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This is to certify that the

thesis entitled

Assessment of Late Blight (Phytophthora infestans)
Among Cultivars and Advanced Lines of Potato
(Solanum tuberosum)

 

 

presented by

Maria Amparo Agnes Bertram

has been accepted towards fulfillment
of the requirements for

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moo momma.“

ASSESSMENT OF LATE BLIGHT (PHYTOPHTHORA INFESTANS) REACTION
AMONG CULTIVARS AND ADVANCED LINES OF POTATO (SOLANUM
TUBEROSUM)

By

Maria Amparo Agnes Bertram

A THESIS
Submitted to
Michigan State University

in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE
Crop and Soil Sciences

1 999

ABSTRACT
ASSESSMENT OF LATE BLIGHT (PHYTOPHTHORA INFESTANS) REACTION

AMONG CULTIVARS AND ADVANCED LINES OF POTATO (SOLANUM
TUBEROSUM)

By

Maria Amparo Agnes Bertram

The spread of metalaxyl-resistant genotypes ofPhytophthora infestans from
central Mexico in the 19805 and 19903 has led to the re-emergenee of late blight as a
major threat to potato production worldwide. To assist a breeder in planning crosses to
increase resistance in lines for future release as varieties, information on the resistance in
available germplasm must be obtained. This study compares data from two years of field
evaluation, greenhouse foliage resistance screening, and inoculated tuber reactions
among potato lines with various levels of resistance to P. infestans. Replicated field trials
were grown under irrigation and inoculated with P. infestans, then rated throughout the
season for defoliation. In the greenhouse, individual plants were grown in pots, moved to
an environment chamber for inoculation, then rated for percent defoliation. Tubers were
injected with cultured P. infestans, incubated, then visually rated for surface degradation
and scanned internally for digital analysis. Five lines (AWN86514-2, 80692-4, B0718-3,
MSG274-3, and Q237-25) with strong foliar resistance were identified based on field trial
results. Greenhouse screens allowed resistant and susceptible lines to be distinguished,
but results did not correlate well with field data. Four highly resistant lines (A08427S-3,
Bzura, MSGOO7-1, and MSGZ97-4RD) were found through tuber evaluation, but tuber
resistance did not correlate with field foliar resistance. Several breeding strategies and

suggestions for future research based on these data are provided.

ACKNOWLEDGMENTS

I thank David Douches, Ray Hammerschmidt, and James Kelly for continuing
guidance and advice. Thanks to Kazimierz JastIzebski, Dilson Bisognin, Christopher
Long, and Kimberley Walters for assistance in collecting data and William Kirk, Brendon
Niemira, and Jeffrey Stein for P. infestans cultures, technical assistance, and valuable
discussion.

This work was supported by Michigan State University, the Michigan Potato

Industry Commission, and the National Potato Council.

iii

TABLE OF CONTENTS

LIST OF TABLES .............................................................................................................. vi
LIST OF FIGURES ........................................................................................................... vii
GENERAL INTRODUCTION
Origin of the potato .................................................................................................. l
Phytophthora infestans ............................................................................................ 2
Host-patho gen interaction ........................................................................................ 4
Breeding for late blight resistance ........................................................................... 5
CHAPTER 1: F OLIAGE SCREENING FOR RESISTANCE TO PHYTOPHH-IORA
INFESTANS .................................................................................................. 7
INTRODUCTION ............................................................................................................... 8
MATERIALS AND METHODS ....................................................................................... 11
Plant material ......................................................................................................... 11
P. infestans inoculum preparation .......................................................................... 12
Field evaluation ...................................................................................................... 12
Greenhouse evaluation ........................................................................................... 13
Field rating ............................................................................................................. l4
Greenhouse rating ...................................... 15
Statistical methods ................................................................................................. 15
RESULTS .......................................................................................................................... 16
Field results ............................................................................................................ 16
Greenhouse P. infestans US8 genotype results ...................................................... 24
Greenhouse P. infestans genotype by variety interaction ...................................... 24
DISCUSSION .................................................................................................................... 28
CHAPTER 2: TUBER SCREENING FOR RESISTANCE TO PHYTOPHIHORA
INFESTANS ................................................................................................ 40
INTRODUCTION ............................................................................................................. 41
MATERIALS AND METHODS ....................................................................................... 42
Plant material ......................................................................................................... 42
P. infestans inoculum preparation .......................................................................... 42
Tuber inoculation ................................................................................................... 43
Tuber disease rating ............................................................................................... 43
Statistical methods ................................................................................................. 44
RESULTS .......................................................................................................................... 46
Surface rating ......................................................................................................... 46
Internal section analysis ......................................................................................... 48
DISCUSSION .................................................................................................................... 52
SUMMARY ....................................................................................................................... 55

iv

LIST OF TABLES

. Relative Area Under the Disease Progress Curve (RAUDPC) for the 1997 Field

Season .................................................................................................................... 17

. Relative Area Under the Disease Progress Curve (RAUDPC) for the 1998 Field
Season .................................................................................................................... 20

. Relative Area Under the Disease Progress Curve for Selected Potato Lines ........ 23

. Greenhouse Defoliation Ratings of 28 Lines Inoculated with Phytophthora

infestans USS Genotype ......................................................................................... 25
. Greenhouse Defoliation Ratings of 8 Lines Inoculated with Phytophtora infestans
U81 and USll Genotypes ..................................................................................... 27
. Field and Greenhouse Results for 28 Selected Potato Lines ................................. 36
. Visual Rating Scale for Phytophthora infestans Infection in Potato Tubers ......... 45
. Tuber Late Blight Resistance Ratings for Selected Potato Lines .......................... 47

. Comparison Between Diseased and Healthy Tuber Flesh Based on Internal
Section Mean Light Intensity ................................................................................. 50

vi

LIST OF FIGURES

Area Under the Disease Progress Curve after 28 days for the highest (MSEOl l-
14) and lowest (MSGZ74-3) rated Michigan State University potato breeding
lines in the 1998 Phytophthora infestans resistance field trial .......................... 14

Relative rankings of 28 selected potato lines in 1997 (vertical axis) and 1998
(horizontal axis), where 1 = most resistant to late blight and 28 = most
susceptible, based on RAUDPC values ............................................................. 29

Breeding strategies for introducing durable resistance to P. infestans into
agronomically acceptable genetic backgrounds. Clones in bold show
resistance, and the underlined are unspecified potato lines. A) Cross selected
progeny of strong resistance sources with difi'erent pedigrees. B) Cross
selected progeny of a strong resistance source with that of a moderately
resistant source ................................................................................................... 32

Relative P. infestans resistance rankings of 28 selected potato lines in
greenhouse tests (horizontal axis) and field trials (vertical axis) where l = most
resistant to late blight and 28 = most susceptible. Greenhouse rankings are
based on percent defoliation and field rankings on the Relative Area Under the
Disease Progress Curve for each line. No correlation Was found between the
greenhouse data and the 1997 field results (top, p = 0.17) or the 1998 field
results (bottom, p = 0.08) ................................................................................... 35

Scanned apical sections of four sample tubers inoculated with P. infestans from
a) GZ97-4RD (mean intensity 183.79), b) G274-3 (mean intensity 136.51), and
c) Nordonna (mean intensity 101.64) ................................................................. 49

Relative P. infestans tuber resistance rankings of 26 selected potato lines
according to surface disease (horizontal axis) and internal light intensity
(vertical axis, top) or percent intensity of healthy flesh (vertical axis, bottom)
where l = most resistant to late blight and 26 = mostsusceptible. Surface
rankings are based on a 1 - 9 scale of increasing disease severity and internal
rankings on average light intensity where 0 = black and 255 = white. Top, r =
0.44, p = 0.023. Bottom, r = 0.54, p = 0.0041 ................................................... 51

Relative rankings of 28 selected potato lines in 1997 (vertical axis) and 1998
(horizontal axis), where 1 = most resistant to late blight and 28 = most
susceptible, based on Relative Area Under the Disease Progress Curve

values .................................................................................................................. 58

vii

Relative P. infestans resistance rankings of 28 selected potato lines in
greenhouse tests based on percent defoliation (horizontal axis) and the 1997
field trial based on Relative Area Under the Disease Progress Curve (vertical
axis) where 1 = most resistant to late blight and 28 = most susceptible ............ 59

viii

GENERAL INTRODUCTION

Origin of the potato

The cultivated potato (Solanum tuberosum L.) originated in the Andes mountains
of South America (Dean, 1994), where it was domesticated by the natives of Peru and
Bolivia as many as 7,000 years ago (Zuckerman, 1998). The potato was valuable as a
staple crop in high altitude regions where maize and beans could not be grown, and the
hardy plant survived even in thin, low fertility soil under drought conditions (Zuckerman,
1998). Out of over 2,000 Solanum species, more than 160 are tuber-bearing wild
potatoes, and many can freely interbreed. They can be found in a range of ploidy levels,
from the more common diploids (2x = 24), with tetraploid species (including S.
tuberosum) second most common, to a small percentage of triploids, pentaploids, and
hexaploids (Burton, 1989). Potatoes are cross-pollinated by wind or insects and can form
fi'uit containing true seeds; however, when grown as a crop they are mainly sown as small
seed tubers or sections of tubers containing one or more eyes.

Spaniards discovered potatoes in the 16th century, most likely around 1537
(Hawkes, 1990), and introduced them to Spain around 1570 (Salaman, 1949). The potato
was classified as a member of the Solanaeeae in 1596, a family that includes the tomato
(Lycopersicon lycopersicum), eggplant (Solanum melongena), sweet pepper (Capsicum
annuum), tobacco (Nicotiana tabacum), deadly nightshade (Atropa belladonna),
mandrake (Mandragorum ofiicinarum), and henbane (Hyoscyamus niger). The
association with poisonous (nightshade) and reputedly supernatural (mandrake) plants,

combined with its use as a staple by New World slaves, gave the potato an unfavorable

reputation in Europe, and it was not widely grown for two centuries (Zuckerman, 1998).
However, the tuber's more valuable qualities eventually became apparent in the poor
country of Ireland.

First, the potato is highly nutritious, particularly in terms of usable protein and
vitamin C, lacking mainly calcium (Burton, 1989), which could be provided by the
addition of milk. Second, it provides more yield per unit area planted than any of the
major grain crops, and it can be eaten with a minimum of preparation time and cooking
utensils, making it an excellent food source for those with little money or land. The
potato thrived under the mild, wet conditions prevailing in Ireland, the short—day adapted
South American varieties producing well during the country's long growmg season.
Finally, unlike other crops with their valuable portion above ground, the tubers were
protected from damage by soldiers in the frequent battles of the time (Zuckerman, 1998).
Phytophthora infestans

A new potato disease appeared in Europe and North America in the early 18408
that caused foliage to blacken and tubers to rot. Observers first noted it in the area
around New York and Philadelphia in 1843, fi'om which it spread west to the Great Lakes
and north into Canada in succeeding years. The disease may have spread fiom there to
Europe in 1844, or both epidemics may have derived fi'om infected tubers imported
separately fi'om the same source (Bourke, 1964). In 1845, this blight spread from
Belgium westward until it reached Ireland, where it resulted in losses to half the year's
crop of potatoes (Robertson, 1991). Another devastating outbreak of late blight in 1846,
combined with the societal structure at the time, resulted in a famine in Ireland so severe

that one million people died and another l'/z million emigrated (Burton, 1989).

Late blight is considered the most important disease of the potato (Hooker, 1981).
Phytophthora infestans (Mont) de Bary, the pathogen that causes potato late blight, is an
Oomycete, a member of the order Chromista, and not a true fungus (Fry and Goodwin,
1997). Its asexual cycle begins with lemon-shaped sporangia, which can either infect
healthy tissue directly or release zoospores possessing flagella for mobility in water.
Once the sporangia or zoospores come into contact with plant tissue, they enter and
spread hyphae that can initiate a new round of sporangia] production in a matter of a few
days (Coffey and Gees, 1991). It is heterothallic, needing two distinct mating types (Al
and A2) in order to undergo sexual reproduction, which results in oospores that can better
tolerate winter conditions than the zoospores, in addition to providing a means for genetic
recombination (Kirk, 1996).

P. infestans is believed to originate in the central highlands of Mexico. The long-
term coexistence of both mating types, the abundance of oospores, and the proliferation
of resistant Solanum species suggest coevolution with the disease (Bourke, 1964;
Goodwin and Drenth, 1997). P. infestans prospers in a humid, cool environment,
particularly Since water helps the spread of its motile zoospores. Primary inoculum is
generally fi'om hyphae that overwinter on infected living tissue, such as tubers in storage
or cull piles. Tuber lesions are irregular, reddish-brown spots of tissue decay. Infected
foliage develops dark, round, water-soaked lesions surrounded by a white fringe of
sporangiophores, most easily observed on the lower surface of the leaf (Dean, 1994;

Franc, Brown, and Kerr, 1996); stems are also susceptible to infection (Robertson, 1991).

I-Iost-pathogen interaction

Potato species display two kinds of resistance to late blight: vertical or specific
resistance and horizontal or general ”field" resistance. Lines with vertical resistance,
controlled by a few major resistance (R) genes, can Show the hypersensitive response, a
rapid necrosis of infected and nearby cells. This kind of resistance is highly specific to a
virulence gene in the pathogen, conferring strong resistance only to certain pathotypes.
Horizontal resistance is regulated by a complex interaction of minor genes and provides
partial resistance to all P. infestans pathotypes (Ross, 1986). It is more strongly affected
than vertical resistance by environmental conditions. Early efforts to breed resistance
into cultivated potato fiom wild relatives focused on the vertical resistance from Solanum
demissum. However, as backcrossed progeny with specific R genes became exposed to
compatible pathotypes, the resistance was quickly overcome (Bradshaw et al., 1995b).
Black et a1. (1953) proposed a system of nomenclature for identifying P. infestans
pathotypes based on virulence gene composition determined by testing against potato
clones with known R genes. A pathotype of P. infestans is said to have virulence gene 1
if it can cause infection on a potato clone with gene R1. Eleven R genes have been
identified (Malcolmson, 1969). This is different from other systems of R gene
interaction, in which pathogens are classified by avirulence genes that provoke a
resistance response in a host plant with a corresponding R gene.

Unless both A1 and A2 mating types are present, P. infestans undergoes asexual
propagation. Lacking sexual recombination, specific strains should form clonal lineages
that can be identified with genetic markers. A method has been proposed to identify

pathogen genotype using electrophoresis to distinguish two allozyme loci, Glucose-6-

phosphate isomerase (Gpr) and Peptidase (Pep) (Goodwin, Schneider, and Fry, 1995).
At least 17 of these clonal lineages have been classified using this method in the United
States alone. The lineages are named US1, US7, US8, and so on. Detailed
characterizations of these lineages to include mating type and resistance to the
phenylarnide fimgicide metalaxyl have been conducted (F ry and Goodwin, 1997).

Prior to the early 19808, USl, an Al mating type lineage sensitive to metalaxyl,
was the predominant genotype, and so late blight could be controlled with metalaxyl
application (Goodwin, Sujkowski, and Fry, 1996). Beginning in 1984, isolates of the A2
mating type resistant to metalaxyl were discovered around the world (Goodwin and
Drenth, 1997). The migration of these new genotypes fiom Mexico posed serious disease
management problems (Goodwin et al., 1994) leading to severe economic losses due to
late blight epidemics in the United States in the 1990s (Goodwin et al., 1998).

Breeding for late blight resistance

Currently, potato production worldwide is surpassed only by wheat, maize, and
rice (Ross, 1986). Despite the crop's importance, there are no acceptable commercial
varieties with adequate resistance to late blight (Landeo et al., 1995; Helgeson et al.,
1998). Many breeding programs around the world have made the development of
resistant cultivars a priority (Bradshaw et al., 1995b; Corsini et al., 1999; Darsow, 1995;
Douches et al., 1998b; International Potato Center, 1984; Kankila et al., 1995).

Since varieties of S. tuberosum subsp. tuberosum, the tetraploid potato most
commonly grown in Europe and North America, are susceptible to the disease, breeders
must use its cultivated and wild relatives in Mexico and South America for sources of

resistance (Bradshaw et al., 1995b; Colon et al., 1995 ; Darsow, 1995). Introduction of R

genes has largely been abandoned as a means of building resistance in favor of the more
complex horizontal resistance because of the ease with which P. infestans develops new
virulence pathotypes (Black, 1970; Bradshaw et al., 1995a; Colon et al., 1995; Dorrance
and Inglis, 1997). One breeding strategy even employs screening and selection to
eliminate any R genes present in breeding lines to ensure that all resistance is horizontal
(Landeo et al., 1995), although some ineffective R genes may be linked to quantitative
horizontal resistance (Ordofiez et aI. , 1998).

Resistance sources such as S. tuberosum subsp. andigena (Black, 1970), the
cultivated tetraploid fi'om Peru and Bolivia (Burton, 1989), cross readily with commercial
varieties through traditional breeding methods. Due to different ploidy levels and
endosperrn balance number, crosses with other wild species necessitate further measures
including embryo rescue, manipulation of parental ploidy level (Bradshaw et al., 1995b),
and somatic hybridization (Helgeson et al., 1998). Central to breeding efforts is the
ability to screen the available germplasm for resistance so that selection is effective.
Many methods are used, including inoculated (Colon et al., 1995a) and naturally infected
field trials (Inglis et al., 1996), intensive field trials in Toluca, Mexico (Helgeson et al.,
1998), greenhouse seedling tests (Dorrance and Inglis, 1997), detached-leaf evaluation
(Goth and Keane, 1997), quantification with transgenic P. infestans (Karnoun et al.,
1998), and both field and laboratory tuber screens (Dorrance and Inglis, 1998).

In this two-year study, over 200 potato varieties and advanced breeding lines were
evaluated under inoculated field conditions. Selected lines were also subjected to
greenhouse foliage testing and tuber resistance screens, and the results were compared to

the field trial data.

CHAPTER 1: F OLIAGE SCREENING

FOR RESISTANCE TO PHYTOPHIHORA INFEST ANS

INTRODUCTION

Potato late blight, which caused the Irish potato famine in the 18405, emerged in
the mid-19808 and 1990s as a new threat to global potato (Solanum tuberosum L.)
production. The pathogen, Phytophthora infestans (Mont) de Bary, had previously been
successfully controlled with the systemic fungicide metalaxyl. The migration of
metalaxyl-resistant strains of P. infestans fiom central Mexico has caused serious potato
production and economic problems worldwide (Fry and Goodwin, 1997).

One of the major goals of the Michigan State University potato breeding program
is to introduce new market-quality varieties with greater levels of late blight resistance
than are currently available (Douches er al., 1998b). These varieties must also possess
agronomic qualities such as high yield, early or moderate maturity, unblemished internal
flesh, high specific gravity, and attractive appearance (Dean, 1994). However, sources of
strong resistance, especially wild Solanum species, do not have commercially desirable
attributes, and so many years of backcrossing and selection must be performed prior to
release (Bradshaw et al., 1995b).

Since disease response differs due to physiological age, resistance to late blight
coincides with extremely late maturity in most sources (Colon et a1, 1995; Dorrance and
Inglis, 1997; Inglis et al., 1996). When highly resistant varieties are used as parents in a
breeding program, they must be crossed to early maturing susceptible lines to generate
progeny with acceptable maturity. The result is offspring with a wide range of resistance
levels. Varieties with intermediate resistance, partially adapted European lines for

example, are less likely to have the agronomic weaknesses of the wild species with the

strongest resistance, and could thus be valuable as parents. Resistance may arise through
several different partially effective mechanisms (Black, 1970; Colon et al., 1995b).
Progress might be made by intercrossing clones with moderate resistance levels derived
fi'om varied sources in an attempt to combine these mechanisms into the same line.
Efficient use of this material depends on accurate resistance evaluation.

Although field screening of potato lines is an effective method to evaluate their
resistance to late blight, a comparable test would be valuable if it uses less space, takes
less time, does not introduce disease inoculum over a large land area, and lacks seasonal
restrictions. Greenhouse disease chamber testing can be useful for many reasons.

The ability to test plants during the winter (Colon et al., 1995a) in addition to the normal
growing season increases the number of lines screened, thereby shortening the selection
period. Greenhouse screening allows a breeder to evaluate large numbers of progeny in a
short amount of time to determine the general or Specific combining ability of the parents
(Black, 1970; Bradshaw er al., 1995a) for planning future crosses. Screening progeny of
resistance crosses in the greenhouse to predict their performance in the field (Brown et
al., 1999) gives the breeder preliminary information to assist in making selection
decisions, so that limited field space can be optimized by planting the most promising
clones (Bradshaw et al., 1995b). Environmental conditions can be controlled to create
uniform infections when performing repeated tests (Helgeson et al., 1998). The
controlled conditions reduce the risk of disease spread from an inoculated disease trial
(Colon, Budding, and Hoogendoorn, 1995). Finally, the breeder can evaluate varietal

resistance to different P. infestans genotypes, including both mating types (Inglis et al.,

1996), which would introduce the danger of sexual recombination if carried out in the
field.

Greenhouse screening is considered an accurate means of predicting a line's field
resistance (Dorrance and Inglis, 1997), though differences in lesion growth rate have
been reported between field- and greenhouse-grown leaves (Colon et al., 1995b). The
environment in an enclosed late blight inoculation chamber, where temperature,
humidity, and light can be controlled, is vastly different from that in the field; it is
possible that the concentrated conditions of the test might break down the resistance of
lines with good field tolerance.

US8 is currently the most prevalent P. infestans genotype in the United States
(Fry and Goodwin, 1997); however, genotypes such as the original, metalaxyl-sensitive
USl and the metalaxyl-resistant USll are also found, particularly on the West Coast
(Goodwin et al., 1998; Dorrance et al., 1999). A variety with the desired durable
resistance should show low infection under all three genotypes of the pathogen.

The primary objective of this study was to conduct a late blight field resistance
screen on current MSU potato breeding lines and the germplasm available fi'om other
locations for use as parents. These data will provide the basis for decisions about future
crosses and for identifying resistant germplasm for commercial release. The second
objective was to conduct greenhouse evaluations of select potato lines according to
Douches et al. (1997) and compare with results obtained in the 1997 and 1998 field trials.
The third objective was to gather and compare data on the resistance of eight potato lines

when inoculated with US l , U88, or USll genotypes of P. infestans.

10

MATERIALS AND METHODS

Plant material

About 170 clones were screened in each field trial, although the composition of that
total changed from 1997 (Table 1) to 1998 (Table 2) as lines were dropped flour or added
to the breeding program. The clones fell into the following main categories:

0 National Late Blight Trial lines-distributed by Dr. K. G. Haynes (USDA) for the
national late blight resistance testing program. These included commercial
varieties, European and unadapted material, and lines with known R genes.
(Haynes et al., 1998)

o Susceptible controls-commercial varieties that are widely grown in the United
States for table or processing markets, including 'Atlantic,‘ '0naway,‘ 'Russet
Burbank,’ 'Russet Norkotah,’ 'Shepody,' 'Snowden,’ 'Superior,’ and 'Yukon Gold'
(Inglis et al., 1996).

0 European varieties-~clones imported for evaluation as potential late blight
resistance sources or commercial cultivars.

o Breeding lines--clones in the process of agronomic testing and selection fi'om
university breeding programs in the North Central region (Douches et al., 1998a).

The US8 greenhouse trials screened a subset of 28 lines from the field trials,

including clones with various levels of resistance and susceptible check varieties (Table
3). Eight of those lines (AWN86514-2, Atlantic, B0718-3, Bzura, MSGZ74-3, Matilda,

Snowden, and Zarevo) were further tested against USl and US11 genotypes.

ll

P. infestans inoculum preparation

Michigan isolates 95-7 (1997) and 97-2 (1998) of the U88 genotype of P. infestans
were maintained in culture on rye or potato dextrose agar. To prepare an inoculum
suspension, each plate of mycelium was flooded with 15-20 ml of distilled water, and the
aerial hyphae were peeled from the media with a plastic scraper. The water and hyphae
were poured into a beaker, and the plate was rinsed into the same beaker with an
additional 10 ml of distilled water to recover as much of the hyphal mass as possible.
The hyphae were agitated on a stir plate for 15 min to disperse the sporangia into the
water. The water was filtered through four layers of cheesecloth to remove excess
hyphae and refiigerated at 4°C for 3 to 4 hr to release the motile zoospores. The
concentration of zoospores was estimated with the aid of a hemacytometer and diluted to
the desired concentration, 103 zoospores/ml. For the field evaluation, the inoculum
suspension was administered (100 ml/7.5m row) through the field's irrigation system on
July 18 (1997) or July 22 (1998).

Inoculum for the greenhouse screen was prepared fi‘om rye cultures as described
above, using the following Michigan isolates at 200 ml inoculum per chamber:

0 U81: 95-5 and 95-6.

0 U88: 94—1, 94-3, 95-7, 97-1, 97-2, and 98-2.

0 U811: 96-1.
Field evaluation

Field tests of cultivars and breeding lines were conducted during the 1997 and

1998 growing seasons at the Michigan State University Muck Soils Research Farm, Bath,

MI. The trials were planted June 3 (1997) or June 5 (1998) in a randomized complete

12

block design with three replications. A guard row of susceptible red potatoes bordered
each replication to maintain a dense canopy in which the disease could spread. Each 1.5
m plot contained four seed pieces at 30 cm spacing, with eight plots per row. A
susceptible red seed piece was planted between plots in 1997; the 1998 trial plots were
modified to leave that space bare so the plots could be more easily distinguished. A 1.5
m aisle between every two rows in the 1998 design allowed plot inspection. The field
was irrigated frequently with a sprinkler system to promote the humidity favored by the

pathogen. No fungicide was applied during the growing season. To ensure that the trial

 

plots would be continuously exposed to the pathogen, 8 kg piles of late blight infected
tubers were left in the field's aisles.
Greenhouse evaluation

Greenhouse screening was performed according to the method described by
Douches et al. (1997). Tubers of the desired lines were hand-harvested from the field
trial following vine senescence in late September and stored in paper bags at room
temperature (about 20°C) until mid-winter to break dormancy. Seed pieces were planted
in 16 cm clay pots in the greenhouse, replicated three times (U88) or ten times
(U81/U811) and allowed to grow for approximately six weeks, until just prior to
flowering. The late blight disease chamber consisted of a metal bench with a plastic tarp
covering it to keep the atmosphere inside the chamber isolated from that of the
surrounding greenhouse. The pots were placed inside the chamber on metal trays in a
completely randomized design. The foliage was sprayed with the inoculum suspension in

the late afternoon or evening, after the tarp cover was closed. A humidifier in the

13

chamber maintained the high humidity (>90%) favoring disease development. Plants
were rated for percent foliar infection after about seven days post inoculation.
Field rating

Beginning on July 30 (1997) or August 6 (1998) and continuing for the next 4
weeks, the percent defoliation of each plot due to the disease was visually estimated
every 3 to 7 d. To compare reactions to the disease over time, the Relative Area Under
the Disease Progress Curve (RAUDPC) for each line was calculated. This is expressed in
terms of the Area Under the Disease Progress Curve (AUDPC, Figure 1), the area under
the linear progression of defoliation from inoculation to the end of the evaluation period
(Colon et al., 1995a), divided by the maximum AUDPC (100 X the total number of days

afier inoculation).

100

 

75

 

 

. MSE01 1-14
I M86274-3

 

50

 

 

 

% Defoliation

25 ——

 

 

1 8 1 5 22
Days After Inoculation
Figure 1. Area Under the Disease Progress Curve after 28 days for the highest

(MSEOI 1-14) and lowest (MSG274-3) rated Michigan State University potato breeding
lines in the 1998 Phytophthora infestans resistance field trial.

Greenhouse rating

Lines were rated about 7 days after inoculation for percent defoliation. Analysis
of variance was performed on the results and the least significant differences calculated
as above. For the U88 screen, percentages were also converted to a 0-5 scale of
increasing severity (Douches et al., 1997) for analysis.
Statistical Methods

Analysis of variance was performed on the RAUDPC values for the field and the
percent defoliation values for the greenhouse, and then the least significant differences
were calculated using the SAS general linear models procedure (SAS Institute Inc., Cary,
NC). Relative rankings of 28 selected lines in 1997 and 1998 were correlated with proc

corr in SAS. Correlations of greenhouse and field results were performed as above.

15

RESULTS

Field results

In 1997 and 1998, P. infestans infection spread evenly and rapidly throughout the
field, with lesions visible by 9 days after inoculation (DAI). Significant differences were
found in 1997 (p < 0.0001) and 1998 (p < 0.0001). Data from the 76 lines common in
both years could not be combined because the variation was significantly greater in 1997
than 1998 (Pom = 4.07). Among the clones tested, eight showed high levels of
resistance in 1997 (Table l) and eight in 1998 (Table 2). Two of the lines, M8G274-3
and Q237-25, had lesions that were not typical of late blight (small radius, dry
appearance, no visible sporulation, leaf wilting and curling) contributing to the total
defoliation ratings; an RAUDPC based strictly on late blight defoliation for those two
lines would be lower than reported. A

The most susceptible line, MSEOI 1-14, reached 100% defoliation by 22 DA].
The commercial varieties used as susceptible controls, such as Atlantic and Russet
Burbank, reached 100% defoliation by 28 DAI. Figure 1 illustrates the difference in
disease progress between a susceptible (M8E011-14) and a resistant clone (M86274-3).

Lines with moderate to high levels of resistance or breeding lines with one
resistant parent were singled out for repeated testing along with susceptible check

varieties (Table 3). Relative field performance remained consistent through two growing

seasons (Figure 2).

l6

 

Table 1. Relative Area Under the Disease Progress Curve
(RAUDPC) for the 1997 Field Season

 

Line RAUDPC1

30692-42 0,4
LBRMULTI 1.1
AWN86514-2 1.1
30767-2 1.2
LBR8 1.3
30233-17 2.5
ms3274-3 4.1
30713-3 5.7
LBRO 13.9
BERTITA 15.4
DORITA 20.6
LBR1R3R4 22.6
BZURA 23.4
00033003-1 25.4
A084275-3 25.6
ROBIJN ' 25.3
LBR3 27.4
LBR1R2R4 27.4
LIBERTAS 23.6
PIMPERNEL 28.6
A080432-1 23.3
STOBRAWA 29.0
31004-3 29.7
ZAREVO 29.9
KRANTZ 29.9
ELBA 30.7
30749-21: 30.9
GRETA 31.6
3031 1-13 34.0
A341 133 34.3
NORDONNA 34.7
OBELIX 35.7
MSE230-6 35.9
MSC120-1Y 36.2
LILY 36.3
MSE018-1 33.1
SNOWDEN 33.6

17

 

Line RAUDPC

80856-4 45.5
ATL 45.5
P63—1 45.6
ALPHA 45.7
MSG227-2 45.8
MSF068-5 45.9
MSF019-1 1 46.1
MSE228—5 46.2
MSG251-10 46.2
HAMPTON 46.3
MSE220-14 46.4
N02676-1 0 46.5
MSGZ97-4RD 46.6
MSGt 39-1 46.8
P32-3 46.9
MSG007-1 47.1
MSBOS7-2 47.2
MSE226-5Y 47.2
PIKE 47.2
P84-9-8 47.9
MSF099—3 48.2
MSE041-1 48.2
MSDOZQ-3Y 48.4
MSG135-12 48.7
P84-12-7 49.2
MSBO73-2 49.2
MSEZZ1-11 49.3
MSG1 19-1 RD 49.3
MSGO10-11 49.3
MSA1 10-2 49.3
MSF373-A 49.4
MSA097-1 Y 49.4
MSG049-4 49.6
YELLOW FINN 49.7
M85228-9 49.7
MSF321-5 49.9
MSEO30-4 50.1

 

Table 1 (cont‘d).

 

 

 

Line RAUDPC1 Line RAUDPC

HINDENBURG 39.3 MSF020—23 50.2
A082611-7 39.3 YUKON GOLD 50.6
ONTARIO 39.4 NY101 50.7
MATILDA 39.5 NY103 50.3
MSE246-5 39.7 M3E215-12 51.0
MSG163-1 39.9 MSA105-1 51.2
MSG083-1RD 40.4 RUS. BURBANK 51.3
IS. SUNSHINE 40.7 1233-13-4 51.4
RUSSIAN BLUE 40.3 M3E223-11 51.5
MSG050-2 40.3 1373-2 51.5
M33076-2 40.9 ONAWAY 51.6
LATONA 41.1 MSE221-1 51.7
MSBO40-3 41.1 MSE202-3RUS 51.3
MSE230-3 41.1 MSG079-2 51.9
MSB107-1 41.3 M3E230-13 52.0
MSF019-2 41.3 MSNT—t 52.0
MSE228-3 41.3 M3E011-10 52.5
A7961-1 41.9 W1313 52.5
309153 42.0 A8495-1 52.6
MSG170-117 42.1 CENTURY RUS. 52.9
MSE009-1 42.1 14330544 53.4
J81 11-23 42.1 MSC148-A 53.5
ALLEGHENY 42.2 MSF014-9 53.3
DALI 42.2 M3E226-4Y 54.0
MSF105-10 42.2 MSE247-2 54.4
FL1879 42.3 LONGLADE 54.4
SHEPODY 42.4 ND860-2 54.9
MSG135-5 42.3 1333-15-1 55.0
MSF001-2 42.9 R. NORKOTAH 55.0
JUL. ROSE 42.9 M3E149-5Y 55.2
IS. SUNSET 43.7 MSE048-1Y 55.6
MN16489 43.7 G8610-2PY 55.6
MSA091-1 44.4 M33296-3 55.6
M3E263-3 44.6 M33106-7 56.0
MSE222-5Y 44.9 MSGO49-7 56.1
W1348RUS 44.9 M80122-A 56.6
MSE263-10 45.1 MN16966 57.0

MSF165-6RY 45.1 N02225-1 58.2

18

Table 1 (cont'd).

 

 

 

Line RAUDPC1 Line RAUDPC
M33027-1 R 45.2 M3E033-1 RD 53.9
MSC103-2 45.2 M33094-1 59.1
FL1833 45.3 MSF087—03 59.9
M3E213-2 45.4 RE3A 61.7
R. NORLAND 45.5 P83-6-18 63.6
DESIREE 45.5 MSE192-8RUS 63.7
MICHIGOLD 45.5 MN16180 66.3
Mean = 42.7

LSDQO5 = 10.6

CV% = 33.5

1Maximum RAUDPC = 100.

2Lineslin bold are considered highly resistant (RAUDPC < 10)
to P. infestans U88 isolate 95-7.

19

 

Table 2. Relative Area Under the Disease Progress Curve
(RAUDPC) for the 1998 Field Season

 

Line RAUDPC1

L3R32 0.6
L3R9 1.1
M33274-3 3.3
30692-4 4.9
0237-25 5.1
AWN86514-2 5.2
30713-3 3.2
L3R0 3.4
BZURA 10.1
ROBUN 121
30233-17 14.1
ZAREVO 16.2
ELBA 17.1
STOBRAWA 17.4
LBR5 13.2
ND02438-7R 19.1
A084275-3 19.3
DORITA 19.4
L3R1R2R3R4 19.9
ARS4219-1 20.3
BERTITA 20.5
GRETA 20.7
A080432-1 21 .3
A841 133 21 .4
L3R7 21.7
3031 1-13 22.2
LBR2 24.3
A08261 1-7 24.4
NORDONNA 251
89922-1 1 25,4
PICASSO 25.6
1333-5-12 25.3
LILY 26.1
MSF105-10 26.5
MSA091-1 26.6

20

 

Line RAUDPC

NY112 32.9
80178-34 33.0
MN17922 33.2
MSF349-1 33.5
MSF060-6 33.7
FAMBO 33.8
MSG1 19-1 RD 33.8
NY1 19 34.1
MSF019-11 34.1
MSE228—9 34.6
ATLANTIC 34.6
MSGt 39-1 34.7
MSH381-6Y 34.8
MSF020-23 35.0
MSGOO7—1 35.0
SNOWDEN 35.0
MSE274-A 35.1
MSEZ45—B 35.1
MN16966 35.2
MN16478 35.4
W1313 35.6
MSE080-4 35.6
MSE221-1 35.6
MSNT-2 35.6
YUKON GOLD 35.8
MSH321-1 35.9
MSH418-1 36.1
MSGZQ7-4RD 36.4
ONAWAY 36.6
AF 1 475-20 37.0
MSHO18-4 37.3
MSE228-1 37.3
MSH106-2 37.4
AF1763-2 37.5
MSE222-5Y 37.7

Table 2 (cont'd).

 

Line RAUDPC1

LBRY 27.0
PIKE 27.1
31004-3 27.2
MSH120-1 27.2
LBR3TBR 27.3
MSH018-3 27.6
MSG124-8P 27.7
MSGOSO-Z 23.0
TURBO 23.5
MATILDA 23.7
MSG104-6 23.7
MIRAKEL 23.3
MSCtO3-2 29.1
W1355-1 29.9
ND5084-3R 29.9
GOLDRUSH 30.0
AF1753-16 30.3
MSF373-8 31.2
MSF099-3 31.3
MSH392-1 31.4
A7961-1 31.5
MSE018-1 31.6
MSH380-3Y 31.7
W1151RUS 31.7
MSH308-2 31.3
MSBO76-2 32.0
DALI 32.2
MN17572 32.3
ND2470-27 32.3
AF1808-18 32.4
w1343RU3 32.5
ERNTESTOLZ 32.5
NORVALLEY 32.5

21

 

Line RAUDPC

MSB106—7 38.1
MSE228—11 38.1
MSNT-1 38.2
MS401-1 38.3
R. NORKOTAH 38.4
MSBO40-3 38.6
NY1 15 38.9
MSE149-5Y 38.9
MSC148-A 39.2
MSGO88—6 39.4
SUPERIOR 39.4
SHEPODY 39.5
MSE230-6 39.5
MSE263—10 39.5
A8495-1 40.0
ACCENT 40.5
MSBOQ4-1 41.6
MSA097—1 Y 41.6
ND4093-4RUS 41 .7
P84-9-8 42.0
MSE040-6RY 42.1
SAGINAW GOLD 42.5
MSE030-4 42.9
MSG141-3 43.0
MSE226-4Y 43.4
MSE226-5 43.6
MSF059-1 43.6
MSH1 12-6 43.8
MSF313-3 44.0
M80086-3 44.0
MSC122-1 44.1
MSE192-8RUS 44.2
ARS4152-1 44.5

Table 2 (cont'd).

 

 

 

Line RAUDPC1 Line RAUDPC
MSC120-1Y 32.7 MSE011-11 44.5
LADY ROSETTA 32.7 MSE033-1RD 45.7
RUS. BURBANK 32.3 MSH351-6 47.3
MSB107-1 32.3 1333-11-5 43.4
MSF420-1 32.3 MSE011-14 50.3
Mean = 31.4

LSDogs = 6.6

CV% = 33.7

 

‘Maximum RAUDPC = 100.

2Lines in bold are considered highly resistant (RAUDPC < 10)
to P. infestans US8 isolate 97-2.

22

Table 3. Relative Area Under the Disease Progress
Curve for Selected Potato Lines

 

 

Line 1997 Season1 1998 Season
3069242 0.4 4.9
AWN86514-2 1.1 5.2
80288-17 2.6 14.1
M86274-3 4.1 3.8
80718-3 5.7 8.2
DORITA 20.6 19.4
BZURA 23.4 10.1
A084275-3 25.6 19.3
ROBIJN 25.8 12.1
A080432-1 28.8 21 .3
STOBRAWA 29.0 17.4
81004-8 29.7 27.2
ZAREVO 29.9 16.2
ELBA 30.7 17.1
GRETA 31 .6 20.7
80811-13 34.0 22.2
A84118-3 34.3 21.4
NORDONNA 34.7 25.2
LILY 36.3 26.1
MSEO18-1 38.2 31.6
SNOWDEN 38.6 35.0
MATILDA 39.5 28.7
MSGOSO—2 40.8 28.1
ATLANTIC 45.5 34.7
M86297-4RD 46.6 36.4
MSG139-1 46.8 34.7
MSGOO7-1 47.1 35.0
RUS. BURBANK 51.3 32.8
Mean 29.4 21 .7
l-SDo.os 10.1 5.4
CV°/o 52.7 47.5

'Table sorted by 1997 Season. Maximum RAUDPC = 100.

2Lines in bold are considered highly resistant
(RAUDPC < 10) to P. infestans U88 isolate 97-2.

23

 

Greenhouse P. infestans US8 genotype results

Although the U88 greenhouse inoculation procedure was carried out multiple
times, several repetitions of the experiment could not be analyzed due to low levels of
infection, abrupt temperature increases that affected plant health, or failure of the
humidifier equipment. Results fiom two separate runs of the experiment, one in 1998
and one in 1999, could be combined (F OMAX = 2.02). Significant differences were
detected between the most susceptible and most resistant lines (Table 4) both in the
individual (1998, p = 0.0032; 1999, p < 0.0001) and combined (p = 0.044) results. No
correlation was found between the relative resistance and susceptibility of the lines based
on combined greenhouse ratings and field RAUDPC scores (Figure 4), although there
was a weak correlation (r = 0.59, p = 0.0009) between the response of the 1998 field
plants and the greenhouse plants grown fiom their tubers in 1999.
Greenhouse P. infestans genotype by potato variety interaction

Eight lines (AWN86514-2, Atlantic, B0718-3, Bzura, MSG274-3, Matilda,
Snowden, and Zarevo) were selected fi'om the greenhouse US8 evaluation for inoculation
with U81 and U811 (Table 5). Infection with combined Michigan U81 isolates 95-5 and
95-6 was uniformly low across all varieties. Of the eight lines tested, only M8G274-3
developed significantly more than zero infection, with a mean of 5.3% defoliation
(LSDaos = 1.8). Inoculation with Michigan U811 isolate 96-1 produced no infection on

any variety, even after repeated trials.

24

 

 

 

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26

Table 5. Greenhouse Defoliation Ratings
of 8 Lines Inoculated with Phytophthora
infestans U81 and US11 Genotypes

 

 

Line us11 US11
ATLANTIC 0.0 0.0
ZAREVO 0.0 0.0
BZURA 0.3 0.0
SNOWDEN 0.4 0.0
80718-3 0.7 0.0
AWN86514-2 1.1 0.0
MATILDA 1.4 0.0
(5274-32 5.3 0.0

1Rating is a visual estimation of percent
defoliation from O - 100%.

2The value in bold is the only significant
rating (p < 0.05). ‘

27

DISCUSSION

Although the inoculum pathotypes of the Michigan isolates have not yet been
completely characterized (N iemira, personal communication), the strong resistance
exhibited by the USDA late blight differential lines (Black et al., 1953; Malcolmson and
Black, 1966) LBRs in 1997 (Table l) and both LBRs and LBR9 in 1998 (Table 2)
suggests that the P. infestans isolate 95-7 lacks at least virulence gene 8 (LBR9 was not
present in 1997) and isolate 97-2 lacks virulence genes 8 and 9. This is consistent with
1997 findings at five separate late blight field testing locations across the United States
(Maine, Minnesota, New York, North Dakota, and Pennsylvania), where LBRs was
resistant to P. infestans US8 genotype inoculation and natural late blight infection
(Haynes et al., 1997). i

RAUDPC ratings between two field seasons for selected lines of interest are
compared in Table 3. There were year-to-year differences in reaction to the disease for
each line, but the overall ranking of susceptibility and resistance remained stable (Figure
2). This suggests that field screening can provide an accurate assessment of a clone's
level of resistance. When measured against standard varieties, rating should remain
consistent regardless of the conditions of the particular growing season (Colon et al.,

1995a; Dorrance and Inglis, 1997).

28

 

 

 

 

 

 

Rank in 1998

 

 

 

Rank In 1997

Figure 2. Relative rankings of 28 selected potato lines in 1997 (vertical axis) and 1998

(horizontal axis), where l = most resistant to late blight and 28 = most susceptible, based
on RAUDPC values.

Lines 30692-4, AWN86514-2 (Corsini et al., 1999), and B0718-3 (Goth and
Haynes, 1997) demonstrated strong resistance, but their late maturity and poor agronomic
qualities make them unsuitable for commercial use. AWN86514-2 may derive its
resistance from the species S. acaule, S. demissum, S. phureja, S. simiplicifolium, S.
stoloniferum, S. stolomferum, and S. tuberosum subsp. andigena, all of which are in the
lineage of its female parent, KSA195-96. It is male sterile, but when used as a female
can transmit strong resistance to progeny (Corsini et al., 1999). BO718-3 is both male
and female fertile and is believed to inherit its resistance from one great-grandparent,
P138347OB (Goth and Haynes, 1997). Initial progeny testing indicates that 30718-3
transmits strong resistance to offspring, and that a high proportion of its progeny retain

favorable agronomic traits when compared to progeny of other resistance sources

29

(Bisognin et al., 1998). These clones may prove valuable as parental lines, though the
use of B0692-4 and AWN86514-2 is limited by poor fertility.

MSGZ74-3 and Q237-25 are breeding lines fi'om Michigan State University and
Cornell University, respectively. MSGZ74-3 is the product of a cross between the late-
maturing resistant Mexican variety 'Tollocan' and the early maturing susceptible
Canadian variety 'Chaleur.’ It is unknown whether the resistance transmitted by Tollocan
derives from R gene interaction, but current speculation based on greenhouse disease
response (below) is that there may be R genes present in its progeny. MSGZ74-3 is a
high yielding advanced selection with good fertility, and it possesses a commercially
acceptable intermediate maturity. It produces a visually attractive, bright-skinned,
oblong tuber with light yellow flesh and low internal defects. It is an acceptable chipper
directly out of the field, but not after storage (Douches et al., 1998a). Q23 7-25 is derived
from a cross with the field resistance source Solanum tuberosum subsp. andigena
(Raman, 1998). Q237-25 has an acceptable appearance, yield, and maturity, and has high
levels of resistance to potato cyst nematode, scab, and Potato Virus Y (Raman, 1998).
Both are under consideration for direct release as varieties. Because both breeding lines
have earlier maturity than most strong resistance sources, their field defoliation scores
late in the season reflected natural senescence in addition to disease pressure, while the
late-maturing varieties did not suffer from that interaction.

One concern when dealing with strong resistance from a limited number of
sources is that the mechanism of resistance in each source, such as 30718-3 and
AWN86514—2, may originate fi'om the same genetic locus. It is unknown whether this is

the case; speculation is based on observations that the strong resistance in both lines

30

holds up similarly in regions across the United States (Haynes et al., 1997), and both
transmit strong resistance to offspring in a manner suggestive of one or a few major
genes (Bisognin et al., 1998). Mapping of resistance genes (Li et al., 1998) and
quantitative trait loci that influence resistance (Meyer et al., 1998) is being performed for
potato, but until more varieties can be studied, there is a risk that resistance in the above
lines may be allelic and crossing them will not lead to further improvement. Varieties
that display more moderate levels of resistance, such as 'Bzura,‘ 'Zarevo,‘ 'Stobrawa,‘
'Bertita,‘ and 'Greta,‘ are less likely to derive their resistance from the same genetic loci.
They should be crossed with progeny of the strong resistance sources in an effort to
combine different resistance genes from a broader background. Further analysis of
strong resistance sources such as 30718-3 and 6274-3 to determine whether they share
markers linked to quantitative resistance (Meyer et al., 1998) will be helpful in devising

breeding strategies (see Figure 3 for an example).

31

 

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32

The greenhouse US8 late blight resistance screen permitted the separation of
resistant and susceptible lines based on percent defoliation (Table 4). Defoliation ratings
were also analyzed using a weighted scale developed by Douches et a1. (1997) in an
effort to reduce variation by distinguishing more precisely between lower amounts of
defoliation than between higher, undesirable levels; however, the scale did not
appreciably improve the separation of means among these data (Table 4).

The results of the greenhouse US8 screen did not correlate well with the field
results (Figure 4). One contribution to the scatter of the data points may be the unusual
disease severity in the 1998 experiment. When the less severe 1999 experiment alone
was compared to the previous season's field ratings, a weak correlation (r = 0.42, p =
0.02) more consistent with earlier greenhouse data (Douches et al., 1997) was found.
There was an interaction between the potato lines tested and the evaluation method.
Lines with strong foliar resistance in the field, AWN86514—2 and 30718-3 (Table 3),
were intermediate for greenhouse defoliation (Table '4). This difference is probably due
to the high disease pressure in the 1998 greenhouse screen, which may have been caused
by P. infestans cultures with greater virulence than those used in 1999. A second notable
variation is between the strong resistance of Zarevo and its progeny MSGZ97-4RD in the
greenhouse compared to intermediate resistance (Zarevo) or susceptibility
(MSG297-4RD) under field conditions (Table 6). Two other Zarevo progeny,
MSGOO7-1 and MSGI39-l, were intermediate in the greenhouse but highly susceptible in
the field (Table 6). The high impact of the environmental conditions on the disease
response of Zarevo and its progeny is characteristic of horizontal resistance.

Environmental factors in the greenhouse that may influence disease development in these

33

lines include a lack of direct sunlight that may alter physiological responses, pots that
constrain root development, and a more controlled temperature.

Another factor that differed between the field and greenhouse was the number of
genotypes of P. infestans isolates used during inoculation. For both field experiments,
only one isolate was used, while greenhouse inoculation involved a mixture of isolates.
The multiple isolate procedure was developed to compensate for lapses in single isolate
aggression that resulted in failed runs of the experiment due to poor infection. Some
isolates lost virulence over time through standard subculture maintenance, so that even
mature, previously virulent cultures did not lead to infection when used as inoculum.
Combining multiple isolates increased the chance of sufficient disease development for
meaningful evaluation. Another option to maintain isolate virulence would have been to
culture the pathogen on living host tissue, but that method required resources unavailable
at the time of this study. Since naturally occurring infection by the U88 genotype is not
limited to specific isolates, the multiple isolate procedure should in theory produce results
that predict field disease response similarly to a single isolate inoculation as long as high
enough levels of infection take place to differentiate between resistant and susceptible
clones. The usefulness of the US8 classification itself relies upon the genetic identity of
the pathogen, so the differences in the US8 isolates used should not have a great effect

upon host response.

34

 

 

 

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i Combined Greenhouse Rank

 

 

 

 

 

 

 

 

 

 

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Figure 4. Relative P. infestans resistance rankings of 28 selected potato lines in
greenhouse tests (horizontal axis) and field trials (vertical axis) where l = most resistant
to late blight and 28 = most susceptible. Greenhouse rankings are based on percent
defoliation and field rankings on the Relative Area Under the Disease Progress Curve for
each line. No correlation was found between the greenhouse data and the 1997 field
results (top, p = 0.17) or the 1998 field results (bottom, p = 0.08).

35

 

 

 

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37

In the US] late blight screen, one clone (MSGZ74-3) was found with significantly
more infection than the other seven tested (AWN86514-2, Atlantic, 80718-3, Bzura,
Matilda, Snowden, and Zarevo), although average defoliation of MSGZ74-3 was only
5.3% (Table 5). Such a low percentage defoliation in itself may not appear to be a matter
for concern, but taken in the context of a genotype with low aggression in an experiment
with an overall low infection rate, the significant difference across ten replications is
worth noting. Since this breeding line generally shows strong resistance to the USS
genotype of P. infestans in both the field and greenhouse (Table 6), the higher
susceptibility to US] compared to other lines may be caused by an R gene interaction.
The virulence pathotypes of the Michigan isolates used in the US] inoculation (95-5 and
95-6) are not yet known.

Although USll is reported to be an aggressive genotype (Miller et al., 1998),
Michigan isolate 96-1 caused no visible infection in two separate inoculations. It is
possible that this particular isolate has a low level of aggression, or that repeated
subculturing since its isolation in 1996 selected for survival on the medium rather than
high aggression on the host (Niemira, personal communication). Isolate 96—1 was the
only USll isolate available at the time of this study, so the technique of combining
different isolates could not be used. Its virulence had not previously been tested, so the
method of increasing virulence by maintenance on a living host plant was not attempted.
This method may be useful if further work is anticipated with the 96-1 isolate.

Stewart et al. (1983) discuss some of the factors that may cause lower correlation

between greenhouse and field tests. Unpredictable disease severity is one of the main

38

limitations of the greenhouse screening method. Other complications arise fi'om different
levels of dormancy among lines, leading to unbalanced replications when some tubers
sprout weeks later than others planted on the same day, and the constraints imposed by
the limited size of the controlled-environment chamber used for inoculation. Variation is
higher in the greenhouse than in the field, so it is preferable to use more replications, yet
an experiment conducted with plants crowded in the chamber will have a different
canopy density and microclimate than one with fewer replications to be screened,
resulting in different rates of disease spread.

A possible strategy to make the most efficient use of the greenhouse screening
method is to conduct a preliminary test of a wide range of lines with few replications to
determine the most resistant and most susceptible. The second stage would then be to
focus on fewer lines based upon those findings and screen them again with more
replications for greater precision. Through greenhouse evaluation, the breeder can
discard the lines that show the most susceptible response early in the selection process,
conserving resources and field space for those that hold the most promise of resistance.
The field performance of each line can then be used as the best estimate of P. infestans
resistance for final selection. This strategy uses the weak correlation of the two separate

methods to its best advantage by concentrating on the strong points of each.

39

CHAPTER 2: TUBER SCREENING

FOR RESISTANCE TO PHYTOPHIHORA INFEST ANS

40

INTRODUCTION

Defoliation of a potato field is a readily visible symptom of Phytophthora
infestans infection. Yield loss due to the death of the plant, however, is not the only
damage caused by this pathogen. Spores fiom the lesions on the foliage can be washed
into the soil to infect the commercially valuable tubers (Bradshaw et al., 1995a). Infected
tubers pose a serious threat to a potato grower because 1) the diseased tubers become
darkened and decayed (Franc et al., 1996), thus decreasing the harvest's marketable
value, 2) late blight infection of tubers allows secondary pathogens to invade more
effectively, speeding rot and decomposition in storage (Lambert and Currier, 1997), and
3) infected tubers used as seed for the following growing season are a primary source of
inoculum, which can spread P. infestans to previously uninfected fields, especially when
transported over long distances (Fry and Goodwin, 1997).

A cultivar's tuber resistance to late blight is not correlated with its foliar resistance
(Black, 1970; Dorrance and Inglis, 1997; Inglis et al., 1996). Therefore, when evaluating
a clone's foliar response to late blight, it is essential to test the tubers for resistance as
well. The objective of this study was to determine the levels of tuber resistance in
selected lines that have exhibited various degrees of foliar resistance and susceptibility in

previous field and greenhouse screens (Table 6).

41

MATERIALS AND METHODS

Plant material
Twenty-six lines were chosen to be included in the tuber resistance screen (Table 6).
These lines fall into four general field foliar resistance categories (Table 3):
0 Highly resistant: AWN86514—2, 8028847, BO692-4, 30718-3, and MSGZ74-3.
o Moderately resistant: A080432-1, A084275-3, Bzura, Dorita, Elba, Greta, Robijn,
Stobrawa, and Zarevo.

a Reduced susceptibility: 31004-8, Lily, Matilda, MSGOSO-Z, and Nordonna.

o Susceptible: Atlantic, MSE018-1, MSGOO7-1, MSGlB9-l, MSGZ97-4RD, Russet
Burbank, and Snowden.

Tubers were hand-harvested fi'om the 1998 foliage disease trial field plots at the
Muck Soils Research Farm (Bath, MI) following vine senescence in late September.
Healthy tubers were stored in paper bags at room temperature (about 20° C) until
February 1999.

P. infestans inoculum preparation

Tubers were inoculated with a Michigan isolate of the USS genotype. A mature
culture of P. infestans grown on rye agar was homogenized by passage through a sterile
syringe without a needle. The resulting mycelial homogenate was then re-loaded into the

syringe and the needle attached for inoculation.

42

Tuber inoculation

Tubers were prepared and injected at the apical end with inoculum according to
the method of Niemira et al. (1999b). Ten replications were inoculated in a completely
randomized design. The tubers were then placed in plastic bags and incubated for 420
degree days (14 d at 7° C followed by 33 d at 12° C) in the dark at 95% relative humidity.
To provide a basis of reference for healthy tuber flesh, ten tubers of each line were
punctured at the apical end with an identical hypodermic needle and incubated under
similar conditions for 425 degree days.
Tuber disease rating

Tubers were assessed visually for an estimate of surface degradation on a 1 - 9
scale of increasing disease severity (Table 7). Each tuber was then sliced into sections
approximately 2 cm fiom the apical and terminal ends and through the middle. Sections
were immediately placed with the cut surface down on a transparent plate and the plate
transferred to a flatbed scanner. Brightness and contrast were manually set at 170 and
190 with the scanner control software (DeskScan II version 2.4; Hewlett Packard Co.)
and photograph-quality black and white scans were taken at a resolution of 150 X 150,
then saved in tagged image format (.tit). The background of each image was black, with
the tuber section images appearing as light reflected from the cut tuber surface. The
image files of the sections were analyzed digitally for internal discoloration with image
analysis software (SigmaScan version 3.0; Jandel Scientific Software, San Rafael, CA).
On the light intensity scale, pure black has an intensity of 0 units and pure white an
intensity of 255 units. The area of each tuber section was selected with the "fill" tool

with the cut-off threshold set to 10 units so that it would exclude any portion of the image

43

darker than 10, thereby isolating each section fi'om the black background. The sofiware
calculated the light intensity of each pixel in the selected region and returned a value for
the average reflective intensity (N iemira et al. 1999b). The internal average of each tuber
was calculated by summing the apical, middle, and terminal values and dividing by three.
Statistical methods

Analysis of variance was performed on the light intensity values and the least
significant differences were calculated using the SAS general linear models procedure
(SAS Institute Inc., Cary, NC). Relative rankings of potato lines based on tuber surface

rating and internal scan data were correlated with proc corr in SAS.

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45

RESULTS

Surface rating

Individual tubers displayed levels of disease ranging fi'om 1 to 8 on the rating scale
used (Table 7), although mean values only varied between 2 and 7 across all lines (Table
8). Visual rating of tuber surface infection severity did allow detection of differences
among lines (p < 0.0001). There was no correlation between relative tuber surface rating
and field foliar late blight resistance (p = 0.17). The lines fell into the following surface
resistance categories (Niemira et al., 1999a):

o Resistant (< 3): Dorita.

o Moderately resistant (3 - 3.99): B0692-4, Bzura, and A084275-3.

o Moderately susceptible (4 - 4.99): A080432-l, Atlantic, AWN86514-2, Lily,

Matilda, MSGZ74-3, MSG297-4RD, Russet Burbank, and Stobrawa.
o Susceptible (> 5): 80288-17, B0718-3, 31004-8, Elba, Greta, MSE018-1,

MSG007-1, MSGOSO-Z, MSGl39-1, Nordonna, Robijn, Snowden, and Zarevo.

Table 8. Tuber Late Blight Resistance Ratings for Selected Potato Lines

 

 

Line Surface1 Apical2 Middle Terminal lntemal Mean3
A084275-3 3.5 175.8 197.1 197.9 190.2
MSG297-4RD 4.5 183.8 193.9 190.5 189.4
MSGOO7-1 6.1 179.9 190.6 192.8 187.8
BZURA 3.1 170.7 187.9 191.0 183.2
DORITA 2.1 166.9 176.1 174.8 172.6
ZAREVO 5.2 151.7 181.0 181.4 171.3
30288-17 5.7 153.4 178.8 177.7 170.0
ATLANTIC 4.3 146.2 181.8 181.0 169.6
31004-8 5.0 163.3 171.9 163.9 166.4
MATILDA 4.6 150.3 170.7 176.9 166.0
LILY 4.6 151.1 170.1 175.8 165.6
306924 3.1 152.8 162.8 159.3 158.3
MSG139-1 6.8 133.1 165.5 164.9 154.5
SNOWDEN 6.0 142.7 154.7 161.6 153.0
AWN86514-2 4.0 137.1 161.5 157.2 151.9
MSG274-3 4.2 136.5 158.8 159.3 151.5
RUS. BURBANK 4.9 146.9 150.1 152.8 149.9
MSEO18-1 6.5 131.4 166.7 151.3 149.8
30718-3 6.4 139.6 151.9 150.7 147.4
A080432-1 4.3 1 15.2 146.9 156.4 139.5
MSGOSO-2 6.3 118.5 135.7 134.0 129.4
GRETA 5.8 116.4 133.1 136.3 128.6
STOBRAWA 4.3 117.3 130.4 131.9 126.5
ELBA 5.5 118.8 132.8 123.2 124.9
ROBIJN 7.0 98.6 121.4 117.8 112.6
NORDONNA 5.8 101.6 110.3 122.5 111.5
Mean 5.0 142.3 160.9 160.9 154.7
LSDODS 0.9 20.2 16.8 19.5 16.7
CV°/o 31.4 22.2 18.1 19.2 18.5

 

1SurfaCe rating is on a 1 - 9 scale of increasing disease severity.

2Section rating represents the average light intensity of a scanned image
with 0 = black (diseased flesh) and 255 = white (healthy flesh).

3Table sorted by internal mean.

47

Internal section analysis

All potato lines showed signs of infection (Figure 5). Differences were found among
lines at the apical, middle, and terminal sections (p < 0.0001 for all tests), and the average
internal intensity was used for comparisons (Table 8). To compensate for varietal
differences in flesh color, average intensities of the diseased tubers were divided by the
average intensities of the control tubers (Table 9), which slightly altered the rankings of
the lines. A weak correlation (Figure 6) was found between surface ranking and ranking

based on mean internal intensity (r = 0.44, p = 0.023) and between surface ranking and

 

ranking based on percentage of healthy flesh (r = 0.54, p = 0.0041), consistent with the i-

.15.

results obtained by Niemira et al. (1999b). There was no correlation between the
resistance of the tubers and the resistance of their foliage fiom the previous field season
(p = 0.9036). Based on internal mean, the lines can be categorized as follows:
0 Resistant (> 180): A084275-3, Bzura, MSG007-1, and MSG297-4RD.
o Moderately Resistant (165 - 179.9): Atlantic, 30288-17, B1004-8, Dorita, Lily,
Matilda, and Zarevo.
o Moderately Susceptible (150 - 164.9): AWN86514-2, B0692-4, MSGl39-1,
MSG274-3, and Snowden.

o Susceptible (< 150): A080432-1, B0718-3, Elba, Greta, MSE018-l, MSG050-2,

 

Nordonna, Robijn, Russet Burbank, and Stobrawa.

48

 

Figure 5. Scanned apical sections of four sample tubers inoculated with P. infestans from
a) G297-4RD (mean intensity 183.79), b) 6274-3 (mean intensity 136.51), and c)
Nordonna (mean intensity 101.64).

49

Table 9. Comparison Between Diseased and Healthy Tuber Flesh
Based on lntemal Section Mean Light Intensity

 

 

Line Diseased Mean1 Healthy Mean % Healthy2
DORITA 172.6 192.7 89.6
MSG297-4RD 189.4 212.1 89.3
A084275-3 190.2 214.6 88.6
BZURA 183.2 210.1 87.2
MSG007-1 187.8 217.7 86.2
ZAREVO 171.3 205.5 83.4
MATILDA 166.0 203.1 81.7
ATLANTIC 169.6 210.0 80.8
LILY 165.6 206.5 80.2
AWN86514-2 151.9 192.0 79.1
80288-17 170.0 214.9 79.1
81004-8 166.4 218.9 76.0
MSG274-3 151.5 204.0 74.3
RUS. BURBANK 149.9 202.0 74.2
MSG139-1 154.5 209.7 73.7
80692-4 158.3 215.8 73.4
SNOWDEN 153.0 213.3 71.7
30718-3 147.4 206.4 71.4
MSEO18-1 149.8 211.4 70.9
GRETA 128.6 193.5 66.5
STOBRAWA 126.5 197.5 64.1
A080432-1 139.5 221.0 63.1
MSG050-2 129.4 213.6 60.6
ELBA 124.9 210.3 59.4
ROBIJN 112.6 193.7 58.1
NORDONNA 111.5 199.5 55.9
Mean 154.7 207.2

LSD,05 16.7

CV% 18.5 5.4

1Section rating represents the average light intensity of a scanned image
with 0 = black (diseased flesh) and 255 = white (healthy flesh).

2List sorted by percent healthy flesh light intensity.

50

 

 

 

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0

 

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Surface Disease Rank I

 

 

 

 

 

 

 

 

 

 

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Surface Disease Rank

 

Figure 6. Relative P. infestans tuber resistance rankings of 26 selected potato lines
according to surface disease (horizontal axis) and internal light intensity (vertical axis,
top) or percent intensity of healthy flesh (vertical axis, bottom) where 1 = most resistant
to late blight and 26 = most susceptible. Surface rankings are based on a 1 - 9 scale of
increasing disease severity and internal rankings on average light intensity where 0 =
black and 255 = white. Top, r = 0.44, p = 0.023. Bottom, r = 0.54, p = 0.0041.

51

DISCUSSION

The difference in internal tuber appearance between a clone highly resistant to P.
infestans (MSG297-4RD), a moderately susceptible line (MSG274-3), and a highly
susceptible line (Nordonna) is illustrated in Figure 5. The resistant MSG297-4RD is an
advanced breeding line derived fiom the foliar resistance source cv. 'Zarevo.‘ Another
highly resistant line, MSG007-1 (Table 8), is an advanced breeding line selected fi'om a
cross between Atlantic and Zarevo. Zarevo itself was only moderately resistant in this

study. The third progeny line fi'om Zarevo, MSG139-l (Snowden X Zarevo), was

 

significantly more susceptible than its half-siblings as well as its resistant parent (based
on internal mean). A more extensive tuber screen among progeny of Zarevo would be
important to determine its value as a source of tuber resistance to P. infestans. The
Zarevo progeny in this study were lines selected both for tuber appearance and potential
foliar resistance, not for tuber resistance, so the segregation of tuber resistance levels
should be random among the three.

Different methods have been developed to screen tubers for late blight resistance,
including assessment of natural field infection, whole tuber assays, and screens involving
tuber pieces or slices (Dorrance and Inglis, 1998). Most common is the whole tuber
assay (Stewart et al., 1992; Wastie et al., 1993), in which an undamaged tuber is dipped
in or sprayed with inoculum to simulate field conditions, where zoospores are washed
fi'om stem and leaf lesions into the soil to infect tubers (Bradshaw et al., 1995a).
However, laboratory inoculations can have higher disease pressure than field tuber
resistance trials (Platt and Tai, 1998), and other environmental factors like the positions

of the tubers under the soil can produce effects that make field resistance vary fi'om

52

 

laboratory screenings (Dorrance and Inglis, 1998). The whole tuber assays generally
discount any disease due to wounding (Stewart et al., 1994), relying on integrity of the
peridenn as a component of resistance, a factor that can alter as tubers age in storage
(Dorrance and Inglis, 1998). The tuber slice technique, in contrast, does not involve the
peridenn, but it is also too variable to be a reliable general test (Dorrance and Inglis,
1998).

Another tuber evaluation method involving injection of inoculum at the apical end
measures disease spread through the tuber flesh to differentiate between P. infestans
isolates based on aggressiveness.(Lambert and Currier, 1997). In that method, the extent
of the damage is quantified by tracing the outline of the rotted portion on filter paper and
then weighing the paper to determine the area affected. Although such a method is
simpler than digital analysis in that it does not necessitate expensive equipment and
software, it also relies more on the observer's subjective judgment when producing the
outline and requires more time to perform than the computer’s analysis, which can be
accomplished quickly for each scanned tuber.

One main weakness of the digital analysis technique is that it is not well suited for
evaluating tubers with dark-colored flesh. Niemira et al. (1999b) detected no significant
differences among varieties for light intensity of healthy flesh, but the three varieties
screened were all white-fleshed cultivars. The average light intensity of control tubers
for each line tested should be measured to account for natural differences. The surface
score does not correlate well with the internal rating, since some lines darken through the

center and in others the visible symptoms are confined to the surface, yet each gives

53

 

valuable information about varietal response to P. infestans infection. The two
assessments should be used to complement rather than predict one another.

Further work in this area needs to be conducted on a wider range of varieties and
breeding lines, as well as progeny studies with lines that display high levels of resistance.
This is especially important for varieties such as Zarevo that also possess moderate or

high foliar resistance, as there is some evidence that general combining ability for foliar

 

F
and tuber resistance may be correlated (Stewart er al., 1994). More extensive screening
is crucial to developing commercially acceptable cultivars with adequate resistance to 1
late blight in both the foliage and tubers. L

The limiting factor for using tuber screening in the selection process is the
availability of enough intact tubers for the desired replications, which can only be
attained after several years of tuber increase. The concern when postponing selection for
tuber resistance long enough to increase tuber number sufficiently for each clone is that
highly tuber resistant lines may be unknowingly discarded in early stages of selection.
The example of the Zarevo progeny in this study is positive evidence that selection for
unrelated characteristics, such as agronomic quality and foliar late blight resistance, still
leaves the breeder with a variety of tuber resistance levels fi'om susceptible to resistant
among the remaining breeding lines. Therefore, it seems the most advisable strategy to
screen for foliar resistance in the earlier stages of selection, which can be done using a

smaller number of tubers, and reserve tuber resistance screening for advanced lines.

54

SUMMARY

The field trial design was modified between 1997 and 1998 to facilitate the
process of rating the infection levels of the plots. The changes included eliminating
susceptible guard plants fiom between adjacent plots, originally intended to separate plots
and maintain infection, so that they would not be mistaken for plants in the experimental
units and rated. Another difference was the widened path between every two rows in the
1998 trial that allowed researchers to evaluate the plots without damaging the spreading
foliage. These changes reflect an attempt to reduce variation in the results due to the
rating procedure itself. Further seasons of investigation should reveal whether the
measures taken to improve the design are effective.

Field results allowed the identification of highly and moderately foliar resistant
lines. By taking advantage of pedigree information and agronomic quality data, a breeder
can plan crosses with these lines that will maximize the likelihood of producing
commercially acceptable cultivars with strong, durable late blight resistance.

Greenhouse screens were widely variable and provided limited information about
the most resistant and susceptible varieties rather than measuring intermediate levels of
resistance. Such screens could be preliminary tools to help focus resources on the lines
showing the most promise, thereby increasing efficiency in field screening.

The most difficult factor to control in the greenhouse testing procedure was the
virulence of the P. infestans isolates used to inoculate the plants being screened.
Infection was greatest when several different isolates could be combined in the inoculum,

as in the experiment with the P. infestans US8 genotype, instead of only one (U S1 1) or

55

 

two (U Sl) isolates. Although the USl and US11 genotype experiments did not produce
strong infection, the results obtained point to a possible influence of R genes in the potato
breeding material tested that should be investigated firrther.

Tuber late blight resistance results did not correlate with field foliar resistance
data, which reinforces the need for both tests to be performed. It is difficult to conduct a
tuber resistance screen during the early stages of selection, when the number of tubers
available for each line is limited, so the procedure is best suited to evaluating advanced
lines and cultivars. The method of assessing surface and internal disease progress
separately provides complementary sets of data about each line's disease response.
Digital analysis of flesh discoloration due to late blight infection removes the element of
subjectivity from the evaluation.

Two progeny selections of the cultivar 'Zarevo' were among the lines displaying
the strongest tuber resistance. A more detailed progeny test for Zarevo as well as a wider
screen of available breeding material is needed to determine which lines would be good

parents when breeding for improved tuber resistance.

56

 

APPENDIX

57

 

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59

LITERATURE CITED

60

 

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66

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