic compatibility, where

a potential pathogen has attained pathogenicity factors in co-evolution

with the plant in order to overcome non-host resistance.

1.15 Biochemical basis of plant pathogen interactions

1.15.1 Biochemistry

Plants are continually exposed to insects, nematodes and other

potentially damaging pests, as well as to a wide variety of parasitic

microorganisms. Yet the majority of plants remain healthy most of the

time. This observation suggests that plants must possess highly effective

mechanisms for preventing parasitism and predation, or at least limiting

their effects.

1.15.2 ROS scavenging machinery in plant cells

Plant cells produce reactive oxygen species during interactions with

potential pathogens. The oxidative burst belongs to the fastest active

defense responses known in plants. In almost all host-fungus

interactions, one of the first events detected in attacked host cells is the

rapid and transient generation of activated oxygen radicals, including

superoxide anion (O2¯), hydrogen peroxide (H2O2) and hydroxyl radical

Chapter 1


(OH-). The association of ROS formation and increased activity of

enzymes participating in their metabolism with the induction of defense

responses has been demonstrated in many plant-pathogen interactions

(Wojtaszek, 1997). H2O2 is generally thought of as the most versatile of

the ROS, with a number of different possible functions in a plant’s

defense strategy. Besides having antimicrobial effects, such as inhibiting

germination of spores of many fungal pathogens, H2O2 has been shown

to be involved in the oxidative cross-linking of cell wall glycoproteins

(Bradley et al., 1992). H2O2 has also been attributed a possible role in

orchestrating the hypersensitive cell death response. The ROS may also

act as secondary messengers for the activation of genes encoding

protective proteins (Lamb and Dixon, 1997). However, the enhanced ROS

production causes oxidative damage, leads to lipid peroxidation and

damages macromolecules such as pigments, proteins, nucleic acids and

lipids (Apel and Hirt, 2004). Under conditions of normal healthy growth,

plant possesses a number of enzymatic and non-enzymatic mechanisms

of detoxification to efficiently scavenge for either the ROS themselves or

their secondary reaction products. Various enzyme systems participate

in ROS metabolism during the pathogen attack in plants (Bolwell et al.,

2002). Major ROS scavenging enzymes such as superoxide dismutase

(SOD), catalase (CAT), guaiacol peroxidase (GPX) and ascorbate

peroxidase (APX) are produced to avoid cellular disintegration by ROS

(Mittler et al., 2004). These enzymes are present with different

isoenzymatic forms in several cell compartments and their expression is

genetically controlled and regulated both by developmental and

environmental stimuli, according to the necessity to remove ROS

produced in cells (Tommasi et al., 2001). The balance between SOD and

APX or CAT activities in cells is crucial for determining the steady-state

level of O2- and H2O2.

1.15.3 Superoxide dismutase

Of all these mentioned scavenging mechanisms, superoxide dismutase,

the first enzyme in ROS metabolism, catalyzes dismutation of O2_ and

Chapter 1


HO2 to H2O2 (Gupta et. al., 1993). Within a cell, the SOD’s constitute the

first line of defence against ROS (Alscher et. al., 2002). O2 is produced at

any location where an electron transport system is present and hence O2

activation may occur in different compartments of the cell (Alscher et. al.,

2002). This being the case, it is not surprising that SOD’s are found

throughout all sub-cellular locations.

1.15.4 Peroxidase and catalase

Plant peroxidases (POX) [donor: hydrogen peroxide oxidoreductase] have

been studied for: 1) important role in lignification and suberization

(Mohan and Kolattukudy, 1990); 2) active participation in the formation

of diphenyl bridges (Pena et al., 1996); 3) the cross linking of hydroxyl

proline rich proteins (extensions) in the cell wall matrix (Brownleader et

al., 1994); 4) control function of redox state in apoplast (Takahama,

1993). The catalase [hydrogen-peroxide: hydrogen-peroxide

oxidoreductase] is present as multiple isoforms in plants (Scandalios,

1994). Catalases contribute towards removing toxic hydrogen peroxide,

production of which is typical in a non-specific defence mechanism in

plant pathogen interactions (Lamb and Dixon, 1997).

1.15.5 Ascorbate peroxidase and Guaiacol peroxidase

Ascorbate peroxidase exists as an isoenzyme and plays an important role

in the metabolism of H2O2 in higher plants. APX reduces H2O2 to water

by utilizing ascorbate as a specific electron donor. Guaiacol peroxidase is

included in different physiological processes like cross-linking of the cell

wall proteins, pectins by diferulic bridges and the oxidation of cinnamyl

alcohols prior to their polymerization during lignin and suberin formation

(Mittler et al., 2004).

1.15.6 β-1, 3 Glucanases

The accumulation of PR proteins upon infection with microbial

pathogens is well documented in plants (Van Loon, 1997). The PR

Chapter 1


proteins include β-1, 3-glucanases (PR-2) and chitinases (PR-3) (Fanta et

al., 2003). β-1, 3-glucanases (βGlu; EC 3.2.1.39) are the hydrolytic

enzymes, which are capable of hydrolyzing the β-1,3-glucans found in

the cell walls of several genera of fungi (Farkas, 1979). β-1, 3-glucanase

exist in multiple forms in a number of plant species including pea,

tobacco, potato, bean, chickpea, tomato and maize. β-Glucanases are

characterized by a molecular mass around 35 kDa and the numerous

isoforms differing in enzymatic activity, structural properties, cellular

localization (vacuolar or apoplastic), and regulation patterns (Esquerŕe-

Tugayé et al., 2000). β-1, 3-glucanases and chitinases are believed to

contribute to plant defense in two ways, responding directly by degrading

the hyphal walls of the pathogen and rendering them susceptible to lysis,

or indirectly by releasing oligosaccharide elicitors from the fungal cell

wall which are detected by the plant host and cause an accumulation of

phytoalexin (White et al., 1996).

1.15.7 Phenylpropanoid metabolism in plants

Phenolics are a class of substances, which contain an aromatic ring with

one, or more hydroxyl groups attached to it. Phenols have been known to

occur in all plants investigated so far. Some of them occur constitutively,

whereas others are formed in response to pathogen ingress and

associated as part of an active defense response in the host (Nicholson

and Hammerschmidt, 1992). The constitutive phenolics are known to

confer resistance either directly or indirectly through activation of post-

infection responses in the hosts (De Vecchi and Matta, 1989). Post

infection activation of phenol metabolism in xylem vessels of tomato has

been demonstrated by De Vecchi and Matta (1989). It has been reported

that correlation exists between degree of resistance and phenol level in

healthy plants. Phenolic acid metabotism is activated through phenyl

propanoid pathway during infection which gives rise to suberin, lignin

and wall bound phenolics.

Phenylalanine ammonia-lyase (PAL) is a key enzyme of phenyl propanoid

metabolism in plants. The enzyme catalyzes deamination of L-

Chapter 1


phenylalanine into trans-cinnamic acid. The acid serves as a precursor of

various secondary metabolites, including phenols, phenylpropanoids and

monomers of lignin and salicylic acid (Creasy and Zucker, 1974). PAL

activity undergoes considerable variations and depends not only on the

genotype of plants, but also on the age and stage of development, organ

and type of plant tissue (Camm and Towers, 1973).

1.16 Overview on genetic diversity in crop plants

Genetic diversity is a statistical concept referring to the variance at

individual gene loci (among alleles of a gene), among several loci or gene

combinations, between individual plants or within plant populations, or

between plant populations. It may be caused and maintained by a variety

of mechanisms including mutation, sexual recombination, migration,

gene flow, genetic drift and genetic selection.

To meet the need for more food, it would be necessary to make

better use of a broader range of the world’s plant genetic diversity

(Farshadfar and Farshadfar, 2008). Most of the breeding programmes

aimed at the crop improvement are based upon selecting desirable

genotypes from the genetic variation available and manipulating all or as

many as possible of the desirable traits like high yielding, hybrid vigor,

disease resistance, more number of branches etc. into one individual to

develop a commercial variety. Analysis of genetic diversity in germplasm

collections can facilitate reliable classification of accessions and

identification of subsets of core accessions with possible utility for

specific breeding purposes. The study on genetic diversity of species is

emphasized because modern breeding practices have narrowed the

genetic diversity of cultivated crops. This reduction in genetic diversity

could severely limit future breeding programs for adaptive traits such as

resistance to biotic and abiotic stresses and reduce the stability of crop

yield (Labdi et al., 1996). Significant emphasis is being paid to

comprehensive analysis of genetic diversity in numerous crops, including

major field crops such as wheat (Triticum aestivum L.), rice (Oryza sativa

L.), maize (Zea mays L.), barley (Hordeum vulgare L.), and soybean

Chapter 1


(Glycine max L. Merr.). Hence, genetic diversity studies usually provide a

significant data that can be utilized in plant breeding programmes,

phylogenetic studies, management of plant population composed of

species that are either endangered or unwanted.

1.16.1 Importance of genetic diversity studies in chickpea

Chickpea (Cicer arietinum L.) is an important food legume providing

protein in human diet. The national average yield of chickpea is low as

compared to other chickpea growing countries (Anonymous, 2006).

Although, major crop improvements have been made in the recent years

through the evolution of high yielding and disease resistant chickpea

cultivars, breeding for improved types is a continuous process and

requires strenuous efforts by breeding. Therefore, chickpea breeding

programmes are focused on improving the genetic potential of the crop

by producing genetic combination that offers protection against key

biotic and abiotic stresses. In particular, the crop is highly susceptible to

the fungal diseases, Fusarium wilt and Ascochyta blight. As an initial

step towards broadening the genetic diversity of chickpea and achieving

better yielding variety, there has been a surge in the comprehensive

understanding of the amount and pattern of natural genetic variation

that exists within and between the available cultivated chickpea

germplasm collections. Chickpea has high variation for different quality

and quantity traits, including ideal plant type (tall type), shape and grain

colour, flower colour, podding, colour of seed coat, earliness, resistance

to disease and pests, which helps breeders to release improved and

advanced lines and varieties.

Plant diversity can be analyzed at various levels such as:

morphological, biochemical, and molecular level (Ahmad and Slinkard,

1992). In the present study, analysis has been carried out at

morphological and molecular level.

Chapter 1


1.16.2 Relevance of seed morphology in diversity analysis

To study the genetic diversity among population, varieties and species,

various morphological traits such as growth type, number of leaflets per

leaf, leaflet size, plant height, days taken for 50% flowering, flower

colour, flowering period, days to maturity, pod size, pods per plant, seed

numbers per pod, seed colour, seed shape and 100 seed weight have to

be considered. Morphological studies were performed earlier in various

crops like mungbean (Vigna radiata L. Wilcjek) (Rahim et al., 2010),

cassava (Moyib et al., 2007) etc. including chickpea (Rao et al., 1994).

Appearance of chickpea seed is a key market factor and acceptability

varies with cultural reference. In particular, larger seed size coupled with

other desirable traits (eg. light colour) commands premium price in a

market dependent manner (Graham et al., 2001). In chickpea, seed size

is considered as an important factor for subsequent plant growth

parameters including germination, seedling vigor and seedling mass

(Dahiya et al., 1985). Upadhyaya et al., (2006) also proposed seed size to

affect seed yield. Importantly, seed weight was also proposed as an

accurate measure of chickpea seed size. Therefore, to produce seed of

specific size and to meet a specific market demand through targeted

breeding, knowledge of genetic variation is required. Seed size variation

exists within and between chickpea genotypes, with some desi types as

large as kabuli types and some kabuli types as small as desi types

(Kumar and Singh, 1995).

1.16.3 Statistics and genetic diversity analysis

A survey of genetic variability with the help of suitable parameters such

as genotypic coefficient of variation, heritability and genetic advance are

absolutely necessary to start an efficient breeding program (Mishra et al.,

1987). Heritability plays a predictive role in breeding, expressing the

reliability of a phenotype as a guide to its breeding value. It is understood

that only the phenotypic value can be measured directly while breeding

values of individuals are derived from appropriate analyses. Heritability

Chapter 1


helps in determining the influence of environment on the expression of

the genotype and reliability of characters. There is a direct relationship

between heritability and response to selection, which is referred to as

genetic progress. The expected response to selection is also called as

genetic advance (GA). High genetic advance coupled with high heritability

estimates offers the most effective condition for selection (Larik et al.,

2000). With the above background information the present investigation

was undertaken to study the genetic parameters among chickpea

genotypes.

1.16.4 Assessment of genetic variability at molecular level

Genetic variation has been reduced drastically in the cultivated species

due to micropropagation, domestication of species and continuous

selection pressure for a specific trait. Therefore it is important to study

the composition of existing germplasm and compare it with the initial

and related species. This information can help in understanding

phylogenetic relationship (Belaj et al., 2007), identifying useful traits and

assessing genetic variability. Plant breeding programmes must

incorporate sufficient genetic diversity. This allows the production of new

varieties that have enhanced crop production and resistance to biotic

and abiotic factors. Superior genotypes are developed from a cross by the

measurement of genetic distance or genetic similarity between parents.

Such studies provide information not only about phylogenetic

relationship but also about the possibility of finding new, economically

important alleles.

1.16.5 Molecular markers in plant genetic diversity analysis

Molecular methods such as RAPDs, AFLPs, SSRs and microsatellites

used for detecting DNA sequence variations are being used as

complementary strategies to traditional approaches for assessment of

genetic diversity (Karp et al., 1997). Molecular markers are the specific

segments of DNA that can be identified within the whole genome at

specific location. These are used to ‘flag’ the position of a particular gene

Chapter 1


or the inheritance of particular characteristic. In the genetic cross, the

characteristic of interest are usually linked with the molecular markers.

Thus, individual can be selected in which the molecular markers are

present since the markers indicate the presence of desired characteristic.

Molecular markers have several applications including germplasm

characterization, genetic diagnostics, characterization of transformants,

study of genome organization and phylogenetic analysis. The DNA

fingerprints of a particular species verify the uniqueness of species that

makes it different from others. The sequence of DNA is different in every

individual. Therefore, a specific sequence of DNA can be utilized as a tool

to screen variations within a population. Moreover, DNA-based molecular

marker technique provides advance and reliable approaches to study

variability. A comparison of commonly used marker systems is shown in

Table 2.

There are certain conditions that characterize a suitable molecular

marker:-

1. Must be highly polymorphic

2. Co-dominant inheritance

3. Random and frequent distribution throughout the genome

4. Reproducible

5. Easy and cheap to detect

Table 2. Comparison of commonly used marker systems

Sr.No. FEATURES RFLPs RAPDs AFLPs Microsatellite SNPs

1. Template requirement (µg) 10 0.02 0.5 0.05 0.05

2. DNA quality High High Moderate Moderate High

3. PCR based No Yes Yes Yes Yes

4. Number of loci tested 1.0-3.0 1.5-50 20-100 1.0-3.0 1.0

5. Ease of use Not easy Easy Easy Easy Easy

6. Reproducibility High Unreliable High High High

7. Development cost Low Low Moderate High High

Chapter 1


1.16.6 Microsatellite markers

Microsatellites are also known as simple sequence repeats (SSR) or short

tandem repeats (STR). The term microsatellite was coined by Litt and

Lutty (1989). These are 1-6bp tandem repeats of mono-, di-, tri- or penta-

nucleotides that occur ubiquitously throughout the eukaryotic genome.

Microsatellites exhibit high degree of polymorphism due to length

variation of repeating sequence. These variations arise due to polymerase

slippage or unequal crossing-over during meiosis. SSR markers have

been characterized in many crops like maize, rice, soybean, brassica,

barley, tomato including chickpea (Gupta et al., 1996). SSR is a PCR

based technique which involves the use of forward and reverse primers in

a PCR reaction. The tri and tetra nucleotide microsatellites are more

popular for sequence- tagged microsatellite site markers (STMS) analysis

because they present a clear banding pattern and gel electrophoresis

(Hearne et al., 1992). However, di-nucleotides are generally abundant in

genomes and have been used as markers e.g. (CA)n, (AG)n, and (AT)n.

The di- and tetra-nucleotides are present in the noncoding regions of the

genome while 57% of trinucleotide repeats are shown to reside in or

around the genes. A very good relation between the number of alleles

detected and a total number of simple repeats within the target

microsatellite DNA has been observed. Thus larger the repeat number in

the microsatellite DNA, the greater is the number of alleles detected in a

large population (Yang et al., 1994). The uniqueness and the value of

microsatellite arise from their multiallelic nature, co-dominant

transmission, ease of detection by PCR, relative abundance, extensive

genome coverage and requirement for only a small amount of starting

DNA.

Chapter 1


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ObjectivesObjectivesObjectivesObjectives

Objectives


OOOObjectivesbjectivesbjectivesbjectives 1. Anatomical investigations on the interactions between Fusarium

oxysporum f. sp. ciceris (Foc) race 2 and Cicer arietinum L. in a

time course based study using light and electron microscopic

methods.

2. Biochemical characterization of defense responses during

interaction between eight distinct genotypes of chickpea with Foc

race 2.

3. To identify the existing race of Foc in the infected chickpea fields

of Junagadh Agricultural University by conducting pathogenicity

tests, protein profile analysis and molecular studies.

4. Identification and characterization of avirulence gene from Foc.

5. To evaluate the genetic diversity exisiting in chickpea genotypes

showing differential reaction to Fusarium wilt by employing

morphological and molecular methodologies.

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