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IntroductionIntroductionIntroductionIntroduction
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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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Ph.D. Thesis; BRD School of Biosciences, Sardar Patel University 17
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
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(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
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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
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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-
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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
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(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.
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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
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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
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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
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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.
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Ph.D. Thesis; BRD School of Biosciences, Sardar Patel University 27
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ObjectivesObjectivesObjectivesObjectives
Objectives
Ph.D. Thesis; BRD School of Biosciences, Sardar Patel University 36
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.