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This is to certify that the
thesis entitled

INHERITANCE OF COLD TEMPERATURE TOLERANCE IN
PROGENY ISOLATES OF PHYTOPHTHORA INFESTANS
FROM MATING

presented by

Pavani Gooty Tumbalam

has been accepted towards fulfillment
of the requirements for the

Master of degree in Department of Plant Pathology
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INHERITANCE OF COLD TEMPERATURE TOLERANCE IN
PROGENY ISOLATES OF PH Y T OPH T HORA INFES T ANS FROM
MATING

By

Pavani Gooty Tumbalam

A THESIS

Submitted to
Michigan State University
in partial fiJlfillment of the requirements
for the degree of

MASTER OF SCIENCE
Department of Plant Pathology

2007

ABSTRACT

INHERITANCE OF COLD TEMPERATURE TOLERANCE IN PROGENY
ISOLATES OF PH Y T OPH T HORA INFESTANS FROM MATING

By
Pavani Gooty Tumbalam
Late blight of potato, caused by Phytophthora infestans, remains a threat to the
production of high quality potatoes. Optimal temperature for mycelial growth is around
10°C with maxima and minima of 30 and 3°C, respectively. Recent studies have
indicated that some isolates of different genotypes of P. infestans can survive about 5
days continuous exposure to -3°C. To observe more variation in the tolerance to cold
temperature, isolates were tested for tolerance at -5°C for 5 days. This phenotypic trait
has serious implications for potato production and disease protection. In this project
mating type is used as a biological tool for enhancing the mutation in the pathogen.
Crosses were carried out between isolates that were highly variable for temperature
tolerance and identified as tolerant, sensitive or intermediate in tolerance of exposure to
-5°C. To determine if temperature tolerance is a genetically inherited trait or occurs as a
result of physiological adaptation of different P. infestans genotypes the progeny isolates
fiom these crosses were assessed by using the phenotypic and genotypic characteristics of
progeny such as mating type, isozyme analysis, Simple Sequence Repeats, metalaxyl
sensitivity, virulence and pathogenicity tests. Progeny isolates from crosses performed
have indicated that cold tolerance is a continuous variable showing both inheritance and
hybridity. These data illustrate the potential variation in different characteristics of the
pathogen after recombination or mutation occurs. Survival potential of inoculum may be

increased under recent reported climatic changes in north-central US production areas.

Dedicated to:

My daughters Laya and Kavya,
My Pride and Joy

iii

ACKNOWLEDGEMENTS

I would like to express my extreme gratitude to several members who have had an impact
on my research. First of all, I thank my advisor Dr. Willie Kirk for his guidance and
encouragement throughout this work. I would also like to thank Dr. Gerry Adams and Dr.
Dave Douches for serving on my committee and participating in many helpful
discussions. Thanks to my fellow lab mates, Justin DeMaagd, Lauren Liddell, Gillian
Young, Robert Schafer and Phillip Wharton, and my fellow graduate students in the

department. Thanks to Dr. Hao, who graciously accepted to review my work.

I would like to express my heart-felt gratitude to my parents and parents-in-law, who
have always supported, encouraged, and respected my choices and to my husband Shrini,
without whom this work would have never been accomplished. I would also like to thank
all my family and friends, who have always encouraged me in my research and were

always interested in finding out more about my work.

Finally, I would like to express my appreciation to Michigan State University and the
Department of Plant Pathology for allowing me to have such a wonderful academic
experience, especially Chairperson Dr. Ray Hammerschmidt, all the professors and
Teaching Assistants who taught me so much and guided my education. Thanks to Project
GREEEN (MSU), Michigan Potato Industry Commission and NPC/ARS who provided

financial support for my research.

iv

Table of contents

List of Figures .................................................................................................................. viii
List of Tables ................................................................................................................... viii
1 Literature review ......................................................................................................... 1
1 . 1 Introduction ......................................................................................................... 1
1.2 Host ..................................................................................................................... 1
1.3 Pathogen .............................................................................................................. 2
1.4 Taxonomy ........................................................................................................... 3
1.5 Host range ........................................................................................................... 4
1.6 Disease cycle ....................................................................................................... 4
1 .6.1 Asexual reproduction .................................................................................. 7
1.6.2 Sexual reproduction .................................................................................... 8
1.7 Non-chemical and chemical Control ................................................................ 10
1.8 Breeding for resistance ..................................................................................... 11
1.9 Characterization techniques .............................................................................. 12
1.10 Phenotypic markers ........................................................................................... 12
1.10.1 Mating type ............................................................................................... 13
1.10.2 Effects of temperature on the disease ....................................................... 13
1.10.3 Fungicide resistance to metalaxyl as a phenotypic marker ....................... 17
1.10.4 The hypersensitive response ..................................................................... 19
1.10.5 Virulence phenotype ................................................................................. 20
1.10.6 Pathogenicity testing ................................................................................. 22
1.11 Genetic markers ................................................................................................ 23
1.11.1 Isozyme analysis ....................................................................................... 24
1.11.2 Nuclear DNA polymorphisms .................................................................. 27
1 . 12 Objectives ......................................................................................................... 29

2 Genotypic characteristics of Phytophthora infestans from mating: Determination of
hybridity or selfing by isozyme analysis and microsatellite markers ............................... 31
2.1 Introduction ....................................................................................................... 31
2.2 Materials and methods ...................................................................................... 36
2.2.1 Selection of isolates .................................................................................. 36
2.2.2 Isozyme analysis ....................................................................................... 36
2.2.3 Microsatellite analysis .............................................................................. 40
2.2.4 Nuclear condition and ploidy levels ......................................................... 43
2.3 Results ............................................................................................................... 46
2.3.1 Isozyme Analysis ...................................................................................... 46
2.3.2 Microsatellites ........................................................................................... 47
2.3.3 Nuclear condition and ploidy levels ......................................................... 48
2.4 Discussion ......................................................................................................... 49

3 Mating type, metalaxyl resistance, and virulence characteristics of Phytophthora

infestans from mating isolates with variable thermal tolerance ........................................ 63
3. 1 Introduction ....................................................................................................... 63
3.2 Materials and methods ...................................................................................... 68

3.2.1 Selection of isolates .................................................................................. 68
3.2.2 Mating type ............................................................................................... 68
3.2.3 Metalaxyl sensitivity ................................................................................. 69
3.2.4 Virulence assessment ................................................................................ 70
3.3 Results ............................................................................................................... 72
3.3.1 Compatibility type .................................................................................... 72
3.3.2 Metalaxyl sensitivity ................................................................................. 73
3.3.3 Virulence assessment ................................................................................ 74
3.4 Discussion ......................................................................................................... 75

4 Cold temperature tolerance of mycelium in progeny isolates of Phytophthora

infestans from mating ....................................................................................................... 88
4. 1 Introduction ....................................................................................................... 88
4.2 Information from previous studies (Kirk lab) that helped in this research ....... 92

4.2.1 Substrate optimization and digital image analysis .................................... 92
4.2.2 Temperature optimization ......................................................................... 93
4.2.3 Determination of developmental stage of sporangia on mycelial plugs after
transfer from parent cultures ..................................................................................... 94
4.2.4 Relation between digital image of samples and mycelial weight ............. 94
4.3 Materials and methods ...................................................................................... 95
4.3.1 Isolate descriptions .................................................................................... 95
4.3.2 Determination of thermal tolerance .......................................................... 96
4.3.3 Image analysis technique .......................................................................... 97
4.3.4 Production of progeny isolates of Phytophthora infestans from mating 100
4.3.5 Data analysis ........................................................................................... 102
4.4 Results ............................................................................................................. 109
4.4.1 Testing of thermal tolerance for potential parental P. infestans isolates 109
4.4.2 Thermal tolerance ................................................................................... 109
4.5 Discussion ....................................................................................................... 1 l3

5 Pathogenicity of progeny isolates fiom crosses of thermally variable Phytophthora

infestans on potato tubers ................................................................................................ 124
5. 1 Introduction ..................................................................................................... 124
5.2 Materials and methods .................................................................................... 125

5.2.1 Pathogenicity test on tubers .................................................................... 125
5.2.2 Data analysis ........................................................................................... 130
5.2.3 Compiling data ........................................................................................ 131
5.3 Results ............................................................................................................. 131
5.4 Discussion ....................................................................................................... 133

6 Executive Summary ................................................................................................ 141

6.1 Future research ................................................................................................ 143

vi

7 Appendix ................................................................................................................. 144

Appendix 1 .................................................................................................................. 145
Appendix 2 .................................................................................................................. 148
Appendix 3 .................................................................................................................. 149
Appendix 4 .................................................................................................................. 150
Appendix 5 .................................................................................................................. 154
Literature Cited ............................................................................................................... 162

vii

List of Tables

Table 2.1 Phenotypic and genotypic characteristic of isolates of Phytophthora infestans
used in crosses ........................................................................................................... 38

Table 2.2 Presumed genotypes at the Glucose phosphate isomerase (GPI) locus in
parental and single oospore progeny isolates of Phytophthora infestans. ................ 52

Table 2.3 Different allelic patterns of parent and progeny isolates of Phytophthora
infestans at microsatellite, Pi4B and Pi02 loci .......................................................... 53

Table 2.4 Phytophthora infestans showing variation in the DNA content of nuclei among
different single zoospores (asexual progeny) of the isolate PI-1055P-MYA1 1 l4 55

Table 2.5 Different isolates of Phytophthora infestans showing variation in levels of
nuclear condition, karyotype and sexuality .............................................................. 56

Table 3.1 Number of progeny isolates in compatibility type phenotype categories
resulting from oospores generated by crossing parental isolates of Phytophthora
infestans with different compatibility types and tolerance to temperature. .............. 76

Table 3.2 Different progeny and parent isolates showing either presence of self-fertile
oospores in pure cultures after one month of incubation resulting from oospores
generated by crossing parental isolates of Phytophthora infestans with different
compatibility types and tolerance to temperature. .................................................... 77

Table 3.3 Number of progeny isolates in metalaxyl sensitivity phenotype categories
resulting from oospores generated by crossing parental isolates of Phytophthora
infestans with different metalaxyl sensitivity. .......................................................... 80

Table 3.4 Parental and progeny isolates of Phytophthora. infestans showing virulence on
R gene potato differential genotypes LB 0 to LB 11. ............................................... 82

Table 4.1 Isolates of Phytophthora infestans tested for thermal tolerance. Isolates were
exposed to -5°C for 5-days and regenerated for selection as potential parental
isolates with different thermal tolerance phenotypes .............................................. 106

Table 4.2 Origin, mating type, thermal tolerance, and CPI-isozyme phenotypes of
isolates of Phytophthora infestans selected for crossing studies. Progeny from these
crosses were isolated to identify if thermal phenotype character was inherited. 107

Table 4.3 Phenotypic and genotypic characteristic of isolates of Phytophthora infestans
used in crosses ......................................................................................................... 108

viii

Table 4.4 Number of progeny isolates in thermal tolerance phenotype categories
resulting from oospores generated by crossing parental isolates of Phytophthora
infestans with different thermal tolerance to -5°C. ................................................. 114

Table 5.1 Differences between the two executions of pathogenicty and virulence of
parental and progeny isolates of Phytophthora infestans with different thermal
tolerance phenotypes based on the Average Reflective Intensity (ARI) of inoculated
potato tubers cv. FL1879. ....................................................................................... 135

Table 5.2 Quantification of virulence of isolates of Phytophthora infestans using a tuber
inoculation technique based on differences in tuber tissue darkening between non-
inoculated control tubers and positive control tubers inoculated with a virulent
isolate. ..................................................................................................................... 136

Table 5.3 Pathogenicty and virulence of parental and progeny isolates of Phytophthora
infestans with different thermal tolerance phenotypes measured using a severity
scale based on the Average Reflective Intensity (ARI) of inoculated potato tubers
cv. F L1879 based on comparison to negative and positive controls. ..................... 137

Table 5.4 Progeny isolates of Phytophthora infestans selected from some crosses

showing inheritance and recombination for phenotypic and genotypic
characteristics. ......................................................................................................... 139

ix

List of Figures

Figure 1.1 Reproduction structure of Phytophthora infestans: Sporangia and Sporangia
releasing zoospores ..................................................................................................... 5

Figure 1.2 Reproduction structure of Phytophthora infestans: Oospores with Antheridum
and oogonium .............................................................................................................. 5

Figure 1.3 Life cycle of potato late blight causing pathogen, Phytophthora infestans. (©
P.S. Wharton) .............................................................................................................. 6

Figure 2.1 Banding patterns of isolates of Phytophthora infestans for differing allozyme
genotypes, and accompanying US designations (Goodwin et al., 1995) at the GP]
locus, as revealed by cellulose acetate electrophoresis ............................................. 42

Figure 2.2 Standard GPI band patterns of Phytophthora infestans isolates ran on a
cellulose acetate gel for 30 min at 200V ................................................................... 42

Figure 2.3 Isozyme analysis (GPI) of isolates of Phytophthora infestans parents and
progeny; From Left to right, Lane 1: 96/100, Pi 95—3; Lane 2: 100/ 1 1 1/ 122, Pi 02-
007; Lane 3: 100/100, Pi 41-02; Lane 4: 100/100, 2-35; Lane 5: 100/122, Pi 98-1;
Lane 6: 100/100, 2-2; Lane 7: 100/100, 2-10; Lane8: 100/100, 2-15 ...................... 51

Figure 2.4 Electropherograrns illustrating isolates heterozygous for the Pi4B and Pi02
loci at alleles from 200 to 226 bp and 160bp respectively. The Y axis represents
relative fluorescence unit (RF U) and the X axis molecular size of the allele marker
in base pairs (bp) size ................................................................................................ 54

Figure 2.5 Representative histograms of the nuclear condition of parent and progeny
isolates of Phytophthora infestans. The typical diploid nuclei of most isolates of P.
infestans produces a fluorescence intensity of 48 — 52 units on the X-axis (on
channel FL3-A) when stained with Propidium iodide. The units are relative and
compared to the standar (Gray peak) of stained chicked red blood cell nuclei. ....... 62

Figure 3.1 An isolate of P. infestans showing levels of sensitivity to metalaxyl at
different concentrations: A= Control or 0; B: 0.1; C= 1; D= 10; E= 100; F= 1000
PPM metalaxyl.For example isolate 1-4 is intermediately resistant to Metalaxyl
with ECso: 5 PPM ...................................................................................................... 79

Figure 3.2 Example of a single replication for assessing the virulence pathotype. LBR
gene leaflets (LBRO to LBRl 1) showing lesions symptomatic of infection with P.
infestans incubated at 18°C in a controlled growth chamber for eight days. The
virulence pathotype indicated that isolate 3A lacked the avirulence genes 0, l, 2, 5,
7, 8, 10, and 11 .......................................................................................................... 81

Figure 4.1 C-l Peltier-effect temperature cabinet (PTC-l) and a FELT-3 Peltier-effect
temperature controller in a controlled growth chamber used to maintain the
temperature in the Peltier chamber at -5°C. .............................................................. 98

Figure 4.2 A germinating oospore of Phytophthora infestans forming mycelium under
10 X magnification. From Cross 3; ID 3_06. ......................................................... 104

Figure 4.3 An oospore of Phytophthora infestans producing germ tube and Sporangia
under 10 X magnification ....................................................................................... 104

Figure 4.4 An example of an isolate of Phytophthora infestans showing tolerance to -
5°C. JPEG images of isolates of Phytophthora infestans were produced in
Photoshop on an Epson Perfection 4870 flatbed scanner. Images were digitally
analyzed to measure the Average Reflective Intensity (ARI) in Light Intensity Units
(LIU) using Sigma Scan software. Left Side: Positive controls, exposed to 18°C;
Center: Test Isolates, exposed to -5°C for 5 days; Right side: Negative controls, non-
inoculated Petri dish ................................................................................................ 105

Figure 4.5 Differences in thermal tolerance of parental isolates used for mating to
produce progeny isolates. Phytophthora infestans growth was measured as %RARI
after exposure of isolates to -5°C for 5 days. %RARI values followed by the same
letter are not significantly different at p = 0.1 (Tukey Multiple Comparison); LSDO_]0
indicated by bar. ...................................................................................................... 1 15

Figure 4.6 Differences in thermal tolerance of progeny isolates from mating of thermally
tolerant parental isolates of Phytophthora infestans Pi 41-02 (A1, thermally tolerant)
x Pi 02-007 (A2, thermally tolerant). Growth was measured as %RARI after
exposure of isolates to -5°C for 5 days (Cross 1). The black bars represent the
parental isolates and the white bars indicate thermal sensitive (Pi 95-3) and
insensitive (Pi 02-007) standards, respectively; Pi 02-007 was a parent in this cross.
%RARI values followed by the same letter are not significantly different at p = 0.1
(Tukey Multiple Comparison); LSDOJO indicated by bar. ...................................... 115

Figure 4.7 Differences in thermal tolerance of progeny isolates from mating of
thermally tolerant with intermediately tolerant parental isolates of Phytophthora
infestans Pi 41-02 (A1, thermally tolerant) x Pi 98-1 (A2, thermally intermediate).
Growth was measured as %RARI after exposure of isolates to -5°C for 5 days (Cross
5). The black bars represent the parental isolates and the white bars indicate thermal
sensitive (Pi 95-3) and insensitive (Pi 02-007) standards, respectively. %RARI
values followed by the same letter are not significantly different at p = 0.1 (Tukey
Multiple Comparison); LSDOJO indicated by bar .................................................... 116

Figure 4.8 Differences in thermal tolerance of progeny isolates from mating of
thermally tolerant parental isolates of Phytophthora infestans Pi 02-007 (A2,
thermally tolerant) x Pi 95-3 (A1, thermally sensitive). Growth was measured as
%RARI after exposure of isolates to -5°C for 5 days (Cross 7). The black bars

xi

represent the parental isolates and the white bars indicate thermal sensitive (Pi 95-3)
and insensitive (Pi 02-007) standards, respectively; Pi 02-007 and Pi 95-3 are

parents in this cross. %RARI values followed by the same letter are not significantly
different at p = 0.1 (Tukey Multiple Comparison); LSDOJO indicated by bar. ....... 117

Figure 4.9 Differences in thermal tolerance of progeny isolates from mating of thermally
sensitive parental isolates of Phytophthora infestans Pi 4-19 (Al, thermally
sensitive) x Pi Atlantic 2N (A2, thermally sensitive). Growth was measured as
%RARI after exposure of isolates to -5°C for 5 days (Cross 9). The black bars
represent the parental isolates and the white bars indicate thermal sensitive (Pi 95-3)
and insensitive (Pi 02-007) standards, respectively. %RARI values followed by the
same letter are not significantly different at p = 0.1 (Tukey Multiple Comparison);
LSDOJO indicated by bar .......................................................................................... l 17

Figure 5.1 Late Blight Development in FL1879 tuber tissue caused by Phytophthora
infestans (isolate Pi 5-16), incubated at at 10°C for 30 days. ................................. 129

Images in this thesis are presented in color

xii

1 Literature review

1.1 Introduction
Phytophthora infestans, the causal agent of late blight remains a major threat to potato

production in the US. This is particularly true in Michigan where conditions are
typically ideal for its spread, with periods of high humidity, frequent rainfall and
moderate temperatures (Kudwa, 1999). Michigan is a major producer of potatoes in the
US. In 1999, 18,000 ha of potatoes were planted in Michigan, of which 82% were round
white cultivars used for table production or potato chipping. Currently the majority of
the commercially available cultivars are susceptible to late blight and control measures
can cost over $287.8 million, or $507 per hectare (Guenthner et al., 2001). Potato late
blight is notoriously difficult to manage resulting in tens of billions of dollars of losses

annually (Erwin and Ribeiro, 1996).

1.2 Host
The potato is believed to have originated in the Andes mountains of Peru where it was

first noted to be a major food source for the Incas around 400 BC (Schumann, 1991).
The potato tubers grow underground and originate from the swollen, underground stems
of the plant. It is a complete food that is rich in carbohydrates, proteins and vitamins. In
the 16th century, potatoes were brought to Europe by Spanish explorers who traveled to
South America in search of treasure. On their return trip home they brought potatoes
with them (Schumann, 1991). The potato was introduced to Spain around 1570 and from
there was distributed to Europe and England. The potato became a staple food crop in
Europe during the 17th and 18th century because it was nutritious, easy to grow, and a

large yield could be obtained from a relatively small plot of land. They were first

introduced into North America in the 17th century when European settlers brought potato
tubers back to the New World. Potatoes have become a staple food crop in Europe. By
the 1800’s the Irish were totally dependent upon the potato for their food. Unfortunately,

it was this dependence that led to the famine that occurred in the mid 1850’s.

1.3 Pathogen
The pathogen was first described and named by Anton de Bary in 1861 (De Bary, 1876)

as the cause of potato late blight. One of the main epidemics caused by Phytophthora
infestans was the Irish potato famine of the 1840’s which killed over 1.5 million people
and almost the same numbers of people migrated from Europe to the United States and
other parts of the world as a result of severe failure of the primary crop (Bourke, 1964).
Late blight is readily transmitted from seed tubers or volunteer tubers in the fields or
from discarded cull-piles. P. infestans survives in tubers as mycelium stored at about 3°C
and produces spores on the tuber surface when temperature increases in storage or in soil.
Optimal temperature for the grth of mycelium and spore production in tubers is 10°C.
Distribution of P. infestans varies worldwide. Phytophthora infestans is a diploid,
heterothallic organism believed to have originated and evolved in the central highlands of
Mexico (Niederhauser, 1991). Until the early 1980’s, Al was the only mating type found
in most of the world, but in Central Mexico both A1 and A2 isolates coexisted in a 50:50
ratio and later spread world-wide. In the US, until the mid 1990s, A1 mating type, US-l
isolates were predominant. The A2 mating type was likely introduced to the United
States and Canada in the early 19908. The US-8 (A2) genotype is widespread in the
United States (Fry 1997). The more aggressive nature of US-8 genotype made it the most

common genotype, surpassing US-l. The different genotypes prevalent in North America

are US-l, US-6, US-7/l6, US—8, US-10, US-l 1, US-12, US-14, and US-17. Mating of P.
infestans threatens potato industries worldwide due to the possible emergence of

recombinant genotypes with increased aggressiveness and wider virulence.

The probability that the disease will emerge from an infected tuber as a seed or from a
cull pile is difficult to estimate because several factors (e.g. temperature) can influence,
survival of mycelium and oospores. Oospores of oomycetous fungi are endogenously
dormant, highly effective survival structures that allow Phytophthora spp. to survive
adverse environmental conditions for relatively long periods of time in plant debris and in
soil (Madden et al., 1991). The production of oospores by P. infestans in the field and
their survival under natural conditions has been intensively studied since the introduction
of A2 mating type isolates and the emergence and spread of new genotypes in North

America.

1.4 Taxonomy
Phytophthora species are classified taxonomically in the kingdom Chromista, phylum

Oomycota, order Peronosporales, family Peronosporaceae and genus Phytophthora
(Birch, 2001). Oomycetes have often been confused by taxonomists with the true fungi
such as ascomycetes and basidiomycetes, because of the filamentous growth.
Phytophthora spp. differ from true fungi because the cell walls are composed of [3— 1, 3
glucan polymers and cellulose rather than mostly chitin. Studies of phylogeny based on
sequencing of the small subunit ribosomal DNA (Forster et al., 1990) suggest the class
shares little taxonomic affinity with filamentous fungi and should be classified instead

with the chrysophytes, diatoms and brown algae (J udelson, 1997).

1.5 Host range
Pathogenesis, which involves asexual growth, displays a high degree of biotrophy and

host specificity, with successful infections mainly limited to potato, tomato, and several
less well known Solanaceous species although more distantly related species such as
petunia may also be hosts (Erwin and Ribiero, 1996). The disease characteristics of P.
infestans are therefore more reminiscent of the downy mildews than most other
Phytophthora species, which are necrotrophs and often exhibit broad host ranges (Erwin

and Ribiero, 1996).

1.6 Disease cycle
Primary infection often occurs after emergence of the pathogen in the spring. Epidemics

are usually initiated by the dispersal of sporangia and zoospores. The source of primary
inoculum has been widely debated. However, most studies show that cull piles are the
major cause of primary infection (Bonde and Schultz, 1943, Zwankhuizen et al., 1998,
Kirk 2003a). Sources of early infection in potato fields have been traced to cull piles,
infected seed tubers and volunteer plants and infested organic potato fields were also

found to be a source of mid-season inoculum in Netherlands (Zwankhuizen et al., 1998).

P. infestans is an aerial pathogen capable of infecting all parts of the plant with the
exception of the roots (F ehrmann and Dimond, 1967). Sporangia are formed on aerial
portions of infected plants and transferred to uninfected plants where direct germination
may occur from a germ tube or by indirect germination by producing zoospores that
swim to an infection site (J udelson, 1997). The germ tube then differentiates into an

appressorium that produces narrow hyphae also called penetration pegs and degrading

 

Figure 1.1 Reproduction structure of Phytophthora infestans: Sporangia and sporangla releasing
zoospores

 

Figure 1.2 Reproduction structure of Phytophthora infestans: Oospores with Antheridum and
oogonium

 

Figure 1.3 Life cycle of potato late blight causing pathogen, Phytophthora infestans. (© P.S.
Wharton)

enzymes that enable invasion of the epidermis of the plant. Once inside the plant cell, the
penetration pegs differentiate into primary infection hyphae, which then colonize host-
plant tissue. P. infestans has a well distinguished life cycle and infection process. It can
reproduce both asexually and sexually in plants and also in vitro. However, the most
predominant means is by asexual reproduction with spores surviving over winter in

infected plant material (Pethybridge, 1911).

1.6.1 Asexual reproduction
Asexual reproduction occurs via formation of sporangia. They are home on stalk-like sporangiophores

(Blackwell, 1949). Sporangia are lemon-shaped, semipapillate, tapered to the base and caducous. Average

size ranges from 36 x 22 um to 29 x 29 um and formation occurs at an optimum temperature of 18-22°C,

where relative humidity exceeds 90% (Erwin and Ribeiro, 1996). Sporangia produced on potato
plants can be dispersed over large distances by wind (Hirst and Stedman, 1960). In the
presence of free water and at temperatures below 18°C biflagellate zoospores produced
within sporangia emerge and can swim in leaf surface or soil water upon emergence to a
new infection site. Zoospores can move from cull piles to field plants in dense fog, rain
puddles, or wind-blown rain (Bonde and Stedman, 1943). The pathogen therefore favors
wet weather with high humidity and temperatures between 16 and 21°C for continual
sporangia] production and zoospore release. The main source of inoculum in an
asexually reproducing population is therefore by infected tubers from the previous
season, with survival possible from infected seed (Pethybridge, 1911), discarded tubers
present in cull piles (Bonde and Schultz, 1943) and from volunteers or groundkeepers

(Hirst and Stedman, 1960).

1.6.2 Sexual reproduction
P. infestans the causal agent of late blight in potatoes is a heterothallic oomycete, with

two mating types or compatibility types and is able to reproduce sexually. Mating types
differ in hormone production rather than in sexual form, with male and female
garnetangia (oogonia and antheridia) produced in response (J udelson, 1997). Sexuality is
independent of mating type with both mating types capable of producing either type of
sexual structure (Brasier, 1992), though isolates will preferentially form either oogonia or
antheridia depending on mating partner (Gallindo and Gallegly, 1960). The mechanism
by which mating type is determined appears to be governed by a single locus, where
heterozygosity results in the A1 mating type and homozygosity results in the A2 mating
type (Judelson et al., 1995). In this system an equal number of A1 and A2 progeny would
be expected from any mating; however, non-Mendelian segregation has been observed
(Judelson et al., 1995). As a result of sexual reproduction, where haploid nuclei from
oogonia and antheridia fuse, they produce diploid thick-walled oospores. Oospores are
spherical in shape and have a thick inner wall composed largely of B-l,3-glucans (Erwin
and Ribeiro, 1996). Oospores are highly effective survival structures that allow
Phytophthora spp. to survive adverse environmental conditions for relatively long
periods of time in plant debris and in soil (Madden et al., 1991). The optimum
temperature for oospore production is 10°C but can occur anywhere between 5 and 25°C
(Drenth et al., 1995). Under high humidity from 8 to 23°C (Cohen et al., 1997), oospores
can germinate and form one or more germ tubes which either commence mycelial growth
directly or produce a sporangium which will proceed to reproduce asexually (Erwin and

Ribeiro, 1996). Oospores of P. infestans have been found to survive in soil during a

winter season in Europe and oospores also have been reported to survive temperatures

between -80 and 35°C (Drenth et al., 1995).

P. infestans can also be homothallic. Oospore production as a result of ‘self-fertilization’
has been well documented, with the first report in pure culture as early as 1911 (Clinton,
1911). Such an occurrence may result from non-disjunction (F yfe and Shaw, 1992) or
the presence of extra copies of the mating type locus in one parent (J udelson, 1996) or by
autogamy (Smart et al., 1998) whereby both male and female sexual structures are
produced. It would appear though that the tendency for self-fertilization is isolated or
even genotype specific (Groves and Ristaino, 2000) with multiple studies suggesting self-
fertility is particularly common in one particular lineage named US-8 (Smart et al., 1998,
Groves, 2002), although reports from other genotypes are common e. g. from the UK
(Tantius et al., 1986) and Japan (Mosa et al., 1989). Oospores may act as a long-term
inoculum source with survival demonstrated for up to two years (Perches and Galindo,
1967). Additionally sexual recombination of genetic factors may generate genetically
more diverse progeny, which could be more pathogenic or aggressive than parental
isolates, leading to drastic changes in genetic structure of the population (Levin et al.,
2001). However, studies of populations where the Al and A2 mating types are known to
co—exist have demonstrated oospores are produced in only small numbers in the canopy
(Drenth et al., 1995). In most parental combinations, a characteristic mating region was
present at the interaction zone between two isolates, where oospores are formed. Self-
fertility could be due to the presence of an extra chromosome containing the mating type

locus (Mortimer et al., 1977). In a study of A1 and A2 mating types of P. infestans strains

from Japan, Mosa and colleagues reported that the A2 isolates could form oospores in
pure culture if the culture was grown in the dark for one month (Mosa et al., 1989). In a
study, Goodwin et al., (1994) observed that a P. infestans strain of the US-8 clonal

lineage was self-fertile in pure culture.

1.7 Non-chemical and chemical Control
For controlling late blight, adequate disease management strategies before, during and

after planting and use of appropriate potato cultivars containing some resistance to potato
blight are required (Garrett and Dendy, 2001). Cultural control usually refers to non-
chemical methods which decrease a population by reduction in survival, dispersal and
reproduction (Kirk et al., 2005). A mixture of non-chemical and chemical measures can
be incorporated into decision support systems (DSS), which integrate information of a
variety of factors to achieve better control and more appropriate fungicide application

with reduced input and longer time intervals between applications (Schepers, 2002).

For non-chemical control, good quality, preferably certified tubers should be used to
minimize infection fi'om planting of infected seed (Bumbawale, et al., 1989). Volunteer
plants should be destroyed by herbicide application or mechanical measures (Leach et al.,
1986) and un-harvested potatoes removed (Sato, 1980). Crop rotation and fallowing have
traditionally been used in potato production to control a variety of diseases and pests such
as the potato cyst nematode (Thurston, 1990). Intercropping with immune crop plants
can also decrease reproduction, usually by planting resistant tubers along with a
susceptible crop either as a mixture, or in blocks can greatly reduce late blight incidence

by presenting a barrier to spread of inoculum, particularly in areas where sporangial

10

levels are relatively low (Garrett and Mundt, 2000). In addition over-fertilization and
early nitrogen application should be avoided with multiple studies observing increasing

infection of P. infestans under such conditions (Phukan, 1993).

For chemical control there are three types of fungicides used. They are protectants,
systemic and translaminar fungicides. Protectants or contact fungicides such as
chlorothalonil, mancozeb, maneb, fluazinam and the copper-based compounds act by
direct contact with fungi through interference with several chemical processes e.g. cell
wall disruption (Kuhn et al., 1991). Both translaminar and systemic fungicides are
single-site inhibitors and are therefore at high risk for resistance development (Gisi et al.,

2002), although as of yet only metalaxyl resistance has been documented in P. infestans.

1.8 Breeding for resistance
Although the production of late blight resistant cultivars is a priority for potato-breeding

programs, no commercial cultivars adapted to North American growing conditions with
good foliar resistance to modern genotypes of P. infestans have been adopted (Douches et
al., 1997). Only a small number of cultivars with some level of general resistance
survived the epidemics of the 1840’s leading to a narrow genetic base for modern potato
breeding (Umaerus et al., 1983). Most potato cultivars grown prior to the epidemics of
the 1840’s were highly susceptible to P. infestans and were derived mainly from Chilean
Solanum tuberosum Group (Gp) Tuberosum with contributions from Gp Andigena
(Pavek et al., 2001). From 1851 to 1910, 380 new cultivars were developed based on
general field resistance which during early potato breeding was the only type available to
the breeder (Stuart, 1905; Davidson, 1928), although none were more resistant than the

traditional ‘Champion’ a cultivar characterized as resistant even up until 1950 (Akeley,

11

1966). Many species have since been used, but the closely related S. demissum has been
by far the most important source particularly during the decades of 1930-1960 along with
the discovery of specific resistance (Salaman, 1929), with many cultivars incorporating
specific R genes into new cultivars such as Pentland Dell in the UK, released in 1961 and
containing three R genes for specific resistance. A cultivar bred in Michigan named
J acqueline—Lee with high foliar resistance to P. infestans has also shown high agronomic
potential due to high yield, excellent cooking qualities and attractive appearance with

bright golden skin, yellow flesh and oval shape (Douches et al., 2001).

1.9 Characterization techniques
Phenotypic and genotypic characterizations of the pathogen are necessary to understand

the pathogen and its population dynamics. There is no single system that can adequately
describe all variation present. There are a range of markers that are applied for
characterizing P. infestans including simple mating type reaction to sophisticated nuclear
DNA analysis. The following section summarizes a few markers that were used for

characterizing P. infestans in this study.

1.10 Phenotypic markers
Phenotypic markers were the first tests used to characterize P. infestans before more

specific genetic markers became prevalent. Phenotypic markers are used to describe

morphological characteristics of the pathogen.

l2

1.10.1 Mating type
Mating type is one of the easiest techniques used to differentiate between two isolates and

is determined when oospores are produced when a tester isolate is paired with an
unknown isolate on media plates. Oospores may be produced by self mating and are
termed “self-oospores”. If the oospores result from apomixis, then oospores contribute
nothing to variability in pathogen population but selfed oospores resulting from meiosis
and recombination are a potential source of variation in this species (Shattock et al.,
1985). The role of selfed oospores is unknown, but they create a mechanism for genetic
variability (Shattock et al., 1985). The genetic basis of sexual reproduction is unclear
despite much investigation. It would appear mating type is controlled by a single locus
and subject to non-Mendelian segregation (Gallindo and Gallegly, 1960; Judelson et al.,
1995). Although it has been proposed mating type is controlled by nuclear genes, a study
of Phytophthora parasitica and Phytophthora capsici supports the view that mating type
in Phytophthora spp. is under control of mitochondrial gene (Gu and Ko, 2005). Mating
type differences can also be detected due to differences in DNA polymorphisms, as
demonstrated by studies using enzyme restriction of amplified fragment length
polymorphisms (AFLP; Kim and Lees, 2002) and amplification of a sequence
characterized amplified region (SCAR) marker (Xuan-Zhe et al., 2006). These

techniques are complicated and time-consuming.

1.10.2 Effects of temperature on the disease
Van Everdingen’s conditions (Dutch rules) for causing an outbreak of potato late blight

epidemics are as follows (Beaumont, 1947): 1. Minimum temperature not less than 17°C

below the dew point. 2. Minimum temperature not less than 10°C. 3. Sunshine of the

13

following day not more than five hours. 4. Rainfall of the following day at least 0.1 cm.
The disease has been associated historically with “cool (near 20°C) wet weather”
(Harrison 1992). The effects of temperature, hmnidity, precipitation, wind and irradiation
on the development of late blight in potato foliage were described by Harrison (1992).
Most developmental structures of P. infestans cannot survive temperatures above 30°C
(Harrison, 1992), but the pathogen is more robust when inside plant tissue and has
survived temperatures of 40°C in stem tissue (Kable and Mackenzie, 1980).
Temperatures below an organism’s grth optimum will also cause reduced growth and
temperatures near 0°C can induce ice nucleation or cellular dehydration causing stress or
death (Pearce, 2001). Humidity, precipitation, wind, and irradiation are environmental
factors that have an effect on leaf wetness and therefore effect germination and infection

(Harrison, 1992).

Economic losses due to late blight result from foliar defoliation as well as tuber infection,
and severe storage losses can occur after tubers infected with P. infestans are held for
processing at temperatures in excess of 7°C (Kirk et al., 2001). Late blight epidemics are
associated with wet conditions and moderate temperatures (Harrison, 1995). There is a
wide variation in the temperature effect on different life stages of P. infestans (Harrison
1992). The optimal temperature for growth of hyphae in foliage is 15-20°C (Harrison
1992). Kadish and Cohen (1992) found that the rate of sporangial development
decreased at temperatures below 10°C. Temperature can influence spore germination,
mycelial growth rate, inoculum production and survival. Potato tubers for seed or table

stock are typically stored at 3°C, and processing tubers at 7 to 10°C (Kirk et al., 2001b).

14

Tubers stored at 10°C are at greater risk for development of late blight because P.
infestans has been shown to develop maximally at this temperature rather than at 3 or 7°C
(Kirk et al., 2001b). Tubers are usually held at 10°C for up to 6 months; therefore

colonization of individual tubers poses a greater risk than spread of disease in storage by

sporangia (Kirk et al., 2001b).

There is generally a greater incidence of soil freezing in Michigan during warm dry
winters due to recurring freeze-thaw episodes (Isard and Schaetzl, 1998). Isolates of P.
infestans vary in their ability to survive winter freezing (Shattock, 1976; Kadish and
Cohen, 1992; Kirk 2003b). More aggressive isolates of P. infestans are able to colonize
potato tissue faster and pose a larger threat during the growing season than their less
aggressive counterparts; however, the aggressive isolates may be at a selective

disadvantage when it comes to over wintering (Shattock, 1976).

Temperatures lower than 20°C, with the optimum between 12-13°C, favor indirect
germination of sporangia, formation of zoospores (5-10 per sporangium) and
temperatures higher than 20°C, with the optimum temperature at 24°C, favor direct
germination, i.e., one sporangium gives rise to a germ tube (Mizubuti and Fry, 1998).
Infection of susceptible plant tissue by P. infestans takes place most readily when
zoospores are formed, and this occurs only when there is a layer of moisture on the leaf.
Zoospores were found to be motile for 22 hours at 5-6°C, while at 24-25°C zoospores

were motile for only 19 minutes (Melhus, 1915a). In different genotypes of P. infestans,

15

temperature may affect sporangial germination and developmental rates which could

have important implications for epidemic development and disease management.

The over wintering of P. infestans is an important epidemiological factor in temperature
agroecosystems (De Bruyn, 1926). Mycelium of P. infestans can initiate crop infections
over successive years by over wintering as mycelium in infected potato tubers or within
cull piles or volunteer tubers. Tolerance of freezing temperatures by mycelium of P.
infestans may be an ecologically important survival mechanism and the increased
tolerance of US-8 and US-14 may explain their predominance in cooler climates such as
north-central United States (Kirk, 2003). It is difficult to estimate the probability that
infected potato stems or foliage will emerge from an infected tuber, as there are several
factors that could influence the fate of infected tuber, temperature being one of the most
important (Monteith, 1977, Kato et al., 1997). Perches and Galindo (1969) found soil to
be infectious 2-years after a late blight infection. Many investigators have used in vitro
and soil assays to study the optimal and lethal thermal temperatures for grth of
Phytophthora spp. (Bollen, 1985, Coelho et al., 2000). No other studies that systemically
examine the ability of P. infestans mycelium to survive at temperatures below 0°C exist,
although De Bruyn showed that a mycelial colony was capable of withstanding
temperatures as low as -25°C for up to 5-days in manure-based substrate and survival at
such low temperatures was enhanced in dried substrates (De Bruyn, 1926). A few P.
infestans isolates with different genotypes were killed by exposure to temperatures of

-20°C and -10°C for more than one hour (Kirk, 2003). Pathogen growth was better after a

sudden change in temperature, from cold to warm than after a gradual change. P.

16

infestans survived best when grown on manure because it encouraged good growth and

strong resistance to cold (De Bruyn, 1926).

Oospores were found to survive in soil for 5 to 7 months at temperatures between 0 and
20°C (Pittis and Shattock, 1994). Temperature is an underpinning factor in both host and
pathogen development and host by pathogen interactions. As the climate changes it is
likely that soil temperature will increase to close to freezing in Northern U.S. production
areas over winter. As it has been shown that P. infestans is tolerant to temperature close
to freezing (Kirk, 2003), this therefore creates a situation for both the host and the
pathogen to survive over winter. This could result in an increase in the emergence of
potato plants carrying initial inoculum that could start a seasonal epidemic. Mizubuti and
Fry (1998) studied the effect of temperature on sporangial germination from US-l, US-7,
US-8 isolates of P. infestans. Their results indicate that US-l isolates germinate better at
higher temperatures (IS-25°C) than US-7 or US-8, which germinated better at 10°C

(Mizubuti and Fry, 1998).

Oospores of other Phytophthora species have shown similar response to temperature.
Oospores of P. cactorum (homothallic) and P. cinnamomi (heterothallic) were not viable
after exposure at 45°C for 30 min while oospores of a heat resistant strain of P.

megasperma (homothallic) survived this treatment Juarez-Palacios et al., 1991).

1.10.3 Fungicide resistance to metalaxyl as a phenotypic marker
The problems caused by potato late blight were exacerbated by the development of

resistance to the most widely used group of systemic fungicides, the phenylamides, and

17

the appearance of new, highly aggressive strains of P. infestans (Gisi and Cohen, 1995).
Phenylarnides were the first compounds to be systemically active against oomycetes, and
the development of resistance has been a major factor in the contemporary severity of
potato late blight (Gisi and Cohen, 1995). The phenylamide metalaxyl (methyl-N- (2-
furoyl)-N-(2,6-xylyl)-DL-alaninate; Ridomil®, Syngenta, Basle, Switzerland) was
introduced by Ciba-Geigy in 1977 (Urech et al., 1977) for control of P. infestans because
of the high protective, curative and eradicative activity of the firngicide (Schwinn and
Staub, 1987). Currently, the predominant isolates of P. infestans in North America are
resistant to metalaxyl. Resistance to metalaxyl is now widespread globally with most
populations outside North America containing both sensitive and resistant isolates (Deahl
et al., 1993; Koh et al., 1994). Metalaxyl—resistant isolates have been shown to infect
more tubers, but tubers infected with metalaxyl-sensitive isolates were more likely to
produce infected plants because they were not completely destroyed by the disease
(Walker and Cooke, 1990). The genetic control of metalaxyl sensitivity is unclear and
multiple genes are likely involved (Shattock, 1988; Shaw, 1991; Lee et al., 1999). If as
suggested by Shattock, (1988) resistance is controlled by a single locus exhibiting
incomplete dominance, heterozygous isolates with both resistant and sensitive alleles
would exhibit intermediate resistance. However backcrosses have not segregated as
expected by this model (Shattock, 1988) and the precise mechanism of inheritance is
unknown. Since resistance is a relatively stable and heritable trait the method can be used

to characterize individual isolates within a population (Forbes et al., 1998).

18

1.10.4 The hypersensitive response
Disease resistance processes in plants are diverse, occurring at the sub-specific or varietal

level (host specific resistance) or at the species or genus level (nonhost resistance). The
hypersensitive response (HR) of plants is often associated with disease resistance (Dangl,
et al., 1996). The HR occurs as a rapid, localized necrosis, a form of programmed cell
death. Early infection by P. infestans is similar in both susceptible and resistant hosts.
Typically it starts when zoospores encyst and germinate on root or leaf surfaces. They
penetrate the epidermal cell by germ tubes and subsequent formation of infection
vesicles. However unlike in sensitive plants, feeding structures known as haustoria are
formed expand from the site of penetration to neighboring cells through the intercellular
space. In resistant plants the major defense reaction is the hypersensitive response (HR).
The extent and timing of the reaction will depend on the interaction of pathogen and plant
genotype and ranges from death of only a few cells with no macroscopic symptoms i.e. in
non-host interactions, to potato cultivars containing R genes or displaying high general
resistance where a group of cells are involved with later retardation of the pathogen and
appearance of macroscopic brownish-black spots (Kamoun et al., 1999). Although the
HR is most often associated with expression of R gene resistance, the response has been
associated with all forms of resistance including partial resistance, although in these cases
the response of the host can be ineffective, with numerous hyphae escaping where the
pathogen remains ahead of the response known as trailing HR (Kamoun et al., 1999).
This would suggest recognition occurs independently of the type of resistance

(V leeshouwers et al., 2000).

19

Elicitins (extracellular protein elicitors), have also been identified in P. infestans which
may play a role in host recognition (Kamoun et al., 1997). It has been suggested the HR
reaction follows recognition by the host of signal molecules known as elicitors, encoded
by avirulence genes in the pathogen (Staskawicz et al., 1995). Signal transduction may
be associated with cytoplasmic aggregation as observed by acceleration of protoplasmic
strand production in incompatible reactions (Tomiyama, 1956). Cytoplasmic aggregation
has been connected with actin filament association, which when repressed inhibits
accumulation of the phytoalexin rishitin, so suggesting the cytoskeleton is of importance

in plant defense responses (F uruse et a1, 1999).

Resistance has also been associated with induced deposition of structural barriers such as
callose and extracellular globules containing phenolic compounds which may function in
cell wall strengthening (V leeshouwers et al., 2004) and with a rapid increase in other
compounds such as phytoalexins and glycoalkaloids (Andreu et al., 2001). Other
observed reactions include generation of superoxide anions (Doke et al., 1991), an
increase in chitinase and beta-1,3-g1ucanase and accumulation of pathogenesis related

proteins (Taylor et al., 1990).

1.10.5 Virulence phenotype
Even though R genes are thought to be ineffective in the field over long periods of time,

there are plausible hypotheses that suggest these genes could mediate durable resistance
(Karnoun et al., 1997). The term ‘virulence’ of P. infestans has been used to refer to
compatibility with single R genes introgressed into the host from wild Solanum species

(primarily Solanum demissum) and corresponding virulence genes in the pathogen

20

(Malcohnson and Black, 1966). This gene for gene recognition system was first
proposed by Black et al., (1953) and to date eleven R genes have been elucidated,
although it has been acknowledged more may exist (Trognitz, 1998). Compatibility with
these known R genes will determine an isolates ‘race’ or ‘virulence’ phenotype (Van der
Plank, 1963) and is assessed by inoculating eleven genetically defined ‘differential’
potato genotypes, each possessing a single R gene with subsequent recording of
compatibility (Cooke et al., 2004). The ‘guard hypothesis’ suggests that normal
resistance occurs when a complex of the plant virulence target and the pathogen Avr gene
product is recognized by a R gene product, with mutation to a state which avoids host
recognition causing virulence to the gene (Dangl and Jones, 2001). Populations have
been monitored for virulence phenotype since break-down of resistance was first
recorded in commercial cultivars incorporating such R genes (Malcolmson, 1969;
Shattock et al., 1977). Multiple studies have demonstrated an increase in virulence
complexity in recent years along with increasing sexual recombination (Drenth et al.,
1994; Cohen, 2002), with virulence against all eleven R genes reported (Goodwin et al.,
1995b). Since it is a long and laborious method of population characterization, virulence
testing has been criticized due to inaccuracies between and within laboratories (Stewart,
1990). Improved understanding of the molecular nature of R genes, new methods, such as
PCR-based approaches to the isolate analogs of R genes from resistant plants, in
combination with reverse genetics and complementation in to the host crop, should

facilitate the cloning and identification of novel R genes 0(amoun et al, 1997).

21

1.10.6 Pathogenicity testing
Pathogen response to resistance in tubers may be genotype specific (Kirk et al., 2001a).

The susceptibility of potato tubers is extremely variable and depends on the interaction
between the cultivar and the isolate of P. infestans involved in addition to environmental
properties and tuber age. Infection of tubers is similar to that of foliage with germ-tubes
penetrating at lenticels, eyes, stolons, buds, wounds or any site incompletely suberized
(Bain and Moller, 1999). Dowley and O’Sullivan (1991) found that healthy tubers
became infected with P. infestans from sporangia after being stored with diseased tubers
at 20°C for up to 35 days. Several studies of blighted tubers in storage have
demonstrated P. infestans spreads fastest and sporulates at highest levels at around 10°C
(Kadish and Cohen, 1992; Walker and Cooke, 1988). Research has shown that the newer
US-8 genotype is more aggressive in potato tubers than the US-l genotype, causing more
surface necrosis and deeper lesions (Medina et al., 1999). Spread of disease between
whole tubers in storage is insignificant because of the barrier of intact skin and because
tuber surfaces are usually dry, inhibiting production of sporangia (Lambert et al., 1998).
A study by Lambert reinforced the results from Medina and showed that some US-6 and
US-7 isolates of P. infestans also cause faster infection spread than US-l (Lambert and
Currier, 1997). Potato tubers are conventionally held in storage at 10°C for up to six
months to enhance sugar accumulation; spread of disease is enhanced under these
conditions (Burton, 1992). Several studies of blighted tubers in storage have
demonstrated P. infestans spreads fastest and sporulates at highest levels at around 10°C

(Walker and Cooke, 1988; Kadish and Cohen, 1992).

22

Gees and Hohl (1987) suggested that the mechanisms by which late blight development
is slowed are different between tubers and foliage, and they may be related to histological
and/or cytological variations in tuber or canopy, biochemical defense responses or a
combination of these and other factors. Studies of tuber tissue degradation in the whole
tubers due to late blight infection have concentrated on the pathogenicity of biotypes as

affected by tuber age and duration of storage periods (Grinberger et al., 1995).

Infected tubers normally carries mycelium of the pathogen and when such tubers are
planted in soil the fungus may produce sporangia through buds and lenticels, which may
attack stems emerging from the infected tuber. A study from Grinberger et al., (1995),
has shown that no oospores were formed in young tubers, reaffirming that some physical
and physiological-biochemical factors of the tubers prevents the fungus from mating and
making oospores. Oospore formation in tuber tissue is very limited but increases with
tuber aging which may be related also to pathogen aging. The number of oospores
produced in tuber tissue was lower compared with that in leaf tissue regardless of the
origin of isolates used (Levin et al., 2001). Viability of oospores as measured by
plasmolysis declined slightly over a period of 18 months whether they were stored in

water at 4°C, in soil under natural field conditions (Mayton et al., 2000).

1.11 Genetic markers
Although the use of phenotypic or biologically significant markers for distinguishing

between isolates and assessing diversity within populations has been ongoing over the
past number of decades and remains highly useful, it is only with advances in genomics

(Shaw, 1991) that more powerful discriminatory tests capable of tracking isolates within

23

a population have become available. Such techniques have become fundamental for
population studies and investigation of mechanisms such as selection, migration and

competition.

1.11.1 Isozyme analysis
Characterization of allozyme loci were the first unambiguous genetic markers for

investigation of populations of P. infestans and have been important for the
characterization and tracking of isolates as well as other investigations including the
study of diploidy (Tooley et al., 1985) and identification of sexually reproduced isolates
(Shattock et al., 1986). GP] isozyme pattern is the most widely used isozyme approach
to characterize isolates of P. infestans, in which GPI isozymes occur in multiple
combinations, as shown by starch gel and cellulose-acetate electrophoresis analyses
(Goodwin et al., 1995a). Isozymes are defined as multiple molecular forms of a single
enzyme and are usually differentiated only by variation in amino acid composition due to
differences in the nucleotide sequence of DNA coding for that protein (Smithies, 1955).
However, this will only occur where there is a difference in net charge (usually due to
large differences in size and shape or substitution of a basic for an acidic amino acid), or
some other change affecting electrophoretic mobility such as a change in tertiary
structure (Micales and Bond, 1995). It has been suggested only one third of all single
amino acid substitutions are electrophoretically detectable and thus use of isozyme
analysis is a very conservative estimate of genetic variability (Micales and Bond, 1995).
As a result gel bands are described as an enzyme phenotype rather than genotype (Hubby
and Lewontin, 1966). The usefulness of isozyme analysis depends on the assumption of

heritability and co-dominant expression i.e. all alleles at any one locus will be expressed

24

(Shaw, 1965). Polymorphic loci encoding glucose-6-phosphate isomerase (GPI),
Peptidase (PEP), malic enzyme (ME), and xanthine dehydrogenase (EH) have been
successfully utilized to unequivocally differentiate multiple individuals within a sexual
population (Tooley et al., 1985) but it is in the GP] and PEP loci where polymorphisms

are most commonly observed (Ospina-Giraldo et al., 2003).

Previous studies have revealed that at least six genotypes can be found in sexual
populations of P. infestans (Tooley et al., 1985), although many more are possible
(Grunwald et al., 2001). These genotypes are known as 83/100, 86/86, 86/ 100, 100/ 100,
100/ 122 and 122/122. These alleles are named according to the electrophoretic mobility
of the isozyme it encodes, in relation to the most common one, which is 100. Each allele
codes for a structurally distinct polypeptide chain with a monomeric enzyme consisting
of only one and a multimeric enzyme with more than one chain and will usually be either
dimeric (two chains) or tetrameric (four chains). Over 50 different isozyme systems have
been tested for P. infestans (Spielman et al., 1990) with the first characterization made by
Tooley et al. (1985) using starch gel electrophoresis. A genetic locus within a population
is described as monomorphic where only one allele is present or polymorphic where
more than one allele is present, with a polymorphic isozyme known as an allozyme. The
number of alleles will depend on ploidy number, genetic make-up (i.e. homozygous or
heterozygous) and nuclear condition (i.e. monokaryotic or dikaryotic), with complexity
of resultant banding patterns varying accordingly (Micales and Bond, 1995). Differences
in form may be distinguished either by number of bands, with complex patterns emerging

from multimeric heterozygous isoforms due to formation of intermediate ‘hybrid’ bands,

25

or by intensity of bands, with differences in ploidy levels distinguishable due to the

additive effects of subunits (Danzman and Bogart, 1982).

Until the late-1980’s, the populations in North America was mostly represented by single
clonal lineages known as US-6, which carried the 100/ 100 genotype, and US-l, which
carried the 86/ 100 genotype (Goodwin et al., 1994). In the early 1990’s two additional
lineages, US-7 and US-8 (GPI genotypes 100/111 and 100/ 111/ 122, respectively) were
also found in the United States and Canada (Goodwin et al., 1995b). In addition GPI
genotype 100/ 122, found in US-14 and US-17, fi'equently observed between 1995 and
1997, and significantly decreased by 1998 (Daayf and Platt, 2000). Much in vitro
evidence suggests new genotypes possess higher aggressiveness and have more efficient
colonization of potato than the ‘old’ US-l genotype (Goodwin et al., 1995; Miller et al.,
1998; Flier et al., 1998). However as new genotypes such as US-8 and many of the
European genotypes are metalaxyl resistant this in itself may have allowed a competitive
advantage against the phenylamide sensitive US-l (Goodwin et al., 1996; Cooke et al,
2006). It is generally accepted that metalaxyl resistance in the US. is the result of
migration rather than mutation and subsequent selection (Goodwin, 1997). Although
most of these genotypic changes have been attributed to migration rather than mutation
(Goodwin et al., 1994), the widespread occurrence of the newest genotypes might
suggest a selective advantage for such genotypes and perhaps, a role for sexual

recombination (Ospina-Giraldo et al., 2003).

26

1.11.2 Nuclear DNA polymorphisms
Alternate forms of a chromosomal locus, either differing in nucleotide sequence or by

variable numbers of repeated nucleotide units are known as DNA polymorphisms.
Differences can arise due to insertions, deletions and substitution of individual
nucleotides, or by chromosomal inversions or translocations (Goodwin, 1997).

Polymorphism can be detected using a range of nuclear DNA markers.

1.11.2.1 Simple Sequence Repeats (SSR’s)
SSR’s are tandemly repeated motifs of one to six bases found in the nuclear genomes of

all eukaryotes tested and are often abundant and evenly dispersed (Tautz & Renz, 1984;
Lagercrantz et al., 1993). These differences between numbers of repetitive units are
known as satellites, minisatellites or microsatellites. Microsatellite sequences are usually
characterized by a high degree of length polymorphism, and are ideal single-locus co-
dominant markers for genetic studies (Lees et al., 2006). Once SSR’s have been
identified through DNA sequencing, appropriate primers flanking the microsatellite can
be developed to amplify the region through PCR (Cooke et al., 2004). DNA fiagrnents
can then be separated on a gel under electrophoretic conditions and sequenced for
number of repeats. Alleles (usually two per locus) appear either as one peak where the

locus is heterozygous or two where heterozygous (Lees et al., 2006).

Although a recent development, with few studies incorporating its use, fifteen
polymorphic SSR markers have now been published for P. infestans (Knapova and Gisi,
2002; Lees et al., 2006), although some show limited variation, with 68 different

genotypes and an average of 3.9 alleles per locus detected amongst fourteen markers

27

tested on a subset of 90 isolates (Lees et al., 2006). SSR analysis is highly specific
usually focusing on specific loci allowing for easier interpretation (comparable with
allozyme genotype) unlike other DNA based techniques, but is also highly powerful,
unambiguously defining length variation (Cooke et al., 2004). There is some suggestion
though that high mutation rate may decrease heritability and therefore usefulness for
certain functions such as tracking within populations (Weber and Wong, 1993). As
commented by Cooke and Lees (2004), a greater emphasis must be put on studies of P.
infestans biology, these studies should include improvements in our understanding of the
relative contributions and rates of mutation, recombination, natural selection, gene flow,
random genetic drift and migration (Burdon and Silk, 1997) to the generation and
maintenance of variation in populations (relative importance of asexual v. sexual

reproduction; Lees etal., 2006).

1.11.2.2 Chromosome ploidy levels
Ploidy is the number of homologous sets of chromosomes in a biological cell. Laser flow

cytometry and RFLP (Restriction Fragment Length Polymorphism) analyses allows
quantitative measurement of nuclear DNA content. Substantial variation exists within
populations of P. infestans with diploid, triploid, tetraploid and aneuploid isolates
identified through the use of chromosome counting, cytophotometric counting and
inheritance studies (Sansome and Brasier, 1974; Sansome, 1977; Tooley and Therrien,
1991). Early cytogenetic studies in P. infestans identified roughly 9 to 12 chromosomes
in some isolates, but twice that amount in others (Sansome and Brasier, 1973; Sansome,
1977). These observations increase confusion in deciding if the genus is naturally haploid

or diploid. It has been suggested that polyploid isolates may have arisen through

28

selection of autotetraploid nuclei, produced occasionally during rapid nuclear division
(Sansome, 1977). These isolates may have been selected through better adaptation to
environmental conditions or by higher fitness due to possession of a higher number of
virulence genes in comparison to diploids (Sansome, 1977). It would appear fi‘om in
vitro studies that mating between ploidy levels is viable, although with variable
chromosome content (Whittaker, et al., 1991). However, studies of natural populations
would suggest such matings are rare (Tooley and Thierren, 1987; Spielrnan et al., 1991)
with an intraspecific post-zygotic barrier to mating hypothesized (Miller and Johnson,
2000). Difference in ploidy between mating partners is not a major barrier to sexual
fertility, although such crosses may yield offspring with varied chromosome content
(Whittaker et al., 1991). This technique may not be feasible for the larger sized P.
infestans chromosomes. Variable ploidy levels could lead to irregularities in segregation

ratios and confusion in inheritance studies.

1.12 Objectives
Although it is highly likely that P. infestans can mutate asexually the rate of production

of distinct phenotypes can be accelerated by the use of enforced sexual recombination
utilizing the different mating types. To test this hypothesis several phenotypic and
genotypic characters were selected some with immediate relevance in epidemiology and
disease management including thermal tolerance, metalaxyl sensitivity, mating type and
aggressiveness and further investigated in terms of genotypic markers such as isozyme

analysis, microsatellite and ploidy analyses.

29

Evaluate survival potential of mycelium of different genotype isolates of P.
infestans exposed to temperature at -5°C for 5 days in vitro using a digital
imaging technique.

Carry out crosses between P. infestans isolates (A1 and A2 mating types) that are
highly variable for temperature tolerance, to determine if temperature tolerance is
inherited or occurs as a result of physiological adaptation of different genotypes
of P. infestans.

The progeny isolates from these crosses will be assessed for temperature tolerance
at -5°C and data will be analyzed to estimate the inheritance of this trait.

Evaluate other phenotypic characteristics of progeny such as mating type,
isozyme analysis, metalaxyl sensitivity and virulence assessment.

Evaluate the progeny using Microsatellite markers to confirm whether they are
selfs or hybrid progeny.

Evaluate if the progeny isolates are pathogenic in potato tubers.

30

2 Genotypic characteristics of Phytophthora infestans from mating:
Determination of hybridity or selling by isozyme analysis and
microsatellite markers

2.1 Introduction
The Chromistan Oomycetous fungus Phytophthora infestans, the causal agent of potato

late blight remains poorly understood relative to fungal pathogens. Since Oomycetes do
not have a close taxonomic affinity with genetically well-characterized fungi such as
Ascomycetes and Basidiomycetes, it is likely that study of P. infestans will yield novel
biological findings (Judelson, 1997). In order to assess and follow selection within an
epidemic it is important to possess the ability to identify and assess both gene and
genotypic diversity. P. infestans causes the devastating late blight disease of potato and
tomato. Re-emergence of late blight during the late twentieth century ensured that this
pathogen continues to be an important threat to agriculture (Fry and Goodwin, 1997).
P. infestans is an obligate biotroph which usually reproduces asexually, therefore the
probability of extinction overwinter is high in most commercial systems (Milgroom,
1996) unless the pathogen overwinters as mycelium in seed, culled or volunteer tubers
(Kirk 2001, 2003a, 2003b). As a result gene and genotypic diversity within local
populations can vary considerably, both temporally and spatially as new sources of
inoculum may be introduced sporadically (Fry and Goodwin, 1997; Day and Shattock,
1997). It is important to have the capability to assess both gene and genotypic diversity to
monitor selection during an epidemic. Gene diversity refers to the frequency of alleles at
individual loci in a population while genotypic diversity refers to the frequency of
genetically distinct individuals within a population (McDonald and Linde, 2002).
Assessment of such diversity is dependent on the availability and potential of precise and

abundant genetic and phenotypic markers (Fry et al., 1992). Cellulose-acetate

31

electrophoresis (CAE) provides an excellent resolution of allozyme genotypes of P.
infestans and has many advantages over traditional starch gel analyses. CAE is faster,
more reproducible, requires less amount of enzyme for an analysis, and can be useful as a

diagnostic tool in field situations.

Allozyme genotyping by cellulose acetate electrophoresis, first used for this pathogen by
Tooley et al., (1985), is one of the easiest and cheapest methods and has now become the
most commonly used technique to characterize populations (Goodwin et al., 1995;
Ospina-Giraldo and Jones, 2003). Polymorphic loci encoding glucose-6-phosphate
isomerase (GPI), Peptidase (PEP), malic enzyme (ME), and xanthine dehydrogenase
(XDH) have been successfully utilized to unequivocally differentiate multiple individuals
within a sexual population (Tooley et al., 1985) but it is in the GP] and PEP loci where
polymorphisms are most commonly observed (Ospina-Giraldo et al., 2003). Despite the
importance of GPI in P. infestans studies, the gene encoding this enzyme has not yet
been characterized (Ospina-Giraldo et al., 2003). Although allozyme polymorphisms are
used widely these markers have been criticized, because migration is expressed in
relative terms and the genetic change involved is largely unknown (Cooke and Lees,

2004).

Amplified Fragment Length Polymorphism (AF LP) and microsatellites or Simple
Sequence Repeats (SSR) markers are unambiguous nuclear tools considered to be highly
useful for population investigations (Knapova et al., 2002). In European studies using

AF LP markers, up to 50% of isolates are shown to have discriminatory patterns

32

(Knapova and Gisi, 2002; Cooke et al., 2003). However AFLP genotyping is also
laborious, time-consuming, and may produce excessive data difficult to interpret in
comparisons among strains (Cooke and Lees, 2004). Additionally AF LP markers are
dominant so that heterozygotes cannot be distinguished and therefore many markers are
required for thorough investigations (J orde et al., 1999). Although the supporting data
are limited, there has been speculation that P. infestans may contain a variable and plastic
genome. This has been proposed to explain the sectoring for traits such as growth rate,
colony morphology, avirulence, and pathogenic aggressiveness reported for certain single
cultures (Caten, 1971; Erwin et al., 1963; Jeffrey et al., 1962; Jinks and Grindle, 1963;
Abu el Samen et al., 2003). An increase in genetic variation at the molecular level and a
more complex structure were observed in new P. infestans populations in Europe (Drenth
et al., 1993b; Knapova and Gisi 2002). SSR’s have been used successfully in recent
investigations to characterize populations in France and Switzerland (Knapova et al.,
2002). SSRs are short fragments of DNA composed of tandemly repeated motifs of 1-6
base pairs, characterized by a high degree of length polymorphism (Lees et al., 2006).
SSR markers focus on individual loci and have been proven to be highly specific (Lees et
al., 2006). Furthermore due to their co-dominant nature homozygotes and heterozygotes
are easily distinguished, although the possible presence of null alleles at heterozygous

loci may be problematic (Knapova et al., 2002).

A genetic locus within a population is described as monomorphic where only one allele is

present or polymorphic where more than one allele is present, with a polymorphic

isozyme known as an allozyme. The number of alleles will depend on ploidy number,

33

genetic make-up (i.e. homozygous or heterozygous) and nuclear condition (i.e.
monokaryotic or heterokaryotic), with complexity of resultant banding patterns varying
accordingly (Micales and Bond, 1995). Differences in number of bands, with complex
banding patterns may result from multimeric heterozygous isoforms due to formation of
intermediate ‘hybrid’ bands, or by intensity of bands, with differences in ploidy levels
producing the additive effects of subunits (Danzman and Bogart, 1982). The chromosome
number of diploid nuclei is 8-12 and in tetraploid nuclei 14-20 (Whittaker et al., 1991).
Knowledge of ploidy is important both for population characterization and all types of
inheritance investigations, since variation can lead to irregular segregation ratios
(J udelson, 1997). Determination remains problematic however as chromosomes are too
small for accurate counting using conventional cytological techniques (Shaw and
Shattock, 1991). As commented by Cooke and Lees (2004), a greater emphasis must be
put on studies of P. infestans biology. These studies should include the relative
contributions and rates of mutation, recombination, natural selection, gene flow, random
genetic drift and migration (Burdon and Silk, 1997) to the generation and maintenance of
variation in populations (relative importance of asexual v. sexual reproduction; Lees et

aL,2006)

Oomycetes including P. infestans are recognized as diploid. However, P. infestans often
exists in populations that may be heteroploid or polyploid (Lee et al., 2004; Sansome,
1985; Tooley & Therrien, 1987, 1989). The genomic size of P. infestans is approximately
237 Mb (Tooley and Therrien, 1987) with at least 51% repeated DNA sequences

(J udelson and Randall, 1998). Two independent analyses indicate that the genome has

34

about 52% GC content. Microscopic analyses indicate that P. infestans has 8-10
chromosomes (Sansome and Brasier, 1973). Due to this considerable genomic size and
complexity, early P. infestans genomics efforts focused on cDNA sequencing and
generated collections of expressed sequence tags (ESTs) from a variety of developmental,
stress, and infection conditions (Kamoun et al., 1999 and Randall et al., 2005). Most of
the extra genome mass has recently been identified as "junk" DNA that is not transcribed
into ribosomes or transcribed and translated into proteins. The DNA content of nuclei of
various isolates of P. infestans and progeny from controlled matings and progeny from
asexual propagation by zoospore formation was assessed by laser flow cytometry.
Variation of chromosomal size among isolates was evaluated with southern

hybridizations, and some virulence genes were located on chromosome fragments.

Sexual crosses have been made in vitro for P. infestans to investigate inheritance of
several genotypic characteristics using allozyme markers (Spiehnan et al., 1990), or
related microsatellite markers (Knapova and Gisi 2002). The characteristic of mating type
is generally considered to be stable, heritable and easily detectable and is a commonly
used and important marker for characterizing and assessing the potential for sexual
reproduction within a population (Brasier, 1992). Although generally considered robust,
mutation, mitotic recombination and treatment with certain fungicides can result in
segregation of the opposite mating type (Smart et al., 1998; Groves, 2002). Additionally
self-fertility can occur amongst US lineages (specifically the US-7 and US-8 lineages),

although fewer oospores are produced than between opposite mating types (Smart et al.,

35

1998; Groves 2002). How far these factors may confound use of mating type as a marker

in selection studies is unknown.

The objective of this study was to determine whether the phenotypic character of
temperature tolerance was genetically inherited or occurred as a result of physiological
adaptation. P. infestans progeny isolates that were highly variable for temperature
tolerance generated from A1 and A2 mating types were evaluated and phenotypes
compared with inheritance of markers for mating type, isozyme analysis, microsatellites,

and characterization of nuclear conditions and ploidy levels.

2.2 Materials and methods

2.2.1 Selection of isolates
In this study, the procedure for selection of isolates to use as parents for crossing to

produce progeny isolates and determine the inheritance was described in chapter 4.

Selected isolates were shown in Table 2.1.

2.2.2 Isozyme analysis
Cellulose acetate electrophoresis (CAE) was used to determine the polymorphic allozyme

loci. All US parental isolates were heterozygous for the GP] (Glucose-6-phosphate
isomerase) allozyme genotype and as a result only this marker (and not PEP) was used
for isolate characterization in this study. GPI was conducted using the protocols of
Goodwin et al., (1995). Isolates used for isozyme analysis were 14 days old cultures,
grown on Rye agar (Appendix 1). Mycelium was scraped from the culture plates using a
rubber policeman and transferred to 1.5 m1 micro-centrifuge tubes and 150-u1 sterile

distilled water was added to each tube. Proteins were extracted by grinding the mycelium

36

in double distilled water using a hand held homogenizer for 1 minute. Tubes were
briefly vortexed and centrifuged at 13,000g for 10 minutes at 4°C. Supernatant was
decanted to leave a pellet and a residual volume of 100 pl. The supernatant containing
proteins was pipetted to a new tube, labeled and stored on ice or at -20°C if not used
immediately. Parent isolates were used as standards for comparison with progeny
isolates. Extracted protein was sufficiently stable for 3 —— 4 electrophoretic assays prior to

onset of enzyme degradation.

2.2.2.1 Cellulose acetate electrophoresis
10 X Tris Glycine (TG) buffer was prepared by adding 0.25 M (30g) Tris base and 0.192

M (144 g) Glycine to l L double distilled water, and adjusting the pH to 8.5 (by adding
either sodium hydroxide pellets or hydrochloric acid), using a pH meter (Orion PerpHect
Meter, Orion Research, INC., Beverly, MA 091915-6199, USA). 180 ml of IX TG
(100ml 10 X TG in 900ml sterile distilled water) was added to each side of the
electrophoresis chamber (Zip Zone® Electrophoresis Chamber; Helena Laboratories, PO
Box 752, Beaumont TX 77704-0752). Paper wicks (220 mm x 30 mm; Helena
Laboratories) were placed in contact with gel plate edge and bridging buffer tray over the
two rails separating the buffer trays for later contact with gel plates. Wicks were reused
until the buffer became contaminated. Cellulose acetate gel plates (94mm x 76 mm;
Titan® 1H; Helena Laboratories) were soaked for at least twenty minutes in appropriate 1
x TG electrode buffer. Plates were used immediately or were soaked for several days
before use in a refiigerator (4°C). Buffer was reused until cloudiness appeared. Gel
plates were blotted between sheets of blotting paper and fixed to the center of the

aligning base of the loading applicator (Super Z-12 Applicator System; Helena

37

Table 2.1 Phenotypic and genotypic characteristic of isolates of Phytophthora
infestans used in crosses.

 

 

Isolate of

Phytophthora Thermal GPI isozyme Mating Metalaxyl

infestans Tolerance‘l genotypeb Type° Responsed R gene phenotypee
Pi 41-02f T 100/ 100 A; R I,3,4,5,6,7,8,9,10,1 1
Pi 02-0073 T 100/1 1 1/122 A2 R 1,2,3,4,5,6,7,8,9,10,l 1
Pi Sl-3g T 100/111/122 A2 I l,2,3,4,5,6,7,8,9,10,11
Pi 98-1g I 100/122 A2 R l,4,5,9,10.l 1

Pi 62-02f T 100/ 100 Al I l,2,3,4,5,6,7,8,9,10,l 1
Pi 95-3g S 86/ 100 A; S 5

Pi Atlantic 2Ng S 100/111/122 A2 I l,3,4,5,6,7,8,9,10,l 1
Pi 4-19h S 100/100 A1 I l,3,4,5,6,7,8,9,10,11

 

8 Comparison of grth of isolates of cultures of Phytophthora infestans for 10 days at 18°C after
exposure at -5°C for 5 days measured by an image analysis technique comparing the growth of
exposed isolates to a positive control (exposed to 18°C for 5 days) or a negative non-inoculated
control with no growth. T= Tolerant; growth not significantly different fi'om the growth of the
positive control cultures of the same isolate; I= Intermediate; growth significantly less than that
of the growth of the positive control cultures of the same isolate but significantly greater than the
negative control; S= Sensitive; growth not significantly different from the negative control.

b Glucose -6- phosphate isomerase allozyme genotype; 86/ 100 (U 81), 100/ 100 (U S-1.7),
100/111/122 (U S-8), 100/ 122 (U S-l4); genotype as designated by Goodwin et al., (1995).

c Mating type determined by production of oospores when grown with isolates of P. Infestans of
known mating type.

d Sensitivity to the fungicide metalaxyl, R = resistant, I= intermediate, S = sensitive determined
by comparing ECso response of the isolates grown on media amended with different
concentrations of metalaxyl with ECso of known resistant and susceptible isolates.

e Virulence pathotype as defined by Muller and Black (1952).

f Isolates of P. infestans from Northern Ireland from collection of Dr. Louise Cooke, Plant
Pathology, Queens University, Northern Ireland.

3 Michigan isolates of P. infestans from collection of Dr. W. Kirk, Plant Pathology, Michigan
State University, East Lansing, MI, USA.

b A progeny isolate obtained from the 4th cross, used as a parent because of its thermal sensitive
phenotype (as defined above in a)

38

Laboratories). The cellulose acetate side was oriented so that it faced upwards, and the
bottom side was marked using a permanent marker, for identification. 10 ul of each
protein extract sample was added to a reservoir of the applicator system (Super Z-12
Applicator Systems; Helena Laboratories). The multi-well applicator was pressed into
the reservoirs three times. The sample was then transferred onto the surface of the
cellulose acetate plate by depressing the multi-well applicator for 10 seconds. Gels were
placed cellulose acetate side downwards onto the electrophoresis chamber in contact with
wicks and the sample side oriented towards the cathodal rail. Microscope cover slips
were placed on top of the gel to ensure contact to the wicks. Current was applied for 30
min at 200 V by using a power supply (E-C. Apparatus Corp., St. Peterburg, Fla., 33709,

USA).

2.2.2.2 Gel overlay preparation and staining
Genotypes of the isolates were determined by staining with agar overlays, prepared from

stock solutions of reagents (Sigma Aldrich Chemical Company, St. Louis, MO-63178).
GPI overlay contained 1.5 ml of Tris-HCl (0.05M, pH 8), 5 drops of Fructose-6-
phosphate (20 mg/ml in double distilled water, stored at -20°C), 1 ml of nicotinamide
adenine dinucleotide (3 mg/ml in double distilled water, stored at-20°C), 5 drops of
Methyl thiazole blue (10 mg/ml in double distilled water, stored at 4°C), 5 drops of
Phenazine methosulphate (2 mg/ml in double distilled water, stored at 4°C) and 3 ul of
Glucose-6-phosphate dehydrogenase (l U/ul, stored at 4°C). Photosensitive chemicals
were added last, prior to staining. Finally all the above reagents were mixed in a glass test
tube with 2 ml of pre-melted 1.6 % agar. This was poured over the cellulose side of the
cellulose acetate plate. Enzymatic activity stained the gel at the location of the allozyme

migration creating bands that started appearing in 1-2 minutes and were then scored.

39

Plates were washed in water to prevent over staining, blotted, and images of the gels were
recorded by scanning them facing down on a flat bed scanner (Epson Perfection 4870
PHOTO; Epson Inc. USA) and the scanned image was imported into Photoshop (Adobe

Inc.), and saved in the J PEG file format.

2.2.2.3 Scoring of hands
Genotypes were determined by comparing the bands of unknowns with those of known

standard isolates, including Pi 02-007 (US-8, GPI 100/111/122), Pi 41-02 (GPI 100/100),
Pi 62-02 (GPI 100/100), Pi SI-3 (US-8, GPI 100/111/122), Pi 98-1 (US-14 GPI
100/122), Pi Atlantic 2N (US-8, GPI 100/111/122), Pi 95 -3 (US-I, GPI 86/100), and Pi 4-

19 (GPI 100/100) (Figure 2.1 and 2.2).

2.2.3 Microsatellite analysis
Simple sequence repeat (SSR) markers are used to identify polymorphisms at specific

microsatellite loci and characterize sample isolates. All the homothallic isolates and few
random isolates (5 from each cross), along with the parents were selected for SSR testing.

A method modified from Knapova and Gisi (2002) and Lees et al., (2006) was used.

2.2.3.1 Microsatellite Primers
Two SSR markers (Pi02, and 48) shown to be polymorphic for specific alleles of DNA

from P. infestans provided by Cooke et al., (2006) were used in this study. These were
derived from sequences documented in the Phytophthora Genome Consortium database
(Waugh, 2000) and by Knapova and Gisi (2002). Primers were labeled at the 5’ end with

the fluorescent dye HEX (4,7,2,4,5,7-hexachloro-6-carboxyfluorescein) supplied by

40

Integrated DNA technologies (IDT, Inc., Coralville, IA, 52241, USA). Sequences of the

primer pairs are:

Name Sequence Size No. of alleles
range
(hp)

Pi 02 (forward) 5’CAGCCTCCGTGCAAGA- 3’ 142 -166 8

Pi 02 (reverse) 5’ AAGGTGCGCGAAGACC- 3’
Pi 4B (forward) 5’ AAAATAAAGCCTTTGGTTCA- 3’ 205 — 217 3

Pi 4B (reverse) 5’ GCAAGCGAGGTTTGTAGATT- 3’

2.2.3.2 Amplification and Sequencing of Alleles
Amplification was done as follows for all primer combinations: 1-10 ng of genomic DNA

(extracted using QIAGEN DNeasy Mini kit, QIAGEN Inc., 27220 Tumberry Lane,
Valencia, Suite 200, CA91355, USA)) was mixed with 20 ul masterrnix (Eppendorf
MasterMix (2.5x), Brinkmann Instruments, Inc., One Cantiague Road, PO. Box 1019,
Westbury, New York 11590-0207, USA). 25 mM microsatellite primers were added, and
finally sterile distilled water was added to make it to total volume of 50 ul. PCR
conditions were as follows; 1 cycle of 95°C for 120 s, 33 cycles of 94°C for 40 s; 56°C
for 40 s and 72°C for 20 s followed by one cycle of 72°C for 10 min. Reactions were
held at 4°C following amplification. All amplifications were performed using a
Thermocycler (Eppendorf Mastercycler, Brinkrnann Instruments, Inc., One Cantiague
Road, PO. Box 1019, Westbury, New York, USA). A negative control (sterile distilled
water instead of DNA) was included in each batch of samples. Samples were submitted
to Genomics Technology Support Facility (GTSF, Michigan State University, 178 Plant

Biology Building, S-18, E. Lansing, MI, USA). Genotypic allocations were based on

41

US US US
U81 U36 U88 10 11 17/14

83-

100- -

111

122

Figure 2.1 Banding patterns of isolates of Phytophthora infestans for differing allozyme genotypes,
and accompanying US designations (Goodwin er al., 1995) at the GP] locus, as revealed by cellulose
acetate electrophoresis.

100/100/111
100/122

          

-"100r 111 r 122

111/111/122

  
   

US-‘l US—l7US-8 US-lO US-ll US-H

Figure 2.2 Standard 61’! hand patterns of Phytophthora infestans isolates ran on a cellulose acetate
gel for 30 min at 200V.

42

electropherogram analysis (denoted by peaks at specific alleles) for individual loci and

were described as having either a homozygous or heterozygous genotype.

2.2.4 Nuclear condition and ploidy levels

2.2.4.1 Sample preparation and flow cytometry
P. infestans isolates, and cultures derived from single zoospore progeny were grown at

18°C on Rye A agar (Caten & Jinks 1968) in Petri plates. The surface of each plate was
flooded with 10 m1 sterile water and a sterile cotton swab was employed to dislodge
mycelium and sporangia. One milliliter of the resulting suspension was used to inoculate
9 cm diameter Petri plates containing 20 ml of pea broth (125 g/L fi'ozen fresh peas,
boiled for l h, filtered, and autoclaved). After 5-10 days depending on the growth rate of

the isolate, mycelium was harvested and centrifuged at 5000 rpm for 10 min. The liquid
was discarded and residual liquid removed by pressing the mycelium against the
centrifuge tube wall. Numerous methods were evaluated for preparation of nuclei of high
quality with stable nuclear staining, few nuclear fragments, no clumping of nuclei, and an
absence of nuclear fragments and cell wall remnants. These methods included releasing
nuclei by enzymatic protoplast of cells, by shearing hyphae or protoplasts in a pressure
bomb using various intensities of pressure, grinding hyphae with and without liquid
nitrogen and filtering the released nuclei, and chopping hyphae with a scalpel blade and
filtering the released nuclei. By trial and error, the most reliable method of nuclear
preparation was found to result from chopping hyphae with a scalpel blade and filtering
the released nuclei. Additionally, pre-staining the suspension of nuclei with propidium
iodide yielded more stable fluorescence than adding the stain just prior to injecting the

suspension into the flow cytometer. Approximately 200 mg of fresh hyphae was chopped

43

with a sterile scalpel blade for 3-4 min in ice-cold lml PBS (pH 7.4) containing 0.1 %
Triton X-100. Cellular debris was filtered through a 30 um mesh nylon membrane to
capture wall debris, and the nuclei passing through the filter were retained. RNase A (50
rig/ml) and 50 ug/ml propidium iodide (Molecular Probes, Inc., Eugene, Oregon) in PBS
(pH 7.4) were added to the suspension of nuclei. The suspension was incubated in the
dark for two hours at room temperature prior to injection into the flow cytometer.
Commercially prepared Singlet Chicken Red Blood Cells (CRBC; BioSure, Grass Valley,
CA) were used as standard nuclei of known DNA content. The CRBC were added to the
oomycete nuclear preparation in quantity to account for 3-5 % of the total fluorescent
nuclei, and stained simultaneously. Additionally, aliquots of the propidium iodide
solution were fed into the flow cytometer sample delivery system prior to sample
analysis. This minimized small fluctuations in sample DNA content caused by leaching
of DNA into the cytometer. The pre-staining and pre-injection of stain yielded flat
measurements of fluctuations in DNA fluorescence over time for both oomycete and

(CRBC) nuclear DNA.

Prepared and stained nuclei of P. infestans and standardized CRBC were delivered into a
Vantage SE laser flow cytometer (BD Bioscience, San Jose, CA). A Spectra-Physics
377G laser (Spectra-Physics, Mountain View, CA) set to a 100 mw 488 argon line
excited the propidium iodide stained nuclei, and fluorescence was collected using a
630/22 band pass filter. CellQuest ver. 3.3 software (BD Bioscience, San Jose, CA) was
used for acquisition and processing of fluorescence signals from the detectors. DNA

fluorescence was collected in linear scaling using single CRBCs as an internal standard

44

for determination of oomycete nuclear DNA content. Fluorescent data for 20,000
oomycete nuclei were collected at a fluorescence threshold excluding only signal noise
while allowing the detection of the lowest fluorescence fiom oomycete nuclei. DNA
fluorescence was statistically analyzed using Modfit LT ver. 3 software (Verity Software
House, Topsham, ME) to distinguish complexities in the population of nuclei, such as
G1, G2, and M phase nuclei present in the cell cycle of eukaryotes. A count gate was set
over the oomycete DNA fluorescence region to insure the ability to count 3 minimum of

12,000 nuclei.

DNA content of singlet P. infestans nuclei was calculated with comparison of the
oomycete G1 peak fluorescence to the G1 peak fluorescence of standard singlet CRBC
nuclei. The Modfit LT calculated the G2/Gl ratio for each sample to optimize the G1
peak to G2+M peak fit. Additionally, DNA area fluorescence versus sample run time
data plotting was monitored to insure that no fluctuation in DNA fluorescence was
occurring during a run (any deviation from a flat DNA fluorescence versus time curve).
DNA integrated area fluorescence versus DNA width fluorescence (time of flight) was
plotted in order to delimit DNA analysis to the optimum fluorescence data for singlet
oomycete nuclei. The plotting produced a histogram of DNA quantity based on area of
fluorescence. The data was further analyzed with the program Modfit LT to determine the
number of nuclear populations present, and then the data was parsed based on best
statistical fit into representative peaks. The numbers of cell cycles present (i.e., diploid,
aneuploid, heteroploid nuclear populations) was determined by analyzing the numbers of

G1 peaks present which also showed corresponding G2 peaks. The Gl/G2 peaks were

45

compared to those of the typical diploid peaks. The Modfit LT program automatically
detected the additional cell cycles after the typical diploid cell cycle peaks were
designated manually. A color code was assigned to each peak using the Modfit LT
software to aid in visual recognition of the nuclear populations in each P. infestans isolate
and progeny. Graphical output was written to a PDF file writer and edited in Adobe

Photoshop CS for display (Adobe Systems Incorporated).

Each flow cytometry analysis of a parent isolate or progeny was repeated three times
using suspensions of nuclei independently prepared from replicate cultures in order to

have statistically supported means for calculations of DNA content per nucleus.

2.3 Results

2.3.1 Isozyme Analysis
The electrophoretic patterns at the CPI loci were identified for all the isolates of parents

and progeny (n=300) isolates from nine crosses (Table 2.2). From the nine crosses, none
of the progeny isolates produced banding patterns consistent with Mendelian ratios
expected from hybridization (sexual recombination) between two parents. Single oospore
cultures gave banding patterns identical to one or the other of the parents (homothallic
reproduction). Parent isolate, Pi 41-02 (with 100/ 100 GP] banding pattern, A1 mating
type, from Northern Ireland) was involved in 6 crosses, 204 of 295 progeny produced this
single banded phenotype, while the rest of them were identical to the other parent. Parent
isolate, Pi 02-007 (with 100/111/122 GPI banding pattern, A2 mating type) was involved
in 4 crosses, 71 of 295 progeny produced this five-banded phenotype, and the remaining

were identical to the other parent. Parental isolate, Pi 81-3 (with 100/111/122 GP]

46

banding pattern, A2 mating type) was involved in 2 crosses, 13 of 101 progeny produced
this five-banded phenotype. Parental isolate, Pi AtlanticZN (with 100/111/122 GPI
banding pattern, A2 mating type) was involved in 2 crosses, and out of 18 progeny none
produced this five-banded phenotype. Parental isolate Pi 98-1 with 100/122 GPI banding
pattern, when crossed with Pi 41-02, produced 7 out of 45 progeny with the double-
banded phenotype and the rest had a 100/ 100 genotype. In cross 7 (Pi 95-3 X Pi 02-007),
there were no progeny with the phenotype identical to the parent Pi 95-3 (96/ 100 GPI).
Parental isolate, Pi 62-02 (with 100/ 100 GP! banding pattern, A1 mating type, from
Northern Ireland) was involved in 1 cross, 17 of 25 progeny produced this single-banded
phenotype. CPI-banding of progeny isolates indicted that that the GP] genotype is
heritable, as only the parental type alleles were identified and hybrids were not produced.
Isolate Pi 41-02 was homozygous for the most common allele ( 100/ 100 GP!) and

majority of the progeny possessed this allele.

2.3.2 Microsatellites
Of the two microsatellite markers used to characterize parental isolates, marker Pi 4B

differentiated well between the isolates (Table 2.3). Parents selected for crosses in this
study have heterozygous alleles using microsatellite markers (Pi 4B and Pi 02; Table
4.3). Using the marker Pi 4B, 8 out of 25 progeny isolates tested, showed patterns of
alleles that were recombinants, differing from the pattern of either parent. Seventeen
progeny isolates had alleles identical to the parents, and therefore many of these offspring

may have resulted from apomixis (selfing). Progeny isolate 2_29 has the alleles at 201,

47

212, 214, 216 and 223 bp that were produced from both the parents Pi 41-02 (201, 212,

214 bp) and Pi 02-007 (210, 223 bp; Fig. 2.5).

The Pi 02 microsatellite marker showed more variation in the banding pattern of the
progeny isolates than marker Pi4B. Out of 25 progeny, only 8 showed banding patterns
identical to a parent and are assumed to be the result of selfing, whereas, 17 progeny were
recombinants (hybrids), Allelic patterns for a few isolates are shown in Fig. 2.5.
Offspring 2_35, 3_50, 5_l2, and 5_37 (from crosses 2, 3, and 5, respectively) were

observed as hybrids using both the markers.

2.3.3 Nuclear condition and ploidy levels
Examination of field isolates of P. infestans using laser flow cytometry revealed that of

16 isolates, 10 contained one nuclear population (G1 peak) that was presumably diploid.
The other 6 isolates were heterokaryotic, containing more than one nuclear population in
their thallus (Table 2.5). Among the typical (homokaryotic) isolates, the DNA content per
nucleus varied considerably among isolates, ranging from a mean of 0.48537 to 0.70260,
: 0.02200 s.d. (Tables 2.5 and 2.6). In heterokaryotic isolates, the nuclear population
with a DNA content similar to the nuclei in the homokaryotic isolates was designated as
"diploid" and other nuclear populations present were designated aneuploid if they had
less DNA content or heteroploid if greater in DNA content (Table 2.5). Five isolates
contained two populations of nuclei (G1 peak) in their thallus, two of these were from
Michigan, one from Minnesota and another fi'om Mauritius. This is the first discovery

that field isolates in Michigan may be heterokaryons. One isolate from Mauritius

48

contained 3 nuclear populations in its thallus, aneuploid, diploid and heteroploid nuclear

populations (Gl peak). No isolate could be demonstrated to be polyploid (i.e. 4N).

Single-zoospore derived cultures of isolate MYA1114 also varied statistically in their
mean DNA content per nucleus, ranging from 0.41 to 0.50 i 0.007 s.d. (Table 2.4). At
least 3 single-zoospore isolates were statistically significantly smaller in DNA content
than the parent. These isolates are presumably aneuploids. Additionally, laser flow
cytometry revealed that the nuclear populations present in the heterokaryotic parents
were not inherited equally by the single-zoospore derived isolates (asexual progeny).
Segregation of nuclear types during asexual propagation was unexpected and represents a
new mechanism of variation in P. infestans field populations that are primarily clonal.
Differences in nuclear condition among field isolates are represented graphically in
Figure 2.6 with G1 peaks color-coded to distinguish aneuploid (yellow), diploid (red),
and heteroploid (orange) nuclear populations. The percentage of single-zoospore derived
isolates of a heterokaryotic parent that inherited the same nuclear condition of the parent,
or just one or the other of the two nuclear populations, are represented in Table 2.5 for

isolates MYA-1114 and isolate Pi 3768.

2.4 Discussion
Two systems of genetic markers were used to study inheritance in field isolates of P.

infestans. They are SSR or microsatellite markers (Pi 4B and Pi 02), and the allozyme
markers or GPI. Additionally, DNA content of nuclei and nuclear condition of isolates
was examined with laser flow cytometry to clarify interpretation of inheritance patterns.

The parental isolates chosen for this study from the US population were composed of

49

three differing genotypes for the CPI locus and this variation in GPI patterns was
sufficient among the parents and progeny for use of allozymes as markers for a study of
sexual inheritance. However, due to the lack of variation in the Pep locus this allozyme
marker was not used in our study and isolates homozygous (100/ 100) for the GP! locus
were excluded fiom the study. All Northern Ireland parental isolates were homozygous
100/100 for the Gpi allozyme genotype and as in this study there are no other parental
isolates with this genotype, PEP marker was not used in this study. P. infestans is a
diploid organism that is usually heterothallic in sexuality and some populations have been
reported to be polyploid (Tooley & Therrien, 1991). When A1 and A2 mating type
isolates are paired, each isolate may produce antheridia, oogonia or both, which later fuse
to form hybrid oospores from outcrossing , however, such a pairing also can initiate a

proportion of self—fertilization events, or inbreeding events (J udelson, 1997).

Outcrossing and selfing can both occur within mating cultures (Judelson, 1997). In our
crosses, approximately 10% of oospore-derived progeny were recombinants (automixis
or sexual reproduction) based on inheritance patterns of codominant SSR markers. The
remaining progeny were products of self fertile oospores (homothallic reproduction or
apomixis). The application of isozyme analysis enabled identification of progeny from
mating cultures of Phytophthora infestans (Cooke & Lees, 2004). In this study, oospore-
derived progeny from all crosses yielded banding patterns and presumed genotypes
consistent with one or the other of the parents but no recombinant patterns. When a
parental isolate with a homozygous allele pattern was crossed with a parent having the

heterozygous three-banded pattern, the homozygous allele was inherited predominantly

50

96 100 100 100 100 100 100 100
I100 /111 I100 I100 /122 I100 I100 I100
I122

 

Figure 2.3 Isozyme analysis (GP!) of isolates of Phytophthora infestans parents and
progeny; From Left to right, Lane 1: 96/100, Pi 95-3; Lane 2: 100/111/122, Pi 02-
007; Lane 3: 100/100, Pi 41-02; Lane 4: 100/100, 2-35; Lane 5: 100/122, Pi 98-1;
Lane 6: 100/100, 2-2; Lane 7: 100/100, 2-10; Lane8: 100/100, 2-15.

51

Table 2.2 Presumed genotypes at the Glucose phosphate isomerase (GPI) locus in
parental and single oospore progeny isolates of Phytophthora infestans.

 

 

Parentsa Genotypeb Progenyc
Pi 41-02 X Pi 02-007 100 / 100 13
100/111/122 24
Pi 41-02aX Pi 02-007a 100/ 100 31
100/111/122 39
Pi41-02XPiSl-3 100/100 43
100/111/122 8
Pi41-02aXPiSI-3 100/100 45
100/111/122 5
Pi4l-02XPi 98-1 100/ 100 38
100/122 7
Pi 02-007 X Pi 62-02 100/ 111 / 122 7
100/ 100 18
Pi 95-3 X Pi 02-007 96 / 100 0
100/111/122 8
Pi AtlanticZN X Pi 41-02 100/ 111 / 122 0
100/ 100 16
Pi Atlantic2nX Pi 4-19 100/111/122 O
100/ 100 2

 

’ Isolates of Phytophthora infestans used as parents in the crosses to produce progeny
isolates derived from single oospores

b Genotypes of parent isolates

c Number of progeny having a respective genotype from each cross

52

Table 2.3 Different allelic patterns of parent and progeny isolates of Phytophthora
infestans at microsatellite, Pi4B and Pi02 loci.

 

 

Crosses Isolate Pi 4B marker Pi 02 marker
Parent 1 Pi 02-007 210,223 157,159

Parent 2 Pi 41-02 201,212,214 150,160,161
Progeny 2_29 201, 210,212,214,223 nd

Progeny 2_35 201 ,210,212,214,223 147, 1 58,160,161
Progeny 2_20 201,210,212,213,223 158,160,145
Progeny 1_13 201,212,214,215,223 145,158,160
Progeny 2_25 201,212,214,223 nd

Progeny 1_9 201,212,215 145,156,159
Progeny 2_51 201,212,214 145,161
Progeny 2_46 210,214,223 nd

Progeny 2_13 210,223 145,158
Progeny 2_21 210,223 145,160
Progeny 2_3 210,223 145,156,159
Progeny 2_9 210,223 143,145,158,159
Progeny 2_33 nd 145,158,159
Progeny 2_2 210,223 145,160
Progeny 2_31 210,223 145,160
Progeny 1_6 210,223 145,156,159
Progeny 1_4 210,223 145,156,159
Progeny 1_7 210,223 145,156,159
Progeny 2_19 210,223 nd

Progeny 2_7 210,223 nd

Parent 1 Pi 02-007 210,223 157,159

Parent 2 Pi Sl-3 210,224 nd

Progeny 3_4 202,213,215 146,160,161
Progeny 3_13 202,213,215 146,160,161
Progeny 3_50 202,213,215 146,160,161
Progeny 4_37 nd 147,161
Progeny 4_1 1 201,212,214 nd

Parent 1 Pi 02-007 210,223 157,159

Parent 2 Pi 95-3 210,214 145,157,159
Progeny 7_8 202,213,215 145,146,160,16l
Progeny 7_17 202,213,215 145,146,160,16l
Parent 1 Pi Atlantic 2N 210,223 145,157,159
Parent 2 Pi 95-3 210,214 145,157,159
Progeny 8-1 202,213,215 145,146,160,161

 

nd = not detected

53

 

VIII'IIII'TrltT'TrIIEIV'er'qrfiU rTUU—IrU—Irqrjjr'firfi" Iii!

130 140 150 160 170 180 190 200 210 220 230 240

 

2_29, Pi48. iii-parental progeny

1500
1000
i All i i...
2_35,Pi02,progony '
4000
20m

Pi 41-02, Pi48, Parent

1000
500

Pi 62-02, P148 Parent __-

_- A___A—-_.A- _ A

Pi Atlantic 2N, Pi48, Parent
Pl 95-3, P148, Parent

Pl 02-007, P148, Parent

Figure 2.4 Electropherograms illustrating isolates heterozygous for the Pi4B and
Pi02 loci at alleles from 200 to 226 hp and 160bp respectively. The Y axis represents
relative fluorescence unit (RFU) and the X axis molecular size of the allele marker
in base pairs (hp) size.

54

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56

. When a parental isolate with a two-banded pattern was crossed with a parent having the
three-banded pattern, the two-banded pattern was predominantly inherited. There were no
progeny with recombinant or hybrid patterns. This is interpreted to imply that the oospore
derived progeny were the result of selfing (homothallic sexuality). Mating types are now
widely distributed (Deahl et al., 1991) and it is probably only a matter of time before
sexual recombination alters the population genetics of P. infestans in North America.
Currently, the clonal populations show correlations between allozyme genotype, mating

type, and fungicide sensitivity (Goodwin et al., 1995).

Mating type markers, isozyme analysis and SSR markers of many of the progeny were
identical to their parents and confirmed that these progeny had resulted from selfing. In
our studies, isozyme analysis could not differentiate the variation as well as SSR markers
(Cooke and Lees 2004), whereas, SSR markers showed variation in some of the progeny
confirming that they were recombinants (hybrids). SSR markers proved to be important
nuclear tools useful in identifying and characterizing the segregation patterns in progeny

(Knapova et al., 2001; Knapova and Gisi, 2000).

SSR markers are important nuclear tools useful to identify and characterize the
segregation patterns in F1 progeny. Isozyme analysis couldn’t differentiate the variation
as well as SSR markers. SSR markers indicated some variation in the progeny.
Microsatellites have shown that some of these isolates may have resulted from selfing.
The isolates that were identified as hybrids using SSR markers were not differentiated

using isozyme analysis. The application of SSR markers has distinguished hybrids from

57

selfs. Mating type, isozyme analysis and SSR genotype of the selfs were identical to their
parents. The presence of three bands is indicative of trisomic linkage and trisomic

progeny were shown to be pathogenic (van der Lee et al., 2001 , 2004).

Progeny isolates showed comparatively minor variation with either a band missing or
presence of an extra band from standard fingerprints. Our data support the conclusion
that P. infestans has the potential to be homothallic and have the ability to self under
certain conditions such as stimulation to form antheridia and oogonia by pairing with the
opposite mating type. Additionally, our data support the conclusion that a limited amount
of sexual recombination occurred which produced progeny with different banding pattern
than the parents by meiotic recombination. Alternatively, these recombinants could be
due to non-mechanisms of variation such as mutation, mitotic recombination or
parasexual recombination in the nuclear DNA. Mutation usually involves the appearance
of new alleles and has likely occurred where an extra single band is detected in the DNA
fingerprint (Drenth et al., 1994; Goodwin et al., 1995). Previous investigations indicate
mutation rates in the nuclear DNA are at a detectable level in P. infestans (Goodwin
1997). The other selfed isolates having GPI and SSR marker patterns similar to a parent

could be a result of apomixis or asexual reproduction.

Microsatellite markers and isozyme markers differentiated well between parental isolates.
A combination of the two microsatellite primers Pi 02 and Pi 48 were able to distinguish
between the isolates and could be useful in future population studies. The variation

between the isolates using the SSR markers did not correlate between the phenotypic and

58

 

genotypic characteristics (data not shown in this chapter). These results agree with
previous work which has shown no correlation between SSR allele identification and
phenotypic markers including mating type, virulence, aggressiveness and metalaxyl

sensitivity (Knapova et al., 2002).

As previously noted, although progeny have identical allozyme genotypes, differing
alleles in SSR markers were detected, which indicated these isolates were recombinants.
Although one study has investigated inheritance during sexual reproduction with
microsatellite markers (Knapova et al., 2002) none have investigated inheritance during
asexual reproduction in Phytophthora infestans or variation between individual zoospore
isolates with SSR’s. However, such studies have been conducted for inheritance of
virulence and metalaxyl sensitivity using AF LP markers (Abu-El Samen et al., 2003). As
distinguished from other genetic markers, SSR analysis can differentiated among parental
isolates thus it would appear that microsatellite markers are easily identifiable, relatively
robust and stable over time. Additionally as co-dominant markers, SSR’s could be
extremely useful in population genetics analysis for to track populations, temporally and
spatially. The usefulness of SSR genotyping is dependent on the heritability and stability
of the marker. Also, the usefulness of SSR markers will depend also on the ability of
such markers to distinguish between clonal lineages and the rate of mutation or crossing

over events which occur.

The inheritance of the progeny with respect to the measured tratits in this study did not

follow Mendelian segregation. The reasons for the distortion are not understood fully, but

59

there are several possibilities described in the literature, including linked deleterious,
recessive alleles, mitotic irregularities, chromosome rearrangements and unsuspected
epistatic loci (Spielman et al., 1990), or something as simple as contamination. This
could be a reason that some progeny showed recombination in one SSR marker but not
the other SSR markers. The variation among single zoospores of an isolate could explain
the undergoing diversity of P. infestans isolates in nature. Our studies revealed that field
isolates varied in DNA content of nuclei which would be assumed to be either due to
variation in number of copies of chromosomes or different amounts of accumulated
"junk" DNA within a genotype. Previous studies in P. infestans suggested that most
hybrids were stable polyploids or parasexual recombinants rather than heterokaryons
(Shattock & Shaw, 1976), however, our results suggest many hybrids are the result of
heterokaryosis, differences in the balance among nuclei in similar heterokaryons, or in
unbalanced inheritance of nuclear types in asexual progeny of heterokaryons. Single-
oospore derivatives of interstrain crosses often display the genetic markers of only one
parent (Shattock et al., 1986; Whittaker, Assinder & Shaw 1994) and such strains are
commonly described as a result of selfing, a type of asexual reproduction. However, this
asexual reproduction method is enabled in mating cultures by the bisexuality of P.
infestans (Judelson, 1997) behavior where hormonal signals can initiate antheridia and

oogonia in both of the paired isolates.

Other mechanisms of variation may act during controlled and natural pairings. Mating

may begin in the normal manner with the multiple nuclei present within each of an

adjacent antheridium and oogonium undergoing meiosis, then defective transmission of

60

the signal that normally induces simultaneous meiosis in both gametangia (Win-Tin &
Dick, 1975) might allow diploid nuclei to pass into the oosphere in which karyogarny
might subsequently occur. Another mechanism might include errors occurring in the
oosphere such that rather than one viable diploid zygote forming and the other nuclei
degenerating, the other nuclei could participate in forming heterokaryons, or participate
in karyogarny and spawn polyploids (Judelson et al., 1998). Within the oomycetes there
is substantial diversity in patterns of garnetangial differentiation and nuclear behaviour
(Dick, 1995) and the assumption that all oosporic nuclei within a species are produced by
the same process may be invalid (Judelson et al., 1998). Further genetic studies of
polyploids might contribute to a better understanding of mating type determination by
indicating the effect of allele dosage on phenotype, since polyploid by diploid crosses
might yield progeny of variable ploidy (Judelson et al., 1998). The generation of
heterokaryons and polyploids may contribute to the evolution and adaptation of natural
populations of Phytophthora. Heterokaryosis and polyploidy also represent a means to
increasing the variability and adaptability of P. infestans through the mechanism of
mitotic crossing-over. There is some suggestion that high mutation rate may decrease
heritability and therefore usefulness for certain functions such as tracking within
populations (Weber and Wong, 1993). Hybrid isolates of Phytophthora infestans may
vary in firngicide sensitivity, virulence and temperature tolerance and enable the
development of strains which could potentially increase damage in potato crop
production. These processes might be rare in nature but if they occur they could result in
shifts within localized populations of P. infestans and produce epidemics that are difficult

to manage.

61

Figure 2.5 Representative histograms of the nuclear condition of parent and
progeny isolates of Phytophthora infestans. The typical diploid nuclei of most isolates
of P. infestans produces a fluorescence intensity of 48 — 52 units on the X-axis (on
channel FL3-A) when stained with Propidium iodide. The units are relative and
compared to the standar (Gray peak) of stained chicked red blood cell nuclei.

8 Pi 3768

 

 

 

  
   

o.
8
E
I; §.
2 I
§I
.— 100 150 200 250
Channels(FL3-A)
c Pi 95—7
OJ
8‘.
2 :
§§1
z :
§J
1
4
50 100 150 200
Chmnels(FL3-A)
e ATCC Single
g zoospore progeny-2
§
gs
2
§
§

 

50 100 150 200
Chamois (FLs-A)

Aneuploid Diploid

b ATCC1114
O
2 8
§
2%
O
8
50 100 150 200 250
ChameluFLS-A)
d ATCC Single

zoospore macaw-1

Number

 

Chamois (FLS-A)
l ATCC Single
2W W3
O
3 8
5 a
50 100 150 200
Chamois (FLa-A)

m Heteroploid Standard

62

3 Mating type, metalaxyl resistance, and virulence characteristics of
Phytophthora infestans from mating isolates with variable thermal
tolerance

3.1 Introduction
Heterothallic species of Phytophthora are bisexual and capable of self-mating and

producing oospores. De Bary (1881) was the first to report the germination of oospores of
a Phytophthora species. Compared to the number of genotypic markers available there
are relatively few phenotypic tests for Phytophthora infestans (Shaw and Shattock,
1991). Of these the most reliable and commonly used are mating type, resistance to
metalaxyl and virulence complexity. Phytophthora infestans has a mating system that is
representative of most heterothallic oomycetes, with two known mating types existing,
A1 and A2. Mating type plays a major role in the phenotypic development of populations.
Each mating type is distinguished by the production of a specific pheromone and
subsequent response to the pheromone of the opposite mating type (Ko, 1988). Mating
type is generally considered to be stable, heritable and easily detectable and as such is a
commonly used and important test for characterizing and assessing the potential for
sexual reproduction within a population (Brasier, 1992). Mating type is controlled by a
single locus, with A1 being heterozygous and A2 homozygous. Most isolates are bisexual
but display relative sexuality, preferentially forming antheridia or oogonia depending on

their mating partner (Gallindo and Gallegly, 1960; Judelson, 1997).

The genetics of mating type in P. infestans is complex and the Al : A2 ratio in progeny
can vary significantly, ranging from close to 1 : 1 to an excess of A1 over A2 (Shaw
1991; Judelson et al., 1995). Oospores are the sexual structures that are produced when

two mating type fuse. Oospores are diploid, contain one viable nucleus and are produced

63

when two haploid nuclei firse in the gametangia. Germinating oospores are considered as
progeny of the A1 or A2 type. Self-fertile progeny are sometimes recovered as well, and
these may result from nondisjunction (Fyfe and Shaw, 1992) or the presence of extra
copies of the mating type locus in one parent (Judelson, 1996b). Efforts to recover
progeny of P. infestans from crosses began in earnest only a few decades ago (Gallindo
and Gallegly, 1960). Most oospore-derived cultures are true progeny although some may
arise from selfing or apomixis (asexual reproduction; Shaw, 1991). If oospores result
from apomixis, then selfed oospores contribute nothing to variability in the pathogen
population. If on the other hand, selfed oospores result from meiosis and recombination,
they would be a potential source of variation in this species (Shattock et al., 1985).
Sexual crosses have been made in vitro for P. infestans in order to investigate inheritance
of several phenotypic characteristics such as resistance to metalaxyl (Shattock 1998; Lee
et al., 1999); allozyme markers (Spielman et al., 1990); mating type (Judelson et al.,
1995) and for avirulence genes (Al-Kherb et al., 1995; Lees et al., 2001). A system with
a single locus, where the alternate mating types are homozygous and heterozygous, has
been proposed for Phytophthora spp. although the mating type ratios observed in the
progeny of crosses are frequently inconsistent with this model (Gallegly 1968; Shattock
et al., 1986; Spielman et al., 1990; Shaw, 1991). Although generally considered robust,
mutation or mitotic recombination as well as treatment with certain fungicides can result
in segregation of the opposite mating type (Smart et al., 1998). Additionally, self-fertility
can occur amongst US lineages (specifically the US-7 and US-8 lineages), although

fewer oospores are produced than between opposite mating types (Smart et al., 1998).

64

How far these factors may influence use of mating type as a marker in selection studies is

unknown.

The problems caused by potato late blight were exacerbated by the development of
resistance to the most widely used systemic fimgicide group (phenylamide, FRAC group
4) and the appearance of new, highly aggressive strains of P. infestans (Gisi and Cohen,
1995). Major improvements and sophisticated procedures have significantly enhanced the
understanding of the biology and pathogenic strategies of P. infestans. Phenylamides
were the first compounds discovered to be systemically active against oomycetes, and the
development of resistance has been a major factor in the contemporary severity of potato
late blight (Gisi and Cohen, 1995). Currently, the predominant isolates of P. infestans in
North America are resistant to the widely used fungicide metalaxyl. Metalaxyl [methyl-
N-(2-furoyl)-N-(2,6-xylyl)-DL-alaninate (Ridomil®, Syngenta, Basle, Switzerland)]
belongs to a class of fungicides known as the phenylamides and was introduced by Ciba-
Geigy in 1977 (Urech et al., 1977) for control of P. infestans due to a high protective,
curative and eradicative activity (Schwinn and Staub, 1987). Metalaxyl-resistant isolates
have been shown to infect tubers, but tubers infected with metalaxyl-sensitive isolates
were more likely to produce infected plants because they were not completely destroyed
by the disease (Walker and Cooke, 1990). Pathogen response to resistance in tubers may
be genotype specific (Kirk et al., 2001a). For example, observations by Flier et al.
(2001) indicate that the gene-for gene pathosystem in potato is not solely responsible for
the differential interaction in tuber infection. A resistance response occurs when an

incompatible race of P. infestans attempts to infect a potato plant containing appropriate

65

R genes. The genetic control is unclear though and multiple genes are likely involved
(Shattock, 1988; Shaw, 1991; Lee et al., 1999). If as suggested by Shattock, (1988)
resistance is controlled by a single locus exhibiting incomplete dominance, heterozygous
isolates with both resistant and sensitive alleles would exhibit intermediate resistance.
However backcrosses have not segregated as expected by this model (Shattock, 1988)
and the precise mechanism of inheritance is unknown. Since resistance is a relatively
stable and heritable trait the method can be used to characterize individual isolates within

a population (Forbes et al., 1998).

Much in vitro evidence suggests new genotypes possess higher aggressiveness and have
more efficient colonization of potato than the ‘old’ US-l (96/100) genotype (Goodwin et
al., 1995; Miller et al., 1998; Flier et al., 1998). However as new genotypes such as US-
8 (100/111/122) and many of the European genotypes are metalaxyl resistant this in itself
may have allowed a competitive advantage against the phenylamide sensitive US-l
(100/100) genotype (Goodwin et al., 1996; Cooke et al., 2006). It is generally accepted
that metalaxyl resistance in the US. is the result of migration rather than mutation and
subsequent selection (Goodwin, 1997). Sensitivity to the fungicide metalaxyl has been
used to characterize isolates since the first detection of resistance in 1980 (Davidse et al.,
1981). Resistance is qualitative in practice with isolates being either sensitive or resistant
with intermediate sensitivity usually infrequent (Gisi and Cohen, 1996). The nature of
this resistance is uncertain though and it is now considered likely that multiple genes are
involved (Judelson and Roberts, 1999). The trait is believed to have originated from
‘naturally’ resistant strains present in the population at a low level prior to introduction of

the fungicide (Gisi and Cohen, 1996).

66

Genetic analysis of resistance derived from wild Solanum species has demonstrated the
existence of a gene-for gene interaction between the host (Solanum tuberosum) and the
pathogen (Phytopthora infestans) with single genes, termed R genes corresponding to
specific virulence genes in the pathogen (Malcolrnson and Black, 1966). Patterns of
inheritance support the hypothesis of Mendelian inheritance with avirulent alleles being
dominant and virulent alleles recessive (Al-Kherb et al., 1995). However, it has been
demonstrated that isolates of a single genotype, as well as single zoospores fi'om a single
isolate can have a high level of variability as regards compatibility with R genes
(Goodwin et al., 1995; Abu-El Samen et al., 2003). To date eleven R genes have been
elucidated, although it has been acknowledged more may exist (Trognitz, 1998).
Detached leaflet assays for quantitative assessments of virulence in P. infestans have
been commonly used in number of studies. Virulence refers to the ammount of
pathogenicity of an organism, or it can also be defined as the relative ability of a microbe
to cause disease. These techniques have been used in preference to whole-plant assays
because of the ease of testing multiple isolates simultaneously, less contamination fiom
airborne spread of inoculum, and ability to control the environmental conditions in
replicated tests consistently. Test isolates of the pathogen which cannot be released into
the field can also be tested in these conditions. Legard et al., (1995), observed that the
detached leaflet assay was a reasonably accurate predictor of the behaviour of isolates

under field conditions.

67

The objectives of this study are:

1. To assess the different phenotypic characteristics a) mating type, b) metalaxyl
sensitivity and c) virulence assessment on the progeny isolates generated from the
nine crosses described in chapter 4.

2. Estimate the inheritance of these traits.

3.2 Materials and methods

3.2.1 Selection of isolates
Parental isolates were selected on the basis of their temperature tolerance at -5°C and the

compatibility type as described in Chapter 2. Two out of seven parental isolates are from
Northern Ireland, UK and the rest fiom the United States (Appendix 2). Progeny were

isolated from single oospore cultures as described in chapter 2.

3.2.2 Mating type
Mating type determination was accomplished by growing the isolates on non-amended

rye agar media (Appendix 1), poured thinly on 15 x 60 mm Petri dishes for a final
volume of 25.0 ml (as described in Chapter 4). Briefly, each sample isolate was paired
with known A1 and A2 isolates of P. infestans referred to as testers. Mycelial plugs (4
mm diameter) were placed 2 cm apart on the agar and incubated at 18°C in the dark for
14 days. Isolates were then assessed microscopically for oospore production where the
two colonies interacted. An isolate was designated A1 if oospores were produced when
paired with the A2 tester and vice versa. Pure cultures of the same test isolate were also

observed under the microscope, to test for any evidence of self-fertilization.

68

3.2.3 Metalaxyl sensitivity
All the isolates were tested for the sensitivity to the systemic fungicide, metalaxyl (new

isomer mefenoxam; Syngenta, Research Triangle Park, NC U.S.A.). The method
followed was as described by Sujkowski et al., (1995) and as modified by Stein and Kirk
(2003). Stock solutions were prepared by adding metalaxyl (Ridomil Gold EC, 47.6%
active ingredient) to the double distilled water. For making 1000 ppm (parts per million)
stock solution, 2.10g metalaxyl was added to 1 L double distilled water. Serial dilutions
were continued to make 100, 10, 5, 1, 0.1 ppm stock solutions. Stock solutions (100, 10,
5, 1, and 0.1 ppm) were added to molten media (10 ml/990 mL) after the media was
cooled to 55°C. All stock solutions were sterilized by passing them through 0.22 “M
millipore filters (Millipore Corp., Bedford, MA, U.S.A.) using 10 ml syringes. Plates
containing 10 m1/liter of filtered water were used as a control (0 ppm). Rye B agar
amended with 0, 0.1, 1, 5, 10 and 100 ppm of fungicide were made and compared with

grth on medium without frmgicide (0 ppm); (Fig. 3.1).

Mycelial plugs of the sample isolates of P. infestans (2 mm diameter) were removed fiom
the edge of an actively growing colony on Rye RAN agar (Appendix 1) and placed on the
center of fungicide amended rye agar. The experiment was replicated three times and
repeated twice. The cultures were incubated at room temperature for 8-days and the
radius of each colony was measured. A separate ECso was produced for each isolate
within all three experiments. The mean ECso was determined for each isolate using all
three experiments. Percent inhibition for each concentration of metalaxyl was calculated
relative to the mean of the control plates within each experiment. EC50 for hyphal growth

(diameter) was calculated using the Pharmacology function standard curves analysis

69

feature of SigmaPlot (Version 9.01, Systat Software, Inc., Chicago, IL, USA). The
sensitivity of the isolates was classified by comparing ECso response of the isolates
grown on media amended with different concentrations of metalaxyl with ECso of known
resistant and susceptible isolates. Isolates were termed as resistant (R) when the ECso was
not significantly different at p = 0.05 from the grth of the resistant reference isolate;
intermediate in resistance (I) when the EC50 was significantly less than that of the ECso of
the resistant reference isolate but significantly greater than that of the sensitive reference
isolate; and sensitive (S) when the ECso was not significantly different from the sensitive

reference isolate.

3.2.4 Virulence assessment
All parent and progeny isolates were analyzed for virulence phenotype by the detached

leaf method using differential plants LBl to LBll (micro-plants of the differentials were
obtained from the USDA Potato Germplasm Unit, WI, US and grown in tissue culture
until needed) carrying single genes for resistance (Rl-Rl 1) and Craig‘s Royal (R0)
containing no R genes was used as the susceptible variety or control. Plants were grown
for about 8 weeks in six 1 L pots (one tuber per pot) containing sterilized soil. They were
maintained in temperature-controlled environment chambers (Environmental Growth
Chambers, Chagrin Falls Ohio, USA) at 18°C, 16/8 h photoperiod and >80% RH and the
illumination was provided by 500 W Philips-HPIT lamps. Greenhouses were also used to
grow the plants and they were maintained at 20°C under light. Healthy secondary leaflets
were cut from the compound leaves and used for all the tests. Isolates were tested on
three replicate leaflets of each differential cultivar (LBl to L81 1) and control (Craigs

Royal). Inoculum was prepared from 12-14 day old cultures grown on Rye agar.

70

Sporangia were collected by adding 10 ml sterile distilled water to each isolate.
Mycelium was scraped from the flooded culture using a rubber-policeman and then the
solution was filtered through three layers of cheesecloth. Sporangia were counted and the
suspension was standardized to approximately 2 x 104 sporangia ml'1 using a
hemacytometer. The spore suspension was incubated in a refiigerator at 4°C for 90
minutes to induce zoospore release. Transparent plastic boxes (13 x 18 x 5 cm) were used
for the assay. A moistened thick paper towel was placed in the bottom of the box to
maintain the humidity at >90%, and then a plastic mesh was placed over the towel, so
that the leaflets did not come into contact with the wet paper towel and predispose the
leaflets to bacterial wet-rot caused by pectolytic bacteria. Leaflets about the same size (2
cm long x 2 cm at the widest point) were excised from potato plants generated from
tissue culture and placed abaxial side up on the plastic mesh. Five leaflets per R gene
differential genotype were inoculated with a droplet of the suspension (20 ul) placed
close to the center of each leaflet (Fig. 3.2). The inoculated leaflets were incubated in a
growth chamber at 18°C and 95% humidity with a 12-h lightzdark cycle and the
illumination was provided by 500 W Philips-HPIT lamps. Leaflets were scored afier
seven days for presence or absence of a sporulating lesion, by giving the numbers 1 and
0, respectively. Each test was repeated twice for a total of 15 leaflets per differential per
parental and progeny isolate tested. The corresponding Avr gene was considered absent if

>85% of leaflets had a sporulating lesion.

71

3.3 Results

3.3.1 Compatibility type
Phytophthora infestans was primarily heterothallic, though some isolates appeared to be

self-fertile. The number of oospores germinated in matings between A1 and A2
compatibility types of P. infestans was significantly different in each cross. Oospores
were produced in all crosses between A1 and A2 compatibility types, the manner of
germination was similar for oospores from all crosses, but the amount of germination
significantly differed among crosses. Germination rate of oospores from some crosses
was greater than 75% and in some crosses the gemrination rate was less than 5%.
Progeny fi'om each cross had a different segregation in the compatibility type, and some
self-fertile progeny were also observed. About 215 oospores germinated when both
parents with the thermal phenotype character “tolerant” were crossed (crosses 1, 2, 3, 4,
and 6; Table 3.1); 45 oospores germinated when thermal tolerant with intermediately
tolerant were crossed (CrossS; Table 3.1); 24 oospores germinated when thermal tolerant
with thermal sensitive parent isolates were crossed (crosses 7 and 8; Table 3.1); 2
oospores germinated when thermal sensitive with sensitive parent isolates were crossed

(cross 9, Table 3.1). In total, 300 progeny isolates were collected from the nine crosses.

3.3.1.1 Inheritance of mating type after 2 weeks of incubation
In cross 1 and cross 2 (Pi 41-02 X Pi 02-007) high numbers of progeny were established

and the ratio of A1:A2 was approximately 1:2. There were eight self-fertile isolates
(oospores found in both the testers) present out of 88 total isolates from cross 1 and cross
2 (Table 3.1). In cross 3 (Pi 41-02 and Pi Sl-3) and cross 7 (Pi 95-3 X Pi 02-007) A1: A2

progeny were in the ratio of approximately 1:1. In cross 4 (Pi 41-02 X Pi 81-3), and cross

72

5 (Pi 41-02 X Pi 98-1) Al: A2 progeny were in the ratio of approximately of 2:1. In cross
6 (Pi 62-02 X Pi 02-007) A1: A2 progeny were in the ratio of approximately of 1:2. There
were three self-fertile isolates out of 51 from cross 3 and one from cross 5 out of 35
isolates. Oospores formed in pure culture were indistinguishable from those produced in
mating involving A1 and A2 mating type strains. Progeny isolates in general had thick fast
growing mycelium, abundant sporangia and oospores compared to the parents. Self-
fertility (oospore production) was found in the progeny isolates from crosses where one
of the parents was the 100/111/122 genotype. Compared to the US-8 genotype, progeny
isolates generated from other genotypes did not form as many self-fertile progeny isolates

in pure culture (Table 3.1).

3.3.1.2 Variation of mating type after 1 month of incubation
All the pure cultures (not crossed with testers) were observed under the microscope for

the presence of oospores (Table 3.2). The number of self-fertile oospores in the progeny
isolates increased with time. In cross 1, 18 out of 27 progeny isolates; cross 2, 27 out of
39; cross 3, 8 out of 19; cross 4, 12 out of 29; cross 5, 7 out of 32; cross 6, 11 out of 18;
cross 7, 3 out of 6; cross 8, 3 out of 6 had self-fertile oospores; and in cross 9, there were

no self fertile oospores observed.

3.3.2 Metalaxyl sensitivity
Parents in this study were either resistant, intermediate in sensitivity or sensitive to

metalaxyl (Table 3.3). Variation was observed in the progeny isolates in metalaxyl
phenotype. From the crosses l, 2, and 5 (metalaxyl resistant X resistant cross), out of 133

isolates, 67 progeny isolates were identical to the parents; 64 isolates were intermediate

73

and 2 isolates were sensitive to metalaxyl and were hybrid progeny. Parents in the crosses
3, 4, 6, and 8 were resistant and intermediate in resistance to metalaxyl. Out of 101
isolates from crosses 3 and 4 (same parents), 18 isolates were resistant and 61 isolates
were intermediate in metalaxyl sensitivity (identical to the parents) and 22 isolates were
hybrids and had a sensitive metalaxyl sensitivity phenotype (Table 3.4). Out of 41
isolates from crosses 6, and 8, recombination was observed in 3 isolates that were
sensitive to metalaxyl. 12 isolates were resistant and 26 isolates were intermediate (Table
3.4). In cross 7, parents were resistant and sensitive to metalaxyl and produced 7
resistant, linterrnediate and 0 sensitive isolates (Table 3.4). In cross 9, both the parents
were intermediate in resistance to metalaxyl and produced two isolates also intermediate
(Table 3.4). In total, 117 isolates were determined as hybrids out of total of 285 progeny

isolates.

3.3.3 Virulence assessment
Virulence testing was completed for all the parents and several progeny isolates selected

at random fi'om the crosses. From crosses 1 through 6, 5 to 10 progeny isolates were
chosen per cross. Among these a few isolates were identical to the parents and some were
recombinants (Table 3.4). The parental isolates chosen were similar in race structure and
by having common virulence genes. Pi 02-007, Pi 62-02 and Pi S1-3 were virulent on all
11 R genes (LBRl to LBRll). Pi 98-1 was virulent on 1, 4, 5, 9, 10, and 11. Pi 41-02
had the virulence factors 1, 3, 4, 5, 6, 7, 8, 9, 10 and 11. Pi 95-3 had only virulence factor
5. Pi Atlantic2N had virulence factors 2, 3, 5, 6, 7, 8, 10, and 11. Progeny were all
pathogenic on at least more than seven LBR differential lines. About 80% of the isolates

were pathogenic on all 11 R gene differentials. All the isolates were shown to be

74

pathogenic by infecting the R0 differential and were able to overcome at least one
virulence factor. Variation was observed in the progeny isolates, by having different R
genes. Isolates with extensive variation because of the absence of R genes were 1_04,

1_05, 1_06, 1_29, 3_14, 3_37, 4_01, 5_12, 5_14, 5_38, 5_41, 5_45, 6_11, and 6_19.

3.4 Discussion
In these studies, progress in understanding the patterns of inheritance was severely

hampered by difficulties in germinating oospores and establishing single-oospore
cultures. For example, Romero and Erwin, 1969 detected high levels of oospore
production in some crosses, but established only 34 single oospore cultures. In another
study with siblings, only 4 of 620 produced oospores were established in pure culture,
thus preventing a study of inheritance in F2 progeny (Romero and Erwin, 1969).
Variation among P. infestans populations in phenotypic characteristics such as growth
rate, colony morphology, aggressiveness and virulence were recognized in the clonal
populations of P. infestans, collected prior to migrations in the 1990’s which were
restricted to asexual reproduction (Graham, 1955; Caten and Jinks, 1968; Zarzycka,
1996). The sources of this variability are not well understood in the absence of sexual
reproduction and genetic markers. Direct observation of germination of selfed oospores
of heterothallic species of Phytophthora has only been reported once before (Brasier et
al., 1975). When A; and A2 mating type isolates are paired, each may produce antheridia,
oogonia or both, which later fuse to form hybrid oospores (Judelson, 1997). Outcrossing
and selfing can both occur within mating cultures (Judelson, 1997). In this study, 12
isolates produced oospores without the presence of the opposite mating type, which were

determined to be self-fertile or homothallic isolates. The genetics of mating type in P.

75

Table 3.1 Number of progeny isolates in compatibility type phenotype categories
resulting from oospores generated by crossing parental isolates of Phytophthora
infestans with different compatibility types and tolerance to temperature.

 

Number of progeny isolates in

 

Thermal Compatibility type categories“
tolerance t e Self fertile

Crossa 0f parent Arc Azd (SF?
1. Pi 41-02 X Pi 02-007 T X T 9 23 5
2. Pi 41-02 XPi 02-007 TXT 17 31 3
3. Pi 41-02XPiSl-3 TXT 27 21 3
4. Pi41-O2XPi Sl-3 TXT 36 14 0
5. Pi41-02XPi 98-1 TXI 29 15 l
6. Pi 62-02 Pi 02-007 T X T 8 l7 0
7. Pi 95-3 X Pi 02-007 S X T 4 4 0
8. Pi 41-02 X Pi AtlanticZN T X S 14 2 0
9. Pi 4-19 X Pi AtlanticZN S X S 2 O O

 

a P. infestans isolates used as parents to produce single oospore progeny (A; X A2).

b Comparison of grth of isolates of cultures of Phytophthora infestans for 25 days at
18°C after exposure at -5°C for 5 days measured by an image analysis technique
comparing the growth of exposed isolates to a positive control (exposed to 18°C for 5
days) or a negative non-inoculated control with no growth. T= Tolerant; grth not
significantly different from the growth of the positive control cultures of the same isolate;
I= Intermediate; growth significantly less than that of the growth of the positive control
cultures of the same isolate but significantly greater than the negative control; S=
Sensitive; growth not significantly different from the negative control.

c Number of isolates produced that were A1 compatibility type.
d Number of isolates produced that wereA2 compatibility type.

° Oospores that were produced with both A1 and A2 tester isolates and also in pure
cultures.

76

Table 3.2 Different progeny and parent isolates showing either presence of self-
fertile oospores in pure cultures after one month of incubation resulting from
oospores generated by crossing parental isolates of Phytophthora infestans with
different compatibility types and tolerance to temperature.

 

 

Cross 1 Cross 2 Cross 3 Cross 4
1.01 03 2m + 3.01 0 4m +
1-02 + 2-03 + 3-05 + 4-02 0
1-03 + 2-05 0 3-06 0 4-03 0
1-06 + 2-06 + 3-07 0 4-04 +
1—07 0 2-07 + 3-09 0 4-05 0
1-08 + 2-08 + 3-1 1 + 4-06 +
1-10 0 2-09 + 3-12 + 4-07 0
1-13 0 2-10 + 3-13 + 4-08 +
1-14 + 2-11 + 3-14 0 4-09 +
l-15 + 2-12 0 3-21 + 4-10 +
1-16 + 2-17 + 3-27 0 4-11 . +
1-17 0 2-19 0 3-37 + 4-12 +
1-19 + 2-20 + 3-41 + 4-13 0
1-20 + 2-23 + 3-42 0 4-14 0
1-21 + 2-24 0 3-44 0 4-15 0
1-22 + 2-25 + 3-46 0 4-16 0
1-23 + 2-26 0 3-47 0 4-17 0
1-24 + 2-27 + 3-48 0 4-18 +
1-26 + 2-28 0 3-49 + 4-29 0
1-27 0 2-30 + 4-32 0
1-28 + 2-32 + 4-34 0
1-29 + 2-33 0 4-36 0
1-30 + 2-34 + 4-40 0
1-31 0 2-35 + 4-41 +
1-34 0 2-36 + 4-44 0
1-36 + 2-37 + 4-45 0
l-A 0 2-38 + 4-46 0

2-39 + 4-48 +
2-40 0 4-51 +
2-41 +
2-44 +
2-46 0
2-47 +
2-48 0
2-49 +
2-50 0
2B.A 0
2B.B +
ZB.C +

 

a “+” = Self-fertile oospores present in pure cultures, “0” =

cultures

77

Self-fertile oospores absent in pure

Table 3.2 continued

 

 

Cross 5 Cross 6 Cross 7 Cross 8 Cross 9
5-04 0 6-06 + 7-01 + 8-02 + 9_01 0
5-05 0 6-07 + 7-03 0 8-03 0 9_02 0
5-06 0 6-08 + 7-05 0 8-05 0
5-07 0 6-1 1 0 7-08 + 8-06 +
5-08 0 6-12 + 7-09 + 8-08 + Parents
5-10 0 6-13 + 7-18 0 8-09 0 Pi 41-02 0
5-11 0 6-14 0 Pi 81-3 0
5-12 0 6-15 0 Pi 62-02 0
5-14 0 6-16 0 Pi98-1 0
5-15 + 6-17 0 Pi 95-3 0
5-16 0 6-18 + Pi 4-19 0
5-17 + 6-19 + Pi 02-007 1
5-19 0 6-20 +
5-20 + 6-22 +
5-21 0 6-23 +
5-23 0 6-21 +
5-24 0 6-24 0
5-25 0 6-25 0
5-26 0
5-27 0
5-29 +
5-31 0
5-32 0
5-33 +
5-35 0
5-36 +
5-38 +
5-39 0
5-40 0
5-41 0
5-42 +
5-43 0
5-46 0

 

‘ “+” = Self-fertile oospores present in pure cultm'es, “0” = Self-fertile oospores absent in pure

cultures

78

 

 

Figure 3.1 An isolate of P. infestans showing levels of sensitivity to metalaxyl at
different concentrations: A= Control or 0; B= 0.1; C= 1; D= 10; E= 100; F= 1000
PPM metalaxyl.For example isolate 1-4 is intermediately resistant to Metalaxyl
with ECso: 5 PPM

79

Table 3.3 Number of progeny isolates in metalaxyl sensitivity phenotype categories
resulting from oospores generated by crossing parental isolates of Phytophthora
infestans with different metalaxyl sensitivity.

 

Number of progeny isolates in
metalaxyl sensitivity categoriesb

 

Metalaxyl Resistant Intermediate Sensitive
Crossa Sensitivity Parentb (B) (1) £3)
1. Pi41-02XPi 02-007 RXR 20 15 2
2. Pi4l-02XPi 02-007 RXR 38 13 0
3.Pi41-02XPiSl-3 RXR 10 31 10
4. Pi41-02XPi $1-3 R’XR 8 30 12
5. Pi4l-02XPi 98-1 . er 9 36 0
6. Pi 62-02 x Pi 02007 R x I 8 16 1
7. Pi 953 X Pi 02—007 s x R 7 1 0
8. Pi Atlantic2N x Pi 41-02 I x R 4 10 2
9. Pi AtlanticZN x 449 I x I o 2 0

 

a P. infestans isolates used as parents to produce single oospore progeny (A1 X A2).

b Metalaxyl sensitivity phenotypes; R= Resistant; 1: Intermediate; S=Sensitive; the
sensitivity of the isolates was classified by comparing ECso response of the isolates
grown on media amended with different concentrations of metalaxyl with ECso of known
resistant and susceptible isolates. Isolates were termed as resistant (R) when the ECso was
not significantly different from the growth of the resistant reference isolate. Isolates were
termed as intermediate in resistance (I) when the EC50 was significantly less than that of
the ECso of the resistant reference isolate but significantly greater than that of the
sensitive reference isolate; Isolates were termed sensitive (S) when the ECso was not
significantly different from the sensitive reference isolate.

80

 

Figure 3.2 Example of a single replication for assessing the virulence pathotype. LBR gene leaflets
(LBRO to LBRll) showing lesions symptomatic of infection with P. Infestans incubated at 18°C in a
controlled growth chamber for eight days. The virulence pathotype indicated that isolate 3A lacked
the avirulence genes 0, 1, 2, 5, 7, 8, 10, and 11

81

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infestans is complex and the A1 : A2 ratio in progeny can vary significantly, ranging from
close to 1:1 to an excess of A1 over A2 (Shaw 1991; Judelson et al., 1995). It was quite
difficult to understand if the progeny isolates were self fertile or hybrid, as they have a
tendency to change their compatibility type, from being heterothallic (A1 or A2) to
homothallic. In the first two weeks after isolation, about 70% were hybrids with
characteristics of the parents and the remainders were self-fertile. After one month, 50%
of the progeny were selfs or homothallic and the other 50% were heterothallic. Further
differentiation of these oospores generated from from outcrossing and from cultures
arising from residues of parental mycelia was attempted by the segregation of SSR

markers (Chapter 2).

In previous studies the frequency of oospore-derived cultures that resulted from selfing
ranged from low (Shattock et al., 1986) to more than half of all cultures (Whittaker et al.,
1994). When antheridia and oogonia from the same parent fuse they form self-fertile
oospores. The gene control of pathogenicity and virulence is not yet defined and the
mechanisms of variation are still ambiguous (Shattock et al., 1985). Early studies
proposed that one locus determined mating type (Galindo and Gallegly, 1960), but this
could not be reconciled with the widely varying ratios of A1 and A2 types observed in
crosses (Shaw, 1991). Later studies using linked DNA markers confirmed the
involvement of a single locus and indicated that it displayed non-Mendelian inheritance
in most isolates (Judelson et al., 1995). In cross 5, the A1 mating type was dominant in
progeny isolates. When this phenotypic character was compared with the genotypic

character for this cross (GPI isozyme type), they both showed that the parent, Pi 41-02

83

(100/100) was more prevalent than Pi 02-007 (Chapter 2). The progeny selfs were either
intermediate or resistant as were the parents, and true sexual recombinants had a sensitive

response to metalaxyl.

Variation between isolates in their resistance to fungicide, aggressiveness on R gene
foliage, and pathogenicity on tubers may lead to an understanding of the different factors
that plants use for defense. In the evolution of field isolates and genetic diversity of
populations, not only outcrossing but also selfing should be considered. The latter process
may be a more likely event in populations, assuming that selfing is similar in frequency

in planta as observed in vitro (Knapova et al., 2002).

Inheritance of resistance to metalaxyl has been described as being controlled by one
single, incompletely dominant gene (Shaw and Shattock, 1991). In another report,
resistance to metalaxyl was associated with one semi-dominant locus combined with the
effect of several minor loci resulting in a continuous segregation for sensitivity in F1
progeny (F abritius et al., 1997). In a separate study, one parent was heterozygous with
one single gene carrying a dominant allele, and the segregation was Is: 1r in F1 progeny
and 1s:3r in F2 progeny (Lee et al., 1999). The distortion from the Mendelian distribution
may be explained by the effects of minor loci (Fabritius et al., 1997) or the presence of
more than one major locus for metalaxyl resistance (J udelson and Roberts, 1999). It is not
known how frequent the various resistant alleles are in resistant individuals of different
origins, but it is apparent that sensitive offspring will always appear afier recurrent sexual

recombination in subsequent generations, and resistant individuals will not disappear

84

independently of the model of inheritance described above (Knapova et al., 2002). In this
study, the progeny from the crosses had different inheritance ratios and did not follow
Mendelian segregation, agreeing with J udelson and Roberts (1999), implying that there is

more than one locus for metalaxyl resistance.

Virulence is usually considered as the most important phenotypic marker in studying
most plant pathogens; however virulence/avirulence loci represent only a small portion of
the total genetic variation that might exist among different races within a plant pathogen
population. The term complex virulence has generally been used to describe isolates
carrying more than one virulence gene (Umaerus et al., 1983). Multiple population
studies have reported an increase in virulence complexity in association with the
emergence of ‘new’ populations of Phytophthora infestans (Goodwin 1997; Dorrance et
al., 1999), although others suggest all known virulence factors were present at a much
earlier stage (Day and Shattock 1995). Isolates from the US-8 and US-14 groups
contained more than six virulence factors, with US-lO containing five and other
genotypes contain less than three. These results agree with other studies where US-8 and
US-14 genotypes on average contain higher numbers of virulence factors than ‘older
genotypes’ such as US-ll (Dorrance et al., 1999) and US-6 (Goodwin et al., 1995).
Isolates originating from Northen Ireland Pi 41-02 and Pi 62-02 are aggressive and also
had 10 and 11 virulence factors, respectively. Since most parents from this study
contained highly complex isolates, any effect of incompatibility with R genes is likely to
be small. Just like the parents, US-8 progeny isolates had all the 11 virulence factors;

several progeny from this parent had all the eleven virulent factors, which could be

85

because of selfing, as they had the same virulence phenotype. From Van der Plank’s
theory of stabilizing selection, races with the fewest unnecessary virulence genes will be
the most fit (Van der Plank, 1968). Since the majority of planted commercial cultivars in
Europe and the United States contain few if any R genes (Fry and Goodwin, 1997) most
virulence genes would be deemed unnecessary and therefore very simple races (such as
100/122 and 86/100 from this study), should be fittest. In disagreement with the theory
Tooley et al., (1986) assessed fitness on simple hosts (i.e. containing few R genes) using
sexual and asexual populations of P. infestans and detected no higher fitness associated
with fewer unnecessary virulence genes. More recent studies have also detected no
evidence of any correlation between fitness and virulence phenotype (Schober and
T urkensteen, 1992; Lebreton et al., 1999). Although an important phenotypic test for
selection studies, R gene testing has certain weaknesses as regards general population and
tracking investigations. Multiple studies have highlighted the importance of evaluation
conditions, with inoculum concentration, temperature, age of plant and state of the
pathogen all shown to influence results (Swiezynski et al., 2000). Findings have been
inconsistent between laboratories and even between individual isolates (Schick and
Schick, 1959; Schober and Turkensteen, 1992). Additionally variability in virulence
among sexual progeny (most likely due to mitotic recombination) has led to the
hypothesis that P. infestans has the capacity for continuous change in R gene
compatibility (Abu-E1 Samen et al., 2002). For all of these reasons only a few progeny
isolates were tested for compatibility with R genes as a means of tracking inheritance in

this study.

86

Sexual recombination will produce new genetic combinations, creating the possibility of
unusually fit or pathogenic individuals or individuals with novel ecological
characteristics. This possibility has been termed the “Red Queen hypothesis (Barton et
al., 1998) and is particularly important for organisms that reproduce both asexually and
sexually (Mayton et al., 2000). Shattock et al., (1985) presented data that indicated that
selfed oospores of A1 isolates of P. infestans produced in vitro by intraspecific
stimulation are viable and will germinate to produce mycelial colonies. If interspecific
stimulation occurs in natural populations of Phytophthora, then selfing may generate
variation in populations of P. infestans of a single compatibility type. Oospores formed at
the beginning of epidemics could therefore contribute to the emergence of novel

phenotypes within a growing season as well as between seasons (Drenth et al., 1995).

87

4 Cold temperature tolerance of mycelium in progeny isolates of
Phytophthora infestans from mating

4.1 Introduction
Potato late blight epidemics caused by Phytophthora infestans are associated with wet

conditions and moderate temperatures (Harrison, 1992). Temperature can influence spore
germination, mycelial growth rate, inoculum production and survival of P. infestans. The
range of temperature at which predominant genotypes of P. infestans in the US and
Europe can develop and grow is relatively wide and recent estimates indicate that the
range for mycelium may be as extensive as -5 to 35°C (Zwankhuizen et al., 1998; Kirk,
2003b). There have been fewer studies of the temperature range for spore development
and other components of epidemic development for these predominant genotypes and
cm'rent estimates are known mainly for pan-global clonal lineages (U S—l; Goodwin et al.,
1995) although this lineage is seldom recovered in nature. The thermal profile for this
pan-global lineage was reported by Harrison (1992); indirect sporangial germination
[formation of zoospores (5 to 10 per sporangium)] was favored below 20°C with the
optimum between 12 to 13°C and direct germination was favored at above 20°C,
(optimum 24°C; Mizubuti and Fry, 1998). Recent reports suggest that predominant
genotypes of P. infestans have variability in the dynamics of thermal responses may
affect epidemic development (Kirk 2003a, 2003b). Many investigators have used in vitro
and soil assays to study the optimal and lethal upper temperatures for growth of
Phytophthora spp. (Bollen, 1985; Juarez-Palacios et al., 1991; Coelho et al., 2000; Snider
et al., 2000). The survival of P. nicotianae chlamydospores in soil was reduced
considerably when temperatures were raised above 45°C (Coelho et al., 2000). Few

studies that systematically examine the ability of P. infestans mycelium to survive at

88

temperatures below 0°C exist although De Bruyn (1926) showed that a mycelial colony
was capable of withstanding temperatures as low as -25°C for up to 5 days in manure-
based substrate and that in dried substrates survival at such low temperatures was
enhanced and Kirk (2003b) showed that several genotypes of P. infestans could

withstand exposure to temperature close to freezing for at least 5 days.

The overwintering of P. infestans is an important epidemiological factor in temperature
agroecosystems (De Bruyn, 1926). Mycelium of P. infestans can initiate crop infections
over successive years by over wintering as mycelium in infected potato tubers or within
cull piles or volunteer tubers (Easton, 1982; Zwankhuizen et al., 1998). Economic losses
due to late blight result from foliar defoliation as well as tuber infection, and severe
storage losses can occur after tubers infected with P. infestans are held for processing at
temperatures in excess of 7°C (Kirk et al., 2001). Tolerance of freezing temperatures by
mycelium of P. infestans may be an ecologically important survival mechanism and the
increased tolerance of US-8 and US-14 may explain their predominance in cooler winter
climates such as north-central United States (Kirk, 2003a). It is difficult to estimate the
probability of infected potato stems or foliage will emerge from an infected tuber, as
there are several factors that could influence the fate of infected tubers, temperature being
one of the most important (Monteith, 1977; Kato et al., 1997). Many investigators have
used in vitro and soil assays to study the optimal and lethal thermal temperatures for
growth of Phytophthora spp. (Bollen, 1985; Coelho et al., 2000). Perches and Galindo

(1969) found soil to be infectious 2-years after a late blight infection. For example,

89

oospores were found to survive in soil for 5 to 7 months at temperatures between 0 and

20°C (Pittis and Shattock, 1994).

An increased risk of potato late blight epidemics in the same region has been suggested
over the period from 1950 to 2000 (Baker et al., 2005). If, seasonal soil temperature is
moderating as speculated (and measured), especially over the winter period the risk of
survival of volunteer tubers in the soil, in culls and in rock piles will increase in Northern
U.S. production areas over winter and the risk of tuber-vectored potato late blight will
increase. As it has been shown that P. infestans is tolerant to temperature close to
freezing (Kirk, 2003a), this therefore creates a situation for both the host and the
pathogen to survive over winter. This could result in an increase in the emergence of

potato plants carrying initial inoculum that could start a seasonal epidemic.

Weather conditions such as temperature and precipitation will affect the survival of P.
infestans during winter months and can potentially affect disease development in the
summer (Easton, 1982). Climate data illustrate that, in Michigan, warmer winters are
accompanied by lower winter precipitation (Isard and Schaetzl, 1998). There is generally
a greater incidence of soil fieezing in Michigan during warm dry winters due to recurring
freeze-thaw episodes (Isard and Schaetzl, 1998). Isolates of P. infestans vary in their
ability to survive winter freezing (Shattock, 1976; Kadish and Cohen, 1992; Kirk,
2003b). It has been suggested that the pathogen population and variability is severely
reduced during winter, but the variability is regenerated each year within the expanding

population (Shattock, 1976).

90

The production of oospores by P. infestans in the field and their survival under natural
conditions has become important since the introduction of A2 mating type isolates.
Oospores of oomycetous fungi are, in general, endogenously dormant, tolerant of adverse
conditions, and capable of long-term survival (Ribiero, 1983). Oospores of P. infestans
have been found to survive in soil during a winter season in Europe and oospores also
have been reported to survive temperatures between -80 and 35°C (Drenth et al., 1995).
Genetic recombination in P. infestans through the sexual process (production of
oospores) has been shown to occur (Lees et al., 1997). The presence of oospores in the
soil as a source of inoculum will drastically change present disease management
strategies (Umaerus and Umaerus, 1994). Oospores of Phytophthora spp. are insensitive
to fungicides (Duncan, 1985b) but sensitive to heat treatments of 45°C (Duncan, 1985a).
Despite many studies (Ribiero, 1983), factors affecting germination of oospores are not
well understood. The objective of the project was to conduct an in vitro study to evaluate
the inheritance of cold temperature tolerance in the oospore generated isolates (progeny)
after mating of P. infestans because of the convenience of using oospores as a tool for
studuyng this inheritance. Given these scenarios the objectives of this study were to
investigate:

I The survival potential of mycelium of different genotype isolates of P. infestans
exposed to temperature at -5°C for 5 days in vitro using a digital imaging
technique

I Carry out crosses between P. infestans isolates (A1 and A2 mating types) that were

highly variable for temperature tolerance, and determine if temperature tolerance

91

was inherited or occured as a result of physiological adaptation of different

genotypes of P. infestans

4.2 Information from previous studies (Kirk lab) that helped in this research

Previous research conducted at the Kirk lab at the department of Plant Pathology,

Michigan State University was utilized to expedite the project.

4.2.1 Substrate optimization and digital image analysis
Sterol-free clarified rye agar (SFCRA) was prepared by washing 100g organically

produced rye seed, boiling for 1 h in dHZO, straining the suspension through four layers
of cheese cloth and adding 15g agar, 7.5g sucrose and deO to bring the solution to 1L
final volume. The optimal volume of sterol-free clarified rye agar (SFCRA) that could be
added to 60 mm diameter Petri dishes in order to measure the reflectance characteristics
by digital image analysis (described below) was determined. Volumes of 5, 10, 15 and 20
ml SFCRA were poured into Petri dishes. The reflectance intensity of the Petri dishes
with different volumes of SFCRA was then determined by digital image analysis. The
scanned images were the SF CRA Petri dishes without cover lids. The Petri dishes were
placed open-surface down on a glass plate, 40 x 30 x 0.2 cm thick and digital images
generated and analyzed as adapted from Niemira et al., (2001). The average reflective
intensity (ARI) of all the pixels within the image gave a measurement of the plate without
any growth of the sample. The calibrations resulted in optimal agar volumes of 10 ml

substrate/60 mm diameter Petri dishes with mean ARI = 90.

92

4.2.2 Temperature optimization
Temperature exposure studies were performed at different temperatures close to 0°C in

Peltier chambers [PTC-l Peltier-effect temperature cabinet (PTC-l) controlled by a
PELT-3 Peltier-effect temperature controller (FELT-3); Sable Systems International,
2887 Green Valley Parkway #299, Henderson, NV 89014]. Temperature equilibration
was measured after the door of the PTC-l cabinet was opened, and at 0°C set

temperature, temperature rose to 5°C after the door was opened but then dropped to

-0.3°C in 1.5 hr. At -3, -5, -10 and -20°C set temperatures, temperature rose to -1.2, -1.5,

-3.5 and -4.8°C and recovered in 1.1, 1.3, 1.5 and 1.4 h, respectively. Sample cultures of
P. infestans were placed in the PTC-l cabinets and exposure times were measured from
when the set temperature was reached. Sample cultures were removed after exposures of
1, 3, 5, and 10 days. Temperature treatments were 0, -3, -5, -10 and -20°C. After plates
were removed from the PTC-l cabinet they were stored in the dark at 18°C. After 25 days
the samples were scanned to determine amount of growth of mycelium. P. infestans
cultures survived -3°C for 5 days, and a few cultures have survived at -5°C afier 1 day

(Kirk, 2003b).

There was not much variation noticed in the temperature tolerance when the isolates were
tested at -3°C for five days and -5°C for one day. Based on these results, to observe more
variation the temperature exposure used in this study to determine temperature tolerance

was -5°C for 5 days.

93

4.2.3 Determination of developmental stage of sporangia on mycelial plugs after

transfer from parent cultures

As the objective of the experiment was to determine the influence of duration of exposure
of temperatures to -5°C on mycelial survival (described below), it was necessary to
determine the length of time for all sporangia to germinate afier transfer of mycelial plugs
from parent cultures. P. infestans cultures of each genotype were grown on Sterol-Free
Clarified Rye Agar (SFCRA) for 14 days in the dark at 18°C. Ten 5 mm-diameter cores
were removed from the growing edge of each of 10 plates. The cores were placed into 10
ml distilled H20 in test tubes and stirred on a magnetic stirrer for 1 hour to dislodge
sporangia. The suspension was strained through four layers of cheesecloth and the
concentration of sporangia was calculated using a hemacytometer. The number of non-
germinated and germinated sporangia was counted at the time of transfer and again two
days after incubation at 4°C. Afier two days only empty sporangia were observed in any
of the genotypes tested and a pre-treatment of two days incubation at 4°C in the dark was

imposed on all freshly transferred cores prior to exposure to temperature treatments.

4.2.4 Relation between digital image of samples and mycelial weight

Mycelium of P. infestans is white and the average reflectance intensity (ARI) of a digital
image of a mature culture measures about 150 - 170 LIU. To determine the relation
between ARI of cultures of P. infestans growing on SFCRA and fresh weight of the
cultures, a further calibration was attempted. Sterile cellophane discs (40 mm diameter)
were placed over SF CRA and 5 mm-diameter plugs of mycelium were transferred to the

center of the plates (11 = 12 per genotype tested). Sample plates were incubated in the

94

dark at 12°C and every two or three days after the transfer until 28 days after the initial
transfer, two plates of each isolate were harvested. The ARI was determined for each
plate. The weight of the culture was determined by weighing the total mycelium plus
cellophane and initial transfer plug and subtracting the weight of the cellophane plus
initial transfer plug. ARI was expressed as a function of the flesh weight of the culture
for each genotype to determine the ARI at which no grth occurred. This threshold was
used as an indication to determine qualitatively if cultures were alive. The relation
between the ARI and weight of individual cultures was determined by linear regression

(SigrnaStat ver. 2.03, J andel Scientific, San Rafael, CA).

4.3 Materials and methods

4.3.1 Isolate descriptions
Cultures of different genotypes of P. infestans were originally isolated from foliage of

potato plants grown in north central/Great Lakes region of the US with symptoms of late
blight and maintained in the Kirk collection at Michigan State University (MSU). The
identity of the genotypes was determined by isozyme analysis (chapter 2), metalaxyl
sensitivity and mating type (Chapter 3). The isolates were maintained on sterile rye seed
stock cultures. For each experiment, isolates were re-propagated from the long-term
storage stock culture. The different isolates tested for the temperature tolerance were

shown in Table 4.1.

In order to increase diversity of the phenotypic characteristics of isolates of P. infestans
some isolates collected from Northern Ireland, UK were used. These isolates were from

the collection of G. Young and were maintained under USDA PPQ permit number P526-

95

061106-014 in the MSU collection in the Kirk laboratory. These isolates had been

previously described and were A1 mating type (Chapter 3).

4.3.2 Determination of thermal tolerance
An image analysis technique developed by Kirk (2003b) was used to determine the

thermal tolerance of the isolates of P. infestans. Several procedures and stages were
followed including substrate optimization; determining the thermal exposure treatments,
and generation of the images; analyses of the images and quantification of the responses.
Cultures were prepared using 10 ml SFCRA in each Petri dish (Appendixl) so that the
distance between the scanner surface and media in the plate was the same throughout all
experiments. This was important to ensure that the brightness of the images was
consistent throughout the experiment since radial growth of the fungus in the images was
to be assessed using image analysis and optimizing the image clarity during the image

analysis process (see section below).

Temperature exposure experiments were done over a five-day period. All the plates were
labeled with culture II) numbers and exposure times, bound together with Parafihn. For
every isolate 15 cultures were prepared; 10-Petri dishes were inoculated with a 5 mm
diameter plug of agar plus mycelium from a stock culture (described above). Plates were
incubated at 4°C for two days in the refrigerator as an acclimatization period. Test
isolates were moved to -5°C in the PTC-l cabinets for five days. Five of these plates were
incubated at 18°C in an incubator (Advanced Intellus Environmental Controller, Percival
Scientific, Inc. Perry, IA 50220 USA) for 30 days as positive controls for statistical

comparisons; five cultures (the test isolates) were placed together on a plastic loading

96

tray and then incubated in PTC-l cabinets set at -5°C located either in a cold-room (set at
4°C) or in a growth chamber set at 4°C. Five negative control plates (non-inoculated)
were prepared by adding a 5-mm diameter plug of SFCRA without any mycelium and
incubated along with the test isolates. On the sixth day, culture-plates were removed fi'om
-5°C environment and moved to the 18°C incubator with the control cultures. All the

cultures were incubated for a further 25 days.

The FELT-3 was set for the exposure temperature 2 h prior to the start of the experiment ‘
(Figure 4.1). The tray with the culture-plates was placed into the PTC-l cabinet quickly
to minimize temperature increase. Temperature equilibration was measured after the
door of the chamber was opened, and at 0°C set temperature, temperature rose to 5°C
after the door was opened but then dropped to 03°C in 1.5 hr. At -5°C set temperature,
temperature rose to -1.5 and recovered in 1.3h. Exposure time was measured from when

the set temperature was reached.

After 25 days the samples were scanned to determine amount of growth of mycelium.
The experiment was repeated twice over the period fiom December 2005 to February

2006 for a total of 15 estimations of thermal survival for each test-isolate of P. infestans.

4.3.3 Image analysis technique
Images of the cultures were scanned and digitally analyzed to measure the Average

Reflective Intensity (ARI) in light intensity units (LIU). The lids of the plates to be
assessed (with and without growing cultures) were removed and placed face down on a

flat bed scanner (Epson Perfection 4870 PHOTO; Epson Inc.) and the image was

97

 

Figure 4.1 C-l Peltier-effect temperature cabinet (FTC-1) and a PELT-3 Peltier-effect temperature
controller ln a controlled growth chamber used to maintain the temperature in the Peltier chamber
at -5°C.

98

scanned, imported into Photoshop (Adobe Inc.), and saved in the JPEG file format. All
images were captured at 11.8 pixels per mm, which gave a final image 2547 pixels wide
by 3509 pixelshigh. The plates were scanned against a black background so that images
were formed from light reflected off the surface of the media. To ensure consistency
between the images, 10 m1 of SCFRA was used in each Petri dish (Appendixl) so that the
distance between the scanner surface and media in the plate was the same throughout all
experiments. Image analysis was carried out using the image analysis software Sigma
Scan Pro 5 (SPSS Inc. IL, USA). The image files created with Photoshop were loaded
into the image analysis soflware. The software was then calibrated on the basis that the
width of the glass screen of the flat bed scanner was 219.5 mm and this was equivalent
to 2547 pixels. The image pixels in the black background have a brightness of 0 light
intensity units (LIU), while pure white pixels have a brightness of 255 (LIU). Using the
“eye dropper” tool in Photoshop it was determined that the brightness of the pixels
making up the image of the PDA media were no brighter than 90 LIU, while the fimgal
hyphae had a brightness above 91 LIU. So the area selection cut-off threshold in the
Sigma Scan Pro 5 software was set to 90 LIU. This effectively allowed the software to
exclude all parts of the image darker than 80 LIU (e. g. the Rye media surrounding the
hyphal growth). By selecting the “fill” tool and clicking on the fungal colonies within the
image all the pixels with an LIU above 80 were selected. The scans were digitally
analyzed to measure the ARI in LIU using Sigma Scan software and this value was then

automatically added to a spreadsheet by the image analysis software

99

 

4.3.4 Production of progeny isolates of Phytophthora infestans from mating

4.3.4.1 Production of oospores
Oospores were produced from the crosses among cold tolerant (T), cold intermediate (1)

and cold sensitive isolates (S). These isolates used to produce oospores were referred to
as parental isolates and were transferred to Petri plates (100 X 15mm diameter)
containing rye agar amended with 0.1g/ltr beta-sitosterol (Spectrum Chemical MFG.
Corp., CA 90248). Mycelial plugs (3 mm diameter) of each of the two selected isolates in
a cross were placed on opposite sides of 100 x 15mm Petri dishes containing 25 m1 rye
agar. Crosses were replicated five times for each cross. Three agar plugs of 2mm
diameter were taken from a fast growing colony (parental isolate 1) and were placed at
the edge of the agar on the Petri plate. Three mycelial plugs from the parental isolate 2
were placed about 3 cm apart at 90, 180 and 270° from parental isolate 1. Using three
plugs of parental isolate 2 increased the potential for oospore production. Plates were
sealed with parafilm and were incubated in dark for 1 to 2 months and were examined
weekly for the presence of mature oospores in the zone of contact between the two
colonies using an Olympus BX41 System microscope (Olympus America Inc., NY

1 1747-3157, USA).

4.3.4.2 Extraction of oospores
Plates with abundant oospores were prepared for extraction. The mating region was

marked with a permanent marker on the bottom of the Petri dish and the mating region
was excised using a scalpel. The agar pieces containing the mating region were
transferred to sterile 50 ml Eppendorf centrifuge tubes, containing 9 ml sterile distilled

water and then ground with a homogenizer (IKA-Ultra-Turrax, T 25 basic, with the

100

 

dispersing tool, S25N — 8G, IKA WORKS, INC. SE, Wilmington, NC 28405) for 2-min
in the laminar flow cabinet, using standard sterile technique. Probes were sterilized for
30-s at 20,000 rpm by mixing in a beaker containing 96% ethanol. The probe was then
rinsed twice with double distilled water. The agar pieces containing the mating region
were blended for 60-8 at 20,000 rpm, making sure that the mixture did not spill out. The
oospores were purified by digesting mycelia with a lysing enzyme (L1412, Sigma; from
Trichoderma harzianum). Lysing solution was prepared by mixing 0.1 g of lysing enzyme
(Ll412 Sigma) in 10 m1 double distilled water and 1 ml of the lysing solution was added
to 9 ml of the oospore suspension. Lysing enzyme was used to digest sporangia and
mycelial fragments. Tubes were incubated at 18°C for 40-h, in the dark. The solution of
oospores was washed three times by adding 8 ml water each time, spinning down the
oospores using a centrifuge (RC5C, DuPont, Sorvall instruments, Newton, CT, USA) for
5-min at 5,000 rpm. Supernatant was poured off carefully after each spin and finally, the
pellet containing oospores was re-suspended with 10 ml double distilled water. Oospore
concentration was quantified by counting oospores in three 50 pl aliquots using a
hemacytometer. The concentration of the solution of oospores was adjusted to 1 x 103
spores/ml and used to inoculate water agar plates with 1 ml of the diluted suspension.
Plates were incubated under continuous fluorescent light at 22°C for 5-d. Plates were
examined for germinating oospores using the BX41 System microscope and were
transferred on to a fresh rye agar plate. Single oospore cultures (50) were collected from
each cross. Each isolate was numbered and the first number referred to the cross it came
from e.g. cross 3 isolate 06 = 3_06, 5_11. These isolates were referred to as progeny

isolates.

101

 

4.3.4.3 Oospore germination
A sample of 1 ml was drawn from the oospore suspension and spread on a 100 x 15mm

Petri plate containing 15 ml water agar (2% agar, Sg/L) amended with 20mg/L
Rifampicin"; SOmg/L polymixin B*; SOmg/L Vancomycin“; 100mg/L Ampicillin*; and
100mg/L Benomyl* (* = dissolved in 100% DMSO). Plates were bound with parafilm
and incubated for l4-d at 20°C under cool blue fluorescent light. Oospore germination
was assessed by inverting the plate and observing if germination had occurred using the
Olympus BX41 System microscope at a magnification of 10X. Gerrninated and well-
separated oospores were marked and transferred to fresh Rye B plates, and then pure
cultures were obtained. Each germinating oospore (Fig. 4.2 and 4.3) was considered as a
single progeny isolate. From each cross 50 oospores were isolated and labeled from 1 to
50, along with the cross number. All the progeny isolates from each cross were tested for
mating type and temperature tolerance and the genotype was determined by isozyme
analysis (Chapter 2). Additional tests such as metalaxyl sensitivity, virulence tests, and

pathogenicity tests were also completed on parental and progeny isolates (Chapter 3 & 5).

4.3.5 Data analysis
To determine if mycelium of P. infestans isolates survived exposure to the thermal

treatment, the ARI of treated mycelium was compared to the ARI of the non-inoculated
control (negative control) and the inoculated control (positive control) grown at 18°C for
comparable periods to determine the relative degree of survival. Combining these
comparative outcomes resulted in a qualitative measure of survival; if the ARI of a
sample was not significantly different at P = 0.01 (NSD) from the negative control the

culture was considered dead and sensitive to exposure (thermally sensitive; S); if NSD

102

A. .rw--..-~.t... " 2.;‘r .“

 

‘. m ‘- .A.

form the positive control the culture was considered unaffected by either temperature
treatment or exposure period (thermally tolerant; T); if significantly different from both
the negative and positive controls the culture was considered affected by the treatment
but not killed (thermally intermediate response; I). The qualitative outcome of
temperature treatments resulted in assignment of the parental isolate to a category for
future crossings with other P. infestans isolates of opposite mating types. Comparisons of
survival potential were made among isolates exposed to different temperatures for
variable durations. An index value was assigned for each progeny isolate based on ARI
of each isolate (as described above) in relation to the negative and positive control and

was described by the following equation:

1— _
%RARI=[ In” —x..,

(I... — x37] . 100

where the variation of each replicate’s ARI value, x,-, with respect to the average positive
control, Em, is compared with the total ARI range between the positive and negative

controls, 3r- po, - Eng. When this ratio was equal to 1, the replicate ARI value is equal to
the mean negative control value. By subtracting the ratio from 1 and multiplying the
difference by 100, the percent relative ARI is calculated (%RARI). This percent relative
ARI quantifies the percentage by which the total range in values between average
positive and negative controls is greater than the variation difference between the
replicate value and the average positive control. Index values of temperature tolerance for
progeny isolates from each cross were analyzed by ANOVA to determine if the %RARI
was significantly different from isolates previously characterized as thermally tolerant or

intolerant as described for ARI above.

103

 

Figure 4.2 A germinating oospore of Phytophthora infestans forming mycelium
under 10 X magnification. From Cross 3; ID 3_06.

 

Figure 4.3 An oospore of Phytophthora infestans producing germ tube and
sporangia under 10 X magnification

104

Control Neg. Control

 

Figure 4.4 An example of an isolate of Phytophthora infestans showing tolerance to
-5°C. J PEG images of isolates of Phytophthora infestans were produced in
Photoshop on an Epson Perfection 4870 flatbed scanner. Images were digitally
analyzed to measure the Average Reflective Intensity (ARI) in Light Intensity Units
(LIU) using Sigma Scan software. Left Side: Positive controls, exposed to 18°C;
Center: Test Isolates, exposed to -5°C for 5 days; Right side: Negative controls, non-
inoculated Petri dish.

105

Table 4.1 Isolates of Phytophthora infestans tested for thermal tolerance. Isolates
were exposed to -5°C for 5-days and regenerated for selection as potential parental
isolates with different thermal tolerance phenotypes.

 

 

Isolate Origin Isozyme Mating Thermal
Genotype Type Tolerance“
Pi 02-007 MI, USA 100/111/122 A2 T
Pi 96-2 MI, USA 100/100 Al I
Pi 458 ND, USA 100/111/122 A2 T
Pi 95-3 MI, USA 96/100 A1 S
Pi 98—1 MI, USA 100/122 A2 I
Pi 30-01 Ireland, UK 100/100 A1 T
Pi 41-02 Ireland, UK 100/100 A1 T
Pi 62-02 Ireland, UK 100/100 A1 T
Pi 107-1 MI, USA 100/111/122 A2 T
Pi 3768 Mauritius 100/100 A1 T
Pi 203-9 MI, USA 100/111/122 A2 T
Pi S4-1 1 MI, USA 100/111/122 A2 T
Pi S1-3 MI, USA 100/111/122 A2 T
Pi NI 43/02 Ireland, UK 100/100 A1 T
Pi 284 ND, USA 100/100/111 A1 I
Pi 365 ND, USA 100/100 A1 I
Pi Banam AK, USA 111/111/122 A2 T
Pi 95-7 MI, USA 100/11 1/122 A2 I
Pi 99-2 MI, USA 100/122 A2 1
Pi 103-4 MI, USA 100/111/122 A2 T
Pi 3765 Mauritius 100/100 Al T
Pi 407-6 MI, USA 100/111/122 A2 T
Pi Atlantic 2N MI, USA 100/111/122 A2 S
Pi 46-02 Ireland, UK 100/100 A1 S

 

a T= tolerant; I= intermediate; S= sensitive; Isolates are considered as Tolerant when the
growth is not significantly different fi'om the growth of the positive control cultures of the
same isolate. Isolates are considered as Intermediate when the growth is significantly less than
that of the growth of the positive control cultures of the same isolate but significantly greater
than the negative control. Isolates are considered as sensitive when the grth is not
significantly different from the negative control.

106

Table 4.2 Origin, mating type, thermal tolerance, and GPI-isozyme phenotypes of
isolates of Phytophthora infestans selected for crossing studies. Progeny from these
crosses were isolated to identify if thermal phenotype character was inherited.

 

 

Cross Thermal
Number Isolatea Origin Tolerance
1 Pi02-007 US T

Pi 41 -02 NI T

2 Pi 02-007 US T
Pi 41 ~02 NI T

3 Pi S 1-3 US T
Pi 41 -02 N1 T

4 Pi S 1 -3 US T
Pi 41 -02 NI T

5 Pi 98-1 US I
Pi 41 -02 NI T

6 Pi 02-007 US T
Pi 62-02 NI T

7 Pi02-007 US T
Pi 95-3 US S

8 Pi Atlantic 2N US S
Pi 41 -02 NI T

9 Pi Atlantic 2N US S
Pi 4-19 US S

 

a The first isolate in the cross is the A2 isolate and the second is the A1 isolate of P.
infestans.

107

Table 4.3 Phenotypic and genotypic characteristic of isolates of Phytophthora
infestans used in crosses

 

 

 

Isolate of
Phytophthora Thermal Gpi Isozyme Mating Metalaxyl R gene
infestans Tolerancea Genotypeb Typec Responsed Phenotype"
Pi 41.02f T 100/100 A1 R l,3,4,5,6,7,8,
9,10,1 1
Pi 02-0078 T 100/1 11/122 A2 R 1,2,3,4,5,6,7 I
8,9,10,1 1
Pi s1-3g T 100/1 11/122 A2 1 1,2,3,4,5,6,7,
8,9,10,11 f
Pi 98-13 I 100/122 A2 R 1,4,5,9,10,11 1
Pi 62.02f T 100/100 A1 1 1,2,3,4,5,6,7,
8,9,10,1 1
Pi 954g 3 86/100 A1 5 5
Pi 4.1911 5 100/100 A1 1 1,3,4,5,6,7,8,
9,10,1 1

 

’ Comparison of growth of isolates of cultures of Phytophthora infestans for 25 days at 18°C afier
exposure at -5°C for 5 days measured by an image analysis technique comparing the growth of
exposed isolates to a positive control (exposed to 18°C for 5 days) or a negative non-inoculated
control with no growth . T= Tolerant; growth not significantly different from the growth of the
positive control cultures of the same isolate; I= Intermediate; grth significantly less than that
of the growth of the positive control cultures of the same isolate but significantly greater than the
negative control; S= Sensitive; growth not significantly different from the negative control.

b Glucose -6- phosphate isomerase allozyme genotype; 86/100 (USl), 100/100 (U S-1 .7),
100/111/122 (U S-8), 100/122 (US-14); genotype as designated by Goodwin et al., 1995.

° Mating type determined by production of oospores when grown with isolates of P. infestans of
known mating type.

d Sensitivity to the fungicide metalaxyl, R = resistant, I= Intermediate, S = sensitive determined
by comparing ECso response of the isolates grown on media amended with different
concentrations of metalaxyl with EC50 of known resistant and susceptible isolates.

° Virulence pathotype as defined by Muller and Black 1952.; f Isolates of P. infestans from
Northern Ireland fi'om collection of Dr. Louise Cooke, Plant Pathology, Queens University,
Northern Ireland; 3 Michigan isolates of P. infestans from collection of Dr. W. Kirk, Plant
Pathology, Michigan State University, East Lansing, MI, USA.

b A progeny isolate obtained fi'om the 4th cross, used as a parent because of its sensitive

phenotype.

108

4.4 Results

4.4.1 Testing of thermal tolerance for potential parental P. infestans isolates
There was variation in the tolerance of parental isolates to exposure to temperature for

different durations at -5°C. Out of 24 parental isolates tested 15 were positive for
tolerance to -50C (Table 4.1). Six isolates were intermediately tolerant to -5°C and only

three were sensitive, completely dead when exposed to -5°C for five days. The following
crosses (Table 4.3) were selected and tested for the following phenotypes; 1) opposite
mating type; 2) difference in thermal tolerance; 3) different genotype; 4) different

tolerance to metalaxyl.

4.4.2 Thermal tolerance
Parental isolates were variable in thermal tolerance at -5°C (Table 4.1 and 4.2). The range

in thermal tolerance measured as % RARI of the parental isolates was shown in Figure
2.5. Pi 02-007, Pi 41-02, Pi 62-02, and PiS1-3 were tolerant to -5°C and were used as
thermally tolerant parents. Pi 98-1 was used as an intermediately tolerant isolate. Pi 95-3
and Pi Atlantic 2N were thermally sensitive isolates. Pi 02-007 and Pi 95-3 were used in
all analyses as standards of thermal tolerant and sensitive isolates, respectively for
comparisons of thermal responses. Pi 02-007 had %RARI value of 100.0 and Pi 95-3 had
%RARI value of 1.35 (Fig. 4.5). The isolates used for the crosses were selected based on
mating type and thermal tolerance. All isolates grew at expected rates and densities in

isolation and formed sporangia (data not shown).

The crosses were selected using a selection of thermally tolerant (T) x T crosses;
thermally sensitive (S) x T crosses; thermally intermediate (I) x T; S x T crosses; and S x

S crosses (Table 4.2). Formation of oospores on Rye B agar occurred after 2 weeks of

109.

incubation for most crosses. The number of oospores germinated in vitro varied greatly
among the nine crosses tested. The highest number of oospores were produced in cross 2,
followed by cross 3, 4, 5, 1, 6, 7, 8 and 9, respectively. Cross 9 (S X S) produced about

20 oospores but only two germinated.

Forty percent of the progeny isolates had intermediate temperature tolerance, but the
other 60% were identical in response to one of the parents. The highest number of
germinated oospores was from the T x T crosses (l and 2). The number of oospores that
germinated decreased as thermal tolerance decreased. Generally, the crosses that
produced the greatest number of viable oospores were the most tolerant to -5°C. A total
of 305 progeny isolates were produced from 9 crosses together. Crosses 1, 2, 3, 4, and 6
were the crosses among T x T parental isolates and produced 206 progeny isolates. Cross
5 was a cross between T and I and produced 33 progeny isolates. Crosses 7 and 8
between T and S parental isolates produced total of 24 isolates. Cross 9 between S x S
parental isolates produced only 2 progeny isolates. Other crosses such as intermediate
with intermediate, intermediate with sensitive were attempted, but did not produce any

viable oospores.

In general, the effects of temperature exposure, isolates and their interactions on %RARI
were significant at P = 0.01 in all the crosses. There was variation in the tolerance
between progeny isolates from similar crosses of T x T isolates and had different ratios of
tolerances, even though they were similar crosses. In almost all the crosses, more than

50% of the progeny retained thermal phenotypes identical to their parents. In cross 5, T x

110

u—u-

 

I, the percentage of thermally sensitive progeny isolates was higher than that of thermally

tolerant and intermediately tolerant isolates.

In cross 1 (T x T), there was no significant difference among isolates with %RARI values
from 123.4 to 77.3, 75.9 to 60.7, 58.8 to 52.8 and 40.9 to 0 (Fig 4.6). The isolates that
were defined as thermally tolerant had RARI values not significantly different (N SD)
from Pi 02-007 (%RARI = 100). The isolates that were defined as thermally sensitive had
RARI values NSD from Pi 95-3 (%RARI = 1.35); the same standards were maintained
throughout the analyses. Out of a total of 29 isolates from cross 1, there were 23 x T
isolates, 2 x I isolates and 4 x S isolates (Table 4.4). There was 21% variation in cross 1
and 79% of the progeny isolates were identical in temperature tolerance (Fig. 2.6). Cross
2 (T x T), there was no significant difference among isolates with %RARI values from
107.2 to 53.4, 50.3 to 45, 46.0 to 1.3 (Appendix 4). Out of total 50 isolates fi'om cross 2,
there were 40 x T isolates, 1 x I isolates and 9 x S isolates. There was 20% variation
occurred in cross 2 and 80% of the isolates were inherited (Appendix 4). Cross 3 (T x T);
there was no significant difference among isolates with %RARI values from 111.2 to
59.5, 49.5 to 44, 44.2 to 1.3 (Appendix 4). Out of total 52 isolates fi'om cross 3, there
were 37 x T, 1x I and 14 x S isolates. There was 29% variation occurred in cross 3 and
71% of the isolates were inherited (Appendix 4). Cross 4 (T x T), there was no significant
difference among isolates with %RARI values from 105.3 to 47.5, and 47.5 to 1.3
(Appendix 4). Out of total 50 isolates from cross 4, there were 37 x T and 13 x S isolates.
There was 1 intermediate isolate (4_49) produced which shared %RARI value with both

tolerant and sensitive control isolates. There was 26% variation occurred in this cross and

111

 

74% of the isolates were inherited. In Cross 5 (T x I), there was no significant difference
among isolates with %RARI values from 100 to 96.4, 45.1 to 36.1, and 24.6 to -1.5 (Fig
4.7). Out of total 29 isolates from cross 5, there was 1 x T, 2 x I and 26 x S isolates.
There was 89% variation occurred in this cross and 11% of the isolates were inherited.
Isolate 5_04 has %RARI value not significantly different fi'om the control intermediate
isolate (Pi 98-1) and sensitive isolates (Pi 95-3). Cross 6 (T x T), there was no significant
difference among isolates with %RARI values from 104.1 to 58.9, 53.2 to 53.1, and 43.9
to 0.0 (Appendix 4). Out of a total of 19 isolates from cross 6, there were 5 x T, 2 x I and
12 x S isolates. There was 74% variation occurred in this cross and 26% of the isolates
were inherited. Cross 7 (T x S), there was no significant difference among isolates with
%RARI values from 115.2 to 82.8, and 14.4 to 0.0 (Fig 4.8). Out of 10 isolates from
cross 7, there were 4 x T and 6 x S isolates. There were no intermediate progeny isolates.
There was no variation occurred in this cross, they all were inherited from one parent or
the other. Cross 8 (T x S), there was no significant difference among isolates with
%RARI values from 100 to 50.6, and 42.9 to 1.3 (Appendix 4). Out of 10 isolates from
cross 8, there were 7 x T and 3 x S progeny isolates. Once again there were no
intermediate progeny isolates. There was no variation occurred in this cross, they all were
inherited from one parent or the other (Appendix 4). Cross 9 (S x S), there was no
significant difference among isolates with %RARI values from 100 to 87.91, 33.38 to 9.7
and 9.84 to 1.35 (Fig 4.9). Out the two progeny isolates in this cross, one was tolerant and

the other was sensitive.

112

 

v- ”-i

4.5 Discussion
Temperature is an important environmental factor that can affect both host and pathogen.

The temperature trends in the Great Lakes region of the United States have displayed an
increase in the annual mean temperature for the area (Baker et al., 2002). Temperature is
an underpinning factor in both host and pathogen development and host by pathogen
interactions. In response to changes in temperature, microorganisms like fiingi adjust the
fatty acid composition of membrane phospholipids. As temperatures decrease, the fatty
acid chains undergo a change in state fi'om fluid to a more ordered crystalline array of
fatty acid chains (Berry and Foegeding, 1997). Cold adapted fungi respond to freezing
temperatures by incorporating fatty acids with lower melting points into their membranes,
lower the temperature of lipid phase transition, and thus maintain membrane fluidity and
cell function. Cold tolerant fungi generally have higher proportions of unsaturated fatty
acids in their membrane phospholipids, and there is a correlation of minimum growth
temperature and fatty acid composition (Berry and F oegeding, 1997). Plants that undergo
cold acclimation experience changes in accumulation of solutes such as sugars, proline,
betaines, and proteins called dehydrins. Solutes and dehydrins have been proposed to
stabilize membranes by interaction with membrane surfaces or by interaction with

surrounding water (Pearce, 2001).

113

 

Table 4.4 Number of progeny isolates in thermal tolerance phenotype categories
resulting from oospores generated by crossing parental isolates of Phytophthora
infestans with different thermal tolerance to -5°C.

 

 

Thermal Number of progeny isolates in

tolerance of thermal tolerance categories'I
Parents‘ Parent” Tolerant ('I‘)c Intermediate (I)‘I Sensitive (S)e
Pi 41-02 X Pi 02-007 T X T 23 2 4
Pi 41-02 X Pi 02-007 T X T 40 l 9
Pi4l-O2XPiS1-3 TXT 37 l 14
Pi 41-02XPi S1-3 TXT 37 1 13
Pi 41-02 X Pi 98-1 T XI 1 2 26
Pi 02-007 X Pi 62-02 T X T 5 2 12
Pi 95-3 X Pi 02-007 S X T 4 0 6
Pi 41-02 x Pi AtlanticZN S X T 7 0 3
Pi Atlantic2n X Pi 4-19 S X S 1 0

 

a P. infestans isolates used as parents to produce single oospore progeny (Al x A2)
° Thermal tolerance phenotype of the parents

c Number of isolates produced were tolerant to -5°C temperature

° Number of isolates produced were intermediately tolerant to -5°C temperature

c Number of isolates produced were sensitive to -5°C temperature

114

 

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P14 19 a ... e
W 4 --— 1 LSDo,1= 39 65 1 c
ms“ r i c
j T r f F I
0 20 40 w 80 100 120
96 RARI

Figure 4.5 Differences in thermal tolerance of parental isolates used for mating to produce progeny
isolates. Phytophthora infestans growth was measured as %RARI after exposure of isolates to -5°C
for 5 days. %RARI values followed by the same letter are not significantly different at p = 0.1 (Tukey
Multiple Comparison); LSDmo indicated by bar.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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1_31 . ab
Pt 02 7 « ab
1_25 ~ - -- -- ~~—m a—c
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1_06 ~ ~ “r M
1_2a 4 b-e
1_05 " A—J- "'7’" g L H
1_12 ~ b—e
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1_07 ~ :1 b-e
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1_17 4 b-e
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1_26 . d—e
1_33 . :9

1_10 4 L800 = 47.05
Pl 95-3 ~ 1 '1 i O
1_14 4 9
0 2O 40 00 80 100 120
96 RARI

Figure 4.6 Differences in thermal tolerance of progeny isolates from mating of thermally tolerant
parental isolates of Phytophthora infestans Pi 41-02 (An thermally tolerant) x Pi 02-007 (A2,
thermally tolerant). Growth was measured as %RARI after exposure of isolates to -5°C for 5 days
(Cross 1). The black bars represent the parental isolates and the white bars indicate thermal sensitive
(Pi 95-3) and insensitive (Pi 02-007) standards, respectively; Pi 02-007 was a parent in this cross.
%RARI values followed by the same letter are not significantly different at p = 0.1 (Tukey Multiple
Comparison); LSDMo indicated by bar.

115

 

 

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% RARI

Figure 4.7 Differences in thermal tolerance of progeny isolates from mating of thermally tolerant
with intermediately tolerant parental isolates of Phytophthora infestans Pi 41-02 (An thermally
tolerant) x Pi 98-1 (A2, thermally intermediate). Growth was measured as %RARI after exposure of
isolates to -5°C for 5 days (Cross 5). The black bars represent the parental isolates and the white bars
indicate thermal sensitive (Pi 95-3) and insensitive (Pi 02-007) standards, respectively. %RARI
values followed by the same letter are not significantly different at p = 0.1 (Tukey Multiple
Comparison); LSDoJo indicated by bar.

116

 

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Figure 4.8 Differences in thermal tolerance of progeny isolates from mating of thermally tolerant
parental isolates of Phytophthora infestans Pi 02-007 (A2, thermally tolerant) x Pi 95-3 (A1, thermally
sensitive). Growth was measured as %RARI after exposure of isolates to -5°C for 5 days (Cross 7).
The black bars represent the parental isolates and the white bars indicate thermal sensitive (Pi 95-3)
and insensitive (Pi 02-007) standards, respectively; Pi 02-007 and Pi 95-3 are parents in this cross.
%RARI values followed by the same letter are not significantly different at p = 0.1 (Tukey Multiple
Comparison); LSDoJo indicated by bar.

 

 

 

 

 

 

 

M 120

%RARI

Figure 4.9 Differences in thermal tolerance of progeny isolates from mating of thermally sensitive
parental isolates of Phytophthora infestans Pi 4-19 (A., thermally sensitive) x Pi Atlantic 2N (A2,
thermally sensitive). Growth was measured as %RARI after exposure of isolates to -5°C for 5 days
(Cross 9). The black bars represent the parental isolates and the white bars indicate thermal sensitive
(Pi 95-3) and insensitive (Pi 02-007) standards, respectively. %RARI values followed by the same
letter are not significantly different at p = 0.1 (Tukey Multiple Comparison); LSDim indicated by
bar.

117

 

‘m. u ._ v

Volunteer tubers are therefore protected from reduction in soil temperature below the
freezing point of water (and tuber tissue). Temperatures at the center and base of cull
piles found in Michigan range from 10°C to 0°C during the winter months (Kirk 2003a).
In addition, insulation of the soil by early snow in northern potato producing states of the
US may result in soil temperatures that only decrease to temperatures at which both
tubers and mycelium of P. infestans can survive. The ability of P. infestans to survive
cold temperatures is therefore important in understanding the possible sources of primary
inoculum. In recent years in northern US potato producing areas the occurrence of soil
temperatures below -5°C at 6 cm depth has been rare (Michigan Automated Weather
Network (MAWN), www.agweather.geo.msu.edu/mawnl) and re-growth from volunteer

tubers has been common throughout MI since 1997 (Kirk pers. comm).

De Bruyn (1926) reported that mycelium can survive exposure to at least -25°C however
this may be due to a physiological mechanism that is different from that which enables
survival at temperatures closer to the fieezing point of water and may be of little
epidemiological significance either in terms of survival of mycelimn between seasons or
of long term ecological implications of the thermal tolerance of populations. P. infestans
cultures survived -3°C for 5 days, and a few cultures have survived at -5°C after 1 day
(Kirk, 2003b). There was not much variation noticed in the temperature tolerance when
the isolates were tested at -3°C for five days and -5°C for one day. To observe more
variation the temperature exposure used in this study to determine temperature tolerance
was -5°C for 5 days. Thermal tolerance of isolates of P. infestans was defined in this

study as the ability of mycelium to re—grow after exposure to -5°C for a duration of 5

118

 

“mi .

days. These considerations and linritations to survival of substrate for the mycelium of P.
infestans dictated the parameters of the current study. This definition may underestimate
the ability of some of the isolates recovered to tolerate even lower temperatures or longer

exposure to -5°C.

The isolates tested by Kirk (2003) had a narrow range of mating types and for the
purposes of this study a wider range of thermal tolerance phenotypes were required that
could also potentially mate, i.e. A1 and A2 mating types were required. In the current
study a wider set of isolates originating from different locations in the US and
internationally representing different genotypes and mating strains (11 = 24) were selected
and tested in replicated experiments at -5°C in order to identify isolates with tolerance to
sub-zero temperatures. Both the Mauritius isolates Pi 3765 and Pi 3768 were tolerant to -
5°C and 80% of isolates from Northern Ireland were temperature tolerant and only Pi 46-
02 was sensitive to exposure to -5°C for five days. Two of the North Dakota isolates
were intermediate and one was tolerant. Alaskan isolate, Pi Banam was tolerant. There
were sensitive, intermediate and tolerant isolates in Michigan. There was no pattern
between the habitat from where the isolates originated and thermal phenotype. There
were cultures from cold and warm regions such as Mauritius that were tolerant to
exposure to -5°C for five days. It can be speculated that thermal tolerance is a variable

phenotype, and can occur in isolates fi'om any location.

Differences in epidemiological factors such as latent period, growth rate and potential
sporangial production among clonal lineages of P. infestans are likely to be greater than

differences among isolates of the same lineage than within distinct lineages (Mizubuti et

119

 

al., 1998). In this study, all the potential parental P. infestans genotypes tested survived
exposure to 0°C for any exposure period but survival was varied after exposure to -3°C
and -5°C, up to 24h exposure. A more stringent exposure period was therefore selected
and thermal tolerance was defined as the ability of an isolate to re-grow after exposure of
5 days to -5°C. This resulted in a diverse group fi'om which to choose parents however
the ability of these genotypes to mate was severely restricted and in many cases oospore
production was not observed. For example, in order to find a thermally sensitive A2
mating type one of the progeny isolated from cross 4 was selected (Pi 4_19) and used in

cross 9; thermally sensitive x sensitive,

In this study, oospores were mostly formed in agar cultures after two weeks of
incubation. Although the parents were of opposite mating type a few crosses did not
produce oospores, and a few crosses produced few oospores. Non-viability in progeny
may be caused by balanced lethal due to a deficiency in essential genes (Judelson et al.,
1995). Furthermore, germination of oospores was low or zero from some crosses. It could
have depended on the combination of parental isolates and tolerance to freezing
temperatures, as crosses with isolates sensitive to temperature produced less progeny
isolates. Continuous variation in thermal tolerance was observed in most of the crosses
however variation was also observed in crosses involving the same parents e.g. cross 1
and 2 although essential they produced similar propoetions of thermally tolerant,
intermediate and sensitve progeny isolates. Physiological mechanisms conferring cold
tolerance in fungi are complex; they include increases in intracellular trehalose

(important storage compound in fungal vegetative cells and spores) and polyol

120

 

concentrations and unsaturated membrane lipids as well as secretion of antifreeze

proteins and enzymes active at low temperatures (Robinson, 2001).

Age of culture, mycelial growth rate, origin of the culture, might also play an important
role in the production and germination of oospores. Multiple studies have highlighted the
importance of evaluation conditions, with inoculum concentration, temperature, age of
plant and state of the pathogen all shown to influence results (Swiezynski et al., 2000). It
is not understood which factors control fertility in P. infestans isolates, but origin and
fitness of parents are likely to play a major role. In general, sexual recombination in P.
infestans may yield a high diversity in progeny isolates which could lead to either

tolerance or sensitiveness to lower temperatures.

As the rate of growth of cultures originating from single spores can vary five replicates
were used in every repetition of the experiment, and each of the experiments was
repeated twice. In total, 252 progeny isolates were successfully revived from a total 300
isolates that were originally produced. Among all the crosses, there were a total of 155
thermally tolerant isolates; 9 intermediate in tolerance isolates and 88 sensitive isolates.
The range in thermal tolerance in progeny isolates from many crosses was continuous
indicating a wide range in thermal tolerance even though the definitions used in the study
were statistically precise. Thermally tolerant isolates may be more likely to survive
moderately cold winters and may initiate epidemics the following season. However, the
epidemiological implications of thermal tolerance in the population of P. infestans are

unclear as survival potential is closely linked with the thermal tolerance of the host tuber

121

 

which may vary within the same cultivar depending on the physiological age or physical
condition e.g. size and shape of the tuber which may in turn vary with cultivar. Some
cultivars that mature later or have different dry matter accumulation potential may have
better survival than tubers of early maturing low dry matter tubers which are prone to
rotting. In addition, parameters such as the timing of infection of the tuber in the soil and
the number or position of infections relative to meristematic tissue may also be more
important in determining the fate of the tuber or ultimately of an epidemic the following
year. The observation that there is potential for variation in thermal tolerance within P.
infestans suggests that there may be epidemiological consequences resulting from these

phenotypes.

The causes of variation in the progeny isolates of P. infestans could have occurred
because of mating, mutation, mitotic crossing over, chromosome number and ploidy
condition. Variation of P. infestans from mating studies has been reported to vary in
terms of fungicide sensitivity (Gisi and Cohen, 1996), and virulence phenotype (Abu el
Samen et al., 2003). In this study the same genetic causes may underpin the wide
variation observed among progeny isolates in terms of temperature tolerance. Whether or

not thermal tolerance poses an increased damage to agriculture is difficult to determine.

Temperature tolerance of pathogen has ecological significance, especially in agricultural
regions where the climate is warming and becoming more conducive for late blight
(Baker et al., 2002). Since 1950, soils have become more likely to freeze in the Great

Lakes region due to climate change, but only to a depth of 5 cm (Isard et al., 1998),

122

which is above the depth that tubers may be buried (Kirk, 2003). Oospores produced
from US strains of P. infestans will apparently survive temperatures encountered in moist
soils in the United States (Fry et al., 1997). Sexual reproduction results in the formation
of oospores which serves as a source of initial inoculum and may with an additional
genetic variation, rendering oospore-infested soil dangerous to potato crops. The effects
of varied temperature on mycelial survival and sporangia germination could have
important implications for epidemic development and disease management. Reports of
increased annual mean daily temperature in the Great Lakes region of the United States
increases the potential for survival of volunteer potatoes in field and cull piles (Baker et
al., 2002; Kirk, 2003). The interaction between climate change, soil temperature during
winter months, thermal tolerance of mycelium of P. infestans and physiological
parameters of tubers of the potato may require more intensive evaluations to determine
the future of potato production in the Great Lakes region and other northern regions of

North America in relation to epidemic potential of potato late blight.

123

5 Pathogenicity of progeny isolates from crosses of thermally variable
Phytophthora infestans on potato tubers

5.1 Introduction
The final determinant of the significance of thermal tolerance was to determine a profile

of aggressiveness of progeny and parental isolates of Phytophthora infestans, used and
generated in this project. Infection of tubers is similar to that of foliage with germ-tubes
penetrating at lenticels, eyes, stolons, buds, wounds or any site incompletely suberized
(Bain and Moller, 1999). To understand if the progeny isolated from several temperature
phenotype crosses are pathogenic, a pathogenicity test was done. Potato tuber tissue
infected with late blight (Phytophthora infestans) was quantified using a digital scanner
and image analysis software. The average reflective intensity of light reflected fi'om the
cut surface of sample tubers measures the darkened, diseased potato tuber tissue in
conjunction with lighter, late blight-free tissue. Qualitative disease rating systems often
assess the disease level on the surface of whole tubers or tuber slices (Douches et al.,
1997, Kirk et al., 1985). The change in the reflective intensity of exposed tuber surfaces
can be determined by digital scanning. This method allows comparing pathogenicity

along with virulence.

Infection of a potato is extremely variable and it depends on the cultivar and the isolate of
P. infestans involved. The pathogen can undergo various changes (recombination), but
the final goal is to identify if the progeny both inherited and recombinants were
pathogenic. In Chapter 2, we found that the phenotype, thermal tolerance inherited a wide
range of variability but we don’t know if this variability was related to the ability to cause

disease. Pathogenicity testing can validate if variation has caused the progeny isolates to

124

be more aggressive. Biotypes of P. infestans that have become dominant since 1992 can
infect tubers more effectively than biotypes of the fimgus present in North America prior
to 1992 (Fry and Goodwin, 1997; Lambert and Currier, 1997). Even though we did not
notice any recombination in the genotypic character, isozyme analysis (Chapter 4); it
would be interesting to find out if the progeny isolates are pathogenic. This could be a
result of recombination occurring in nature, which shows that it is important to study
recombination and it’s effects on pathogenicity, to prepare us with better management
strategies. We noticed that the isolates from thermal tolerance crosses are also variable in
terms of mating type, metalaxyl sensitivity and virulence phenotype (Chapter 3), but we

don’t know if there is variability in terms of pathogenicity.

The objective of this study was to evaluate if the progeny isolates from the different
thermal phenotype crosses are pathogenic, and to estimate if the progeny isolates have

inherited the parental characteristics or if variation has occurred in this phenotype.

5.2 Materials and methods

5.2.1 Pathogenicity test on tubers
Five isolates were selected at random from each cross along with the parents for

pathogenicity and aggressiveness testing. Methods developed by Niemira et al., (1999)
and Kirk et al., (2003 b) using whole tubers were adapted for this study. Tubers of cv.
FL1879 were obtained from Walthers and Sons Potato Farms, Newberry, Michigan.
Potato tubers were harvested during the previous growing seasons and were stored at 3°C
in the dark at 90% relative humidity until used. Tubers for the experiments were within

the size grade range 50-150 mm diameter (any plane). Visual examination of a random

125

.(anY'q

 

 

sample of tubers (n = 20) for disease symptoms indicated that tubers were free from late
blight and other diseases such as Fusarium dry rot. The sample was further tested with the
ELISA immune-diagnostic Alert Multi-well kit (Alert Multiwell Kit - Phytophthora sp.
Neogen Corporation, Lansing, MI, USA); P. infestans was not detected in any of the
tubers. Prior to inoculation, all tubers were washed with water to remove soil. The tubers
were then surface sterilized by soaking in 2% sodium hypochlorite (Clorox) solution for
30 min and then rinsed with water. Tubers were dried in a controlled environment with
continuous airflow (5950 l min") at 15°C in dry air (30% relative humidity) for four

hours prior to inoculation.

The sample isolates of P. infestans (parents and progeny) selected for testing were grown
on Rye agar plates (Appendix 1) for 14 to 20 days. A sample from each of the cultures
was measured to determine sporangial production using a hemacytometer. Each culture
produced about 2 x 10’5 sporangia per ml of solution indicating that the cultures were
viable and of similar maturity. Non-growing cultures were discarded and were not used
for the evaluation. The washed, surface-sterilized tubers were inoculated by removing a
tuber plug of 5mm diameter x depth fi'om the apical end of the tuber about 0.5 cm from
the dominant apical sprout at the bud end of the tuber to a maximum depth of 5 m using
a sterile cork borer. A plug (4 mm diameter) was removed from the colony edge of the
sample culture and placed in the plug-hole of the tuber and the tuber plug was re-placed
over the inoculum. The non-inoculated control tubers were inoculated with a sterile plug
of rye agar. Vaseline was placed over the wound to prevent the plug from falling out and
desiccating the inoculum. Aerated plastic boxes (46 x 28 cm Akro-N.S.T boxes, Akro-

Mils, Akron, Ohio 44309, USA) were prepared by placing a wet paper towel in the base

126

 

”twin-25;)“ .) .
A a

of the boxes followed by placing a plastic mesh to separate sample tubers from the wet
towel to avoid direct contact between the wet towel and the tubers and minimize the
contamination or rotting of the tubers. Five tubers were inoculated for each isolate and
the whole experimented was repeated twice to give a total of 15 tubers for the estimation
of aggressiveness. The tubers were stored in a temperature-controlled environment
chamber, 1.8 m3 volumes (Environmental Growth Chambers, Chagrin Falls Ohio, USA)
at 10°C for 30 days. Relative humidity was maintained at 90% within the chamber.
Disease development rates within tubers in relation to storage temperature were known
from previous experiments (Kirk et al., 2001) and a single sampling date was selected

about 30 days after inoculation (DAI).

A digital image analysis technique was used to assess tuber tissue infection similar to the
method described for estimation of growth of cultures of P. infestans on Petri dishes
(Chapter 4). The scanned surface was the cut face of a sample tuber. A sharp knife was
used to ensure a smooth cut face. Fresh-cut tuber sections were placed cut surface down
on a glass plate, 40 x 30 cm and 2 mm thick. The glass plate was used to prevent surface
contamination of the scanner glass and permitted multiple samples to be prepared and
moved to the scanner for image production. The plate was transferred to a flatbed scanner
(HP ScanJet 4c, Hewlett-Packard Co., Houston, TX) controlled by an IBM-compatible
PC. Scanner control software (Desk Scan 11 ver. 2.4, Hewlett-Packard, Co., Houston,
TX), generated an image of the cut tuber surfaces against a black background. The image

was formed from light reflected fi'om the cut tuber surfaces.

127

 

The brightness value of the image controlled the light intensity of every pixel in the
image. The contrast value controlled the differences between light and dark regions of the
image. While the scanner control software was able to automatically adjust the brightness
and contrast of the image by comparing the relative size of the pale tuber surfaces against
the black background, the settings were manually set to 180 units (brightness) and 200
units (contrast) to ensure consistent readings. A photograph-quality image was taken and
stored for analysis (e.g. Fig. 5.1). A typical image in Tagged Image Format (*.tif)
occupies 1 megabyte. Typical ARI values for a range of infected and uninfected cut tuber
surfaces were shown on Table 5.2. The image files created with the scanner software
were loaded into the image analysis software (SigrnaScan ver. 3.0, Jandel Scientific, San
Rafael, CA). The black background has 0 light intensity units (LIU), while pure white
has 255 LIU. Disease-free and blemish-free tuber tissue is pale. Diseased or blemished
tuber tissue is darkened. The image of the cut tuber surface was selected for analysis, and
isolated from the adjacent regions of the image. The image was carefully cropped for
irregularly shaped tubers to remove the image of the adjacent tuber skin, and the image of
the cut surface was unedited. The area was selected with the fill tool, which
encompassed all pixels within a given area brighter than the cut-off threshold. The area
selection cut-off threshold was set to 10 LIU, effectively allowing the software to exclude
all parts of the image darker than 10 LIU, e.g. the black background. The average
reflective intensity (ARI) of all the pixels within the image gave a measurement of
disease severity of the tuber tissue of each sample. The ARI was measured in sections

from the apical, middle and basal regions of the tuber, approximately 25% (apical), 50%

128

 

Middle

   

Figure 5.1 Late Blight Development in FL1879 tuber tissue caused by Phytophthora
infestans (isolate Pi 5-16), incubated at at 10°C for 30 days.

129

(middle) and 75% (basal) of the length of the tuber (respectively) as measured from the
apical end. The amount of late blight infected tissue per tuber was expressed as a single
value (Mean ARI) calculated as the average ARI of the apical, middle and basal sections
(total images, n = 45 per Mean ARI). ‘ The lower Mean ARI values indicate relatively
greater disease severity than those with values close to that of the non-inoculated control.
The presence of P. infestans in sample tubers was confirmed by isolating pure cultures of
P. infestans from the infected tuber tissue and successful re-inoculation of potato tubers

and leaves.

5.2.2 Data analysis 2
Pathogenicity and virulence of the progeny isolates of P. infestans produced from mating

different thermal phenotypes were compared using a technique based on statistical
comparison using the ANOVA procedure in IMP (version 5.01; SAS). The quantification
of virulence of isolates of Phytophthora infestans was based on the presence or absence
of significant differences measured at P = 0.05 in tuber tissue darkening between non-
inoculated control tubers and positive control tubers inoculated with a virulent isolate.
The procedure was summarized in Table 5.1; the outcomes were absence or presence of
tuber tissue discoloration, and further resolved by the degree of tuber tissue discoloration
in comparison to the controls. Discolored tubers were classified into three categories
dependent on the comparison of the Mean ARI values to those of the negative and
positive controls, non-inoculated and inoculated with Pi 02-007, respectively. The
negative and positive controls had discrete values for comparisons at the apical, middle
and basal regions of the test tubers. Numerical values 0, 1, 2 or 3 (severity scores) were

assigned to the isolates dependent on classification using the criteria described in Table

130

TV

 

5.1. The total severity value (TSV) for each isolate was attained by adding the severity

score calculated from each of the three regions on the test tuber.

5.2.3 Compiling data
Data on temperature tolerance from Chapter 2 (temperature tolerant (A), intermediate (B)

or sensitive (C) to -5C); Mating type (A1, A2, or self fertile); metalaxyl sensitivity
(Resistant (R), intermediate (1), or sensitive (S) to fungicide metalaxyl), and virulence
assessment on foliage (LBR l to 11 genes) from Chapter 3; isozyme analysis (GP!) from
Chapter 4; and pathogenicity test (TSV = 0 — 4) from Chapter 5 were all put together in
Appendix 4. Finally, using the parental and progeny isolates of P. infestans selected for
the pathogenicity tests comparisons of their other phenotypic and genotypic
characteristics were compiled in a single table (Table 5.4). If the progeny has the
phenotype or genotype character from either of the parent, it was given a value = 0,
meaning the character was inherited, and if it had the character different from the parents,
it was given a value = 1, meaning that it was a recombinant and isolates with value = -l

meant that isolates were less virulent than the parents. (Table 5.4).

5.3 Results

As an example, tubers of the FL1879 inoculated with Phytophthora infestans isolate Pi 5-
16, (U S8 genotype) incubated at 10°C for 30 days resulted in significant tuber infection
and range of ARI values (Figure 5.1). The mean ARI values of the negative control (non-
inoculated) and the positive control (Pi 02-007) were 112.2, 114.8 and 117.3 (TSV = 0)

and 90.2, 107.9 and 115.7 (TSV = 2) at the apical, middle and basal sections of the

131

sample tubers 30 days after inoculation, respectively (Table 5.3). The range in
susceptibility of the tubers to late blight was heterogenous (Table 5.3) over the two
executions of the experiment and the data were therefore combined by taking an average.
The most aggressive isolates, 2-20 had apical, middle and basal ARI values of 69.3, 79.2
and 100.5 and isolate 3-14 had apical, middle and basal ARI values of 80.7, 92.7, 97.9.
The parental isolates Pi 62-02, Pi 95-3 and Pi Atlantic 2N were pathogenic (showing
some darkening on some tubers) but were avirulent using the criteria of the pathogenicity
and virulence test. Pi 02-007, Pi 41-02 and Pi S1-3 were moderately virulent (TSV = 2).
Out of total 49 isolates tested, 11 were pathogenic but were avirulent, showing little tuber
tissue darkening around the plug core, and had TSV = 0. The other 38 isolates had
varying degrees of virulence, with TSV = 1-4. Two isolates (2-20 and 3-13) were the

most virulent isolates, with TSV = 4, more than the positive control (TSV=2).

We observed that, out of 21 isolates tested for mating type, thermal tolerance phenotype,
metalaxyl sensitivity, virulence phenotype and Gpi isozyme genotype, 2 (9.5%), 11
(52.4%), 9 (42.9%), 16 (76.2%), 12 (57.1%) isolates showed recombination respectively
(Table 5.4). The isolates inherited discrete characteristics from the parent for example an
isolate may have a different thermal tolerance phenotype but not differ in mating type or
metalaxyl sensitivity. Therefore there is continuous heterozygosity which means a very
diverse population. So, if clonality exists for one phenotype or genotype it is unlikely to
be clonal with respect to the other phenotypes. In total, 139 isolates have attained
phenotypes that are not inherited from either parent. 49% progeny were recombinants

(139 / 284 isolates (Appendix 4). All the parents in the 9 crosses were heterothallic

132

isolates which produced out of a total of 285 progeny isolates 13 self-fertile or
homothallic isolates (oospores were produced in both testers). Recombination occurred in
4.5% of the isolates (13 / 284 isolates, Appendix 4). After one month, all the pure
cultures (parental and progeny) were observed under microscope, and about 50% of the
isolates produced self-fertile oospores (Table 3.3, Chapter 3). In the metalaxyl sensitivity
test, out of 284 isolates, 117 (41%) had a metalaxyl sensitivity phenotype different fi'om
the parents indicating that many of the progeny were recombinants (Appendix 4). In the
virulence phenotype assessment, out of 37 isolates tested, 22 (59.4%) of them had
different virulence fi'om parents and that progeny may have been recombinants
(Appendix 4). In the isozyme analysis, all the progeny inherited the genotype from the
parents and from this trait recombination did not occur. Out of 54 progeny isolates tested
for pathogenicity, 31 (57%) were more or less aggressive than the parents, suggesting

that 43% of the progeny were recombinants.

5.4 Discussion
The variability in pathogenicity and virulence of both parental and progeny isolates of

Phytophthora infestans with different thermal tolerance phenotypes was clear from this
study. The observation that three of the parental isolates and nine of the progeny isolates
were non-pathogenic on tubers of FL1879 suggests a strong varietal component and a
potential confounding factor of this technique. Susceptibility of foliage and tubers to the
same genotype of P. infestans can vary within the same cultivar (Kirk et al., 2003).
Therefore when conducting this type of analysis it would be prudent to use tubers of at
least three cultivars known to be susceptible to P. infestans. The pathogenicity and

virulence technique utilized the response of tuber tissue darkening during development of

133

tuber blight. By introducing the inoculum to only the bud or apical end of the tuber it was
possible to measure the spread of the disease through the sample tubers by sampling at
three points through the length of the tuber from the apical to the basal end with ARI
generally increasing with distance from the inoculation site. Nienrira et al., (1999)
optimized this technique and reported that using three surfaces cut across the longitudinal
plane of the tuber about 25, 50 and 75% of the distance from the apical to the basal end
gave equivalent results to taking as many as 10 slices (Kirk pers. comm). Differences in
section thicknesses and shapes resulted in significant, though small, differences. Thinner
tuber sections may allow greater transmission of light through the section, thereby
reducing the light reflected from the section surface. This would return a lower (darker)
ARI rating than sections which reflect light more effectively. The optimal thickness for
slices was reported to be >5 mm (Niemira et al., 1999). Darker infected tissue results in
lowered average reflective intensity because of the reduced light reflectance. In this
study, the use of replication and repetition resulted in 15 estimations of tuber disease
severity for each isolate tested. Pathogenicty of the test isolates was confirmed by the
presence of at least a single cut surface showing darkening typical of successful infection.
The strict statistical conditions imposed to measure virulence indicated that many of these
isolates although pathogenic were nearly avirulent. To improve this technique, an
increased range of potato cultivars should be used and also a greater sample size for the
estimations. However, to put the technique into perspective the number of tubers used for
a comparison of 50 isolates was 750 and 2250 cut surfaces were evaluated. Some of the
non-pathogenic progeny even from crosses between two pathogenic parental isolates

could be the result of significant chromosomal heterogeneity or many recessive

134

 

Table 5.1 Differences between the two executions of pathogenicty and virulence of
parental and progeny isolates of Phytophthora infestans with different thermal
tolerance phenotypes based on the Average Reflective Intensity (ARI) of inoculated
potato tubers cv. FL1879.

 

 

Isolate Replicates Isolate Replicates
1_06 A 5_43 B
1_07 A 5_46 B
1_13 A 6_06 B
1_20 A 6_1 l B
1_36 A 6_14 A
2_03 A 6_19 B
2_09 B 6_20 A
2_20 A 7_01 A
2_33 A 7_1 8 B
2_34 A 7_03 A
3_01 A 7_08 B
3_05 B 7_09 A
3_13 A 8_02 A
3_41 B 8_05 B
3_41 A 8_08 A
4_01 B 8_09 A
4_05 B 9_01 B
4_09 A 9_02 A
4_12 A Pi Atlantic 2n B
4_19 B -ve Control A
4_21 A Pi 02-007 A
4_29 A Pi 95-3 B
5_16 A Pi S1-3 A
5_19 A Pi 41 -02 A
5_36 A Pi 62-02 B

 

The first digit of the isolate name is the cross number and the 2-digits following the hyphen are
the numbers of the single oospore-generated progeny isolate.

Isolate names starting with Pi are parents.

A= No significant difference in disease severity in between the two replicates at p=0.05;

B= significantly different in between the two repetitions at p=0.05.

-ve control = Non-inoculated tubers.

135

Table 5.2 Quantification of virulence of isolates of Phytophthora infestans using a
tuber inoculation technique based on differences in tuber tissue darkening between
non-inoculated control tubers and positive control tubers inoculated with a virulent
isolate.

 

Comparison criteria based on ARI' values of tuber

 

Condition tissue from apical, middle and basal tuber sections Severi
Non-inoculated (-ve Inoculated (+ve control)c Value
control)

No tissue NSD° (ARI = -ve
discoloration control) 0
Tissue SDf (ARI < -ve control) SD (ARI > +ve control)
discolored 1
Tissue NSD (ARI = +ve control)
discolored 2
Tissue SD (ARI < +ve control)
discolored 3

 

“ ARI = Average Reflectance Intensity [measured as Light Intensity Units (LIU)] of all
the pixels within the scanned cut tuber surface image gave a measurement of severity of
tuber tissue darkening due to P. infestans from each sample.

° The non-inoculated control had a specific ARI value to which all ARI values of
inoculated tuber slices could be compared (a unique value was measured for apical,
middle and basal sections).

° The positive control was inoculated with a known aggressive isolate of P. infestans (Pi
02-007) which produced a specific ARI value to which all ARI values of inoculated tuber
slices could be compared (a unique value was measured for apical, middle and basal
sections).

° The numerical value was assigned dependent on the outcome of the statistical
comparisons and each value generated from the outcome criterion form apical, middle
and basal evaluations was summed and used to produce an overall severity score with
maximum value of 9.

° NSD = not significantly different from the critical ARI value measured at p = 0.05.

f SD = significantly different from the critical ARI value measured at p = 0.05.

136

Table 5.3 Pathogenicty and virulence of parental and progeny isolates of
Phytophthora infestans with different thermal tolerance phenotypes measured using
a severity scale based on the Average Reflective Intensity (ARI) of inoculated potato
tubers cv. FL1879 based on comparison to negative and positive controls.

 

 

Apical Middle Basal Total
Isolate ARI Severity ARI severity ARI Severity severity
1_01 71.7 2 91.0 1 104.5 0 3
1_05 74.0 2 92.4 1 104.2 0 3
1_10 87.0 2 102.4 0 106.9 0 2
1_06 83.3 2 99.8 0 100.6 0 2
1_07 97.0 0 106.2 0 94.9 0 O
1_13 82.2 2 104.2 0 105.9 0 2
1_18 75.9 2 91.2 1 107.7 0 3
1_20 71.7 2 91.0 1 104.5 0 3
1_29 93.7 1 103.6 0 108.1 0 1
1_36 78.4 2 91.9 1 107.7 0 3
2_03 76.4 2 105.5 0 110.8 0 2
2_09 75.7 2 90.2 1 108.7 0 3
2_13 76.8 2 94.5 1 110.6 0 3
2_20 69.3 2 79.2 2 100.5 0 4
2_33 79.2 2 89.9 1 103.6 0 3
2_34 79.9 2 99.9 0 107.4 0 2
3_01 83.6 2 95.8 1 106.1 0 3
3_06 88.0 2 102.7 0 107.9 0 2
3_14 80.7 2 92.7 1 97.9 1 4
3_37 82.9 2 92.9 1 105.9 0 3
3_41 82.6 2 92.4 1 100.6 0 3
3_42 84.9 2 93.3 1 104.2 0 3
3_51 81.5 2 93.5 1 104.9 0 3
4_01 79.4 2 99.1 0 1 10.5 0 2
4_05 89.6 2 100.5 0 1 1 1.4 0 2
4_09 87.0 2 97.9 0 1 10.1 0 2
4_12 90.4 2 103.3 0 106.6 0 2
4_19 88.1 2 106.0 0 103.1 0 2
4_21 91.3 1 99.0 0 1 11.6 0 l
4_29 82.3 2 98.3 0 101.1 0 2
5_12 82.9 2 91.4 1 99.6 0 3
5_16 89.8 2 110.7 0 117.9 0 2
5_19 100.8 0 116.0 0 111.5 0 0
5_36 87.4 2 101.0 0 111.3 0 2
5_37 79.8 2 93.8 1 106.8 0 3
5_41 91.7 1 103.0 0 109.1 0 1
5_45 104.2 0 116.9 0 112.9 0 0
6_06 84.1 2 104.8 0 107.4 0 2
6_11 92.1 0 107.1 0 108.8 0 0
6_14 77.8 2 92.5 1 111.6 0 3
6_19 82.5 2 93.9 1 106.7 0 3
6_20 88.9 2 107.0 0 1 10.3 0 2
7_01 92.6 0 105.9 0 115.3 0 0
7_02 92.4 0 105.4 0 1 13.8 0 0
7_03 82.7 2 95.1 1 106.2 0 3
7 08 88.8 2 106.4 0 108.7 0 2

l

 

137

Continued

 

Table 5.3 Continued

 

 

Apical Middle Basal Total
Isolate ARI Severity ARI severity ARI Severity severity
7_09 98.3 0 105.6 0 109.3 0 0
8_02 80.8 2 94.9 1 107.8 0 3
8_05 86.8 2 104.1 0 108.9 0 2
8_07 86.4 2 104.0 0 1 10.3 0 2
8_08 86.8 2 104.6 0 1 10.1 0 2
9_01 80.5 2 90.2 1 107.4 0 3
9_02 103.7 0 111.2 0 101.2 0 0
Parental and reference isolates
PiAtlanticZN 108.2 0 1 18.3 0 113.5 0 0
Pi 41-02 84.1 2 104.8 0 106.1 0 2
Pi 62—02 104.5 0 103.5 0 109.8 0 0
Pi 95-3 107.4 0 115.1 0 108.0 0 0
Pi S1-3 91.0 2 107.5 0 107.5 0 2
Pi 02-007
(+ve control) 90.2 2 107.9 0 115.7 0 2
-ve control 112.2 0 114.8 0 117.3 0 0

 

Total severity = Apical severity + Middle severity + Basal severity.

138

Table 5.4 Progeny isolates of Phytophthora infestans selected from some crosses
showing inheritance and recombination for phenotypic and genotypic
characteristics.

 

Mating Temperature Metalaxyl Virulence
Isolatea type° Tolerancec Sensitivity° Assessmente Pathogenicityr Genotypeg

 

1_01 0i 0 1 1 0
1_05
1_06
1_10
1_13
1_18
1_29
2_13
3_01
3_37
3_51
4_01
4_12
4_29
5_12
5_16
5_41
5_45
6_11
6_19
6_20
Total 9 12

% 9.5 52.4 42.9 76.2 57.1 0

O

h

1

0

0

l

1

-1

1

l

l

1

0

0

0

1

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a The first digit of the isolate name is the cross number and the 2-digits following the hyphen are
the numbers of the single oospore-generated progeny isolate.

° Mating type, determined by production of oospores when grown with isolates of P. infestans of
known mating type (testers).

° Temperature tolerance, Comparison of growth of isolates of cultures of P. infestans for 25 days
at 18°C after exposure at -5°C for 5 days.

° Metalaxyl sensitivity determined by comparing ECSO response of the isolates grown on media
amended with different concentrations of metalaxyl with ECso of known resistant and susceptible
isolates.

e Virulence assessment, Virulence pathotype as defined by Muller and Black, 195 2.

f Pathogenicity where 1 indicated more virulent than parental isolates.

h Isolates were less virulent than the parents.

3 Genotype is the genotypic characteristic Glucose ~6- phosphate isomerase allozyme genotype;
designated by Goodwin et al., 1995.

‘ For all assessments “0” = the respective character was inherited; and
character was not inherited (recombinant) fi'om either of the parental isolates.

68199

= the respective

139

defective alleles. Significant chromosomal heterogeneity might also exist within the
species. This would also explain why the frequencies of oospore germination in different
crosses ranged from nearly 0 to 100% and why mating between pathogenic parents may
yield significant numbers of nonpathogenic progeny or progeny with decreased virulence

(Al-Kherb et al., 1995).

140

6 Executive Summary
The digital method of assessment of survival of mycelium of P. infestans in vitro is based

on light reflectance from developing mycelium (Kirk, 2003). Image analysis is
quantitative and objective and scanned images can be stored for future references (Kirk,
2001). The pathogenicity and virulence testing were vital to check if progeny isolates
differed from the parental isolates in their ability to cause disease and if they were more
or less aggressive than parental isolates. P. infestans has been shown to have several
variable characteristics such as those tested in this study and such variations may have
important survival and disease management implications. In this study, the main goal was
to evaluate variation in several phenotypic and genotypic characteristics after mating
between parental isolates with similar or different thermal tolerance phenotypic
characteristics. The continuous variation in thermal tolerance among progeny isolates
from tolerant to sensitive to thermal exposure to -5°C for five days indicated that there
was a high likelihood that progeny isolates could readily adapt to exposure to
temperature. The phenotypes with most variability were e.g. tempareture tolerance,
virulence phenotype and the most conserved was genotype. The implications of this
finding are serious in that it is likely that given this improved thermal tolerance within
the population of the pathogen during times when climate change studies indicate warmer
aerial and therefore soil temperatures in potato growing areas of the north central region
of the US, that the pathogen will likely survive during winter. Even if oospores were
produced in natural conditions in soil or in the plant material (tubers, stem, leaves), it is
hard to speculate and differentiate or characterize the parents that are producing the
oospores, as there could be only one or more than one genotype present. To eliminate

this problem, and understand more about this pathogen this study was done in vitro. Even

141

though our main objective was to check the inheritance of thermal tolerance, the research
has interested us to include few other phenotypic and genotypic tests like compatibility or
mating type, metalaxyl sensitivity, virulence on foliage, pathogenicity and aggressiveness
on tubers, isozyme analysis. Microsatellites, DNA content and ploidy level tests were
also tested on some isolates, to prove that recombination had occurred and to reveal the

complexity of DNA content in P. infestans.

The thermal tolerance study indicated that if mating occurred, producing progeny that are
more tolerant to cold temperature (-5°C) they could also be more aggressive to potatoes.
In terms of survival and initiation of epidemics these characteristics may balance as more
aggressive isolates are likely to kill the host tubers quickly and therefore reduce the
potentially infected meristematic tissue that will survive to the following season.
Alternatively, if isolates can tolerate reduced soil temperatures (within host tuber tissues)
freezing conditions but are moderately aggressive then the risk of surviving winter is
greater and the initiation of epidemics the following season more likely. As noted in
Chapter 3, the continuous variation noted in terms of metalaxyl sensitivity is interesting
in that Ridomil-based products are really used for control of potato late blight in North
America as it is widely assumed that all isolates are insensitive to metalaxyl. The wide
variation in sensitivity may result in populations in the future developing that are even

partially sensitive to metalaxyl and may justify the re-use of this class of fungicides.

P. infestans is likely to continue surviving, so it is important to expand our knowledge of

the survival potential of different P. infestans genotypes under different temperature

142

settings. It is essential to predict changes in P. infestans population structure over time,

especially when extraordinary weather conditions prevail.

6.1 Future research
1. Expand our knowledge to find out the biochemical basis and physiological basis

of thermal tolerance in Phytophthora infestans.
2. Characterize genes that are involved for the complexity of Phytophthora

infestans, in mating type, thermal tolerance, and metalaxyl sensitivity.

143

7 Appendix

144

Appendix 1

Rye A Agar

Rye A Agar was used for the isolation of Phytophthora infestans, modified from (Caten
and Jinks, 1968 #35). For 1 liter media, 100.0g of pesticide free rye seed (Soldan's Feed
& Pet Supplies, 5200 S Martin L King Jr. Blvd, Lansing, MI 48911) was washed before
use by placing seed into a beaker, covering the opening with cheesecloth secured with a
rubber band and running under water for 10-15 minutes. Washed seed was placed into a
stainless steel container with an appropriate amount of distilled water and was boiled for

one hour. Adequate amount of water is necessary to prevent burning.

After boiling, solution was transferred into a graduated cylinder by filtering through two
layers of cheesecloth (equals four layers) placed over the mouth of a funnel. All the seed
was brought to the beaker and additional distilled water is added and procedure is
repeated until desired final volrnne is attained. Rye broth is evenly distributed among the
different proper sized flasks. Following chemicals were added to the broth before it’s
autoclaved. 10.0g of sucrose (Sigma-Aldrich, Inc., 3050 Spruce Street, St. Louis, MO-
63103 USA) and 15.0g of Baccto Agar (Sigma-Aldrich, Inc., 3OSOSpruce street, St.
Louis, MO-63103 USA) were added to the media. For enhancing sporulation, 0.05g of
Beta-sitosterol (Spectrum Chemical MFG. Corp., CA 90248) was added. Media was kept
in the autoclave for 30 minutes for the sterilization. Hot liquid media was poured into 60
X 15mm Petri plates (Falcon Petri dish, Becton Dickinson Lab ware, Becton Dickinson
and company, Franklin Lakes, NJ — 07417-1886, USA) with a 10m] pipette tip, using the

automatic pipettor (Eppendorf Easypet 4421, Eppendorf North America, One Cantiague

145

Road, PO. Box 1019, Westbury, New York 11590-0207, USA). While pouring the agar

the flask is placed on a stirrer plate to keep agar homogenous.

To eliminate contamination, 500ul RAN antibiotic stock solution was added for 1 Liter
Rye media, which is selective for Oomycetes containing 20 mg/ml Ampicillin, 75 mg/ml
Nystatin, and 75mg/ml Rifarnycin B (Sigma-Aldrich Co., P.O.Box 14508, St.Loius MO-
63178, USA) in lml DMSO (Dimethylsulfoxide, JT Baker, Mallinckrodt Baker, Inc.,
Phillipsburg, NJ 08865). RAN was added to media after autoclaving and prior to pouring
in to the plates. Sterile Rye agar plates amended with RAN were stored in the
refiigerator, with no lights since the antibiotics are both heat and light sensitive. Aliquots

of RAN were stored in the freezer, -20°C.

Long-term storage of isolates
Cultures were stored for periods of up to five to six months in three methods.

1. In 60 X 15mm Petri plates, each plate containing 25 ml Rye A agar (based on
Caten and Jinks, 1968). Antibiotics were included in Rye A agar as needed.
Isolates were maintained at 18°C, in the dark.

2. In 25ml vials (Research Products International Corp.) with 8gms of Rye seed (See
above) and 10 ml sterile distilled water. After autoclaving add 2 plugs (3mm) of
freshly growing P. infestans mycelium. After observing the growth, store them at
10°C in the dark.

3. Cultures were also maintained in long-term storage on slopes of Rye A agar using

25 ml vials. Isolates were maintained at 10°C, in the dark.

146

Isolates were sub-cultured as required by removing a section of mycelium from the
actively growing edge of a fungal colony, using a No. 3 cork borer and a scalpel and

transferring to another fresh medium.

147

Appendix 2

Origin, mating type, thermal tolerance, and GPI-isozyme phenotypes of isolates of
Phytophthora infestans selected for crossingitudies.

 

 

Cross Thermal
number Isolate Origin Mating type tolerance
l Pi02-007 US A2 ”1°
Pi 4102 M“ A] T
2 Pi 02-007 US A2 T
Pi 41-02 NI Al T
3 Pi Sl-3 US A2 T
Pi 41-02 NI A] T
4 Pi S1-3 US A2 T
Pi 41-02 NI A, T
5 Pi 98-1 US A2 11)
Pi 41-02 NI A1 T
6 Pi 02-007 US A2 T
Pi 62-02 NI A; T
7 Pi02-007 US A2 T
Pi 95-3 US A2 Sc
8 Pi Atlantic 2N US A2 S
Pi 41-02 NI Al T
9 Pi Atlantic 2N US A2 S
Pi 4-19 US A1 S

 

Origin, mating type and thermal tolerance of different parent isolates used in nine different
crosses. Progeny from theses crosses were isolated to identify if thermal phenotype character is
inherited. a = Tolerant; ° = Intermediate; c = Sensitive; ° = Northern Ireland

148

Appendix 3

Phenotypic and genotypic characteristics of Phytophthora infestans used in crosses.

 

Thermal Gpi isozyme Mating Metalaxyl R gene
Isolate tolerancea genotype° typec response° phenotype°
Pi 41-02f T 100/100 A, R 1,3,4,5,6,7,8,
9,10,1 1
Pi 02-0078 T 100/1 1 1/122 A2 R 1,2,3,4,5,6,7
8,9,10,1 1
Pi S1-3g T 100/11 1/122 A2 I 1,2,3,4,5,6,7,
8,9,10,l 1
Pi 98-13 I 100/122 A2 R 1,4,5,9,10,11
Pi 152—02f T 100/100 A, 1 1,2,3,4,5,6,7,
8,9,10,1 1
Pi 95-3g S 86/100 A, S 5
Pi 4-19h S 100/100 A, I 1,3,4,5,6,7 ,8,
9,10,1 1

 

3 Comparison of growth of isolates of cultures of Phytophthora infestans for 25 days at 18°C after
exposure at -5°C for 5 days measured by an image analysis technique comparing the growth of
exposed isolates to a positive control (exposed to 18°C for 5 days) or a negative non-inoculated
control with no growth . T= Tolerant; grth not significantly different from the growth of the
positive control cultures of the same isolate; I= Intermediate; growth significantly less than that
of the growth of the positive control cultures of the same isolate but significantly greater than the
negative control; S= Sensitive; growth not significantly different fiom the negative control.

b Glucose -6- phosphate isomerase allozyme genotype; 86/100 (USl), 100/100 (U S-1 .7),
100/111/122 (U S-8), 100/122 (U S-14); genotype as designated by Goodwin et al., 1995.

c Mating type determined by production of oospores when grown with isolates of P. infestans of
known mating type.

d Sensitivity to the fungicide metalaxyl, R=resistant, I=Intermediate, S=sensitive determined by
comparing ECso response of the isolates grown on media amended with different concentrations
of metalaxyl with EC50 of lmown resistant and susceptible isolates.

° Virulence pathotype as defined by Muller and Black 1952. f Isolates of P. infestans from
Northern Ireland from collection of Dr. Louise Cooke, Plant Pathology, Queens University,
Northern Ireland. 8 Michigan isolates of P. infestans from collection of Dr. Kirk, Plant
Pathology, Michigan State University, East Lansing, MI, USA. h A progeny isolate obtained from
the 4th cross, used as a parent because of its sensitive phenotype.

149

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120

Differences in thermal tolerance of progeny isolates from mating of thermally tolerant
parental isolates of Phytophthora infestans Pi 41-02 (A,, thermally tolerant) x Pi 02-007
(A2, thermally tolerant). Growth was measured as %RARI after exposure of isolates to -
5°C for 5 days (Cross 2). The black bars represent the parental isolates and the white bars
indicate thermal sensitive (Pi 95-3) and insensitive (Pi 02-007) standards, respectively; Pi
02-007 was a parent in this cross. %RARI values followed by the same letter are not
significantly different at p = 0.1 (Tukey Multiple Comparison); LSDo,,o indicated by bar.

150

 

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isolates of Phytophthora infestans Pi 41-02 (A,, thermally tolerant) x Pi Sl-3 (A2, thermally tolerant).
Growth was measured as %RARI after exposure of isolates to -5°C for 5 days (Cross 3). The black
bars represent the parental isolates and the white bars indicate thermal sensitive (Pi 95-3) and
insensitive (Pi 02-007) standards, respectively. %RARI values followed by the same letter are not
significantly different at p = 0.1 (Tukey Multiple Comparison); LSD“, indicated by bar.

151

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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isolates of Phytophthora infestans Pi 41-02 (A,, thermally tolerant) x Pi Sl-3 (A2, thermally tolerant).
Growth was measured as %RARI after exposure of isolates to -5°C for 5 days (Cross 4). The black
bars represent the parental isolates and the white bars indicate thermal sensitive (Pi 95-3) and
insensitive (Pi 02-007) standards, respectively. %RARI values followed by the same letter are not
significantly different at p = 0.1 (Tukey Multiple Comparison); LSD“, indicated by bar.

152

 

 

 
 

 

 

 

 

 

 

 

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Differences in thermal tolerance of progeny isolates from mating of thermally tolerant parental
isolates of Phytophthora infestans Pi 62-02 (A., thermally tolerant) x Pi 02-007 (A1, thermally
tolerant). Growth was measured as %RARI after exposure of isolates to -5°C for 5 days (Cross 6).
The black bars represent the parental isolates and the white bars indicate thermal sensitive (Pi 95-3)
and insensitive (Pi 02-007) standards, respectively; Pi 02-007 was a parent in this cross. %RARI
values followed by the same letter are not significantly different at p = 0.1 (Tukey Multiple
Comparison); LSD...” indicated by bar.

 

 

 

 

 

 

 

 

 

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Piss-3 1 I I 1 o
o 20 40 no so 100 120

Differences in thermal tolerance of progeny isolates from mating of thermally tolerant parental
isolates of Phytophthora infestans Pi 41-02 (A1, thermally tolerant) x Pi Atlantic 2N (A2, thermally
sensitive). Growth was measured as %RARI after exposure of isolates to -50C for 5 days (Cross 8).
The black bars represent the parental isolates and the white bars indicate thermal sensitive (Pi 95-3)
and insensitive (Pi 02-00‘7) standards, respectively. %RARI values followed by the same letter are not
significantly different at p = 0.1 (Tukey Multiple Comparison); LSDO.10 indicated by bar.

153

Appendix 5

Phenotypic and genotypic characteristic of progeny isolates of Phytophthora
infestans produced from 9 different thermal phenotype crosses.

 

Mating

Temp-

Metal-

 

 

Isolate m era ture axyl Vlrulence Pathogen1c1ty Isozyme
1101 A1 A R 0’1’2’31’15’6’10’ 3 100/100
1102 A2 A R Nd Nd 100/100

0,1,2,3,4,5,6,7

1103 A1 A R 39,10,“ Nd 100/100

1104 A1,A2 A 1 o Nd 100/111/122
1105 A2 A 1 o,1,2,3,4,5 3 100/111/122
1-06 A1,A2 A R 1,2 2 100/111/122
1417 A1,A2 A 1 Nd 1 100/111/122
1-08 A2 A 1 Nd Nd 100/111/122
1109 A1,A2 B 1 Nd Nd 100/100

0,1,2,3,4,5,6,7

1110 A2 B 1 33,10,” 2 100/111/122
1-11 A2 B 1 Nd Nd 100/100

1.12 A2 B R Nd Nd 100/100

1-13 A1,A2 B R 0’1’3’1’15’6’10’ 2 100/111/122
1114 A1 C R Nd Nd 100/111/122
1115 A2 A R Nd Nd 100/111/122
1-16 A2 A R Nd Nd 100/111/122
1117 A2 A R Nd Nd 100/111/122
1-18 A1 c R 0’1’2’3f‘11’6’7’8 3 100/111/122
1119 A2 B R Nd Nd 100/111/122
1.20 A1 A s Nd 3 100/111x122
1121 A2 A s Nd Nd 100/111/122
122 A1 B 1 Nd Nd 100/111/122
1123 A1 A I Nd Nd 100/111/122
1124 A2 A 1 Nd Nd 100/111/122
1125 A2 B 1 Nd Nd 100/111/122
1-26 A2 C R Nd Nd 100/1 1 1/122
1127 A2 A R Nd Nd 100/111/122
1-28 A2 A R Nd Nd 100/111x122

Continued

154

 

Mating Temp- Metal-

 

 

Isolate type era ture axyl Virulence Pathogenicity Isozyme
1129 A2 A 1 3’5’6’71’9’10’1 1 100/100
130 A1 A R Nd Nd 100/100
1131 A2 A R Nd Nd 100/100
1132 A2 B I Nd Nd 100/100
1133 A2 B R Nd Nd 100/111/122
1-34 A2 A 1 Nd Nd 100/100
1135 A2 B R Nd Nd 100/111/122
1-36 A2 A 1 Nd 3 100/100
1-51 A1 B R Nd Nd 100/100

0,1,2,3,4,5,6,7
201 A1 A 1 33,10,“ Nd 100/100
202 A1 B R Nd Nd 100/100
2103 A1,A2 B R Nd 2 100/100
204 A2 B R Nd Nd 100/100
2105 A1 B 1 Nd Nd 100/100
2-06 A1 A R Nd Nd 100/100
O,1,2,3,4,5,6,7
207 A2 B R 3,1 0,11 Nd 100/100
2-08 A2 B R Nd Nd 100/100
2419 A1,A2 c R Nd 3 100/100
2110 A1 A R Nd Nd 100/100
211 A2 A R Nd Nd 100/100
2112 A2 A R Nd Nd 100/100
2113 A2 B 1 1’3’5’fi’17’8’10’ 3 100/111/122
2114 A2 A R Nd Nd 100/111/122
2115 A2 A R Nd Nd 100/100
2-16 A1 B R Nd Nd 100/100
217 A2 A R Nd Nd 100/100
2-18 A1 A I Nd Nd 100/100
2119 A1 B 1 Nd Nd 100/100
220 A1 A R Nd 4 100/100
221 A2 B R Nd Nd 100/100
2122 A2 B R Nd Nd 100/100
223 A2 B R Nd 3 100/100
2124 A2 A 1 Nd 2 100/100
2125 A2 B 1 Nd Nd 100/100
2-26 A2 A I Nd Nd 100/111/122
2127 A1 A R Nd Nd 100/100
Continued

'155

 

Mating Temp- Metal-

 

 

Isolate type era ture axyl Virulence Pathogenicity Isozyme
2-28 A1 B R Nd Nd 100/100
2129 A2 A R Nd Nd 100/100
230 A2 B R Nd Nd 100/111/122

0,1,2,3,4,5,6,7
2131 A2 B R 3,9,1 0,“ Nd 100/100
2132 A2 B R Nd Nd 100/111/122
2133 A1,A2 B R Nd 3 100/100
234 A1 B 1 Nd 2 100/100
2135 A2 c R Nd Nd 100/111/122
2-36 A2 B 1 Nd Nd 100/111/122
2137 A2 B R Nd Nd 100/111/122
2.311 A1 B R Nd Nd 100/100
2139 A2 B 1 Nd Nd 100/111/122
240 A1 B R Nd Nd 100/100
241 A2 A R Nd Nd 100/111/122
242 A2 B I Nd Nd 100/1 11/122
243 A1 B R Nd Nd 100/111/122
244 A2 B R Nd Nd 100/111/122
245 A2 A R Nd Nd 100/111/122
2-46 A2 A R Nd Nd 100/111/122
247 A2 c R Nd Nd 100/111/122
243 A1 B R Nd Nd 100/111/122
249 A2 A R Nd Nd 100/111/122
2150 A2 A 1 Nd Nd 100/111/122
2151 A1 c R Nd Nd 100/111/122
3_OA A1 A R 0’1’263’151’7’8’1 3 100/111/122
0,1,2,3,4,5,6,7
301 A1 A R 3’10,“ 3 100/100
3102 A2 A I Nd Nd 100/111/122
3413 A1 A R Nd Nd 100/100
3414 A1,A2 B R Nd Nd 100/100
3415 A1 A I Nd 2 100/100
3-06 A2 A R 1’2’36‘1’151’7’8’1 Nd 100/100
3417 A2 A 1 Nd Nd 100/100
3-08 A2 A I Nd Nd 100/100
3109 A2 B 1 Nd Nd 100/100
3110 A2 A 1 Nd Nd 100/100
3.11 A2 A 1 Nd Nd 100/100
Continued

156

 

Mating

Temp-

Metal-

 

 

Isolate type era ture axyl Virulence Pathogenicity Isozyme
3-12 A2 B I Nd Nd 100/100
3-13 A1,A2 A I Nd Nd 100/100
3-14 A2 A S 0,1,2,3,6 4 100/100
3-15 A1 B I Nd Nd 100/100
3-16 A1 B S Nd Nd 100/100
3-17 A2 C S Nd Nd 100/100
3-18 A1 A I Nd Nd 100/100
3-19 A1 B S Nd Nd 100/100
3-20 A1 A S Nd Nd 100/ 100
3-2 1 A2 A I Nd Nd 100/ 100
3-22 A1 A R Nd Nd 100/100
3-23 A1 A I Nd Nd 100/100
3-24 A1 B I Nd Nd 100/ 100
3-25 A1 A I Nd Nd 100/100
3-26 A1 A S Nd Nd 100/100
3-27 A1 A I Nd Nd 100/100
3-28 A1 B I Nd Nd 100/100
3-29 A1 A I Nd Nd 100/100
3-30 A1 A I Nd Nd 100/100
3-31 Al B I Nd Nd 100/100
3-32 A2 I Nd Nd 100/100
3-33 A2 B I Nd Nd 100/100
3-34 A1 B I Nd Nd 100/100
3-35 A1 B I Nd Nd 100/100
3-36 A2 B S Nd Nd 100/100
3-37 A2 B S 0,1,2,3,5,6 3 100/111/122
3-38 A2 B I Nd Nd 100/111/122
3-39 A2 B I Nd Nd 100/111/122
3-40 A1 B I Nd Nd 100/111/122
341 A1 A I Nd 3 100/100
3-42 A2 A R Nd 3 100/111/122
343 A1 B I Nd Nd 100/100
3-44 Al B S Nd Nd 100/100
3-45 A1 A S Nd Nd 100/100
346 A2 B R Nd Nd 100/111/122
347 A2 A R Nd Nd 100/100
3-48 A2 B R Nd Nd 100/100
349 A1 A I Nd Nd 100/100
3-50 A1,A2 A I Nd Nd 100/100
4-01 A1 B I 1,3,5,9,11 2 100/100

Continued

157

 

Mating Temp- Metal-

 

 

Isolate type era ture axyl Virulence Pathogenicity Isozyme
4-02 A2 A I Nd Nd 100/100
4-03 A1 A I Nd Nd 100/100
4-04 A1 A I Nd Nd 100/100
4-05 A1 A R Nd 2 100/ 100
4-06 A1 A R Nd Nd 100/100
4-07 A1 A R Nd Nd 100/100
4-08 A1 A R Nd Nd 100/100
4-09 A1 A S Nd 2 100/100
4-10 A2 A R Nd Nd 100/100
4111 A1 c 1 0’1’33’151’7’9’1 Nd 100/100
0,1,2,3,4,5,6,7
4-12 A1 C I ,8,10,ll 2 100/100
4-13 Al B I Nd Nd 100/100
4-14 Al B I Nd Nd 100/100
4-15 A1 C R Nd Nd 100/100
4-16 Al B I Nd Nd 100/100
4-17 A1 B I Nd Nd 100/100
4-18 A1 B I Nd Nd 100/100
4-19 A1 C I Nd 2 100/100
4-20 A1 B I Nd Nd 100/100
4—21 A1 A S Nd 1 100/100
4-22 A1 A S Nd Nd 100/ 100
4-23 A1 B I Nd Nd 100/ 100
4-24 A1 B I Nd Nd 100/100
4-25 A1 A S Nd Nd 100/100
4-26 A1 A I Nd Nd 100/100
4-27 A1 B I Nd Nd 100/100
4-28 A1 A I Nd Nd 100/ 100
O,1,2,3,4,5,6,7
4—29 A2 B S 33,10,“ 2 100/111/122
4-30 A2 A S Nd Nd 100/ 100
4-3 1 A1 A R Nd Nd 100/100
4-32 A2 B I Nd Nd 100/ 100
4-33 Al B I Nd Nd 100/ 100
4-34 A2 B I Nd Nd 100/100
4-35 A2 A I Nd Nd 100/100
436 A1 A S Nd Nd 100/100
4-37 A2 A S Nd Nd 100/ 100
4-38 A1 A S Nd Nd 100/100
Continued

158

 

Mating

Temp-

Metal-

 

Isolate type era ture axyl Virulence Pathogenicity Isozyme
4139 A2 A s Nd Nd 100/100
440 A1 A I Nd Nd 100/100
441 A1 B s Nd Nd 100/100
442 Al B I Nd Nd 100/100
443 A2 B 1 Nd Nd 100/111/122
444 A1 C 1 Nd Nd 100/100
445 A1 B 1 Nd Nd 100/100
4-46 A2 A 1 Nd Nd 100/100
447 A2 B s Nd Nd 100/111/122
4-48 A2 B R Nd Nd 100/100
449 A1 B 1 Nd Nd 100/111/122
4150 A2 B 1 Nd Nd 100/111/122
4151 B Nd Nd
501 A1 B R o,1,2,3,4,5,6 Nd 100/122
5412 A1 B 1 1’2’36‘3’151’6’7’1 Nd 100/100
5413 A1 C 1 Nd Nd 100/100
5104 A1 B 1 Nd Nd 100/100
5415 A2 C 1 Nd Nd 100/100
5-06 A2 B R Nd Nd 100/1110
5107 A1 A 1 Nd Nd 100/100
5418 A2 A 1 Nd Nd 100/122
5419 A1 I 1’2’3’465’6’7’1 Nd 100/100
5110 A1 C R Nd Nd 100/100
541 A1 B R Nd Nd 100/100
5112 A1 1 o,1,2,3,4,5,6 3 100/100
5-13 A2 A 1 Nd Nd 100/100
5114 A1 C 1 3,5,7,8,9 Nd 100/100
5115 A2 A R Nd Nd 100/100
5-16 A1 A R 0111 2 100/100
5117 A1 1 Nd Nd 100/100
5-18 A2 R Nd Nd 100/100
5119 A1 C I 0’1’2’3’?’7’8’9 0 100/100
5120 A1 1 Nd Nd 100/100
5121 A2 C 1 Nd Nd 100/100
5122 A1 1 Nd Nd 100/100
523 A1 1 Nd Nd 100/100
524 A2 C R Nd Nd 100/122

Continued

159

 

 

Isolate M133? grim Maia]- Virulence Pathogenicity Isozyme
5-25 A1 A I Nd Nd 100/100
5-26 A1 A I Nd Nd 100/100
5-27 A2 I Nd Nd 100/100

0,1,2,3,4,5,7,8
5-28 A1 A I 3,10,“ Nd 100/122
5-29 A2 A I Nd Nd 100/100

0,1,2,3 ,4,5,6,7
5-30 A1,A2 A I ,9,10,1 l Nd 100/100
5-31 A1 I Nd Nd 100/100
5-33 A1 A I Nd Nd 100/100
5-34 AI C I Nd Nd 100/100
5-35 A1 C I Nd Nd 100/100
5-36 A1 A I Nd 2 100/100
5-37 A2 C I Nd 3 100/122
5-38 Al B I 1,3,5,6,8 Nd 100/100
5-39 A2 C I Nd Nd 100/100
5-40 A1 C I Nd Nd 100/122
5-41 A2 A I l 1 100/100
5-42 A1 C I Nd Nd 100/100
5-43 A2 B I Nd 1 100/100
544 A1 B R Nd Nd 100/100
5-45 A2 C I 1,4 0 100/122
5-46 A1 C I Nd 0 100/100
6-01 Al B R Nd Nd 100/100
6-02 A1 B I Nd Nd 100/100
6-03 Al B I Nd Nd 100/100
6-04 A1 B R Nd Nd 100/100
6-05 A2 B I Nd Nd 100/1 11/122
6-06 A2 A R Nd 2 100/111/122
6-07 A2 B I Nd Nd 100/111/122

0,1,2,3,4,5,6,7
6-08 A2 B I ,8,9,10,11 Nd 100/111/122
6-09 A2 C I Nd Nd 100/1 1 1/122
6-10 A2 B R Nd Nd 100/111/122
6-11 A1 B S 1,3,5,7,10,11 0 100/100
6-12 A2 C R Nd 0 100/111/122
6-13 A2 A R Nd Nd 100/111/122
6-14 A2 C I Nd 3 100/111/122

Continued

160

 

 

Isolate M133: g 2:3; Maifil' Virulence Pathogenicity Isozyme
6-15 A2 A I Nd Nd 100/111/122
6-16 A2 B I Nd 3 100/111/122
6-17 A2 B 1 Nd 2 100/111/122
6-18 A2 B 1 Nd Nd 100/111/122
6-19 A1 B I 3’4’5'fi’17’8’10’ 3 100/111/122
6-20 A1 A R 0’1’2’31’15’7’10’ 2 100/100
6-21 Al B I Nd Nd 100/100
6-22 A2 C R Nd Nd 100/111/122
6-23 A2 A I Nd Nd 100/1 11/122
6-24 A2 C I Nd Nd 100/111/122
6-25 A2 C 1 Nd Nd 100/111/122
741 A2 A R Nd 0 100/111/122
7412 A2 B R Nd 0 100/111/122
7103 A2 C R Nd 3 100/111/122
7104 A2 B R Nd Nd 100/111/122
7415 A1 A R Nd Nd 100/111/122
7-06 A1 C R Nd Nd 100/111/122
7107 A1 C R Nd 0 100/111/122
7-08 A1 A 1 Nd 2 100/111/122
8-01 A1 B R Nd Nd 100/100
8-02 A1 C I Nd 3 100/100
8-03 A1 B s Nd Nd 100/100
8-04 A1 A I Nd Nd 100/100
8-05 A1 A s Nd 2 100/100
8-06 A1 B I Nd Nd 100/100
8-07 A1 A 1 Nd Nd 100/100
8-08 A1 B R Nd 2 100/100
8-09 A1 A 1 Nd 2 100/100
8-10 A1 A I Nd Nd 100/100
8_1 1 A2 A I Nd Nd 100/100
8_12 Al C R Nd Nd 100/100
8_13 A1 C R Nd Nd 100/100
8_14 A1 A 1 Nd Nd 100/100
8_15 A2 A I Nd Nd 100/100
8_16 A1 C 1 Nd Nd 100/100
9_01 A1 T I Nd 3 100/100
9_02 A1 s 1 Nd 0 100/100

 

161

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