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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
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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
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DEDICATION
To my affectionate parents
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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.
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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
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I n the name of
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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
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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
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LIST OF FIGURES
Figure
4.1
Infection frequencies (IF)% of isolates Phytophthora infestans
during 2011 and 2013.
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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
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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
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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
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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
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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
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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%.
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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).
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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.
79
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.
206
Biol., 16 (4): 807-812.