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Application of molecular

biology to conventional disease

strategies

Paper : Assignment

Supervisor:- Dr. Roopam Kapoor

Submitted by:- Satya Prakash

M.Phil. (Botany), 2013-14

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CONTENTS: Pg. no.

1. Introduction 3

2. Resistance breeding 3

2.1 Artificial selection 4

2.2 Hybridization 4

2.3 Polyploidy 4

2.4 Induced Mutations 4

3. Solutions to problems associated with plant breeding: 5

3.1 Tissue Culture 5

3.2 Marker- Assisted Breeding 6

3.2.1 Advancement: QTL-Mapping 9

3.2.2 Identification and application of resistance genes 10

4. Use of chemicals for disease control 11

4.1 Fungicides 12

a) Negative impacts of fungicide on the membrane 12

b) Effects on amino acid and protein synthesis 13

c) Effects on signal transduction 13

d) Effects on respiration 13

e) Effects on mitosis and cell division 13

f) Effects on Nucleic Acids Synthesis 13

4.2 Antiboitics 14

4.3 Nematicides 14

4.4 Implementation of molecular biology to design chemical structures 15

(agrochemicals) with increased effectiveness

Use of elicitors 15

Induction of systemic defense 15

4.5 Chemical immunization of plants against diseases 16

5. Summary 17

6. References 14

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Application of molecular biology to conventional disease strategies

1) Introduction:

As resistance to disease in plants is genetically controlled, molecular tools like breeding

resistant cultivars has been an intensively used approach for crop protection since near beginning

of human civilization, the time when we did not know its molecular aspects. Even today,

molecular biology is applied in multiple ways to control plant diseases. Some of which are

breeding, tissue culture, marker assisted breeding, QTL- mapping, identification of novel

resistance genes etc. With the commencement of advanced technologies in the recent past, we

are now able to genetically modify a plant without wasting a lot of time and avoiding problems

of sexual incompatibility which we encounter in breeding programs.

Any problem with a plant that causes a reduction in yield or appearance is called plant disease.

Plant diseases caused by biotic agents like viruses and bacteria are a threat to the food security of

developing countries, causing serious crop and income losses for people whose livelihoods

depend on farming. Demand for food is influenced by a number of forces, including population

growth, income levels, urbanization, lifestyles, and preferences. Almost 80 million people are

likely to be added to the world's population each year during the next quarter century, increasing

world population by 35 percent from 5.7 billion in 1995 to 7.7 billion by 2020 ( UN 1996). More

than 95 percent of the population increase is expected in developing countries, whose share of

global population is projected to increase from 79 percent in 1995 to 84 percent in 2020. Over

this period, the absolute population increase will be highest in Asia. With such rapidly increasing

human population and simultaneous rise in food requirement, scientists across the world are

aimed to increase yield percentage by several ways including reduction of loss in net output due

to plant diseases.

Modern science offers humankind a powerful instrument to assure food security for all. Through

enhanced knowledge and better technologies for food and agriculture, science has contributed to

astonishing advances in feeding the world in recent decades. If we are to produce enough food to

meet increasing and changing food needs, we must put all tools of modern science to work.

In this assignment, some of these tools of molecular biology are discussed with examples. The

cost associated with technologies and the possible solutions have also been discussed in brief.

2) Resistance breeding:

Plant breeding is the science of adapting the genetics of plants for the benefit of humankind. The

overall aim of plant breeding is to improve the quality, performance diversity of crops with the

objective of developing plants better adapted to human needs. This can be accomplished in

several ways including selection and propagation of superior individuals from the field, crossing

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different varieties to get improved one, use of tissue culture to get superior varieties, introduction

or modification of resistance gene/s in plant concerned etc.

2.1) Artificial selection:

As mentioned above, this kind of breeding approach is been in use since the time human started

agriculture. Many elite crop varieties that we see today are the result of thousands of years`

breeding practices. The art of recognizing desirable traits and incorporating them into future

generations is very important in plant breeding. Breeders scrutinize their fields and travel long

distances in search of individual plants that exhibit desirable traits. It is regarded as environment

friendly crop management.

Advantages:

a) We get cultivar with improved desirable trait. b) Resultant varieties are stable and uniform. c) No or reduced use of agrochemicals. d) Require no expertise.

e) Advanced technologies not needed.

Disadvantages:

a) It narrows genetic diversity.

b) Lack of genetic diversity favors spread of new pathogens and epidemic development.

c) Since focus stays on specific gene/s, other important gene/s may be lost in the process.

d) Relatively complex and time consuming method.

2.2) Hybridization:

The most frequently employed plant breeding technique is hybridization. The aim of

hybridization is to bring together desired traits found in different plant lines into one plant line

via cross- pollination.

Steps:

a) Generation of homozygous inbred lines.

b) Outcrossing

c) Progeny selection

d) Backcrossing

A homozygous inbred line is generated by continuous episodes of self-pollination until a pure

line is achieved. This step is followed by crossing of this pure line with another such kind of

inbred line. Thus, traits of two inbred lines are combined in one. However, not all the progenies

carry desired combinations of traits and hence, a selection procedure is required.

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Demerits:

a) If a trait from a wild relative of a crop species, e.g. resistance against a disease is to be

brought into the genome of the crop, a large quantity of undesired traits (like low yield, bad

taste, low nutritional value) are transferred to the crop as well. These unfavorable traits must

be removed by time-consuming back-crossing, i. e. repeated crossing with the crop parent.

b) Potential of hybridization is limited by sexual incompatibility between distant groups of

plants.

Merit:

a) Combining two highly inbred lines often result in heterosis i.e improved or increased

function of any biological quality in a hybrid offspring.

2.3) Polyploidy:

Ploidy is the number of sets of chromosomes in the nucleus of a biological cell. Most plants are

diploid. Plants with three or more complete sets of chromosomes are common and are referred to

as polyploids. The increase of chromosomes sets per cell can be artificially induced by applying

the chemical colchicine, which leads to a doubling of the chromosome number. Generally, the

main effect of polyploidy is increase in size and genetic variability.

Demerit: Polyploids often have lower fertility and grow more slowly.

2.4) Induced Mutations:

Mutations can be induced with the application of mutagens like radiation and mutagenic

chemicals. Instead of relying on wild relatives, we can induce mutations and look for desired

traits. This saves time and intensive work of backcrossing.

Demerits:

a) Mutagens cannot induce mutation at specific sites only.

b) Mutants often carry undesirable traits.

3) Solutions to problems associated with plant breeding:

3.1) Tissue culture:

As mentioned above, a very strong tool of plant breeding, hybridization is limited by phenomena

of sexual incompatibility making it very difficult to transfer genes from an incompatible plant to

commercial species. Tissue culture has opened up ways to alleviate such problems with

conventional breeding programmes. Protoplast fusion can be used for plants that might be

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impossible to cross by classical breeding methods and can be a way to incorporate resistance

genes. These plants then can be regenerated and taken through backcrossing programmes.

Added advantage: Tissue culturing has been shown to result in greater variability in progeny

plants, a phenomenon known as somaclonal variation. These variants often show more resistance

than their progenitors. For example, in experiments where plants were generated in tissue culture

from a line of potatoes susceptible to phytophthora infestans and Alternaria solani, 2.5 % were

found to be resistant to P. infestans , and 1% to A. solani .

3.2) Marker-assisted breeding:

Major problems with conventional breeding programmes include the time taken between

generations and the need to test with pathogens to select for the resistant progenies after each

cross. It becomes more problematic when pathogen only infects mature plants, or is difficult to

work with and maintain, perhaps because it is not an indigeneous organism and needs to be

maintained under strict license constraints.

This problem led to implementation of marker-assisted breeding strategy. Marker-assisted

breeding combines classical plant breeding with the tools and discoveries of molecular biology

and genetics, most specifically the use of molecular markers. A marker, in this context, is an

identifier (sometimes called a “tag”) of a particular aspect of phenotype and/or genotype; its

inheritance can easily be followed from generation to generation. Thus, marker used in a

breeding programme may be morphological (e.g. flowering time), biochemical (e.g. isozymes) or

molecular (e.g. microsatellites).

The following four stages should be followed carefully during MAB programmes aiming at the

introgression of stress resistance:

I. Identification of marker trait Association for stress resistance using the concept of

linkage mapping

II. Validation of marker trait association in different genetic background using cross

breeding population

III. Test the usefulness of marker in characterization core germplasm

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IV. Implementation of the validated marker in the breeding programmeme

Prepare simple genotyping protocol (marker assay) for the

marker detection and its allele size

↓

Provide the marker data along with allelic information to the

molecular breeders for the MAB for stress resistance

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Strategy:

Figure 1

The rationale behind using molecular markers in breeding programmes. M is a molecular marker tightly linked to

the resistance gene (see text for details). Following each cross, progeny containing the resistance gene are selected

by pathogen screens or by detection of the tightly linked marker (M) if using marker-assisted breeding. ( Source:

M.Dickinson (2005) Molecular Plant Pathology. BIOS Scientific Publishers, New York, USA).

Gao et.al, 2013, did Marker-assisted breeding for rf1, a nuclear gene controlling A1 CMS in

sorghum (Sorghum bicolor L. Moench). MAS study was conducted on the offspring population of

two crosses between a maintainer line, BTx-622, and two sweet sorghum lines, BJ-299 and Lunen-2, to

test the effectiveness of the MAS method and develop maintainer lines with sweet and juicy stalks and

corresponding cytoplasmic nuclear male sterility (CMS) lines. The simple sequence repeat marker

Xtxp18 exhibited a high accuracy (95.098 %) for selecting recessive homozygotes for the rf1 gene. The

segregation ratio matched the expected ratio calculated according to the reported genetic distance in

the F2 population of the two crosses used. Finally, four excellent maintainer lines/CMS line pairs

(F5/BC3) with high stalk juice and stalk juice sugar contents were developed. The MAS method based on

Xtxp18 for the sorghum rf1 gene could be used for hybrid breeding programs at a low cost in the future.

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3.2.1) Advancement: QTL-Mapping

Quantitative trait locus (QTL)-Mapping is one of the new techniques adopted to improve plant

breeding methodologies. Many important traits for crop improvement are quantitative (e.g.

yield). This technique allows us to monitor multiple locus instead of relying on single gene

breeding. This mapping approach is a highly effective approach for studying genetically complex

forms of plant disease resistance. With QTL mapping, the roles of specific resistance loci can be

described, race-specificity of partial resistance genes can be assessed, and interactions between

resistance genes, plant development, and the environment can be analyzed. Outstanding

examples include: quantitative resistance to the rice blast fungus, late blight of potato, gray leaf

spot of maize, bacterial wilt of tomato, and the soybean cyst nematode. These studies provide

insights into the number of quantitative resistance loci involved in complex disease resistance,

epistatic and environmental interactions, race-specificity of partial resistance loci, interactions

between pathogen biology, plant development and biochemistry, and the relationship between

qualitative and quantitative loci. QTL mapping also provides a framework for marker-assisted

selection of complex disease resistance characters and the positional cloning of partial resistance

genes.

Hatakeyama et,al ,2012, Identified and Characterized Crr1a, a Gene for Resistance to Clubroot

Disease (Plasmodiophora brassicae Woronin) in Brassica rapa L. Clubroot disease, caused by the

obligate biotrophic protist Plasmodiophora brassicae Woronin, is one of the most economically

important diseases of Brassica crops in the world. Although many clubroot resistance (CR) loci

have been identified through genetic analysis and QTL mapping, the molecular mechanisms of

defense responses against P. brassicae remain unknown. Fine mapping of the Crr1 locus, which

was originally identified as a single locus, revealed that it comprises two gene loci, Crr1a and

Crr1b.

Q. How Is QTL Analysis Conducted?

o The very first requirements are two or more varieties of a plant that differ genetically with

regard to trait of interest (e.g. plant height). These are termed parental lines.

o Second, researchers also require genetic markers that distinguish between these parental

lines. Common markers used are single nucleotide polymorphisms (SNPs), simple

sequence repeats (SSRs, or microsatellites), restriction fragment length polymorphisms

(RFLPs), and transposable element positions.

o Cross between parental lines gives heterozygous individuals (F1).

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o F1 individuals are then crossed using one of the different schemes.

o Finally, the phenotypes and genotypes of the derived (F2) population are scored. Markers

that are genetically linked to a QTL influencing the trait of interest will segregate more

frequently with trait values, whereas unlinked markers will not show significant association

with phenotype.

3.2.2) Identification and application of resistance genes:

Molecular genetics is the boon helping with the identification of novel resistance genes and

aiding in breeding of resistant cultivars.

Strategies:

a) Cloned resistance genes and primers to the conserved regions of resistance genes can be

used in PCR to identify similar genes in wild relatives that might be potentially useful.

Homologues of mlo resistance genes have been identified in a number of plant

species.

b) Identification of proteins required for full pathogenicity and then their transient

expression in a wild crop to see hypersensitive response . The gene(s) responsible for

this response can then be transferred to commercial cultivars.

Transient expression of extracellular fungal protein EPC2, required for full

pathogenicity in tomato genotype. A dominant gene, the Cf-ECP2 gene was

identified and transferred to commercial cultivars. ( EPC2 expressing PVX system

can be used at each step of breeding and backcrossing programme)

c) DNA shuffling techniques can also be used. This involves random generation and testing

of mixed sequences from resistant genes and sequences from such genes and

identification of combination that gives best results.

There is therefore, enormous potential for molecular biology to be used in improvements to crop

protection methods based on resistance mechanisms with or without the use of transgenic plants.

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Figure 2

The use of the PVX transient expression system to isolate a resistance gene that recognises the ECP2 protein from

Cladosporium fulvum. ECP2 cDNA has been fused to the PVX coat protein gene so that a fusion protein is

expressed in the PVX system.

4) Use of chemicals for disease control:

Chemical disease control employs the use of chemicals that are either generally toxic and used as

disinfectants or fumigants or chemicals that target specific kinds of pathogens, as in the case of

fungicides, bactericides (or antibiotics) and nematicides. These chemical agents can be sold as

dusts, concentrated solutions, wettable powders, granules or emulsions. India ranks four in

agrochemical production in the world. This industry is important for Indian economy. In India,

there are about 125 technical grade manufacturers (10 multinationals), 800 formulators, over

145,000 distributors. 60 technical grade pesticides are being manufactured indigenously.

Properties of Ideal chemical agent:

a) It should be effective at concentrations that will not harm the plant.

b) It should have low risk to humans and animals.

c) It should have minimal effect on the normal microflora on the plants and in the soil.

d) There should be little chance of the pathogen quickly developing resistance to it.

e) It should be suitable for long periods of storage in ambient conditions.

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4.1) Fungicides:

Fungicides are chemicals used in the control of fungal diseases. (Whole live or dead organisms

that are efficient at killing or inhibiting fungi can sometimes be used as fungicides)

Examples: - Neem oil, tea tree oil, copper sulfate pentahydrate, hexachlorobenzene etc.

Types:

Contact – Fungicides that are not taken up into the plant tissue.

Translaminar – These fungicides redistribute the fungicide from the upper, sprayed leaf surface

to the lower, unsprayed surface.

Systemic – Fungicides that are taken up and redistributed through the xylem vessels.

Mode of action:

a) Negative impacts of fungicide on the membrane of microorganisms were found to alter

the structure and function of soil microbial communities.

o For example, fungicides of the Aromatic Hydrocarbons (AH) group can modify

the lipid structure.

i. Dicloran (2,6-dichloro-4-nitroaniline) treatment results in increased

sensitivity of treated fungi to solar radiation , which then destroys the

structure of Linoleic acid.

ii. Etridiazole (5-ethoxy-3(trichloromethyl)-1,2,4-thiadiazole), causes the

hydrolysis of cell membrane phospholipids into free fatty acids and

lysophosphatides , leading to the lysis of membranes, in fungi.

o Sterols are another important component of cell membrane in fungi.

Demethylation-inhibiting (DMI) fungicides inhibit sterol biosynthesis in

fungal cells. Triadimefon ((RS)-1-(4-chlorophenoxy)-3,3-dimethyl-1-(1H-

1,2,4-triazol-1-yl)butan-2-one) demethylated at C-14, introduced a double

bond at C-22, and reduced a double bond at C-24 in the carbon skeleton of

sterols in a fungal membrane, causing disfunction and cell lysis.

o Some fungicides target fungal intracellular membrane systems and their

biological functions. A widely used fungicidal compound, acriflavine (3,6-

diamino-10-methylacridin-10-ium chloride), increases mitochondrial

permeability and releases cytochrome c in fungal cells, repressing plasma

membrane receptor activation, disordering proton stream and collapsing the

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electrochemical proton gradient across mitochondrial membranes. As a

consequence, ATP synthesis is decreased leading to cell death.

b) Effects on amino acid and protein synthesis:

o It can lead to misincorporation of amino acid ( e.g streptomycin in E. coli,

caused misincorporation of an isoleucine molecule in the phenylalanine

polypeptide chain associated with 70S ribosomes) .

o Inhibiting protein synthesis by interfering the binding of RNA complex with

accepter site of ribosome (e.g. Oxytetracycline).

c) Effects on signal transduction:

o Affecting the genes involved in two-component signal transduction system

(e.g. fludioxonil).

d) Effects on respiration: Several fungicides with different modes of action were reported

to inhibit microbial respiration.

o By inhibiting NADH oxidoreductase, Complex I (e.g. Diflumetorim), succinate-

dehydrogenase, Complex II (e.g. boscalid (2-chloro-N-(4′-chlorobiphenyl-2-yl)

nicotinamide), carboxin (5,6-dihydro-2-methyl-1,4-oxathiine-3-carboxanilide),

and flutolanil(α,α,α-trifluoro-3′-isopropoxy-o-toluanilide) and cytochrome bc1 ,

Complex III .

o By uncoupling oxidative phosphorylation.

i. The metabolic state of mitochondria in target cell was found to be

inhibited after exposure to fluazinam, which may be caused by the

conjugation of the chemical with glutathione, in mitochondria.

e) Effects on Mitosis and Cell Division: The methyl benzimidazole carbamate (MBC)

fungicides are known to impact mitosis and cell division in target fungi.

o Inhibiting polymerization of tubulin into microtubules.

f) Effects on Nucleic Acids Synthesis :

o Inhibiting the activity of the RNA polymerase I system.

i. For example, metalaxyl (methyl N-(methoxyacetyl)-N-(2,6-xylyl)-DL-

alaninate), a widely used PA fungicide, inhibits uridine incorporation into

the RNA chain.

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Picture of some commercially available fungicides in India.

4.2) Antibiotics:

Relatively few antibiotics are routinely used to control plant diseases. Antibiotics are chemical

produced by micro-organisms, which destroy or injure living organisms, in particular, bacteria.

Streptomycin is effective against a few fruit pathogens, such as blights and cankers, and

cyclohexamine can be used to control some fungal pathogens of crops, particularly powdery

mildews and rusts.

Disadvatage: Bacteria, as well as fungi, have the ability to develop resistance to antibiotics.

Examples: streptomycin, Tetracyclines , cyclohexamide etc.

4.3) Nematicides:

A nematicide is a type of chemical pesticide used to kill plant-parasitic nematodes. The use of

nematicides is confined largely to high-return horticultural crops, because they are expensive.

Additionally, they are all highly toxic, and alternative measures for controlling nematodes are

being investigated.

Example: Aldicarb, polysulfide etc.

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4.4) Implementation of molecular biology to design chemical structures (agrochemicals) with

increased effectiveness:

To make use of chemicals for plant disease control more effective, we can identify metabolic

pathways & biochemical processes that are essential for pathogenicity. One potential approach is

insertion mutagenesis of pathogens. Essential factors thus identified can be targeted selectively

to control plant diseases. For example there are chemical agents that can be used to attack cell

wall component, chitin in fungi.

Important: the processes which are targeted must be non-essential in host plant and animals, to

ensure that any agrochemical thus obtained is specific to pathogen and is not harmful to host

plant and consumers.

Some examples of pathways and processes that can be targeted are:

a) Important steps in fungal penetration (e.g. formation of appressoria, melanisation).

b) Genes involved in toxin production.

c) Biosynthesis of specific cell wall components ( e.g. chitin in fungi). Etc.

Thus, after identification of gene(s), the corresponding protein`s/ proteins` structure can be

analyzed. Here, use of computer programs and software is required. Three dimensional

modelling of the encoded protein(s) can be undertaken to help with the identification of active

site and domains that are significant for the activity of protein(s) concerned. These significant

portions of protein can be utilized generate and test the chemical structures made to disrupt these

active sites or domains.

The different potential approaches we can use to target significant locations on proteins are:

a) Chemical structures can be designed that disrupt target domain or active site.

b) Recombinant DNA technology (e.g. Phage display technique and Yeast two hybrid

methods) can be employed to identify structures that interact and inhibit the target

protein.

4.5) Chemical immunization of plants against diseases:

Plant immunization is the process of activating natural defense system present in plant induced

by biotic or abiotic factors. As pesticides contribute to the problem of environmental

deterioration which in turn has a marked influence on the economy, health, and the quality of

life, immunizing plants chemically against plant diseases is now popular. Although, the use of

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agrochemicals is still continued and growing, but making use of natural defense mechanism has

been in practice for many years. A popular example is discovery of phytoalexins resulted in

strategies in which these were sprayed onto plant to control diseases.

Examples: Rishitin and Phytuberin against potato blight and isoflavonoid phytoalexins against

rusts and mildews.

Disadvantages:

Fungicides are more effective.

Phytoalexins are ineffective as eradicants.

Economically not viable.

Suffer from problems of reliability.

Use of elicitors:

Rather than using a single component (e.g. phytoalexins) of defense pathway, we can induce the

battery of defense mechanism by employing the use of elicitors.

Examples:

Oligogalacturonides like Chitins, chitosan, etc.

Mechanism: rapid and transient membrane depolarization (e.g. harpin produced by Erwinia

amylovora has been developed into commercial product in the USA).

Induction of systemic defense:

Chemicals like salicyclic acid, jasmonic acid and ethylene can induce systemic defence in plants.

Problem:

These chemicals are effective in laboratory study but in many cases commercial

applications have not been feasible.

Diversion of biochemical pathways into defense sometimes result in low yield.

Technical problems are also there.

However, a benzathiodioazole (BTH) compound has been developed which mimics the effects of

salicylic acid as an inducer of systemic acquired resistance, and this has been marketed under the

tradename Bion as an effective chemical for the control of certain diseases in tobacco, tomato,

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Arabidopsis, wheat, rice and cucumber, and when appropriate concentrations are used no

harmful effects on the health/productivity of crop is observed.

5) Summary:

Crop improvement for diseases resistant is necessary to increase food production to feed rapidly

growing world population. Breeding for disease resistance has been practiced by humans since

the time human civilization evolved. Many problems associated with conventional plant breeding

strategies have been omitted with the introduction and development of molecular biology. Use of

molecular markers has made breeding much more efficient and easy as it reduces the number of

crosses required. With the introduction of tissue culture, now people are able to cross even the

incompatible plants which were earlier impossible to cross with the aided advantage of increases

variation due to somaclonal variation in some cases. Beside breeding of resistance, a wide array

of chemicals, both inorganic and organic are currently used in crop protection, and new

agrochemicals are constantly being produced and evaluated. These chemicals include fungicides,

bactericides, nematocides and virocides as well as insecticides that can be used to control vectors

for viral and bacterial diseases and herbicides that can be used on parasitic plants and weeds.

As with the use of resistance breeding, persistent use of chemicals can influence the way the

pathogen populations evolve and result in resistance developing or increasing in pathogen

populations against the chemical, compromising the effectiveness of the control. Strategies for

using mixtures of chemicals with differing modes of action, and combining the use of chemicals

with methods such as disease-resistant cultivars into integrated control programmes, can increase

the long-term effectiveness of chemicals and also reduce the quantities required.

Rather than the somewhat hit-and-miss approaches that have been used in the past for

agrochemical identification, molecular biology has the potential to be used in a more targeted

approach. The aim would be to identify, potentially through insertion mutagenesis of pathogens,

metabolic pathways and biochemical processes that are essential for pathogenicity. Once a

pathway and/or gene has been identified, three-dimensional modelling of the encoded protein(s)

can be undertaken to help with identification of the active site and other domains that are

significant. Recently, the use of chemicals like salicylic acid , jasmonic acid and elicitors of plant

defense response has opened up new ways of increasing plant productity.

All these approaches in an integrated manner has led to revolution resulted in development of

new strategies for crop improvement.

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