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2 LITERATURE REVIEW

2.1 Tea: An overview

The word “tea” is derived from “t’e”, the Chinese Fukien dialect. Going by an ancient

Chinese dictionary, revised during 350 AD by Kuo P’o, tea-drinking originated in China

(Ukers, 1935). Tea plant is supposed to be discovered by Robert Bruce in 1823 from some

hills near Rangpur (near present Sibsagar), then the capital of Assam (Ukers, 1935).

According to Wight’s nomenclature (Wight, 1959 and Wight, 1962), tea can be classified

into three races: 1) Camellia sinensis L. or the China tea plant, 2) Camellia assamica

(Masters) or the Assam tea plant and 3) Camellia assamica sub sp. lasiocalyx (Planch. MS)

or the Cambodiensis or Sounthern form of tea plant. Tea plant exhibit open cross

pollination making them genetically complex species. The genus Camellia with its 82

species (Sealy, 1958) accounting for more than 325 species (Mondal, 2002) belongs to the

family Theaceae. It is reported that 600 varieties of tea are cultivated worldwide with

unique traits such as high caffeine content, drought tolerance, blister blight disease tolerant

etc. (Mondal, 2004).

Tea is the second most consumed beverage in the world, after water. The beverages include

black, green, oolong, white and yellow teas (Basu, 2003). However, black and green tea

account for the principal types of tea produced and consumed in the world, with small

amounts of other types (International Tea Committee, 2003). The tea plant is indigenous

throughout the forests of south-east Asia, where in its natural state, grows to a height of 30-

40 feet. The tender leaves are used to make tea. Its centre of origin is thought to be the

indefinite belt to the south-east of the Tibetan plateau encompassing Sze-Chuan, Yu-Nan,

North Vietnam, Burma, Siam and Assam in north-east of India. The tea plant has been

introduced into and become naturalized in many areas of the world and is currently found

in many continents. It can be found growing near all old trading routes between China and

India and in the islands of south-east Asia, Japan, Europe, North and South America, Africa

and Australia. It is cultivated as far north as Georgia (42°N) on the eastern shores of the

black sea in southern Russia and as far south as Argentina (27°S) in South America and

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South Africa (Weatherstone, 1992). The tea industry plays a significant role in the economy

of India where the crop is the leading foreign exchange earner and export commodity.

India, Sri Lanka and Kenya produce most of the black tea while the other countries produce

green tea and other varieties. India is the second largest tea producing country in the world

after China (FAO statistical year book 2009).

2.2 Fungal diseases of tea

The following list of pathogens known to affect tea, are listed below:

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1) Anthracnose

Colletotrichum theae-sinensis�(Miyake) Yamamoto

(=�Gloeosporium theae-sinensis�Miyake)

2) Armillaria root rot

Armillaria mellea�(Vahl:Fr.) Kummer

(=�Armillariella mellea�(Vahl:Fr.) P. Karst.)

Armillaria heimii�Pegler

(=�Armillaria fuscipes�Petch)

3) Bird's eye spot

Cercoseptoria ocellata�Deighton

(=�Cercospora theae�(Cavara) Breda de Haan)

Pseudocercospora theae�(Cavara) Deighton

(=�Septoria theae�Cavara

=�Cercoseptoria theae�(Cavara) Curzi)

4) Black blight

Cylindrocladium lanceolatum�Peerally

5) Black root rot

Rosellinia arcuata�Petch

Rosellinia bunodes�(Berk. & Broome) Sacc.

6) Black rot

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Ceratobasidium�sp.

Corticium invisum�Petch

Corticium theae�Bernard

7) Blister blight

Exobasidium vexans�Massee

8) Botryodiplodia root rot

Lasiodiplodia theobromae�(Pat.) Griffon & Maubl.

(=�Botryodiplodia theobromae�Pat.)

9) Brown blight

Glomerella cingulata�(Stoneman) Spauld. & H. Schrenk

(anamorph:�Colletotrichum gloeosporioides�(Penz.) Penz. &

Sacc. in Penz.

=�Colletotrichum camelliae�Massee)

10) Brown root rot

Phellinus noxius�(Corner) G.H. Cunningham

(=�Fomes noxius�Corner)

11) Brown spot

Calonectria colhounii�Peerally

(anamorph:�Cylindrocladium colhounii�Peerally)

12) Brown zonate leaf blight

Ceuthospora lauri�(Grev.) Grev.

13) Bud blight

Phoma theicola�Petch

14) Charcoal stump rot

Ustulina deusta�(Hoffm.:Fr.) Lind

(anamorph:�Ustulina zonata�(Lév.) Sacc.)

15) Collar and branch canker

Phomopsis theae�Petch

16) Collar rot

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Rhizoctonia solani�Kühn

(teleomorph:�Thanatephorus cucumeris�(Frank) Donk)

17) Copper blight

Guignardia camelliae�(Cooke) E.J. Butler

18) Damping-off

Cylindrocladium floridanum�Sobers & Seymour

(teleomorph:�Calonectria kyotensis�Terashita)

Hypochnus centrifugus�(Lév.) Tul.

19) Dieback

Leptothyrium theae�Petch

Nectria cinnabarina�(Tode:Fr.) Fr.

20) Gray blight

Pestalotiopsis theae�(Sawada) Steyaert

(=�Pestalotia theae�Sawada)

Pestalotiopsis longiseta�(Spegazzini) Dai et Kobayashi

(=�Pestalotia longiseta�Spegazzini)

21) Gray mold

Botrytis cinerea�Pers.:Fr.

22) Gray spot

Phyllosticta dusana�Hara

23) Horse-hair blight

Marasmius crinisequi�Müller ex Kalchbrenner

(=�Marasmius equicrinus�Müller)

24) Leaf spot

Calonectria pyrochroa�(Desmaz.) Sacc.

(=�Calonectria quinqueseptata�Figueiredo & Namekata)

(anamorph:�Cylindrocladium ilicicola�(Hawley) Boedijn &

Reitsma)

Calonectria theae�C.A. Loos

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(anamorph:�Cylindrocladium theae�(Petch) Subramanian)

Cochliobolus carbonum�Nelson

Hendersonia theicola�Cooke

Pestalotiopsis adusta�Ellis & Everh.

Phaeosphaerella theae�Petch

Pleospora theae�Speschnew

25) Leaf scab

Elsinoe theae�Bitancourt & Jenkins

26) Macrophoma stem canker

Macrophoma theicola�Petch

27) Net blister blight

Exobasidium reticulatum�Ito & Sawada

28) Pale brown root rot

Pseudophaeolus baudonii�(Pat.) Ryv.

29) Phloem necrosis

Phloem necrosis virus (Camellia Virus 1)

30) Phyllosticta leaf spot

Phyllosticta erratica�Ellis & Everh.

Phyllosticta theae�Speschnew

31) Pink disease

Corticium salmonicolor�Berk. & Broome

32) Poria root rot and stem canker

Poria hypobrunnea�Petch

33) Purple root rot

Helicobasidium compactum�(Boedijn) Boedijn

34) Red leaf spot

Phoma theicola�Petch

35) Red root rot

Ganoderma philippii�(Bresad. & P. Henn.) Bresad.

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Poria hypolateritia�(Berk.) Cooke

36) Red rust (alga)

Cephaleuros virescens�Kunze

(=�Cephaleuros parasiticus�Karsten)

37) Rim blight

Cladosporium�sp.

38) Root rot

Cylindrocarpon tenue�Bugnicourt

Cylindrocladiella camelliae�(Venkataramani & Venkata

Ram) Boesewinkel

(=�Cylindrocladium camelliae�Venkataramani & Venkata

Ram)

Cylindrocladium clavatum�C. S. Hodges & L.C. May

Fomes lamaoensis�(Murr.) Sacc. & Trott.

Ganoderma applanatum�(Pers.) Pat.

Ganoderma lucidum�(Curtis:Fr.) P. Karst.

39) Rough bark

Patellaria theae�Hara

40) Sclerotial blight

Sclerotium rolfsii�Sacc.

(teleomorph:�Athelia rolfsii�(Curzi) Tu & Kimbrough

(=�Corticium rolfsii�Curzi)

41) Shoot withering

Diplodia theae-sinensis�Lui & Li

42) Sooty mold

Capnodium footii�Berk. & Desmaz.

Capnodium theae�Boedijn

Meliola camelliae�(Cattaneo) Sacc.

43) Stump rot

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Irpex destruens�Petch

44) Tarry root rot

Hypoxylon asarcodes�(Theiss.) Mill.

45) Thorny stem blight

Tunstallia aculeata�(Petch) Agnihothrudu

46) Thread blight

Marasmius tenuissimus�(Junghuhn) Singer

47) Twig blight

Patellaria theae�Hara

48) Velvet blight

Septobasidium bogoriense�Pat.

Septobasidium pilosum�Boedijn & B.A. Steinman

Septobasidium theae�Boedijn & B.A. Steinman

49) Violet root rot

Sphaerostilbe repens�Berk. & Broome

50) White root rot

Rigidoporus microporus�(Sw.:Fr.)

51) White scab

Elsinoe leucospila�Bitancourt & Jenkins

(=�Sphaceloma theae�Kurosawa)

52) White spot

Phyllosticta theifolia�Hara

53) Wood rot

Hypoxylon nummularium�Bull.:Fr.

Hypoxylon serpens�(Pers.:Fr.) J. Kickx

Hypoxylon vestitum�Petch

54) Xylaria root rot

Xylaria�sp.

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2.3 Blister blight disease of tea

Blister blight is the most serious disease affecting shoots of tea and is is known to occur in

India since 1855 (Venkataram, 1967) which is capable of causing enormous crop loss.

Blister blight of tea is caused by the obligate biotrophic fungal pathogen, Exobasidium

vexans. The pathogen attacks harvestable tender shoots, inflicting enormous yield loss and

in absence of any control measures distinct crop loss due to blister blight could as high as

43% (Ordish, 1952). The disease is endemic to most tea-growing areas of Asia but is not

known to occur in Africa or America. Cloudy, wet weather favors infection. Shan or Indian

varieties of tea are somewhat resistant to this disease. The disease is predominantly

prevalent among tea plantations of Darjeeling. There are also reports of its occurrence in

North East India particularly in the hilly regions in inflicting a crop loss of upto 24%

depending on the severity and duration of the disease (Satyanarayana, 1980).

Figure I: The systematic position of Exobasidium vexans (Massee, 1898). Note: the

tea leaf infected with E. vexans.

Kingdom: Fungi

Phylum: Basidiocota

Class: Exobasidiomycetes

Order: Exobasidials

Family: Exobasidiaceae

Genus: Exobasidium

Species: E. vexans (Massee,

1898)

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2.3.1 Epidemiology and symptoms:

2.3.1.1 Easily recognizable symptom

The young leaf forms shiny gray or white color spot which usually swells more on the

lower surface of the leaf. This spot is called blister which can be easily recognized. Above

the blister, the upper surface of the leaf sunkens (Figure II).

2.3.1.2 Eventually developed symptoms

The disease is first seen on leaves, younger than one month old, as a small spot. At this

stage, the size of the spot is about the top of a needle. Multiple spots may be present on a

single leaf. The spot quickly increases its size becoming transparent with the color of

chicken-fat or light brown. Often pink or red powder is formed at the center of the spot.

Besides young leaves, blisters may sometime form on young branches and even green

fruits.

A)

B)

C)

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Figure II: Different stages of blister blight lesions in the leaves of susceptible tea clone

(Adapted from Premkumar et al., 2008).

A: tiny translucent spots;

B: lesions on upper surface of the leaf;

C: matured sporulating lesions on lower surface of the leaf. Arrows indicate

single lesions.

About 7 days after the appearance of the first spot, blister swells out from the lower surface

of the leaf. The superficial surface of the blister becomes gray, then white. At last the

blister bursts, releasing a white or pale-pink powder called spores (“seeds”) of the fungus.

After the release of the spores, the diseased spot becomes violet then brown, and finally the

leaf shrinks. Leaves and shoots with multiple blisters perish and drop from the bush. The

growth of the plant is slowed and sometimes cannot be harvested until 2 months after the

disease.

2.3.1.3 Disease cycle

Spores from the blisters are dispersed by the wind to the leaves of other tea plants. The

spores are unstable in drought or bright sunlight. But if a spore lands on a leaf that is

covered with a film of water or dew, the spore will germinate within 2-5 days, which

produces a thin thread mycelium that grows into the leaf. The mycelium branches and

grows to produce a mass of threads called mycelia inside the leaf. After about 10 days, the

fungus develops inside the leaf as a chicken-fat colored translucent spot measuring about

0.2-0.5 mm in diameter. The fungus in due course causes the leaf to swell into a blister, and

grows spores inside the blister. Life cycle from germination to sporulation takes about 28

days.

2.3.1.4 Favourable conditions for infection

The disease grows best in moderate temperatures (17-22 degrees) and humidity ranging

from 60-100% (as shown in Table I). The disease becomes graver in the years with warmer

spring and showery rain during February-April. Hot temperature (25-27 degrees or more)

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inhibit the growth of the fungus. The problem is aggravated in tea fields with heavy shade.

So the event is more serious at the bases of the hills with poorly-drained areas and in dense

bushy tea plantations rather than in well-drained, well-ventilated and well-spaced

plantations.

Table I: Favourable conditions that make the disease worse.

2.3.1.5 Effect on tea quality:

Diseased buds are black and when processed, tea gives a bitter taste. Tea manufactured

from blister blight infected leaves lowers the quality of tea (Satyanarayana and Baruah

1983; Baby et al., 1998). With increasing disease severity total phenols, catechin(s), total

nitrogen, amino acids, and chlorophylls, as well as polyphenol oxidase activity declines in

tea shoots which attributes to the quality of tea (Gulati et al., 1999). Likewise, theaflavins,

thearubigins, caffeine, aroma components as well as total liquor colour, brightness and

briskness also declines in orthodox tea prepared from infected leaves (Gulati et al., 1999).

Similarly, catechins content, flavour component 2-phenylethanol, as well as prephenate

dehydratase enzyme activity decreases in the tea shoots infested by E. vexans (Sharma et

al., 2011). Thus, the disease plays a significant role in gross quality deterioration and lower

market valuation of tea.

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2.3.1.6 Control measures:

Blister blight is controlled mainly by therapeutic approach with the application of toxic

chemicals for over 50 years (Saravanakumar et al., 2007). To control the disease within the

economic threshold level, protectant fungicides, systemic fungicides and antibiotic

solutions in combination with protectant fungicides are sprayed at regular intervals (TRI

Advisory Circular 2002). Protective copper fungicide formulations, namely copper

oxychloride, eradicant nickel chloride and systemic fungicides, such as hexaconazole,

tridemorph, propiconazole and bitertanol are recommended. These fungicides are sprayed

at 7-days interval throughout the disease season with an average of 26 rounds of spray to

keep the disease at bay (Ajay and Baby, 2010). However, the use of fungicides to control

blister blight becomes less accepted due to phytotoxicity and fungicide residual effect

besides environmental pollution and human health hazards (Saravanakumar et al., 2007).

Use of such chemicals increases the potential for the build-up of resistance in E. vexans

against fungicides, decreases species diversity in given ecosystem, increases risk of residue

built up in made tea as well (Khan et al., 2006). Residues may be present in the final

product due to massive applications of fungicides, sometimes exceeding the fixed

maximum residue limits (1-2 ppm for systemic fungicides) of the international health

standards (Balasuriya and Kalaichelvan 2000).

Attempts have also been made by the application of biological control agents like

Trichoderma harzianum, Gliocladium virens, Serratia marcescens, Pseudomonas

fluorescens and Bacillus subtilis (Premkumar, 2001, 2002, 2003; Balasubramanian, et al.,

2006) but the results were not found to be sound effective. Use of resistant varieties can be

one of the components of Integrated Pest Management (IPM) but there no report of any

resistant variety (Jeyaramraja et. al., 2005). Besides non availability of resistant cultivar

developed through conventional breeding, it is time-consuming and labour intensive due to

perennial nature and long gestation period (4-5 years) of tea plant. Since, most of the

known quality clones or cultivars of tea are susceptible to blister blight, and there is no

single method including chemical, cultural or biological control seems to provide complete

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control of the disease, transgenic approach can be key strategy towards the development of

blister blight resistant/ tolerant plant materials.

2.4 Plant-pathogen interactions: An overview

Plants are the source of food and abode for number of parasites, including bacteria, fungi,

viruses, nematodes, insects and even some plants. Plants are sessile, lack of locomotion,

which let to evolve some defense mechanisms like: preformed and induced defense

responses. In preformed responses, also known as nonspecific responses, plants depend

either on presynthesized structural elements like cell walls or presynthesized secondary

metabolites intended to reduce or restrain pathogen attack (Osbourn, 1996). And in induced

responses, on the other hand, plants co-evolve with pathogens in order to overcome the

preformed responses. These responses initiate on the recognition of the invading pathogen

and then stimulate a signal/multiple cascade(s) pathway that induces changes in gene

expression consisting of large fraction of a plant genome. Bevan and co-workers (1998)

have revealed that 14% of Arabidopsis genome is devoted to this activity.

2.4.1 Types of fungal plant pathogens:

Based on trophic nature, there are three types of fungal plant pathogens: necrotrophic,

biotrophic and hemitrophic (Glazebrook, 2005). Necrotrophic pathogens kill and destroy

plant tissue by producing toxic chemicals and cell wall degrading enzymes. For instance:

Rhizoctonia solani. Biotrophic pathogens, on the other hand, complete their lifecycle in a

living host plant. Here, the pathogens do not allow their host to die but the growth of the

plant is severely affected. An example of a biotrophic fungal pathogen affecting tea plant is

Exobasidium vexans causing blister blight disease. Hemitrophic pathogens are usually

biotrophic in nature at the initial stage of their infection cycle, which become necrotrophic

at the end.

2.4.2 Activation of plant defense response:

Induction of defense response in plants initiates on recognition of the invading pathogen.

This is possible through specific recognition of elicitors produced by the pathogen, such as

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peptides or other compounds (Scheel, 1998). The phenomenon is called a gene for gene

resistance, where a single dominant gene (Avr, avirulence gene) is recognized by a single

dominant resistance (R) gene (Flor, 1972) inhibiting the pathogen to initiate disease and

resulting incompatible interaction. This type of pathogen is known as avirulent pathogen.

But the effectivity of the response depends on how quickly the plant can react once the

pathogen has been detected (Yang et al., 1997). On the other hand those avirulence genes

which escape recognition allow the pathogen to interact with the plant and cause disease

thus resulting into compatible interaction.

Avr-R gene interaction triggers oxidative responses with the production of active oxygen

species (AOS), like O-, H2O2 and OH

- which results in calcium and ion fluxes (Wu et al.,

1997). Transcriptional reprogramming occurs due to AOS which may also lead to

programmed cell death (Belkhadir et al., 2004). The growth of biotrophic pathogens is

thought to be restricted by the hypersensitive response (HR) preventing the pathogen from

getting its food source as plant tissue (Thatcher et al., 2005). The mechanism happens to be

detrimental in case of necrotrophic pathogens as it helps the pathogen to obtain its nutrition

(Glazebrook 2005). On the other hand, when avr-R recognition interaction fails the plant

activates its basal defense response, involving compounds like flagellin or liposaccharides.

This defense mechanism is called pathogen associated molecular pattern (PAMPS)

(Gomez-Gomez and Boller 2002). However, PAMPS is slower and weaker in response

compared to avr-R interaction where colonization is not prevented but the spread of

pathogen is restricted (Glazebrook et al., 1997).

2.4.3 Defense-signalling pathways

After the recognition of pathogen by plants three defense-signaling pathways become

activated: salicyclic acid (SA), nitric oxide (NO), ethylene (ET) and jasmonic acid (JA)

dependent pathways. These pathways either act synergistically or antagonistically.

In necrotrophic pathogen interaction the plant defense response is JA and ET signaling

dependent (Glazebrook, 2005). It has been reported that JA and ET dependent SA

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independent defense response is induced by wounding in plant system (Leon et al., 2001).

While in case of biotrophic interaction the plant defense response is SA dependent

(Glazebrook, 2005). Gene for gene interaction and hypersensitivity reaction are the stimuli

responsible for this pathway. The increase in SA level has been reported to be associated

with pad4 and eds1 genes in Arabidopsis thaliana (Christine et al., 2001; Falk et al., 1999).

This increase in SA also induces the expression of a chain of defense response genes like

PR1, PR2, and PR5 families (Jeong et al., 2011).

2.5 Mechanism of activated plant defense response

The signal transduction pathway is induced following the recognition of the pathogen

which in turn leads to the transcriptional activation of numerous genes involved in defense

response. The resultant pool of proteins can then give rise to primary and secondary

defense response. The primary role includes cell reinforcement, hypersensitivity response

leading to programmed cell death, the release of toxic metabolites and defense related

proteins (Jones, 2001). The secondary immunity role helps in proliferating systemic

acquired resistance (SAR) which gives to a broad-spectrum immunity from the local area of

infection to other parts of the plant (Ryals et al., 1996).

2.5.1 Primary defense mechanism against fungal pathogen in plants:

There are numbers of molecules and proteins found to be involved (either directly or

indirectly) in pathogen defense response. These include pathogenesis related proteins,

ribosome inactivating proteins, small cysteine rich proteins, lipid transfer proteins,

polygalacturonase inhibiting proteins and antiviral proteins.

2.5.2 Pathogenesis related proteins

Plants express a wide variety of genes in response to pathogen/pest infection. Such genes

are referred to as pathogenesis-related (pr) genes (Bowles, 1990). PR proteins were first

identified and defined as proteins that are missing in healthy plants but are over expressed

in pathogen infected plants (Van Loon and Van Kammen 1970). Since then PR proteins

have been found in more than 40 plant species (Van Loon and Van Strien 1999) being

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expressed as part of local and systemic response (Heil and Bostock 2002). The production

of PR proteins in the remote uninfected parts of plants can lead to the occurrence of

systemic acquired resistance, protecting the affected plants from further infection (Ryals et

al., 1996; Delaney, 1997).

At first PR proteins were grouped into five main classes based on biological and molecular

characterization (Van Loon and Van Strien 1999; Edreva 2005). Currently there are

seventeen recognized families of PR proteins based on amino acid sequence similarity and

also biological and enzymatic similarity (Van Loon and Van Strien 1999; Van Loon et al.,

2006). This classification is based on two criteria:

a) The protein must be induced by a pathogen in tissues that do not normally express

the protein and

b) The induced expression must be shown in at least two events of plant-pathogen

interactions or expression must be confirmed in at least two independent research labs.

PR proteins can be either acidic or basic in nature, although possessing similar biological

function. Normally acidic PR proteins are localized in intercellular spaces while basic PR

proteins are found in intracellular spaces such as vacuoles (Van Loon and Van Strien

1999). PR proteins include �-1, 3-glucanases, chitinases, thaumatin-like proteins,

peroxidases, ribosome-inactivating proteins, defensins, thionins, nonspecific lipid transfer

proteins, oxalate oxidase, and oxalate oxidase-like proteins (Van Loon et al., 1994; Van

Loon, 1997; Van Loon and Van Strien, 1999; Görlach et al., 1996; Okushima et al., 2000;

Christensen et al., 2002; Van Loon et al., 2006; Sels et al., 2008).

PR protein expression is specially induced based on a particular signaling pathway that

activates it. This allows the plant to act specifically, producing specific PR proteins that

either target biotrophic or necrotrophic pathogens (Glazebrook, 2005). It has been reported

that in Arabidopsis the biotrophic pathogen Peronospora parasitica induces the activation

of PR-1, PR-2 and PR-5 due to SA mediated pathway. The necrotrophic pathogens such as

Alternaria brassicola and Botrytis cinerea induce the activation of PR-3 and PR-4 for

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induced resistance which is jasmonic acid signal pathway dependent (Penninckx et al.,

1996; Thomma et al., 1998). There are so many reports about the transgenic approaches

using genes encoding pathogenesis-related (PR) proteins which are able to confer resistance

to fungal pathogens (Broekaert et al., 1995, 1997; Cammue et al., 1992; Gao et al., 2000;

Hoshikawa et al., 2012).

2.5.2.1 Plant chitinases

The best characterized genes belonging to PR protein group are those that encode the

hydrolytic enzymes known as chitinases (EC 3.2.1.14) and �-1,3-glucanases (EC 3.2.1.39).

Chitinases catalyze the hydrolysis of �-(1,4)-linkages between N-acetylglucosamine (2-

acetami-do-2-deoxyglucopyranoside) residues in the linear homopolymer, chitin. They are

widely distributed enzymes found in microorganisms, plants and animals. A role for these

enzymes in plant defense mechanism against fungal attack is suggested by the absence of

chitin in higher plants (Abeles et al., 1970), its presence in fungal cell walls (Bartnicki-

Garcia, 1968), and the finding that the plant chitinases inhibit in vitro spore germination

and mycelial growth of certain fungi (Roberts and Selitrennikoff, 1988). Thus, these

enzymes have the ability to hydrolyze the chitin present in the fungal cell wall and prevent

the entry of fungal pathogen into leaf tissue. This enzyme is called b-protein or

pathogenesis-related protein (Tuzun et al., 1989). Furthermore, oligomeric products of

digested chitin can act as signal molecules to stimulate further defense responses. These

lytic enzymes have attracted much attention and have become very important resources in

the genetic engineering of crop plants for disease resistance (Muthukrishnan et al., 2000).

Chitinase genes exist as seven classes: class I, II, II, IV, V, VI, & VII (Collinge et al., 1993;

Meins et al., 1994; Neuhaus, 1999).

In general, class I chitinases have the highest antifungal activity, perhaps due to the

presence of a chitin-binding domain (Sela-Buurlage et al., 1993). They also have higher

specific activities compared to other classes of chitinases. All other chitinase classes have

lower to no antifungal activity as compared to class I chitinases. Based on these

observations, most transgenic work to produce plants with elevated chitinase activity has

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utilized class I chitinase gene(s). A bean (Phaseolus vulgaris) chitinase gene under the

control of CaMV 35S promoter was introduced into tobacco plants through Agrobacterium

mediated transformation. A high level of chitinase activity of 20-40 folds as compared to

control was observed in the transgenic plants. Transgenic plants showed increased

resistance to infection by pathogenic fungi Rhizoctonia solani and delayed development of

disease symptoms. In one study, oilseed rape (Brassica napus) transgenic plants

transformed with a tomato chitinase gene were grown and challenged with three different

fungal pathogens at two field locations (Grison et al., 1996). Over a period of 52 days, the

protection level against three fungi was 23% to 79% with both delayed appearance of

symptoms and reduced lesion numbers. A rice chitinase gene transformed into rice showed

enhanced resistance to sheath blight caused by Rhizoctonia solani (Datta et al., 2001); a

tobacco chitinase gene transformed into peanut showed enhanced resistance to leaf spot

disease caused by Cercospora arachidicola (Rohini and Rao, 2001); a rice chitinase gene

transformed into grapevine increased resistance of these plants against powdery mildew

caused by Uncinula necator (Yamamoto et al., 2000). The transgenic wheat showed

resistance to powdery mildew and leaf rust diseases. Shin et al. (2008) has reported about

the development of transgenic wheat expressing a barley class II chitinase gene with

enhanced resistance against Fusarium graminearum.

2.5.2.2 Plant 1,3-�-glucanases

This pr gene is one of the best characterized pr genes that encodes the hydrolytic enzyme

known as �-1,3-glucanases (EC 3.2.1.39). Beta-glucanases are widely distributed among

bacteria, fungi and higher plants. There are 2 types of glucanases. The first type, exo-1,3-�-

glucanases hydrolyse laminarin by sequentially cleaving glucose residues from the non-

reducing end of polymers or oligomers. Consequently, the sole hydrolysis products are

glucose monomers. The second type, endo1,3-�-glucanases cleave �-1,3-linkages at

random sites along the polysaccharide chain releasing smaller oligosaccharides (Cohen-

Kupiec et al., 1999).

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Plant glucanases have a major role in defence mechanism against fungal pathogens, along

with some importance in cell differentiation as well (Donzelli et al., 2001; Jin et al., 1999).

The exclusive substrate of these enzymes is �-1,3-glucans found as callose and laminarin in

fungal cell walls. They are induced by pathogen attack or environmental stress. These

enzymes have an important nutritional role in saprophytes and mycoparasites. In addition,

these enzymes release elicitors after hydrolysis of pathogen cell walls for the induction of

the defence response (Keen and Yoshikawa 1983). Beta-1,3-glucanase genes have also

been part of tissue specific and developmentally regulated non-pathogen induced

expression (Hird et al., 1993). In growing plant tissues, these enzymes, participate in the

dissolution of the tetrad callose wall and the release of the young microspores into the

anther locules (Kotake et al., 1997).

Table II: The family of pathogenesis-related proteins (Adapted from Van Loon and Van

Strien, 1999).a

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a Genes considered for present study are circled.

2.5.2.3 Plant Defensins

Defensins are small positively charged, antimicrobial peptides (~5 kDa in size) and some of

them exhibit potent antifungal activity. Complete cDNA containing an ORF of 243 bp of a

defensin of mustard was cloned. The deduced amino acid sequence of the peptide showed

more than 90% identity to the amino acid sequence of the well-characterized defensins,

RsAFP-1 and RsAFP-2 of Raphanus sativus. Transgenic tobacco and peanut plants

constitutively expressing the mustard defensin was generated and characterized. Transgenic

tobacco plants were resistant to the fungal pathogens, Fusarium moniliforme and

Phytophthora parasitica pv. nicotianae. Transgenic peanut plants showed enhanced

resistance against the pathogens, Pheaoisariopsis personata and Cercospora arachidicola,

which jointly cause serious late leaf spot disease. These observations indicate that the

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mustard defensin gene can be deployed for deriving fungal disease resistance in transgenic

crops (Anuradha et al., 2008). Overexpression of pepper pathogen induced genes CAPIP2,

CASAR82A and RAV1 in transgenic plants resulted in disease resistance and abiotic stress

tolerance (Lee and Hwang 2006; Lee et al., 2006; Sohn et al., 2006).

Membrane permeabilization induced by plant defensins occurred at concentrations that

correlated with the inhibition of fungal growth (Thevissen et al., 1999). In vitro antifungal

activity of a defensin from Trigonella foenum graecum was tested against some fungal

pathogens (Olli and Kirti 2006). Defensins have been found to display antimicrobial

activity not only against plant and insect pathogens, but also against human fungal

pathogens including Candida and Aspergillus sps, and they are employed as novel leads in

antifungal therapeutics (Thevissen et al., 2007). Over expression of these genes will

therefore cause higher levels of the enzymes on the plant cell surface, which might lead to

a faster and more effective interaction with and neutralization of the invading pathogen

(Schlumbaum et al., 1986; Simmons et al., 1994; Terakawa et al., 1997; Terras et al., 1992;

Toubart et al., 1992).

Some studies revealed that plants transformed with �-1, 3-glucanase alone did not exhibit

resistance to certain pathogens or showed less resistance compared to plants co-transformed

with �-1, 3-glucanase and chitinase genes. Similarly, plants transformed with chitinase

gene alone also did not show an adequate level of resistance. In addition, like �-1, 3-

glucanase, chitinases inhibit only a limited number of fungal species. However, when the

two enzymes are combined, a synergic effect can usually be observed. For example, co-

transformation of tobacco plants with barley class II basic chitinase and barley class II basic

�-1, 3-glucanase gene showed enhanced resistance against Rhizoctonia solani as compared

to plants transformed with a single gene (Jach et al., 1995). In another experiment, tomato

plants expressing tobacco class I �-1, 3-glucanase and chitinase transgenes showed reduced

susceptibility to infection by Fusarium oxysporum f.sp. lycopersici (Jongedijk et al., 1995).

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2.6 Transgenic tea:

Transgenic technology has immense potential for genetic improvement of tea; however,

until 2000 there was not any report of transgenic tea. The initial challenge was to develop a

protocol for gene transfer. Taking reporter genes there are a few reports about the

development of transgenic tea (Mondal et al., 2001, Lopez et al., 2004, Jeyaramraja &

Meenakshi 2005, Sandal et al., 2007). Transgenic tea with silenced caffeine synthase was

done by Mohanpuria and co-workers (Mohanpuria et al., 2010; Mohanpuria et al., 2011).

There is also a recent report about the transgenic tea with improved stress tolerance and

higher quality (Bhattacharya et al., 2013).

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