Biotechnological Aspects of Fungal Diseases … · · 2015-07-22Biotechnological Aspects of Plant Resistant for Fungal Diseases ... and their management requires the use of techniques

American-Eurasian Journal of Sustainable Agriculture, 2(1): 1-18, 2008

ISSN 1995-0748

© 2008, American Eurasian Network for Scientific InformationThis is a refereed journal and all articles are professionally screened and reviewed

ORIGINAL ARTICLE

1

Corresponding Author: Dr. Wafaa Mohamed Haggag, Plant Pathology, National Research Centre, Egypt.

Biotechnological Aspects of Plant Resistant for Fungal Diseases Management

Wafaa Mohamed Haggag

Plant Pathology, National Research Centre, Egypt.

Wafaa Mohamed Haggag,: Biotechnological Aspects of Pant Resistant for Fungal Diseases

Management, Am.-Eurasian J. Sustain. Agric., 2(1): 1-18, 2008

ABSTRACT

The future of sustainable agriculture will increasingly rely on the integration of biotechnology with

traditional agricultural practices. Diseases can be caused by a variety of plant pathogens including fungi, others

and their management requires the use of techniques in transgenic technology, molecular biology, and genetics.

These include: genes that express proteins, peptides, or antimicrobial compounds that are directly toxic to

pathogens or that reduce their growth in situ; gene products that directly inhibit pathogen virulence products

or enhance plant structural defense genes, that directly or indirectly activate general plant defense responses;

and resistance genes involved in the hypersensitive response and in the interactions with a virulence factors.

The following review discusses the key approaches to managing of fungal pathogens within the context of

recent developments in biotechnology.

Key words:

Introduction

Plant pathogens represent real threat to world agriculture. More than 70% of all major crop diseases are

caused by fungi (Agrios, 2005). Crops of all kinds often suffer heavy losses. Fungal plant diseases are usually

managed with applications of chemical fungicides. For some diseases, chemical control is very effective, but

it is often non-specific in its effects, killing beneficial organisms as well as pathogens, and it may have

undesirable health, safety, and environmental risks (Manczinger et al., 2002 and Cavrilescua and Chisti, 2005).

Control of disease is a subject of great interest for biotechnologists. Biotechnology will enhance our

understanding of the mechanisms that control a plant ability to recognize and defend itself against disease-

causing fungi (Punja, 2007). The future of sustainable agriculture will increasingly rely on the integration of

biotechnology with traditional agricultural practices.

Biotechnology: Origin and Definitions

Biotechnology is a technology based on biology, especially when used in agriculture and food science.

Various definitions are given for the term biotechnology. Biotechnology, an abbreviation of “biological

technology”, has been defined as “the application of scientific and engineering principles to the processing of

materials by biological agents to provide goods and services” (Alan, 1985). Elsewhere, Jones (1990) described

biotechnology as comprising a continuum of technologies, ranging from traditional to modern biotechnologies

(Figure 1). It is in the context of technologies that the OTA (1989) described biotechnology as ‘any technique

that uses living organisms, or substances from these organisms, to improve plant for specific uses’. According

to Persley (1992) traditional biotechnology covers well established and widely used technologies based on the

commercial use of living organisms. These include the biotechnologies currently employed in brewing,

fermentation, and many others (Amalu, 2004). Modern biotechnology on the other hand encompasses the uses

of more recently developed technologies, particularly those based on the use of: recombinant DNA technology;

2Am.-Eurasian J. Sustain. Agric, 2(1): 1-18, 2008

monoclonal antibodies; and new cell and tissue culture techniques, including novel bio-processing

(Monti, 1992). The major techniques of biotechnology are tissue or cell culture, cell fusion, embryo transfer,

recombinant DNA technology, etc and the age-old techniques of fermentation.

Fig. 1: Biotechnology continuum, from traditional biotechnologies to modern biotechnologies.

In recent years, bioinformatics is an interdisciplinary field which addresses biotechnology and biological

problems using computational techniques, and makes the rapid organization and analysis of biological data

possible. The field may also be referred to as computational biology, and can be defined as, "conceptualizing

biology in terms of molecules and then applying informatics techniques to understand and organize the

information associated with these molecules, on a large scale (Baxevanis et al., 2007). Bioinformatics and

computational biology involve the use of techniques including applied , informatics, statistics, computer science,

and biochemistry to solve biological problems usually on the molecular level. Major research efforts in the

field include sequence alignment, gene finding, protein structure alignment, protein structure prediction,

prediction of gene expression, and the modeling of evolution ( Baxevanis and Ouellette, 2005).

Biotechnology Aspects of Plant Resistance for Fungal Diseases Management

Plant biotechnology is a precise process in which scientific techniques are used to develop molecular- and

cellular-based technologies to improve plant productivity, quality and health; to improve the quality of plant

products; or to prevent, reduce or eliminate constraints to plant productivity caused by diseases, pest organisms

and environmental stresses (Azhaguvel et al., 2006). Plant biotechnology involves the modification of plant

performance for a particular purpose. Genome segments from plant pathogenic fungi are widely used as vectors

into which genes are inserted to make transgenic plants. This is of paramount importance to ensure efficacy

and genetic integrity of the product and to protect intellectual genetic modification. This can be achieved in

a number of ways include:

C increasing or decreasing the activity of genes that are naturally present in an organism,

C or transferring genes between individuals of the same species from one organism to another of different

species across different types of living things.


Strategies and Applications of Plant Biotechnology

Natural Transfer of Genes Between Species

Natural movement of genes between species, often called horizontal gene transfer or lateral gene transfer,

can occur because of gene transfer mediated by natural processes. This natural gene movement between species

has been widely detected during genetic investigation of various natural mobile genetic elements, such as

transposons, and retrotransposons that naturally translocate to new sites in a genome, and often move to new

species over an evolutionary time scale. There are many types of natural mobile DNAs, and they have been

detected abundantly in food crops such as rice, a species resistant to blast disease (Feschotte 2005).

Synthetic Transfer of Genes Between Species

Callus and Suspension Cultures

Callus is an unorganized, proliferative mass of differentiated plant cells, and usually occurs naturally as

wound response (Dhekney et al., 2007). It can be induced through culture of plant tissue on a medium usually

containing relatively high levels of auxin, especially 2,4-D (Gamborg, 2002). As a general rule, mutation

breeding is difficult to control and frequently yields mosaics and deleterious changes. However, in some cases,

beneficial changes can occur, for example, in sugar cane, plants were obtained that had resistance to the “Fiji”

disease, which was a significant disease in the production of sugar cane (Pierik, 1997). Suspension cultures

can be produced from non- embryogenic or embryogenic callus, and are commonly used these days by

molecular biologists for transformation research. With many species the development of regenerable

embryogenic cell suspensions has provided the opportunity for the production of transgenic plants. One of the

most promising developments is the expression in banana and plantain of antimicrobial peptides, some of which

have been shown to exert in vitro fungistatic activity to Mycosphaerella fijiensis and Fusarium oxysporum , the

causal agents of black sigatoka and panama wilt disease, respectively (Pierik, 1997).

Protoplast

Protoplasts are produced by the enzymatic removal of the cell wall, through the use of mixtures of fungal

cellulases, pectinases and hemicellulases in a solution of high osmotic potential. Removal of the cell wall then

facilitates genetic manipulation of some kind. Fusion of protoplasts can be induced chemically or by

electrofusion. After fusion has occurred the resulting culture usually contains a mixture of fusion products and

parental types, and so some method has to be adopted to distinguish between these two. Plants with a crown

and root system from different genotypes frequently form commercial plantations of Citrus. Recently there have

been reports of somatic hybrids formed from protoplasts isolated from Citropsis reticulata and C. gilletiana,

a species resistant to collar rot and tolerant to a number of diseases including tristeza (Pierik , 1997).

Embryo Culture (Plant Germplasm)

In vitro culture techniques have important applications for the collecting, exchange and conservation of

plant germplasm (Dhekney et al., 2007). Production of transgenic groundnut germplasm resistant to rust and

late leaf spot diseases has been reported (Mace et al., 2006). Because of its large size, and its immediate

germination after seed maturation, pant -collecting missions can be problematic. For germplasm exchange, the

FAO/IBPGR technical guidelines for the safe movement of coconut Germplasm recommend that coconut

germplasm be distributed as zygotic embryos in vitro. In the mid-90s the crop was wiped out with the

introduction of Phytophthora colocasiae, (taro leaf blight). With the importation of taro leaf blight resistant

material to Samoa, taro is now being grown again (Tien et al., 2000).

Plant Breeding

Production of transgenic plants in wide-crosses by plant breeders has been a vital aspect of conventional

plant breeding for about a century. Without it, security of our food supply against losses caused by crop

diseases such as peanut Sclerotinia blight, rusts, and mildews would be severely compromised

(Chenault et al., 2005 and Huang et al., 2005). In the 20 century, the introduction of alien probing intoth

common foods was repeatedly achieved by traditional crop breeders through artificially overcoming fertility

barriers. Novel genetic rearrangements of plant chromosomes, such as insertion of large blocks of rye genes

into wheat chromosomes ('translocations'), has also been exploited widely for many decades and rust disease

management (Huang et al., 2005).


Genetically Engineered Plants

Genetic engineering refers to artificial techniques capable of transferring genes from other organisms

directly to recipient organisms (Gold, 2003). The techniques of genetic engineering can be used to manipulate

the genetic material of a cell in order to produce a new characteristic in an organism. Genes from plants, and

microbes can be recombined and introduced into the living cells of any of these organisms (Azhaguvel et al.,

2006). Transgenic recombinant plants are generated by adding one or more genes to a plant's genome, and the

techniques frequently called transformation (Newell, 2000). Transgenic recombinant plants are identified as a

class of genetically modified organism (GMO); usually only transgenic plants created by direct DNA

manipulation are given much attention in public discussions (Osusky, 2004). Genetic engineering has the

potential to provide a cornucopia of beneficial plant traits, particularly an enhanced ability to withstand or resist

attack by plant pathogens (Chenault et al., 2005 and Punja, 2007). New approaches to plant disease control

are particularly important for pathogens that are difficult to control by existing methods. Genetic engineering

can help farmers increase crop yields and feed even more people (Amalu, 2004). The percentage of GMO plant

resistant to diseases is approximately about 2% of total cultivated GMO plants (Gold, 2003).

The Use of Molecular Markers

In molecular biology, DNA molecular-marking techniques have been used for some time. Genetic

fingerprinting is used to identify particular desirable genes required for specific breeding plants

(Azhaguvel et al., 2006). The DNA of the off spring from an F1, a segregating population, is examined using

different marker technology (AFLPs, RFLPs, RAPDs). Since the gene markers also segregate in these

segregating populations, researchers try to find out which gene marker correlates best to disease resistance,

allowing the location of resistance genes on a genetic map of the plant (Azhaguvel et al., 2006).

Further germplasm lines can be evaluated for resistance by using the marker and without putting the plants

into a field trial. Existing successes include identifying markers for powdery mildew (Erisyphe graminis) and

Rhynchosporium secalis in barley and apple, Fusarium wilt, ascochyta blight in chickpea (Weeden, 1993) and

peanut Sclerotinia blight (Chenault et al., 2005).

How are Genes Transferred to Crop Plants?

C Gene transfer by Agrobacterium tumefaciens is another powerful tool for plant genetic engineering

(Azhaguvel et al., 2006 and Van de Valde et al., 2003). Transformation is usually achieved using gold

particle bombardment or a soil bacterium (Agrobacterium tumefaciens) carrying an engineered plasmid

vector, or carrier of selected extra genes. The process is used routinely to move genes into dicotyledonous

plants.

C The “Gene Gun” is a popular tool used world-wide for genetically engineering plant cells (Kikkert and

Reisch, 1996) .

C Tissue Culture: Tissue culture is a major component of plant genetic engineering .For example, banana

tissue culture is a first-generation plant biotechnology tool used to curtail the spread of diseases, especially

fungal wilt disease, by making disease-free planting material available for the propagation of new crops.

Simple tissue culture techniques such as shoot-tip and embryo culture are well-developed in Africa and

have greatly improved banana breeding, whereby the shoot meristem is extracted from the male flower

and aseptically multiplied into hundreds of shoots for eventual planting in fields (Gamborg, 2002).

C Traditional breeding combines many genes from two parents at once. Several backcrosses may be needed

to remove undesired genes. Breeding by biotechnology, a single new gene is added to the genome .Since

the late 1960s, all varieties of wheat released from one or more genes for resistance to stripe rust

(Line, 2002). Two kinds of resistance have been in use: a) race-specific, single-gene, immunity expressed

at all stages of plant development, in which the genetics of the host-pathogen interaction follows the gene-

for-gene model;

C race-nonspecific, multiple-gene partial-type resistance expressed largely or entirely in adult plants, and in

response to high temperatures, also known as high-temperature, adult-plant resistance. Race-specific, single-

gene immunity is readily defeated by the pathogen, with the result that each new gene deployed in a new

variety selects eventually, and sometimes quickly, for a new race of the pathogen. Approximately

ninety races of the stripe-rust pathogen now exist whereas only one was known in the 1960s.

Nevertheless, through a combination of varieties with high-temperature adult-plant resistance, the use of

several sources of single-gene resistance in isolines mixed to provide a multi-line and deployment of

combinations of single genes as stacked genes, stripe rust remains largely under control through plant

breeding (Line, 2002).


Fig. 2: Transgenic plants with enhanced disease resistance have been engineered to express gene

products to control attack fungal virulence products (from hypha on left), enhanced expression

of plant-derived gene products (inside of cell) or gene products from non plant sources

(outside of cell).

The cells are then grown out to whole plants to make sure that the trait is actually expressed as wanted.

The methods to incorporate the spliced genes are not precise– in fact they are such that the DNA can be

inserted in rather random ways. The insertion of the genetic material may cause other genes to be turned on

or off which can cause major problems within the plant ( Azhaguvel et al., 2006).

Mode of Action of Plant Diseases Resistance Genes

Several other researchers grouped transgenic plants into five general categories (Melchers and Stuiver

2000 and Rommens and Kishore 2000) (Figure 2):

C The expression of gene products that are directly toxic to pathogens or that reduce their growth. These

include pathogenesis-related proteins (PR proteins) such as hydrolytic enzymes (chitinases, glucanases),

antifungal proteins (osmotin- and thaumatin-like), antimicrobial peptides (thionins, defensins, lectin),

ribosome inactivating proteins (RIP), and phytoalexins.

C The expression of gene products that destroy or neutralize a component of the pathogen arsenal such as

polygalacturonase, oxalic acid, and lipase.

C The expression of gene products that can potentially enhance the structural defenses in the plant. These

include elevated levels of peroxidase and lignin.

C The expression of gene products releasing signals that can regulate plant defenses. This includes the

production of specific elicitors, hydrogen peroxide (H2O2), salicylic acid (SA), and ethylene (C2H4).

C The expression of resistance gene (R) products involved in the hypersensitive response (HR) and in

interactions with avirulence (Avr) factors.


The selection of genes to genetically engineer into plants to protect against fungal diseases has been based,

in part, on evaluation of the toxicity of the gene product to fungal growth or development in vitro, and to the

prominence of the particular gene(s) in a disease resistance response pathway as follows:

Hydrolytic Enzymes

The most widely used approach has been to overexpress chitinases and glucanases, which belong

to the group of PR proteins and have been shown to exhibit antifungal activity in vitro and in vivo

(Giannakis et al., 2005). Since chitins and glucans comprise major components of the cell wall in many groups

of fungi, the overexpression of these enzymes in plant cells is postulated to cause the hyphae to lyse and

thereby reduce fungal growth (Mauch and Staehelin, 1989). However, following expression of different types

of chitinases in a range of transgenic plant species, the rate of lesion development and the overall size and

number of lesions were reduced upon challenge with many fungal pathogens, including those with a broad host

range, such as Botrytis cinerea and Rhizoctonia solani. A few transgenic crop species expressing chitinases

have been evaluated in field trials and it was demonstrated that disease incidence was reduced (Melchers and

Stuiver, 2000). Increasing expression of individual and multiple PR-proteins in various crops have demonstrated

some success in enhancing disease resistance in particular pathogens (e.g., in rice against Rhizoctonia solani,

the sheath blight pathogen). A recent research shows a chitinase gene from an anti-fungal biocontrol fungus

species (Trichoderma viride) confers transgenic resistance against the rice sheath blight pathogen. There are

fewer examples of the expression of glucanases in transgenic plants but the results have generally been similar

to that for chitinase expression. The combined expression of chitinase and glucanase in transgenic carrot,

tomato, and tobacco was much more effective in preventing development of disease due to a number of

pathogens than either one alone, confirming the synergistic activity of these two enzymes reported

(Melchers and Stuiver, 2000 and Giannakis et al., 2005). As a general rule, the deployment of genetic

engineering approaches that involve the expression of two or more antifungal gene products in a specific crop

should provide more effective and broad-spectrum disease control than the single-gene strategy (Melchers and

Stuiver, 2000 ).

Pathogenesis-Related Proteins

Host plants contribute an enormous number of diseases resistance genes such as those encoding

pathogenesis-related (PR) proteins, which have been used against fungal diseases (Van Loon and Van

Strien, 1999). PR protein genes appear to be a very potential source for candidate genes for fungal resistance.

These proteins may play a direct role in defense by attacking and degrading pathogen cell wall components.

PR proteins that exhibit antifungal activity, including osmotin- and thaumatin-like proteins (TLP), and some

uncharacterized PR proteins have been engineered into crop plants. The PR-5 proteins induce fungal cell

leakiness, presumably through a specific interaction with the plasma membrane that results in the formation

of transmembrane pores (Kitajima and Sato, 1999). Osmotin has been shown to have antifungal activity in vitro

and, when tested in combination with chitinase and â-1,3-glucanase, showed enhanced lytic activity

(Lorito et al., 1996). When expressed in transgenic potato, osmotin was shown to delay expression of disease

symptoms caused by Phytophthora infestans. Thaumatin-like proteins are also expressed in plants in response

to a range of stress conditions and were demonstrated to have antifungal activity in vitro against several

pathogens, including Botrytis, Fusarium , Rhizoctonia, and Sclerotinia (Koiwa et al. 1997).

Antimicrobial Proteins, Peptides, and Other Compounds

The overexpression of defensins and thionins in transgenic plants was demonstrated to reduce development

of several different pathogens, including Alternaria, Fusarium , and Plasmodiophora, and provided resistance

to Verticillium on potato under field conditions (Gao et al., 2000). Ribosome-inactivating proteins are plant

enzymes that have 28S rRNA N-glycosidase activity, which depending on their specificity, can inactivate

conspecific or foreign ribosomes, thereby shutting down protein synthesis. An antimicrobial protein with

homology to lipid transfer protein was shown to reduce development of Botrytis cinerea when expressed in

transgenic geranium (Bi et al., 1999). Antimicrobial peptides have been synthesized in the laboratory to

produce smaller (10–20 amino acids in length) molecules that have enhanced potency against fungi

(Cary et al., 2000). In addition, a synthetic cationic peptide chimera (cecropin–melittin) with broad-spectrum

antifungal activity has been produced (Osusky et al., 2000). When expressed in transgenic potato and tobacco,

these synthetic peptides have provided enhanced resistance against a number of fungal pathogens, including

Colletotrichum, Fusarium , and Phytophthora. These peptides may demonstrate lytic activity against fungal


hyphae, inhibit cell wall formation, and (or) enhance membrane leakage. The ability to create synthetic

recombinant and combinatorial variants of peptides that can be rapidly screened in the laboratory could

provide additional opportunities to engineer resistance to a range of pathogens simultaneously (Dhekney et al.,

2007). MsrA3, a temporin analogue, as well as MsrA2, derived from Dermaseptin, and combinations

thereof, provided a greater degree of disease resistance against a broad range of pathogens (Misra , 2005).

When MsrA2 was used to inhibit the growth of agronomically important fungal pathogens, including Fusarium ,

Alternaria, Rhizoctonia, Phytophthora, and Pythium sp., its activity was far superior to the activities of other

probiotic peptides tested. The estimated concentration of MsrA2 in leaf tissue was 1 – 5 µg/g of fresh tissue,

which, although not high, seems to be sufficient to protect the potato plants from the attack of pathogens

(Osusky, 2004) . In addition, MsrA2 peptide was shown to control Fusarium head blight (FHB) of wheat and

barley grains caused by Fusarium graminearum . Trichothecenes genes- the virulence factors produced by the

fungus - were introducing into wheat in order to increase the FHB defense mechanism in wheat spikes and

reduced or prevented the initial infection (Osusky, 2004).

The synthetic gene was also introduced into two potato (Solanum tuberosum L.) cultivars, Desiree and

Russet Burbank, stable incorporation, and expression confirmed by reverse transcription (RT)-PCR and recovery

of the biologically active peptide .The morphology and yield of transgenic Desiree plants and tubers was

unaffected.

Phytoalexins

Many gene products belong to the group of PR proteins, while others are involved in phytoalexin

biosynthetic pathways and in enhancing plant structural defenses. These are low molecular mass secondary

metabolites produced in a broad range of plant species, which were demonstrated to have antimicrobial

activity and are induced by pathogen infection and elicitors (Grayer and Kokubun 2001). Using transgenic

plants, it has been possible to show that the overexpression of genes encoding certain phytoalexins, such as

trans-resveratrol and medicarpin, resulted in delayed development of disease and symptom production by a

number of pathogens on several plant species (Dixon et al., 1996).

Inhibition of Pathogen Virulence Products

The plant cell wall acts as a barrier to penetration by fungal pathogens and numerous strategies have

evolved among plant pathogens to overcome this (Walton, 1994). Production of phytotoxic metabolites, such

as mycotoxins and oxalic acid, by fungal pathogens has been shown to facilitate infection of host tissues

following cell death. Degradation of these compounds by enzymes expressed in transgenic plants could provide

an opportunity to enhance resistance to disease. Expression of a trichothecene degrading enzyme from

Fusarium sporotrichioides in transgenic tobacco reduced plant tissue damage and enhanced seedling emergence

in the presence of the trichothecene (Muhitch et al., 2000). Their activity on the substrate oxalic acid results

in the production of CO2 and H2O2; the latter can induce defense responses in the plant and enhance

strengthening of cell walls . Expression of oxalate oxidase in transgenic hybrid poplar enhanced resistance to

Septoria, while oxalate decarboxylase expression enhanced resistance of tomato to Sclerotinia sclerotiorum

(Thompson et al., 1995). These results indicate that the inactivation of specific pathogen virulence factors, such

as toxins, by gene products expressed in transgenic plants has the potential to reduce development of specific

fungal pathogens.

Alteration of Structural Components

Lignification of plant cells around sites of infection or lesions has been reported to be a defense response

of plants that can potentially slow down pathogens spread (Nicholson and Hammerschmidt, 1992). The enzyme

peroxidase is required for the final polymerization of phenolic derivatives into lignin and may also be involved

in suberization or wound healing. A decrease in polyphenolic compounds, such as lignin, in potato tubers by

redirection of tryptophan in transgenic plants through expression of tryptophan decarboxylase rendered tissues

more susceptible to Phytophthora infestans (Yao et al., 1995), illustrating the role of phenolic compounds in

defense. Lignin levels were significantly higher following expression of the H2O2-generating enzyme glucose

oxidase in transgenic potato and by expression of the hormone indoleacetic acid (IAA) in transgenic tobacco

(Sitbon et al., 1999). In the former case, tolerance to several fungal pathogens was enhanced. A reduction in

large callose deposits surrounding haustoria of Peronospora parasitica infecting Arabidopsis thaliana was

indirectly achieved in transgenic plants not accumulating Salicylic acid (SA) by expression of the enzyme

salicylate hydroxylase (Donofrio and Delaney , 2001). These plants also had reduced expression of the PR-1


gene and exhibited significantly enhanced susceptibility to the pathogen, suggesting that callose deposition

during normal defense responses of the plant was influenced by the reduced levels of SA.

Activation of Plant Defense Responses

Plant resistance to pathogen infection can be achieved by systemic regulation of the defense-related genes

that respond to specific systemic signals. One activator of host defense responses are elicitor molecules from

an invading pathogen. These can trigger a network of signalling pathways that coordinate the defense responses

of the plant, including PR protein, and phytoalexin production (McDowell and Dangl, 2000). A gene encoding

the elicitor cryptogein from the pathogen Phytophthora cryptogea was cloned and expressed in transgenic

tobacco under control of a pathogen-inducible promoter (Keller et al., 1999). Other activators of plant defense

responses include signaling molecules such as SA, ethylene, and jasmonic acid (Dempsey et al., 1998

and 1999). The roles of SA as a signal molecule for the activation of plant defense responses to pathogen

infection and as an inducer of systemic acquired resistance (SAR) have been extensively studied (Metraux,

2001). Using transgenic plants, evidence for the role of SA in defense response activation has been obtained.

Plants expressing the SA-metabolizing enzyme salicylate hydroxylase, a fungal protein that converts SA to the

inactive form catechol, did not accumulate high levels of SA and had enhanced susceptibility to pathogen

infection (Donofrio and Delaney, 2001). The over expression of SA in transgenic tobacco was recently shown

to enhance PR-protein production and provide resistance to fungal pathogens (Verberne et al., 2000).

Expression of tobacco catalase, an enzyme with SA-binding activity, in transgenic potato enhanced defense

gene expression leading to SAR and enhanced tolerance to P. infestans (Yu et al., 1999). At the same time,

ethylene and jasmonic acid appear to be signals used in response of plants to necrotrophic pathogen attack and

that work independently of, and possibly antagonistic to, SA-mediated responses (Lee et al., 2001). Ethylene

and jasmonic acid may be involved in signaling the defense responses in the gene-for gene interaction

(Dong, 1998). In wheat leaves sprayed with 20 or 30 mM methyl jasmonate, the soluble protein and total

phenol greatly increased compared with control (Haggag, Wafaa and Abd-El-Kareem, 2007) (Figure 3).

Endogenous levels of free putrescine, spermidine and spermine increased in response to the elicitor. Perchloric

acid-soluble conjugated polyamines and polyamine biosynthetic enzymes of ornithine decarboxylase and

polyamine oxidase, peroxidase and chitinase, were also stimulated and reduced wheat yellow rust disease

compared with untreated plants. Number of cDNA clones encoding various PR genes including an acidic class

III chitinase (VvChi3), a basic class I glucanase (VvGlub) and a thaumatin-like protein (VvTl2) were expressed

in grapevine leaves and berries in response to ethephon and salicyclic acid treatments (Giannakis et al., 2005).

Expression of PR genes in grapevine reduced powdery mildew disease incidence. Mutant or transformed plants

non responsive to either jasmonate or ethylene were found to be more susceptible to infection by root- and

foliar infecting fungi (Thomma et al., 1999), confirming a role for these signals in certain host– pathogen

interactions (Figure 4). Whether or not reduced or elevated levels of hormones, such as auxins, cytokinins,

gibberellins, and jasmonate, can lead to the development of transgenic plants with enhanced disease resistance

remains to be seen, given their broad range of physiological effects on plant development. Altered auxin or

cytokinin expression has the potential to also affect mycorrhizal colonization of plant roots (Barker and Tagu,

2000). To elucidate defense responses in chickpea (Cicer arietinum L.) against fungal pathogens, Ascochyta

rabiei causing ascochyta blight and Fusarium oxysporum f.sp. ciceri causing fusarium wilt, expression patterns

of defense-related genes in chickpea after pathogen inoculation and exogenous treatments with systemic signals

such as SA and Me-JA were reported (Cho and Muehlbauer, 2004). Two blight differentia germplasm lines,

FLIP84-92C(2) (blight resistant and SA- and Me-JA-sensitive) and PI359075(1) (blight susceptible and SA-

and Me-JA-insensitive) showed significant differential expression patterns of the defense-related genes after

Arabidopsis rabiei inoculation and exogenous treatment with SA and Me-JA. Ahn et al. (2005) studied the role

for Vitamin B1 (thiamine) as a plant defense activator that induces SAR. Thiamine activates SAR-related genes

in rice, tobacco, tomato, cucumber, and Arabidopsis and prevents several diseases caused by semibiotrophic

and biotrophic pathogens. The effects of thiamine on disease resistance are prevented in Arabidopsis mutants

3impaired in SA accumulation as well as by treatment with the calcium channel blocker LaCl , demonstrating

that thiamine induces SAR in plants through the SA- and Ca -related signaling pathways. Challenge inoculation 2+

with a range of fungi induced the HR as well as several defense genes, and growth of the pathogens was

concomitantly restricted. Resistance to the pathogens was not complete, possibly because of the time needed

for production of the transgenic elicitor following initial infection (Keller et al., 1999). Antisense inhibition

of catalase, a H2O2-degrading enzyme, resulted in development of necrotic lesions and PR-protein

accumulation (Takahashi et al., 1997). While these and other reports indicate that induction of the HR and

necrosis, with the resulting activation of general defense pathways, could potentially result in broad-spectrum

disease resistance (Melchers and Stuiver, 2000), the use of such an approach would require tight regulation


Fig. 3: Protein bands of wheat plants treaded with different concentrations of methyl josmonate and

inoculated with yellow rust Puccinia recondita f. sp. tritici.

Fig. 4: Induction of grapevine PR genes in leaves and berries of Sultana plants by ethephon (A) and

salicyclic acid (B) treatments.

of the expressed phenotype, in addition to ensuring that no deleterious side effects, such as abnormal

or suppressed growth, occurred on the transgenic plants. Another activator of defense responses that

has been engineered in transgenic plants is H2O2 generated through expression of genes encoding for

glucose oxidase. H2O2 has been shown to directly inhibit pathogen growth and to induce PR

proteins, SA, and ethylene, as well as phytoalexins (Mehdy, 1994). It is produced during the early

oxidative burst in plant cell response to infection and can trigger the HR, strengthen cell walls, and

enhance lignin formation (Wu et al., 1997). Expression of elevated levels of H2O2 in transgenic cotton,

tobacco, and potato reduced disease development due to a number of different fungi, including

Rhizoctonia, Verticillium , Phytophthora, and Alternaria; high levels can, however, be phytotoxic (Murray et

al., 1999). Therefore, the widespread induction of cell death in a transgenic plant to induce disease

resistance has to be approached with caution. Expression of canola plants catalase katE - transformed via

Agrobacterium - mediated in the chloroplasts, enhanced defense gene expression leading to SAR and enhanced

resistant to Peronospora parasitica causing downy mildew and Erysiphe polygon causing powdery mildew (El-

Awady et al., 2007) (Figure 5). HR-mediated cell death is triggered sequentially through an increase in the

intracellular cytosolic Ca concentration by an influx of external Ca and the secretion of Ca from the2+ 2+ 2+

calcium stores into the cytoplasm (Chung et al., 2004), a burst of reactive oxygen species, changes in the

extracellular pH and membrane potentials , and variations in protein phosphorylation patterns (Tsukamoto et

al., 2005). Finally, key mediators such as SA accumulate and resistance is induced systemically (Pieterse and

Van Loon, 2004).


Fig. 5: Over-expression of catalase gene in canola exhibits enhanced resistance

(A) to Peronospora parasitica (B)and Erysiphe polygoni (C)

Resistance Genes (R-gene)

There are several examples of the expression of R genes in transgenic plants. The R-gene products that

have been cloned from tomato, tobacco, rice, flax, Arabidopsis, and several other plant species shared one or

more similar motifs: a serine or threonine kinase domain, a nucleotide binding site, a leucine zipper, or a

leucine-rich repeat region, all of which may contribute to recognition specificity (Takken and Joosten, 2000).

The authors proposed that multiple factors may be involved in determining the resistance response, or that the

resistance may be HR-independent. The plant's resistance (R) gene product acts as a signaling receptor for the

pathogen's avirulence (Avr) gene product in the presence of resistance-regulating factors such as RAR1 and

SGT1, leading to a form of cell death termed hypersensitive response (Rowland et al., 2005). AVR genes was

isolated from Bgh, a representative of the powdery mildews as the third major group of obligate biotrophic

parasites (Ridout et al., 2006). The results indicated that the mildew fungus has a repertoire of AVR genes,

which may function as effectors and contribute to parasite virulence. Multiple copies of related but distinct AVR

effector paralogues might enable populations of Bgh to rapidly overcome host R genes while maintaining

virulence. A combination of several interacting genes, similar to that for the antifungal proteins, will likely be

required. An enhanced understanding of R-gene structure and function could, however, make it possible to

modify functional domains in the future to tailor R genes for use in providing broad-spectrum resistance to

diseases in transgenic plants (Dempsey et al., 1998).

Examples of Transgenic Crop Varieties Resistant to Fungal Diseases

Genes can be identified that confer resistance to fungal pathogens. For instance, many genes have been

found that provide resistance to specific races of each pathogen (Gold, 2003). In many cases, resistance genes

are available in the gene pool of cultivated plants and can be transferred to them. In the African region, two

initiatives for fungal resistant transgenic varieties are reported, a field trial of transgenic strawberry with

phytoalexin synthesis genes on transgenic maize for resistance to cob rot , both in South Africa (Amalu, 2004).

The three initiatives for development of fungal resistant transgenic varieties reported in Eastern Europe were

all in Bosnia and Herzegovina where laboratory testing of transgenic potato for resistance to Fusarium,

Verticilium and Rhizoctonia has been initiated. In the Asian region, there is a field trial underway in China

for transgenic cotton with resistance to Verticilium and Fusarium . A few countries in Latin America, mainly

Argentina, Brazil and Cuba, are carrying out a number of activities on transgenic resistance to fungi,

particularly on tropical fruit trees, with some results already being tested in the field. In this region, most of


the activities for transgenic fungal resistance are reported in Cuba, in particular involving field trials of

transgenic potato for late blight resistance, and fungal-resistant sugar cane. Other field trials in the Latin

America region for transgenic fungal resistance are reported for maize, sunflower and wheat in Argentina, and

tobacco in Mexico. Other countries involved in transgenic fungal resistance research are Argentina on alfalfa,

Brazil on rice, barley and cocoa, Chile on grape and apple, Colombia on tree tomato, Peru on potato for late

blight resistance and Venezuela on sugar cane (Gold, 2003). Some examples of molecular genetic successes

and contributions to plant disease-resistance vegetable crop varieties are listed in Table (1).

Table 1: GM Disease-Resistant Crops

Crop Variety Comments

French Bean Aigullion Resistant to bean anthracnose

Broad Bean Futura Tolerant of chocolate spot

Brussels Cavalier Resistant to light leaf spot

Braveheart F1 Resistant to powdery mildew and light leaf spot

Cabbage Stonehead F1 Resistant to mildew

Carrot Fly Away F1 Bred for resistance to carrot fly

Newmarket Good resistance to splitting

Cucum ber Cum laude F1 Tolerant of powdery mildew

M edia F1 Tolerant of powdery mildew

Lettuce Alexandria Highly resistant to downy mildew and bolting

Lisbusa Resistant to downy mildew

Sylvestra Resistant to downy mildew

Barcelona Resistant to mildew

Iglo Resistant to downey mildew

Smile Resistant to mildew

Parsnip Avonreister Very good resistance to canker

Gladiator F1 High resistance to canker

Pea Ambassador Resistant to powdery mildew and fusarium wilt

Cavalier Good resistance to powdery mildew.

Greenshaft Resistant to downy mildew and fusarium wilt

Rondo Resistant to fusarium wilt

Potato (first early) Colleen Good resistance to blight and scab

Premiere Resistant to blight and spraing

Swift Resistant to golden eelworm and tolerant to blackleg

Potato Cosmos Resistant to blight and common scab

Osprey Very resistant to scab

Potato (maincrop) Harmony Very resistant to scab and partially resistant to white and golden eelworm

M ilva Blight resistant

Remarka Good all around disease resistance

Adm iral Good resistance to blight and scab

Tomato Alicante Good resistance to greenback and m ildew

Retrieved from "http://en.wikipedia.org/wiki/Disease_resistance_in_fruit_and_vegetables

Late Blight resistant (GM) Potato as A New Approach

Late blight, caused by the oomycete pathogen Phytophthora infestans, is the most devastating potato

disease in the world. All plant-breeding attempts to increase the plant’s resistance have so far had little success

and have repeatedly been threatened by Phytophthora itself because the fungus is incredibly flexible and has

developed lots of different strains. Today, in order to prevent the spread of Phytophthora, potato plants must

be treated several times with various chemical fungicides as a precaution. In Europe, research to develop a

genetically engineered potato has been carried out in Germany and Switzerland. In a German study, potatoes

were transformed with the addition of genetic material from a soil bacterium. The transformed potatoes produce

a fungal inhibitor, which leads to the death of the plant cell, thus preventing further spread of the disease

(Strittmatter, 1995). In a Swiss experiment, potatoes were transformed by introducing a wheat gene that

encodes for an enzyme that degrades oxalic acid into carbon dioxide and hydrogen peroxide. Hydrogen

peroxide in the plant tissue is a defense against the blight fungus. One focus of current research is a wild plant

species related to potato from Mexico. This species, Solanum bulbocastanum coevolved with the late blight

fungus and has exhibited durable race non-specific resistance to the late blight fungus (Naess, 2002).

However, S. bulbocastanum is largely sexually incompatible with potato due to differences in endosperm

balance numbers. One alternative to sexual crosses is the uniting of diverse genomes via somatic fusion, which

has been effectively used to capture the late blight resistance from S. bulbocastanum and has then been passed

on to potato breeding lines (Helgeson, 1998). The progeny of the somatic hybrids were grown in Mexico where

nearly every race of the fungus is found. The resistance to late blight of S. bulbocastanum has been

mapped to chromosome 8. This single gene, RB, has been previously cloned and transformed into Katahdin,

a highly susceptible potato cultivar. Cloning of the resistance gene and the transformation of potato cultivars


with its insertion could result in complete resistance to late blight in commercial potatoes (Van der

Vossen, 2002). Transgenic potato lines containing the RB gene have showed strong late blight resistance,

comparable to the backcrossed progenies derived from the somatic hybrids between potato and S.

bulbocastanum (Jones and Simko, 2005).

Genetic Engineering of Grapevines

Grapevine diseases- include mildews, grey mould and root rots- cause growers to invest millions of dollars

and numerous hours on various techniques to reduce losses (Dhekney et al., 2007). Kikkert and Reisch (1996)

described the use of genetic engineering in which genes that code for desired traits are inserted into a plant.

Gene transfer technology became routine in the mid 1980's for easily manipulated non-woody plants such as

tobacco. However, it has only been in the last few years that genetically transformed grapevines have been

produced. Currently, transformed grape varieties are being tested in France and in the United States. Genes

for resistance to fungi work in different ways. Some genes that have been isolated from plants and higher

fungi, code for enzymes (such as chitinase) that degrade a major component of the outer protective walls of

certain fungi. Other genes act by creating holes in the membrane of fungal cells. To insert genes into

embryogenic cultures, most researchers working on grape transformation rely upon modified strains of

Agrobacterium , which transfers genes into plants as part of its normal life cycle. In a Geneva laboratory,

researcher have taken a different approach. They rely upon the biolistic process, whereby DNA-coated particles

of extremely minute size are used to carry foreign genes into grapevine cells. DNA coding for the genes of

interest is coated onto the minute tungsten-micro projectiles. These are accelerated at extremely high speeds

into the cultured cells using a biolistic device, also known as the "gene gun". There are usually several genes

transferred into each cell penetrated by a micro projectile. One of these genes might be the gene of interest

coding for a desired trait. Another gene is used to help separate the transformed cells from the remaining

normal cells. This is important because usually less than 5% of the cells receive and maintain the genes long-

term. To select transformants, genes for antibiotic resistance are usually used. A gene was used which confers

resistance to the antibiotic, kanamycin. Selection for the transformed cells takes place in a medium containing

kanamycin, on which the transformed cells are able to grow and develop into embryos. Normal cells without

the newly inserted gene will die on medium with kanamycin, so that the only growth observed should originate

from cells with the newly inserted genes.

Development of Dominant Rice Blast Resistance Gene

Rice blast disease caused by the fungus is one of the most devastating diseases worldwide. Twenty

resistance genes have been identified by extensive genetic studies (Jia et al., 2002). Pi-b and Pi-ta, two major

resistance genes, introgressed from indica cultivars, have recently been molecularly characterized. Both Pi-b

and Pi-ta encode predicted nucleotide binding site type proteins that are characteristics of products of major

resistance genes . Molecular markers linked to resistance genes have been used for selection at the early

seedling stages, and genotypes can be easily raised. A PCR-based Pi-ta gene marker is useful in marker-

assisted selection breeding since it is the part of resistance gene, and is simple, rapid and inexpensive and can

be used for analyzing large numbers of samples (Jia et al., 2002).

Biotechnology for Rust Resistance in Wheat

Diseases of wheat, are mostly caused by fungal pathogens. Leaf rust caused by Puccinia recondita f. sp.

tritici is the most widespread and regularly causing rust on wheat. Various wheat breeding programs throughout

the world have had mixed results in producing cultivars with long-lasting, effective resistance to leaf rust

(Huang and Gill, 2001). Genetic resistance is the most economical method of reducing yield losses due to leaf

rust. Genetic resistance to leaf rust can be most fully utilized by knowledge of the identity of resistance genes

in commonly used parental germplasm and released cultivars. Identification of the leaf rust resistance genes

allows for efficient incorporation of different genes into germplasm pools, thus helping to avoid the release

of cultivars that are genetically uniform. Resistance gene expression is dependent on the genetics of host-

parasite interaction, and interaction between resistance genes with suppressors or other resistance genes in the

wheat genomes. Genes expressed in seedling plants have not provided long-lasting effective leaf rust resistance.

Adult-plant resistance genes Lr13 and Lr34 singly and together have provided the most durable resistance to

leaf rust in wheat throughout the world.


Management of Fungal Diseases in Barley

Rhizoctonia root rot, the most important root disease affecting direct-seeded barley, is caused by

Rhizoctonia solani AG8 and R. oryzae. Extensive screening of the gene pools of wheat, barley, and their

relatives has failed to produce a useful source of resistance (Smith et al., 2002). Dasapyrum villosum , a distant

relative of wheat, shows evidence of some resistance (Smith et al., 2002), but, thus far, it has not been

possible to transfer it into wheat by conventional methods, including by chromosome substitution and

manipulation. Transgenic resistance to both species of Rhizoctonia has been produced in barley by transfer of

the ThEn42 gene from the fungus Trichoderma harzianum (Wu, 2003). This gene encodes an endochitinase

that softens the cell walls of the pathogens. Barley was selected as the vehicle for testing this gene

because a) it is more susceptible than wheat to Rhizoctonia root rot, b) being a diploid, the genetics are less

complicated than that of hexaploid wheat. Lee et al., (2007) was purified a novel antifungal protein from blast

fungus (Magnaporthe grisea)-treated rice leaves using consecutive chromatographies on CM-Sepharose ion-

change, Affi-gel blue, and HPLC gel filtration columns. After cloning two cDNAs encoding OsPEX5L and

OsPEX5S genes that are splice variants of OsPEX5 from a rice leaf cDNA library, they investigated their

antifungal properties. The recombinant proteins were expressed in Escherichia coli and found to significantly

inhibit cell growth of various pathogenic fungal strains. mRNA expression of the OsPEX5L gene was induced

by diverse external stresses such as rice blast fungus, fungal elicitor, and other signaling molecules including

H2O2, abscisic acid, jasmonic acid, and salicylic acid. These results suggested that the peroxisomal receptor

protein, OsPex5p, plays a critical role in the rice defense system against diverse external stresses including

fungal pathogenic attack.

At the same time, stem rust caused by Puccinia graminis was among the most devastating diseases of

barley in the world. The barley stem rust-resistance gene Rpg1 confers resistance to many pathotypes of the

stem rust fungus Puccinia graminis. The Rpg1 gene identity was confirmed by high-resolution genetic and

physical mapping, sequencing of multiple alleles from resistant and susceptible lines, a fortuitous recombination

that combined parts from the resistant and susceptible parents, and exclusion of other potential candidate genes

by sequence analysis. The Rpg1 gene encodes a receptor kinase-like protein with two tandem protein kinase

domains, which is a novelstructure for a plant disease resistance gene. It does not contain a strong membrane-

targeting motif and known receptor sequences, suggesting a mechanism of action similar to the tomato Pto

gene. The barley Rpg1 gene may be useful for durable stem rust-resistance in other species (Gurel and

Gozukirmizi (2003).

Issues Associated with Genetically Modified Plants

Benefits and Risks

The list of plants and plant-derived products made as a result of modern biotechnology is ever- increasing.

There is public concern over the safety of eating GM foods, there is no scientific reason to suspect that food

from GM crops which have been licensed by the various regulatory bodies, presents any more of a safety

problem than that from non-GM crops. Many of the potential hazards, such as food allergies, are also

associated with traditional breeding techniques. The concerns that are being raised of environmental and food

safety risks of biotechnology through gene exchange and evolution of new pathogens, or from putative

increased or unexpected allergenicity are legitimate risks that will be addressed as have similar potential risks

with any new plant or plant product. Assessment and management of these and other risks of new technologies

in a formal process is appropriate, and must be conducted in a science-based manner and also include

economic, human and animal health, and ecological consequences. However, these risks and concerns are not

limited to plants and plant products produced through biotechnology and thus must be placed in perspective.

The consequences of foregoing the use of biotechnology for improving plant health and sustainable plant

productivity must also be considered.

Potential risks associated with transgenic plants include

C Introduction of allergenic or otherwise harmful proteins into foods

C Transfer of transgenic properties to viruses, bacteria, or other plants

Benefits of plant biotechnology

The application of biotechnology in agriculture has resulted in benefits to farmers, producers and

consumers. Plant biotechnology has helped make diseases control safer and easier. Some of the potential

benefits from using transgenic plants include:


C Reduced crop production costs and increased yields

C Reduced environmental impact from farming and industry

C Increased food availability for underdeveloped countries

Legal and Regulation

All genetic modification must comply with strict regulations (Vines, 2002), For example, organic standards

reflect a "zero tolerance" policy concerning transgenic products and organisms. Organic food producers are

taking precautions to minimize the risk of unintentional contamination of their products with transgenic ones.

In the United States, the Federal level, the Food and Drug Administration, the Environmental Protection

Agency, and the Department of Agriculture extensively review products of biotechnology to ensure that they

are safe for public use and the environment . In UK and Europe, the acceptance of GM crops by the

European Union has been more reserved. Europe currently favors labeling of all GM foods and a system that

would allow for "identity preserved" processing in which foods would be guaranteed to contain no genetically

modified products.

Safety Considerations

Safety considerations surrounding plant biotechnology focus on food safety and environmental safety.

All countries with functioning biosafety regulatory systems have developed these systems gradually, usually

beginning with voluntary guidelines and standards developed cooperatively by stakeholders in academia,

industry, and government. A national biosafety policy or strategy provides a set of principles to guide the

development and implementation of a biosafety system and should describe the goals and objectives of the

regulatory framework. Direction on many of the fundamental issues and public policy choices that must be

considered during the development of regulations can be provided by such a strategy. Examples of these issues

include the extent to which social, ethical, and economic factors should be considered, the social acceptability

of biotechnology and its products, and linkages with other national policies on food, agriculture, and economic

development. For, example, in United States, GM foods require labeling only if they differ significantly in

safety, composition, or nutritional content when compared to their non-GM counterparts.

Future Prospects

Achievements today in plant biotechnology have already surpassed all previous expectations, and the future

is even more promising. The future of biotechnology will depend on how the consumers accept this technology

and those to come. As the technology evolves toward the use of tissue-specific or pathogen-inducible

promoters, the expression of engineered traits that are effective against a broad range of pathogens, and the

utilization of synthetically derived peptides and of R genes, the impact on disease management will be

enhanced. Evaluation of these transgenic plants for response to disease will need to be extended to field trials

and appropriate agronomic data collected to ensure that this technology can be successfully implemented in

farmer’s fields to augment on-going disease management practices. Transgenic plants with enhanced disease

resistance can become a valuable component of a disease management program in the future. The next three

or four of years will provide an answer to how biotechnology will continue to be used in crop production.

The possibility, in the short term, is that there will be some sort of labeling of the foods that have been created

with GMO material and the consumers will make their determination of what they think by the buying of the

product or not. The farmers/producers will have the ability of trying to determine whether it will be more

profitable to produce the traditional or biotech types of crops. They may have many reasons to want to use

the biotech varieties.

Conclusions

Agricultural biotechnology is the application of molecular biology and, especially, molecular genetics to

solving agricultural problems and exploiting agricultural opportunities. Biotechnology research leads to new

and improved agricultural inputs. Biotechnology provides new opportunities to build pest- resistance into

plants. Such developments may reduce the demand for fungicides. Biotechnology research will produce crop

varieties with special characteristics that increase their value as grain and forage. Growers can justify greater

inputs of chemicals when producing higher value crops. This, in turn, will increase the demand for

biotechnology.


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