Chapter 1 Introduction
Chickpea (Cicer arietinum L.) a member of sub family, papilionaceae (family-
leguminaceae) originated from middle East, subsequently spread to 45 countries having arid/
semi-arid and sub-tropical environment. There are two main types recognized as desi small
size brown colour seed which accounts nearly for 90% and Kabuli with bold creamy seeds is
grown in about 10% of the total area.
Chickpea is the third important crop after dry beans (Phaseolus vulgaris L.) and dry
peas (Pisum sativum L.). It contains 17-22% protein, 60-64% carbohydrate and 3-4% fat
(Sindhu et al., 1974) and is a good source of calcium, phosphorus, vitamins and minerals. The
growing leaves of chickpea contain malic and osealic acids, which bear medicinal value in
The production of chickpea was 12.33 m tonnes with an area of 9.49 m ha during 2015-
16 in India. In Madhya Pradesh it occupied an area of 2.46 m ha with a production of 2.41 m
tonnes of grain, the productivity being 1280 kg/ha (Krishak Jagat, 2008).
Low yield of chickpea is attributed to several diseases and insect. The crop suffers from
a number of soil borne diseases like dry root rot (Rhizoctonia solani), wet root rot (Rhizoctonia
bataticola), Collor rot (Sclerotium rolfsii) and wilt (Fusarium oxysporum f. sp. ciceri). Despite
of different diseases, Fusarium wilt disease is most important disease of chickpea causes severe
damage of crop. Vascular wilt caused by an important obligate biotroph Fusarium oxysporum
f. sp. ciceri (Padwick) (F.O.C), is consider one of the limiting factor for its low productivity.
Although the disease is wide spread in the chickpea growing areas of the world, it is most
prevalent in the Mediterranean Basin and the Indian subcontinent (Jalil and Chand, 1992).
The fungus is a primarily soil borne pathogen, however, few reports indicated that it
can be transmitted through seeds (Haware et al., 1978). The pathogen can infect at all stages of
plant growth with more incidences in flowering and pod filling stage. The wilt appeared in field
within three to four weeks after sowing, if the variety is susceptible (Haware, 1990). Early
wilting causes more loss than late wilting, but seeds from late wilted plants are lighter, rough
and dull than those from healthy plants (Haware and Nene, 1980). Relatively high temperature
with drought may cause up to eighty percent plant mortality (Govil and Rana, 1994).
The pathogen is facultative saprophytic and it can survive as mycelium and
chlamydospores in seed, soil and also on infected crops residues, buried in the soil for up to
five to six years (Haware et al., 1986). If the disease inoculums establish in the soil, it is difficult
to check the disease or eliminate the pathogen except by following crop rotation for more than
six years (Gupta, 1991). Under favourable condition, the wilt infection can damage the crop
completely and cause 100% yield loss (Navas-Cortes et al., 2000). Annual yield loss due to
Fusarium wilt were estimated at 10% (Trapero-Casas and Jiménez-Díaz, 1985). The better way
to manage the pathogen in eco-friendly approach is consider the economic way for
management of the disease instead of costly and hazards chemicals.
Biological management is considered to be antagonistic to many soils borne and plant
pathogenic fungi (Prasad et al., 2002). Chary et al., 1984 reported that some of the toxic
substances obtained from various plant species have been reported to manage a number of
fungal diseases of crop plants. A number of plant species have been reported to possess some
natural substances which are toxic to many fungi causing plant diseases (Mishra and Dixit,
1977). Therefore, the present study was carried out to evaluate the bio-agents and phyto-extract
against the growth of Fusarium oxysporum f.sp. ciceri (F.O.C) inciting agent of chickpea wilt,
under in-vitro and in-vivo condition.
The most efficient economical and ideal way of managing chickpea wilt, is the use of
resistant cultivars (Karimi et al., 2012). Chemical control of wilt is not feasible and economical
because of the soil-borne nature of the pathogen. The most practical and cost-efficient method
for management of Fusarium wilt of chickpea is the use of resistant cultivars (Nene and Reddy,
1987; Bakhsh, 2007).
The use of resistant cultivars is one of the most practical and cost-effective strategies
for managing Fusarium wilt, but deployment of resistant varieties has not been extensive
because of undesirable agronomic characteristics. Moreover, the high pathogenic variability in
F.O.C may limit the effectiveness of resistance. Pathotypes have been differentiated into two
groups based on the distinct yellowing or wilting syndromes (Jimenez-Gasco et al., 2004).
Presently, eight races of the pathogen (race 0, 1A, 1B/C, 2, 3, 4, 5 and 6) have been
identified by reaction on a set of differential chickpea cultivars. Races 0 and 1B/C induce
yellowing symptoms, whereas the remaining races induce wilting (Jimenez-Gasco et al., 2001).
The eight races have distinct geographic distribution also. Races 1-4 have been reported from
country, whereas 0, 1B/C, 5 and 6 are found in the Mediterranean region and USA.
The genus Trichoderma is widespread in soil and on decaying wood and vegetable
matter. As saprophytic organisms, Trichoderma spp. are able to use a wide range of compounds
as carbon and nitrogen sources and secrete a variety of enzymes to break down recalcitrant
plant polymers into simple sugars for energy and growth. The high degree of ecological
adaptability shown by strains within the genus Trichoderma is reflected its worldwide
distribution, under different environmental conditions, and its survival on various substrates.
This considerable variation, coupled with their amenability of cultivation on
inexpensive substrates, makes Trichoderma isolates attractive candidates for a variety of
biological control applications (Harman, 2006). Several modes of action have been proposed
to explain the biocontrol of plant pathogens by Trichoderma; these include production of
antibiotics and cell wall degrading enzymes, competition for key nutrients, parasitism,
stimulation of plant defence mechanisms and combination of these possibilities.
Trichoderma spp. generally grows in its natural habitat on plant root surfaces and
therefore it controls root diseases in particular. The dual role of antagonistic activity against
plant pathogens and plant growth promoter make Trichoderma strains appealing alternatives
to hazardous fumigants and fungicides. Trichoderma spp. were shown to be very efficient
producers of extracellular enzymes, and some of these have been implicated in the biological
control of plant diseases (Monte, 2001; Harman, 2006).
Plants are capable of producing an immune response after primary pathogen infection,
which is known as systemic acquired resistance (SAR) (van Loon et al., 1998). The activation
of SAR correlates with the expression of pathogenesis-related (PR) genes, including acidic and
basic ?-1,3-glucanase and chitinase, which supposedly act against the cell walls of the pathogen
(Ferreira et al., 2007). Non-pathogenic rhizobacteria and fungi, such as Trichoderma spp. can
induce systemic resistance in plants that is phenotypically similar to SAR (Yedidia, 2000). No
single biocontrol strain is known to possess all of the mechanisms discussed above, and the
genetic and biochemical bases for their efficacy are still explored.
A number of Trichoderma isolates produce a wide variety of fungal cell wall-degrading
enzymes, such as chitinase, ?-1,3-glucanase ?-1,6-glucanases and proteases, when grown on
polysaccharides, fungal cell walls, or autoclaved mycelium as a carbon source (Lorito 1998;
Kucuk 2007; Kuck & Kivanc 2008; Singh et al., 2008). These observations, together with the
fact that chitin and ? -1,3-glucan are the main skeletal polysaccharides of fungal cell walls
(except from oomycetes and cellulose), suggest that chitinase and ? -1,3-glucanases act as key
enzymes in the lysis of phytopathogenic fungal cell walls during the antagonistic action of
Trichoderma. Hence, fungal cell wall-degrading enzymes of Trichoderma spp are of special
importance in plant defence mechanisms. Rice bran, crab shell powder and neem cake
combination was one of the suitable formulation for the induction of maximum levels of fungal
Several studies have shown that phenolics activity is induced in chickpea upon
treatment with pathogen (Karthikeyan et al., 2006; Arfaoui et al., 2007). Enzyme activity was
increased by the treatment with strain L1, implicating it in induced defence responses against
wilt disease in chickpea. Induction of Trichoderma spp. biological agents has been reported in
several host-pathogen combinations (Karthikeyan et al.,2006). Meena et al., (2000) reported
that P. flurescence induced the activities of Trichoderma response to infection by Cercospora
personnatum in groundnut. Chen et al., (2000) reported that various rhizobacteria and Pythium
aphanidermatum induced the Trichoderma activity in cucumber root tissues. Induction of plant
defence enzymes and phenolics by treatment with plant growth-promoting harmones
rhizobacteria Serratia marcescens has been reported (Lavania et al., 2006). Trichoderma
overexpressing transgenic tomato plants exhibited high resistance to Pseudomonas syringae,
the causative agent of speck disease, when compared with control plants (Li and Stiffens,
Therefore, although potential biocontrol agents with suitable antagonistic
characteristics may be readily found, they must be screened carefully for other traits relevant
to their use in a given application. In the present study we have screened a local isolate of
Trichoderma harzianum strain L1 for secretion of some lytic enzymes, and report its
exploitation as a biocontrol agent against chickpea wilt, a soil-borne fungal disease caused by
Singh et al., (1986) reported wilt incidence varying from 10 to 100 % covering many
parts in different states of the country. In Malwa Plateau and adjoining region 20 to 25% wilt
incidence has been observed. The work on variability of F.O.C to identify the pathogenic races
in different areas has not been systematically done so for in Malwa and Nimar. It is therefore,
proposed to explore the variability in the pathogen along with its biological management with
the following objectives:
1. Collection of isolates of Fusarium oxysporum f. sp. ciceri (F.O.C) from important chickpea
2. Isolation and quantification of chitinases and glucases from Trichoderma species
3. Comparison of Trichoderma species with chemicals and phyto chemicals for management
of Fusarium wilt of chickpea.