1

CHANGES IN FOLIAR GLYCOALKALOIDS OF POTATO

INDUCED BY LATE BLIGHT DISEASE AND STUDY OF THE

AGGRESSIONS OF 15 ISOLATES OF PHYTOPHTHORA

INFESTANS

ABDUL MAJEED

2

DEPARTMENT OF BOTANY HAZARA UNIVERSITY MANSEHRA

2016

CHANGES IN FOLIAR GLYCOALKALOIDS OF POTATO

INDUCED BY LATE BLIGHT DISEASE AND STUDY OF THE

AGGRESSIONS OF 15 ISOLATES OF PHYTOPHTHORA

INFESTANS

By

3

ABDUL MAJEED

The thesis submitted to the department of Botany, Hazara University, Mansehra

in partial fulfillment of the requirements for the degree of the Doctor of

Philosophy in Botany

DEPARTMENT OF BOTANY HAZARA UNIVERSITY MANSEHRA

2016

CHANGES IN FOLIAR GLYCOALKALOIDS OF POTATO

INDUCED BY LATE BLIGHT DISEASE AND STUDY OF THE

AGGRESSIONS OF 15 ISOLATES OF PHYTOPHTHORA

INFESTANS

4

SUBMITTED BY: ABDUL MAJEED

PhD Scholar

SUPERVISOR: DR. ZUBEDA CHAUDHRY

Associate Professor

Department of Botany

Hazara University,

Mansehra

CO-SUPERVISOR: PROF. DR. IRFANULHAQ

5

Chairman

Department of Plant Pathology

Arid Agriculture University,

Rawalpindi

DEPARTMENT OF BOTANY

HAZARA UNIVERSITY MANSEHRA

2016

6

DEDICATION

To my affectionate parents

7

ACKNOWLEDGEMENT

In the name of Allah, the most merciful and most compassionate, whose

innumerable blessings enabled me to complete this task. All my tributes are to

the Holy Prophet Hazrat Mohammad (P.B.U.H) for enlightening the universe

with teaching and thoughts of the Holy Quran.

The credit of my research work really goes to my honorable supervisor Prof. Dr.

Zubeda Chaudhry, Associate Professor, Department of Botany Hazara University,

who guided and encouraged me in all phases of research work.

8

I find no words to express my feelings in offering whole hearted thanks to my co-

supervisor Prof. Dr. Irfanulhaq for his affectionate behavior and guidance.

Lab facilities for some part of this work were provided by Dr. Zahir Muhammad,

Assistant Professor, Department of Botany, University of Peshawar. His active

support and useful suggestions are thankfully acknowledged.

Sincere thanks are extended to Dr. Louise R. Cooke (Agri-Food and Biosciences

Institute) for critical evaluation and her valuable suggestions which helped me to

amend the final draft of this dissertation. She also provided me some of her

valuable papers and SOP which were very helpful in culturing procedure. I am

also indebted to Dr. William E. Fry (Cornell University) and Dr. Mendel

Friedman (Agricultural Research Service, U.S. Department of Agriculture) for

providing their important papers and book chapters on late blight disease and

potato glycoalkaloids.

I am extremely indebted to Higher Education Commission (HEC), Government

of Pakistan for funding my PhD through Indigenous 5000 Ph. D. Fellowship

program (Batch IV). I feel no hesitation to mention that without the financial

support of HEC, I would not have been able to complete my Ph.D.

Abdul Majeed

9

I n the name of

10

The Most Merciful The Most

Beneficent

CONTENTS

Title Page No.

LIST OF

TABLES………………………………………………………………………………XI

LIST OF

FIGURES…………………………………………………………………………....XII

LIST OF

ABREVIATIONS…………………………………………………………………..XV

ABSTRACT………………………………………………………………………………………

1

Chapter-1

INTRODUCTION………………………………………………………………......4

11

1.1. POTATO (SOLANUM TUBEROSUM l.)………………………….…………………….4

1.1.1 Potato cultivation and production in Pakistan………………………................5

1.1.2 Challenges to potato production…………………………………………….…..6

1.2 POTATO LATE BLIGHT – THE DISEASE AND THE PATHOGEN…………...........7

1.2.1 Life cycle of Phytophthora infestans………………………………………..……..8

1.2.2 Control of the disease………………………………………………………..…..10

1.2.3 Removal of the potential sources of inoculum…………………………..……11

1.2.4 Use of disease free potato seeds………………………………..……………….11

1.2.5 Cultivation of resistant varieties………………………………………………..11

1.2.6 Control of potato blight by application of fungicides……………………..…12

1.2.7. Resistance development in Phytophthora infestans to Metalaxyl

fungicides……………………………………………………………………………….1

3

1.2.8. Late blight in

Pakistan…………………..……………………………………....14 1.3 AGGRESSIVENESS

OF NEW STRAINS OF THE PATHOGEN: CHANGES IN

THE GLOBAL POPULATION STRUCTURE OF PHYTOPHTHORA INFESTANS15

1.4 GLYCO-ALKALOIDS IN POTATO AND THEIR ROLE IN PLANT DEFENSE..…18

12

Chapter-2 REVIEW OF LITERATURE…………………………………………………...…23

2.1. STUDIES ON THE AGGRESSIVENESS OF P.INFESTANS ISOLATES….………...23

2.2 ROLE OF LEAF GLYCOALKALOIDS IN DISEASE…………………………....….....40

2.2.1. Studies on the relationship between glycoalkaloids and potato

pathogens/pests………………………………………………………………………..4

0

2.2.2. Studies on association between glyco-alkaloids and late blight and other

fungal

diseases………………………………………………………………………….42 Chapter-3

MATERIALS AND METHODS…………………………………………………46

3.1 COLLECTIONS OF INFECTED LEAVES………………………………...………….46

3.1.1 Maintenance of

isolates…………………………………………………….……46

3.1.2 Culture of Phytophthora infestans isolates………………………………………47

3.2 EXPERIMENT I- VARIABILITY IN AGGRESSIONS OF P. INFESTANS ……....48

3.2.1 WHOLE PLANT LEAVE………………………………………………….……48

3.2.2 DETACHED LEAFLET ASSAY…………………………………………...……49

13

3.2.3 TUBER DISC

ASSAY…………………………………………………………….50

3.2.3 MEASUREMENT OF AGGRESSIVENESS PARAMETERS..……………..…51

3.3 EXPERIMENT II- CHANGES IN TOTAL GLYCOALKALOIDS OF POTATO

LEAVES INDUCED BY LATE BLIGHT …………..SEVERITY…………………………53

3.3.1 Determination of glycoalkaloids……………………………………………….54

3.4 STATISTICAL

ANALYSIS…………………………………………………….......55

Chapter-4

RESULTS………………………………………………………………………...…67

4.1 EXPERIMENT I- VARIABILITY IN THE AGGRESSIONS OF DIFFERENT `

PHYTOPHTHORA INFESTANS ISOLATES………………………………...………67

4.1.1 WHOLE PLANT

LEAVES…………………………...….………………67

4.1.2 DETACHED LEAFLET

ASSAY…………………………………...……82

4.1.3 TUBER DISC ASSAY……………………………………………...……..95

4.1.4 COMPARISON BETWEEN EXPERIMENTAL MODELS…………..104

14

4.2 EXPERIMENT II- EFFECT OF LATE BLIGHT SEVERITY ON TOTAL

GLYCOALKALOIDS OF POTATO

LEAVES……………….………….….…...118

Chapter-5

DISCUSSION……………………………………….………………...…123

5.1 EXPERIMENT I- VARIABILITY IN AGGRESSIONS OF P.INFESTANS’

ISOLATES……………………………………………………………...………...…134

5.2. EFFECT OF LATE BLIGHT DISEASE SEVERITY ON FOLIAR

GLYCOALKALOIDS OF

POTATO……………………………………………….139 CONCLUSION AND

RECOMMENDATIONS……………………..…...……145 REFERENCES

………………………………………………………………..….…148

ANNEX…………………………………………………………………………....…17

8

15

LIST OF TABLES

Table

3.1

Details of visited locations of KPK and sample collection 56

Table

4.1

Analysis of variance for IF (%), LIP (days), LA (mm2), ALER

(mm2 day-1), AULEC and CAI for Whole plant Model (M1)

68

Table

4.2

Aggressiveness parameters on whole plant leaves caused by

different isolates of Phytophthora infestans collected from

different locations

69

Table

4.3

(A)

Aggressiveness level and composite aggressiveness indices of

P.infestans’ isolates (Whole plant experiment) during 2011

80

16

Table

4.3

(B)

Aggressiveness level and composite aggressiveness indices of

P.infestans’ isolates (Whole plant experiment) during 2013

81

Table

4.4

Mean squares of the analysis of variance for IF (%), LIP (days),

LA (mm2), ALER (mm2 / day), AULEC and CAI for detached

leaflet assay

83

Table

4.5

Values of different aggressiveness parameters of different

isolates of P. infestans collected from various locations on

detached leaflets assay during

84

Table

4.6

Aggressiveness level and composite aggressiveness indices of

P.infestans’ isolates (Detached leaflet assay)

94

Table

4.7

Mean squares of the analysis of variance for IF (%), LIP (days),

LA (mm2), ALER (mm2 / day), AULEC and CAI for Tuber disc

assay

95

Table

4.8

Aggressiveness components on tuber discs of potato caused by

different isolates of Phytophthora infestans collected from various

locations

96

Table

4.9

Aggressiveness level and composite aggressiveness indices of

P.infestans’ isolates (Tuber disc assay)

104

17

Table Varaibility in infection frequency of different isolates of P. 107

4.10 infestans on whole plant leaves, detached leaflet and tuber discs

Table

4.11

Latent infection period (days) of isolates of Phytophthora

infestans on whole plant leaves, detached leaflets and tuber disc

109

Table

4.12

Lesion areas (LA) mm2 of isolates of P. infestans on whole plant,

detached leaflets and tuber disc assay

111

Table

4.13

Average lesion expansion rate (ALER) mm2/day of different

isolates of P. infestans on whole plant leaves, detached leaflets

and tuber disc assay

113

Table

4.14

Area under lesion expansion curve for different pathotypes on

whole plant, detached leaflets and tuber discs assay

115

Table

4.15

Composite aggressiveness index (CAI) on whole plant leaves,

detached leaflets and tuber discs

117

Table

4.16

Analysis of variance of mean square for disease severity (%) on

potato leaves inoculated with Phytophthora infestans

121

Table

4.17

Effect of disease severity on total glycoalkaloids (TGA) of potato

leaves determined at different inoculation periods

121

18

LIST OF FIGURES

Figure

4.1

Infection frequencies (IF)% of isolates Phytophthora infestans

during 2011 and 2013.

70

Figure

4.2

Latent infection period (days) of different isolates of

Phytophthora infestans during 2011 and 2013.

72

Figure

4.3

Lesion area (mm2) of different isolates of P. infestsns collected

from fifteen locations during 2011 and 2013.

73

19

Figure

4.4

Average lesion expansion rate (ALER) mm2 day-1 for

different isolates of P. infestans during 2011 and 2013.

75

Figure

4.5

Area under lesion expansion curve (AULEC) for isolates of

Phtophthora infestans collected from different locations during

2011 and 2013.

77

Figure

4.6

Composite aggressiveness indices (CAI) for different isolates

of Phytophthora infestans during 2011 and 2013.

78

Figure

4.7

Infection frequency (IF) calculated as percentage of diseased

portion of different isolates of Phytophthora infestans

85

Figure

4.8

Latent infection period (LIP) days of different isolates of

Phytophthora infestans on detached leaflet assay.

87

Figure

4.9

Lesion area (LA) mm2 of different isolates of Phytophthora

infestans on detached leaflet experiment

88

Figure

4.10

Average lesion expansion rates (ALER) mm2/day of isolates of

P. infestans sampled from fifteen areas (detached leaflet

experiment).

89

Figure

4.11

Area under lesion expansion curve (AULEC) of different

isolates of P. infestans on detached leaflet assay

91

20

Figure

4.12

Composite aggressiveness indices (CAI) for different isolates

of P. infestans on detached leaflet assay.

92

Figure

4.13

Tuber disc assay revealing infection frequency (IF) for

different isolates of P. infestans

97

Figure

4.14

Latent infection period in days (LIP) for sampled isolates of P.

infestans on tuber disc assay

98

Figure

4.15

Lesion area (LA) mm2 for isolates of P. infestans sampled from

different locations on tuber disc assay

99

Figure

4.16

Average lesion expansion rates (ALER) mm2/day on tuber

disc assay for different isolates of P. infestans

100

Figure Area under lesion expansion curve (AULEC) on tuber disc 101

4.17 assay for sampled isolates of P. infestans

Figure

4.18

Composite aggressiveness index (CAI) of Phytophthora

infestans’ isolates sampled from fifteen different locations of

Khyber Pakhtunkhwa on tuber disc experiment.

103

Figure

4.19

Infection frequency of isolates on whole plant leaves, detached

leaflet and tuber disc assay

108

Figure

4.20

Latent infection period (days) of different isolates of P. infestans

on whole plant leaves, detached leaflets and tuber discs

110

21

Figure

4.22

Lesion area (mm2) of different isolates of P. infestans on whole

plant leaves, detached leaflets and tuber disc assay

112

Figure

4.23

Average lesion expansion rate (ALER) of isolates of P. infestans

on whole plant leaves, detached leaflets and tuber disc assay

114

Figure

4.24

Values of area under lesion expansion curve (AULEC) for

different isolates of Phytopthora infestans on whole plants

leaves, detached leaflets and tuber discs

116

Figure

4.25

Composite aggressiveness index (CAI) for different isolates of

Phytophthora infestans on whole plant leaves, detached leaflets

and tuber discs assay

118

Figure

4.26

Late blight disease severity (% defoliation) on potato leaves

determined at different days after inoculation with P.infestans

122

Figure

4.26

Effect of disease severity on total glycoalkaloids (TGA)(mg

100-1 g fresh weight)

122

22

LIST OF ABBREVIATIONS

ALER Average lesion expansion rate

ANOVA Analysis of variance

AUDPC Area under disease progress curve

AULEC Area under lesion expansion curve

CAI Composite aggressiveness index

23

CIP International Potato Centre

DAI Days after inoculation

FAOSTAT Food and Agriculture Organization Statistical database

Gpi glucose-6-phosphate isomerase

HPLC High Performance Liquid Chromatography

IF Infection frequency

IP Incubation period

LA Lesion area

LIP Latent infection period

LB Late Blight

LP Latent period

LS Lesion size

LSD Least significant differences

mt DNA Mitochondrial DNA

PCSIR Pakistan Council for Scientific and Industrial Research

RAUDLEC Relative area under lesion expansion curve

RFLP restriction fragment length polymorphism

SP Sporulation potential

SSR Simple Sequence Repeats

TGA Total glycoalkaloids

24

CHANGES IN FOLIAR GLYCOALKALOIDS OF POTATO INDUCED BY

LATE BLIGHT DISEASE AND STUDY OF THE AGGRESSIONS OF 15

ISOLATES OF PHYTOPHTHORA INFESTANS

ABSTRACT

Late blight of potato, triggered by Phytophthora infestans (Mont.) de Bary, is a

pathogenic disease of significant importance and regarded as one the most

challenging biological constraints to crop productivity of potato (Solanum

tuberosum L.), tomato (Lycopersicon esculentum L.) and several other genera in the

family Solanaceae. The disease is particularly devastating in cold and humid

agricultures where crop losses are substantial. Since 1980s, population of P.

infestans has changed drastically comprising of more aggressive and virulent races

which make control strategies more difficult. In this study, potato fields of fifteen

different localities of Khyber Pakhtunkhwa were visited during 2011 and 2012 and

naturally late blight infected leaves were collected. Collecte isolates of Phytophthora

infestans were tested for aggressiveness parameters in; (i) whole plant (intact

leaves) experiment; (ii) detached leaflets assay and; (iii) tuber disc method.

Infection frequency (%), lesion area (mm2), latent period (days), lesion expansion

rate (mm2 day-1) and area under lesion expansion curve (AULEC) were used as a

criteria for aggressiveness of isolates of P. infestans after they were inoculated into

whole leaflets, detached leaflets and tuber discs of potato (cultivar Desiree).

Analysis of variance (ANOVA) revealed significant differences for the studied

25

parameters which demonstrated that variability occurred in the aggressiveness of

the sampled isolates. Degree of aggressiveness was determined using the

composite aggressiveness index (CAI) which also showed variability among

sampled isolates. It was observed that isolates of P. infestans from Nathya Gali,

Bara Gali, Ayyubia, Batakundi and Sharan resulted in greater values of infection

frequency, lesion area, lesion expansion rate and area under disease progress

curve but lesser values of latent period; hence they were strongly aggressive

among the studied isolates. Isolates recovered from Kalam, Shangla, Shankiari,

Mahaban, Balakot, Shoghran and Naran revealed almost similar values for the

aggressiveness parameters among themselves but lower than strongly aggressive

isolates and they fell into mildly aggressive category. When compared to highly

and moderately aggressive isolates, isolates sampled from Shabqadar and

Mansehra regions showed relatively lower values of aggressiveness parameters

and they were grouped as weakly aggressive. Whole plant experiment was

repeated in 2013 which showed that pathogen races had different aggressiveness

potentials as compared to 2011 experiment. Experimental models e.g., whole plant,

detached leaflet and tuber disc assay also revealed consistent values regarding

aggressions of the studied isolates.

Second experiment was conducted to assess the effect of late blight severity on

total glycoalkaloids (TGA) of leaves. Disease severity was determined as

percentage area of foliage infected by P. infestans inoculated into leaves while

26

glycoalkaloids were worked out by High Performance Liquid Chromatography

(HPLC) at four different disease severity levels. There were no significant changes

in leaf glycoalkaloids in response to late blight disease severity level on 3rd, 6th, 9th

and 12th day after inoculation (DAI) with P. infestans and no relationship between

disease severity and total glycoalkaloids of leaves was established. ANOVA

demonstrated that total glycoalkaloids contents of diseased leaves with different

late blight severity i.e., 5.3, 23.2, 49.3 and 70.1 % calculated at 3, 6, 9 and 12 th day

after inoculation with P. infestans respectively, did not significantly vary from total

glycolakaloids of control plants (29.3, 42.1, 49.3 and 52.2 mg 100g-1 Fresh Wt.)

which were inoculated with sterile distilled water. However, a slight but

statistically non-significant increase in TGA contents was observed in diseased

plants when compared to the control. Results of this study demonstrated that leaf

TGA contents were not affected by late blight disease severity level; however, age

of plant and length of inoculation period corresponded to higher glycoalkaloids

contents and disease severity of leaves. Highest TGA concentration (52.8 mg 100g-

1 Fresh Wt.) was observed on 12th day where disease severity was 70.1%.

27

Chapter-1

INTRODUCTION

1.1 IMPORTANCE OF POTATO

Potato (Solanum tuberosum L.) in the family Solanaceae is an annual herbaceous

plant which is cultivated in tropics and subtropics, although the crop is best

propagated in temperate conditions. It is principal and economically important

vegetable crop grown in Pakistan and throughout the world. It is ranked the third

most widely consumed non-legume crop after rice and wheat (Hermansen et al.,

2012), contributing to regional and global dietary needs, nutrition security and

economic development (He et al., 2012; DeFauw et al., 2012). In the last two

decades, potato production has been significantly increased both in developing as

well as in developed countries, which demonstrates its significance both as a food

crop and trade commodity (Brich et al., 2012). During 2014, global potato

production exceeded 385 million tons, with China being the leading potato

producer followed by India, Russia, Ukraine and the United States (FOASTAT

2016). In developing countries, potato production has been reached to a record

level during the last decade, producing 165.41 million tons of potatoes as

compared to 159.86 tons produced by the developed countries (FAOSTAT, 2008).

28

The increasing trend in potato production and consumption in the developing

countries is because of high nutrient contents of the crop, easy cultivation of potato

in wide range of climatic conditions and increasing trend in the prices of food

commodities during the current decade (Haverkort, 1990; Brich et al., 2012;

Godfray et al., 2010). In Pakistan potato production is not encouraging relevant to

other potato producing countries (Abbasi et al., 2012).

1.1.1. Potato cultivation and production in Pakistan

In Pakistan, potato is an important dietary and staple vegetable food crop grown

by farmers on a vast land of area. Estimated average yield of potato in Pakistan

during 2007-08 was approximately 23 tons/hectare with major shares from Punjab

(94.79%) followed by Khyber Pakhtunkhwa (4%), Balochistan (1.43%) and Sindh

(0.9%) respectively (Anonymous, 2008). Owing to its high energy contents,

sufficient carbohydrates, minerals and vitamins (Folgado et al., 2013) and low labor

inputs for cultivation, if the yields of potato are to be improved, it may play a very

imminent role in stabilizing food-shortage issues and can contribute towards

economic development of regions.

However, during 2010 and 2011, potato production and area under cultivation for

potato crop in the country has risen significantly. Agricultural Statistics of

Pakistan revealed that during 2008-09 potato crop was grown on an area of 125 ×

29

103 hectares with more than 200 thousand tons potatoes produced annually those

years (Younis et al., 2009). However, during 2010 and 2011, area under potato

cultivation in the country increased up to 160 × 10 3 hectares which contributed in

increasing potato production reaching 3.5 million tons (Anon., 2012). Province wise

production has also significantly increased.

1.1.2 Challenges to potato production

Despite of steady growth rate in terms of production, there are still numerous

challenges which delimit potato productivity in several regions of the world

(Hidalgo, 1998). Major abiotic and biotic constrain which delimit potato

productivity are low temperature, inadequate soil type, drought and salinity

stresses, unavailability of irrigation water, lack of healthy and diseases free seed

tubers, various insect and pest and pathogenic diseases (Raman and Radcliffe,

1992; Oerke et al.,1994; Evers et al., 2012; Holgado and Magnusson, 2012; Folgado

et al., 2013). The most commonly reported pathogenic diseases of potato are scab

(Streptomyces spp.), black scurf (Rhizactonia solani), early blight (Alternaria solani),

stem cankers (R. solani), brown rot (Pseudomonas spp), Charcoal Rot (Macrophomina

phaseolina), soft rot (Ervinia spp.) and late blight of potato (Phytophthora infestans).

Moreover, several types of nematodes particularly root-knot nematodes, cyst

nematodes, rot nematodes and lesion nematodes cause significant reduction in

30

yields as well in quality of tubers. Among the potato diseases, late blight is

potentially threatening to potato production throughout the world.

1.2 POTATO LATE BLIGHT – THE DISEASE AND THE PATHOGEN

Potato late blight is among the many pathogenic diseases of potato crop and other

members of family Solanaceae, corresponding to significant crop damages in

almost all regions of the world where potato cultivation is carried out (Fry et al.,

2009; Forbes et al., 2011). Symptoms of the disease are the appearance of water

soaked lesions and small necrosis on the foliage, causing defoliation and wilting

of the whole plant in severe cases and when the environment is conducive for the

disease development (Henfling, 1987; Fry, 2008). In favorable conditions (low

temperature i.e., below 20 ºC and high relative humidity above 90 %) the disease

development may be accelerated and may cause complete destruction of potato

plants within a few days (Haynes et al., 2002; Nemanda et al., 2004).

The disease is triggered by an oomycete hemibiotroph pathogen Phytophthora

infestans (Mont.) de Bary which also parasitize other members of family Solanaceae

(Fry, 2008; Fry et al., 2009; Forbes et al., 2011). P. infestans is considered to have the

origins in the central highlands of Mexico, then migrated to south America and

then to north America followed by migrations to Europe in the 1840s (Andrivon,

1996). In 1845, P. infestans caused severe crop losses in Ireland and resulted in the

notorious ‘Great Irish Potato Famine’ which caused poverty and starvation, the

31

death of one million and migration of 1.5 million Irish people to other countries.

Thereafter, late blight pathogen is frequently cited in the literature for its

devastations and several epidemics of the disease it had has caused; still,

remaining one of the many challenges to the world’s agriculture with pronounced

crop losses and monetary expenditures (CIP, 1996; Fry, 2008).

1.2.1 Life cycle of Phytophthora infestans

Phtophthora infestans is regarded as a pseudo-fungus dealt with taxonomically in

the Oomycetes (Drenth et al., 1993). It mainly reproduces by asexual means,

although sexual recombination has also been reported in recent years. Asexual

structures are lemon-shaped sporangia which are borne on erect hyphae - the

sporangiophores (Hansen and Shattock, 1998; Visker, 2005). Sporangia are the

means by which P. infestans spread the disease. Under cold and moist

environment, sporangioppores arise from the diseased leaflets which form

sporangia. The sporangia are released with help of rain and are carried next to

uninfected plants thus causing fresh infections (Drenth et al., 1993; Fry, 2008).

When a successful infection occurs, a lesion is appeared on leaf which has the

caharaterestic appearance of greenish darkened spot (Drenth et al., 1993).

Favorable conditions of higher moisture facilitates the release of sporangia which

helps in completeing cycle causing further infection development when there is

sufficient humidity and temperature is also low, release of sporangia occur and

32

which help in completing asexual cycle by germination through germ tube

(Dahlberg et al., 2002; Batista et al., 2006; Andrade-Piedra et al., 2005;

http://bayercropscience.co.uk).

Prior to 1970s, the global populations of LB pathogen (except Mexico) exhibited

limited genetic diversity and comprised of one pathotype – the US-1 or A1 mating

type - which could reproduce only by asexual means (Spielman et al., 1991; Fry

and Goodwin, 1997). Then in the first half of 1980s, scientists reported the

introduction of A2 into the global population of P. infestans alongside A1 (Hohl

and Iselin, 1984). Several reports are documented about the presence of A2 mating

type showing that A2 had not only spread to Europe but also to many other

countries worldwide and this widespread appearance of A2 mating type was

regarded because of several migrations of Phytophthora, sexual reproduction and

evolutionary changes in the late blight pathogen (Tantius et al., 1986; Kadir and

Umaerus, 1987; Mosa et al., 1989). In mid 1970s, larger quantities of potato were

shipped from Mexico, where A2 mating type is common; to Europe and other

countries and this might have helped the new genotype to migrate globally

(Niederhauser, 1991; Ko, 1994). It is generally assumed that the “new” population

where both mating type coexists have changed the “old” population where only

A1 type existed on a global scale (Derie and Inglis. 2001; Fry, 2008). Moreover, A2

mating type serves as a compatible partner for A1, hereby easing the sexual contact

33

between the two as one of the mating type produces antherida and the other

oogonia – male and female sex organs respectively

Asexual reproduction cycle, as described above, can be contimued by the sexual

cycle when both the opposite genotype occur in a population; one type develop

male sex organs ‘antheridia’ while the other produces female sex organs ‘oogonia’

, resulting in sexual contact and the production oospores (sexual spores) (Fry et al.,

2001; Kirk et al., 2004; fry, 2008). The sexual spores (oospores) show resistance to

unsuitable environments and can survive in the diseased stems, tubers, leaves or

free in soil for many years, thus acting as additional source of inoculum (Andrivon,

1995). In the absence of both compatible forms, overwintering is overcome by the

pathogen in the form of vegetative hyphae on the affected plant parts e.g., stems,

tubers or leaves (Kirk et al., 2004; Hannukkala et al., 2007).

1.2.2 Control of the disease

Late blight of potato is managed by employing an integrated disease management

approach for reducing crop losses. One way to control the disease is the use of

healthy and disease-clear seed potatoes for cultivation. Use of varieties which may

possess some resistance genes to late blight pathogen is perhaps the best option;

however, not all the varieties of potato are completely resistant to P. infestans.

Sanitation and appropriate timing for sowing of potato may help in reducing

disease damage. An efficient and quick method to contest late blight disease is the

34

application of synthetic chemicals (fungicides) which eradicate the pathogen or at

least slow down disease frequency. Following methods are generally applied in

agriculture to minimize late blight challenges.

1.2.3 Removal of the potential sources of inoculum

Plant parts like tubers, stems and leaves which were previously infected by P.

infestans may harbor mycelium, sporamgia or oospores of the late blight pathogen.

These infected parts can provide potential inoculum for fresh potato crop (Kirk et

al., 2004). Similarly, during grading of potato tubers, waste heap may also contain

P. infestans and may cause late blight in newly planted potatoes (Andrivon, 1995;

Fry et al., 2001). Proper handling and removing of such a source of inoculum may

reduce the chances for pathogen to infect healthy plants.

1.2.4 Use of disease free potato seeds

Use of healthy and disease-free seed is another strategy to control late blight. Seed

potatoes obtained from unkown sources may harbor the late blight fungus and can

potentially carry the disease with them. As it is very difficult to diagnose which

potato tubers are infected in initial infections; there are greater chances that late

blight pathogen may deceive the farmers while they prepare to grow potatoes.

Thus to prevent potato-borne disease out break, seed potatoes should be obtained

35

from disease free resources. Growing disease free seed potatoes is a preventive

strategy and it may not help in the later disease establishment (Caldwell, 1998).

1.2.5 Cultivation of resistant varieties

Cultivation of blight resistant varieties is a good option for potato growers to

minimize the losses caused by P.infestans. Potato cultivars which have resistance to

P. infestans can reduce the impact of late blight disease to a significant extent (Kirk

et al., 2004; Fry, 2008). Host resistance againt the pathogen has significant

importance in fighting the disease damage. There are some wild species of solanum

such as S. mochiquense, S. bulbocastanum, S. berthaultii, S. demissum, etc. which possess

late resistant genes (Ewing et al., 2000; Ballvora et al., 2002; Smilde et al., 2005). Genes

from wild Solanum species which have exhibited resistance to late blight have been

extensively studied and have been tried to incorporate them into cultivated Solanum

tuberosum by potato breeding and molecular technologies for the purpose of

creating resistance to the notorious pathogen and which has been proven effective

to some extent in developing potato varieties showing some degree of blight

resistance (Walker et al., 2003; Ewing et al., 2000; Ballvora et al., 2002; Vossen et al.,

2005). Thus, for potato growers, to avoid crop and monetaory losses in response to

P. infestans and environmental problems posed herein by the use of chemicals,

growing varieties which have some sort of resistance to the pathogen is a good

alternative.

36

1.2.6 Control of potato blight by application of fungicides

Manipulation of resistant genes in potato varieties is a valuable and risk free

method; however, such genetic manipulation is either not possible because of

breeding/crossing barriers with wild Solanum species or due to enhanced

virulence shown by increased number of P. infestans pathotypes to the resistance

genes (Ballvora et al., 2002; Vossen et al., 2005). Therefore, the primary and efficient

method to reduce crop yield losses triggered by late blight disease is to use

synthetic fungicides (Runno-Paurson et al., 2011). Late blight may be effectively

controlled when application of fungicides is made before infection because most

fungicides have protective effects and they lose efficacy after P. infestans once

establish itself in plant tissues (Schwinn and Margot, 1991; Stevenson, 1993).

1.2.7. Resistance development in Phytophthora infestans to Metalaxyl fungicides

In the last few years, several studies have revealed new strains of P. infestans which

show insensitivity to metalaxyl containing fungicides (James and Fry, 1983; Gisi

and Cohen, 1996; Inglis et al., 1996; Fry et al., 2001). Although, metalaxyl containing

fungicides rveveales good results against P. infestans (Gisi and Cohen, 1996) but

due to continuous applications, these fungicides has resulted in resistance

development in P. infestans (Clayton and Shattock, 1995; Colon et al., 1995) and a

considerable proportion of metalaxyl-insensitive genotypes of P. infestans has been

documented from different regions worldwide. In 1980, phenylamide resistant

37

races of late blight pathogen were reported from Ireland, Switzerland and

Netherlands and and the detection of these resistant isolates were associated with

a decline in the disease control (Jmour and Hamada, 2006). Since then various

workers have investigated the occurrence of resistant isolates of P. infestans from

different potato growing regions.

1.2.8. Late blight in Pakistan

Pakistan is an agricultural country blessed with a wide range of agro-climatic

conditions. Potatoes are grown in both plains and hilly areas of the country almost

round the year. Late blight disease was first noticed in Sawat valley in 1984 (Khan

et al., 1985) and later on it has been frequently found in all potato growing regions

of Pakistan which includes both hilly areas and plains of the four provinces

(Ghazanfar et al., 2010). In hilly areas, frequency of rainfall and low temperature

provides favorable conditions for late blight and many other pathogenic diseases.

So the disease is more common in hilly areas and regions with low temperature

and huge rainfalls. In general, major potato production occurs in five zones of the

country which include zone two, zone four, zone five and zone six (Ahmad et al.,

2008). These zones are occupied by potato production area from Punjab, Khyber

Pakhtunkhwa, Balochistan and Sindh, Northern areas, Azad Jammu and Kashmir

(Ahmad et al., 2008; Anonymous, 2008). These zones have varied climatic

conditions and potato blight is a common disease here. Although, the most

38

suitable environment for P. infestans to cause disease is provided by hills and moist

temperate regions such as Kaghan, Naran and Sawat; however, the disease have

frequent appearance form plains of the other zones as well. After the first incidence

of late blight of potato in 1984 in Sawat valley, the disease has been spread to

almost all potato growing regions of the country and there are numerous reports

in scientific literature regarding the presence of diverse races of the pathogen from

different geographic location of the country (Batool et al., 1998; Ahmad and Mirza,

1995; Ahmad et al., 2008). It is not well understood that where the pathogen

actually came from into Sawat valley; however, it may be inferred that potato

import from countries where the disease was already present may be a possible

mean for introducing the pathogen in local environments.

1.3 AGGRESSIVENESS OF NEW STRAINS OF THE PATHOGEN: CHANGES

IN THE GLOBAL POPULATION STRUCTURE OF PHYTOPHTHORA

INFESTANS

Prior to 1980, in many regions of the world, P. infestans’ population comprised of

one type commonly referred to as A1 or US1 while in Mexico, the population

structure was based on mixed presence of A1 and A2 , occurring in almost equal

proportions (Fry, 2008). Hohl and Iselin (1984) documented in their paper the first

ever report of A2 mating type in 1981 from Switzerland in 1981 and thereafter Fry

et al. (1993) and Goodwin et al. (1998) demonstrated the presence of A2 pathotype

from America and Europe. Their reports concluded that the emergence of A2 type

39

in other parts resulted in population changes of the late blight pathogen. They

further stated that the new population of P. infestans include both A1 and A2

mating types which facilitated sexual reproduction and genetic recombination

which has resulted in the emergence of more fitter and aggressive races of P.

infestans which show broad genetic diversity than their old parental races (Drenth

et al., 1993; Fry, 2008; Flier et al., 2007; Day and Shatock, 1997). Since the appearance

of A2 mating type in scientific literature, studies have been conducted to monitor

variability in worldwide population of P. infestans. For this purpose, several

screening techniques such as glucose-6-phosphate isomerase, mitochondrial DNA

assay, simple sequence repeats and restriction fragment length polymorphism are

practiced by researchers to study variability in the population structure of the

pathogen (Cooke and Lees, 2004; Pule et al., 2013). These techniques are expensive

as well as require greater care and time. In addition to these techniques, disease

parameters for aggressiveness such capacity of infection, latent and incubation

period, lesion size development, spore production and area under lesion

expansion curve, however, provide easy to handle, simple and effective choices

for scientists to study variation in the aggressiveness of different genotypes of P.

infestans. Moreover, these practices can be carried out on detached leaflets and

tubers with reliable results.

Aggressiveness sometimes reffered to as pathogenicity is the capability of a

pathogen or its races to cause quantitative damage on susceptible hosts

40

(Vanderplank, 1963). For measurement of aggressiveness of P. infestans isolates

and many other plant pathogens’ races, different disease parameters such as spore

production capacity, frequency of infection, incubation period, latent period, area

under lesion expansion curve, lesion growth and lesion area on infected hosts are

used to designate a particular pathogen or its races as highly aggressive or weakly

aggressive depending on disease damage outcome (Miller et al., 1998; Flier and

Turkensteen, 1999; Pliakhnevich and Ivaniuk, 2008; Pariaud et al., 2009). These

aggressiveness parameters are generally used according to sample size of isolates,

duration of study, experimental conditions, types of experiment wither whole

plant or detached leaves. Reliable results have been reported using one or more of

the aggressiveness parameters used for determination of aggressiveness of

pathtypes of P. infestans and for monitoring changes in the population of this

pathogen worldwide (Fry, 2008; Pliakhnevich and Ivaniuk, 2008). Infection

frequency, lesion area, latent period, lesion growth rate, area under lesion

expansion curve (AULEC) and composite aggressiveness index are important

aggressiveness parameters which demonstrate the pathogen’s capacity to cause

quantitative damage on the host. On the basis of variability in these components,

different genotypes of the pathogen under study may be considered as strongly

aggressive or weakly aggressive. The evidence that new populations of P. infestans

studied after the appearance of A2 mating types are more aggressive and diverse

than the old population has been frequently documented in the literature. Miller

41

et al. (1998) used aggressiveness parameters to report that population of P. infestans

of the US showed variability in different sampling regions in their study. Irish

population of P. infestans races exhibited significant variation for aggressiveness

parameters such as area under lesion expansion curve, infection frequency, spore

capacity, latent period (Carlisle et al., 2002). Suassuna et al. (2004) studied Brazilian

isolates of P. infestans for aggressiveness and host specificity. They reported

variation in the aggressiveness parameters of the studied isolates i.e., infection

frequency, latent period, period for incubation, area of lesion, rate of lesion

expansion and capacity for sporulation for tomato and potato. Population changes

of late blight pathogen on the basis of variability in the aggressiveness parameters

form other parts of the world such from Ecuador (Chacon et al., 2007), Belarus

(Pliakhnevich and Ivaniuk, 2008) and South America (Andrade-Piedra et al., 2005)

have been documented.

1.4 GLYCOALKALOIDS IN POTATO AND THEIR ROLE IN PLANT

DEFENSE

Potato, like other plants, produce secondary metabolites which have no direct role

in growth, development or reproductive potential. Among the secondary

metabolites produced in potato and some other members of family Solanaceae are

glycoalkaloides which are produced in variable concentrations in different plant

parts (Osman, 1983). These compounds are toxic to pathogenic fungi, insects and

pests, herbiovres and are known to play some role in offereing resistance to

42

pathogens, herbivores, insects and pests (Tingey, 1984; Matthews et al., 2005;

Friedman, 2006). Alpha-chaconine and alpha-solanine are major classes of

glycoalkaloids found in potato (Freidman and McDonald, 1997). Glycoalkaloids

levels of potato are dependent on different factors such as potato germplasm, plant

age, biotic and abiotic stress, attacks of pathogens and herbivory (Sinden et al.,

1984; Friedman, 2006). Higher concentrations of glycoalkaloids are usually observed

in those plant parts which have high metabolic activity. Young leaves, buds and

reproductive organs posses higher glycoalkaloids which become declined when

plant progress towards maturity (Freidman and McDonald, 1997).

Distribution of glycoalkaloids is variable in different parts of potato; however,

leaves have more glycoalkaloids than tubers, sprouts and stems, generally

controlled by several factors such as potato cultivars, age of plant, environmental

stresses and phyto-pathogens (Sinden et al., 1984; Friedman, 2006). Based on

previous studies of Friedman and Dao (1992) and Deahl et al. (1993), glycoalkaloids

in leaves may range between 50 and 145 mg 100g-1 fresh weight. Similarly, in

whole tubers their concentration in many studies, have been reported between 10

and 150 mg 100g-1 fresh weight (Gelder et al., 1988; Mathews et al., 2005). However,

these results may be challenged elsewhere because concentrations of these

compounds are variable in different varieties and in different environments.

Higher concentrations of glycoalkaloids in leaves than tubers is attributed to the

fact that leaves are generally more exposed to sunlight and phytopathogens in

43

addition to many other abiotic stresses than tubers and other parts of potato

(Friedman, 2006).

Glycoalkaloids are potentially toxic for human consumption; therefore, potato

breeders always opt for obtaining breeding lines of potato with low and acceptable

level of these compounds (Friedman, 2006). However, their toxic nature is

considered by several researchers as blessing for host defense against different

herbivores, nematodes, insects and pests and a wide range plant pathogens

(Tingey, 1984; Matthews et al., 2005). Mode of action of glycoalkaloids like other

secondary metabolites in protection of host against plant pathogens is not well

understood; however, possible elucidation for the protective role of these

compounds might be the production of defense proteins, complex formation with

pathogen cell wall by the glycoalkaloids and activation of pathogen specific

catalyzing enzymes for degradation cell wall of the pathogen (Osbourn, 1996).

Although role of potato glycoalkaloids in minimizing damages caused by

notorious insects and pests such as Colorado potato beetle (Jonasson and Olsson,

1994; Lorenzen et al., 2001), potato aphid (Guntner et al., 1997), white cyst

nematode (Forrest and Coxon, 1980), wireworm (Jonasson and Olsson, 1994) and

snail (Smith et al., 2001) is well established; however, few studies have been

conducted to point out the role of glycoalkaloids found in potato leaves and tubers

in disease resistance against P. infestans with variable reports (Friedman, 2006;

44

Khan et al., 2013). According to Deahl et al. (1973) ther was no association between

late blight disease resistance and leaf and tuber glycoalkaloids of 15 potato lines.

Frank et al. (1975) also demonstrated potato leaves glycoalkaloids did not play a

significant role in disease resistance against P. infestans in field conditions. Andreu

et al. (2001), on the other hand found that glycoalkaloids and some other

phytoalexins accumulated in leaves and potato tubers when they were inoculated

with P.infestans, however, there was lack of a clear and significant relationship

between glycoalkaloids and disease resistance.

The increased prevalence of late blight epidemic in different parts of the world

during 2000s indicated that newer strains of P. infestans have emerged which

exhibited variation in aggressiveness. Thus, monitoring of population changes of

P. infestans on the bases of variation in the aggressiveness parameters of a

geographic region is very important for formulating adequate control measures

for reducing crop losses caused by the disease. Similarly, understanding the role

of glycoalkaloids in defence and resistance against P. infestans will appeal potato

breeders for the development of potato varieties which possess appropriate and

acceptable concentration of glycoalkaloids for human consumption and with

potential role in protection against late blight pathogen. To my knowledge, studies

regarding variability in aggressiveness of P. infestans isolates for the

aggressiveness parameters and correlation between glycoalkaloids and late blight

45

disease are have not been conducted so far under local environmental conditions

of Pakistan.

Therefore, specific objectives of the study were:

1. To determine variability in the aggressiveness of 15 isolates of Phytophthora

infestans collected from fifteen different locations of the Khyber

Pakhtunkhwa province on the basis of aggressiveness parameters

2. To examine and compare changes in variability of aggression of the studied

isolates

3. To test the efficacy of three models of experiments i.e., whole plant, detached

leaflet and tuber disc assay for disease parameters of P. infestans isolates

4. To diagnose possible changes in foliar glycoalkaloid of potato induced by

late blight disease severity

46

Chapter-2

REVIEW OF LITERATURE

2.1. STUDIES ON THE AGGRESSIVENESS OF P.INFESTANS ISOLATES

Aggressiveness determination of a pathogen’s races is important area to monitor

changes in its population structure. In regard to variability of the occurrence of

diverse pathotypes of P. infestans population throughout the world when from

Switzerland A2 mating type of late blight pathogen was reported in 1980s (Hohl

and Iselin, 1984), extensive studies mainly based on genotypic markers such as

mtDNA haplotype, peptidase, restriction fragment length polymorphism, simple

sequence repeats and Glucose-6-phosphate isomerase analysis of different isolates

from different sampling regions have been carried out (Cooke and Lees, 2004; Lees

et al., 2006; Pule et al., 2013). Few researchers have worked on aggressiveness as a

criterion for characterization of P. infestans strains for monitoring global

population changes of the late blight pathogen.

47

Legard et al. (1995) evaluated different genotypes of P. infestans collected from

USA, the Netherlands, Canada and Mexico for aggressiveness on potato and

tomato leaves in growth chamber and field experiments. The studied

epidemiological components, lesion area and sporangia per lesion varied

significantly for different isolates. Generally, US-1 genotype was more aggressive

on potato leaves while US-6 on tomato hereby causing greater lesion diameter and

more sporangial production.

Sujkowski et al. (1996) carried out mating type, allozyme, and DNA sequencing for

aggressiveness and virulence of 95 isolates collected from Poland during 19851991.

They detected new variants in Polish population representing new strains which

were not present in Poland before the studied period. Isolates were divided into

three groups: first group comprised 22 isolates (PO-1) which showed 5.5 specific

gene resistances / isolate. The second group had 30 isolates (PO-4) completely new

to Poland having average virulence 6.5 per isolate. The third group contained 43

isolates which showed mean virulence 6.7 per isolate. They showed that Polish

population of P. infestans had new migrants with more aggressiveness and

virulence not detected before 1988 in the country.

Day and Shattock (1997) evaluated 618 isolates of blight pathogen recovered from

England and Wales during 1978 and 1995 for aggressiveness, mating type,

metalexyl insensitivity and mitochondrial DNA haplotype. They observed both

48

pathotypes with infrequent occurrence of A2 in the studied population, increased

insensitivity to metalexyl, replacement of mt DAN haplotype Ib by Ia and IIa and

varied level of aggressiveness which was measured by infection frequency and

number of sporangia per lesion. Generally, pathotypes with I-a and II-a type of

mitochondrial DNA were more aggressive than Ib type.

Miller et al. (1998) determined the aggression potential of twenty two genotypes of

P. infestans collected from Washington and Oregon in petriculture assay and whole

plant experiment. Significant variability was recorded among isolates for disease

parameters (AUDPC, AULEC, IP, LP, SP, LS). Their study indicated that

genotypes US-11 and US-8 exhibited strong aggressiveness by yielding higher

AULEC values, lesion size and sporulation when compared to aggressiveness

values of other genotypes US-7, US-6 and US-1. Among the tested pathotypes, US6

genotype was less aggressive which resulted in relatively lower values of

aggressiveness parameters.

Flier and Turkensteen (1999) tested thirty six strains of P. infestans sampled in the

Netherlands for infection efficiency, latent period, sporulation intensity and

maximal growth rate. The studied populations originating from three different

regions showed significant variations for aggressiveness parameters and

aggressiveness indices.

49

Lebreton et al. (1999) reported high degree of variations for the aggressiveness

parameters in French population of the pathogen; characterization was performed

on both detached and whole plant experiment. A2 mating type was more

aggressive than A1 corresponding to higher lesion rates, infection efficiency and

disease progress curves on both detached and whole plants. Similarly, isolates

hosted on tomato showed more aggressions than those hosted on potato.

Peters et al. (1999) worked on the aggressiveness potential of P.infestans’ isolates

collected from Canada during 1995-1997. They documented that multilocus

isolates, G40, G29, G26, G11, US8, US7 were strongly aggressive than US1

genotypes because they caused greater aggressiveness parameters such as

composite aggressiveness index, lesion depth and surface necrosis on potato

tubers than US1.

Jaime-Garcia et al. (2000) reported major changes in Mexican’s population of P.

infestans where previously the two mating strains existed in equal frequency; but

enhance proportion of A1 was documneted during their study period (1994-1997).

The study based on characterization of different isolates P. infestans on potato and

tomato plants for aggressiveness, mating types, metalaxyl sensitivity and

genotypic markers during the years 1994 to 1997.

Daayf et al. (2001) reported highly pathogenic diversity in Canadian genotypes of

P. infestans recovered from potato and tomato cultivated fields in different regions

50

during 1997; which they characterized for aggressiveness (size of lesions), mating

type, metalaxyl sensitivity, Gpi-allozyme and RAPD analysis.

Carlisle et al. (2002) demonstrated that significant diversity in aggressions existed

between P.infestans isolates originating from different regions of Northern Ireland.

Their studies were based on aggressiveness parameters, latent period, infection

frequency, sporulation capacity and area under lesion expansion curve measured

on petridish experiment containing leaflets of three varieties of potato viz Cara,

Bintje and Stirling after inoculation with P. infestans. They concluded that Irish

population of P. infestans comprised of significantly variable isolates with different

aggressiveness capacity.

Knapova and Gisi (2002) characterized one hundred and thirty four P. infestans’

pathotypes sampled from Swis and French fields of tomato and potato during

1996-1997. Characterization of genotypes was worked out on phenotypic markers

(aggressiveness, differential potato virulence, mating strains, response to

fungicides (phenylamide), and fitness of pathogen races) and genotyping (mtDNA

haplotype). They recorded highly variable results among isolates for

aggressiveness parameters (latent period, lesion size, sporulation capacity and

fitness index) and other phenotypic and genotypic markers.

Cooke et al. (2003) stated that Scottish population of P. infestans contained broad

spectrum genetically diverse group of pathotypes during 1995 and 1997. Their

51

finding came from characterization of 500 isolates collected from commercial fields

and gardens for mating, response to metalexyl fungicide and DNA AFLP analysis.

The pathogen’s population in Scotland consisted of both strains in 4:1 ratio (4A1:

1A2); equal proportion of metalexyl resistant isolates in 1995 but increased number

intermediate isolates in 1996 and 1997. Cluster analysis revealed that half of the

studied isolates had unique DNA patterns.

Ghimire et al. (2003) studied 280 isolates during 1999-2000 collected from different

fields in Nepal. Mitochondrial and nuclear DNA polymorphism was carried out

which suggested diverse genotypes categorized into four clusters with Gleason

index 1.78. Ia and Ib mt DNA haplotype were recorded in 88 and 12 % respectively.

They concluded that the displacement of old population by new ones and three

migration events to Nepal.

Samen et al. (2003) evaluated thirty two US-8-relatives isolates in USA for genetic

variability through DNA-RAPD and AFLP. Results showed high level of genetic

diversity and polymorphism in the studied isolates.

Day et al. (2004) established that a total of 2691 isolates collected from Scotland,

England and wales during 1995-1998 revealed 3 % of A2 and rest of A1 mating

types, increased fungicides resistance (intermediate) during the study period,

increased frequency of mt DNA haplotype I-a (91%) and about 30 multilocous

RFLP fingerprints. Three fingerprints, RF-006, RF-039, RF-002 were more common

52

and were representative of A-1 clones whereas, RF-040 was assigned to A-2.

Isolates RF-002 were metalexyl-non-responsive and were more frequent in

Scotland than other sampled regions.

Suassuna et al. (2004) characterized Brazilian isolates of P. infestans for

aggressiveness and host specificity in 1998. They reported variation in the

aggressiveness parameters of the studied isolates i.e., incubation period

sporulation capacity, lesion area, infection frequency, latent period, lesion area

and lesion expansion when experiments were performed on tomato and potato.

Gotoh et al. (2005) carried out characterization of 401 isolates originating from

Korea, Taiwan India, Nepal, Indonesia, Thailand, Japan and China for mating

type, sensitivity to metalexyl, mt DNA haplotyping, RG57 fingerprinting, Gpi, Pep

and malic enzyme polymorphism. They identified twenty multilocous with 14

new genotypes. Some multilocus strains e.g., JP3, JP1 and JP2 showed metalexyl

resistance. Samples from Japan, Nepal, China (Central) and Thailand revealed

coexistence of multiple locus pathotypes in the period 1998-2000, 1997 and 1994

respectively.

Atheya et al. (2005) reported marked genetic diversity in 67 P.infestans’ strains

collected from fields of Himalaya and Indian subtropical regions assayed through

genetic markers on the basis of geographic regions. They observed 161

polymorphic fragments which were generated by 10 decamer primers. Isolates

53

originating from planes significantly differed from those collected from

Himalayan hills in regard to polymorphism; however, there were no

differentiation among isolates for mating type.

Sliwka et al. (2006) analyzed 93 P. infestans isolates sampled from naturally infected

fields of potato in Poland during 2002-2004 for aggressiveness, metalaxyl

response, mating type and pathotypic virulence on leaves of potato cultivars Bintje

and Tarpan. Aggressiveness component used in the study was lesion size. They

reported that P. infestans populations in Poland comprised of highly to moderately

aggressive isolates.

Chacon et al. (2007) evaluated four strains of the late blight pathogen obtained from

field as well as wild species of potato in Ecuador during 2003 for aggressiveness

measures e.g., lesion growth rate, infection frequency, lesion size, latent period

and incubation period and relative area under the lesion expansion curve. Their

results revealed significant variations between isolates for aggressiveness

parameters.

Flier et al. (2007) documented extensive variations in genetics and pathogenicity of

LB pathogen isolates originating from France, Norway, Switzerland and the

United Kingdom based on their studies during 2001 and 2002 in which they used

mitochondrial DNA haplotyping, mating types and different aggressiveness

parameters such as sporulation density, lesion growth rate and infection efficiency

54

as a criteria for characterization of aggressiveness on detached leaves of potato

cultivars Bintje and Sante. Isolates varied in pathogenicity and genotypic markers

indicating considerable diversity of pathotypes in the studied regions.

Ahmad et al. (2008) tested 178 late blight genotypes sampled from different potato

zone in Pakistan for Metalexyl-sensitivity. They found Pakistani population of the

pathogen comprised sensitive, intermediate and resistant strains. Population of

zone 2 revealed 50.17 % while zone 6b had 33.33% resistant types.

Pliakhnevich and Ivaniuk (2008) documented that variability existed in P. infestans’

genotypes surveyed in Belarus during 2006 and 2007. Isolates were characterized

for metalaxyl resistance and aggressiveness parameters (infection frequency,

lesion area, incubation period, latent period, sporulation capacity and composite

aggressiveness indices). They found isolates had greater diversity in regard to

metalaxyl response but insignificantly differences were recorded for

aggressiveness components.

Chen et al. (2009) stated that Taiwanis population of P. infestans consisted of

genetically diverse and aggressive isolates during 1991 to 2006 after they

investigated 655 P. infestans’ pathotypes sampled tomato and potato and

characterized for allozyme analysis, aggressiveness potential, metalaxyl-response,

mating type and mt-DNA fingerprinting.

55

Li et al. (2009) revealed significant variations among Chinese’ late blight isolates

characterized for virulence, mating type, metalaxyl-responses and genotypic

markers during 1998-2007.

Vargas et al. (2009) characterized Columbian population of P. infestans collected

from two hosts (S. tuberosum and P. peruviana) for aggressiveness, isozyme analysis

and RFLPF assay. They reported extensive variations among the isolates for

studied parameters.

Guo et al. (2010) studies genetic and pathotypic properties of 100 isolates from

China in 1998 and 2004. Their analysis was basically for mating types, resistance

to metalexyl, mitochondrial DNA haplotype, allozyme and RFLP fingerprinting

which revealed significant variation among the studied isolates indicating that

Chinese population contained highly diverse genotypes.

Montarry et al. (2010) collected 220 isolates from 20 different fields in France during

2004 and 2005 and characterized them for mating types and multi locus

genotyping. They stated that French population consisted of genetically diverse

isolates with the co-existence of both compatible strains.

Runno-Paurson et al. (2010) performed characterization of Estonian (n=432)

genotypes to test mating type, mitochondrial DNA haplotype, virulence and

metalexyl response. They concluded that population in Estonia comprise

genetically variable isolates, contain both mating types with the ability to produce

56

oospores and varied degree of response to metalexyl fungicides. They further

stated that A2 type was present in the range 3-71 %, virulence for resistant gene

R5 was with low frequency (3-17 %) and three i.e., I-a, II-a and II-b mt-

DNAhaplotypes were present with varied percentages (7-51 %).

Schultz et al. (2010) carried out phenotypic (medial growth, pathogenicity, mating

types and metalexyl responses) and genotypic (isozymes, mt DNA haplotypes and

genomic profiling) studies on isolates surveyed from 2005 to 2007 in Florida. They

presented their results which revealed A2 were the only mating strains and high

proportion of mt DNA I-a, varied level of genetic diversity and no response toward

metalexyl were observed.

Brurberg et al. (2011) assessed 200 isolates obtained from different fields of

Norway, Finland, Denmark and Sweden for genotypic variation with nine SSR

markers. 169 multilocus genotypes were observed in 191 isolates with Shannon

diversity index 0.95 for the studied geographic locations. They observed large

variation in genetic patterns within the four countries. Results revealed 60 % A1

and 40 % A2 strains respectively.

Gisi et al. (2011) reported considerable variation in aggressiveness and genetic

characteristics of late blight genotypes sampled from eight European countries in

1997, 2006 and 2007. They recorded that A2 mating type increased in number

during 1997 to 2007 and increased occurrence of isolates resistant to mefenoxam.

57

Aggressiveness was determined by lesion expansion x sporulation capacity which

revealed higher values for all pathotypes. However, French and British genotypes

were more aggressive in 1997 and 2007 respectively than isolates sampled from

other countries. Aggressiveness of French isolates declined in 2007. The study

established six SSR genotype families (I-VI) of which I, III and IV were dominant

in 1997 but declined in 1997 and were replaced by families II and V.

Blandon-Diaz et al. (2012) worked on analysis of Nicaraguan population of late

blight pathogen to study virulence, mating type, fungicides response, mt DNA and

microsatellite genotyping during 2007-2010. A total of 132 isolates analyzed

revealed only A2 types and I-a dominant mt DNA haplotype and most pathotypes

showed no response phenylamide fungicide (propamocarb hydrochloride). Great

variations were observed among isolates for virulence which revealed that out of

82 isolates, 13 different races were found with the most common occurrence of

R111. The researchers concluded that Nicaragua population is dominated by NI-1

genotypes.

Hu et al. (2012) collected 178 isolates from potato and tomato fields from eastern

US, Midwestern US and eastern Canada and find out analysis allozyme, DNA

fingerprints, mating type and fungicides resistance during 2002-2009. The study

revealed the presence of three new genotypes not previously reported in the

region in addition to commonly found strains. Samples from NC revealed US-8

58

and US-20 were the most commonly occurring isolates which parasitized potato

and tomato respectively during 2002-2007 but only in 2005 in Florida. US-22 hosted

upon both tomato and potato in New York and Tennessee in 2007 and 2009, while

US-23 in Delaware, Pennsylvania, Virginia and Maryland in 2009. Results also

showed that both strains were presents equally in proportion on both hosts in

Pennsylvania and Virginia. Genotypes US-20 and US-8 showed resistance, US-21

was intermediate while US-24, 23, 22 responded sensitively to the test fungicide.

Cooke et al. (2012) documented the more aggressive and virulent genotype of P.

infestans ‘13_A2’ in eupropean population and stated that the documented lineage

rapidly displaced other races which resulted in 75 percent increase in its

population in Britain in less than three years. The 13-A2 was found more

aggressive on cultivated potato and showed remarkable polymorphism and

effector genes. It was further showed that 13_A2 carry proteins which disturbs the

immunity responses of potato and thus overcoming resistance genes of the host.

Chowdappa et al. (2013) assessed 19 isolates recovered from tomato fields in

Karnataka (India) in 2009-2010 for aggressiveness, mating, metalexyl insensitivity,

mitochondrial DNA, DNA analysis by SSR and RFLPs with RG57 probe. They

documented that A-2 strains were the only genotypes in the study, resistant

metalexyl compounds, I-a mtDNA haplotype and largely aggressive on tomato.

59

The examined characters of isolates revealed many races of 13_A2 type which were

not previously detected in India.

Harbaoui et al. (2013) studied Tunisian population of P. infestans for mating type,

aggressiveness, virulence, metalexyl resistance and radial growth of mycelium

during 2006 to 2008. They characterized 165 pathotypes obtained from

tomato/potato fields with natural infections in Tunisia and revealed highly

variable differences among isolates for the studied attributes.

Li et al. (2013) published their report about first ever presence of blue_13 isolates

of P. infestans in China. Genetic analysis mitochondrial assay revealed 68 different

lineages and I-a, II-a and II-b haplotypes inside different geographic regions. In

nortehn and southeastern provinces of China, A1 was dominant but in other

provinces both A1 and A2 co-existed present indicating migration of asexual

pathotypes into the country and sexual recombination in Chinese population.

Danies et al. (2013) determined variability in germination rate, mating type,

sensitivity to mefenoxam and variation in aggressiveness of the US pathotypes.

A1/A2 was found in the studied population. Mefenoxam sensitivity were shown

by genotypes US-24, US-22 and US-23, however US-8 was not sensitive.

Pathogenicity differed between genotypes and host crops; US-8 and US-24 showed

pathogenicity on potato while US-22 and US-23 on tomato. Increasing tendency

toward realeasing zoospores were detected in US-24 than US-22 and US-23.

60

Delgado et al. (2013) studied 66 P. infestans’ pathotypes recovered from potato

fields in Ecuadorian provinces (Loja, Chimborazo and Carchi). Their analysis

showed that all the isolates belonged to A1 group. Genetic variations were

determined by SSR which revealed 31 multilocus genotypes which comprised 49

various races. Large aggressiveness variations were recorded in Ecuadorian

population exhibiting virulence on 4-11 R genes. Also, increased sub-clonal

differences were detected in EC-1 lineage in comparison to other clones found in

Netherland and Nicaragua.

Han et al. (2013) stated that 70 isolates were A1 and 15 A2 of a total of 85 isolates

collected from potato fields during 2007 in Gansu province of China. DNA

haplotyping established 25 % Ia and 75 % IIa with 54 % metalexyl resistance in the

province. 26 genetically variable genotypes were detected by SSR technique.

Seidl and Gevens (2013) sampled and analyzed 143 isolates from different counties

of Wisconsin during 2009-2012 for clonal lineages, mating type, resistance to

mefenoxam, and Gpi profile. Genotypes US-22, 23 and 24 were found new to

Wisconsin not previously reported. US-22 was of A2 mating and sensitive to

mefenoxam which had Gpi 100/122 while US-23 and 24 were of A1 which showed

intermediate sensitivity to test fungicide.

In Russia, Stasyuk et al. (2013) worked on virulence, mating and metaleyl response

of 1097 isolates of P. infestans during 2000-2011. They detected increased frequency

61

of A2 genotypes, stability in metalexyl sensitivity of isolates and increased

frequency of all but virulence gene 2 of the total detected 11 vir genes.

Chmielarz et al. (2014) conducted experiment on characterization of 96 Polish

isolates of the late blight pathogen for aggressiveness, mating, metalexyl effect,

polymorphism and haplotyping in 2006, 2008 and 2009. A1 mating group

dominated the population, I-a haplotype was the common mt DNA and most of

the pathotypes were metalexyl responsive. Pathogenicity factors contrasting the

host immune-resistance-responses were detected in most of the studied

genotypes. Substantial genetic variations were observed in the Poland population.

Out of 96, 66 genotypes had unique genetic patterns. Similarly, novel geneotype

13_A2 were also present in Poland.

Chowdappa et al. (2014) determined aggressiveness, metalexyl sensitivity, mating

type, DNA fingerprints and mitochondrial DNA profile of 157 isolates of late

blight pathogen sampled from potato (n=63) and tomato (n=94) from south India

during 2010-2012. Results confirmed all the studied isolates belonged to A2 type

amd most were resistant to test fungicide. Dominant DNA haplotype was I-a. SSR

and RG57 probe revealed prominent clonal lineage was13_A2 with sub-variants

13_A2_1, 13_A2_3c, 13_A2_3b and 13_A2_3a. Aggressiveness was measured by

lesion diameter which revealed high aggression potential of all isolates on tomato

and potato.

62

Genotypic diversity through SSR markers, mitochondria DNA and mating

category and aggressiveness of 165 isolates for collected from Tunisia were studied

during 2008 and 2009 (Harboui et al., 2013). The researchers revealed that

dominant clonal lineage was NA-01 and A1 type. Frequent DNA haplotype was

Ia. These isolates showed less virulence on R genes. In some regions, more diverse

and aggressive strains comprising both mating type were also detected.

Peters et al. (2014) characterized 138 isolates sampled from tomato and potato foliage

from 11 Canadian provinces for DNA RFLP, metalexyl response, mit. DNA

haplotyping, mating type and allozyme-assay during 2011. Their findings were that

A1 and A2 both were found in Canadian population, dominant pathotype was US-

8 although, US-22, US-11, US-23 and US-24 had established in different provinces.

Rojas et al. (2014) worked on the epidemic characters of twelve P, infestans isolates

on potato tuber using lenticel infection and AUDPC as a criterion for measuring

aggressiveness of the test pathogen collected from northern United States in 2009

and 2010. They found that US isolates 8 were more aggressive than US isolates 22.

Similarly, differential responses of hosts (potato and tomato) to pathogen’s

aggressiveness were also observed.

Runno-Paurson et al. (2014) demonstrated that diversity occurred among 386

isolates of P. infestans in Estonia and 671 isoaltes in Finland during 2001 and 2007.

They mainly tested aggressiveness pathotyping, mating types and metalexyl

63

responses. Variable strains were recovered in population belonging to both

countries; in Estonia AI and A2 occurred in equality while in Finland A2 were

dominant races. Moreover, they observed increased sensitivity of isolates towards

metalxxyl in Estonia but it decreased in Finish isolates.

Shan et al. (2015) determined that there were low genetic diversity among 269 races

of P. infestans which the recovered from nursery germplasms of potato in China

during 2010-2011 and assayed for mating types, mitochondrial dna and

aggressiveness against eleven R-genes. The reported both AI and A2 types were

present; there were higher number of isolates which showed virulence towards 11

R genes and pronounced resistance among isolates to metelexyl compound.

Tian et al. (2015a) analysed pathogenic diversity among 125 isolates of P. infestans

recovered from different fields of Shaanxi, China in 2009. Phenotypic and

genotypic variation, mating type diversity, virulence variability were significant

among the isolates. A total of 94 pathotypes were regarded as A1 type, many of

the pathotypes did not possess a-virulence genes and IIa was dominant mt DNA

haplotype.

Tian et al. (2015b) demonstrated that 959 isolates of P. infestans collected from

Chinese fields during 2009-2011 were different in their mating types,

mitochondrial DNA, and pathogenicity. They observed that mating pattern of

isolates changed drastically over time and majority of the isolates were self fertile.

64

Similarly, 74 isolates were reported to show virulence against late blight resistance

genes R 1-11.

2.2 ROLE OF LEAF GLYCOALKALOIDS IN DISEASE

2.2.1. Studies on the relationship between glycoalkaloids and potato

pathogens/pests

Published reports on the possible role of potato glycoalkaloids in host defense

against economically important Colorado potato beetle, nematodes and other pests

demonstrate variable results. In many cases, positive roles of glycoalkaloids in

deterring feeding, attack, growth and development of certain potato pests has been

well established.

Forrest and Coxon (1980) demonstrated a lack of correlation between tuber total

glycoalkaloids content of potato clones derived from the cross between S. vernei ×

S. tuberosum and common nematode of potato (G. pallida).

Tingey (1987) reported insecticidal and pesticidal activity of potato glycoalkaloids

against leafhopper of potato (E. fabae) and Colorado beetle (L. decemlineata) and

regarded these compounds as the natural resistance factors to insects and pests.

Jonasson and Olsson (1994) performed field experiments and bio-assays to

evaluate the influence of total glycoalkaloids of different varieties of potato which

had differential concentrations of glycoalkaloids to test wether these varieities

65

were resistant to wireworm (Agriotes obscurus). They recorded significant

retardation in wireworm and larval feeding on potato with high levels of total

glycoalkaloids contents.

Guntner et al. (1997) demonstrated variable responses of potato aphid

(Macrosiphum euphorbiae) to glycoalkaloids and their aglycones. Lower

concentrations of α-chaconine were stimulatory to feeding behavior of the aphid

but lethal at higher concentrations.

Rangarajan and Miller (2000) revealed that potato hybrids derived from Solanum

chacoense with different levels of leptine concentrations effectively controlled

feeding by Colorado potato beetle (Leptinotarsa decemlineata).

Yencho et al. (2000) evaluated the impact of leptine glycoalkaloids concentration

on defoliation and feeding behavior of Leptinotarsa decemlineata (Colorado

potato beetle) on progenies of tetraploid S. chacoense x S. tuberosum. They reported

significantly decreased defoliation by Colorado potato beetle (up to 26%) at higher

concentrations of leptine in foliage of potato hybrids.

Lorenzen et al. (2001) published their findings regarding correlation between foliar

glycoalkaloids of tetraploid potato and resistance to Colorado potato beetle. They

reported that potato hybrids with high foliar glycoalkaloids resulted in delayed

development of neonate L. decemlineata and 75 % inhibition of larval weight gain.

66

Smith et al. (2001) reported inhibition in feeding of snail (Helix aspersa L.) by potato

glyco-alkaloids, α-solanine and α-chaconine in a filter paper disc method.

Chaconine was found more effective in deterring snail than solanine.

2.2.2. Studies on association between glycoalkaloids and late blight and other

fungal diseases

Possible role of glycoalkaloids (foliar as well as tuber) in potato to offer resistance

to diverse fungal diseases including late blight of potato has been reported in the

literature. Different researchers have reported different results regarding the

correlation between glycoalkaloids and late blight pathogen and some other

fungal pathogens. According to Friedman (2006), studies conducted in Russia and

USA on relationship between glycoalkaloids and infection by P.infestans in

revealed contrasting results. However, of the published reports, many studies

have not established any possible relationship between these compounds and late

blight disease.

Ishizaka and Tomiyama (1972) reported accumulation of glycoalkaloids (solanine)

when potato cut surface was inoculated with a non-compatible race of

Phytophthora infestans; however, only traces of these compounds were found when

inoculation was done with suitable races of late blight pathogen.

Dehal et al. (1973) assessed fifteen potato clones for finding possible association

between late blight disease resistance and total glycoalkaloids contents of leaves

67

and tubers. Their results indicated no relationship between the disease severity

and total glycoalkaloids contents.

Frank et al. (1975) revealed that there was no correlation between late blight disease

resistance and leaf glycoalkaloids of ten potato cultivars in their field trials

conducted in Maine during 1972 and 1973. Similarly, other potato diseases, early

blight, common scab and verticillium wilt had no association with the

glycoalkaloids of tested potato cultivars

Morrow and Caruso (1983) documented no relationship between total

glycoalkaloids and the severity of Rhizactonia solani infection on potato plants with

glycoalkaloids levels between 1.6 and 32.8 mg/100 g fresh tissue.

Olsson (1987) studied the impact of glycoalkaloids levels of potato tuber on

resistance to soft rot of Fusarium solani and Phoma exigua in different genotypes.

He determined no relationship between disease resistance and glycoalkaloids

contents of the studied potato clones.

Fewell and Roddick (1993) documented antifungal activity of potato

glycoalkaloids against different fungal pathogens such as Phoma medicaginis,

Alternaria brassicicola, Rhizoctonia solani and Ascobolus crenulatus. They observed

that 1:1 mixture of solanine and chaconine had strong inhibitory effects on the

studied fungi; however, inhibition varied with type of fungi, concentration of

glycoalkaloids and pH.

68

Andreu et al. (2001) demonstrated the accumulation phytoalexins, glycoalkaloids

and phenols in leaves and tubers of different potato varieties after inoculation with

P. infestans. Late blight susceptible variety Bintje had lower level of glycoalkaloids,

phenols and phytoalexins than resistant variety Pampeana INTA, suggesting

possible role of those compounds in field resistance against P. infestans.

Andrivon et al. (2003) found low but significant correlation between the

concentration of glycoalkaloids (α-solanine) of some potato clones and late blight

resistance parameters i.e., incubation period and spore production per unit lesion

area, suggesting a possible role of these compounds against P. infestans.

Henriquez et al. (2012) studied changes in foliar metabolic compounds of potato

when P. infestans was introduced into potato foliage and infection occurred. They

reported that potato varieties differing in immunity to late blight pathogen

showed differential response in regard to accumulation of metabolic compounds

following infection by P. infestans. A significant suppression in levels of rutin and

catechin was observed in variety Russet Burbank in response to P. infestans while

in Defender, levels of flavonol-glycoside and catechin were elevated. Similarly, the

infection resulted in higher level of an unknown terpene. They suggested the

possible role of secondary metabolites in host defense against P. infestans.

69

Chapter-3

MATERIALS AND METHODS

3.1 Collections of infected leaves

Cultivated potato fields of different areas of the Khyber Pakhtunkhwa province,

Kaghan, Naran, Sharan, Shougran, Batakundi, Balakot, Ayyubia, Bara Gali,

Mahaban, Shabqadar, Shinkiari, Shangla, Kalam, Nathya gali and Mansehra were

visited during 2011-12 potato growing season. A minimum of thirty samples of

late blight infected leaves (natural infection) were obtained from each sampled

locality (Table 3.1). Collected samples were placed in black polythene bags. They

were then transferred to Plant Pathology Laboratory, Department of Botany,

University of Peshawar, Peshawar, for further studies.

70

3.1.1 Maintenance of isolates

Method described in Laboratory Manual for P. infestans (CIP, 1997) was followed

for maintaining different isolates of P. infestans. In order to maintain isolates of P.

infestans alive for the experiment establishment, potato tubers were washed with

water and cut into slices. Collected leaves with late blight infection (lesions) were

washed with tap water so dust and other impurities were removed. Lesions were

cut with the help of sterile razor and placed beneath tuber slices in petridishes

without culture media separately for each sample. Petri plates were incubated for

5-6 days at 18 °C. After incubation, sporulation formed as white cottony plug on the

upper side of slices. Sporangia were picked with help of sterile forceps and were

placed along with a drop of water on fresh tuber slices for re-inoculation purpose.

These steps were repeated many times until experiments for aggressiveness

determination and glycoalkaloids determination were ready.

3.1.2 Culture of Phytophthora infestans isolates

Rye agar media B was used for culturing sampled isolates of P. infestans. Culturing

was performed at Plant Pathology Laboratory, Department of Botany, Arid

Agriculture University, Rawalpindi. Rye agar medium was prepared following

the method of Caten and Jinks (1968) with minor changes as listed on Fry’s Labs

(http://www.plantpath.cornell.edu/). In order to prepare Rye B agar medium,

first, 60 g of Rye grains were soaked in 1000 ml deionized distilled water for 24

71

hours. After 24 h, the supernatant was poured off the grains and preserved in a

separate beaker. The soaked grains were provided with distilled water (one inch

above the grains) and boiled at 68 °C for one hour. The boiled grains were filtered

through quarter folded cheese cloth and squeezing the softened grains into a

beaker. The supernatant separated initially from the soaked grains was mixed with

filtrate. Then 20 g of sucrose, 0.05g β-sitosterol and 15 g of agar were added to it

by adjusting its volume to 1000 ml and then autoclaving for 20 minutes at 15 psi.

Infected tissues of collected leaves (single lesion) from each locality were placed in

100 x 15 mm petri-dishes containing rye agar medium which was amended 20mg/L

rifamycin, 200mg/L Ampicilin and 100mg/L vancomycin antibiotics obtained

from Standard Sales, Peshawar. For sporangial initiation, petridishes were placed

in an incubator at 18 ºC in dark for four days. After four days of incubation,

sporangia were visible and well developed. Sporangia were transferred by sterilized

glass rod to fresh rye agar medium without antibiotics and were re-incubated at 18

°C for 14 days in dark. 40 ml of distilled water was added to freshly formed

sporangia in each petriplate and they were extricated with the help of sterile glass

rods. Thus, sporangial suspensions were obtained. A double layer cheese cloth was

used for filteration of sporangial suspensions in order to remove mycelial

fragments. With the help of haemocytometer, concentration of sporangia was

adjusted to 6 × 104 sporangia/ml. in order to release zoospores from sporangia and

to use them for further inoculation studies, sporangial suspensions were kept in

72

referigerator at 4ºC for two hours as previously documented (Mukalazi et al., 2001;

Pliakhnevich and Ivaniuk, 2008)

(Plates 3.1-3.3).

3.2 EXPERIMENT I- VARIABILITY IN AGGRESSIONS OF DIFFERENT P.

INFESTANS ISOLATES

3.2.1 WHOLE PLANT LEAVES

Seed potatoes (tubers) of cultivar Desiree were obtained from Hazara Agricultural

Resaerch Station, Abbottabad. They were grown in four row plots which were 3

meter long provided with space of 70 cm between rows and 30 cm within rows in

cultivated field near Tarnol, Rawalpindi during October – December 2011. The

experiment was repeated in October- December 2013. During each experimental

period, full expanded leaves of two months old potato plants were randomly

selected for inoculation purpose. Five middle leaves of each plant were randomly

selected and each isolate from the fifteen sampled areas were inoculated onto leaves

as a single drop of 20 µl of zoospore suspension. Inoculation was performed during

evening. Zoospore suspensions were provided to the central right side of midrib of

each leaf. Thus each plant had five inoculation sites. In order to provide conducive

environment for pathogen establishment, potato plants were overhead irrigated

with a hand sprayer twice a day. There was no rainfall recorded in 10 days of

experimental period in each year. Temperatures ranged between 15-20 °C.

73

The experiment was arranged in a randomized completely block design (RCBD).

A single plant was regarded as one experimental unit with four replications each.

Aggressiveness parameters were determined every 24 hours after first inoculation

till 10 th day of inoculation.

3.2.2 DETACHED LEAFLET ASSAY

For detached leaf assay, expanded leaves of 60 days old potao plant (cultivar

Desiree) were detached. Uniform size leaflets were choosen for inoculation

purpose. Leaflets were washed with distled water for removing dust and dried

with clean paper. Five leaflets were put in a single petri dish (100 x 15 mm)

containing moist filter paper with adaxial side of leaflets for inoculation (Plate 3.4,

3.6). Each of the 15 sampled isolates was provided onto five leflets each as a single

drop of 20µl zoospores suspension transferred to the midrib of each leaflet in the

center on adaxial surface. Petri dishes were incubated at 18ºC in an incubator with

12 hour photoperiod for 10 days. The detached leaf experiment was laid out in a

completely randomized design (CRD) manner, considering each petri dish a single

experimental unit which was further replicated four times. Detached leaf

experiment was performed in Plant Pathology laboratory, University of Arid

Agriculture, Rawalpindi during 2011.

74

3.2.3 TUBER DISC ASSAY

Medium sized healthy potato tubers (cultivar Desiree) were washed for 5 minutes

to remove any dust particles. They were cut into slices. Tuber discs of 25 mm

diameter and 5mm depth were prepared with the help of cork-borer. Five discs

were placed in a single 100 x 15 mm petri dish lined with moist filter paper without

agar media. 20 µL of zoosporangial suspension of each isolate collected from

fifteen locations were provided to the center of each tuber disc with the help of

micro pipette. Each petri plate was replicated four times and the experimental

design was randomized complete block. Petri plates were incubated for 10 days at

18 C in an incubator following the method of Miller et al. (1998).

3.2.4 MEASUREMENT OF AGGRESSIVENESS PARAMETERS

After inoculation, observations were made with naked eye for every 24 hours for

field experiment (whole plant), detached leaflet assay and tuber disc assay for 10

days. The aggressiveness parameters those described by Spielman et al., 1992; flier

et al., 2007; Carlisle et al., 2002; miller et al., 1998; Lebreton et al., 1999 were

determined for whole plant leaves, detached leaflet assay and tuber disc assay as:

75

(a) Percent infection frequency (IF)

It was measured at the 10th day after inoculation as no infection on leaf = 0%,

infection on one leaf = 20%, three leaves infected = 60%, four leaves infected = 80%

and five leaves infected = 100% following the method of Carlisle et al. (2002).

(b) Latent period (days)

LIP was calculated as time taken (days) from the start of infection till the

development of sporangia on the infection site of leaf or tuber disc (Kato et al.,

1997; Chacon et al., 2007). For calculating LIP, fully developed lesions from tuber

discs, detached leaflet and whole plant for each isolate were randomly selected

from the replicates and observed with naked eyes from the day of inoculation till

the appearance of sporangia.

(c) Lesion area (LA) (mm2)

Lesion size was calculated as lesion area (mm2) by measuring length and width of

each lesion in each experiment. Observations were made with the naked eye every

24 hours after inoculation. Lengths and widths were measured using a calibrated

ruler. Lesion area was calculated by LA =1/4π × length × width following the

method of Colon et al. (1995) and Vleeshouwers et al. (1999).

76

(d) Average Lesion expansion rate (ALER)

Lesion area was used for calculation of average lesion expansion rate (ALER) (mm2

day-1) by dividing the final lesion area on number of days of inoculation period as

documented by Colon et al., 1995 and Vleeshouwers et al., 1999.

(d) Aarea under the lesion expansion curve (AULEC)

AULEC was measured according to Carlisle et al. (2002) using the following

relation

∑ [(LA i+1 +LA i)/2] [d i+1 – d i]

Where LAi is lesion area at ith observation; di is inoculation period in days at ith

observation

(e) Composite aggressiveness index (CAI)

In order to determine the aggressiveness level (%) of isolates, composite

aggressiveness index (CAI) was calculated by CAI = (IF x LA)/LIP as reported by

Montarry et al. (2007). Isolates with CAI ≥100 were considered as strongly

aggressive; 100 ˂CAI ˃50 as mildly aggressive and CAI ≤ 50 as weakly aggressive.

77

3.3 EXPERIMENT II- CHANGES IN TOTAL GLYCOALKALOIDS OF

POTATO LEAVES INDUCED BY LATE BLIGHT DISEASE SEVERITY

In order to determine total glycoalkaloids of leaves of healthy and late blight

infected potato leaves, seed potato of cultivar Desiree were grown in four row

plots, 3 meter long with spacing of 70 cm between rows and 30 cm within rows at

Botany Department, Hazara University Mansehra during 2011. Experimental

design was randomized complete block design and four replications were used. A

20 µl zoosporangial suspension of P. infestans isolate collected from Sharan was

placed at the midrib of mature leaf after 45 days of planting. Five leaves from each

plant were inoculated in this manner. Sterile distilled water was used to inoculate

control plants. In order to protect control plants from late blight infection, they

were sprayed with Mandy Prompide (Revus), a contact fungicide after every 72

hours. Disease severity was determined as percentage of foliar infected area after

3rd, 6th, 9th and 12th days of inoculation (Lebreton et al., 1999). Green leaves were

carefully assessed visually for late blight infection at the specified days. The

appearance of brown spots indicated late blight infection. Green and non-green

(brown spots) portion of leaves were compared and disease severity was

calculated as percentage of infected portion of leaf of the total leaf area.

78

3.3.1 Determination of glycoalkaloids

For glycoalkaloids detremination, infected and healthyleaves (each leaf weighing

approximately 300mg) were detached from middle of plants from water

inoculated control and late blight diseased plants at 0, 3, 6, 9 and 12 day after

inoculation. Glycoalkaloids contents were analysed by high performance liquid

chromatography (HPLC) at Pakistan Council for Scientific and Industrial Resaerch

(PCSIR) Laboratory complex, Peshawar using HPLC Shimadzu SC – 6A System

following the method of Dao and Friedman (1996) with minor modifications. After

3, 6, 9, and 12th days of inoculation, infected leaves with different severity levels

were collected from each plot inoculated with P. infestans. Healthy leaves were

collected from control plants which were inoculated with distilled water. Leaves

for each treatment (control and diseased) were collected from middle parts of

plants and they were fully expanded. Each leaf weighed approximately 300 mg.

Leaf samples with different disease severity and water inoculated control leaves

were blended separately in 20 mL aqueous acetic acid (5%) solution for ten

minutes to obtain a homogenious mixture. The homogenious mixture was filtered

through no. 4 filter paper in a beaker and pH of the filtrate was adjusted to 10 by

ammonium hydroxide solution. The filtrate from control and diseased leaves was

used for total glycoalkaloids contents determination by HPLC technique at PCSIR

Laboratory complex, Peshawar using HPLC Shimadzu SC – 6A System. Total

glycoalkaloids were determined as mg 100g-1 fresh weight sample.

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3.4 STATISTICAL ANALYSIS

For whole plant experiment and detached leaflet assay, five leaflets per potato

plant were used for inoculation and they were regarded a single treatment. Each

treatment was replicated four times. Similarly, tuber disc experiment was

performed considering five tuber slices (discs) as a single treatment, each

replicated four times. For each experiment, i.e., whole plant, detached leaflet and

tuber disc assay, 15 isolates of phytophthora infestans from fifteen locations, were

inoculated into respective leaflets and tuber discs. Analysis of variance (ANOVA)

was performed on collected data under randomized completely block design with

four replicates using SPSS software v. 19 (IBM Corp, 2012) for measuring variation

among the studied isolates for the aggressiveness parameters. Significant

differences among mean were evaluated by Least Significant Difference (LSD) at

p ≤ 0.05.

For data analysis regarding changes in foliar glycoalkaloids induced by late blight

disease, ANOVA was applied to the collected data. The experimental design was

randomized completely block with four replications. Least Significant Differences

(LSD) Test was used for accepting significant differences between means at p ≤

0.05.

Table 3.1 Details of visited locations of KPK and sample collection

80

Locations Fields

visited

Samples

collected

Isolates

cultured

Abbreviation

assigned to

isolates

Kaghan 5 80 15 Ka

Naran 3 50 17 Nr

Sharan 4 54 16 Sh

Shougran 6 60 17 Sg

Batakundi 3 40 15 Bt

Balakot 4 40 17 Bl

Ayyubia 2 30 15 Ab

Bara Gali 3 55 20 Bg

Mahaban 2 30 14 Mb

Shabqadar 9 40 19 Sb

Shankyari 3 50 18 Sk

Shangla 4 30 16 Sn

Kalam 5 35 6 Kl

Nathya Gali 2 40 10 Nt

Mansehra 4 30 16 Mn

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Plate 3.1. Preparation of Rye Agar medium

Plate 3.2. Culturing of isolates of Phytophthora infestans on rye agar plates

82

Plate 3.3. Sporangial suspensions of Phytophthora infestans isolates

Plate 3.4. Whole plant (intact leaves) 2011

83

Plate 3.5. Whole plant (intact leaves) 2013

Plate 3.6. Detached leaflet assay

84

Plate 3.7. Tuber disc assay; A- Before inoculation; B- After inoculation

Plate 3.5. Tuber disc assay; A - before inoculation; B - after inoculation with P. infestans

B

A

85

A

B

Plate 3.8. Detached leaflet assay; A- before inoculation; B- after inoculation with different isolates of Phytophthora infestans

86

A

B

Plate 3.8. Continued…..

87

A

B

Plate 3.8. Deatached leaflet assay; A- before inoculation; B- after inoculation with P. infestans isolates

88

A

B

Plate 3.9. Tuber disc assay; A- before inoculation; B- after inoculation with P. infestans

89

A

B

Plate 3.9. Continued……

90

A

B

Plate 3.9. Tuber disc assay; A- before inoculation; B- after inoculation with isolates of Phytophthora infestans collected from

different locations.

91

Chapter-4

RESULTS

4.1 EXPERIMENT I- VARIABILITY IN THE AGGRESSIONS OF DIFFERENT

PHYTOPHTHORA INFESTANS ISOLATES

4.1.1 WHOLE PLANT LEAVES

Analysis of variance (ANOVA) demonstrated that data for infection frequency,

latent period, lesion area, lesion expansion rate, area under lesion expansion curve

and composite aggressiveness index for whole plant (intact leaves) varied

significantly (p≤0.05) among isolates of P. infestans sampled from various locations

(Table 4.1 & 4.2).

92

Table 4.1. Analysis of variance for IF (%), LIP (days), LA (mm2), ALER (mm2 day-

1), AULEC and CAI for Whole plant Model (M1)

Source d.f IF (%) LIP

(days)

LA

(mm2)

ALER (mm2

day-1)

AULEC CAI

Replications 3 16.36ns 0.029ns 0.090ns 0.002ns 0.084ns 0.33ns

Isolates 14 564.63* 6.79* 5.23* 0.048* 33.73* 5451.81*

Error 42 15.50 0.020 0.033 0.001 0.021 0.114

Total 59

Coefficient

of variation

(%)

4.99 1.74 2.59 4.63 1.18 0.49

ns = non-significant; * significant

d.f. = degrees of freedom; IF = infection frequency; LIP = latent period; LA =

lesion area; ALER = average lesion expansion rate; AULEC = area under lesion

expansion curve; CAI = composite aggressiveness index

Table 4.2. Aggressiveness parameters on whole plant leaves caused by different

isolates of Phytophthora infestans collected from different locations

93

Isolates IF (%) LIP (days)

LA (mm2)

ALER

(mm2

day-1)

AULEC CAI

1. Sharan (Sh) 94.43a 6.40c 8.70a 0.86a 14.80b 124.9d

2. Batakundi (Bt)

94.35a 6.50c 8.60ab 0.86a 15.13a 126.2c

3. Ayyubia

(Ab) 94.38a 6.52c 8.47ab 0.79b 14.75b 126.7b

4. Bara Gali

(Bg) 94.32a 6.57c 8.35ab 0.83ab 15.02a 126.7b

5. Nathya gali (Nt)

94.07a 6.50c 8.50ab 0.85a 15.05a 128.9a

6. Kaghan (Ka)

73.72b 8.47b 6.50c 0.65c 12.63c 59.65e

7. Naran (Nr) 73.70b 8.47b 6.55c 0.65c 12.43cde 59.35e

8. Shougran (Sg)

73.57b 8.32b 6.47c 0.64c 12.52cd 59.45e

9. Balakot (Bl)

73.55b 8.35b 6.47c 0.64c 12.50cde 58.22f

10. Mahaban

(Mb) 73.40b 8.37b 6.50c 0.65c 12.45cde 59.30e

11. Shankyari

(Sk) 73.07b 8.47b 6.50c 0.65c 12.30e 58.13f

12. Shangla

(Sn) 73.43b 8.35b 6.45c 0.64c 12.40de 57.80f

13. Kalam (Kl)

72.80b 8.45b 6.55c 0.65c 12.38de 59.20e

14. Shabqadar

(Sb) 58.70d 10.40a 5.60d 0.55d 5.72g 31.83g

15. Mansehra

67.13c 10.32a 5.37d 0.53d 6.02f 31.40g

5.618 0.2018 0.2592 0.0451 0.2068 0.5415

IF = infection frequency; LIP = latent period; LA = lesion area; ALER = average lesion

expansion rate; AULEC = Area under lesion expansion curve; CAI = composite

aggressiveness index.

Means bearing different alphabets are different significantly from each other at p≤0.05.

94

4.1.1.1 Infection Frequency (IF) %

Highest infection frequencies 94.4, 94.3, 94.3, 94.3 and 94.0 % were observed for

isolates collected from Sharan (Sh), Batakundi (Bt), Ayyubia (Ab), Bara Gali (Bg)

and Nathia Gali (Nt) respectively during November 2011. However, during 2013,

however, these isolates resulted in increased values of infection frequencies which

were 95.3, 96.5, 95.7, 98.0 and 97.6 % (Fig. 4.1).

Fig. 4.1. Infection frequencies (IF)% of isolates P. infestans during 2011 and 2013.

IF values for isolates Kaghan (Kg), Naran (Nr), Shougran (Sg), Balakot (Bl),

Mahaban (Mb), Shankyari (Sk), Shangla (Sn), Kalam (Kl), Shabqadar (Sb) and

Mansehra (Mn) were recorded in the order 73.7, 73.7, 73.5, 73.5, 73.4, 72.8, 58.7 and

95

67.13 % during 2011 and 85.8, 86.2, 94.5, 82.6, 95.9, 85.4, 90.8, 98.5, 78.9 % during

2013 respectively. Almost all the studied isolates resulted in significantly higher IF

values in the year 2013 as compared to 2011. Increase in IF values was however,

much pronounced for isolates Sg, Mb, Sn, Kl and Sb which showed drastic

increases in 2013 when compared to 2011. It was noted that during 2011, highest

IF (94.4%) was caused by isolates Sh while the lowest was recorded for Sb (58.7%)

but during 2013, Sb was highly aggressive by causing 98.5 % infection frequency

with the lowest being observed for Mn (78.9 %) in that year (Fig. 4.1).

4.1.1.2 Latent period (LP) (days)

Fig. 4.2 depicts data regarding latent period (LPs) for sampled isolates (whole plant

experiment) during 2011 and 2013. Results indicated that isolates of P. infestans

exhibited significant variation in latent period both during 2011 and 2013;

however, relatively lower LPs were observed for different isolates during 2013.

During 2011, maximum LP was found for isolates Mn (10 days) followed by Sb

(9.8 days), Kl, Nr, Sk (8.4 days), Mb (8.37 days) and Sg, Bl, Sn (8.3 days). Lowest

latent period were calculated for isolates collected from Bara Gali (Bg) and Nathia

Gali (Nt) which revealed 6.5 days each. During 2013, a significant decrease in LIPs

occurred for almost all the studied isolates when compared to LIPs of the studied

isolates during 2011. Highest LP (7.8 days) was observed for isolate Ka in 2013

where it was 8.4 days for the same isolates in 2011. Unlike LPs calculated for

isolates during 2011, there was no specific sequence of increase or decrease in LPs

96

for isolates during 2013. In 2011, isolates Mn resulted in LPs of days while during

2013 it drastically dropped to 6.8 days. Shabqadar isolates (Sb) had yielded 9.8

days LP which reduced to 5.6 days in 2013. Bg and Nt resulted in 6.5 days LIP

during 2011 which were 5.8 and 5.4 respectively for the studied isolates in 2013.

Decreases in LPs during 2013 were also evedent for isolates collected from Naran,

Shougran, Mahaban, Shangla, Shabqadar and Kalam when the data were

compared to those in 2011 (Fig. 4.2).

Fig. 4.2. Latent period (days) of different isolates of P. infestans during 2011 and

2013. LIP were calculated in days.

4.1.1.3 Lesion area (mm2)

Data for lesion area (LA) of different isolates of P. infestans is shown in Fig. 4.3.

97

From the results it was observed that lesion area (LA) varied significantly for

studied isolates of P. infestans in the study period. During 2011, isolates collected

from Sharan (sh) caused maximum damage on host leaves which resulted 8.7 mm2

lesion area; however, during 2013 it was not as destructive and values for LA

dropped significantly which were 5.4 mm2. Sampled islaotes from Batakundi (Bt)

resulted in 8.6 mm2 lesion area in 2011 but 6.7 mm2 in 2013. Isolates obtained from

Ayyubia, Bara Gali and nathia gali reveled almost similar values for lesion area

which ranged between 8.3-8.5 mm2 and 6.2-6.3 mm2 during 2011 and 2013

respectively.

Fig. 4.3. Lesion area (mm2) of different isolates of P. infestsns collected from fifteen

locations during 2011 and 2013.

Isolates originated from Kaghan, Naran, Shoughran, Balakot, Mahaban, Shankyari

and Kalam had lesion areas in the range 6.4-6.5 mm2 during 2011 but 5.2-6.7 mm2

98

in 2013. It was noted that isolates collected from shabqadar and mansehra had the

lowest lesion development during 2011 which were 5.6 and 5.3 mm2 respectively.

During 2013, isolates from shabqadar caused 7.4 mm2 while those from Mansehra

resulted in 55 mm2 lesion area (Fig. 4.3).

4.1.1.4 Average Lesion Expansion Rate (mm2 day-1)

Results for whole plant experiment showed that average lesion expansion rate

(ALER) (mm2 day-1) for different isolates significantly varied among collected

isolates. Similarly, during each of the experimental period i.e., 2011 and 2013,

isolates yielded different ALER values (Fig. 4.4). During 2011 experimental period,

isolates obtained from Sharan, Batikundi, Bara Gali, Nathia gali and Ayyubia were

found to be more aggressives causing significantly greater ALER values which

were recorded as 0.86, 0.86, 0.79, 0.83 and 0.85 mm2 day-1 respectively; however,

lesion expansion rates were significantly lower for these isolates during 2013

which were recorded as 0.54, 0.67, 0.62, 0.63 and 0.62 mm2 day-1 respectively.

Isolates collected from Kaghan, Naran, Shougran, Balakot, Mahaban, Shankiari,

Shangla and Kalam caused 0.65, 0.65, 0.64, 0.64, 0.65, 0.65, 064 and 0.65 lesion

expanasion rates in 2011 but 0.55, 0.58, 0.60, 0.52, 0.68, 0.56, 0.65 and 0.67 mm2/day

in 2013. These isolates were relatively moderate in aggression because they caused

somewhat lower ALER than those isolates collected from Sharan, Batakundi,

Ayyubia, Bara Gali, and Nathia Gali. Isolates obtained from shabqadar and

Mansehra regions resulted in 0.55 and 0.53 mm2 day-1 average lesion expansion

99

rates in 2011 and 0.74 and 0.55 mm2 day-1 in 2013. It was noted that isolates

collected from Shabqadar and Mansehrawere lowestly aggressive during 2011

because their ALER values were the lowest among all the studied isolates;

however, Sb isolates caused highest ALER (0.74 mm2 day-1) in 2013 and hence it

was strongly aggressive (Fig. 4.4).

Fig. 4.4. Average lesion expansion rate (ALER) mm2 day-1 for different isolates of

P. infestans during 2011 and 2013.

4.1.1.5 Area under lesion expansion curve (AULEC)

Two year data for area under lesion expansion curve (AULEC) for different

isolates is presented in Fig. 4.5. Results confirmed that area under lesion expansion

100

curve (AULEC) values for different isolates were variable in both experimental

years (2011 and 2013) as well as variable among the studied isolates. In 2011,

highest AULEC values were observed for isolates originated from Sharan (14.8),

Batakundi (15.13), Ayyubia (14.75), Bara Gali (15.02) and Nathyagali (15.05) which

demonstrated the strong aggressiveness potential of these isolates. Isolates

obtained from Kaghan, Naran, Shougran, Balakot, Mahaban, Shangla and Kalam

fell into moderately aggressive catagory because their AULEC readings were

almost similar ranging between 12.3-12.6. The lowest AULEC were recorded for

isolates from Shabqadar and Mansehra which corresponded to 5.72 and 6.02

respectively. During 2013, drastic changes in AULEC of different isolates were

found. Isolates with highest to lowest AULEC were noted in the order as Mahaban

(42.0), Shangla (38.1), Batakundi (37.19), Shougran (34.9), Shabqadar (32.4), Kalam

(30.7), Ayyubia (28.15), Bara Gali (24.35), Saharan (24.5), Shankyari (22.5), Nathia

Gali (22.2), Balakot (20) and Mansehra (20). Interestingly, isolates from Shabqadar

and Mansehra which had lower AULEC in 2011 (5.72 and 6.02) resulted in

significantly higher values of AULEC (32.4 and 20) in 2013 (Fig. 4.5).

101

Fig. 4.5. Area under lesion expansion curve (AULEC) for isolates of Phtophthora

infestans collected from differen locations during 2011 and 2013.

4.1.1.6 Composite aggressiveness index (CAI)

Purpose of composite aggressiveness index (CAI), calculated as IF×LA/LIP, was

to determine the aggressiveness level of different isolates of P. infestans collected

from fifteen locations of Khyber Pakhtunkhwa. From the results presented in Fig.

4.6, it was observed that isolates coming from Sharan, Batakundi, Ayyubia, Bara

Gali and Nathia Gali had maximum composite aggressiveness index values during

2011 which ranged between 124.9 and 128.9. This order was followed by isolates

collected from Kaghan, Naran, Shougran, Balakot, Mahaban, Shankiari,

102

Shangla and Kalam which resulted CAI values in the range 57.8-59.65. During

2013, however, composite aggressiveness index values showed drastic changes for

the studied isolates. Unlike 2011, greater CAI values were recorded for isolates

which were obtained from Batakundi, Ayyubia, Bara Gali, Nathia Gali, Mahaban,

Shangla and Shabqadar regions which had composite aggressiveness indecis as

105.9, 103.8, 106.4, 112.3, 105.2, 111.3 and 134.9. On the other hand, isolates from

Sharan, Kaghan, Naran, Shougran, Balakot, Shankiari, Kalam and mansehra had

comparatively lower values of CAI ranging between 60.0 and 97.5 (Fig. 4.6).

Fig. 4.6. Composite aggressiveness indices (CAI) for different isolates of P. infestans

during 2011 and 2013.

Aggressiveness level of isolates on the basis of composite aggressiveness index

103

(CAI) is shown in Table 4.3 A & B. On the basis of composite aggressiveness

indices, studied isolates were classified as; (a) strongly aggressive (CAI ≥ 100), (b)

mildly aggressive (100 ˂CAI ˃50) and (c) weakly aggressive (CAI ≤ 50). In 2011,

isolates collected from Sharan, Batakundi, Ayyubia, Bara Gali and Nathya gali

resulted in higher CAI values (CAI≥100) and they were strongly aggressive.

Isolates obtained from Kaghan, Naran, Shougran, Balakot, Mahaban, Shankyari,

Shangla and Kalam were mildly aggressive (100 ˂CAI ˃50) while isolates from

Shabqadar and Mansehra were found to be weakly aggressive which had CAI˂

50. Overall, 43.33 % isolates were strongly aggressive, 53.33 % mild aggressive and

13.33 % were recorded as weakly aggressive (Table 4.3 A). However, during 2013,

47% isolates (Batakundi, Ayyubia, Bara Gali, Nathya gali, Mahaban, Shangla) were

strongly aggressive with CAI values ≥100 while 53 % isolates (Sharan, Kaghan,

Naran, Shougran, Balakot, Shankyari, Kalam, and Mansehra) were mildly

aggressive which had 100 ˂CAI ˃50 (Table 4.3 B).

104

Table 4.3 A. Aggressiveness level and composite aggressiveness indices of

P.infestans’ isolates (Whole plant experiment) during 2011

Aggressiveness

level of isolates

Isolates from

sampled

locations

Composite

aggressiveness

index (CAI)

Percentage of

isolates based

on

aggressiveness

Group A

Strongly

Aggressive

(CAI ≥ 100)

Sharan (Sh)

Batakundi (Bt)

Ayyubia (Ab)

Bara Gali (Bg)

Nathya gali (Nt)

124.9 126.2

126.7 126.7

128.9

33.33 %

Group B

Mildly

aggressive

(100 ˂CAI ˃50)

Kaghan (Ka)

Naran (Nr)

Shougran (Sg)

Balakot (Bl)

Mahaban (Mb)

Shankyari (Sk)

Shangla (Sn)

Kalam (Kl)

59.65

59.35

59.45

58.22

59.30

58.13

57.80

59.20

53.33 %

Group C

Weakly

aggressive

CAI ≤ 50

Shabqadar (Sb)

Mansehra (Mn)

31.83

31.40

13.33 %

105

Table 4.3 B. Aggressiveness level and composite aggressiveness indices of

P.infestans’ isolates (Whole plant experiment) during 2013

Aggressiveness

level of isolates

Isolates from

sampled

locations

Composite

aggressiveness

index (CAI)

Percentage of

isolates based

on

aggressiveness

Group A

Strongly

Aggressive

(CAI≥ 100)

Batakundi (Bt)

Ayyubia (Ab)

Bara Gali (Bg)

Nathya gali (Nt)

Mahaban (Mb)

Shangla (Sn)

Shabqadar (Sb)

105.9

103.8

106.4

112.3

105.2

111.3

134.9

47 %

Group B

Mildly

aggressive

(100 ˂CAI ˃50)

Sharan (Sh)

Kaghan (Ka)

Naran (Nr)

Shougran (Sg)

Balakot (Bl)

Shankyari (Sk)

Kalam (Kl)

85.2

60.14

75.3

94.5

60.0

65.8

97.5

53 %

Mansehra (Mn) 63.8

106

4.1.2 DETACHED LEAFLET ASSAY

Analysis of variance (ANOVA) and data for different isolates of P. infestans under

detached leaflet experiment are shown in Table 4.4 and Table 4.5 respectively.

Analysis revealed that isolates collected from fifteen different locations exhibited

significant variability for different aggressiveness parameters. Six computed

epidemiological parameters i.e., infection frequency, latent period, lesion area,

average lesion expansion rate, area under lesion expansion curve and composite

aggressiveness index were significantly different among different isolates of P.

infestans.

Table 4.4. Mean squares of the analysis of variance for IF (%), LIP (days), LA

(mm2), ALER (mm2 day-1), AULEC and CAI for detached leaflet assay

107

Source d.f IF (%) LIP

(days)

LA

(mm2)

ALER (mm2

day-1)

AULEC CAI

Replications 3 0.01ns 0.01ns 0.02ns 0.00ns 0.04ns 0.38ns

Isolates 14 483.56* 5.95* 4.01* 0.4* 73.49* 6010.26*

Error 42 0.021 0.009 0.009 0.000 0.028 0.624

Total 59

Coefficient

of variation

0.17 1.24 1.17 1.32 1.02 0.82

ns = non-significant; *

Significantly different

d.f. = degrees of freedom

IF = infection frequency

LIP = latent period

LA = lesion area

ALER = average lesion expansion rate

AULEC = area under lesion expansion curve

CAI = composite aggressiveness index

Table 4.5. Values of aggressiveness parameters of different isolates of P. infestans

collected from various locations on detached leaflets assay during

108

Isolates IF (%) LIP (days)

LA (mm2)

ALER (mm2/day)

AULEC CAI

1. Sharan (Sh) 95.63c 5.65h 9.50a 0.95a 20.68d 152.4c

2. Batakundi (Bt)

96.35ab 5.85g 9.47a 0.94a 20.58d 155.3b

3. Ayyubia

(Ab) 96.22b 5.87g 9.50a 0.95a 21.08c 156.6a

4. Bara Gali

(Bg) 96.30ab 5.77gh 9.47a 0.95a 21.52b 156.7a

5. Nathya gali (Nt)

96.45a 7.85cd 7.97bcd 0.79de 22.00a 86.50d

6. Kaghan (Ka)

78.72d 7.47f 7.92bcd 0.72fg 15.43h 84.53e

7. Naran (Nr) 78.72d 7.85cd 8.00bcd 0.80d 15.65gh 80.47f

8. Shougran (Sg)

76.93i 7.97c 7.95bcd 0.79de 15.93ef 80.55f

9. Balakot (Bl)

77.75g 7.93cd 8.05b 0.80d 15.93ef 80.22fg

10. Mahaban

(Mb) 77.32h 7.82de 7.87d 0.78ef 16.00ef 79.72fgh

11. Shankyari

(Sk) 78.15f 7.85cd 8.02bc 0.81b 15.98ef 79.33gh

12. Shangla

(Sn) 77.93g 7.55f 7.90cd 0.79de 15.88fg 78.65h

13. Kalam (Kl)

78.50e 7.70e 7.87d 0.78ef 16.15e 78.63h

14. Shabqadar

(Sb) 64.53k 9.62a 6.37e 0.63g 7.85j 47.50i

15. Mansehra

(Mn) 66.15j 9.40b 6.32e 0.63g 8.22i 45.70j

LSD values (p≤0.05)

0.2068 0.1354 0.1354 0.0045 0.2388 1.127

IF = infection frequency; LIP = latent period; LA = lesion area; ALER = average lesion

expansion rate; AULEC = area under lesion expansion curve; CAI = composite

aggressiveness index.

109

Means bearing different alphabets are different significantly from each other at p≤0.05.

4.1.2.1 Infection Frequency

In detached leaflet assay, values of infection frequency (IF) varied among different

isolates. Highest infection frequency (96.45%) was caused by Nathia Gali isolate

while the lowest IF was observed for Shabqadar isolate (64.53%). On the basis of

IF, isolates fell into three categories. Isolates obtained from Sharan, Batakundi,

Ayyubia, Bara Gali and Nathia gali had highest infection frequencies i.e., 95.6,

96.35, 96.2, 96.3 and 96.4 % respectively. Isolates originated from Kaghan, Naran,

Shougran, Balakot, Mahaban, Shankiari, Shangla and Kalam revealed relatively

lower infection frequencies which were recorded as 78.72, 78.72, 76.93, 77.75, 77.32,

78.15, 77.93 and 78.5 respectively. On the other hand lowest infection frequencies

were observed for isolates from Shabqadar and Mansehra which were read as

64.53 and 66.15% respectively (Fig. 4.7).

Fig. 4.7. Infection frequency (IF) calculated as percentage of diseased portion of

different isolates of P. infestans collected from fifteen locations of Khyber

Pakhtunkhwa province.

110

4.1.2.2 Latent period (days)

Latent period is an important parameter which show the aggression potential of

pathogenic races. Lower values of LIP denote higher while larger values of LIP

correspond to lower aggression potentials of pathogens. Data for latent period (LP)

of isolates on detached leaflet experiment is presented in Fig. 4.8. Data revealed

that LIP values were different for different isolates. Considerable variations were

observed isolates groups i.e., those from Saharn, Batakundi, Ayyubia and Bara gali

which had lowest LIP values; Nathia Gali , Kaghan, Naran, Shougran, Balakot,

Mahaban, Shankiari, Shangla and Kalam group which resulted in moderate LIP

data; and Shabqadar and Mansehra group had highest LIP values. From the results

of this study it was found that lowest LIP was calculated for isolates from Sharan

(5.65%) followed by slightly increased LIP by Batakundi

(5.85%), Ayyubia (5.87%) and Bara Gali (5.77%). Isolates from Nathia gali, Kaghan,

Naran, Shougran, Balakot, Mahaban, Shankyari, Shangla and Kalam revealed

significantly increased LIP values which were recorded as 7.85, 7.47, 7.85, 7.97,

7.93, 7.82, 7.85, 7.55 and 7.7 % respectively. Greater LIP values were obtained for

isolates Shabqadar and Mansehra which yielded 9.62 and 9.4% LIP respectively

(Fig. 4.8).

111

Fig. 4.8. Latent period (LP) days of different isolates of P. infestans on detached

leaflet assay.

4.1.2.3 Lesion area (mm2)

Like other parameters of aggressiveness, lesion area also exhibited significant

variations among different isolates of Phytophthora infestans (Fig. 4.9). Isolates

obtained from regions Sharan, Batakundi, Ayyubia and Bara Gali caused

maximum damage on detached leaflets. Pathotypes from Nathia Gali , Kaghan,

Naran, Shougran, Balakot, Mahaban, Shankyari, Shangla and Kalam had relatively

less severe damage on host leaflets while than the above mentioned isolates while

weak aggressions were shown by isolates from Shabqadar and

112

Mansehra. Highest lesion area (LA) were found for isolates Sharan, Batakundi,

Ayyubia and Bara Gali revealing 9.5, 9.47, 9.5 and 9.4 mm2 LA respectively. Those

isolates obtained from Nathia Gali, Kaghan, Naran, Shougran, Balakot, Mahaban,

Shankiari, shangla and Kalam resulted in 7.97, 7.92, 8, 7.95, 8, 7.87, 8.02, 7.9 and

7.87 mm2 LA respectively. Lowest values of lesion area were computed for isolates

collected from Shabqadar and Mansehra which had 6.37 and 6.32 mm2 LA

respectively (Fig. 4.9).

Fig. 4.9. Lesion area (LA) mm2 of different islates of P. infestans on detached leaflet

experiment

113

4.1.2.4 Average lesion expansion rate (ALER) mm2 day-1

Data in Fig. 4.10 elucidates average lesion expansion rates (ALER) of isolates of P.

infestans belonging to fifteen different sampled areas on detached leaflet

experiment calculated as mm2 day-1. From the results it was evident that isolates

from some of the sampled locations had greater average lesion expansion rates

than isolates of other sampled areas and there was significant variability in ALER.

Isolates from Manshra and Shabqadar had the lowest ALER readings which were

recoreded as 0.63 mm2 day-1. Isolates sampled from Kalam, Shangla, Shankiari,

Mahaban, Balakot, Shougran, Naran, Kaghan and Nathia Gali had almost similar

ALER readings which were 0.78, 0.79, 0.81, 0.78, 08, 0.79, 0.8, 0.72 and 0.79

mm2/day. On the other hand, isolates recovered from Bara Gali, Ayyubia,

Batakundi and Sharan caused the highest average lesion expansion rates which

ranged between 0.94 and 0.95 (Fig. 4.10).

114

Fig. 4.10. Average lesion expansion rates (ALER) mm2 day-1 of isolates of P.

infestans sampled from fifteen areas (detached leaflet experiment).

4.1.2.5 Area Under Lesion Expansion Curve (AULEC)

Area under lesion expansion curve (AULEC) is another important feature of

aggressiveness representing the virulence capacity and disease progress of

pathogenic races. Data for AULEC of P. infestans isolates on detached leaflet

experiment is depicted in Fig. 4.11. It was clear from the results of this study that

isolates from fifteen localities had different values of AULEC. Those collected from

Mansehra and Shabqadar regions had AULEC 8.22 and 7.85 respectively which

were the lowest. Isolates belonging to Kalam, Shangla, Shankiari, Mahaban,

Balakot, Shougran, Naran, Kaghan resulted in 16.15, 15.88, 15.98, 16, 15.93, 15.65

115

and 15.43 AULEC values which demonstrated that these isolates had almost

similar aggressiveness potentials. Isolates from Sharan, Batakundi, Ayyubia, Bara

Gali and Nathia Gali revealed highest AULEC among all the isolates which

corresponded to 20.68, 20.58, 21.08, 21.52 and 22 respectively (Fig. 4.11).

Fig. 4.11. Area under lesion expansion curve (AULEC) of different isolates of P.

infestans on detached leaflet assay

4.1.2.6 Composite aggressiveness index (CAI)

Composite aggressiveness index (CAI) demonstrates the aggressiveness level of

different isolates of pathogen. Data for CAI of different isolates of P. infestans on

detached leaflet assay is presented in Fig. 4.12. Results suggested that fifteen

116

sampled locations had isolates with different CAI values. Four out of fifteen

locations i.e., Sharan, Batakundi, Ayyubia and Bara Gali had isolates with highest

CAI values which were recorded as 152.4, 155.3, 156.6 and 156.7 respectively.

Isolates from two sampled areas i.e., Shabqadar and Mansehra had the lowest CAI

47.5 and 47.7 respectively. While isolates sampled from Nathia Gali , Kaghan,

Naran, Shougran, Balakot, Mahaban, Shankiari, Shangla and Kalam had

intermediate CAI readings which were as 86.5, 84.53, 80.47, 80.55, 80.22, 79.72,

79.33, 78.65 and 78.63 respectively (Fig. 4.12).

Fig. 4.12. Composite aggressiveness indices (CAI) for different isolates of P.

infestans on detached leaflet assay.

Data for the aggressiveness index was used to calculate the aggressiveness level of

isolates of P. infestans. Isolates which had composite aggressiveness index ≥ 100

117

were regarded as strongly aggressive; those with CAI values above 50 but below

100 (100 ˂CAI ˃50) were mildly aggressive while isolates with CAI ≤ 50 were

grouped as weakly aggressive. Aggressiveness level of studied isolates is

presented in Table 4.6. It was foound that isolates sampled from Sharan,

Batakundi, Ayyubi and Bara Gali had CAI ≥ 100 and they fell into strong

aggressive category. Nathya gali, Kaghan, Naran, Shougran, Balakot, Mahaban,

Shankiari, Shangla and Kalam had CAI values between 50 and 100 (100 ˂CAI ˃50)

and thus they were moderately aggressive. On the other hand, isolates sampled

from Shabqadar and Mansehra had CAI ≤ 50 and corresponded to weakly

aggressive group. Out of fifteen sampled locations, 27% isolates represented

strongly aggressive category, 60% were moderately aggressive while 13% were

found to be weakly aggressive on detached leaflet assay (Table 4.6).

118

Table 4.6. Aggressiveness level and composite aggressiveness indices of

P.infestans’ isolates (Detached leaflet assay)

Aggressiveness

level of isolates

Isolates from

sampled

locations

Composite

aggressiveness

index (CAI)

Percentage of

isolates based

on

aggressiveness

Group A

Strongly

Aggressive

(CAI ≥ 100)

Sharan (Sh)

Batakundi (Bt)

Ayyubia (Ab)

Bara Gali (Bg)

152.4 155.3

156.6.

156.7

27%

Group B

119

Mildly

aggressive

(100 ˂CAI ˃50)

Nathya gali (Nt)

Kaghan (Ka)

Naran (Nr)

Shougran (Sg)

Balakot (Bl)

Mahaban (Mb)

Shankiari (Sk)

Shangla (Sn)

Kalam (Kl)

86.5

84.53

80.47

80.55

80.22

79.72

79.33

78.65

78.63

60%

Group C

Weakly

aggressive

CAI ≤ 50

Shabqadar (Sb)

Mansehra (Mn)

47.5

45.7

13%

120

3 TUBER DISC ASSAY

Like whole plant (intact leaves) and detached leaflet assay, results for tuber disc

assay confirmed that there were significant variations among isolates for studied

parameters. Analysis of variance (ANOVA) showed that infection frequency,

latent period, lesion area, average lesion expansion rate, area under lesion

expansion curve and composite aggressiveness indices were significantly different

(p≤0.05) among different isolates. For tuber disc assay, mean squares of ANOVA

and aggressiveness parameters of different isolates sampled from fifteen locations

are presented in Table 4.7 and Table 4.8.

Table 4.7. Mean squares of the analysis of variance for IF (%), LIP (days), LA

(mm2), ALER (mm2 / day), AULEC and CAI for Tuber disc assay

Source d.f IF (%) LIP

(days)

LA

(mm2)

ALER

(mm2/day)

AULEC CAI

Replications 3 0.05ns 0.016ns 0.17ns 0.003ns 0.082ns 0.049ns

Isolates 14 290.87* 8.02* 48.79* 0.54* 154.66* 21279.8*

Error 42 0.068 0.049 0.032 0.006 0.055 0.094

Total 59

Coefficient

of variation

0.31 2.69 1.68 7.04 0.81 0.25

ns = non-significant; * significant

d.f. = degrees of freedom; IF = infection frequency; LIP = latent period; LA =

lesion area; ALER = average lesion expansion rate; AULEC = area under lesion

expansion curve; CAI = composite aggressiveness index

4.1.

121

Table 4.8. Aggressiveness parameters on tuber discs of potato caused by different isolates of Phytophthora infestans collected from various locations

Isolates IF (%) LIP (days)

LA (mm2)

ALER (mm2/day)

AULEC CAI

1. Sharan (Sh) 95.25ab 6.12c 15.52b 1.59a 37.42a 243.3a

2. Batakundi (Bt)

95.25ab 6.37c 15.63b 1.56b 37.00b 226.2c

3. Ayyubia

(Ab) 95.00b 6.37c 15.93a 1.59ab 37.15ab 237.1b

4. Bara Gali

(Bg) 95.38a 6.37c 15.70ab 1.57b 37.10ab 223.1d

5. Nathya gali (Nt)

71.25f 10.30a 6.33e 0.63d 19.55d 41.45j

6. Kaghan (Ka)

83.38de 8.52b 9.77c 0.97c 28.10c 95.30g

7. Naran (Nr) 84.25c 8.35b 9.60cd 0.96c 28.30c 92.57i

8. Shougran (Sg)

83.25e 8.45b 9.75cd 0.97c 28.30c 95.63g

9. Balakot (Bl)

84.38c 8.25b 9.50d 0.95c 28.30c 97.38e

10. Mahaban

(Mb) 83.25e 8.47b 9.77c 0.97c 28.27c 96.85f

11. Shankyari

(Sk) 84.13c 8.33b 9.72cd 0.97c 28.38c 97.50e

12. Shangla

(Sn) 84.38c 8.55b 9.60cd 0.96c 28.35c 93.45h

13. Kalam (Kl)

83.63d 8.40b 9.65cd 0.96c 28.25c 92.55i

14. Shabqadar

(Sb) 70.63g 10.38a 6.17e 0.62d 19.48d 41.63j

15. Mansehra

70.88g 10.25a 5.90f 0.59d 19.50d 40.10k

0.3721 0.3159 0.2553 0.1105 0.3347 0.4375

122

IF = infection frequency; LIP = latent period; LA = lesion area; ALER = average lesion

expansion rate; AULEC = area under lesion expansion curve; CAI = composite

aggressiveness index.

Means bearing different alphabets are different significantly from each other at p≤0.05.

3.1 Infection frequency (IF)

Results for tuber disc assay revealed significant differences for infection

frequencies of the studied isolates. Isolates from sampled regions Sharan,

Batakundi, Ayyubia and Bara Gali revealed 95.25, 95.25, 95 and95.38 % IF

respectively. Those isolates collected from Nathia Gali, Kaghan, Naran, Shougran,

Balakot, Mahaban, Shankiari, Shangla, Kalam, Shabqadar and Mansehra yielded

relatively moderate IF on tuber discs. Out of fifteen sampled areas, isolates

belonging to Nathia Gali, Shabqadar and Mansehra had lowest infection

frequencies which ranged between 70.63-71.25% (Fig. 4.13).

4.1.

123

Fig. 4.13. Tuber disc assay revealing infection frequency (IF) for different isolates

of P. infestans

4.1.

124

3.2 Latent period (LP) days

Data for latent period (LP) for studied isolates on tuber disc assay is given in Fig.

4.14. Results demonstrated that sampled isolates showed variation in LIP values.

Pathotypes from Shabqadar, Mansehra and Nathia Gali regions caused highest

latent periods 10.38, 10.25 and 10.3 days respectively. Isolates collected from

Kalam, Shangla, Shankyari, Mahaban, Balakot, Shougran, Naran and Kaghan

resulted in 8.4, 8.5, 8.3, 8.4, 8.2, 8.4, 8.3 and 8.5 days of latent period. Lowest LIP

were found for isolates sampled from Bara Gali , Ayyubia, Batakundi and Sharan

which revealed 6.3, 6.3, 6.3 and 6.1 days respectively (Fig. 4.14).

Fig. 4.14. Latent period in days (LP) for sampled isolates of P. infestans on tuber

disc assay

4.1.

125

3.3 Lesion Area (LA)

Lesion area (LA) mm2 was found to be significantly variable for different isolates

of sampled areas. Results indicated that Sharan, Batakundi, Ayyubia and Bara Gali

based isolates had higher lesion areas on tuber discs. LA for these isolates were

15.52, 15.63, 15.93 and 15.7 mm2 respectively. Isolates from Nathia Gali, Shabqadar

and Mansehra resulted in lowest lesion areas i.e., 6.33, 6.17 and 5.9 mm2

respectively. On the other hand, relatively intermediate values of lesion areas were

computed for isolates collected from Kaghan, Naran, Shougran, Balakot,

Mahaban, Shankiari, Shangla and Kalam which revealed 9.77, 9.6, 9.75, 9.5, 9.77,

9.72, 9.6, 9.65 respectively (Fig. 4.15).

Fig. 4.15. Lesion area (LA) mm2 for isolates of P. infestans sampled from different

locations on tuber disc assay

4.1.

126

3.4 Average lesion expansion rate (ALER) mm2 day-1

Data for average lesion expansion rate (ALER) is presented in Fig. 4.16. Data

revealed that lesions expansions in different isolates were different. Isolates from

four regions Sharan, Batakundi, Ayyubia and Bara Gali had highest lesion

exapansion rates 1.59, 1.56, 1.59 and 1.57 mm2 day-1 respectively. Relatively mild

expansion rates in lesion were observed for isolates obtained from Kaghan, Naran,

Shougran, Balakot, Mahaban, Shankiari, Shangla and Kalam which had ALER

0.97, 0.96, 0.97, 0.95, 0.97, 0.97, 0.96 and 0.96 mm2 day-1 respectively. Contrarily,

lowest lesion expansions per day were recorded for isolates sampled from Nathia

Gali, Shabqadar and Mansehra which were as 0.63, 0.62 and 0.59 mm2 day-1

respectively (Fig 4.16).

4.1.

127

Fig. 4.16. Average lesion expansion rates (ALER) mm2/day on tuber disc assay for

different isolates of P. infestans

3.5 Area under lesion expansion curve (AULEC)

Results presented in Fig. 4.17 confirmed that area under lesion ezpansion curve

(AULEC) were different for different isolates. Isolates from Sharan, Batakundi,

Ayybia, and Bara Gali demonstrated highest AULEC readings which ranged

between 37.0-37.42. Lowest AULEC were obtained for isolates of P. infestans from

Nathia Gali, Shabqadar and Mansehra which resulted in 19.55, 19.48 and 19.5

AULEC respectively. Moderately AULEC values were computed for isolates

originated from Kaghan, Naran, Shoughran, Balakot, Mahaban, Shankiari,

Shangla and Kalam which ranged between 28.1-28.3 (Fig. 4.17).

4.1.

128

Fig. 4.17. Area under lesion expansion curve (AULEC) on tuber disc assay for

sampled isolates of P. infestans

3.6 Composite aggressiveness index (CAI)

For tuber disc assay, data regarding composite aggressiveness index (CAI) is

presented in Fig. 4.18. Results demonstrated that lowest CAI were calculated for

isolates sampled from Nathia Gali, Shabqadar and Mansehra which were 41.45,

41.63 and 40.1 respectively. Isolates from Sharan, Batakundi, Ayyubia and Bara

Gali revealed highest CAI values which were in the range 223.1 and 243.3. On the

other hand, intermediate CAI values were computed for isolates sampled from

Kaghan, Naran, Shoughran, Balakot, Mahaban, Shankiari, Shangla and Kalam

which ranged between 92.55 and 97.5 (Fig. 4.18).

Aggressiveness level of isolates was determined on the basis of CAI above 100 as

strongly aggressive; CAI between 50 and 100 as mildly aggressive and CAI below

50 were considered as weakly aggressive. Data shown in Table 4.9 demonstrates

the aggressiveness level of sampled isolates. It was observed that out of fiteen

locations, four regions i.e., Sharan, Batakundi, Ayyubia and Bargali had isolates

with strong aggressiveness potential. They were found to have CAI ≥ 100. Isolates

belonging to Kaghan, Naran, Shoughran, Balakot, Mahaban, Shankiari, Shangla

and Kalam were mildly aggressive which had composite aggressiveness indices

4.1.

129

between 50 and 100 (100 ˂CAI ˃50). On the other hand, isolates sampled from

Shabqadar, Mansehra and Nathia Gali were recorded as least aggressive with CAI

≤ 50. It was further observed that on the basis of CAI values, 27 % isolates were

130

strongly aggressive, 60% were mildly aggressive while 13% of the studied isolates

were weakly aggressive (Table 4.9).

Fig. 4.18. Composite aggressiveness index (CAI) of P. infestans isolates sampled

from fifteen different locations of Khyber Pakhtunkhwa on tuber dicc experiment.

Table 4.9. Aggressiveness level and composite aggressiveness indices of

P.infestans’ isolates (Tuber disc assay)

131

Aggressiveness

level of isolates

Isolates from

sampled

locations

Composite

aggressiveness

index (CAI)

Percentage of

isolates based

on

aggressiveness

Group A

Highly

Aggressive

(CAI ≥ 100)

Sharan (Sh)

Batakundi (Bt)

Ayyubia (Ab)

Bara Gali (Bg)

243.3

226.2

237.1

223.1

27 %

Group B

Moderately

aggressive

(100 ˂CAI ˃50)

Kaghan (Ka)

Naran (Nr)

Shougran (Sg)

Balakot (Bl)

Mahaban (Mb)

Shankyari (Sk)

Shangla (Sn)

Kalam (Kl)

95.30

92.57

95.63

97.38

96.85

97.50

93.45

92.55

60 %

Group C

Weakly

aggressive

CAI ≤ 50

Shabqadar (Sb)

Mansehra (Mn)

Nathya gali (Nt)

41.63

40.10

41.45

13 %

132

4.1.4 COMPARISON BETWEEN EXPERIMENTAL MODELS (WHOLE

PLANT LEAVES, DETACHED LEAFLET AND TUBER DISC ASSAYS)

Results showed that three methods of assessment (whole plant leaves, detached

leaflet and tuber disc assay) revealed almost similar results for the aggressiveness

parameters, although in some cases differences were observed. Moreover, values

for aggressiveness parameters e.g., infection frequency, latent period, lesion area,

average lesion expansion rate, area under lesion expansion curve were

significantly variable for experimental methods. Overall, tuber disc assay and

detached leaflet assay resulted in consistent non-significant results for the

aggressiveness levels of isolates as compared to whole plant leaves experiment

which showed slight deviation from the two methods. Values of the studied

aggressiveness parameters on whole plant leaves were significantly lower than the

detached leaflet and tuber disc assay. These variation in experimental models have

been discussed in discussion section of this thesis.

4.1.4.1 Infection frequency

Infection frequencies caused by isolates from Sharan, Batakundi, Ayyubia and

Bara Gali revealed similar tendency on whole plants leaves, detached leaflets and

tuder discs which were found between 94 and 96 % with slight variations. Nathia

133

Gali isolates on the other hand had lowest IF values on tuber disc assay as

compared to whole plant leaves and detached leaflet assay (Table 4.10 and Fig.

4.19). Isolates from other regions such as Kaghan, Naran, Shoughran, Balakot,

Mahaban, Shankiari, Shangla, Kalam, Shabqadar and Mansehra resulted in

consisten infection frequencies on whole plant leaves and detached leaflets but

much larger values were observed for same isolates on tuber disc method. Lowest

infection frequency for isolates Shabqadar and Mansehra were 58.7 and 67.13% on

whole plant; 64.53 and 66.15% on detached leaflets but 70.63 and 70.88% on tuber

disc assay. Similar tendency was observed for other isolates where IF values were

larger on tuber disc method than whole plant and detached leaflets. Only Nathia

Gali isolate was found to have lower IF on tuber which was recorded as 71.25%

on tuber disc assay when compared to whole plant and detached leaflets where IF

values were 94.07 and 96.4% respectively (Table 4.10; Fig. 4.19).

134

Table 4.10. Varaibility in infection frequency of different isolates of P. infestans on

whole plant leaves, detached leaflet and tuber discs

Isolates Experimental model (M) Isolates

means Whole plant

leaves model

(M1)

Detached

leaflet model

(M2)

Tuber disc

model (M3)

Sharan (Sh) 94.43a 95.63a 95.25a 95.10a

Batakundi (Bt) 94.35a 96.35a 95.25a 95.32a

Ayyubia (Ab) 94.38a 96.22a 95.00a 95.20a

Bara Gali (Bg) 94.32a 96.30a 95.38a 95.33a

Nathya gali

(Nt)

94.07a 96.45a 71.25d 87.26b

Kaghan (Ka) 73.72d 78.72c 83.38b 78.61c

Naran (Nr) 73.70d 78.72c 84.25b 78.89c

Shougran (Sg) 73.57d 76.93c 83.25b 77.92c

Balakot (Bl) 73.55d 77.75c 84.38b 78.56c

Mahaban (Mb) 73.40d 77.32c 83.25b 77.99c

Shankyari (Sk) 73.07d 78.15c 84.13b 78.45c

Shangla (Sn) 73.43d 77.93c 84.38b 78.57c

Kalam (Kl) 72.80d 78.50c 83.63b 78.31c

Shabqadar (Sb) 58.70f 64.53e 70.63d 64.62e

Mansehra

(Mn)

67.13e 66.15e 70.88d 68.05d

Experimental

model Means

78.97c 82.38b 84.28a ---------

LSD values (p≤0.05) for Experimental models (M) = 0.8240; Isolates (I) =1.843 and

interaction of isolates (I) × Experimental model (M) = 3.191.

Values bearing different alphabets in each column and row show significant

differences at p≤0.05.

135

Fig. 4.19. Infection frequency of isolates on whole plant leaves, detached leaflet

and tuber disc assay

4.1.4.2 Latent period

Combined data for latent period (LP) of isolates on whole plant leaves, detached

leaflets and tuber discs is presented in Table 4.11 and Fig. 4.20. Among the

experimental methods, detached leaflet revealed the lowest LIP (5.65-9.62 days)

when compared to whole plant leaves (6.4-10.4 days) and tuber disc (6.12-10.38

days) respectively. In whole plant leaves, Sharan, Batakundi, Ayyubia, Bara Gali

and Nathia gali caused lowest LIP (5.4-6.5 day) which gradually increased for

other isolates raching to maximum 10.4 and 10.32 day for shabqadar and Mansehra

136

isolates. When compared to whole plant and detached leaflets, tuber disc assay

revealed highest LIP values for studied isolates (Table 4.11; Fig. 4.20).

Table 4.11. Latent period (days) of isolates of Phytophthora infestans on whole plant

leaves, detached leaflets and tuber disca

Isolates Experimental model Isolates

means Whole plant

model (M1)

Detached

leaflet model

(M2)

Tuber disc

model (M3)

Sharan (Sh) 6.40h 5.65j 6.12i 6.06e

Batakundi (Bt) 6.50h 5.85j 6.37h 6.24d

Ayyubia (Ab) 6.52h 5.87j 6.37h 6.25d

Bara Gali (Bg) 6.57h 5.77j 6.37h 6.24d

Nathya gali

(Nt)

6.50h 7.85ef 10.30a 8.21c

Kaghan (Ka) 8.47cd 7.47g 8.52c 8.16c

Naran (Nr) 8.47cd 7.85ef 8.35cd 8.22c

Shougran (Sg) 8.32cd 7.97e 8.45cd 8.25c

Balakot (Bl) 8.35cd 7.92ef 8.25d 8.17c

Mahaban (Mb) 8.37cd 7.82ef 8.47cd 8.22c

Shankyari (Sk) 8.47cd 7.85ef 8.32cd 8.21c

Shangla (Sn) 8.35cd 7.55g 8.55c 8.15c

Kalam (Kl) 8.45cd 7.70fg 8.40cd 8.18c

Shabqadar (Sb) 10.40a 9.62b 10.38a 10.13a

Mansehra

(Mn)

10.32a 9.40b 10.25a 9.99b

Experimental

model Means

8.03b 7.47c 8.23a -------

LSD value (p≤0.05) for Experimental models (M) = 0.05823; Isolates (I) = 0.1302 and

interaction of Isolates (I) × Experimental model (M) = 0.225.

137

Values bearing different alphabets in each column and row show significant

differences at p≤0.05

138

Fig. 4.20. Latent period (days) of differen isolates of P. infestans on whole plant

leaves, detached leaflets and tuber discs

4.1.4.3 Lesion area (mm2)

Lesion area (LA) mm2 was found to be significantly greater for all the studied

isolates on tuber discs as compared to whole plants and deatched leaflets assays.

On tuber disc assay, isolates from Shabqadar and mansehra caused lowest LA as

6.17 and 5.9 mm2 while highest LA was observed for Ayyubia (15.93 mm2). On

139

whole plant leaves and detached leaflets, isolates from Shabqadar and Mansehra

revealed almost similar values. However, isolates from Sharan, Batakundi,

Ayyubia, Kaghan, Naran, Shoughran, Balakot, Mahaban, Shankiari, Shangla and

Kalam more or less similar results on whole plant and detached leaflets; however

significantly lower than LA of isolates on tuber discs (Table 4.12 and Fig. 4.21).

Table 4.12. Lesion areas (LA) mm2 of isolates of P. infestans on whole plant,

detached leaflets and tuber disc assay

Isolates Experimental model Isolates

means Whole plant

model (M1)

Detached

leaflet model

(M2)

Tuber disc

model (M3)

Sharan (Sh) 8.70f 9.50de 15.52b 11.24a

Batakundi (Bt) 8.60f 9.47e 15.63b 11.23a

Ayyubia (Ab) 8.47fg 9.50de 15.93a 11.30a

Bara Gali (Bg) 8.35g 9.47e 15.70ab 11.18a

Nathya gali

(Nt)

8.50fg 7.97h 6.32ij 7.60c

Kaghan (Ka) 6.50i 7.92h 9.77c 8.06b

Naran (Nr) 6.55i 8.00h 9.60cde 8.05b

Shougran (Sg) 6.47i 7.95h 9.75c 8.05b

Balakot (Bl) 6.47i 8.05h 9.50de 8.01b

Mahaban (Mb) 6.50i 7.87h 9.77c 8.05b

Shankyari (Sk) 6.50i 8.02h 9.72cd 8.08b

Shangla (Sn) 6.45i 7.90h 9.60cde 7.98b

Kalam (Kl) 6.55i 7.87h 9.65cde 8.02b

Shabqadar (Sb) 5.60l 6.37ij 6.17j 6.05d

Mansehra

(Mn)

5.37l 6.32ij 5.90k 5.86e

Experimental

model Means

7.04c 8.14b 10.57a --------

140

LSD value (p≤0.05) for Experimental models (M) = 0.06043; Isolates (I) = 0.1351 and

interaction of Isolates (I) × Experimental model (M) = 0.2341.

Values bearing different alphabets in each column and row show

significant differences at p≤0.05

Fig. 4.22. Lesion area (mm2) of different isolates of P. infestans on whole plant

leaves, detached leaflets and tuber disc assay

4.1.4.4 Average lesion expansion rate (mm2/day)

Wole plant, detached leaflets and tuber disc assay revealed that average lesion

expansion rate (ALER) mm2/day were consistent in experimental methods but

values were significantly greater in tuber disc assay in comparison to whole leaves

and detached leaflets. Lowest values of ALER of studied isolates were found in

141

whole plant leaves which ranged between 0.53-0.86 mm2/day followed by

detached leaflets (0.63-0.95 mm2/day) with significantly larger ALER readings

recorded in tuber discs which ranged between 0.59-1.59 mm2/day (Table 4.13 and

Fig. 4.23).

Table 4.13. Average lesion expansion rate (ALER) mm2/day of different isolates

of P. infestans on whole plant leaves, detached leaflets and tuber disc assay

Isolates Experimental model Isolates

means Whole plant

model (M1)

Detached

leaflet model

(M2)

Tuber disc

model (M3)

Sharan (Sh) 0.862d 0.950c 1.69a 1.16a

Batakundi (Bt) 0.680de 0.947c 1.56b 1.12b

Ayyubia (Ab) 0.797ef 0.950c 1.59b 1.11b

Bara Gali (Bg) 0.835def 0.947c 1.57b 1.11b

Nathya gali

(Nt)

0.850def 0.797ef 0.636gh 0.760d

Kaghan (Ka) 0.650gh 0.792f 0.977c 0.806c

Naran (Nr) 0.655g 0.800def 0.960c 0.805c

Shougran (Sg) 0.647gh 0.795f 0.970c 0.804c

Balakot (Bl) 0.647gh 0.805def 0.950c 0.801c

Mahaban (Mb) 0.650gh 0.787f 0.977c 0.805c

Shankyari (Sk) 0.650gh 0.810def 0.972c 0.810c

Shangla (Sn) 0.645gh 0.790f 0.960c 0.798c

Kalam (Kl) 0.655g 0.787f 0.965c 0.802c

Shabqadar (Sb) 0.557ij 0.637gh 0.617ghi 0.604e

Mansehra (Mn) 0.537j 0.632gh 0.590hij 0.586e

Experimental

model Means

0.700c 0.815b 1.066a

142

LSD value (p≤0.05) for Experimental models (M) = 0.01615; Isolates (I) = 0.03611

and interaction of Isolates (I) × Experimental model (M) = 0.6255.

Values bearing different alphabets in each column and row show significant

differences at p≤0.05

Fig. 4.23. Average lesion expansion rate (ALER) of isolates of P. infestans on whole

plant leaves, detached leaflets and tuber disc assay

4.1.4.5 Area under lesion expansion curve

In each experiment, data for area under lesion expansion curve (AULEC) showed

consistency for the aggressions of studied isolates although great variation was

observed among values of AULEC in different experiment. Minimum values of

AULEC of different isolates of P. infestans were recorded on whole plant leaves

which revealed lowest (5.72) for Mansehra while the highest (15.13) for Batakundi

143

isolates. On detached leaflet assay, 7.58 AULEC was found for Mansehra while

21.52 for Baragli. Significantly larger AULEC were found in tuber disc assay which

revealed 19.5 for Mansehra and 37.42 for Sharan (Table 4.14 and Fig. 4.24).

Table 4.14. Area under lesion expansion curve for different pathotypes on whole

plant, detached leaflets and tuber discs assay

Isolates Experimental model Isolates

means Whole plant

model (M1)

Detached

leaflet model

(M2)

Tuber disc

model (M3)

Sharan (Sh) 14.80op 20.68h 37.42a 24.30b

Batakundi (Bt) 15.13n 20.58h 37.00b 24.23b

Ayyubia (Ab) 14.75p 21.08g 37.15b 24.33b

Bara Gali (Bg) 15.02no 21.52f 37.10b 24.55a

Nathya gali

(Nt)

15.05no 22.00e 19.55i 18.87cd

Kaghan (Ka) 12.63q 15.43m 28.10d 18.72d

Naran (Nr) 12.43qr 15.65lm 28.30cd 18.79cd

Shougran (Sg) 12.52qr 15.93jk 28.30cd 18.92c

Balakot (Bl) 12.50qr 15.93jk 28.30cd 18.91c

Mahaban (Mb) 12.45qr 16.00jk 28.27cd 18.91c

Shankyari (Sk) 12.30r 15.98jk 28.38c 18.88c

Shangla (Sn) 12.40qr 15.88kl 28.35cd 18.88c

Kalam (Kl) 12.38qr 16.15j 28.25cd 18.92c

Shabqadar (Sb) 5.71v 7.85t 19.48i 11.01f

Mansehra (Mn) 6.03u 8.23s 19.50i 11.25e

Experimental

model Means

12.41c 16.59b 28.90a ------

144

LSD value (p≤0.05) for Experimental models (M) = 0.06947; Isolates (I) = 0.1553 and

interaction of Isolates (I) × Experimental model (M) = 0.2691.

Values bearing different alphabets in each column and row show significant

differences at p≤0.05

145

Fig. 4.24. Values of area under lesion expansion curve (AULEC) for different

isolates of Phytopthora infestans on whole plants leaves, detached leaflets and

tuber discs

4.1.4.5 Composite aggressiveness index

Data for composite aggressiveness index (CAI) of different isolates of Phytophthora

infestans computed for whole plant leaves, detached leaflets and tuber disc assay

is presented in Table 4.15 and Fig. 4.25. reselts revealed consistency in three

experimental methods for aggressiness of isolates with slight variability. In whole

plant assay, lowest CAI values were observed for isolates Mansehra and

Shbaqadar which were same in detached and tuber disc methods. Similarly,

highest CAI in whole plant was recorded for Ayyubia and Bara Gali which were

consistent with deatched leaflet assay however different from tuber disc assay. In

tuber disc assay, highest CAI was observed for Sharan and Ayyubia isolates. Other

isolates followed similar pattern of aggressiveness in whole plant, detached leaflet

146

and tuber disc assay; although values of CAI on tuber disc assay were larger than

detached leaflets and whole plant leaves (Table 4.15; Fig. 4.25).

Table 4.15. Composite aggressiveness index (CAI) on whole plant leaves, detached

leaflets and tuber discs

Isolates Experimental model Isolates

means Whole plant

model (M1)

Detached leaflet model (M2)

Tuber disc

model (M3)

Sharan (Sh) 124.9j 152.4g 243.3a 173.5a

Batakundi (Bt) 126.2i 155.3f 226.2c 169.2b

Ayyubia (Ab) 126.7i 156.6e 237.1b 173.5a

Bara Gali (Bg) 126.7i 156.6e 223.1d 168.9b

Nathya gali

(Nt)

128.9h 86.50o 41.45y 85.62c

Kaghan (Ka) 59.65u 84.53p 95.30l 79.82d

Naran (Nr) 59.35u 80.47qr 92.57n 77.47f

Shougran (Sg) 59.45u 80.55q 95.63l 78.54e

Balakot (Bl) 58.22v 80.22qr 97.38k 78.61e

Mahaban (Mb) 59.30u 79.72rs 96.85k 78.63e

Shankyari (Sk) 58.13v 79.33st 97.50k 78.32e

Shangla (Sn) 57.80v 78.65t 93.45m 76.63g

Kalam (Kl) 59.20u 78.63t 92.25n 76.79g

Shabqadar (Sb) 31.83z 47.50w 41.63y 40.32h

Mansehra (Mn) 31.40z 45.70x 40.10z 39.07i

Experimental

model Means

77.85c 96.19b 120.90a --------

LSD value (p≤0.05) for Experimental models (M) = 0.1941; Isolates (I) = 0.4341 and

interaction of Isolates (I) × Experimental model (M) = 0.7519.

Values bearing different alphabets in each column and row show significant

differences at p≤0.05

147

Fig. 4.25. Composite aggressiveness index (CAI) for different isolates of P. infestans

on whole plant leaves, detached leaflets and tuber discs assay

4.2 EXPERIMENT II- EFFECT OF LATE BLIGHT SEVERITY ON TOTAL

GLYCOALKALOIDS OF POTATO LEAVES

Foliar disease severity (% defoliation) was recorded at different periods i.e., 3, 6, 9

and 12 days after inoculation (DAI) with P.infestans. ANOVA revealed significant

effects of days after inoculation on disease severity (Table 4.16). Three days after

inoculation (DAI) with P.infestans, leaves showed 5.26 % disease severity level (Fig

4.26). Progress in DAI resulted in severe infections on leaves, increasing percent

infected foliage area. Significant effects of days intervals after inoculation on foliar

disease severity were observed. After 6 DAI, disease severity was measured 23.14

148

% followed by 9 DAI causing 49.305 % leaflet infection and 12 DAI which severely

affected potato leaves showing maximum disease severity 70.135 % respectively

(Fig. 4.26).

Data on foliar total glycoalkaloids (TGA) under the influence of late blight disease

severity is represented in Table 4.17 and figure 4.26. TGA of leaves were

determined five days after the emergence of plants (0 days after inoculation) and

thereafter TGA values were determined after 3DAI, 6DAI, 9DAI and 12DAI in

both control and inoculated plants. Control plants were inoculated with sterile

distilled water and total glycoalkaloids contents determined in these plants were

used for comparison with diseased plants inoculated with P.infestans. In control

plant at 0 DAI, total glycoalkaloids of leaves were recorded as 29.43 mg/100g fresh

weight followed by 42.14, 49.33, 52.18 and 74.28 mg/100g fresh weight at 3, 6, 9

and 12 days after inoculation (DAI) respectively. Results showed significant

differences in glycoalkaloids concentration measured at different DAIs in control

plants (Table 4.17). After 3rd day of inoculation, foliar glycoalkaloids significantly

increased from 29.43 to 42.14 mg/100g fr. Wt. and to 49.33 mg/100g fr. Wt at 6DAI.

These differences were statistically significant. At 9DAI only slight

(nonsignificant) elevation in glycoalkaloids concentration (52. 18 mg/100g fr. Wt.)

was recorded which was almost consistent with glycoalkaloids concentration

determined at 6DAI. Statistically maximum total glycoalkaloids concentration

149

74.28 mg/100g fresh weight was recorded at 12 DAI (Table 4.17). These results

showed that days after inoculation (DAIs) had significant effects on foliar total

glycoalkaloids levels.

In diseased plants inoculated with P. infestans, TGA values were consistent with

those of control plants. At 3 DAI, leaves of diseased plants resulted in 43.08

mg/100g fr. Wt. of total glycoalkaloids followed by 50.80, 52.81, 75.67 mg/100 g

fr. Wt at 6, 9, and 12 DAI respectively. Significant increases were observed at

different assessment period. At 6 DAI, TGA significantly increased from 43.08 to

50.80mg/100 g fr. Wt. However, at 9 DAI no significant increase was observed in

TGA showing only sight elevation than 6DAI. Maximum increase in TGA was

recorded at 12 DAI where their values were 75.67 mg/100 g fr. Wt.

Results of this study indicated that total glycoalkaloids contents in diseased plants

at different DAIs increased slightly; however these increments were not significant

and thus disease severity had no significant effects on TGA contents determined

at different period. Compared to control at 3DAI, TGA contents in diseased plants

increased by 2.230 %, at 6DAI by 2.970 %, at 9 DAI by 1.207 % and at 12 DAI by

1.87 % (Fig. 4.27).

Table 4.16 Analysis of variance of mean square for disease severity (%) on potato

leaves inoculated with Phytophthora infestans

Variation

Source

Degrees of

freedom

Sum of

squares

Mean

square

F-values Probability

Replications 3 0.01 0.003 4.95 0.0268

Treatment 3 9795.06 3265.020* 5966131.65 0.0000

150

Error 9 0.00 0.001

Total 15 9795.07

*Significantly different

Table 4.17. Effect of disease severity on total glycoalkaloids (TGA) of potato leaves

determined at different inoculation periods

Disease

severity (%)

Days after inoculation

(DAI)

Foliar TGA (mg 100 g-1 fresh

Wt.)

LSD value at

p=0.05

Inoculated

with P.

infestans

Control

(inoculation

with distilled

water)

0 0 29.43a 29.43a -

5.263 3 43.08b

(2.230) 42.14b

1.204

23.14 6 50.8c

(2.970) 49.33c

2.86

49.308 9 52.81c

(1.207) 52.18c

0.987

70.135 12 75.67d

1.870) 74.28d

2.023

LSD value

at p=0.05

5.129 4.71

-

Values in parenthesis in the third column represent percent increase in TGA to control

151

Fig. 4.26 Late blight disease severity (% defoliation) on potato leaves determined

at different days after inoculation with P. infestans

Fig. 4.27 Effect of disease severity on total glycoalkaloids (TGA)(mg 100-1 g fresh

weight)

Chapter-5

5.263

23.14

49.308

70.135

0

10

20

30

40

50

60

70

80

3 DAI DAI 6 9 DAI 12 DAI

Days after inoculation (DAI)

0

10

20

30

40

50

60

70

80

90

0 DAI 3 DAI 6 DAI 9 DAI 12 DAI

Days after inoculation (DAI)

control inoculated with P. infestans

152

DISCUSSION

5.1 EXPERIMENT I- VARIABILITY IN AGGRESSIONS OF P.INFESTANS

ISOLATES

The findings from this study indicate that different potato growing regions of

Khyber Pakhtunkhwa possess diverse pathotypes of P. infestans which showed

variation in the aggressiveness. Variation in the aggressiveness of the studied

isolates may be assigned to the presence of both mating types in the studied

locations, to sexual recombination and possible mutational events which could

have lead to greater variability in aggressiveness. The results of this study are

consistant with those reported by Miller et al. (1998), Carlisle et al. (2002), Cooke et

al. (2003), Day et al. (2004), Flier et al. (2007), Pliakhnevich and Ivaniuk (2008),

Lehtinen et al. (2009), Runno-Paurson et al. (2010) and Peters et al. (2014).

Aggressiveness is influenced by different parameters such as infection efficiency,

lesion size, growth of lesion day-1, incubation period, latent period, sporulation

capacity, maximal growth curve, and area under lesion expansion curve are used

as criteria for studying the aggressiveness of different races of P. infestans and other

pathogens (Miller et al., 1998; Flier and Turkensteen, 1999; Pliakhnevich and

Ivaniuk, 2008; Pariaud et al., 2009). Characterization of isolates for aggressiveness

is usually based on sampling locations, type of study i.e., whole plant experiment,

detached leaflet assay or tuber disc method and aims of the experiment. Many

researchers have documented that aggressiveness compnents are reliable tools for

studying changes in the population dynamics of P. infestans and they have stated

153

that changes in pathogenicity and aggressiveness of P. infestans pathotypes have

replaced previous population by new population of this pathogen worldwide (Fry,

2008; Pliakhnevich and Ivaniuk, 2008). Infection frequency, latent period, lesion

expansion rate day-1, lesion area and relative area under lesion expansion curve

are important aggressiveness parameters which help in evaluation of disease

causing capacity of different races of a pathogen particularly P. infestans. On the

basis of variability in aggressiveness parameters, pathogenic races of P. infestans

may be regarded as highly aggressive, moderately aggressive or least aggressive.

Moreover, composite aggressiveness index is also used widely to study

aggressiveness potential among various races and pathotypes of P. infestans

(Pliakhnevich and Ivaniuk, 2008). In addition to aggressiveness components,

molecular methods such as mitochondrial DNA-haplotypes, restriction fragment

length polymorphism, peptidase, simple sequence repeats markers and Glucose6-

phosphate isomerase are also commonly used in determination of aggressive

strains of P. infestans and for studying population (Cooke and Lees, 2004; Lees et

al., 2006; Pule et al., 2013). Higher infection frequency of pathotypes represents

higher aggressiveness. Similarly, a pathogen with low LIP values means that it has

high level of virulence and aggressiveness. Lesion area, average lesion expansion

rate and area under lesion expansion curve have been described as important

aggressiveness parameters which can depict the pathogen’s capacity for virulence

and aggressiveness. Pliakhnevich and Ivaniuk (2008) found that larger infection

154

frequency, lesion area, composite aggressiveness index and lower latent period

corresponded to aggressiveness of isolates of P. infestans. Similarly, lesion sizes

were greater in aggressive strains of P. infestans collected from Poland during 2002

and 2004 (Sliwka et al., 2006). Previously several researchers have described the

relative importance of aggressiveness parameters of different isolates for

monitoring changes in population structure of P. infestans (Chacon et al., 2007).

Carlisle et al. (2002) considered sporulation intensity as the most consistent

aggressiveness component for measuring variation among different pathotypes of

P.infestans. On the other hand, sporulation capacity of various pathotypes is a good

indicator for detecting their aggressions potentials (Suassuna et al., 2004) For Miller

et al. (1998), lesion size, sporulation intensity, latent period and RAULEC were the

most effective parameters for detecting changes in P. infestans isolates. Lebreton

et al. (1999) suggested that infection frequency and sporulation intensity were

important epidemiological parameters for determination of variations among

different isolates of the late blight pathogen. They found infection frequency,

sporulation intensity, and lesion expansion were greater for aggressive isolates of

P. infestans on tomato and potato leaves. Significant variation among isolates for

aggressiveness were found in their report. Sharma et al. (2010) stated that area

under lesion expansion curve (AULEC) could be regarded as reliable

aggressiveness parameter. In their study they found isolates with greater

sporulation capacity, infection efficiency and AULEC were higly aggressive which

155

caused maximum disease damage on host plants. Cooke et al. (2011) argued that

lesion area and latent period were important determinants in documenting the

aggressiveness capacity of pathogen races. They found that different isoltes of P.

infestans collected from European countries had different lesion areas and latent

period. Those isolates which had shorter latent period and larger lesion areas were

highly aggressive while those with lower lesion area and longer latent periods

were described as least aggressive. Thus results of this study are similar to findings

of Miller et al. (1998), Lebreton et al. (1999), Suassuna et al. (2004), Chacon et al.

(2007), Sharma et al. (2010) and Cooke et al. (2011).

In regard to different views of different researchers on various aggressiveness

components, composite aggressiveness indices (CAI) were calculated in addition

to five aggressiveness parameters i.e., latent period, lesion area, area under lesion

expansion curve, infection frequency and lesion expansion rate. All these

aggressiveness parameters revealed almost consistent results in both detached and

whole plant but slight variable results in tuber disc assay as well as when

experiments were repeated on whole plant leaves. Variations in aggressiveness

level of studied isolates were apparent in this study. Results of this work are

parallel to findings of Kato et al. (1997) which revealed that most commonly

occurring genotypes in USA i.e., US1, US7 and US8 showed significant variations

for causing lesion area, latent period and sporulation. In a detached leaflet assay,

Miller et al. (1998) documented that 22 isolates of P. infestans were different in

156

aggressiveness capacity. Area under lesion curve, lesion size, sporulation and

composite aggressiveness were recorded higher for US-8 and US11 genotypes

compared to other tested isolates. Flier and Turkensteen (1999) observed

considerable differences in the aggressions of different pathotypes of P. infestans

collected from the Netherlands on the basis of infection frequency, latent period

and composite aggressiveness index. The results are also similar to work described

by Lebreton et al. (1999) which reflected significantly different aggressiveness

parameters for different pathotypes of P. infestans sampled from different areas of

France on two hosts (potato and tomato). Results of Gisi et al. (2011) reveals similar

trend in aggressiveness parameters of 241 isolates from France, Britains and other

European countries during 1996, 2006, 2007. French isolates were more aggressive

than Brtish isolates during 2007 for causing high sporulation.

P. infestansa is heterothallic and diploid pathogen which means it has two

compatible thalus types, A1 and A2, both morphologically similar but different in

genetic traits. The two compatible forms are different in their behavior and release

of compatibility hormone which is responsible for bringing the two different thali

together to go for mating and developing antheridia and oogonia (Fry, 2009).

When either of the thalus type come across the opposite thalus hormone,

development of antheridia and oogonia begins in close proximity. It is inferred

that oogonia sticks and pierce into antheridia resulting in the union of two

opposite thali and hence in formation of oospore (Fry, 2009). Unlike asexually

157

reproducing sporangia or zoospores, oospores are hard structures which can

withstand unfavorable environmental conditions for years. Moreover, sporangia

or zoospores can only survive as inoculum on leaves, stem or on host’s debris

while oospores can manage to live in soil. After months or even years, the oospore

may germinate to form either A1 or A2 thalus types (http://oomyceteworld.net).

This sexual union brings genetically diverse hyphae of the pathogen which show

differences in aggressiveness and virulence towards its host. Prior to 1980s, P.

infestans population throughout the world except Mexico (which is considered as

assumed origin of P. infestans and where both mating type co-exists) consisted of

US-1 clonal lineage which is regarded as genetically less diverse, of A1 type and

with asexual reproduction capability (Fry et al., 1992; Samen et al., 2003). Hohl and

Iselin (1984) documented first record of the presence of A2 hypha of P. infestans

from Switzerland. Their different views regarding the emergence of A2 hyphae in

1980s. Shaw (1987), Spielman et al. (1991), Ko (1994) and Goodwin and Drenth

(1997) assume that; (a) even before the 1980s, both mating hyphae were existed in

other parts of the world but they remained un-noticed; (b) shipments of potato

from Mexico to other countries lead to migration of A2 thali to other parts; (c)

mitosis recombination or mutation events resulted in transformation of A1 to A2

type; (d) mating type changes occurred as a result of fungicides application or due

induction of self-fertilization. When A2 hyphae type was reported out of Mexico

for the first time in the 1980s, the pathogen’s population has ever since drastically

158

changed throughout the world (Knapova and Gisi, 2002). New races of P. infestans

has changed the old population with increased level of aggressiveness; mating

types are unequally distributed and comprises diverse genotypes which are

resistant to phenylamide fungicides (Drenth et al., 1994; Carter et al., 1991; Gisi and

Cohen, 1996). Many authors attribute increased aggression of new population to

the emergence of A2 mating type (Corbieri et al., 2010). A population, which

consists of only one mating type reproduces asexually through sporangia

formation, is hypothesized to have little genetic diversity in the absence of sexual

reproduction (Samen et al., 2003). Sexual reproduction, which needs the presence

of both mating types, and which results in the formation of oospore, brings about

recombination and serves a potent source of genetic variability (Fry, 2008). Thus,

the progenies developed through sexual reproduction may have greater genetic

variation including pathogenicity and aggressiveness. Moreover, oospores have

the advantage of being survived in soil or in other material for many years in

contrast to asexually reproducing structures sporangia or zoospores which have

shorter survival period and whose life span depends on the source of inoculum

(Nowicki et al., 2012; Fry, 2008). The increased virulence and frequent reports of

the new strains with higher aggressiveness potential during the later years of the

1980s has been generally assigned to migration events that might have occurred

from its origin center (Mexico) to different areas of the world producing diverse

genotypes more fitter than those belonging to the old population and bringing

159

significant genetic variability and diversity of allelic loci in the new strains of P.

infestans (Sujkowski et al., 1994; Randall et al., 2002; Fry, 2008). Additionally, some

authors have documented that the new population of P. infetans comprise diverse

strains which carry increased number genes involved in the pathogenicity and

fitness than the old population; European populations for instance, have been

repoted to possess considerable proportion of genes which are virulent to its hosts

and show more aggressions than original Mexican populations of the pathogen

(Sujkowski et al., 1994; Drenth et al., 1994). It is widely accepted in the literature

that high degree of aggressiveness of the isolates is the result of genetic complexity

in new population of P. infestans that may have evolved through sexual

reproduction between the two compatible strains (Fry, 2008). Results of this study

are consistent with those reported by Carlisle et al. (2002). They found that

variability in 20 pathotypes of P. infestans was significantly high for sporulation

capacity, infection frequency, lesion expansion curves and latent period. The

isolates in their study originated from various fields in Northern Ireland and

worked out the aggressiveness parameters in petri dish containing leaves of three

varieties of potato e.g., Bintje, Cara and Stirling. Chacon et al. (2007) also

documented significant variability for aggressiveness parameters viz lesion area

curves, infection frequency and incubation period but not for size of the lesion size,

latent period and growth rate of lesion of four Ecuadorian isolates (EC-1 lineage)

of late blight pathogen.They assigned differences in variability for disease

160

parameters to environmental conditions, host resistance and inoculation density.

Flier et al. (2007) observed significant differences between P. infestans isolates

sampled from Switzerland, Norway, France and the United Kingdom for

sporulation density, growth rate of lesion and infection efficiency. They stated that

variability was as a consequence of coexistence of both mating types, highly

genetic diversity and to sexual reproduction which might have occurred

periodically in population of the studied areas. Peters et al. (2014) assigns

genotypic variations for aggressiveness among isolates of P. infestans to

propagation and widespread migration of clonal lineages like US-24, US-23, US22,

US-11 and US-8 and to the co-occurrence of both mating strains hereby bringing

genetic complexity and emergence of virulency in the population. On the other

hand, Suassuna et al. (2004) correlates the difference in aggressiveness between

isolates of P. infestans to host specification in different geographic location. They

revealed that variability existed in Brazilian isolates of late blight pathogen for

disease parameters IF, IP, LP, LA, LER and sporulation; however, variability was

host (potato and tomato) dependent.

Evolution of the pathogen is one of the most important reasons which contribute

to greater pathogenicity, aggressiveness, invasion and fitness. McDonald and

Linde (2002) attribute that capability of pathogens to overcome resistance genes to

high mutation rates, compatible reproduction system, gene flow and larger

population sizes. Similarly, climatic conditions and cultural practices are also

161

important determinants in creation of new races of pathogen. One of the many

climatic factors influencing the pathogen’s mode of infection are temperature and

humidity. In case of P. infestans, temperature and humidity play a pavitol role in

disease outbreak, infection rate and successful interaction with the host. If

temperature of a region where potato are grown is ideal, then late blight pathogen

will easily make infection on the host and will be able to propagate the disease

further by producing more sporangia. Likewise, high rate of moisture in the host

area is also essential for infection rate and disease progress. If both mating type

are present under favorable climatic conditions, sexual reproduction will proceed

with consequent appearance of new genotypes through genetic recombinations of

which, some strains may have the potential virulence over their parental types

(Fry, 2008). If environment of the growing fields is not ideal for P. infestans for

longer periods, it will be difficult for the pathogen to survive and will tend to

migrate to some favorable conditions. Coakley et al. (1999) states that climatic

conditions alters the pathogen’s capacity of infection, migration patterns,

hostpathogen interactions and host resistance. Pariaud et al. (2009) are of the view

that humidity and temperature markedly affect the physiological conditions of P.

infestans corresponding to greater variation in aggressiveness potentials.

Chakraborty et al. (2000) say that change in climate is a driving force for changing

geographical distribution of both pathogens and their hosts. Garret et al. (2006)

argue that climatic factors strongly influence leaf density, water and resource

162

utilization of host plant which has direct or indirect impact on the rate of infection,

interaction with host, migration, aggressiveness and in the long term on the

evolution of the pathogen. Shaw and Osborne (2011) relates the geographic

distribution of plant pathogens to occurrence of hosts, environmental conditions,

susceptibility of hosts and management practices of crops. Furthermore, cultural

practices such as cultivation patterns, land use and fungicides application are

other important contributors towards changing population structure of late blight

pathogen. Chmielarz et al. (2014) suggests that small fields surrounded by forests

or other barriers are usually vulnerable to late blight outbreak and greater chances

of sexual reproduction, hence increased genetic diversity. Likewise, cultivation of

susceptible host means that even less aggressive genotypes will be able to cause

considerable damage to crop under favorable conditions thereby reducing the

chances for asexual transmission since source of inoculum will not be available to

act as survival agents for carrying the inoculum for the next season. In such a case,

if both mating types were present, chances of sexual reproduction are greater with

oospore development which can survive unfavorable conditions (Fry, 2008;

Andrivon et al., 2013) with result of next out break and more aggressive genotypes.

Similarly, limited or excessive use of fungicides contributes to selection pressure

of particular genotypes either by elimination or establishment of aggressive strains

in population of late blight pathogen (Goodwin et al., 1998; Cooke and Lees, 2004;

Shaw and Osborne, 2011). On the basis of results for the composite aggressiveness

163

indices and other disease parameters, different aggression levels were recorded

for different isolates. On each of three assays, isolates of P. infestans originating

from Sharan, Batkundi, Ayubia, Bara gali and Nathya gali caused highest IF, LA,

LER and AULEC but lowest LIP values. These isolates were grouped as strongly

aggressive (CAI ≥ 100). Those isolates sampled from Kaghan, Naran, Shougran,

Balakot, Mahaban, Shankiari, Shangla and Kalam revealed mild aggressive

potentials with 100 ˂CAI ˃50. Lowest aggressions were shown by isolates

originated from Shabqadar and Mansehra were found to be weakly aggressive

(CAI ≤ 50). Thus, variation among sampled isolates for aggressiveness parameters

were found in this study. Similarly when whole plant expereiment was repeated

in 2013, 47 % of the total isolates (batakundi, Ayyubia, Nathyagali, Bara Gali ,

Mahaban, Shangla, Shabqadar) were grouped as strongly aggressive while 53 %

(Sharan, Kaghan, Naran, Shoughran, Balakot, Shankyari, Kalam and Mansehra)

were mildy aggressive. There were no category of weakly aggressive isolates in

2013. On the other hand, during 2011, 33 % sampled isolates (Sharan, Batakundi,

Ayyubia, Bara Gali , and Nathyagali) had maximum disease parameters and were

grouped as strongly aggressive. But 53 % of isolates (Kaghan, Naran, Shougran,

Balakot, Mahaban, Shankyari, Shangla and Kalam) were found to have moderate

aggressiveness potentials and they were categorized as mildly aggressive. Isolates

from Shabqadar and Mansehra accounted for 26 % of the total studied isolates to

have weak aggressions and were classified as weakly aggressive. These results

164

suggests that in Khyber Pakhtunkhwa, genetically diverse races of P. infestans with

variable aggressiveness potentials existed during studied perioed. The study also

concludes that population structure of P. infestans showed changes in 2013 because

aggressiveness shift of pathotypes was recorded in 2013 experiment when

compared to 20111

Although mating type and metalaxyl resistance tests were not conducted in this

study, however, from the results of this work it may be inferred that population

P.infestans’ population in Khyber Pakhtunkhwa possess genetically diverse

pathotypes with varying level of aggressiveness measured by IF, LIP, LA, LER

and AULEC. Differences among P.infestans’ pathotypes originating from different

potato fields of Khyber Pakhtunkhwa for different aggressiveness parameters in

this study may possibly be assigned to the presence of diverse genotypes of

studied pathogen in the province. The presence of P. infestans in Pakistan was

documented in 1984 for the first time in Northern Pakistan (Khan et al., 1985) while

the report about almost equal proportions of A-2 and A-1 mating type in the

country was published in 1995 (Ahmad and Mirza, 1995). It may be inferred that

A1 and A2 mating type may have undergone through sexual reproduction causing

genetic complexity in the progenies with differential aggressiveness behavior. It is

also possible that mutation may have resulted in more aggressive strains of the

pathogen in the later years. Agricultural practices, frequent use of fungicides and

climatic conditions are also important contributors towards selection of fitter and

165

aggressive genotypes of the late blight pathogen in a geographic region (Pariaud

et al., 2009). Lehtinent et al. (2008) states that if reproduction potential - controlled

by several factors e.g., climate, physiological state of pathogen, host susceptibility

– is high in late blight epidemics, then migration of genotypes is favored which

brings about population changes and dominance of a particular strain in a given

area.

Results of current work indicated that aggressiveness parameters of 15 isolates

computed on three different assays e.g., whole plant leaves, detached leaflet and

tuber disc assays revealed that isolates exhibited similar aggression tendency

under experimental assays. Stronger aggressiveness potentials were shown by

isolates collected from Sharan, Ayyubia, Bara Gali, Nathyagali and Batakundi on

whole plant, detached leaf and tuber disc while mild aggressiveness was recorded

for isolates originated from Kaghan, Shankyari, Shoghran, Mahaban, Naran,

Kalam, Shangla, and Balakot. Isolates from Mansehra and Shubqadar regions were

found to have low aggression on each assay. However, values of aggressiveness

parameters were significantly larger on tuber disc assay when compared to whole

plant and detached assay. Lowest values of aggressiveness of the studied isolates

were recorded for whole plant experiment. Lower values of aggressiveness

parameters on whole plant leaves in this study may be assigned to the reason that

in field conditions when host plant is growing may offer maximum resistance to

pathogen. When P. infestans attacks on potato leaves in natural environment, the

166

plant defense system tries to tackle the attacking pathogen. Enzyme system of host

become activated and production of phytoalexins is triggered which in turn

provide maximum resistance to the growth mycillium of P. infestans and spread of

disease is slowed down. In lab conditions, leaves and tuber slices are placed in

petridishes which is an artificial environment and test organs are deprived of

natural defense system in case of intact tissues. So virtually, the pathogen may find

it conducive to colonize detached tissues more rapidly and aggressively than in

the case of intact living tissues. Moreover, enzymes involved in catalyzing the cell

membrane of attacking pathogen may become less active in detached leaves and

tuber slices as a result of degradation after detachment from living potato plant.

Thus it is apparent that P. infestans might face difficulty in colonization, mycelial

growth and disease spread in living host tissues where strong enzyme system,

defensive system and phytoalexins are present. While in detached leaves and tuber

discs such a strong defense response might have not been shown which resulted

in greater infection, lesion sizez, ALER, AULEC, CAI but low latent period when

compared to whole plant leaves in this study. These results are in good agreement

with Sedegui et al., (1999) who documented that there was no significant

differences for aggressiveness parameters of isolates of P. infestans on whole plant,

detached leaves and tuber disc assays although they reported that lesion

development on detched leaves was rapid than on detached leaves. They

suggested that tuber disc assays and detached leaflet assays yielded reliable results

167

for aggressiveness tests and recommended these methods for general screening of

aggressiveness of P. infestans isolates. On the other hand Dorrence et al. (1997)

suggests that field experiment (intact leaves) are ideal methods for studying

aggressiveness of pathotypes of P. infestans. Miller et al. (1998) reported that most

of tested isolates of P. infestans particularly US1, US7, US8 and US11 in their study,

lesion area, infection frequency and AULEC were similar on whole plant and

detched leaves but different in sporulation, incubation period and AUDPC. They

stated that ineffectivity of isolates through upper leaf surface of whole plant (intact

leaves) play an import role in aggressiveness. In case of detached leaflets, the

pathogen’s isolates are effective in expanding their mycelia and causing rapid

lesion development. Flier et al. (2001) concluded that physiological age, cortical

tissue and medulla of tuber in tuber disc assay and whole tuber assay are

important determinant in aggressiveness studies.

Overall, results of this work confirm the findings of other studies conducted by

(Chacon et al., 2007) which reported that variation in aggressiveness existed in

isolates of P. infestans collected from Ecuador. Similar results were also reported

from European countries where varaiability in aggressiveness of different

pathotypes were evident (Carlisle et al., 2002; Flier et al., 2007; Lehtinen et al., 2009),

Belarus (Pliakhnevich and Ivaniuk, 2008). Isoltes different in aggressiveness

potential were recorded from differen districts of USA (Miller et al., 1998), Canada

(Peters et al., 2014), and Great Britain (Day et al., 2004; Cooke et al., 2003) which

168

agree with results of this study. Similarly, Runno-Paurson et al. (2010) observed

that Estonian population of P. infestan comprised severely aggressive and

moderately aggressive strains of P. infestans which showed different

aggressiveness parameters in their study. Variability in aggressiveness of different

races has also been reported from Asia (Gotoh et al., 2005) and South America

(Andrade-Piedra et al., 2005) where the occuerence of aggressively divere isolates

of P. infestans exist.

5.2. EFFECT OF LATE BLIGHT DISEASE SEVERITY ON FOLIAR

GLYCOALKALOIDS OF POTATO

Results of this study indicated that disease severity had no effect on glycoalkaloids

of leaves. Deahl et al. (1973) and Frank et al. (1975) determined the effect of late

blight disease and some other pathogenic fungi on total glycoalkaloids of different

potato clones but were unable to find association between late blight disease and

leaf TGA contents. Andreu et al. (2001) reported that glyco-alkaloids, phenolic

compounds and phytoalexins slightly but insignificantly increased in leaves of

potato when they were inoculated with P.infestans; however, asoociation between

disease seveirity and glycoalkloids could not be established in their studies. The

findings of these researchers support the results in this study.

169

On the other hand, Andrivon et al. (2003) reported low but significant correlation

between the concentration of glycoalkaloids (α-solanine) of some potato clones

and late blight resistance parameters i.e., incubation period and spore production

per unit lesion area, suggesting a possible role of these compounds against P.

infestans. Also, Henriquez et al. (2012) studied changes in secondary metabolic

compounds in leaves of potato leaves when they were inoculated with P. infestans.

They reported differential responses of varieties Defender and Russet Burbank

towards accumulation of secondary metabolites following infection by P. infestans.

metabolic compounds rutin and catechin levels showed suppression in potato

variety Russit Burbannk following the infection P. infestan while in variety

Defender, levels of flavonoid glycoside exhibited elevation. Similarly, another

terpenoid compound was reported to have been increased after disease

development in Defender. They suggested the possible role of secondary

metabolites in host defense against P. infestans. Their findings are in contradiction

with the results reported in this study. Differences in results regarding the

relationship between late blight disease and foliar glycoalkaloids documented by

different workers and in this study may possibly be due to different experimental

conditions and different cultivars of potato used in the studies.

Potato, like other plants, produce secondary metabolites which have no direct role

in growth, development or reproductive potential. Among the secondary

metabolites produced in potato and some other members of family Solanaceae are

170

glycoalkaloides which are produced in variable concentrations in different plant

parts (Osman, 1983). These compounds are toxic to pathogenic fungi, insects and

pests, herbiovres and are known to play some role in offereing resistance to

pathogens, herbivores, insects and pests (Tingey, 1984; Matthews et al., 2005;

Friedman, 2006). Alpha-chaconine and alpha-solanine are major classes of

glycoalkaloids found in potato (Freidman and McDonald, 1997). Glycoalkaloids

levels of potato are dependent on different factors such as potato germplasm, plant

age, biotic and abiotic stress, attacks of pathogens and herbivory (Sinden et al.,

1984; Friedman, 2006). Higher concentrations of glycoalkaloids are usually

observed in those plant parts which have high metabolic activity. Young leaves,

buds and reproductive organs posses higher glycoalkaloids which become

declined when plant progress towards maturity (Freidman and McDonald, 1997).

Distribution of glycoalkaloids is variable in different parts of potato; however,

leaves have more glycoalkaloids than tubers, sprouts and stems, generally

controlled by several factors such as potato cultivars, age of plant, environmental

stresses and phyto-pathogens (Sinden et al., 1984; Friedman, 2006). Based on

previous studies of Friedman and Dao (1992) and Deahl et al. (1993), glycoalkaloids

in leaves may range between 50 and 145 mg 100g-1 fresh weight. Similarly, in

whole tubers their concentration in many studies, have been reported between 10

and 150 mg 100g-1 fresh weight (Gelder et al., 1988; Mathews et al., 2005). However,

these results may be challenged elsewhere because concentrations of these

171

compounds are variable in different varieties and in different environments.

Higher concentrations of glycoalkaloids in leaves than tubers is attributed to the

fact that leaves are generally more exposed to sunlight and phytopathogens in

addition to many other abiotic stresses than tubers and other parts of potato

(Friedman, 2006).

Glycoalkaloids are potentially toxic for human consumption; therefore, potato

breeders always opt for obtaining breeding lines of potato with low and acceptable

level of these compounds (Friedman, 2006). However, their toxic nature is

considered by several researchers as blessing for host defense against different

herbivores, nematodes, insects and pests and a wide range plant pathogens

(Tingey, 1984; Matthews et al., 2005). Mode of action of glycoalkaloids like other

secondary metabolites in protection of host against plant pathogens is not well

understood; however, possible elucidation for the protective role of these

compounds might be the production of defense proteins, complex formation with

pathogen cell wall by the glycoalkaloids and activation of pathogen specific

catalyzing enzymes for degradation cell wall of the pathogen (Osbourn, 1996).

Although role of potato glycoalkaloids in minimizing damages caused by

notorious insects and pests such as Colorado potato beetle (Jonasson and Olsson,

1994; Lorenzen et al., 2001), potato aphid (Guntner et al., 1997), white cyst

nematode (Forrest and Coxon, 1980), wireworm (Jonasson and Olsson, 1994) and

172

snail (Smith et al., 2001) is well established; however, few studies have been

conducted to point out the role of glycoalkaloids found in potato leaves and tubers

in disease resistance against P. infestans with variable reports (Friedman, 2006;

Khan et al., 2013). According to Deahl et al. (1973) ther was no association between

late blight disease resistance and leaf and tuber glycoalkaloids of 15 potato lines.

Frank et al. (1975) also demonstrated potato leaves glycoalkaloids did not play a

significant role in disease resistance against P. infestans in field conditions. Andreu

et al. (2001), on the other hand found that glycoalkaloids and some other

phytoalexins accumulated in leaves and potato tubers when they were inoculated

with P. infestans, however, there was lack of a clear and significant relationship

between glycoalkaloids and disease resistance.

The mechanism of the potential role of glycoalkaloids in disease resistance and

host defense is not well understood. However, it is assumed that changes in

glycoalkaloids or other phytoalexins after infection by pathogen is triggered by

chemicals released from the pathogen or it may be due to the host and pathogen

interaction. Defense response of the host to pathogenic attack results in the

production or induction of changes (increase or decrease) in phytoalexins which

may correlate with the pathogen in a positive or negative way (Hammerschmidt,

1999). The defense response of the host is generally initiated by elicitors- molecules

released by the pathogen or produced by the host in response to pathogen

interaction (Hammerschmidt, 1999; Sharma et al., 2011). In turn, the host may

173

possibly produce toxins to the pathogen’s growth and feeding, or may trigger

other metabolites and enzymes of the host for a prompt resonse to the pathogen.

Many studies conducted on the antifungal and pesticidal activity of glycoalkaloids

elucidates that these compounds are toxic, anti-feedent and deterrent to pest and

fungal growth (Lorenzen et al., 2001; Fewell and Roddick, 1993; Martin and

Douglas, 1997; Yencho et al., 2000). The antifungal and pesticidal activity of

glycoalkaloids particularly α-solanine and α-chaconine, are presumed to be

because of their ability to bind with and disrupt cell membranes of the pathogens

(fungi, insects, pest) having high sterols (Martin and Douglas, 1997). Sterol

binding, destabilization of cell membranes and inhibition of enzymes by

glycoalkaloids in different studies have been confirmed (Roddick et al., 1988a,

2001b). Differences in sensitivity of glycoalkloids of potato to different pathogens

may possibly be because of differential concentrations of cell membrane’s sterol

contents in different pathogens (Martin and Douglas, 1997). Thus one of the

possible answers for the lack of correlation between glycoalkloids present in

potato foliage or tubers and late blight disease as indicated in many studies, may

probably be due to low sterols contents in cell membrane of P. infestans; hence low

binding and membrane disruption capacity of these compounds with P. infestans

(Nes et al., 1983; Roddick et al., 1987).

174

CONCLUSION AND RECOMMENDATIONS

In conclusion, isolates of Phtophthora infestans collected from fifteen different

locations of KPK were tested for aggressions in three experimental models i.e.,

whole plant experiment (intact leaves), detached leaflets and tuber disc assay

during 2011. Whole plant experiment was repeated in 2013. All the sampled

isolates showed significant variations for aggressiveness parameters computed by

latent period, infection frequency, area of lesion size, rate of lesion expansion, area

under lesion expansion curve and composite aggaressiveness index. On the basis

of results for the composite aggressiveness indices and other disease parameters,

different aggression levels were recorded for different isolates. On each of three

assays, isolates of P. infestans originating from Sharan, Batkundi, Ayubia, Bara gali

and Nathya gali caused highest IF, LA, LER and AULEC but lowest LIP values.

These isolates were grouped as strongly aggressive (CAI ≥ 100). Those isolates

sampled from Kaghan, Naran, Shougran, Balakot, Mahaban, Shankiari, Shangla

and Kalam revealed mild aggressive potentials with 100 ˂CAI ˃50. Lowest

aggressions were shown by isolates originated from Shabqadar and Mansehra

were found to be weakly aggressive (CAI ≤ 50). Thus, variation among sampled

isolates for aggressiveness parameters were found in this study. Similarly when

whole plant expereiment was repeated in 2013, 47 % of the total isolates

(batakundi, Ayyubia, Nathyagali, Bara Gali , Mahaban, Shangla, Shabqadar) were

grouped as strongly aggressive while 53 % (Sharan, Kaghan, Naran, Shoughran,

175

Balakot, Shankyari, Kalam and Mansehra) were mildy aggressive. There was no

category of weakly aggressive isolates in 2013. On the other hand, during 2011, 33

% sampled isolates (Sharan, Batakundi, Ayyubia, Bara Gali, and Nathyagali) had

maximum disease parameters and were grouped as strongly aggressive. But 53 %

of isolates (Kaghan, Naran, Shougran, Balakot, Mahaban, Shankyari, Shangla and

Kalam) were found to have moderate aggressiveness potentials and they were

categorized as mildly aggressive. Isolates from Shabqadar and Mansehra

accounted for 26 % of the total studied isolates have weak aggressions and were

classified as weakly aggressive. It may be concluded from the results of this study

that in Khyber Pakhtunkhwa, genetically diverse races of P. infestans with variable

aggressiveness potentials existed during studied period. The study also concludes

that population structure of P. infestans showed changes in 2013 because

aggressiveness shift of isolates was recorded in 2013 experiment when compared

to 2011. Similarly, results also revealed that three experiments showed almost

similar and reliable results for aggressiveness. Since most of the strongly

aggressive pathotypes were present in Sharan, Batkundi, Ayyubia, Nathyagaly,

Bara Gali, Mahaban, Shangla and Shabqadar, it is recommended that farmers

should use early maturing varieties of potato, and should use broad specrum

protectant fungicides. It is further recommended that larger scale studies should

be initiated to study population structure of P. infestans isolates sampling from all

potato growing regions of Pakistan.

176

Based on the results of experiment II conducted for determination of the effect of

late blight disease severity on foliar glyco-alkaloids, it is concluded that late blight

disease severity had no effect on foliar glycoalkloids of potato. Rather, increase in

days of inoculation significantly elevated glycoalkaloids levels in potato leaves.

Thus there is no need for potato breeders to develop breeding varieties of potato

with elevated level of glycoalkaloids as they played no role in disease resistance

in this study.

177

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APPENDIX

Papers published from the thesis

1. Majeed, A., Z. Chaudhry, I. Haq, Z. Muhammad H. Rasheed. 2015. Effect of

aqueous leaf and bark extracts of Azadirachta indica A. Juss, Eucalyptus

citriodora Hook and Pinus roxburghii Sarg. on late blight of potato. Pak. J.

Phytopathol., 27: 13-18.

2. Majeed, A., Z. Chaudhry and Z. Muhammad. 2014. Changes in foliar

glycoalkaloids levels of potato (Solanum tuberosum L.) triggered by late

blight disease severity. Int. J. Agric. Biol., 16 (3): 609-613.

3. Majeed, A., Z. Chaudhry and Z. Muhammad. 2014. Variation in the

aggressiveness of Phytophthora infestans pathotypes collected from

different potato fields of Khyber Pakhtunkhwa (Pakistan). Int. J. Agric.

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Biol., 16 (4): 807-812.