2. review of literature - shodhgangashodhganga.inflibnet.ac.in/bitstream/10603/9638/11/11_chapter...

A Study on Biological control of leaf spot of Lycopersicon esculentum…….. 9

2. REVIEW OF LITERATURE

Tomato is the most popular vegetable in the world because of its taste, colour and

high nutritive value and also for its diversified use. There are many factors involved in

low yield of tomato; among them are infestations by fungi, bacteria, nematodes or viruses

and the competing weeds are predominant. The most urgent need is to develop biocontrol

agents and varieties of tomato that can resist the ravage of important fungal diseases like

early blight, late blight and wilt because, crop rotation, breeding for resistant plant

varieties and application of pesticides are insufficient to control. Hence earlier research

reports relevant to present attempt are collected and presented here.

2.1. Isolation and Characterization of Pathogen

In the vast and interesting history of crop cultivation, before the dawn of the 20th

century, there is little question that the work of Charles Darwin and Gregor Mendel

created the scientific foundation for plant breeding that led to its explosive impact over

the past 150 years (Aziz, 2009). Lycopersicon esculentum (tomato) is one of the

important "protective foods" both because of its special nutritive value and widespread

production. It is the world's largest vegetable crop after potato and sweet potato, but it

tops the list of canned vegetables (Babu et al., 2004).

The Lycopersicon esculentum (Tomato) has been a good model plant to analyse

plant pathogen interactions and its prospects for the future are promising. Tomato is one

of the most popular vegetables worldwide however; its cultivation has been limited by an

abundant attack of pathogens. In order to establish effective control methods; analysis of

tomato pathogen interactions are important (Tsutomu et al., 2007).

A leaf blight disease was first observed in the field of tomato during February 2004

at NIAB, (Nuclear Institute for Agriculture and Biology, Faisalabad), Pakistan.

Symptoms on affected plants started with yellowing and browning of the lower leaves,

progressing upwards under high humidity conditions. Symptoms often developed from

the leaf tips and along the margins of the leaf petiole. Under severe infection, lesions

enlarged and coalesced causing blighting of the leaves (Akhtar et al., 2004). This disease


produces brown to black, target-like spots on older leaves that may coalesce into larger

lesions. Affected leaves turn yellow then wilt, leaving the fruit exposed to sunburn. If

severe, the fungus also attacks stems and fruit. Fruit shows freckles, spots or lesions;

often the spots are not evident until a few days after the fruit is harvested.

Two distinct phenotypes of Alternaria alternata are known to cause a brown spot

disease on young leaves and fruits of certain cultivars of citrus fruits. Both phenotypes

produced host specific toxins (HSTs) in spore germination fluids and in liquid culture.

The HST may be useful to test for susceptibility of prevalent citrus cultivars without

risking the introduction and spread of the pathogen. This might also be useful in plant

breeding (Kohmoto et al., 1991). Trichoderma harzianum showed dominance on contact

over A. alternata at all testing temperatures and water activities tested except at 0.95 aW

and 15° C, at which T. harzianum inhibited A. alternata at a distance. Temperature and

water activity significantly influenced fungal growth rate (Sempere and Satamarina,

2007).

The main effect of early blight was premature defoliation, which was linearly

related to the percentage of leaf area showing symptoms. As inoculum concentrations,

(conidia ml−1) increased from 6·2 to 11·5, the percentages of leaf area affected and

defoliation increased linearly. Four hrs of leaf wetness after inoculation were sufficient to

initiate the disease on plants. As wetness duration increased up to 24 hrs, there was an

increase in the percentage leaf area showing symptoms and in the percentage of

defoliation, but thereafter there was no significant increase in either parameter (Manjula

et al., 2004).

Scanning electron microscopy of wild beet and cotton leaves infected by an

aggressive isolate of Alternaria alternata revealed that conidiophores of the pathogen

emerged only from necrotic areas of leaf tissues. Sporulation occurred on leaves only

during periods of high relative humidity (>95%) and temperatures ranging from 20 to

28°C. Under low relative humidity (60% at 22-25°C), mycelium penetrated into internal

tissues of the leaf or emerged through the stomata. A less virulent isolate did not develop

surface mycelium on inoculated leaves; but sporulation was detected on the leaf veins.


Plants in several cotton fields adjacent to the diseased wild beet plants were also found to

be infected by the pathogen early in the growing season (Bashan and De Bashan, 2005).

Field trials were conducted during the rainy seasons of 1993 and 1994 for chemical

control of tomato leaf spot using a single spray. A fungicide mixture of carbendazim

0.05%+ mencozeb 0.2% was applied once in different treatments. The yield data were not

significantly different in 1994. Spray for 40 days in 1993 and 50 days in 1994 gave

maximum returns. The cost of treatment did not vary significantly (Chandra et al., 1998).

. 2.2. Isolation and characterization of Antagonist

Bacteria are the predominant inhabitants of newly expanded leaves, while yeasts

and filamentous fungi dominate later in the growing season (Kiran and Dilip, 2003). The

genus Pseudomonas has been heterogeneous since Migula first named it in 1894. He

designated and described the species associated with genus in 1895. They were

aggressive colonizers of the rhizosphere of various crop plants, and have broad spectrum

antagonistic activity against plant pathogens. Some exhibit antibiosis by producing

inhibitory compounds; that make them to be good candidates for seed inoculants and root

dips for biological control (Srivastava and Shalini, 2008)

The fast-growing rhizobacteria might out-compete fungal pathogens by

competition for carbon energy sources, which would provide a basis for biological

control. Fluorescent pseudomonads owe their fluorescence to an extracellular diffusible

pigment called pyoverdin or pseudobactin. Another pseudomonad siderophore,

pyochelin, has been identified as an antifungal antibiotic. Some unrelated compounds to

typical siderophores like phenazines, phloroglucinols, pyoluteorin, pyrrolnitrin, cylic

lipopeptides and HCN are produced by these bacteria (Ligon, 2000)

Bacteria are common residents of leaves. Some phyllosphere communities have

been found to consist of more than 78 bacterial species representing 37 bacterial genera

among which includes Pseudomonas spp., (Gwyan and Lindow, 1999). Fluorescent

Pseudomonas spp., equipped with multiple resistant mechanisms for phytopathogens and

plant growth promotion, are being used widely. They produce a variety of antibiotics,


chitinolytic enzymes, and growth promoting hormones, siderophores, HCN and catalase,

and can solubilize phosphorous (Yogesh et al., 2005). Colonization of plant root system

can lead to reduced pathogen attack, directly through production of antimicrobial

substances or competition for space, nutrients and ecological niches, and indirectly

through induction of systemic resistance (Harrllen et al., 2004).

Among different cell concentrations (1000, 2000, 3000, 4000 and 5000 μg/ml) of

Pseudomonas fluorescens used, maximum spore germination of fungus was inhibited at

4000 and 5000 μg/ml. The result indicated that all the strains of Pseudomonas

fluorescens presented a most significant value against Alternaria alternata and

Curvularia lunata (Rachana and Shalini, 2009). Siderophore-producing Pseudomonas

spp. lowered the maximum cell population of Shewanella putrefaciens; when the strains

were grown on fish muscle blocks at 0˚C (Gram and Melchiorsen, 1996).

A detailed understanding of plant immune function will result in crop improvement

for food, fiber and bio-fuel production (Jonathan et al., 2006). Among the PGPR strains

few demonstrated biological control activity, some exhibited antibiosis in- vitro towards

the pathogens but others did not, suggesting that the bio-control PGPR strains consists of

two groups: those that control disease by antagonism to the pathogens, and those that

control the disease by mechanisms that do not involve production of toxic compounds,

but through substrate or site competition or induced resistance (Wei et al., 1991).

Formulation of bio-control agents have been designed to promote their survival in

soil (Kohlmann et al., 1991) and colonization of the rhizosphere, effective disease

suppression (Temple et al., 2004; Wang et al., 2009). Mechanisms of biological control

are complex, however, and often cannot simply be explained by a single factor. Results

of previous studies on biological control of Fusarium wilt by Pseudomonas spp.

incarnation indicated involvement of mechanisms other than merely competition for iron

(Van Peer et al., 1991). Seed dressing and foliar application of the Pseudomonas

fluorescens effectively controlled leaf spot, rust and increased the yield of tomato in

greenhouse and field trials (Meena et al., 2003).


King's medium B used for the production of the antifungal metabolite from

Pseudomonas fluorescens was extracted by hexane. The extracted metabolite appeared as

one spot on TLC and was characterized by using infra- red spectrum (IR), nuclear

magnetic resonance (NMR), mass spectrum (MS) and elemental analysis. The compound

was considered as a derivative of phenazine-1-carboxamide PCN (zag1) (Hassanein et

al., 2009). The addition of 0.1mM FeCl3 to a defined culture medium induced the

bacterial epiphyte Pseudomonas fluorescens strain A506 to produce an antibiotic toxic to

the fire blight pathogen, Erwinia amylovora. Disease suppression by A506 has been

proposed to occur by the mechanism of competitive exclusion for site and nutrients

essential for epiphytic growth and subsequent infection of the host plant (Leeman et al.,

1996).

Molecular communication between plants and potential pathogens, determines the

ultimate outcome of their interaction. The direct delivery of microbial molecules into and

around the host cell, and the subsequent perception of these by the invaded plant tissue

(or lack thereof), determines the difference between disease and disease resistance. In

theory, any foreign molecule produced by an invading pathogen could act as an elicitor of

the broad physiological and transcriptional re-programmings, indicative of a plant

defense response (Nimchuk et al., 2003).

The production of defense enzymes upon challenge was higher in the non

bacterized plants compared to the bacterized plants, indicating the lesser requirement of

defense enzymes in the bacterized plants, upon encounter with the pathogen. There also

found a relatively higher quantity of lignification (30-100% over control) in the

bacterized roots compared to the plants untreated, which resulted in significant root rot

suppression (Leeman et al., 1995).

2.3. Synergistic study of plant extract as an alternative for carrier formulation

Commercial application of Pseudomonas fluorescens for control of diseases

depends upon development of commercial formulations in which the bacteria can survive

for a considerable length of time, development of suitable method of application to


control early and late stages of disease development, and assessment of field efficacy of

these formulations in the control of the disease, as well as increasing the yield (Satish et

al., 1999). Plant metabolites and plant based pesticides appear to be one of the better

alternatives as they are known to have minimal environmental impact and danger to

consumer in contrast to the synthetic pesticides (Verma and Dubey, 1999).

Among the estimated 250,000-500,000 plant species, only a small percentage has

been investigated phytochemically and the fraction submitted to biological or

pharmacological screening is even smaller (Mahesh and Satish, 2008). Purple nut sedge

(Cyperus rotundus) was identified as the world’s worst weed. In most of the countries, it

was reported as the serious principal or the common weed, competing with many major

crops (Dieter and Genevieve, 2005). Purple nut sedge is a problem throughout the

thermal belt. It is the weed of importance of 52 crops in more than 90 tropical and sub

tropical countries of the world. Low temperature and moisture are the two dominant

environmental factors limiting its distribution (Nandi et al., 2002).

Cyperus rotundus dies, becomes invisible, and is carried through extremely dry

periods by the tuber system which draws its water from a root system that may penetrate

deeply into the soil". It is a major weed of cultivated crops and of gardens, but only a

minor weed elsewhere. It is encouraged by frequent cultivation and grows best in moist

fertile soils, but does not grow well in shade. Prevalent in disturbed areas and lawns, very

persistent once established (Lay and Anderson, 2005).

It is also a common weed, for the rare Himalayan species, Ativisha (Aconitum

heterophyllum). The petroleum ether extract of the Cyperus rotundus roots showed anti-

inflammatory activity against carrageen in induced edema in albino rats. The active

fraction was identified as a triterpenoid. A fraction tested on aconitine-induced writhing

in mice showed mild analgesic activity. Extracts of rhizomes were inhibitory to the

growth of fungi depending on species. Antibacterial activity of oil and its fractions from

this weed have been demonstrated against a number of organisms (Bateman, 2005).


Antioxidant activity of Cyperus rotundus rhizomes extract (CRRE) was evaluated

in a series of in- vitro assay involving free radicals and reactive oxygen species and IC50

values were determined. CRRE exhibited its scavenging effect in concentration

dependent manner on superoxide anion radicals, hydroxyl radicals, nitric oxide radical,

hydrogen peroxide, and property of metal chelating and reducing power (Nagulendran et

al., 2007). A new antimicrobial sesquiterpenoid from Cyperus rotundus, 8α-

geloyloxycichoralexin; in addition cichoralexin and 10α-hydroxycichopumilide, were

isolated and identified. These sesquiterpenoids exhibited antifungal activities against

Pericularia oryzae, Pellicularia sasakii, Alternaria kikuchiana, and others (Lay and

Anderson, 2005).

The Cyperus rotundus rhizome meal affected the mycelial growth and sporulation

of all the tested fungi, viz. Alternaria solani, Aspergillus spp., Colletotrichum spp.,

Curvularia pallescens, Fusarium udum, Helminthosporium spiciferum, Heterosporium

spp., Leptoxyphium axillatum, Mucor spp. and Penicillium spp. The, sporulation was

stimulated in Alternaria solani, Curvularia pallescens, Helminthosporium speciferum,

Leptoxyphium axillatum and Mucor spp., at 1 and 2% concentrations, however, at higher

concentrations (3%, 4% and 5%), sporulation was reduced. (Maurya et al., 2006).

Parthenium hysterophorus a native of tropical and subtropical America, is the most

recent invader in Kathmandu valley. It has already threatened grassland ecosystems of

Australia and India to a large extent (Stephen and Sowerby, 1996). The allelopathic effect

of Parthenium hysterophorus, coupled with the absence of natural enemies like insects

and diseases, are responsible for its rapid spread in introduced ranges. Growth inhibitors

like lactones and phenols are released from this plant into the soil through leaching,

exudation of roots and decay of residues. These growth inhibitors suppress the growth

and yield of native plants. Parthenium absorbs nutrients even from nutrient deficient soil

and in cropped land resulting in reduction of yield up to 40% (Rajagopal et al., 2009).

Huge amount of locally available Parthenium hysterophorus can be utilized, as a source

of organic matter to prepare its compost resulting, we can control its exotic weed and

sustain the soil health (Kumar et al., 2003)


Parthenium hysterophorus is reported to have insecticidal, nematicidal and

herbicidal properties. It is also used for composting. The odor of the plant is apparently

disagreeable to bees and they can easily kept be away by carrying a handful of

Parthenium hysterophorus flower heads. A root decoction of the plant is used in treating

amoebic dysentery. Parthenin is also found to be pharmacologically active against

neuralgia and certain types of rheumatism. In the Caribbean and Central America,

Parthenium is applied externally on skin disorders and a decoction of the plant is often

taken internally as a remedy for a wide variety of ailments. In Jamaica, the decoction is

used as a flea-repellent, both for dogs and other animals (Schneider and Keith, 1989).

Parthenium hysterophorus can be used as a bio-herbicide but still needs extensive

study, to fully explore its potential against different summer and winter weeds (Khan and

Zaidi, 2002). The growth of all the three test pathogenic species were generally inhibited

by lower concentrations viz. 10, 20, 30 and 50% of the Parthenium extracts while

aqueous extracts of higher concentrations (60 and 70%) stimulated biomass production of

test fungal species (Ruksana et al.,, 2004).

The combined effect of N through Parthenium compost (PCN) and urea (U) along

with Azotobacter chroococcum on growth and yield of Triticum aestivum L., revealed

that 100% N through Parthenium compost is detrimental to wheat. Judicious use of 50%

N through each of Parthenium compost and urea along with Azotobacter chroococcum

was found to be beneficial for better growth and higher yields of wheat. Increasing

temperature of compost pit could not destroy 100% viability of Parthenium seeds (Kishor

et al., 2005).

The majority of the small scale farmers are reluctant to leave chemical fertilizers;

because of the unassured crop returns owing to the high incidence of fungal diseases and

unpredictable terminal drought. In this context, there is greater scope for development

and popularization of bioinoculants in ground nut and tomato cultivation (Kishore et al.,

2005).


2.4. In vivo study for host response and effective delivery system

Beneficial effects of the introduction of specific microorganisms on plant growth

have been reported for numerous crops, including tomato (Lycopersicon esculentum)

grown under field or green house conditions in organic media (Guo et al., 2004). Seed

and root exudates of tomato, grown in solarized soil contained lower amounts of sugars

and higher amounts of amino acids, which were less favorable for growth of bacteria and

fungi in culture, compared with seed and root exudates of tomato grown in nonsolarized

soil. Addition of exudates from germinating seeds into solarized soil, however, increased

populations of fluorescent pseudomonads and decreased populations of fungi compared

with nonsolarized soil. Establishment of fluorescent pseudomonads in the rhizosphere of

plants in solarized soils was due to an improved capacity of these bacteria to compete for

exudates (Gamliel et al., 1992).

Pseudomonas fluorescens strain 134 delivered as both dry and liquid formulations

was able to colonize cotton root at a population density of about 10(8) CFU/g fresh root,

15 days after sowing (Kumiko et al., 2002). Seed treatment, followed by foliar

application of talc formulated bio-control agent’s triggered systemic resistance in

Lycopersicon esculentum (tomato). As a response of ISR, the activities of defense related

enzymes viz., phenylalanine ammonia lyase (PAL), peroxidase (PO), polyphenol oxidase

(PPO) and β-1-3-glucanase were enhanced and the accumulation of phenols were also

noticed in the tomato upon challenge inoculation with Alternaria alternata, the causal

agent for leaf blight in Lycopersicon esculentum (Chandrasekharan and Kamat, 2000).

Pseudomonas fluorescens strain (PfT-8) was capable of producing high levels of

chitinase, β-1,3-glucanase, cellulase, fungitoxic metabolites and siderophores. Seven

different carrier formulations including a talc-based powder, lignite-based powder, peat-

based powder, lignite + fly ash-based powder, wettable powder, bentonite-paste and

polyethylene glycol (PEG) paste were prepared utilizing PfT-8. Shelf life was evaluated

for up to 6 months of storage at ambient room temperature (28°C).


Among the formulations, peat, lignite, lignite + fly-ash and bentonite paste based

formulations maintained higher propagule number than others and also showed greater

biocontrol potential (Arora, 2006).

Efficient strains of Pseudomonas fluorescens identified were evaluated for their

efficacy in inducing defense enzymes in black pepper. Increased levels of peroxidase

(PO), catalase, phenylalanine ammonia lyase (PAL) and poly phenol oxidase (PPO) were

induced in leaves, apart from the roots of treated plants indicating the systemic protection

offered to black pepper, by the strains exploring the prevention of even foliar infection by

the pathogen, Phytophthora capsici (Paul and Sharma, 2006).

Activities of phenylalanine ammonia-lyase (PAL), Peroxidase (PO) and polyphenol

oxidase (PPO) increased in bacterized tomato root tissues challenged with the pathogen

after one day and the activities of PAL and PO reached maximum at the 4th day while

activity of PPO reached maximum at the 5th day after challenge inoculation (Pieterse et

al., 1998). Similarly, β-1,3 glucanase, chitinase and thaumatin-like proteins (TLP) were

induced to accumulate at higher levels betwen 3-5 days of challenge inoculation in

bacterized plants. Western blot analysis showed that, chitinase isoform Chi2 with a

molecular weight of 46 kDa was newly induced due to Pseudomonas fluorescens isolate

Pf1 treatment challenged with the pathogen. The induction of defense enzymes involved

in phenylpropanoid pathway, accumulation of phenolics and PR-proteins might have

contributed to restriction of invasion by Fusarium oxysporum f. sp. lycopersici in tomato

roots (Ramamoorthy et al., 2002).

The effects of tomato seed treatments with Pseudomonas fluorescens in the control

of bacterial wilt under greenhouse conditions revealed that, the treatment protected plants

against soil-borne infections of the bacterial wilt organism. Seed treatment with

antagonistic Pseudomonas fluorescens strain, significantly improved the quality of seed

germination and seedling vigour. The disease incidence was significantly reduced in

plants raised from Pseudomonas fluorescens treated seeds, followed by challenge

inoculation with Ralstonia solanacearum (Vanitha, 2009). Pseudomonas fluorescens

controlled downy mildew disease both by seed treatment and foliar application, but


efficacy was significantly higher when seed treatment was followed by a foliar

application. Seed treatment was better than foliar application alone (Manjula and Podile

2005).

The most effective treatment for controlling all tested pathogens was seed coating

(Singh and Mehrotra, 1980). A marked increase in shoot and root length was observed in

Pseudomonas fluorescens treated plants. The isolates elicited systemically induced

resistance against fusarium wilt of chickpea caused by Fusarium oxysporum f.sp. ciceri

(FocRs1), and significantly (P = 0.05) reduced the wilt disease by 26-50% as compared to

control (Saikia et al., 2003). The lack of colonization of tomato leaves by strain 89B61

suggests that the observed induced systemic resistance (ISR) was due to systemic

protection by strain 89B61 and not attributable to a direct interaction between pathogen

and biological control agent (Anfoka and Buchenauer, 1997).

Four isolates of Pseudomonas fluorescens, GB 4, GB 8, GB 10 and GB 27, and

Trichoderma viride pq 1 were identified as potent antagonists of Sclerotium rolfsii.

Trichoderma viride pq 1 produced extracellular chitinase and parasitized the mycelium of

Sclerotium rolfsii. Under controlled environment conditions, Pseudomonas fluorescens

GB10, GB 27, Trichoderma viride pq 1 and the systemic fungicide Thiram reduced the

mortality of Sclerotium rolfsii inoculated to bean seedlings by 58.0%, 55.9%, 70.0% and

25.9%, respectively compared to control (Zdor and Anderson, 1992). In vitro growth of

Pseudomonas fluorescens GB 10 and GB 27 was compatible with Trichoderma viride pq

1 and Thiram. Integrated use of these two bacterial isolates with Trichoderma viride pq 1

or Thiram improving their bio-control efficacy. Combined application of either GB 10 or

GB 27 with Trichoderma viride pq 1 was significantly effective than that with Thiram in

protecting groundnut seedlings from stem rot infection (Manjula and Podile, 2001).

Growth and efficacy of two biological control strains, Pseudomonas fluorescens P-

5 and P-6, were evaluated in combinations of two carbon (sucrose & molasses) and two

nitrogen (urea & yeast extract) sources to optimize control of Rhizoctonia solani, which

is a causal agent of bean damping-off. In greenhouse conditions, the influence of the

media on the biocontrol efficacy of P-5 and P-6 was the same and Pseudomonas


fluorescens P-6 in molasses + yeast extract (in two different ratios) reduced the severity

of disease from 90.9% to less than 28 % (Johri et al., 2003).

Pseudomonas fluorescens F113, which produces the antimicrobial compound 2,4-

diacetylphloroglucinol and inhibits soil-borne phytopathogenic fungi, might also affect

non-target resident microorganisms involved in the cycling of soil nutrients. Analyses of

soil nutrients and crop foliage were used to assess possible residual effects of the

pseudomonad on soil fertility. The F113Rif treatment had no effect on the 20 foliage

parameters studied except a small (15%) transient decrease in foliage chlorine at the first

harvest. EUF soil analysis and foliage analysis will be useful in future risk assessment

studies related any biocontrol agent (Markus and Christoph, 2001).

The culture conditions like inoculum concentration, incubation time, pH,

temperature, carbon sources, nitrogen sources and metal ions were optimized. The

optimum conditions found for protease production was 37°C at pH 9, with 2% inoculum

in the medium for 24 h. Wheat bran and peptone stimulated the production of protease.

Magnesium sulphate was less inhibitory among the metal ions tested (Kalaiarasi and

Sumita, 2009). Pseudomonas aeruginosa was less effective in production of indole acetic

acid than Pseudomonas fluorescens. Inoculation of rice seeds with Pseudomonas

fluorescens and Pseudomonas aeruginosa showed a good level(2.30 pmol/ml and 2.1

pmol/ml) of indole acetic acid compared to uninoculated seeds (1.6 pmol/ml) (karnwal,

2009).

Pseudomonas fluorescens 2-79RN10 produced the antibiotic phenazine-1-

carboxylic acid (phz+), and was a bio-control of take-all of wheat caused by

Gaeumannomyces graminis. A positive relationship between root colonization by 2-

79RN10 and bio-control of take- all was demonstrated. Phenazine-1- carboxylic acid was

a major factor in suppression of G. graminis during primary infection of roots because

strain, which is Phenazine deficient, failed to suppress lesion formation (Cornelina et al.,

2005).


Pretreatment of radish (Raphanus sativum) through induction of systemic resistance

not only against the fungal root pathogen Fusarium oxysporum f. sp. raphani, but also

against the avirulent bacterial leaf pathogen Pseudomonas syringae pv. Tomato and the

fungal leaf pathogens Alternaria brassicicola and Fusarium oxysporum (Hoffland et al.,

1996). Among the 50 isolates of Pseudomonas fluorescens only three i.e., Pf S2, Pf Wt3

and PfW1 showed inhibition against the growth of the pathogen. Bacterization of tubers

with isolates Pf S2, Pf Wt3, and PfW1, significantly reduced 59.83% incidence of

bacterial wilt, compared to the pathogen-inoculated control and also increased plant

growth (plant height and dry weight) by 59.83%, 76.89% and 28.44%, respectively. This

suggested the importance of the studied isolates as plant growth-promoting rhizobacteria

(Kiran and Dilip, 2003).

When chickpea seeds were treated with talc-based formulations, Pseudomonas

fluorescens survived on the seeds for at least 180 days. When the treated seeds were sown

in soil, the antagonist moved to the rhizosphere and survived well in it. Biopriming of

seeds increased rhizosphere population (Rao et al., 2007). The use of 1/10 strength

nutrient broth-yeast extract, compared with standard nutrient broth-yeast extract,

amended with glucose and/or glycerol resulted in dramatically increased accumulation of

PHL, pyochelin and salicylic acid, indicating that the ratio of carbon source to nutrient

concentration played a key role in metabolic flow (Brion et al., 1999; Srivastava et al.,

2008).

2.5. Induction of systemic resistance using SA, challenge inoculation and disease

symptoms

During ISR, colonization with rhizobacteria leads to an enhanced expression

(Priming) of jasmonate-inducible genes. With the help of various biosynthesis and

perception mutants for plant signal molecules, it was possible to demonstrate that ISR

requires functional jasmonate and ethylene signaling. N-acyl-L-homoserine lactone

(AHL) signal molecules are utilized by Gram-negative bacteria to monitor their

population density (quorum sensing) and to regulate gene expression in a density-

dependent manner (Van Loon and Bakker, 2003; Pieterse et al., 1998).


New insights into the phenomenon of systemic acquired resistance have been

gained in recent years, by the use of techniques in molecular genetics and biology that

have replaced the largely descriptive approach of earlier work (Esmaeilzadeh et al.,

2008). A detailed understanding of plant immune function will underpin crop

improvement for food, fibre and biofuels production (Jonathan et al., 2006).

Siderophores, including salicylic acid, pyochelin and pyoveridine, which chelate

iron and other metals, also contribute to disease suppression by conferring a competitive

advantage to bio-control agents for the limits in to the supply of essential trace minerals

in natural habitats. Antibiotics and siderophores produced by Pseudomonas fluorescens

act as stress factors or signals including local and systemic host resistance (Brion et al.,

1999).

The action of proteinase inhibitors from plants upon the enzymes from pathogenic

microorganisms and viruses was reviewed by Chang et al., 2007. According to them

induction of proteinase inhibitors in plants, in response to the invasion of pathogens is an

important plant protection mechanism; based on which, transgenic plants with enhanced

resistance to diseases were under scanner. HCN synthase gene diversity may reflect the

adaptive radiation of HCN+ biocontrol fluorescent pseudomonads. Positive correlations

were found between HCN production in vitro and plant protection in the

cucumber/Pythium ultimum and tomato/Fusarium oxysporum f. sp. radicis-lycopersici

pathosystems in vivo (Mayer et al., 2002).


The importance of SA in rhizobacteria-mediated suppression of root-knot

nematodes, biocontrol potential of SA-negative or SA-overproducing mutants against

Meloidogyne javanica was evaluated with their respective wild type counter parts.

Culture supernatant of 7NSK2, CHA0 and their respective mutants caused significant

mortality of Meloidogyne javanica juveniles in vitro. In pot experiments, the bacterial

strains applied in unsterilized sandy loam soil markedly reduced final nematode

population densities in roots and subsequent root-knot infection in tomato seedlings

(Siddiqui and Shoukat, 2005).

Selected strains of nonpathogenic rhizobacteria can induce a systemic resistance in

plants, that is effective against various pathogens. In bean plants, the determinants of the

rhizobacterium Pseudomonas aeruginosa 7NSK2, important for induction of resistance to

Botrytis cinerea were investigated. By varying the iron nutritional state of the bacterium

at inoculation, it was demonstrated that induced resistance by Pseudomonas aeruginosa

7NSK2 was iron-regulated (Pierson and Weller, 1994).

Most of the secondary metabolites produced by PGPR Pseudomonas fluorescens

have completely arrested conidial germination and mycelial growth of the fungus. The

results suggest that the metabolite production may play a role in antagonism/induced

systemic resistance against the pathogen (Khan and Zaidi, 2002). The systemic induced

susceptibility (SIS) is in direct contrast to the well studied avirulence/R gene-dependent

resistance response known as the hypersensitive response that elicits systemic acquired

resistance. It has been found that Pseudomonas syringae-elicited SIS is caused by the

production of coronatine (COR), a pathogen-derived functional and structural mimic of

the phytohormone jasmonic acid (JA) (Jigna and Sumitra, 2008)

The effect of salicylic acid by foliar application at the concentration of 1 mM

significantly reduced the leaf blight disease intensity and increased the tomato yield

under greenhouse conditions. Changes in the activities of phenyl alanine ammonium

lyase, chitinase β-1, 3 glucanase and in phenolic content on tomato after application of

salicylic acid and inoculated with Alternaria alternata were studied. In salicylic acid

treated leaves an increase in phenolic content was observed 5 days after challenge


inoculation with Alternaria alternata in tomato plant (Chitra et al., 2006). Root feeding

200µM SA to tomato plant can (1) induce PR-IB gene expression, (2) significantly

elevate the foliar SA levels and (3) activates SAR that is effective against Alternaria

solani (Spletzer and Enyedi, 1999).

SA may serve as an endogenous signal molecule required for SAR expression. SA

may also specifically inhibit the catalase activity of certain SA-binding protein, resulting

in an increase of H2O2, which in turn induces the expression of genes associated with

SAR (Leeman et al., 1996). Pseudomonas fluorescens bacterium produces three

siderophores under iron-limiting conditions (pyoveridine, pyochenine and salicylic acid)

and can induce resistance to plant diseases caused by Botrytis cinerea on bean and

tomato. In Pseudomonas aeruginosa, SA is produced from chorismate via the shikimate

biosynthesis pathway (Kris et al., 2002). Evidence for the involvement of bacterial SA in

ISR is still circumstantial (Meyer et al., 1992).

Well-characterized examples of SAR have been recorded in both dicotyledonous

and monocotyledonous plants like increased glucose and fructose concentrations in

systemic tissue, and accumulation of fungi toxic β-ionone derivatives, induction of

lipoxygenase, antimicrobial fatty acid derivatives, phenylalanine ammonia lyase,

phytoalexins and hydroxyl-proline rich glycoprotein (Glyn, 2001; Leeman et al., 1995).

SA injected into stems, sprayed onto the entire plant or applied to the roots is a

potent inducer of systemic resistance and pathogenesis related proteins, therefore SA

produced by PGPR in the rhizosphere may be involved in ISR. SA produced by

Pseudomonas aeruginosa was important in the induction of systemic resistance against

Bacillus cinerea in bean (Maurehofer et al., 1998). The effect of salicylic acid was

examined in a split root system of Lycopersicon esculentum; where one half of the

seedling root system was drenched with salicylic acid and the activation of defense

responses was measured subsequently on remaining roots. Salicylic acid treated plants

exhibited enhanced systemic resistance with a significant reduction in disease severity

when compared to untreated plants (He and wolyn, 2005).


The activation of plant defense by salicylic acid production was mimicked by

applying nanograms amounts of exogenous salicylic acid to been roots. These results

clearly demonstrate that the bacterial production of SA by Pseudomonas fluorescens

leads to activation of SA-dependent defense response in plants (Kris et al., 2002).

Salicylic acid (SA) levels were increased in leaves when AHL-producing bacteria

colonized the rhizosphere. No effects were observed when isogenic AHL-negative

mutant derivatives were used in these experiments (Gardner et al., 2005).

It is well documented that treatment of plants with various agents (e.g. virulent or

avirulent pathogen, nonpathogens, cell wall fragments, plant extracts and synthetic

chemicals) can lead to the induction of resistance to subsequent pathogen attack, both

locally and systematically.


Resistance induced by these agents is broad spectrum and long lasting, but rarely

provides complete control over the infection with many resistance elicitors providing

between 20 and 85% disease control. In the field, expression of induced resistance is

likely to be influenced by the environment, genotype and crop nutrition (Walters et al.,

2005).

2. review of literature - shodhgangashodhganga.inflibnet.ac.in/bitstream/10603/9638/11/11_chapter...

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