Introduction

1

Indian spices are well known all over

the world for their taste and strong aromatic

flavor. There are around 80 types of spices

grown throughout the world but India alone

produces about 50 types of them

(Anonymous). The history of spice is almost

as old as human civilization. Among all,

most popular is cumin. Being medicinally so

valuable and greatly used while cooking

around the globe. But this important crop is

threaten from the diseases caused by various

pathogenic fungi. To protect cumin crop

from disastrous diseases and increasing the

yield to meet up the demand easily, cumin

was selected as a model plant in this study

(Fig. 1). Fig 1: Healthy growing cumin plants

History:

The English "cumin" derives from the French "cumin", which was borrowed

indirectly from Arabic "آمون" Kammon via Spanish comino during the Arab rule in

Spain in the 15th century. Cumin is known differently in international languages.

Such as Cumino in Spanish, Cumin blanc in French, Kreuzkummel in German,

Komijn in Dutch, Cumino in Italian, Cuminto in Portuguese, Kmin in Russian, Kumin

in Japanese, Siu waih heing in Chinese and Jeera in Hindi.

Cumin is the second most popular spice in the world after black pepper. It has

been grown and used as a spice since ancient times. Seeds, excavated at the Syrian

site Tell ed-Der, have been dated to the second millennium BC. It is a native of the

Levant and Upper Egypt and known to Egyptian even in 5000 BC era as the seeds

have been found in the old Kingdom Pyramids. The spice is especially associated with

Morocco, where it is often smelt in the abundant street cookery of the medians.

Originally cultivated in Iran and the Mediterranean region, was even known in ancient

Greece and Rome. The Romans and the Greeks used it medicinally and cosmetically

to induce a pallid complexion. It is now mostly grown in Morocco, Egypt, India,

Introduction

2

Syria, Iran, North Africa, North America, Indonesia, Sudan, Algeria and China. India

is the world’s largest producer and consumer of cumin (Indian Commodity Exchange,

2009).

Cuminum cyminum L. is an important seeds spice crop at world level and,

India ranks first in terms of the acreage and production. Within India, it is extensively

cultivated in Gujarat, Rajasthan and in some parts of Madhya Pradesh as rabi crop.

The flavour of cumin seeds is due to the presence of a volatile oil. In indigenous

varieties of cumin, this volatile oil is present up to 2.5%-3.5%. Cumin is generally

grown as a mono crop. The increasing population load and globalization has created

high demand of cumin both in domestic and international markets.

Biology:

Cumin is the dried seed of the herb Cuminum cyminum, a member of the

apiaceae family. The genus Cumin has single species cyminum. The synonyms are

Cuminum odorum Salisb. or Selinum cyminum L. Krause.

Kingdom : Plantae

Division : Magnoliophyta

Class : Magnoliopsida

Order : Apiales

Family : Apiaceae

Genus : Cuminum

Species : Cuminum cyminum f. sp. Cumin

Common name : Jeera

The cumin plant grows to 30–50 cm (0.98–1.6 ft) tall and is harvested by

hand. It is an herbaceous annual plant, with a slender branched stem 20-30 cm tall.

The stem is green, angular and much branched. It bears 5-10 cm long, bluish-green,

dissected leaves that have clasping leaf bases. They are pinnate or bipinnate, thread-

like leaflets. The lamina consists of 2 lateral and one terminal, dichotomously

branched segments that are 0.5-0.6 mm wide (Fig. 2). The flowers are small, white or

Introduction

3

pink and bloom in small umbels. The tap root goes only 10-15 cm deep in the soil.

There are 2 varieties being cultivated in India, viz., the dwarf variety and the tall

variety (Ilyas, 1980)

Cumin seed is commonly known as “jeera” in India. The fruit is a lateral

fusiform or ovoid achene 4-5 mm long, containing a single seed. Cumin seeds (Fig. 2)

are similar to fennel and anise seeds in appearance, but are smaller and darker in

colour. They are oblong in shape, thicker in the middle, compressed laterally about 5

inch long, resembling caraway seeds, but lighter in colour and bristly instead of

smooth, almost straight, instead of being curved. They have nine fine ridges,

overlapping as many oil channels or vittae. The odour and taste are somewhat like

caraway, but less agreeable (Grieve, 1995). Cumin seeds are used as a spice for their

distinctive bitter flavour and strong, warm aroma due to their essential oil content

(Hudani, 2007).

Cumin seeds are rich in oil, it contains powerful compounds. The predominant

compound, cuminaldehyde, accounts for up to 40% of the oil content. Aldehydes are

rich in naturally occurring oxygen compounds

which can interact with human cells to make

profound beneficial changes. Other natural

compounds include terpenes, terpinenes,

cymene, limonene, farnesene and carophyllene.

These natural constituents possess remarkable

antioxidant, antitoxic, anti-microbial, anti-

fungal, anti-parasitic, anti-spasmodic and

diuretic actions. The yield of the oil depends

upon the quality and age of the seed; the older

seeds contain less oil. The cuminaldehyde is

also used in perfumery. Fig 2: Umbels, flower and seeds

The seeds of Cuminum cyminum contains (in percentage): moisture 11.9;

protein 18.7; ether extractive 15.0; carbohydrates 36.6; fiber 12.0; mineral matter 5.8;

calcium 1.08; phosphorus 0.49%; iron 31.0 mg/100 g; carotene calculated as vitamin

A 870 1.0 U/100 g; and vitamin C 3.0 mg/100 g. The seeds on distillation yield a

volatile oil (2.0-4.0%) having an unpleasant characteristic odor, spicy and somewhat

Introduction

4

bitter taste. The oil is colorless or yellow in appearance. The chief constituent of the

volatile oil is aldehyde, cuminaldehyde which forms nearly 20-40% of the oil. Besides

the aldehyde, oil contains p-cymene, pinene, dipentene, cumene, cuminic alcohol,

beta.-phellandrene and alpha.- terpineol. The residue left after the volatile oil

extraction contains 17.2% protein and 30.0% fat. Other chemical constituents reported

are apigenin-7- glucoside, apigenin-7-diglucoside, apigenin-7,4'-diglucoside,

apigenin-7- digalacturonide, apigenin-7- galacturonylglucoside, apigenin-7-

digalacturonide-4'-glucoside, apigenin- 6,8-di-C-glucoside, luteolin-glucoside,

luteolin-7-diglucoside, luteolin-7,3'- diglucoside, luteolin-7,4'-diglucoside and

luteolin-7-galacturonide-4'- glucoside and chrysoeriol glycoside. In addition, the

seeds yield about 22% fats, numerous free amino acids. The cuminaldehyde content

varies considerably, depending on the source of the oil (fresh vs. ground seeds). Fine

grinding of the seed can result in the loss of up to 50% of the volatile oil, with the

greatest loss occurring within 1 hour of milling. Another major component of the oil

is monoterpene hydrocarbons. Sesquiterpenes constitute minor constituents. The seeds

contain cuminol, cymine, hellandren, carvone, cuminique alcohol3. The chief

components of the characteristic aroma of unheated whole seeds are menthen- 7al and

cuminaldehyde in combination with other related aldehydes. Cumin contains safrole,

a natural mutagenic compound, which gets degraded on cooking (Agarwal et al.,

2010).

Nutritional Value:

Cumin is very good source of iron and manganese as per the ratings of the

World’s Healthiest Foods (Table 1) (Azeez, 2008). This makes it good for

haemoglobin in the body and for boosting the immune system

(www.diethealthclub.com).

Introduction

5

Table 1: Nutrition value of cumin seeds per 100 gram

Uses:

Cumin seeds are used as a spice for their distinctive aroma, popular in

Nepalese, Indian, Pakistani, North African, Middle Eastern, Sri Lankan, Cuban,

northern Mexican cuisines, central Asian Uzbek cuisine, and the western Chinese

cuisines of Sichuan and Xinjiang. Cumin was also used heavily in ancient Roman

cuisine. It can be used ground or as whole seeds and helps to add an earthy, warming

feeling to cooking, making it a staple in certain stews and soups, as well as curries and

chilli. These are largely used as condiment and form an essential ingredient in all

mixed spices and curry powders for flavoring soups, pickles, curries, and for

seasoning breads, cakes and so on.

Cumin has varied uses in traditional medicines. It is used to treat hoarseness,

jaundice, dyspepsia and diarrhoea. Its seeds have stomachic, diuretic, carminative,

stimulant, astringent and abortifacient properties. It has various physiological effects

on gastrointestinal, reproductive, nervous and immune systems. It also has

Cumin seeds ( Nutritional value per 100 g (3.5 oz)

Energy 1,567 kcal

Carbohydrates 44.24 g

Sugars 2.25 g

Dietary fiber 10.5 g

Fat 22.27 g

Saturated 1.535 g

Protein 17.81 g

Water 8.06 g

Vitamin A equiv. 64 μg (7%)

Riboflavin (Vit. B2) 0.327 mg (22%)

Niacin (Vit. B3) 4.579 mg (31%)

Vitamin B6 0.435 mg (33%)

Cumin seeds ( Nutritional value per 100 g (3.5 oz)

Folate (Vit. B9) 10 μg (3%)

Vitamin B12 0 μg (0%)

Vitamin C 7.7 mg (13%)

Vitamin E 3.33 mg (22%)

Vitamin K 5.4 μg (5%)

Calcium 931 mg (93%)

Iron 66.36 mg (53%)

Magnesium 366 mg (99%)

Phosphorus 499 mg (71%)

Potassium 1788 mg (38%)

Sodium 168 mg (7%)

Zinc 4.8 mg (48%)

Introduction

6

hypoglycaemic and hypolipidaemic properties. It is a very good source of iron and

essential oil shows antimicrobial and antioxidant properties (Azeez, 2008).

It is skin friendly herb, reduces itching and also purifies blood. It is supposed

to increase lactation and reduce nausea in pregnancy. Cumin seeds act on female

reproductive system by reducing inflammation of uterus. Extract from cumin has also

been shown to inhibit arachidonate-induced platelet aggregation (Srivastava, 1989).

Cumin seeds also have anticarcinogenic properties, as they protect laboratory animals

from developing stomach or liver tumors, this may stem from cumin’s free radical

scavenging abilities and its ability to enhance the liver’s detoxification enzymes

(Gagandeep et al., 2003).

The essential oil of cumin exhibits strong antimicrobial activity against

Escherichia coli, Staphylococcus aureus and Listeria monocytogenes (Gachkar et al.,

2007). Essential oils of cumin inhibited the total aflatoxin production of A. parasiticus

at relatively low concentrations (Farag et al., 1989). Pseudomonas aeruginosa causing

serious infections in burned patients have been shown to be inhibited by essential oils

from cumin (Hosseini et al., 2008).

The tea/infusion (often combined with other digestive herbs) has been used for

minor digestive upsets, abdominal spasms, migraine (associated with digestive

upsets), and diarrhea. Commonly cooked with beans and fried food to prevent gas.

The tea/infusion (or 2 to 3 drops of the oil on a sugar cube) has also been used to treat

nervous irritability.

In Chinese Traditional Medicine it is considered a stimulant and

antispasmodic and believed to benefit the heart and uterus. It has also been used

externally in liniments to stimulate circulation thus bringing warmth to the affected

area of aching muscles and joints. Also the leaves have been crushed and rubbed into

the painful area. A fomentation has been used to treat painful bruises and injuries.

Breeding:

Cumin is cross pollinated crop and bees often help in pollination. The scented

flowers are hermaphrodite (have both male and female organs) and are pollinated by

Introduction

7

Insects. The flowers being small and slender, artificial pollination is rather difficult

and varieties are developed by sib mating in enclosed chambers. Most of the varieties

available today are selections. The basic chromosome number in cumin is X=7, cumin

is diploid in nature so total chromosome No. 2n= 14 (Azzez, 2008). Based on

selections focused on local conditions a number of varieties are released for farmers

in each country.

Cumin variety:

There are two types of cumin generally found around the globe: 1) Black

Cumin, Kala or shah zeera, is quite rare and expensive and 2) White cumin, safed

zeera, is the seed more commonly available in ethnic shops and supermarkets (UCX,

2010).

A variety of cumin differs from area to area. There are many varieties of

cumin grown by the farmers in the world. But particularly in Gujarat, GC-1 (Gujarat

Cumin-1), GC-2, GC-3 and GC-4 etc. evolved by Agriculture Universities of Gujarat,

having higher yield potential are used for cultivation.

Among these varieties GC-1variety is most susceptible to disease and has very

low yield and not cultivated much by farmers. Whereas GC-2 was released in 1992,

became popular among the farmers because of its high yield potential and better grain

quality. Another variety GC-3 was released in 1998 having resistance to wilt and high

oil content. It became popular in wilt affected areas in spite of its seed splitting habit.

Recently in 2003 another variety GC-4 has been released. This variety has very high

yield potential, better seed quality and posses resistance to wilt (Main Spice Research

Station, Jagudan). The maturity varies between 110 and 115 days depending on the

variety.

Ecology and growing conditions:

Cumin is a tropical plant but it grows well in sub-tropical climate too. Cumin

requires a moderately cool and dry climate for its growth, with temperatures between

25 ˚C and 30 ˚C. High humidity during flowering and fruit set, causes fungal diseases

in this crop. It is badly affected by the diseases. The crop is highly sensitive to rain,

Introduction

8

and any rain during harvesting time reduces yield and crop quality. The crop is most

vulnerable to frost damage, especially at flowering and early seed formation stages. It

cannot grow in the shade.

Cumin can be cultivated in all types of soils but well drained sandy loam and

medium soils with a pH range of 6.8 to 8.3 are suitable for the crop. More acidic and

alkaline soil reduce yield (ICEX, 2009). Based on type of soil, crop requires 4-6

rounds of irrigation. Cumin plant has a good tap root system that makes it a drought

resistant plant.

Cumin requires a mild climate and grows from sea level to 3,300 m altitude. It

cannot stand excess of heat or moisture, therefore it is not grown in regions where it is

very hot and rainy. It is grown in north India as a winter crop, while in south India it

is grown both as late (winter) crop and early (summer) crop (Ilyas, 1980)

Cultivation:

Jeera is grown as a rabi crop in India. Sowing season of the cumin starts

during the month October continues till November to first week of December

sometimes. Cumin plant requires less water and more cold for its better growth with

ideal temperature of 25 to 30˚C. Jeera crop is highly sensitive to rain, if rain occurs

during harvesting time (February to March) quality of the jeera will be badly affected

besides quantity damage due to fungal diseases. It will turn black and will fetch the

lowest price in the market (Hudani, 2007).

The period of cultivation of cumin varies in different regions (Table 2, 3). A

well cultivated warm area is selected for sowing. The field is ploughed 4-5 times and

25 tons of compost or farmyard manure per ha are applied before the second

ploughing. The field is brought to a fine tilth by planking and then levelled to ensure

uniform irrigation and also to avoid washing away of the seeds to one side of the

field. The field is then divided into beds separated by irrigation channels. The seeds

are sown broadcast or drilled in rows 22.5 to 30 cm apart. Drilling of seeds requires

fewer seeds. Occasionally the seeds are mixed with sand to make them heavier and to

bring about uniform distribution when broadcasted in the field. The seeds are put into

Introduction

9

the soil, keeping 4-6 inch distance and covered with a little more soil. They are raked

to a depth of 1 cm into the soil.

Table 2: Period for cumin cultivation in different countries

India Syria Turkey Iran

Sowing Oct-Nov Nov-Dec Mar-May Mar-May

Harvesting February May July June

Peak Arrivals March-April July Mid July Mid July

Table 3: Cultivation period of cumin in India

Region Cultivation Period Harvesting Period

Saurashtra 20 Oct. to 20 Nov. 15 Jan to 15 Feb.

North Gujarat 01 Nov. to 10 Dec. 01 Feb. to 15 March

Rajasthan 10 Nov. to 10 Dec 01 March to 31 March

Seed rate vary from 12 to 15 kg / ha, depending upon method of sowing and

type of soil. Soaking of seeds for 8 hours before sowing is helpful in getting good

germination. Soaked seeds should be dried in shade to facilitate broadcasting. Sowing

at higher depth affects the germination of seeds adversely. Crop rotation should be

followed to avoid incidence of pest and diseases.

The field is irrigated immediately after sowing. The seeds are kept moist until

they sprout tiny plants. Seedlings emerge in 5-9 days. Sometimes it takes up to three

weeks to emerge out depending upon climatic condition and seed dormancy. The

second irrigation is done 6 days after sowing (DAS), in order to ease emergence of the

seedlings. The crop needs moderate, well regulated irrigations at 1-2 week intervals.

The subsequent irrigations are needed at tillering stage (30 DAS), milk stage (50

DAS) and at grain development stage (70 DAS). It does not require hoeing, but needs

weeding, once 2-3 weeks after sowing and another time 3 weeks later. The weeds

locally known as "jeerada/jeeralu" and “parjan” (Ilyas, 1980).

Cumin is harvested when the seed heads start changing colour to brown,

thresh and dry like the other Umbelliferae. The seeds are generally harvested

manually in the month of february-march. Generally, cumin crop takes about 120-125

Introduction

10

days to reach maturity. Harvesting should be done early in the morning by

cutting/uprooting the whole plants. Harvested crop should be dried in the threshing

yard, thrashed to separate the seeds. Seeds should be cleaned by winnowing (ICEX).

Storage:

Storage of cumin is very crucial as its quality preservation is important. Cumin

is packed in gunny bags and each bag contains 55 kg weight of cumin seed. Before

packing the cumin in the jute bags it is cleaned by machines in order to remove the

stalks, other foreign material, stones, dust etc. Generally cumin is harvested manually,

and that is why before packaging it is required to be cleaned properly and cut by

machine. During storage Cumin absorbs moisture immediately, therefore it is

advisable to store it in one layer plastic coated bags. Cumin is also packed sometimes

in cloth, paper or polythene bags depending on the requirements of the buyer. It is

preserved at least one to two feet away from the walls in order to save it from

moisture. The jute bags are staked over wooden carton or plastic sheet for more

security.

Cumin essential oil:

Cumin has about 2–4.5% of volatile oil and about 10% fixed oil, together with

tannins, oleoresin, mucilage, gum, protein compounds and malates. The characteristic

cumin odour is due to the presence of its essential oil. This odour and flavour is due

principally to the aldehydes present i.e, cuminaldehyde or cuminol, p-menth-3-en-7-ol

and pmentha 1,3-dien-7-ol). Studies of the chemical composition of cumin oil showed

the presence of the following components: pinene (0.5%), myrcene (0.3%), limonene

(0.5%), 1-8-cineole (0.2%), p-menth-3-en-7-ol (0.7%), p-mentha-1, 3-dien-7-ol

(5.6%), caryophyllene (0.8%), bisabolene (0.9%), pinene (13.0), P-cymene (8.5%),

phellandrene (0.3%), D-terpinene (29.5%), cuminaldehyde (32.4%), cuminyl alcohol

(2.8%), farnesene (1.1%) together with much smaller quantities of phellandrene,

terpinene, cis and trans sabinene, myrtenol, terpineol and phellandral.

The specific characteristics of cumin oil (essential oil) are:

• Colourless or pale yellow

Introduction

11

• Specific gravity (25º/25ºC), 0.905 to 0.925

• Optical rotation (20ºC), + 3 to + 8

• Refractive index, 1.501 to 1.506

• Solubility (80% ethanol), 8 vol

• Aldehydes (as cuminic aldehyde) 40 to 52%.

Cumin oil is a pale yellow to brownish yellow liquid; it occasionally displays

a greenish tint. Cumin essential oil can be adulterated in several ways. The oil is quite

sensitive to daylight, air, moisture, and metals as well as to alkali (Amin, 2000). It is

used in perfumes in trace amounts to introduce green-spicy and green woody top

notes. It is also used for "special effects" in modern aldehydic fragrances (Beis et al.,

2000).

Moreover, cumin oil shows a high antifungal activity against various

pathogenic fungi, and effective high antibacterial activity. Therefore, it is also used as

a fumigant or additive in the storage of foodstuffs. Principal constituent of the oil is

cuminaldehyde. Cuminaldehyde has also been introduced as the characteristic

constituent of the seed which acts as an aldose reductase and a-glucosidase inhibitor.

Cumaldehyde constitutes 20-40% of this oil while the rest is a mixture of

cymene, pinene, dipentene, cumine, cumic alcohol, phellandrene and terpineol.

Cumaldehyde separated from the oil is converted to thymol. The essential oil is

carminative and is used for flavoring liquors. The seed residue is rich in proteins and

fats and can be used as an animal feed (Ilyas, 1980).

Trade in cumin spice around the globe:

Global Scenario:

India is the largest procedure, exporter and consumer of cumin seeds in the

world, with Syria, Turkey and Iran being its main competitors in the world market

(Fig. 3). India’s share is 79% to global production, while that of Syria, Turkey, China

and Iran has been 9%, 4%, 4% and 4% respectively. The average annual production

of cumin in Syria is 10,000-20.000 tonnes; in Turkey, it`s 7000-15,000 tonnes; in

Iran, 5000-10,000 tonnes and in China 8000 tonnes. The cumin harvest in these

Introduction

12

countries is carried out by June and July, whereas February to March is the harvesting

period in India. The major consumer of cumin are China, Indonesia, UAE, UK, US,

Singapore, Malaysia, Bangladesh and Nepal beside India.

Fig. 3: Major producer and exporter of cumin worldwide

USA, Brazil, European Union, Middle East, Asia are the major export markets

for Indian cumin Seed. In the international market, Nepal and Sri Lanka is the major

importer preferring Cumin Seed of 95% to 96% purity, whereas the European market

has a strong preference for 100% purity for machine clean stocks. Both whole seeds

and powdered seeds are internationally traded. Cumin essential oil is also becoming

popular in the western hemisphere (ICL, 2009).

India faces stiff competition from Syria, which exports about 80% of its

produce. Other countries giving stiff competition are Turkey and Iran, as the bulk of

their production is exported and their prices are much lower than India’s. Cumin seeds

produced in Iran, Syria and Turkey are only for exports. Syria and Turkey consume

only 10% of their crops domestically. Though India is largest producer of cumin seed,

part of its produce consumed locally and the rest is exported. The global consumption

of cumin seeds is quite low compared to India and other Asian countries. Day by day

the demand for the best quality cumin seeds from India are increased (Fig. 4, 5).

UAE is the largest importer of cumin approximately 6 thousand tonnes (62.24

crore) from India followed by United States, Nepal, U.K. and Brazil (UCX, 2010).

Introduction

13

Fig 4: Country wise export of cumin

Fig 5: Year wise export of cumin from India

Indian Scenario:

The major seed spices growing area in India is concentrated in semi arid to

arid areas of Gujarat and Rajasthan. Therefore, both the states are esteemed as “seed

spice Bowl of India”. Out of 20 seed spices crops, cumin, coriander, fennel,

fenugreek, dill and ajwain contributed more than 95 % towards area and production.

Cumin and fennel are dominant seed spices of Gujarat while coriander and fenugreek

are important in Rajasthan.

Introduction

14

In India, cumin is cultivated in almost all the states, but leading states are

Rajasthan, Gujarat. West Bengal, Uttar Pradesh, Madhya Pradesh, Punjab, Tamil

Nadu Andhra Pradesh, Bihar, Orissa, Assam, Karnataka and Haryana also make

significant contribution to Indian output (UCX, 2010).

Within India, Gujarat is the largest producer of cumin seeds contributing about

60% of the country’s total production, followed by Rajasthan (Fig. 6). Gujarat and

Rajasthan together contribute more than 90 percent of the total cumin produced in the

country (Fig. 7). The annual production ranged between 1.5 to 2 lakh metric tons. A

significant percentage of the total production is used for domestic consumption (ICL,

2009).

Fig. 6: State wise cumin production in India

Fig 7: Area and production in Gujarat and Rajasthan

Introduction

15

In Gujarat the prominent districts are Sabarkantha, Banaskantha, Mehsana,

Patan, Junagadh, Porbandar, Jamnagar, Rajkot, Bhavnagar, Amreli, Surendranagar,

and Ahmedabad where as Barmer, Jalore, Nagaur, Pali, Ajmer, Bilwara, Tonk,

Jodhpur, Jaisalmer, Sirohi, Sikar and Bikaner districts in Rajasthan produce larger

amount of cumin (Fig. 8). Nimach and Mansoor districts of Madhya Pradesh also

contributes significant production.

Fig 8: District wise cumin production in Gujarat and Rajasthan.

Unjha in Gujarat is the main trading centre for jeera in the country. Mandi in

Nagaur is the largest centre for cumin seed along with Niwai and Kekri in Rajasthan

(Hudani, 2007). Delhi, Jaipur and Rajkot are also known as major terminal markets

for jeera. The Indian exports came down to just 8,000 tons in 2003-04, but exports

picked up in the following years to 13,750 tons. India also exports cumin powder and

oleoresin extracted from cumin seeds (NCDEX, 2011).

Area under jeera is persistently declining in Rajasthan since 2002-03 onwards,

as the crop is highly vulnerable to weather condition in the state and the yield is less.

With this, the output has also taken a hit and ruled at 0.3 tons in 2008-09 compared to

higher output in 2003-04 of about 1.2 tons. On the other hand, Gujarat state has

witnessed continuous increase in acreage and output. The state has average area and

output of about 2.36 lakh hectares and 1.13 tons respectively during the period

between 2002-03 and 2008-09 (Fig. 9) (ICEX).

Introduction

16

Fig 9: Area (lakh ha.) and production (lakh ton) of jeera in Gujarat and Rajasthan

From the given data above, it can be stated that production of cumin is varying

from year to year. There has been a recent increased demand on cumin while its

production is limited and decreased. Significant loss in cumin yield can be attributed

to the adverse effect caused by pathogenic attack and climate change (Omar et al.,

1997). There are several pathogens which are responsible for diseases in cumin, like

Fusarium oxysporum f. sp. cumini causes Fusarium wilt disease and Alternaria

burnsii which cause Alternaria blight disease in cumin. The exports from India have

declined over the past few years just because Syria and Turkey are selling cumin with

a lesser price compared to India. This scenario, with higher production yet lower

exports, is proving to be very harmful for the prices. Several biotic and abiotic

affecting factors have made the farmers to make their mind to switch over to the other

crops.

Biotic and abiotic factors affecting cumin production:

Cumin is very weather sensitive plant and it gets affected by the slightest of

change in weather. Dried cumin seeds must be stored in moisture-proof containers

away from direct sunlight. High humidity during flowering and fruit set, causes fungal

diseases in this crop. Fungal diseases, inappropriate sowing dates and successive

planting are the main factors reducing cumin yield, with 38% contributed by fungal

diseases, 30% by sowing date and 5% by successive planting (Kamkar et al., 2011).

Introduction

17

The average yield of the crop is low due to lack of superior varieties, scientific

crop production technology and vulnerability to diseases like wilt and blight. Wilt is

most common disease, results in yield losses upto 35% in cumin in some districts of

Gujarat and Rajasthan (Vyas and Mathur, 2002).

Various insects and pests are also found to be the major devastating agents

leading to significant drop in cumin yield. It includes Aphids, a major pest of cumin

crop which sucks the sap of tender parts and leaf eating caterpillar, causes damage to

the foliage of plants, both leading to the reduction in crop yield

(www.indianspices.com).

But, the major crop damage is caused by fungal diseases and the major fungal

pathogens affecting cumin yield and growth are Fusarium oxysporum f. sp. cumini

and Alternaria burnsii that causes wilt and blight respectively. The other disease

found in cumin is powdery mildew caused by Erysiphe polygoni. Its effect in the field

of India is not too much. Moreover, it can be easily controlled by controlling agents

immediately. But wilt and blight diseases of cumin are devastating.

Diseases:

1. Wilt of cumin:

Cumin cultivation has received a serious threat from F. oxysporum f. sp.

cumini, a causative agent of wilt syndrome, which devastates the total standing crop

(Patel et al., 1957). The wilt disease manifests from the seedling stage itself and

continues till the maturity of the crop and toxin from the fungus is thought to be

responsible for the disease (Gour and Agrawal, 1988; Gour and Agrawal, 1988). It

causes a severe disease of cumin causing pre-mature plant death. Disease symptoms

included sudden red-brown shriveling of the foliage, growth retardation and pre-

harvest plant death (Pappas and Elena, 1997). A total six isolates of Fusarium

oxysporum f. sp. cumini, isolated from different cumin growing areas of Rajasthan,

had cultural and morphological variability on different agar and broth media.

Maximum mycelial growth and conidia formation was observed on Czapek Dox agar

followed by PDA. The isolates were classified on the basis of their pathogenic

Introduction

18

variability. The most virulent strain showed 8% wilt incidence (Bardia and Rai,

2008).

This disease is caused by Fusarium oxysporum f. sp. cumini, was reported for

the first time as the causative agent of cumin wilt by Patel and Prasad, (1963). Wilt

disease of cumin by F. oxysporum has also been reported in the Island of Chios,

Greece (Pappas and Elena, 1997).

Fusarium oxysporum f. sp. cumini:

Fusarium oxysporum strains are ubiquitous soil inhabitants that have the

ability to exist as saprophytes and degrade lignin and complex carbohydrates

associated with soil debris. They are also pervasive plant endophytes that can colonize

plant roots. Other than cumin it also infects potato, chickpea, sugarcane, garden bean,

cowpea, prickly pear, cultivated zinnia, pansy, Assam rattlebox, Baby's breath and

Musa spp.

Scientific Classification:

Kingdom : Fungi

Division : Ascomycota

Class : Sordariomycetes

Order : Hypocreales

Family : Nectriaceae

Genus : Fusarium

Species : F. oxysporum f. sp. cumini

Distribution and Biology:

The distribution of Fusarium oxysporum is known to be cosmopolitan.

However, the different special forms (f. sp.) of F. oxysporum often have varying

degrees of distribution and varying appearances. F. oxysporum is an abundant and

active saprophyte in soil and organic matter, with some specific forms that are plant

Introduction

19

pathogenic. Its saprophytic ability enables it to survive in the soil between crop cycles

in infected plant debris (Gonsalves and Ferreira, 1993).

In general, the aerial mycelium first appears white, and then may change to a

variety of colours ranging from pinkish red to dark purple according to the strain of F.

oxysporum. In the solid culture media, such as potato dextrose agar (PDA), the aerial

mycelium first appears white and then may change to a variety of colors ranging from

violet to purple according to the strain of F. oxysporum (Smith et al., 1988). The

optimum temperature for growth on artificial media is between 25-30°C and the

optimum soil temperature for root/stem infection is 30°C or above. However,

infection through the seed can occur at temperatures as low as 14°C. The fungus can

survive either as mycelium or as spore (Gonsalves and Ferreira, 1993).

F. oxysporum produce three types of asexual spores; microconidia,

macroconodia and chlamydospores (Agrios, 1988). Most of the species of F.

oxysporum exist and survive in the soil in the form of chlamyospores (Smith et al.,

1988). F. oxysporum f. sp. cumini also produces all three types of asexual spores;

microconidia, macroconidia and chlamydospores (Agrios, 1988). Most species of F.

oxysporum exist and survive in the soil in the form of chlamydospores (Goyal et al.,

1973). Microconidia, one or two celled spores are most abundantly and frequently

produced by the fungus under all conditions. It is also the type of spore most

frequently produced within the vessels of infected plants. Macroconidia are three to

five celled, gradually pointed and curved toward the ends. These spores are

commonly found on the surface of plants killed by this pathogen as well as in

sporodochia like groups. Chlamydospores are round, thick-walled spores, produced

either terminally or intercalary on older mycelium or in macroconidia. These spores

are either one or two celled (Patel et al., 1957).

Disease cycle:

The fungal pathogen Fusarium oxysporum is an abundant and active

saprophyte in soil and organic matter with some specific forms that are plant

pathogenic (Smith et al., 1988; Garrett, 1970). F. oxysporum is present in every type

of soil all over the world (Burgess, 1981) and is also considered a normal constituent

of fungal community in rhizosphere of plants (Gordon and Martyn, 1997). The

Introduction

20

chlamidiospores of Fusarium may persist in soil for many years in the absence of a

susceptible host (Agrios, 1991). It affects a wide variety of hosts at any age. Tomato,

tobacco, muskmelon, watermelon, glycine max, ginger, legumes, cucurbits, guava

(Gupta et al., 2010), capsicum (Kelaniyangoda et al., 2011; Srivastava et al., 2011),

sweet potatoes, Radish (Toit et al., 2003) and bananas (Daly, 2006) are a few of the

most susceptible plants, including cumin. Fusarium wilt disease usually increases in

warm areas and under dried conditions (Tawfik and Allam, 2004a).

Fig. 10: Life cycle of Fusarium oxysporum

Spores of the fungus in the soil germinate and grow towards the nearby roots

of plants in response to chemicals exuded from the root. Infection takes place on the

secondary and finer roots and proceeds into larger, primary roots through xylem

vessels before entering the rhizome (Daly et al., 2006). Wet weather favours the

spread of disease. The Fusarium wilt fungi invade the root systems or other

underground parts of their host plants through wounds that are caused naturally by the

growth of young rootlets through the soil and by wounds in older roots that are made

during transplanting and cultivating or by root-feeding organisms, such as insects or

nematodes. It can also enter from the formation point of lateral roots. The fungus can

invade a plant either with its sporangial germ tube or mycelium.

Introduction

21

Once within the plant, the fungus grows and multiplies in the vascular system

of the roots. It grows through the root cortex intercellularly. When the mycelium

reaches the xylem, it invades the vessels through the xylem's pits. At this point, the

mycelium remains in the vessels, where it usually advances upwards toward the stem

and crown of the plant. As it grows the mycelium branches and produces

microconidia, which are carried upward within the vessel by way of the plants sap

stream. The spores (macroconidia and microconidia) that are transported in the sap

stream become lodged and germinate and affect new plant parts. When the

microconidia germinate, the mycelium can penetrate the upper wall of the xylem

vessel, enabling more microconidia to be produced in the next vessel. Thus the fungus

extends its colonization as it grows in the vascular tissue of the host. Due to the

growth of the fungus within the plant's vascular tissue, the plant's water supply is

greatly affected. The normal flow of liquids and nutrients from the roots to the foliage

is greatly reduced or stopped because the conducting tissue becomes partially plugged

or killed by fungal mycelium and spores, or by the overgrowth of neighboring cells.

This lack of water induces the leaves stomata to close, the leaves wilt, and the plant

eventually dies. It is at this point that the fungus invades the plant's parenchymatous

tissue, until it finally reaches the surface of the dead tissue, where it sporulates

abundantly. The resulting spores can then be used as new inoculum for further spread

of the fungus (Gonsalves and Ferreira, 1993). Toxic substances are believed to be

secreted by interaction of the fungus and the host plant. These materials apparently

cause the wilting and eventual death of the plant (Fig. 10).

Fusarium oxysporum is primarily spread over short distances by irrigation

water. It is also spread by contact and transportation contamination through

contaminated seeds, soil, plants, equipments, training stakes, and packing crates or

shoes. The fungus can be spread over long distances either in infected transplants or

in soil. Although the fungus can sometimes infect the fruit and contaminate its seeds

but the spread of the fungus by way of the seed is very rare (Agrios, 1988). Besides

cumin other host plants affected by this fungus are banana, sweet potato, cucurbits,

tomato, cowpea, sugarcane, garden bean, chickpea etc. A month old plants are the

most affected with this disease and stem is the most affected part.

Introduction

22

Symptoms:

Typical symptoms of Fusarium wilt include a drooping and yellowing of the

leaves, often starting on one side and stunting of the plants. Disease symptoms often

commence at the base of the stem and progress upwards, causing the leaves and

flower heads to wilt, wither, and die. Lower parts of the stem are dark and

discoloured.

In field condition the infected plants first show changes in colour of leaves

from green to yellow, beginning from oldest leaves and extending upward to the

younger leaves leading to wilting of the entire plant which ultimately dries up and

could easily be pulled out of the soil (Gour and Agrawal, 1988).

Infected cumin plants show peculiar symptoms of dropping and yellowing

leaves, leading to mortality of the entire plant. Attack of wilt is severe in younger

plants. Fusarium wilt first appear as slight vein clearing on the outer portion of the

younger leaves, followed by epinasty (downward drooping) of the older leaves. At the

seedling stage, plants infected by F. oxysporum may wilt and die soon after symptoms

appear. In older plants, vein clearing and leaf epinasty are often followed by stunting,

yellowing of the lower leaves, formation of adventitious roots, wilting of leaves and

young stems, defoliation, marginal necrosis of remaining leaves and finally death of

the entire plant (Gonsalves and Ferreira, 1993).

The disease generally appears by the end of December or when the crop is

about a month old. Fusarium wilt disease usually increases in warm areas and under

dried conditions (Tawfik and Allam, 2004a). The pathogen can cause disease to the

plant at any stage of growth and development. The disease generally appears as

patches and is characterized by wilting of affected plants. After the appearance of

wilting, the whole plant dries up (Patel et al., 1957). The toxins of Fusarium spp have

been considered to be the active factors in causing wilt syndrome (Gour and Agrawal,

1988).

There is no chemical control for this disease. Crop rotation and use of Neem

cake are helpful in checking spread of the fungus vis-Ã -vis disease. Seeds collected

from disease free plots should only be used for sowing. Affected cumin plants in early

Introduction

23

stages show minute whitish spots on leaves, petiole, stem pedicel and seeds. In severe

condition, it looks as the plants have been dusted with white powder. At later stages

of attack seeds become white, shriveled, and light in weight.

2. Blight of cumin:

This disease caused by Alternaria burnsii, was first reported as the causative

agent of cumin blight (Uppal et al., 1938). In Pakistan, Alternaria burnsii causing

blight of cumin was recorded for the first time by Shakir et al., (1995). The disease is

also known as black blight. The disease occurs during flowering stage (Uppal et al.,

1938; Patel et al., 1957; Gemawat and Prasad, 1971).

Alternaria burnsii:

Alternaria burnsii grow as mycelium and produces conidiophores vertically

celled, light in colour. Conidia are born at the tip of conidiophores and they are

obolavate in shape, having transverse longitudinal or oblique septa. Conidia have long

beak and pale dirty brown colour. Freshly produced conidia readily germinate in

nutrient medium. Off seasonal rain, cloudy weather with dew formation and a

temperature of 23-28 ˚C are favourable conditions for germination of A. burnsii. The

optimum pH for its germination lies between pH 6-7. In warm-wet conditions the

disease spreads in the whole field within a short period causing complete failure of the

crop. In such conditions the crop must be protected with frequent application of

fungicides and farmers start fungicide application from one month after sowing and

continue up to the maturity of the crop (Jadeja and Pipliya, 2008).

Scientific Classification:

Kingdom : Fungi

Phylum : Ascomycota

Subdivision : Pezizomycotina

Class : Dothideomycetes

Order : Pleosporales

Family : Pleosporaceae

Introduction

24

Genus : Alternaria

Species : Alternaria burnsii

Distribution and biology:

Species of the Alternaria genus are examples of ubiquitous plant pathogens

that may contaminate a wide variety of crops in the field and cause post-harvest decay

of various fruits, grains, and vegetables (Chelkowski and Visconti, 1992;

Weidenborner, 2001).

Plant pathogenic Alternaria species survive between crops as spores and

mycelium in infected plants residues or in and on seeds. Most often, the fungus grows

and sporulates on plants residues during periods of rain, heavy dew and under

condition of good soil moisture (Laemmlen, 2001). A temperature of 23-28˚C are

favourable condition for germination of A. burnsii. The optimum pH for its

germination lies between pH 6-7 (Jadeja and Pipliya, 2008).

On potato dextrose agar, the pale olive-green hyphae are 1.7 to 5.8 µ. in

diameter; the solitary or fasciculate, erect, simple or branched, straight or bent,

geniculate conidiophores, sometimes with a single terminal scar, are 3- to 5-celled.

The spores of Alternaria species are often beaked and always multicelled. The cells

are divided longitudinally and transversely. The spores are dark and borne singly or in

chains. Formation of chlamydospores has been reported in A. burnsii (Uppal et al.,

1938).

Disease Cycle:

According to report on plant disease by Department of Crop Sciences,

University of Illinois, Alternaria fungus overwinters as dormant mycelium in diseased

and partly decayed crop refuse, in weeds and possibly in the soil. Fungus conidia can

survive under warm, dry conditions for several months. Conidia produced on diseased

plants or crop refuse may be blown by the wind for long distances. Clothing, tools and

other equipment, running and splashing water are other means of spread. The

germinating spores penetrate susceptible tissue directly or through wounds and soon

produce a new crop of conidia that are further spread by wind, splashing rain, tools, or

Introduction

25

workers. At least 18 hours of high relative humidity, producing leaf wetness, is

required before infection can occur (Uppal et al., 1938).

The disease also spread in the field through seeds. Secondary spread of the

diesease is through wind borne conidia. Disease development require high relative

humidity (90%) for three days and temparature of 23˚C to 28˚C. The disease take

severe form if high humidity persists and in case of rains after infection. The period

between infection and the appearance of symptoms varies from 3 to 12 days. The

optimum temperature for the development of A. burnsii is 26˚C to 30˚C and optimum

pH for growth is 6.0-7.0. The fungus is highly pathogenic to its own host (in the

presence of moisture only) and cross-inoculation experiments on eight other plants

gave negative results (Uppal et al., 1938).

Figs 11: Life cycle of Alternaria burnsii

Alternaria generally attacks the aerial parts of its host. In the leafy vegetables,

symptoms of Alternaria typically start as a small, circular, dark spot. As disease

progresses, the circular spot may grow to ½ inch (1 cm) or more in diameter and are

usually gray, gray-tan or near black in color. The lesion is often covered with a fine,

black, fuzzy growth. This growth is the fungus sporulating on the dying host tissues.

Dark, sunken lesions are usually the expression of Alternaria infections on roots,

Introduction

26

tubers, stems and fruits. Many Alternaria species also produce the toxins that diffuse

into the host tissues ahead of the fungus (Fig. 11) (Laemmlen, 2001).

The cumin plant is highly susceptible to blight only after flowering stage. This

is due to absence of maltose and sucrose before flowering while their concentration

gets increased at flowering stage. Sucrose and maltose are good source of carbon and

helps in growth and sporulation of fungus. In addition to sugars, DL-serine is found to

be present only after flowering stage and phenylalanine level rises soon after

flowering and declines with the progress of disease. Apart from this, Maltose is found

to be the best carbon source and DL-serine and phenylalanine is observed to be good

nitrogen sources, both for growth and sporulation during physiological studies of the

pathogen. During the flowering maltose and sucrose levels appears to be higher in

plants which facilitate infection by A. burnsii (Gemavat and Prasad, 1971). To save

the crop from infection of this fungus, the crop should be kept free from weeds. Crops

requiring more irrigation and mustard crop should not be grown in the vicinity of this

crop.

Besides cumin Alternaria spp. also affects various ornamental plants, tomato,

onion (Karthikeyan et al., 2005), crucifers (Kucharek, 1994), carrot and basils (Taba

et al., 2009).

Symptoms:

Blight affected plants show minute, whitish, necrotic areas which turn purple,

later brown, and finally black, the disease ultimately leads to the death of the whole

plants or only of the affected parts (Uppal el al., 1938). Mostly diseased plants fail to

produce seeds and if seeds are produced they remain shriveled, light in weight and

dark in colour. During disease in the field, all the above-ground parts of plants appear

as burnt by fire (Shakir et al., 1995).

Control of cumin wilt and blight:

Most of the damage to cumin is done by Fusarium and Alternaria. Hence,

several physical, chemical and biological methods have been employed worldwide to

control the both pathogenic fungi of cumin.

Introduction

27

• Physical methods:

Soil solarization and soil fumigation with methyl bromide are two common

physical methods adopted by farmers for the control of Fusarium wilt and Alternaria

blight disease (Tawfik and Allam, 2004a). Soil solarisation, residue incorporation and

summer irrigation resulted in significant reductions in cumin wilt by Fusarium

oxysporum f. sp. cumini (Israel et al., 2005). Similarly, Brassica amendments (mustard

oil-cake) and summer irrigation when employed together also resulted in significant

reduction in F. oxysporum populations and wilt incidence on cumin (Mawar and

Lodha, 2002; Champawat and Pathak, 1988 and Sharma et al., 1995). Soil solarization

with 25 μm LLDPE plastic cover for 15 days in summer have been proved most

effective in reducing wilt incidence to 26.27% (Jadega and Nandoliya, 2008). Crop

rotation and use of Neem cake are two other options to control the Fusarium wilt

disease (TNAU, 2008).

Soil fumigation with methyl bromide (Larkin and Fravel, 1998) can provide a

control measure against the disease but may be of limited application value for large

scale production systems in the open field. Moreover, methyl bromide is considered

an ozone-depleting compound and has potential risk on the living environment and

human health.

Limited efficiency has been suggested in controlling the disease using

combined crop rotation and other cultural practices including plough, irrigation and

fertilization (Arafa, 1985; Champawat and Pathak, 1990).

• Chemical methods:

Various fungicides like Carbendazim, Thiophonate methyl, Carboxin,

Chlorothalonil and Captan have been shown to protect cumin crop against Fusarium

wilt with varying potential. Maximum inhibition of Fusarium oxysporum f sp. cumini

has been observed with carbendazim and thiophonate methyl (Bardia and Rai, 2007).

Similary, application of carbendazim granules one month after sowing was found to

be effective in controlling cumin wilt (Jadeja and Nandoliya, 2008). Prochloraz,

thiram, toclofos-methyl, hymexazol, azoxystrobin and carboxin have also been used

as fungicides and their inhibitory activities against the pathogen by mycelial growth

Introduction

28

inhibition have promising effect in the field (Song et al., 2004). Trimethyl thiuram

disulphide (0.1 g g-1 seeds) was found to be the best fungicide which is on par with

propiconazole, carbendazim and copper oxychloride (0.1 g g-1 seeds) for the control

of wilt (Deepak and Lal, 2009). Chemical fertilizer like NPK at 40kg/ha have also

shown positive results (Deepak and Lal, 2009). Salicylic acid, acetylsalicyclic acid

and rizolex-T have also been proved useful in inhibiting Fusarium spp and other

pathogenic fungi affecting different crops in field.

Soaking cumin seeds or soil drenching with antioxidant solutions like

salicylic, ascorbic, coumaric, benzoic acids, and propylgalate before planting resulted

in resistant cumin seedlings against infection with the Fusarium oxysporum f. sp.

cumini and Acremonium egyptiacum (Monaim and Ismail, 2010).

The cumin blight caused by A. burnsii is the threatening disease which can be

controlled chemically by using two fungicides, mancozeb and curzat mainly. It is

concluded that five sprays of mancozeb (0.25%) or curzat (0.1%) at 10 days interval

after initiation of flowering were effective to manage the cumin blight in field (Jadeja

and Pipliya, 2008). Other fungicides like captan, thiram and dithane-M-45 are also

used to control Alternaria blight disease. Seed treatment and spraying of 0.2%

solution of Dithane-M-45 4 times at 10 days interval commencing from 40 days after

sowing is recommended. 1 ml soap solution/ liter water for better efficiency of

fungicide is added. Seed treatment with Captan or Thiram, 4 g/kg of seed and

spraying of 0.2% solution of Dithane-M-45 for four times at 10 days interval

commencing from 40 days after sowing is recommended (TNAU, 2008). Substituted

tetra hydro-pyrazolo-[3,4-c]-pyrazole and dihydro-3H-pyrazolo-[3,4-c]-isoxazole

have also been found to show antifungal activity against Alternaria burnsii (Sareen et

al., 2011).

But the continuous use of such chemical fungicides on plants has been shown

to have negative impact on soil, plant and beneficial microorganisms. Moreover,

limited effectiveness and environmental issues has made the use of chemical

fungicides very less. Apparently, there is a need for an efficient control measure that

complies with the recent crop production trends of the environmentally sustainable

agriculture. Therefore, great emphasis has been currently directed, towards the use of

biological control agents that combats efficiently with the pathogenic fungi and also

Introduction

29

possess no environmental hazards. It includes the use of antagonistic microorganisms,

biological elicitors and commercial suspension of biofungicides or bioelicitors.

• Biological methods:

There are numerous plant associated fungi and bacteria that have been shown

to have antagonistic activity against F. oxysporum and A. burnsii and are thus utilized

as biological control. Trichoderma harzianum, T. humatum, T. viride and Bacillus

subtilis exhibited the antagonistic activity on F. oxysporum. The lowest percentage of

infection was found by pre-sowing treatment of cumin seeds with T. harzianum and

B. subtilis (Tawfik and Allam, 2004a). Similarly, infestation of T. harzianum strain in

the soil was very much effective in reducing wilt incidence of cumin crop during field

experimentations (Israel et al., 2005; Gangopadhyay and Gopal, 2010). T. harzianum,

T. viride and Aspergillus niger are reported for their antagonistic activity against A.

burnsii. Out of these, T. harzianum have most effective antagonistic effect on A.

burnsii (Deepak et al., 2008). Also T. harzianum and T. viride have been shown to

result in 100% inhibition of A. burnsii (Jadeja and Pipliya, 2008). Peanut haulms

compost as a carrier of three Trichoderma species i.e. T. harzianum, T. hamatum and

T. koningii exhibited different morphological and chemical characteristics shown its

ability to control Fusarium wilt of cumin prominently (Haggag and Abo-Sedera,

2005). Soil infestation with a native heat tolerant strain of Aspergillus versicolor has

been shown to have higher potential than T. harzianum in reducing F. oxysporum

propagules in the soil (Israel and Lodha, 2005).

Field studies conducted under artificial inoculation conditions revealed that

two sprays of aqueous extracts (10%) of Calotropis procera, Azadirachta seed kernel

or Azadirachta leaf suppressed Alternaria blight and enhanced seed yield in cumin

(Gangopadhyay et al., 2010). Alternaria burnsii, the causal organism of blight disease

of cumin have been shown to get inhibited by 5% and 10% extract of garlic cloves

and ginger rhizomes (Jadeja and Pipliya, 2008). Antifungal activity against F.

oxysporum f. sp. cumini has also been shown by leaf extracts of Datura stramonium

and Calotropis procera (Sharma and Trivedi, 2002). Treatment with carbendazim in

combination with T. harzianum against F. oxysporum resulted in significantly lowest

disease incidence and highest yield (Bardia and Rai, 2007). Seed extract of

Introduction

30

Trachyspermum copticum, leaf extract of Lavandula angustifolia and flower extract

of Rhjeum ribes effectively inhibited the radial growth and spore germination of F.

oxysporum in in vitro studies (Ghorbany and Salary, 2005).

Treatment of cumin seeds with cumin oil at 4% concentration caused

reduction of disease symptoms, which resulted from inoculation with F. oxysporum

and F. solani compared to control. A significant increase was recorded in plant height,

number of branches, fresh weight of plant and root weight after 4 weeks from

inoculation (Hashem et al., 2010).

Looking in to the reports and work of several researchers, in the present study

Trichoderma harzianum and Bacillus subtilis were used to control the disease causing

pathogens in cumin plants.

It is the employment of natural enemies of pests or pathogens in the

eradication or control of their population. It could also be in the form of induction of

plant resistance using non-pathogenic or incompatible micro organisms. The most

recent methods of managing plant diseases, use of pathogen produced toxic

metabolites which is responsible for disease condition in plants. There are number of

instances where the use of culture filtrate and extracellular enzymes have proved to be

more durable in their effects and these have the advantage of not requiring repeated

periodic applications as in the case with chemical fungicides (Amusa and Odunbaku,

2007).

Biological seed treatment offers a potential alternative for chemical

management of soil borne pathogenic fungi (Handelsman and Stabb, 1996). Few of

the metabolic changes elicited by biocontrol agents are structural alterations in plant

cell wall, synthesis of phytoalexins, induction of pathogenesis related proteins and

other defense related enzymes (Kishore et al., 2006).

The genus Trichoderma is widespread in soil and on decaying wood and

vegetable matter. It is able to secrete a variety of enzymes to breakdown recalcitrant

plant polymers into simple sugars for energy and growth. Several modes of action

have been proposed to explain the biocontrol of plant pathogens by Trichoderma.

These include the production of antibiotics and cell wall degrading enzymes,

Introduction

31

competition for key nutrients, parasitism and stimulation of plant defense

mechanisms. The dual role of antagonistic activity against plant pathogen and plant

growth promoter make Trichoderma strains appealing alternatives to hazardous

fumigants and fungicides (Jayalakshmi et al., 2009). Root colonization by

Trichoderma strains results in massive changes in the genome and metabolome.

Plants recognize microorganisms by a variety of mechanisms and the Trichoderma-

plant interaction might be similar to plant-pathogen interactions that are governed by

pathogen-derived Avr genes and corresponding resistance genes in the plant host

(Chet et al., 2006; Reino et al., 2008). Changes in plant metabolism lead to

accumulation of antimicrobial compounds.

Trichoderma harzianum is a fungus that is also used as a fungicide. It is used

for foliar application, seed treatment and soil treatment for suppression of various

disease causing fungal pathogens. It grows tropically towards the hyphae of other

fungi, coil about them in a lectin mediated reaction and degrade cell walls of the

target fungi by the secretion of different lytic enzymes. This process (mycoparasitism)

limits growth and activity of plant pathogenic fungi. Some Trichoderma rhizosphere-

competent strains colonize the entire root surface and usually limited to the 1st and 2nd

layer of cells and only in the intercellular spaces of the root tissue (Chet et al., 2006).

In addition to these direct effects on other fungi, recent evidence indicate that many

Trichoderma spp., including T. virens, T. atroviride and T. harzianum, can induce

both localized and systemic resistance in a range of plants to a variety of plant

pathogens by inducing resistance and inactivation of pathogen’s enzymes (Harman,

1998). Trichoderma spp. produce at least three classes of compounds that elicit plant

defense responses: peptides, proteins and low-molecular weight compounds. The

main interest is in those compounds that exhibit antibiotic activity since they are more

likely to be implicated in the effectiveness of the strain producing them as a biological

control agent (Reino et al., 2008).

Bacillus subtilis, known as the hay bacillus or grass bacillus, is a Gram-

positive, Catalase-positive bacterium commonly found in soil. It is rod-shaped, and

has the ability to form a tough, protective endospore, allowing the organism to

tolerate extreme environmental conditions. Unlike several other well-known species,

B. subtilis has historically been classified as an obligate aerobe (Wikipedia).

Introduction

32

Bacillus is the most common types of bacteria isolated from soil samples and

can account for up to 36% of the bacterial populations. The rhizosphere, which

comprises the region close to the surfaces of roots, and the root surface itself, the

rhizoplane, are colonized more intensively by micro-organisms than the other regions

of the soil. Rhizobium bacteria, Bacillus spp., Pseudomonads and Mycorrhiza fungi

are among the best-known colonizers of this region. Many micro-organisms from the

rhizosphere can influence plant growth and plant health positively and are therefore

often referred to as “Plant Growth Promoting Rhizobacteria (PGPR)” (Schippers,

1992). Beneficial action of culture supernatants of B. subtilis on plant growth

resembles that of phytohormones mainly enhancing growth (Bochow et al., 2001;

Idris et al., 2004)

The antagonistic effects of Bacillus subtilis may be attributed to the

competition which occurs between the two organisms require the same nutrients and

the use of these nutrients by one reduce the amount available to the other (Abo-

Elnaga, 2006). The antagonistic effect may also be attributed to toxins or antibiotics

secreted in the growth medium as well as the production of antifungal volatiles.

Experiments done by Kilian et al., (2000); provided direct evidence of the

involvement of resistance-inducing mechanisms in the biological efficacy of Bacillus

subtilis. Bacillus subtilis can contribute in different degrees to the reduction of disease

and the enhancement of yields, depending on the plant, the environmental conditions,

the application form, and the time of application (Kilian et al., 2000). The various

effects produced by Bacillus subtilis and the mechanisms proposed for this effect as

well as the interactions between them is shown in the figure 12.

Fig 12: Effect of B. subtilis in pathogen control

Introduction

33

Direct use of antagonistic microorganisms or antimicrobial plant

extracts/cakes in controlling the pathogen spread have not seen to be effective much

on plant’s growth and development. In the present study, Headline and monitor, two

commercially available biofungicides have been used to check their efficacy in

controlling the disease and induction of plant immunity. Headline contains

Pyraclostrobin a compound that helps in disease control and plants growth where as

monitor contains T. viride as controlling bioactive compound. Moreover, there are

number of instances where the use of biological elicitor or culture filtrates have

proved to be more durable in their effects and signals to induce plant’s resistance and

development. These measures have advantage of not requiring repeated periodic

applications as with the chemical fungicides (Amusa and Odunbaka, 2007).

Headline:

Headline 20% WG is strobilurin group of fungicide. Pyraclostrobin is the

active ingredient of headline which has been commercialized as biofungicide by a

very famous chemical company Baden Aniline and Soda Factory (BASF, Germany).

Pyraclostrobin is a new broad-spectrum strobilurin fungicide. Until recently, the

fungicides focused on control of phyto-pathogens with the sole purpose of reducing

inoculum. After the launching of strobilurin, and with the evolution of this group of

chemical products, the concept of disease control gained new perspectives, especially

when considering the advantages obtained by the action of positive physiological

effects on plants. Strobilurins are natural products isolated from mushrooms

(basidiomycetes) genera Strobilurus.

These strobilurins are biosynthesized in fungus from phenylalanine via the

shikimic acid cycle. Natural strobilurins have been isolated by chromatographic

techniques, and their molecular formulae have been identified by high resolution mass

spectrometry. Many scientists excluded the idea of looking for fungicides in fungus.

However, once Anke et al., (1977) isolated Strobilurin-A from the liquid cultures of

the mushroom Strobilurus tenacellus many scientists looked for other strobilurins in

many other fungi. Strobilurins were named in the order of their discovery as

strobilurin-A followed by strobilurin-B, C, D etc. one of the synthetic analogue is

pyraclostrobin. Pyraclostrobin-carbamate was discovered by BASF scientists in 2000,

and announced the same year and marketed as Headline. The structure of

Introduction

34

pyraclostrobin (Fig.13) is characterized by its nature as a derivative of carbamate as

the toxiphoric group. The most important attribute of Pyraclostrobin is its wide range

of fungicidal activity (Ammermann et al., 2000).

Fig. 13: Pyraclostrobin (Headline) used in agriculture (Balba & Hamdy, 2007)

The physiological effect of Pyraclostrobin was reviewed under several levels

of complexity, from the greening effect frequently mentioned and the enhancement of

stress factors in field and under controlled conditions, to the influence of hormonal

regulation and assimilation of carbon and nitrogen by the plant (Balba and Hamdy,

2007).

Mode of action of Headline (Pyraclostrobin):

Fig. 14: Mode of action of Headline in plant cell

Introduction

35

Pyraclostrobin is most strongly lipophilic or attracted to waxes on and within

the leaf. According to BASF, pyraclostrobin penetrates the leaf within minutes of

application and is stored primarily in the waxes of the leaf cuticle. This property

allows being rainfast two hours after application. Because of the high lipophilic

properties and quick local penetration of pyraclostrobin, thorough coverage in

application is very important. The Pyraclostrobin fungicides are also known as QoI

fungicides because they bind to ubihydroquinone reduction site, the quinol oxidation

(Qo) site (or ubiquinol site) of cytochrome b and thereby stop electron transfer

between cytochrome b and cytochrome c1, which halts reduced nicotinamide adenine

dinucleotide (NADH) oxidation and adenosine triphosphate (ATP) synthesis (Fig.14)

(Becker et al., 1981; Von Jagow and Becker, 1982; Von Jagow and Link, 1986;

Brandt et al., 1993 and Ammermann et al., 2000). This leads to the stopping of the

energy production and the fungus will eventually die. They are more correctly

referred to as the QoI fungicides. The mode of action of these compounds was

reviewed by Sauter et al. (1995) and Leroux (1996). Their rapid activity is

concentrated on the life cycle of the fungi.

The plants show a clear increase in biomass of about 20%, two weeks after the

fungicide’s application. The most remarkable change is the inhibition of ethylene

biosynthesis by the reduction of the activity of ACC synthase. Together with the

increase in endogenous auxin, this change in hormonal balance would explain the

retarded senescence of leaves and enhancement in the tolerance to stress. Also,

pyraclostrobin stimulated the levels of ABA and this might favour tolerance to cold

and adaptation to conditions of water shortage.

No evidences of strobilurin effects in plants of any direct interaction of

pyraclostrobin with enzymes of receptor systems other than mitochondrial respiration

(Koehle et al., 2003). The increase in the biomass and production obtained by

application of pyraclostrobin, even in plants not affected by fungi, is of special

interest in agriculture (Rous et al., 1974).

The strobilurin Kresoxim-methyle proved to inhibit the biosynthesis of

ethylene through reduction of the activity of 1-aminocyclopropane 1- carboxylic acid

(ACC) - synthase in plants (Grossmann and Retzlaff, 1997) which delays the

Introduction

36

senescence of leaves and as a result, photosynthetic activity of green tissue is

prolonged (Koehle et al., 1997; Grossmann et al., 1999).

It's not a systemic fungicide; pyraclostrobin simply moves across the leaf

blade from the treated leaf surface to the other surface. Thus, applicators should strive

for excellent spray coverage to get the most out of this product. As is typical of the

strobilurin fungicides, pyraclostrobin is effective at low use rates and has low

mammalian toxicity (Dixon and Vincelli, 2004).

Application of strobilurin gives the plant an additional beneficial effect by

adding more healthy green color to the plant. This phenomenon is known as the

“greening effect” and that was paired with significant high crop harvest both in

volume and quality (Koehle et al., 2002; Venancio et al., 2003). Over the past years,

there has also been increasing evidence for direct influences of strobilurins on plant

physiology. Strobilurins are easily degradable compounds (Jewess et al., 1999), this is

an essential characteristic because of their rapid degradation in the environment and in

the plant’s metabolism. In addition to their fungicidal activity, strobilurins might also

enhance the capability of plants to ward off pathogens i.e. through the induction of

systemic acquired resistance in plants (Herms et al., 2002). So in this study Headline

is selected to check its efficiency of inducing systemic acquired resistance in cumin

and compared with fungal elicitors.

Monitor:

Monitor is product manufactured by Agriland Biotech Limited, Gujarat. It is a

suspo-imulsion form of fungicide used for the control of wide range of seed and soil

borne pathogens occurring in agriculture crop. Monitor is available in suspo-emulsion

formulation. It is also useful for over 200 fruit, vegetable, field, plantation, forest,

green house, ornamental, landscape, turf and other agricultural crops. It is active in

decomposing raw organic substances and solubilizing phosphorus that has direct

impact on soil fertility and productivity. It has the property of dissolving excessive

soil salts by decreasing soil pH and thereby playing an important role in the

enhancement of the quality of the soil.

Introduction

37

Monitor has broad antifungal activity. It contains Trichoderma viride as a

active ingredient which is active against large spectrum of fungal genera viz.

Fusarium, Sclerotium, Pythium, Rhizoctonia, Macrophomina, Phytophthora Botrytis

and many higher and lower fungi. T. viride (Monitor-S) application at sowing time in

crop rotation with kharif sorghum combination produced significantly higher seed

yield against control (Jadeja and Nandoliya, 2008).

Biocontrol mechanism of monitor:

Trichoderma spp. is free-living fungi that acts as biocontrol agent, are

common in soil and root ecosystems. At least some strains establish robust and long-

lasting colonization of root surfaces and penetrate into the epidermis and a few cells

below this level. The Trichoderma may suppress the growth of the pathogen

population in the rhizosphere through competition, mycoparasitism and thus reduce

disease development. They produce or release a variety of compounds that induce

localized or systemic resistance responses, and this explains their lack of

pathogenicity to plants. It also produces antibiotics and toxins such as trichothecin

and a sesquiterpine, trichodermin, which have a direct effect on other organisms.

These root–microorganism associations cause substantial changes to the plant

proteome and metabolism. The antagonist hyphae either grow along the host hyphae

or coil around it and secrete different lytic enzymes such as chitinase, glucanase and

pectinase that are involved in the process of mycoparasitism. Examples of such

interactions are T. harzianum and T. viride acting against Fusarium oxyporum, F.

roseum, F. solani, Phytophthara colocaciae, Sclerotium rolfsii and Alternaria species.

Plants are protected from numerous classes of plant pathogen by responses

that are similar to systemic acquired resistance and rhizobacteria-induced systemic

resistance. Root colonization by Trichoderma spp. also frequently enhances root

growth and development, crop productivity, resistance to abiotic stresses and the

uptake and use of nutrients (Harman et al., 2004). Strains of Trichoderma spp. can

produce extracellular enzymes and antifungal antibiotics and also may be competitors

to fungal pathogens and promote plant growth (Haggag and Abo-Sedera, 2005). In

addition, Trichoderma enhances yield along with quality of produce, boost

germination rate, increase in shoot and root length by solubilizing various insoluble

forms of phosphates. It promotes healthy growth in early stages of crop and increase

Introduction

38

dry matter production substantially. The silent role of this agent is to provide natural

long term immunity to crops.

All these methods of controlling fungal diseases in plants using biocontrol

agents and various plant extracts seems to be very effective on first instance but none

of them is long lasting. They impart sufficient level of resistance against pathogens

but for a limited period of time. So there is a need to find out effective alternative

which systematically protects the entire plant against wide range of pathogens for

long duration and also promotes enhanced growth of the plant.

Moreover, potential genetic variability available to conduct conventional

breeding for resistance against the pathogen has been found very limited in cumin

(Tawfik and Allam, 2004b). Apparently, there is a need for an efficient control

measure that complies with the recent crop production trends of the environmentally

sustainable agriculture. Therefore great emphasis has been currently directed, towards

the use biological elicitors and commercial suspension of biofungicides. Systemic

acquired resistance is such approach that meets all the above said needs. And so,

several researchers are working over this strategy on several crop plants.

Systemic acquired resistance (SAR):

Systemic acquired resistance (SAR) is a mechanism of induced defense that

confers long-lasting protection against a broad spectrum of microorganisms (Durrant

and Dong, 2004) and is induced in response to infection by necrotizing pathogen

(Hunt and Ryals, 1996; Neuenschwander et al., 1996; Ryals et al., 1996). Ross first

coined the term “SYSTEMIC ACQUIRED RESISTANCE” which refers to the

resistance. It is not transmitted through seeds (Gozzo, 2003).

Plants are hosts to thousands of infectious diseases caused by vast array of

pathogenic fungi, bacteria and nematodes. There are various measures by which plant

can be protected but thoughtful application of the plant’s own defense mechanism

combined with understanding of the complex colony of the real world disease

process, can lead to more effective protection against plant pathogen (Baker et al.,

1997). Although, plant in nature are constantly exposed to diverse array of pathogenic

micro-organisms (Ryals et al., 1994), but relatively small proportion of pathogens are

Introduction

39

able to infect (Baker et al., 1997). Plants are equipped with pre-formed constitutive

chemical and mechanical barriers as well as with inducible defense systems to defend

themselves against attack from the vast array of viruses, bacteria, fungi, nematodes

and insects (Montesano et al., 2003).

1. Constitutive (passive) resistance: It is due to the presence of preformed physical and chemical factors (Table 4).

Table 4: Elaboration of constitutive resistance

2. Induced (active) resistance: Induce resistance is a state of enhanced defensive

capacity developed by the plant when appropriately stimulated. Induced resistance

can be triggered by certain chemical, avirulent forms of pathogen, incompatible race

of pathogen or by virulent pathogens under circumstances where infection is held up

owing to environmental conditions.

Induced or active resistance is also characterized in two main systems. One is

induced systemic resistance (ISR), triggered by selected strains of non-pathogenic

rhizobacteria or antagonistic fungi and does not require salicylic acid but does depend

on the responsiveness of the plant to jasmonic acid (JA) and ethylene (Van loon et al.,

1998). ISR response is localised and doesn’t last for longer period so chances of

pathogen attack may increase during its ending period.

Thickness or hardness of cuticle (Dangle

and Jones, 2001).

Amount and quality of wax that cover the

epidermal cells (Nuernberger and Lipka,

2005).

Physical

factors

Size and shape of stoma and root pericycle

(Keen, 1990).

Presence of preformed secondary metabolite

and peptides (Heath, 2000; Dixon, 2001).

Constitutive resistance

(Preformed/Passive

resistance)

Chemical

factors High amount of alkaloids and phenolics

Introduction

40

The other is Systemic acquired resistance (SAR). In SAR, the defensive

capacity is increased not only in the primary infected plant parts, but also in non

infected, spatially separated tissue. Because of this systemic character, induced

resistance is commonly referred to as systemic acquired resistance. SAR depends on

the synthesis of salicylic acid (SA) by the host and is effective against pathogens that

are restricted by salicylic acid-dependent basal resistance responses, such as tobacco

mosaic virus in tobacco. Several well-characterized defense reactions such as

hypersensitive reaction (HR) (Zang et al., 2004), oxidative burst (Yaeno et al., 2004),

reinforcement of cell wall structures through lignification or callose deposition (Zhao

et al, 2005; Soylu, 2006), accumulation of antimicrobial phytoalexins and induction of

defense-related proteins with antifungal properties (Andreu et al., 2006;

Chandrashekar and Satyanarayana, 2006) have been extensively reported in many

plant species. It differs from animal immunity in both its apparent lack of specificity

(absence of antibody induction) and its lesser efficacy and duration (Ryals et al.,

1994).

Fig. 15: Comparison between SAR and ISR

Both SAR and ISR are effective against pathogens; induction of both types of

induced resistance in the same plant additively enhances protection against disease

causing pathogens (Fig. 15) (Valled and Goodman, 2004). Induced resistance is

Introduction

41

generally systemic, because defensive capacity is increased not only in the primary

infected parts, but also in non-infected, spatially separated tissue. Because of this

induced resistance is referred as systemic acquired resistance (Kothari and Patel,

2004).

Although originated from a type of natural resistance against certain strains of

the pathogen recognized by the plants through a gene-for-gene process, SAR is

generally expressed against a broad spectrum of pathogen that may include virus,

bacteria and fungi. Once established, SAR may last for a certain period of time (from

weeks to months), during which any attempted invasion by a virulent pathogen is

hampered and in this case symptoms expression reduces and infection is hardly

apparent (Van loon, 1997). Such a specific immunization of plant is not transmitted

through seeds (Gozzo, 2003). The key roles in activating SAR in plants are pathogen

derived elicitors.

Pathogen derived elicitors:

The first biotic elicitor was described in the early 1970 (Keen, 1990). Several

evidences state induction of defense response in plants by pathogen derived elicitor.

Elicitors are the low molecular compounds which triggers plant immune response by

activating signal cascade. Elicitors are classified in two types i.e. pathogen derived

elicitors (exogenous elicitor) and plant derived elicitors (endogenous elicitor). The

exogenous elicitors of the plant defense responses differ widely in their chemical

nature and include protein, oligosaccharides, glycoprotein and lipids. Most of

pathogen derived elicitor is nonspecific. Inducible defense responses can be activated,

not only upon the challenge of the plant tissue by pathogen, but also upon the

exposure to elicitor. Therefore, elicitors are now widely used to study the molecular

mechanism of defense responses (Suzuki, 1999).

Several workers have reported the effect of fungal component in induction of

defense in plants. Host defense mechanisms can be elicited by using elicitors

produced from the pathogen/host. The induction of host defense through biotic and

abiotic elicitors has been considered as a focus of research in recent years. These

elicitors lead to induce defense reactions in host plants. However, the extent and

course of increase varies according to the inducer and host plant. During the plant

Introduction

42

pathogen interaction, the molecules derived either from plants or pathogen known as

elicitors induce the plant defense genes that ultimately lead to broad spectrum

resistance (Ricci, 1997). Fungal culture filtrate (FCF) used as an elicitor might induce

the defense related enzyme and defense mechanism in plants. Moreover elicitor and

biocontrol agent has its own benefits of cost reduction being eco friendly, not

affecting non targeted organism, when compared to chemical controlling agents

(Nandini et al., 2010). Culture filtrate has been used effectively to screen genotype for

resistance variety or susceptible variety (Zemanek et al 2002; Cerato et al., 1993).

Culture filtrate presumed to contain toxic metabolites or phytotoxin unique to the

pathogen that may induce plant defense response. Moreover, cell wall fractions

extracted during preparation of fungal culture filtrate also plays significant role in

provoking resistance mechanism in plants upon elicitor treatment. Elicitor derived

from Fusarium oxysporum have induce resistance in banana plants (Patel et al. 2004;

Thakkar et al., 2007). Nandini et al., (2010) reported induction of defense activities in

Arachis hypogaea L. upon treatment with pathogenic fungus Sclerotium rolfsii

derived elicitors. Elicited level of resistance was seen in potato after treatment with

fungal elicitor derived from Phytopthora infestance (Bariya et al. 2011). In maize,

elicitor derived from Aspergillus flavus and A. parasiticus have induced the defense

response of plants upon their treatment (Mahapatra et al., 2015). The pathogen

derived elicitors eventually are responsible for the transcription of defense genes in

plants. Elicitors derived from the soyabean pathogenic fungus Phytophthora sojae

elicited resistance system in plants via activating defense related genes (Becker et al.,

2000). The use of elicitors in plant defense is considered an important potential

component in plant disease management. Utilization of SAR in inducing resistance in

plants would be of more benefit in conventional agriculture as well as organic farming

practices (Eldoksch and El-Sebae, 2009).

Mode of action:

For the induction of resistance in the plant, recognition of elicitor by the plant

is must. Elicitor recognition by the plant is assumed to be mediated by specific

receptors in the plant cell, been localized either on the cell surface of number of

fungal elicitors (Teniente, 2010) which initiate signalling process that activate plant

defense by inducing systemic acquired resistance in plant (Fig. 16).

Introduction

43

Fig. 16: Mode of action of pathogen derived elicitor on plant

General mechanism for biotic elicitation in plant include

• Binding of the elicitor to the plasma membrane receptor

• Hypersensitive response

• Altered ion fluxes across the plant cell membrane

• Increase lignifications of cell wall, callus deposition

• Rapid change in protein phosphorylation this play a role in activation of

protein kinase from which MAP kinase and Ca-dependent kinase are best

characterized

• Accumulation of pathogenesis related protein.

• Stimulate the plant defense molecules such as tannins and Phytoalexins

• Synthesis of salicylic acid and jasmonic acid as secondary metabolites

Gene for gene theory and hypersensitive response (HR):

Associated with SAR is the expression of a set of genes called SAR genes.

Induction of these marker genes is tightly correlated with the onset of SAR in

uninfected tissue (Ward et al., 1991). However, not all defense related genes are

expressed during SAR, and the particular spectrum of gene expression distinguishes

the SAR response from other resistance responses in plants. The SAR signal

Introduction

44

transduction pathway appears to function as a potentiator or modulator of other

disease resistance mechanisms (Ryals et al., 1996).

The activation of SAR depends on a theory proposed by Flor (1971) known as

gene for gene principle. According to this principle, for avirulence (Avr) gene

expressed by the pathogen there is a corresponding R gene in the plant that recognizes

the Avr gene. Avr genes from the different pathogen classes are structurally very

diverse and have different primary function in the biology of these organisms. R

genes are genes responsible for single gene mediated resistance in plants found on

membranes as well as in the cytoplasm (Flor, 1971). It has been predicted that Avr

gene products functions as ligands and host R gene products function as receptor in an

interaction leading to plant resistance to disease (Fig. 17) (Baker et al., 1997).

Fig. 17: Gene-for-gene principle (Flor, 1971)

Once the initial recognition process that involve detection of certain unique

signal molecules of elicitor by the receptor like molecule in plants, which results in

cascade of biochemical events and susceptibility to the disease. The biochemical

events include activation of protein kinase, stimulation of oxidative bust (Kothari and

Patel, 2004), induction of ion fluxes across the cellular membrane, increased cell wall

lignifications, callose formation tyloses formation, activation of salicylic acid a key

signaling intermediary that triggers systemic acquired resistance in plants,

accumulation of PR proteins and induction of other defense related enzymes (Meena

Introduction

45

et al., 2001), synthesis of phenolics and production of Phytoalexins (M’Piga et al.,

1997; Chen et al., 2000, Montesano et al., 2003).

Plant recognizes and resists many invading phytopathogens by inducing a

rapid defense response termed as hypersensitivity response (HR). The HR results in

localized cell and tissue death at the site of infection which constrains further spread

of infection. This local response often triggers nonspecific resistance throughout the

plant, a phenomenon also known as systemic acquired resistance (SAR). SAR is

typically effective against a wide range of pathogens including taxonomically

unrelated to the original inducing organism (Kiefer et al., 2003).

The concept of SAR has been widely recognized and studied for the past 100

years in relation to increasing resistance to fungal, bacterial and viral pathogens of

economically important crop plants. Any plant reacts to physical stresses such as heat,

frost, drought, salt, inoculation with pathogenic or nonpathogenic microorganisms and

chemical molecules of natural or synthetic origin by expressing defense reactions. The

earliest responses activated after host plant recognition of an Avr protein or non-host

specific elicitor is the oxidative burst, in which level of reactive oxygen species

(ROS) rapidly increases. These ROS are predominantly the superoxide anion (O2) and

hydrogen peroxide (H2O2) which arise from successive one electron reduction of

molecular oxygen. ROS generation is localised to the point of infection and appears in

the form of hypersensitive reaction. It has been proposed that H2O2 acts as a second

messenger of SA in SAR signalling. An SA binding protein was identified as catalase;

SA was found to inhibit the catalase activity of this protein, leading to elevated levels

of H2O2. Furthermore, H2O2 was found to cause induction of PR-1 gene expression

and was postulated to induce SAR (Ryals et al., 1996).

After establishment of SAR, the tissue becomes competent for rapid elicitation

of an oxidative burst at the site of pathogen attack, as opposed to a slower response in

tissues in which SAR has not been established. In infected leaves, high concentrations

of SA around the site of infection may inhibit catalase and other oxidoreductases

(Ryals et al., 1995). Inhibition of catalase activity could prolong the half-life of H202

and lead to an amplification of the oxidative burst (Ryals et al., 1996). H2O2

generation have direct antimicrobial activity inhibiting germination of spores of many

Introduction

46

fungal pathogens and participation in the formation of phenoxyl-radicals during

phenol-polymerization within the plant cell wall.

In the past decade several authors have published evidence suggesting that

salicylic acid (SA), a metabolite downstream to the biosynthetic pathway initiated by

phenylalanine ammonia lyase (PAL), plays a role of endogenous signal when plants

are primed to resist pathogens (Gozzo, 2003). Salicylic acid (SA) is a phytohormone

that plays a central role in defense signaling. It is required for both basal defense and

SAR. SA is one of the most important signal molecules for plant defense. Pathogenic

elicitor induced SA synthesis and accumulation are required for both local resistance

and SAR (Zhang at al., 2010). Plants that can no longer accumulate SA are

compromised in their ability to withstand pathogen infection. Thus, SA-dependent

signal transduction plays a central role in plant defense against pathogens and will

undoubtedly serve as a paradigm for defense signaling in plants (Ryals et al., 1995).

The activation of the phenylpropanoid pathway in disease resistance may generate SA

via trans-cinnamic acid (tCA) and benzoic acid (BA) at least in tobacco (Yalpani et

al. 1993), cucumber (Meuwly et al., 1995) and potato (Coquoz et al., 1998).

Phenylalanine ammonia-lyase (PAL) catalyzes the first step in the pathway in which

phenylalanine is converted into tCA. PAL is transcriptionally activated in response to

pathogen attack, elicitor treatment, mechanical damage, and a variety of abiotic

stresses (Dixon and Paiva, 1995). Induction of defense responses against Alternaria

rot caused by Alternaria alternate by SA as elicitor in harvested pear fruit was

reported (Tian et al., 2006). SA seed treatment induced systemic resistance in

Chickpea against fusarium wilt in wilt sick field (Sarwar et al., 2010). Enhanced

defense related enzymatic activity in tomato fruits as a response to SA as elicitor was

also recorded (Hortensia et al., 2007). The other signaling molecules which also

mediate the defense response are Jasmonic acid (JA) and Ethylene (ET) (Montesano

et al., 2003). But JA and ET based pathways are induced in case of induced systemic

resistance (ISR), not in systemic acquired resistance (Fig. 18).

Introduction

47

Fig. 18: Specific signaling molecules inducing SAR and ISR in plants

Salicylic acid (SA) based phenyl propanoid pathway:

The first evidence of systemic protection induced by a microorganism was

reported by Kuc (1987), who found that cucumber Cucumis sativus protection against

Colletotricum orbiculare after pre-inoculation of the cotyledons with this same

pathogen. Present study is based on such pathogenic originated elicitor compounds

which elicits salicylic acid based phenyl propanoid pathway in plants to boost

immunity.

Accumulation of salicylic acid (SA) is required for the induction of SAR.

However, SA is apparently not the translocated signal but is involved in transducing

the signal in target tissues. In addition to playing a pivotal role in SAR signal

transduction, SA is important in modulating plant susceptibility to pathogen infection

and genetic resistance to disease (Ryals et al., 1995). The resistant state is

characterized by an increase in endogenously synthesized SA at the onset of SAR

(Malamy et al., 1990; Metraux et al., 1990). It has been suggested that SA is

synthesized at the site of pathogen-induced necrosis and is translocated to induce SAR

Introduction

48

in uninfected leaves (Willits and Ryals, 1998). Subsequent studies have established a

strong correlation between SA accumulation and SAR induction in tobacco, cucumber

and A. thaliana. The contribution of salicylic acid to systemic acquired resistance was

investigated in transgenic tobacco plants harboring a bacterial gene encoding

salicylate hydroxylase, which converts salicylic acid to catechol. Transgenic plants

that express salicylate hydroxylase accumulated little or no salicylic acid and were

defective in their ability to induce acquired resistance against tobacco mosaic virus.

Thus, salicylic acid is essential for the development of systemic acquired resistance in

tobacco (Gaffney et al., 1993).

Systemic acquired resistance can also be induced or enhanced, by exogenous

application of SA or elicitor compounds that may have similar or more powerful

effects (Hammerschmidt, 1999). Elicitors could be used as enhancers of plant

secondary metabolite synthesis and could play an important role in the biosynthetic

pathways to enhance production of commercially important compounds (Angelova et

al., 2006). Pre treatment of plants with SA or other elicitors, gave rise to strongly

enhanced production of coumarins and incorporation of phenols into their cell walls.

This means that the phenylpropanoid biosynthetic pathway, which is responsible for

the production of phytoalexins, lignins, and SA itself, is altered to respond more

rapidly to the pathogen challenge by a pre-treatment with the chemical inducers. Such

treatments SA enhances the expression of defense genes (Gozzo, 2003).

The SA induced pathways are characterised by the production of a cascade of

PR proteins and defense related enzymes. These include antifungal chitinases,

glucanases thaumatins and oxidative enzymes such as peroxidases, polyphenol

oxidases and lipoxygenases. Low molecular weight compounds with anti-microbial

properties (phytoalexins) can also accumulate which play vital role against array of

pathogens. Moreover, SA also plays role in flowering, seed germination, stomata

functioning and gravity sensing. It initiates PAL synthesis in phenyl propanoid

pathway thereby switch on the defense mechanism.

Defense Mechanism:

Among the main mechanisms associated with plant defense, it is worthy of

notice the mechanism of SAR mediated by the interaction known as “gene-for gene”

Introduction

49

relationship. This genetic mechanism of resistance involves the molecular recognition

between the products of complimentary genes, present in the plant (R genes or

resistance genes) and in the pathogen (avr genes or avirulent genes). After the

interaction between the products of these genes, it becomes evident the establishment

of a series of alterations which include:

1) Fast production of reactive oxygen intermediates (ROIs), such as: superoxide

anions (O2-), hydrogen peroxide (H2O2) and hydroxyl radical (OH). The production of

hydrogen peroxide and other ROIs presents direct antimicrobial effect and also drive

oxidative cross-linking of the cell wall.

2) Alterations in the cell wall constitution. The cell wall polymers present lateral

phenolic chains, like the tyrosine residues of extensins and the residues of ferulic and

p-coumaric acids linked to polysaccharides, playing an important role in the

architecture of the cell wall, through the formation of cross-linkages among those

polymers. The elevated concentration of hydrogen peroxide promotes the formation

of cross-linkages among the tyrosines of extensions present in the cell wall. These

cross-linkages turn the cell wall less susceptible to the attacks promoted by the

hydrolytic enzymes secreted by the pathogen during its penetration.

3) Induced accumulation of antimicrobial, low molecular weight secondary

metabolites known as phytoalexins.

4) Activation and synthesis of defense peptides and proteins also known as defense

related enzymes.

5) Cell death as a result of establishing the hypersensitivity response (Castro and

Fontes, 2005).

If defense mechanisms are triggered by a stimulus prior to infection by a plant

pathogen, disease can be reduced. This is the basic theory of systemically induced

resistance, one of the most intriguing forms of resistance, in which a variety of biotic

and abiotic treatments prior to infection can turn a susceptible plant into a resistant

one (Heath, 1996). Plant resistance and induced forms of resistance are generally

associated with a rapid response, and the defense compounds are often the same.

Introduction

50

Fig. 19: Schematic overview of branch pathways of shikimate, phenylpropanoid

metabolism and flavonoid biosynthesis in plants leading to the development of SAR

A cascade of molecular and biochemical events underlies the expression of

SAR. It is initiated by perception of inducers (pathogens, chemicals, elicitors)

resulting in generation of signal molecules translocated at long distance, and

switching on of diverse processes contributing to the development of the defense

potential of plants realized upon elicitor treatment or pathogen infection. Perception

of inducers is effectuated through binding of pathogen-derived molecules (elicitors) or

Introduction

51

chemical products with receptor sites on plant membranes or cell walls (Fig. 19).

Generation and nature of signals, the mode of their translocation and interactions are a

matter of intensive research (Edreva, 2004).

The SAR defense signaling networks appear to share significant overlap with

those induced by basal defenses against pathogen-associated molecular patterns

(PAMPs). Basic resistance involves the recognition of PAMPs by pattern recognition

receptors (PRRs), whereas SAR responding leaves must decode one or more unknown

mobile signals. The nature of the molecule that travels through the phloem from the

site of infection or elicitation to establish systemic immunity has been sought after for

decades. Accumulation of salicylic acid (SA) is required for SAR, but only in the

signal perceiving systemic tissue and not in the signal generating tissue (Vernooij et

al., 1994). Reactive oxygen species (ROS), nitric oxide (NO), jasmonic acid (JA),

ethylene and lipid-derived molecules are all implicated in local and sometimes in

systemic signaling.

Salicylic acid is commonly recognized as a signal molecule or a prerequisite

for signal production in SAR; jasmonic acid and ethylene are involved in signalling

upon expression of resistance induced by biocontrol microorganisms (Schneider et al.,

1996; Van Loon et al., 1998). Nitric oxide (NO) has well-known biological functions

and it is apparent that NO serves as a signal for plant growth, development and

defense (Neill et al. 2002). The Avr factors from plants and biochemical agents

stimulate NO production, and this NO promotes disease resistance of the plants,

collaborating with ROS in the oxidative burst. Some studies have shown that NO is

involved in production of secondary metabolites. Plants treated with headline use NO

as a modular to trigger resistance (Mishra et al., 2012).

Salicylic acid (SA) is a well-known inducer of plant systematic acquired

resistance (SAR) in plant-pathogen interaction or pathogenic elicitor treatment, but it

is not a universal inducer for production of plant defensive metabolites. The SA

quickly accumulates at the site of infection during pathogen attack and plant

hypersensitive reaction, and it spreads to other parts of the plant to induce a wide

range of defense responses. Fungal elicitors also stimulate SA accumulation in plant

cell cultures. However, SA induces gene expression related to biosynthesis and

Introduction

52

production of some classes of secondary metabolites and defense related enzymes in

plants (Mishra et al., 2012).

SAR-mediating signal pathways may act simultaneously, thus providing an

additive effect and enter signal-transducing cascades involving MAP kinases (Gozzo,

2003). Then interaction with gene promoters or other regulatory factors triggers the

expression of the so-called SAR-genes (Ward et al., 1991). The term “SAR-genes” is

used to collectively designate this family of nine genes whose expression is correlated

with the onset of SAR. The SAR-genes code for pathogenesis related proteins (PR

proteins) (Ward et al., 1991). The involvement of PR-proteins in SAR could be

related to their characteristic functions. Thus, some PR-proteins exert hydrolytic

action (glucanase, chitinase), suggesting a lytic effect on pathogen cell walls built-up

of glucans or chitins (Van Loon et al., 1998; Gozzo, 2003). Some other PR proteins

have membrane permeabilizing activity due to interaction with membrane

components, leading to conformational changes, dissipation of pH membrane

gradient, and formation of pores in membranes (Edreva, 2004).

The elicitors that switch on the cascade of reactions can be cell wall

fragments, insect saliva, and products of specific recognition genes, which initiate the

biochemical signaling pathways for activation of defense genes. In the

phenylpropanoid pathway secondary metabolites are derived from primary

metabolites first of all amino acids and carbohydrates through methylation,

hydroxylation, and glycosylation biochemical pathways. Up to date, a few thousands

of different secondary metabolite structures have been identified in plants; the largest

of them are the phenylpropanoids (PPs), isoprenoids and alkaloids. By chemical

structure, secondary metabolites in plants are divided in several major classes such as:

- terpenes (isoprenoids, terpenoids) - PPs, phenylpropanoids and their derivatives

(flavonoids, tannins, glycosides, and lignins) - nitrogen-containing compounds

(alkaloids and heterocyclic aromatics). PPs belong to a large class of plant phenols

produced through shikimic acid pathway. The synthesis of PPs has a common initial

step – deamination of phenylalanine to cinnamic acid catalyzed by phenylalanine

ammonia lyase (PAL). Several factors are known to affect the expression and activity

of PAL. They are light, wounding, disease, gamma-ray irradiation, germination,

development and differentiation, and the application of certain macromolecules.

Introduction

53

Many of plant-derived phenolic compounds (flavonoids, isoflavonoids, coumarines,

and lignans) are secondary products of PPs metabolism. First, cinnamic and then,

hydroxycinnamic acid are formed, both acids belonging to PPs. Then, chalcone

synthase uses 3 cinnamoil radicals to produce flavonoids. Stilbene synthase uses 2

cinnamoil radicals to produce trans-3,4’- trihydroxystilbene, known as resveratrol.

Resveratrol is a phytoalexin used by plants to protect themselves from fungi. Lignins

are phenolic polymers playing an important role by reducing the permeability of the

cell wall to water, by increasing the rigidity of cell wall, which is a part of the

pathogen resistance mechanism (Korkina, 2007).

These plant polymers are products of the oxidative coupling of PPs

monomers: peroxidase catalyses the oxidation of PPs to their phenoxyl radicals, and

the subsequent nonenzymatic coupling controls the pattern and extent of

polymerization that results in a vast structural diversity of natural lignins.

An almost ubiquitous feature of plant responses to incompatible pathogens or

to elicitors is the activation of PPs metabolism in which PAL catalyses the first

committed step of the core pathway of general PP metabolism. Branch pathways lead

to the synthesis of compounds that have diverse defensive functions in plants such as

cell wall strengthening and repair (lignin and suberin), antimicrobial activity

(furanocoumarin, pterocarpan and isoflavonoid phytoalexins), and signaling

compounds (salycilic acid).

The resulting phenolics are often converted into more reactive species by

phenol oxidases and peroxidases. There are several PPs-based mechanisms of defense

against pathogens, for example, construction of structural lignin containing barriers

preventing the pathogen penetration into the plant tissues. Another mechanism is the

use of phytoalexin and scopoletin, which could act as broad-range antibiotics.

Additionally, scopoletin being an efficient peroxidase substrate may act as scavenger

of reactive oxygen species and thus prevent, or reduce, oxidative damage to infected

plant cells (Korkina, 2007).

Peroxidase which is also systemically induced is essential for cross-linking

and reinforcement of cell walls, the latter being a marker of the induced state.

Oxidative burst is proposed to mediate SAR expression (Schneider et al., 1996; Kuc,

Introduction

54

2001; Gozzo, 2003). It may be assumed that the deployment of SAR related events

allows the plant to respond more rapidly and effectively to a subsequent, “challenge”

inoculation (Edreva, 2004)

Various components with diverse functions are involved in SAR. After the

contact with the pathogen a series of peptides, proteins and enzymes are expressed by

plants and many of these compounds present direct antimicrobial properties (Castro

and Fontes, 2005). Mahapatra et al. (2015) reported induction of systemic acquired

resistance in Zea mays L. by using Aspergillus flavus and A. parasiticus derived

elicitors which in turn helped in inducing defense related enzymes and biochemical

parameters. Induction of defense reaction in sugar beet and wheat after the treatment

with elicitor derived from Pithium oligandrum was reported by Takenaka et al.

(2003). SAR is successfully achieved in sugarcane against smut disease (Armas et al.,

2007), Vicia faba in response to infection with Botrytis spp. (Soylu et al., 2002;

Hargreaves et al., 1977), banana against fusarium wilt (Companioni et al., 2006),

pepper to control fusarium root rot (Silver et al., 2008), Asparagus densiflorus against

fusarium crown and root rot (He et al., 2001), tobacco and Chinese cabbage by a

novel protein from Phytophthora boehmeriae (Wang et al., 2003). SAR is practiced

and resistance is obtained in plants such as mustard against alternaria blight

(Vishwanath et al., 1999; Sharma et al., 2010), sunflower by Alternaria helianthi

culture filtrate (Rao and Ramgopal, 2010), chickpea against Fusarium wilt (Singh et

al., 2003), banana against fusarium wilt (Thangavelu et al., 2003; Patel et al., 2004;

Thakkar et al., 2007), onion against the infection of leaf blight pathogen (Karthikeyan

et al., 2005), groundnut against stem rot (Nandini et al., 2010); hot pepper against

damping-off (Nakkeeran et al., 2006), black pepper to control soil borne diseases

(Paul and Sarma, 2005), red pepper for the management of Phytophthora infection

(Sriram et al., 2009). Referring to these studies defense related enzymes and

biochemical parameters were checked in this study to confirm the induction of SAR

in cumin plants after treatment with selected bioelicitors.

Pathogenesis Related Proteins (PR Proteins):

Higher plants developed different mechanism to protect themselves against

biotic and abiotic factors including pathogen attack, wounding, salinity, drought and

air pollution (Agrios, 1997). Molecularly, SAR is characterized by the increased

Introduction

55

expression of a large number of pathogenesis related proteins (PR genes), in both

local and systemic tissue. PR proteins are the ultimate effectors of SAR induction.

Production of pathogenesis related proteins is most important because they can lead to

the increased resistance of the whole plant against a pathogenic attack (Adrienne and

Barbara, 2006).

Antoniw et al., (1980) coined the term “pathogenesis related proteins”, which

have been defined as “proteins encoded by the host plant but induced only in

pathological or related situations”, the latter implying situations of non-pathogenic

origin. PR proteins were first described by Van Loon, who observed accumulation of

various novel proteins after infection of tobacco with TMV (Van Loon and Van

Kammen, 1970). It is generally thought that SAR results from the concerted effects of

many PR proteins rather than a specific PR protein.

Table 5: Recognized and proposed families of pathogenesis-related proteins (Van

Loon and Van Strien, 1999)

Family Type member Properties

PR-1 tobacco PR-1a antifungal, 14-17kD

PR-2 tobacco PR-2 class I, II, and III endo-beta-1,3-glucanases, 25-35kD

PR-3 tobacco P, Q class I, II, IV, V, VI, and VII endochitinases, about

30kD

PR-4 tobacco R antifungal, win-like proteins, endochitinase activity,

similar to prohevein C-terminal domain, 13-19kD

PR-5 tobacco S antifungal, thaumatin-like proteins, osmotins, zeamatins,

permeatins, similar to alpha-amylase/trypsin inhibitors

PR-6 tomato inhibitor I protease inhibitors, 6-13Kd

PR-7 tomato P69 Endoproteases

PR-8 cucumber

chitinase

class III chitinases, chitinase/lysozyme

PR-9 lignin-forming

peroxidase

peroxidases, peroxidase-like proteins

PR-10 parsley PR-1 ribonucleases, Bet v 1-related proteins

Introduction

56

PR-11 tobacco class V

chitinase

endochitinase activity

PR-12 radish Ps-AFP3 plant defensins

PR-13 Arabidopsis

THI2.1

Thionins

PR-14 barley LTP4 nonspecific lipid transfer proteins (ns-LTPs)

PR-15 barley OxOa

(germin)

oxalate oxidase

PR-16 barley OxOLP oxalate-oxidase-like proteins

PR-17 tobacco PRp27 Unknown

Many PR proteins have antimicrobial properties in vitro (Van Loon and Van

Strein, 1999) and their function in the defense response has been widely known. It is

generally thought that SAR results from the concerted effects of many PR proteins

rather than a specific PR protein.

Number of PR-1 proteins have been identified in Arabidopsis, Hordeum

vulgare (barley), Nicotiana tabacum (tobacco), Oryza sativa (rice), Piper longum

(pepper), Solanum lycopersicum (tomato), Triticum sp. (wheat) and Zea mays (maize)

(Liu and Xue, 2006). Plant β-1,3 glucanase is also a pathogenesis related protein and

classified as member of the PR-2 family (Cheong et al., 2000). It appears to be

coordinately expressed along with chitinase following pathogen infection, wound,

ethylene treatment and chemical stress (Masuda and Yamamoto, 1970; Krishnaveni et

al., 1999). Peroxidase has potential role in the lignifications, metabolism of indole 3-

acetic acid, defense against infection and protection of cell from oxidative damages

by H2O2 (Takahama and Egashira, 1991). All these PR proteins and defense related

enzymes together confer immense immunity to plants which can’t be achieved

through chemical or physical agents.

β-1,3 glucanase have been well investigated in plants at physiological and

molecular level due to their widespread role in plant defense response (Meins et al.,

1992; Simmon et al., 1992). β-1,3 glucanase induction in several plants have been

seen including pea, bean, tomato, tobacoo, maize, soyabean, etc. Induction of β-1,3

Introduction

57

glucanase in response to various pathogens and their elicitation has been well

investigated in plants by various researches (Vogel and Barz, 1993).

Elicitor receptor interaction are presumed to generate signals that activate the

nuclear genes involved in the plant defense response leading to induction of defense

related enzymes like Phenylalanine Ammonia Lyase (PAL), Peroxidase (POX) and

Polyphenol oxidase (PPO). Analysis of SAR proteins showed that many belong to the

class of pathogenesis-related (PR) proteins. The set of SAR markers consists of at

least nine families comprising acidic forms of PR-1 (PRla, PR-lb, and PR-lc), β-1, 3

glucanase (PR-2a, PR-2b, and PR-2c), class II chitinase (PR-3a and PR-3b, also called

PR Q), hevein-like protein (PR-4a and PR-4b), thaumatin-like protein (PR-5a and

PR5b), acidic and basic isoforms of class III chitinase, an extracellular β-l, 3

glucanase (PR-Q'), and the basic isoform of PR-1 (Ward et al., 1991). In addition,

accumulation of phenols, production of antimicrobial phytoalexins and the

reinforcement of cell walls at invasion site through synthesis and deposition of callose

and lignin compounds contribute to multilayered plant defense system (Karthikeyan,

et al., 2006). The mechanism, time and trend of maximum induction of phenols plays

significant role in governing plant resistance to disease (Gogoi et al., 2001).

Role of defense related enzymes:

1. Phenylalanine ammonia lyase (PAL):

Phenylalanine ammonia lyase (PAL) is the initial entryway enzyme and it

plays an important role in biosynthesis of various defense chemicals phenolics,

phytoalexins and lignin content, which have been considered key components in

disease resistance (Vidhyasekaran, 1988) by phenylpropanoid metabolism. PAL also

serves as a precursor for salicylic acid biosynthesis. Phenylalanine ammonia-lyase

(PAL) catalyzes the nonoxidative deamination of L-phenylalanine to form trans-

cinnamic acid and a free ammonium ion. PAL has been extensively studied because

of its role in plant development and its response to a wide variety of environmental

stimuli. The importance of this enzyme in plant metabolism is demonstrated by the

huge diversity and large quantities of phenylpropanoid products found in plant

materials. PAL has been isolated and characterized from a number of plant species,

some fungi and few bacterial sources. Source tissues used for PAL isolation are

Introduction

58

diverse i.e seedlings, shoots, leaf-sheath, cell culture, fruit, mycelium, and prokaryotic

cells (Hyun et al., 2011). Many secondary metabolic products in plants, such as

anthocyanins, flavonoids, ultraviolet (UV) protectants, antimicrobial

furanocoumarins, isoflavonoid phytoalexins, lignins and wound phenolic esters are

phenylpropanoid derivatives and PAL as the first enzyme in phenylpropanoid

derivative metabolism catalysis to produce all such metabolites. The production of

phenylpropanoid compounds is important in plant development, plant-microbe

signaling and plant defense. The PAL enzyme has been considered to be the one of

the key enzymes in the biosynthesis of flavonoids and plant stress response (Xu et al.,

2012).

PAL activity may also be induced by elicitors present in cell walls or culture

filtrates of both phytopathogenic and nonpathogenic microorganisms, and by

structurally unrelated abiotic elicitors, mechanical damage, or environmental factors

such as light (Camm and Towers, 1973). Increased PAL activity has been found to be

associated with resistance of barley to powdery mildew (Shiraishi et al., 1995).

Further evidence for the importance of PAL in defense of barley to fungal diseases

has been provided by enzyme inhibitory studies (Carver et al., 1994). PAL was

induced in cultured parsley cells (Petroselinum hortense) by treatment with elicitor, a

cell-wall fraction of the fungus Phytophthora megasperma f. sp. glycinea (Kuhn et al.,

1984). The induced PAL activity in both leaf and root tissues of three black pepper

varieties to confer resistance was determined in Phytophthora capsici filtrate treated

plants (Jebakumar et al., 2001).

2. Peroxidase (POX):

Peroxidases are frequently associated with plant defense against pathogens.

The induction of plant peroxidase appears to be an early event in plant-microbe

interaction (Cook et al., 1995). Peroxidase is heam containing protein that catalyses

the reduction of hydrogen peroxides, especially hydrogen peroxide to water.

Peroxidases are involved in several plant defense response including lignifications

(Kuvalekar and Gandhe, 2010) as well production of antimicrobial radicals (Peng and

Kuc, 1992).

Peroxidases catalyze the oxidation of substrates like phenol and its derivates,

by hydrogen peroxide. They are responsible for the radical dehydrogenation of

Introduction

59

sinapilalcohol and koniferylalcohol during the lignin synthesis. Peroxidases

participate in the synthesis of flavons, stilbens and other phenolic secondary

metabolites. They are represented by many of the isoenzymes. Peroxidase

polymorphism could be also used as a biochemical marker related to the different

levels of field resistance. Peroxidase participates in processes which occur in the

extracellular matrix. Their association with the cell wall was confirmed. Peroxidases

remove the toxic hydrogen peroxide from tissues, participate in synthesis of phenolic

compounds and in the building of intermolecular bonds during the organisation of the

cell wall at the sites of infection by pathogens. Peroxidase also participates in the

synthesis of ethylene the concentration of which increases frequently in pathogenesis

process. Generally, peroxidases enhance their activity after a pathogen attack, because

they participate in defensive lignification and synthesis of phenolic compounds

effective against pathogens (Lebeda et al., 1999).

Induction in PR protein and defense related enzyme profiles of plants occurs

in response to fungal elicitor treatment. Two pathogenesis-related peroxidases in

greengram (Vigna radiata (L.) wilczek) leaves and cultured cells induced by

Macrophomina phaseolina (Tassi) Goid. and its elicitor offered superior resistant

against the disease (Ramanathan et al., 2001). Efficient resistant was noticed after

involvement of a class III peroxidase in oxidative burst upon treatment of moss plants

with a fungal elicitor (Lehtonen et al., 2012). Induction of peroxidase activity and

thereby defense responses in cucumber plants (Cucumis sativus L.) by the biocontrol

agent Trichoderma harzianum have suggested the importance of peroxidase in SAR

(Yedidia et al., 1999). Peroxidase activity induced in In vitro propagated banana

(Musa paradisiaca) by Fusarium derived elicitor has confirmed the onset of SAR

(Patel et al., 2004).

3. β- 1,3 glucanase:

It degrades the fungal cell wall and cause lysis of fungal cell wall. The glucan

oligomers released during degradation of the fungal cell wall act as elicitor that elicit

various defense mechanism in plant (Rivera et al. 2002; Karthikeyan et al., 2005). It

also decomposes excess callose deposited near plasmodesmatal opening for better

transportation. Levy et al. (2007) confirmed the role of β- 1,3 glucanase and called

them as plasmodesmal gate keepers for intercellular communication. Moreover its

Introduction

60

induction in response to fungal elicitor to induce SAR in various plants has also

studied broadly. Pathogenesis-related functions of plant β-1,3 glucanases has been

investigated by antisense transformation (Beffa and Mains, 1996). Fungal elicitor

induced over-expression of many isoforms of β -1,3 glucanases, both acidic and basic

forms have been reported from many plant species including tomato, tobacco, maize,

pepper, wheat and melon (Joosten and DeWit, 1989; Ward et al., 1991 ; Lozovaya et

al., 1998; Egea et al., 1999; Kemp et al., 1999; Rivera et al., 2002).

4. Polyphenol oxidase (PPO):

  It is widely distributed within the plant and act in defense response. PPO

activity in disease resistance is by hydrolysing monophenol to O-diphenols, O-

diphenols is then oxidize to quinones which is toxic to microorganisms than other

phenolic compounds (Poiatti et al., 2009). PPO activity was found induced in SA

treated ginger plants and provided significant immunity against Pythium infection

(Ghosh, 2015). Jasmonate-dependent induction of polyphenol oxidase activity in

tomato foliage is important for defense against Spodoptera exigua (Bosch et al.,

2014). Polyphenol oxidases (PPOs) are widespread enzymes which oxidize plant

phenolic compounds. In tomato leaves, PPO is systemically wound-induced, regulated

by the tomato wound signal systemin and appears to function as an anti-nutritive

defense against folivore insect pests and certain fungi (Constabel and Ryan, 1998).

The pathogen, Fusarium-induced PPO activity from wild oat was attributed to a

soluble isoform of the enzyme that appeared to result from proteolytic activation of a

latent PPO isoform and this inducible enzyme-based biochemical defenses represent a

fundamental mechanism of seed survival and longevity in the soil (Fuerst et al.,

2014).

5. Catalase (CAT):

Catalase is included in defense plant reactions. This enzyme occurs in

peroxizomes and decomposes the hydrogen peroxide to water and oxygen. There are

only a few results related to the role of catalase in plant defense processes. This

enzyme is the competitor of peroxidase, because they use the same substrate (Lebeda

et al., 1999). Karthikeyan et al., (2007) found that the use of diazotrophs Azospirillium

and Azotobacter increased the germination rate along with increase in superoxide

Introduction

61

dismutase, POX and catalase activities. Catalase plays a key role in maintaining H2O2

homeostasis in cells and has been implicated in ROS signaling in response to

pathogen attack. Catalase was involved in maize resistance to either A. flavus,

aflatoxin accumulation, or both. Catalase activity was significantly higher in resistant

lines, and H2O2 levels were lower. A. flavus produces aflatoxin in response to

oxidative stress (Kim et al., 2005).

6. Nitrate reductase (NR) and Nitrite reductase :

Nitrate reductase (NR) was recently shown to play an important role during

phytopathogenic interaction by providing substrates for the synthesis of nitric oxide

(NO), a key signal for plant defense responses (Oliveira et al., 2010). The role of NO

in relation to the signaling pathways involved in stomatal movement, plant growth

and senescence, in the frame of its interaction with abscisic acid, auxins, gibberellins,

and ethylene (Simontacchi et al., 2013). NO is a highly toxic gas with a broad

chemistry that involves an array of interrelated redox forms with different chemical

reactivity. The NO modulates both the R gene-mediated resistance and the basal

resistance (Delledonne et al., 2005). Studies on the Arabiodpsis-avirulent P. syringae

system have indicated that NO might play a crucial role as an intracellular signal that

functions in the cell-cell spread of the HR (Zhang et al. 2003). The balance between

NO and H2O2 appears to be important for the induction of the HR cell death

(Delledonne et al. 2005). This is validated by the observation that the reduced

production of H2O2 in the transgenic plants overexpressing a peroxidase results in

protection from a NO-induced cell death (Murgia et al. 2004). The general PAMP

elicitors of plant defense, can trigger a strong and rapid NO burst in Arabidopsis,

which is mainly dependent on the activity of a nitric oxide synthase (Guo et al. 2003).

In addition, significant amounts of NO are produced after wounding or an exogenous

application of JA or pathogenic elicitors in the different plant species (Delledonne et

al., 2005), indicating its role in the defensive response to fungi, insects via various

enzymes. Nitrate reductase serves plants, algae, and fungi as a central point for

integration of metabolism by governing flux of reduced nitrogen by several regulatory

mechanisms (Wilbur et al., 1999). Regulation of nitrate reduction was studied in

wheat leaves by Naik et al. (1982) and in rice by Hemalatha (2002).

Introduction

62

7. Phenolics:

Phenolics are well known antifungal, antibacterial and antiviral compounds

occurring in plants (Mandal et al., 2010). According to the Matern and Kneusal

(1988), the first step of the defense mechanism in plants involves a rapid

accumulation of phenols at the infection site, which restrict or slow the growth of the

pathogen and play a important role in disease resistance (Karthikeyan et al., 2005).

Culture filtrate of Lasiodiphlodia theobromae has been reported to increase

the levels of PAL, hydrogen peroxide, and salicylic acid in Brassica nigra plants

(Thakkar et al., 2004). Induction of PAL and POX activity were also determined in

rice plants after the foliar spray and seed treatment with Magnaporth grisea derived

elicitor to check the efficacy of bioelicitor on controlling the disease (Qing et al.,

2007). Similarly, seed and foliar spray treatment to rice with methylobacterium sp.

strain PPFM-Os-07 showed induction of PR-protein and also promoted the plant

growth (Madhaiyan et al., 2004). Higher levels of polyphenol oxidase (PPO),

phenylalanine ammonia-lyase (PAL), β-1, 3-glucanase (PR-2) and phenolics were

observed in roots and shoots of resistant cultivar than that of susceptible cultivar of

chickpea on treatment with elicitors and pathogen. Induction of defense proteins and

accumulation of phenolics might have contributed to restrict the invasion of F.

oxysporum f. sp. ciceri, in resistant cultivar of chickpea (Raju et al., 2008).

Accumulation of phenolic compounds, PAL and POX enzymes were seen in

sugarcane plant after treatment with elicitor derived from phathogenic organism

Ustilago scitaminea (Armas et al., 2007). Pre-treatment of elicitor derived from F.

oxysporum resulted in the elevation of PAL, POX, PPO, β-1.3- glucanase, chitinase

and phenolics in banana plant (Thakker et al., 2007). Foliar application with elicitors

derived from Sclerotium rolfsii, Aspergillus flavus and A. parasiticus also resulted in

induction of several defense related enzymes in groundnut plants (Nandini et al.,

2010). Netted melon fruit treated with bioelicitor and inoculated with F. oxysporum

activated the natural defense response as measured by the increase in PAL, chitinase

activities and phenolic acid synthesis in a similar way as the infection by the living

pathogen (Estrada et al., 2009).

The application of biocontrol agent by foliar spray and seed treatment with

Alternaria alternate on groundnut significantly reduced the leaf blight incidence both

Introduction

63

under green house and field condition. The groundnut plants treated with biocontrol

agents lead to significant increase in activity of defense related enzymes like

peroxidase and polyphenol oxidase (Chitra et al., 2006). Pre treatment with

Pseudomonas putida and Micrococcus luteus isolates in the rhizospher could trigger

induced systemic resistance in aerial part of cucumber plants against anthracnose

caused by Colletotrichum orbiculare (Jeun et al., 2004). In Cucumber and

Arabidopsis, compost and compost water extract was used to induce systemic

acquired resistance (Zhang et al., 1998). Use of Acibenzolar -S- methyl against

bacterial spot disease in pepper plants induced of systemic acquire resistance (Roberto

et al., 2002). Induction of systemic resistance by selected plant growth-promoting

rhizobacteria (PGPR) strains of P. fluorescens was involved in the suppression of

Fusarium wilt of radish in a special rook-wool bioassay (Leeman et al., 1995). SAR

in Carica papaya L. is induced by benzothiadiazole (BTH) and fungal elicitor and the

response is manifested by increased tolerance to infection by the virulent pathogen

Phytophthora palmivora (Yun et al., 2004). In tomato and tobacco plants, the salicylic

acid dependent defense pathway is effective against different pathogens (Achuo et al.,

2004). In cereal crops like barley and rice, biotic and abiotic elicitors have been used

to induce SAR against powdery mildew and blast (Sengupta and Sinha, 1987;

Manandhar et al., 1998; Kloepper et al., 1992; Du et al., 2000). Induction of various

defense related enzymes in Zea mays L. by Aspergillus flavus and A. parasiticus

derived elicitors was reported (Mahapatra et al., 2015). Induced systemic resistance

thereby increased level of defense related enzymatic activity was reported in

groundnut by foliar application of Headline (Pyraclostrobin 20% WG) (Amin et al.,

2015). Other defense related enzymes, phytoalexins and cell wall barriers that get

induced after effective treatment helps in controlling the disease. Disease control by

plant’s own defensive mechanism has been widely accepted and practiced by workers

all over the world. Better control of the pathogen and disease spread can be achieved

by inducing systemic acquired resistance in plants.

In cumin, it is very much difficult to control Fusarium wilt and Alternaria

blight diseases because pathogens spread in the field very rapidly. Ample efforts have

been made by various workers to control both the diseases using chemical fungicides,

leaf extracts or microbial agents and trying several physical methods.

Introduction

64

Seed dressing and soil drenching with methyl thiophanate and carbendazim

decreased wilt incidence and increased seed yield. It was found that the herbicide

inhibited the mycelial growth of Fusarium oxysporum f. sp. cumini (Patel and Patel,

1993). Aghnoom et al. (1999) studied the effect of fungicides and found that benomyl,

iprodione + carbendazim, carboxim + thiram and captan reduced mycelial growth of

Fusarium oxysporum f. sp. cumini. Trimethyl thiuram disulphide (0.1 g g-1 seeds), the

best fungicide for the control of wilt was suggested by Deepak and Lal, (2009) when

its effect was compared with propiconazole, carbendazim and copper oxychloride (0.1

g g-1 seeds) because the disease incidence was minimum under the soil treatment

with neem cake. Several chemical fungicides, oilcakes and manures were tried either

alone or in combination to control wilt of cumin in field and integrated treatments

resulted in good yield and low disease incidence over control as compared to

individual application (Deepak and Lal, 2009). Seed treatment with ceresan, captan,

thiram, dithane-M-45 supported by sprays of chemical fungicides mancozeb and

curzat at regular interval is recommended to control blight (Pipliya and Jadeja, 2008).

Pyrazole and isoxazoles chemical components have also been developed by Sareen et

al., (2011) to build up control over blight disease but environmental concerns and

issues regarding floral and soil toxicity has limited the use of such chemical agents.

Reduction in the pathogenic propagules of F. oxysporum f. sp. cumini was

attributed to combined effects of volatiles and enhanced microbial activity including

antagonism. (Sharma et al. 1995).Despite proven efficiency of soil solarization or

amending soil with mustard oil-cake (2.5 t ha-1), these approaches did not find

acceptance by the resource deficient farming community of the Indian arid region

because of high cost of polyethylene film or mustard oil cake. Thus, a need was felt to

improve the efficiency of mustard pod residue by integrating with other easily

available, costeffective practical management strategies. Therefore, effects of soil

solarization, residue incorporation, summer irrigation and biocontrol agents alone or

in combination on survival of F. oxysporum f. sp. cumini were ascertained (Israel et

al. 2005). Combining sub- lethal heating, residues of mustard (2.5 t ha-1) and an

obnoxious weed Verbisina encelioides (0.5 t ha-1) and summer irrigation resulted in

almost equal control that was achieved when mustard residues were combined with its

oil cake (Israel et al. 2011). Soil solarization has been found to be an effective

technique in reducing soil population density of the plant pathogens and induced

Introduction

65

diseases in the field, where ample solar radiation and the soil temperatures are

available during crop free period (Katan 1981). Jadeja and Nandoliya, (2008)

suggested that soil solarization with 25 μm LLDPE plastic cover for 15 days in

summer proved most effective in reducing wilt incidence to 26.27% as against

44.90% in non-solarization and increasing yield to 396 kg ha-1 as against 286 kg ha-1

in non-solarized plots. Soil solarization and soil fumigation with methyl bromide are

two physical methods of control of Fusarium wilt disease and Alternaria blight

disease (Tawfik and Allam, 2004a). Other physical methods viz., Summer irrigation

and crop rotation are also useful against Fusarium wilt (Mawar and Lodha, 2005).

Soil solarisation and soil fumigation with methyl bromide can provide a control

against the disease but may be of limited application value for large scale production

systems in the open field. In addition, methyl bromide is considered an ozone-

depleting compound and has potential risk on the living environment and human

health.

Many experiments were carried out to assess possible use of bio-agents,

biological materials for several years to find out the effective retardation of the

growth of two cumin fungal pathogens under in vitro and field conditions. Control of

wilt and blight diseases of cumin through antagonistic isolates of T. harzianum under

in vitro and field conditions by Deepak et al. (2008). They find it substantial in

controlling the disease. On the basis of in vitro and field studies, Bardia and Rai,

(2007) suggested that Trichoderma harzianum isolate I6 and carbendazim were the

best treatments in inhibiting the growth of F. oxysporum f. sp. cumini. Use of fresh

leaf extracts of Datura stramonium and Calotropis procera retarded the growth of F.

oxysporum f. sp. cumini (Sharma and Trivedi, 2002). Seed extracts of Trachyspermum

copticum, leaf extracts of Lavandula angustifolia and flower extracts of Rheum ribes

effectively inhibited the radial growth and spore germination of F. oxysporum f. sp.

cumini (Ghorbany and Salary, 2003). Various concentrations of leaf extracts of

different medicinal plants were applied in vivo and in vitro, to check the growth

retardation of A. burnsii (Gangopadhyay et al., 2010). Various microbial bioagents

viz. Trichoderma spp., Bacillus subtilis, Pseudomonas fluorescens and Rhizobium

spp. were checked for growth retardation of F. oxysporum f. sp. cumini (Sharma and

Trivedi, 2005). Trichoderma harzianum, T. humatum, T. viride, Bacillus subtilis and

commercial biocide were used as seed coating agents and checked their effect on

Introduction

66

pathogen growth retardation and yield parameters (Mawar and Lodha, 2002; Tawfik

and Allam, 2004a,b). Effectiveness of peanut haulms compost as a carrier of species

of Trichoderma (T. harzianum, T. hamatum and T. koningii) in controlling wilt which

improved the plant growth yield, nitrogen content and seeds oil content was studied at

Egypt (Haggag and Abo-sedera, 2005). The studies were conducted by Shekhawat et

al. (2013) on the management of Alternaria blight of cumin in Rajasthan which

revealed that mancozeb completely inhibited the mycelial growth of the isolate Ab03,

while the other isolates were less sensitive to mancozeb. Tebuconazole completely

inhibited the mycelial growth of all the selected isolates of A. burnsii, followed by

azoxystorbin, carbendazim and mancozeb. Neem formulations Azadirachtin was also

found effective in vitro. Under pot culture, combination of tebuconazole and

Azadirachtin was found effective when applied as mixed foliar spray. A field study

has been conducted at National Research Centre on Seed Spices, Ajmer by Sharma et

al. (2013) on the management of Alternaria blight of cumin using 10 new chemical

fungicides in three replications following randomized block design. Among them

Propiconazole was on par with carbendazim + iprodione and chlerothalonil and

recorded significantly least percent disease index than all other treatments.

Kumari and Deshwal (2012) reported two genera namely Pseudomonas and

Bacillus having biocontrol potential among 54 isolated rhizobacteria that were found

in cumin rhizosphere. Other 8 isolates possessed antifungal activity but with less

intense. Jadeja and Pipliya, (2008) indicated that 5% and 10% extracts of garlic cloves

and ginger rhizomes were effective in inhibiting the growth of A. burnsii. Evaluation

of 11 isolates of Trichoderma spp. against the blight causing pathogen in the

laboratory indicated that T. harzianum and T. viride were significantly effective. Tea

waste was found to be good for mass multiplication of T. harzianum isolate

antagonistic to wilt causing organism F. oxysporum f. sp. cumini (Sharma and Trivedi,

2005). Aspergillus versicolor isolated from the cruciferous residue amended soil was

found to parasitize F. oxysporum f. sp. cumini in hot arid climate of India. In F.

oxysporum f. sp. cumini and A. versicolor infested soil, population of F. oxysporum

drastically declined after 15 days while with T. harzianum infested soil reduction was

less (Israel and Lodha, 2005). Sharma and Bohra (2003) found that leaf extract of

Boerhavia diffusa was effective in inhibiting the growth of F. oxysporum f. sp. cumini

and also reduced cumin wilt. Vyas and Mathur (2002) demonstrated that Trichoderma

Introduction

67

spp. effectively inhibited the growth and/or sporulation of F. oxysporum f. sp. cumini

in vitro through production of volatile and non-volatile antibiotics.

Direct use of antagonistic microorganisms in controlling the pathogen spread

have not seen to be effective much on plant’s growth and development. Use of

physical, chemical and biological modes have been applied to restrict the disease

spread in field since many years in the search of an influential option to strike against

the pathogens but response were not so reasonable.

Moreover, no accounts state the use of elicited defensive capacity of plants

after induced with bio-stimulators to hamper the blight and wilt disease. Elicitation of

the phenylpropanoid pathway is being evaluated upon the application of elicitors for

disease management in crop. Moreover, these phenylpropanoids not only control the

disease but also helps in better growth and development of the plant. Therefore,

looking into the importance of SAR, the present study was carried out to check the

induction of various defense related enzymes in cumin plants after treatment with

Fusarium oxysporum f. sp. cumini and Alternaria burnsii culture filtrates and

comparison was made with several other known biocontrol agents such as B. subtilis,

T. harzianum; commercially available agents i.e. headline and monitor.

The following objectives were undertaken for the present study:

• To study the morphology of the selected fungi and bacteria microscopically.

• To prepare fungal culture filtrates (FCFs) from selected fungi and cell free

culture filtrate (CCF) from selected bacteria and standardize treatment

concentration and time.

• To purify and characterize the active principle compound from the fungal

culture filtrates (FCFs) of both the fungi Fusarium oxysporum f. sp. cumini

and Alternaria burnsii.

• Bioassay of different elicitors on cumin plants to standardize the best mode of

treatment.

• To estimate different enzyme activities i.e. Phenylalanine ammonia lyase

(PAL), Peroxidase (POX), Polyphenol oxidase (PPO), β-1, 3 glucanase, nitrate

reductase (NRA), nitrite reductase (NIR) and catalase in plants treated with

various FCFs and biocontrol agents as well as untreated plants.

Introduction

68

• To estimate total protein and total phenol content in elicitor treated and

untreated plants.

• To screen the total protein profiling by SDS-PAGE and total phenol profiling

by HPTLC.

• To conduct bioassay of extracted phytoalexin from treated and untreated plant

samples against selected pathogenic fungi to check its efficacy and isozyme

staining.

• To extract the essential oil from treated and untreated harvested seeds and

quantitative estimation of cuminaldehyde.