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LIBRARY
Michigan State
University

This is to certify that the
thesis entitled

“COMBINING HOST PLANT RESISTANCE
AND MANAGED FUNGICIDE
APPLICATIONS FOR CONTROL OF
LATE BLIGHT IN POTATOES”

presented by

JEAN-BAPTISTE MUHINYUZA

has been accepted towards fulfillment
of the requirements for the

 

 

 

 

MASTER OF degree in PLANT PATHOLOGY
SCIENCE
Major Professor’s Signature
5 I I ’L. ‘ ”L o o 3
Date

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PLACE lN RETURN BOX to remove this checkout from your record.
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6/01 cJCIRC/DateDue.p65-p.15

 

COMBINING HOST PLANT RESISTANCE AND MANAGED FUNGICIDE
APPLICATIONS FOR CONTROL OF LATE BLIGHT IN POTATOES

By

J can-Baptiste Muhinyuza

A THESIS
Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE
Department of Plant Pathology
2003

ABSTRACT

COMBINING HOST PLANT RESISTANCE AND MANAGED FUNGICIDE
APPLICATIONS FOR CONTROL OF LATE BLIGHT IN POTATOES

By

J can-Baptiste Muhinyuza

Fluazinam, a new protectant fungicide with excellent residual activity and active
in relatively small doses, was applied on potato foliage in combination with cultivar
resistance. Three rates [33, 66 and 100% of the manufacturer’s recommended application
rate (MRAR)] of fluazinam were applied on 5, 7, 10 and 14-day application intervals in
2001; two rates [50% and 100% MRAR] on 5, 10 and 15-day application intervals in
2002 and examined for foliar late blight control. The results of this study showed that
reduced amounts of fluazinam were either partially or fully effective at all application
rates tested on all cultivars compared to the non-treated controls at P: 0.05. On
susceptible cultivars, applications of fluazinam at 10-day spray intervals or above were
partially effective for controlling late blight at the doses tested. All cultivars treated at
100% and 50% MRAR of fluazinam were effectively protected against late blight on a 5-
day spray interval and partially protected at all other rates and application intervals.
Application of fluazinam at reduced rates and frequencies could successfully be used into
a control program using host resistance. The impact of plant age and leaf position on
susceptibility to potato late blight infection was also assessed. Susceptibility to potato late
blight infection decreased with plant age and leaf position; from young plants to the old

ones and from leaf 5 to leaf 15 in the cultivars tested.

To my daughter Delitha G. Muhinyuza and mom Winifi'ied Kangabe,

I dedicate this thesis

iii

ACKNOWLEDGEMENTS

I would like to thank with gratitude my major adviser Dr William W. Kirk who

with pleasure suggested and guided this research.

I would also want to express my thanks to other members of my guidance
committee, Dr Raymond Hammerschmidt and Dr David Douches for their advice on the
preparation of this thesis.

This research would not have been possible without the financial support of
PEARL Project. I would like to thank Dr Daniel Clay and Mr. David Weight,
respectively Director and Coordinator of Pearl project who fully supported financially my
formation at Michigan State University (MSU).

I would like to express my appreciation and thanks to MSU and the Department
of Plant Pathology for the academic experience developed. It is a pleasure to express my
extreme gratitude to all the professors who taught me and guided my learning and
research during my formation at MSU. I recognize with esteem all their efforts to provide
good quality teaching.

Finally to my family, my parents, my wife, daughter and son for their

encouragement and love, I would like to express my satisfaction and thanks.

J can-Baptiste Muhinyuza

iv

TABLE OF CONTENTS

Page
LIST OF TABLES vii
LIST OF FIGURES viii
GENERAL INTRODUCTION 1
CHAPTER ONE 4
LITERATURE REVIEW 4
POTATO LATE BLIGHT 4
IMPORTANCE 4
DISEASE DEVELOPMENT 5
CONTROL AND MANAGEMENT 6
INTEGRATED DISEASE MANAGEMENT 6
CHEMICAL CONTROL 8
Non-systemic fungicides 8
Systemic fungicides 12
CONCLUSION 15
LITERATURE CITED 16
CHAPTER TWO 21
COMBINING HOST PLANT RESISTANCE WITH MANAGED FUNGICIDE
APPLICATIONS FOR CONTROL OF POTATO LATE BLIGHT 21
INTRODUCTION 21
MATERIALS AND METHODS 24
PATHOGEN PREPARATION AND lNOCULATlON 25
PLANT MATERIAL 26
FUNGICIDE APPLICATIONS 26
EXPERIMENTAL DESIGN, DISEASE EVALUATION AND
DATA ANALYSIS 27
MICROCLIMATE MEASUREMENTS 29
RESULTS 30
2001 EXPERIMENT 30
2002 EXPERIMENT 41
DISCUSSION 53

LITERATURE CITED 56

CHAPTER THREE

IMPACT OF PLANT AGE AND LEAF POSITION ON SUSCEPTIBILITY

TO LATE BLIGHT
INTRODUCTION
MATERIALS AND METHODS

EXPERIMENTAL DESIGN AND PLANT MATERIAL

Experiment 1
Experiment 2
Experiment 3

PATHOGEN PREPARATION AND INOCULATION
DISEASE EVALUATION AND DATA ANALYSIS

RESULTS
EXPERIMENT 1
EXPERIMENT 2
EXPERIMENT 3

DISCUSSION

LITERATURE CITED

CHAPTER FOUR
CONCLUSIONS AND SUMMARY
LITERATURE CITED

vi

59

59
59
61
61
61
62
62

63
64
65
65
67
69
72
74

76
76
80

LIST OF TABLES

Table 1. Recommended protectant fungicides for the management of potato
late blight.

Table 2. Recommended systemic fungicides for the management of potato
late blight.

Table 3. Environmental conditions for late blight development at Muck
Soils Research Farm in 2001.

Table 4. Summary of efficacy results: fluazinam applied at reduced rates
and frequencies on potato cultivars and advanced breeding lines from
North Central US potato breeding programs, MSU 2001.

Table 5. Environmental conditions for late blight development at Muck
Soils Research Farm in 2002.

Table 6. Summary of efficacy results: fluazinam applied at reduced rates
and frequencies on potato cultivars and advanced breeding lines from
North Central US potato breeding programs, MSU 2002.

Table 7. Summary of susceptibility results: average ntunber of sporangia
of Phytophthora infestans produced per leaf disc per plant age per cultivar
(all leaves together) and per leaf position (all plant ages together)

for the six cultivars tested in the three experiments.

vii

Page

11

14

30

40

41

52

71

Figure l. Susceptibility expressed in terms of relative area under the disease
progress curve values of different cultivars and advanced breeding lines
(untreated) to Phytophthora infestans in 2001 growing season.

Bars represent LSDp=0.05 = 4.60.

Figure 2. Range of RAUDPC values for W 1355-1 treated with

LIST OF FIGURES

different rates of fluazinam in 2001 growing season.

Bars represent LSDp=005 = 4.60.

Figure 3. Range of RAUDPC values for cultivar Snowden treated

with different rates of fluazinam in 2001 growing season.

Bars represent LSDp=o_os = 4.60.

Figure 4. Range of RAUDPC values for cultivar MNA 157-4 treated with

different rates of fluazinam in 2001 growing season.

Bars represent LSDp=0.0S = 4.60.

Figure 5. Range of RAUDPC values of cultivar Dakota Pearl treated with

different rates of fluazinam in 2001 growing season.

Bars represent LSDpzogs = 4.60.

Figure 6. Range of RAUDPC values for Dakota Rose treated with

different rates of fluazinam in 2001 growing season.

Bars represent LSDp=005 = 4.60.

Figure 7. Range of RAUDPC values for MN 19350 treated with

different rates of fluazinam in 2001 growing season.

Bars represent LSDp=0,05 = 4.60.

Figure 8. Susceptibility expressed in terms of relative area under the disease
progress curve values of different cultivars and advanced breeding lines
(untreated) to Phytophthora infestans in 2002 growing season.

Bars represent LSDp=005 = 3.42.

Figure 9. Range of RAUDPC values for cultivar Dakota Pearl treated with

different rates of fluazinam in 2002 growing season.

Bars represent LSDp=0_05 = 3.42.

viii

Page

33

34

35

36

37

38

39

43

44

Figure 10. Range of RAUDPC values for W 1355-1 treated with
different rates of fluazinam in 2002 growing season.
Bars represent LSDp=0_05 = 3.42.

Figure 11. Range of RAUDPC values for cultivar Dakota Rose treated with
different rates of fluazinam in 2002 growing season.
Bars represent LSDpzogs = 3.42.

Figure 12. Range of RAUDPC values for MN 19350 treated with
different rates of fluazinam in 2002 growing season.
Bars represent LSDp=o_05 = 3.42.

Figure 13. Range of RAUDPC values for W 1386 treated with
differentrates of fluazinam in 2002 growing season.
Bars represent LSDp=005 = 3.42.

Figure 14. Range of RAUDPC values for MN 1915 treated with
different rates of fluazinam in 2002 growing season.
Bars represent LSDp=005 = 3.42.

Figure 15. Range of RAUDPC values for cultivar Snowden treated with

different rates of fluazinam in 2002 growing season.
Bars represent LSDp=o_05 = 3.42.

ix

45

46

47

48

49

50

GENERAL INTRODUCTION

Phytophthora infestans (Mont) Anton de Bary (1876), an oomycete that causes
late blight of potato (Solanum tuberosum L.), is the most important potato disease
globally (16). P.infestans is thought to have originated in South America (1, 3).
Worldwide spread of late blight is enhanced by global trade (3). The pathogen attacks
potatoes both in the field and during storage (2). Late blight is also an important disease
of tomato (Lycopersicon esculentum Mill.) (18) and is also found on other wild plant
species, mainly Solanaceous hosts (14).

Potatoes are very important worldwide, the fourth most important food crop and
are essential for providing nutrition in a growing world population (39). It produces much
energy, protein and contains substantial amounts of vitamins and minerals (48). At the
turn of 21St century world potato production was estimated at 135 million tons (24).

However, potato late blight is considered to be the major constraint to potato
production all over the world. The economic impact of potato late blight is compounded
by costly firngicides; their application costs and the limited acceptance of late blight
resistant/tolerant varieties because they do not meet the qualities that growers and
consumers expect such as high yield, multiple resistances and cooking qualities (47).

Unchecked, potato late blight spreads very quickly given the polycyclic
production of secondary inoculum, which can result in great yield losses (51). In
developing countries, yield loss due to late blight was estimated at US $2.75 billion each
year (7). Late blight is mainly controlled by fungicide applications (37). For example, in
the US. the annual total cost of fungicides to protect potato crOpS against late blight

disease is estimated at $77.1 million (20) and $740 million in developing countries (7).

It was estimated that 25% of total fungicide usage worldwide was for the control of
potato late blight (14).

Chemical control of potato late blight with fungicides is becoming more challenging
due to the appearance of new more aggressive forms of P. infestans that are difficult to
control because they are insensitive to commonly used fungicides (18, 40, 44). In
addition, there is an increasing concern about the intensive use of pesticides. Therefore,
control of potato late blight has to be compatible with the current trend to use an
integrated approach to disease management. This approach incorporates legislative,
cultural, and chemical intervention as well as host resistance and is intended to minimize
pesticide usage to appTOpriate levels in order to avoid negative effects of pesticides on the
environment and human health. To address these issues, modern fungicides need to be
effective at a high level and long duration of efficacy, in addition to minimizing
environmental impacts.

Breeding for resistance has resulted in varieties more tolerant to P. infestans but
complete resistance has not yet been achieved to control the disease without additional
protection with fungicides (5, 47). There are some varieties with partial resistance but
fully resistant varieties with desirable agronomic qualities have not been developed (47).
Following the occurrence of new and more virulent strains of P. infestans, Fry (15, 16),
observed that the use of protective fungicides could complement cultivar resistance to
reduce foliar potato late blight. Kirk et a1. (26) stated that reduced application rates and
frequencies of a protectant residual fungicide could be successfully incorporated into a

control program using host resistance.

The objectives of this research were to determine effective reduced fungicide rates
and increased application intervals to control potato late blight using potato germplasm
with a range of susceptibility to late blight and to assess resistance to late blight in
relation to foliage maturity such as plant age and leaf position in some resistant and
susceptible potato cultivars grown both in controlled and field environment.

Although some resistant cultivars are available, they need to be protected from an
early stage of development. It has been reported that leaves of even more resistant
cultivars can be susceptible to late blight (10) and therefore the application of fungicides
may be justified because their resistance is not complete. Similar work has been done
with some European cultivars (10); however, it was necessary to complete a limited
version of the European experiment with US cultivars and a local isolate of P. infestans.

The approach developed in the fungicide trials will potentially be applied later in
Rwanda to evaluate the efficacy of fungicides on resistant/tolerant varieties under the
conditions of Rwanda with the purpose to be incorporated later into an integrated potato
late blight control program in Rwanda. Potatoes grow well in several parts of Rwanda
and play an important role in the economy of Rwanda as a cash crop and the diet of
Rwandan people (38). In most areas at least two major crops are planted each year but the
production is severely limited by late blight, the major constraint to the potato crop

production in the country (3 8).

CHAPTER ONE
LITERATURE REVIEW
POTATO LATE BLIGHT
IMPORTANCE

Potato late blight is best known historically as the cause of the great Irish potato
famine during the 18408 (37). The disease devastated the potato crop, the main staple
crop in Ireland at that time. Potato tubers rotted in the field and in storage and this led to
starvation and death of about 10% of the population (1.3 million). Many emigrated from
Ireland to the United States and elsewhere in North America (2, 37).

At that time, microorganisms were believed to be the result rather than the cause
of diseases. The role of fungi as the causative agents of plant diseases was not fully
understood. Following the Irishpotato famine, Anton de Bary (1876) confirmed that the
oomycete Phytophthora infestans was the primary cause of the disease, potato late blight
(34, 37).

Despite many efforts made by plant pathologists since to control the disease,
potato late blight is still a major challenge in potato production. This is thought to be due
to the appearance of new genotypes of P. infestans that are difficult to control in potato
plants (15, 16). The appearance of novel genotypes is thought to have occurred during the
1970’s through the import of potatoes from Mexico [the origin of P. infestans (18)] to

Europe and during the early 1990’s to North America (11, 33).

Control strategies developed over the past 150 years have failed to eliminate
potato late blight completely. Thus, control of potato late blight relies on well-timed
application of protectant fungicides in susceptible cultivars and moderately resistant

varieties to prevent great yield losses (3 7).

DISEASE DEVELOPMENT

Phytophthora infestans is a biotrophic pathogen and over-winters as mycelium in
potato tubers in storage to be used for seed, tubers that are inadvertently not harvested
and left in fields, and in discarded piles of culled potatoes (12). P. infestans can also
survive in soil as a sexually originated oospore (12, 35). However due to the
predominance of the A2 mating type in North America, this over-wintering source is
thought to be of limited importance. The thick walled oospore is a sexual spore resulting
from conjugation of two mating types normally designated Al and A2 (2, 45). Mycelia
within potato tubers and possibly but rarely oospores serve as the primary inoculum for
seasonal epidemics of potato late blight. Both asexual and sexual stages produce
sporangia. Sporangia produced on infected leaves are carried by wind or splashed by
water to the foliage of new plants, which germinate and initiate an epidemic (2).

Potato late blight is a temporally sporadic disease that occurs only when
microclimate conditions within the canopy are favorable and inoculum is present (28).
Conducive environmental conditions include air temperatures between 7 and 27°C and
relatively long periods (10 hours or more) of leaf wetness. Favorable conditions for
infection and development coincide with periods of high moisture (RH>90%), prolonged

periods of leaf wetness and moderate temperatures (1 5-25°C); ( 2, 22).

During these conditions, sporangia can cause new infections and lead to a
polycyclic epidemic due to the rapid production of asexual generations of secondary
inoculum of the pathogen (51). Crop loss occurs through destruction of foliage and
consequent reduction of photosynthetic capacity and tuber infection by spores that are
washed down from leaves into the soil to enter tubers through wounds, lenticels and

directly through the periderm of the tuber.

CONTROL AND MANAGEMENT

INTEGRATED DISEASE MANAGEMENT

No single measure used alone can successfully control late blight in potatoes (37).
Integrated strategies such as utilization of resistant and/or tolerant varieties; certified late
blight free-seed, limitation and avoidance of conducive environments, climatic
monitoring and disease prediction are some agronomic practices that can be utilized for
control of potato late blight. The use of resistant or tolerant varieties combined with
additional control strategies such as cultural practices and well-applied fungicides can
together limit crop and yield losses and maintain potato late blight at acceptable
economic threshold levels (17, 39).

Breeding for resistance to P. infestans has resulted in resistant varieties and new
resistant cultivars are continuously being released (47) but varieties that have durable and

consistent resistance has not fully succeeded.

P. infestans is a genetically versatile organism and has overcome both vertical and
horizontal resistance in Solanum tuberosum. Therefore cultivars with intermediate
resistance would need to be complemented with additional protection by fungicide
treatment in an integrated manner to provide better control of potato late blight (47).

Cultural practices include destruction of crop residues, disposal of culled tubers,
management practices to avoid leaving volunteer tubers in the field at harvest, and
appropriate rotations. All these practices are intended to reduce potential sources of
inoculum (17, 39). Additional cultural practices include irrigation management to
minimize the duration of leaf wetness periods, hilling of rows to increase depth of tubers
in the soil to provide a mechanical barrier to spore penetration, planting when
environmental conditions are least conducive to late blight development from seed and
harvesting techniques that reduce the risk of spore deposition on tubers.

The application of fimgicides is also an integral component of late blight
management and possibly key to the reduction of the effects of potato late blight and
contribute to prevention of yield loss. Fungicides have to be applied preventatively to
increase their effectiveness, but also judiciously to limit their impact on the environment.
Thus, scouting and weather-based disease models such as BLIGHTCAST (29), a climate-
based model that combines a system based on relative humidity and temperature with a
system based on rainfall and temperature are very important in potato late blight
management to reduce the misuse of fungicides that encourage application when needed

(14, 17,39).

Microclimate—based late blight models were established originally in the 1940’s and
fungicide spray programs and other management decisions were based on the
accumulation of ‘blight favorable days’, or thresholds of the disease severity values
(DSV) in the 1960’s (4, 29, 49, 50). The 90% relative humidity criterion is used for
potato late blight monitoring since early analysis of canopy based records of the physical
environment in relation to disease development (49).

Average air temperatures and duration of wetting period are combined in the daily
assignment of Wallin disease severity values, which have become standard in estimating
potato late blight risk (29). BLIGHTCAST model recommends the initiation of fungicide
applications afier the accumulation of either ten consecutive favorable days or 18 DSV

following emergence (29).

CHEMICAL CONTROL

Non-systemic fungicides

Non-systemic or protectant fungicides are chemical compounds that are not taken
up by the plant and are subject to weathering-decrease. This necessitates frequent and
regular application to maintain protection. Protective fungicides are only active on the
plant surface after germination of spores but before the pathogen has entered the host
tissue. Therefore these fungicides must be applied before infection to prevent disease
(13). They are therefore less effective when the pathogen has already penetrated leaf
tissue because they are out of contact with the ftmgus and unable to interact with the

pathogen inside the host (13, 14).

Contact fungicides with residual properties are applied to the plant surface to protect
plants before the arrival of inoculum of the pathogen and prevent infection from
occurring (Table l).

The first chemical compound used for crop protection was Bordeaux mixture, an
inorganic fungicide consisting of copper sulfate and calcium hydroxide (2). Bordeaux
mixture adheres well to potato leaves even after rainfall. However, like all other copper-
based fungicides, Bordeaux mixture can be phytotoxic due to copper ions, which are
potentially toxic to plants (13, 14).

Copper-based chemicals were widely applied on potato crops until recently when
they have been substituted by modern organic protectant fungicides that are less toxic to
the foliage (13).

The dithiocarbamates were the first group of organic fungicides used to control
potato late blight. The dithiocarbamate anion interacts withthiol groups of enzymes in
the pathogen, affecting both spore germination and mycelial growth and prevents
penetration of the host (13, 39). The dithiocarbamates continue to be used in potato late
blight disease control programs and include compounds such as mancozeb, maneb,
metiram and zineb. Known as ethylenebisdithiocarbamates (EBDCS), they are
extensively used in mixtures with systemic fungicides to supplement efficacy (13).

Following the discovery of the dithiocarbamates, several classes of other
protectant fungicides including tin compounds and phthalonitriles were developed.

Among the organic tin compounds, the most important are fentin acetate and

fentin hydroxide, triphenyltin is the active component.

They are considered effective as protectant fungicides and are somewhat curative against
actively sporulating P. infestans on foliage and may therefore prevent tuber blight (13).
They have antisporulant activity and provide tuber protection from sporangia spread but
they can be phytotoxic when applied to developing foliage (13). Triphenyltins are mixed
with the dithiocarbamates and applied late in the season when an active disease epidemic
occurs during the late stages of potato crop development (13, 14).

Among the phthalonitriles, chlorothalonil is the only commercial product
available. Chlorothalonil is a protectant foliar fungicide that can be used in reduced rates
in combination with a dithiocarbamate to provide effective control of foliar blight by
preventing spore germination and penetration (13).

Fluazinam is the most commonly used fungicide in the recent group of
pyridinamines (13). It is a protectant fungicide with an excellent residual activity (13).
Fluazinam is used for control of a wide group of pathogenic fiIngi and water molds
including white mold (Sclerotinia minor) on peanuts, white mold (Sclerotim‘a
sclerotiorum) and late blight (P. infestans) in potatoes (46). It is active in relatively small
doses and inhibits spore germination and infection (16).

F luzinam is a broad spectrum fungicide with a multi-site mode of action. It is a
powerful uncoupler in oxidative phosphorylation and inhibits proton transfer across the
mitochondrial membrane (21). It may be used to prevent or delay resistance to single-site
fungicides. It is also reported to have better persistence on the leaf surface compared to
other contact fungicides (46). When applied to potato foliage in combination with
cultivar resistance, fluazinam has been shown to be active at reduced rates and increased

spray intervals to control potato late blight (13, 26).

10

The product was recently registered in the US and it is still being tested for its efficacy to

control plant diseases including potato late blight.

Table 1. Recommended protectant fungicides for the management of potato late blight.

 

Chemical class

Mode of action

 

Coppers
- copper oxychloride 50% WP
- copper hydroxide 50%
- copper oxide 50% WP

Dithiocarbamates
- EBDCs (mancozeb, maneb, metiram ,zineb)

Organic tins
- Fentin acetate
- Fentin hydroxide

Phthalonitriles
- Chlorothalonil

Pyridinamines
Fluazinam

 

Nonspecific denaturation of proteins
and enzymes. Inside the spore, the
Cu2+ may link to different chemical
groups such as imidazols, carboxyls,
phosphates, sulphidryls, aminae or
hydroxyls to produce a toxic effect
that disturbs the normal cellular
activities.

Inactivate —SH groups in amino acids,
proteins and enzymes. The
dithiocarbamate anion interacts with
thiol groups of enzymes affecting both
spore germination and mycelial
growth.

Inhibition of oxidative
phosphorylation in fungal cells.

Cell membrane toxicity

Multi-site mode of action. Uncoupler
of oxidative phosphorylation and
inhibitor of proton transfer across the
mitochondrial membrane.

 

ll

._ F .._._

Systemic fungicides

A systemic fungicide is a chemical compound that can be absorbed by the plant
tissue and moves within the plant either acropetally, basipetally or both (Table 2). Unlike
protectants, systemic fimgicides penetrate and act inside the plant to stop the progression
of the disease and are not subjected to washing off by rain or irrigation (14). Inside the
plant, systemic fungicides interfere with the pathogen, act against the mycelium of the
fungus to stop further spread of the disease whereas protectants act upon surface
inoculum against spore tissue penetration. Some systemic fungicides act as both
protectant and curative. They are able to protect uninfected potato and cure pre-
established infections (Table 2).

Systemic fungicides have a specific mode of action and act at a Specific
biochemical sites controlled by one or few genes. Thus simple changes in target enzyme
gene sequence may result in the appearance of populations able to overcome the activity
of the fimgicide, known as firngicide resistance (43).

In the 1970’s systemic fungicides such as the phenylamides for the control of
oomycete—caused diseases were developed (14).

The following chemical groups, cyanoacetamide-oximes, phosphonates,
carbamates and phenylamides were introduced for the control of oomycetes.

Carbamates such as propamocarb hydroxide are not well translocated and thus
their systemic activity is limited (1 7, 36).

Phosphonate compounds such as fosetyl-Al and phosphorous acid are more

effective on Phytophthora tree root diseases than on P. infestans (14).

12

Cymoxanil is an acetarnide compound, trade name Curzate (® DuPont), and the
only commercial fungicide available in the cyanoacetamide-oximes group. Cymoxanil is
used to protect and to cure potato foliage from P. infestans infection. This foliar fungicide
penetrates the potato leaf locally and is effective against P. infestans (l4). Cymoxanil
also acts as a protectant fungicide and inhibits zoospore release and formation of
appressoria. Cymoxanil is moderately curative when it acts as a systemic ftmgicide and
inhibits hyphal growth (25). The fungicide is quickly metabolized in plant tissue and it is
used in mixtures with non-systemic fungicides to provide a synergistic effect (14).
Cymoxanil controls potato late blight during its incubation period and limits potato late
blight damage to the crop.

The phenylamide fungicides are highly and specifically active on the members of
Peronosporales. They have been very efficient against P. infestans Since their
introduction in commercial use in 1978. Potato growers all over the world relied on them
to control potato late blight. Metalaxyl is the most important phenylamide used to control
potato late blight (14). When applied on roots or lower foliar portions of potato plant, the
fungicide rapidly penetrates the plant tissue and it is acropetally translocated within the
plant to protect all the foliage including new grth against the pathogen (9). Metalaxyl
inhibits fungal grth and controls both previous and post infection in potato plants (6).
Phenylamide sprays are recommended in the early season or during the period of active
vegetative growth of the crop. Novel genotypes of P. infestans resistant to metalaxyl are

now reported in the US and elsewhere in the world (l8, 19, 23, 27, 31, 32, 41, 42).

13

‘ 'F- .-__

Following the development of resistance to the phenylamides, dimethomorph, a
new curative fungicide was introduced which is effective against some species of
Phytophthora including P. infestans (14). The product is a morpholine fungicide known
by the trade name Acrobat (® BASF). It has been reported to have excellent antisporulant
activity and can prevent sporangia and oospore production (13). It is active against
metalaxyl-resistant isolates of P. infestans. Dimethomorph acts on the biogenesis of the
pathogen cell wall and has been shown to be most effective in mixture with a protectant
fungicide such as chlorothalonil or mancozeb to control P. infestans (8, 14).

Table 2. Recommended systemic fungicides for the management of potato late blight.

 

 

Chemical class Mode of action
Cyanoacetamide-oximes Inhibition of multiple cellular processes
- Cymoxanil leading to inhibition of zoospore release,
formation of appressoria and hyphal
growth.
Phenylamides Inhibition of RNA synthesis.
- Metalaxyl
Morpholine Inhibition of sterol production; disruption
- Dimethomorph of fungal cell wall formation.

 

 

14

CONCLUSION

Potato late blight remains the most important and challenging potato disease
worldwide and was the cause of the Irish famine in the 1840’s. From that time, many
efforts have been made to contain the disease and this gradually reduced the impact of
late blight in the world potato growing areas.

An integrated approach for control of potato late blight including application of
fungicides, host resistance, cultural practices and climate-based prediction models have
been developed over the past 150 years.

The emergence in the 1970-90’s of new more aggressive strains of P. infestans
able to overcome previously tolerant potato varieties and resistant to frequently used
fungicides augmented efforts to search for better replacement products and develop new,
agronomically acceptable, late blight-tolerant potato cultivars. Novel compounds
effective against potato late blight such as fluazinam were released during the last decade
in order to overcome the new genotypes of the pathogen.

Disease forecasting methods have improved both the timing of application and
type of fungicide appropriate to maximize late blight control.

It is clear that no single approach to a universal control method for late blight will
be successful, however an integrated approach based on all known and partially

successful management techniques should result in a more stable production system.

15

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10.

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19

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20

CHAPTER TWO

COMBINING HOST PLANT RESISTANCE WITH MANAGED FUNGICIDE
APPLICATIONS FOR CONTROL OF POTATO LATE BLIGHT

INTRODUCTION

Potato late blight, caused by Phytophthora infestans, continues to cause losses in
potato production areas throughout the world. This situation is in part due to the
resistance developed by some strains of P. infestans to phenylamide fungicides that were
previously efficient and widely applied to control the pathogen (15). The increased
aggressiveness and phenylamide resistance of novel genotypes of P. infestans (22)
resulted in an intensification of research on new control strategies based mainly on the
development of host resistance combined with the managed application of foliar
fungicides. The problem is compounded by global demand to decrease reliance on
pesticides and their perceived impact on environmental quality. Moreover, the cost of
fungicides used to protect potato crops against potato late blight is relatively high (5, 11,
19). Therefore, there is a need to find effective control methods to manage potato late
blight in a safe, environmentally benign manner and at low cost.

It has already been observed that a combination of cultivar resistance and regular
applications of protective fungicides based on forecasting techniques reduces foliar
potato late blight and chemical input in agricultural systems (l3, l4, 16, 21). However,
the active ingredients of the commonly used fungicides, such as chlorothalonil and
mancozeb, have recently been identified as B2 carcinogens and their use may be curtailed

or discontinued in food crops in the future (18).

21

In response, the agrochemical industry in co-operation with legislative bodies
within governments, such as the Environmental Protection Agency (EPA) in the US, have
recently adopted an approach of developing crop protection products that are active at
low rates, durable and have a broad spectrum of targets.

Fluazinam, is such a product and was registered in the US as a product likely to
have less environmental impact than some older chemistries (26). In addition, the mode
of action of fluazinam, which is an uncoupler of the oxidative phosphorylation in the
respiration chain involving protonation/deprotonation has a minimal risk of resistance
development and was internationally introduced for control of several plant diseases
including potato late blight (P. infestans) and white mold (Sclerotinia sclerotiorum); (2,
25). Recently, fluazinam was compared to the commercial standard fungicide,
chlorothalonil, applied at reduced rates and increased application intervals on selected
potato cultivars and advanced breeding lines (ABL) from the Michigan State University
(MSU) potato breeding program with a range of susceptibility to late blight.
Chlorothalonil and fluazinam were not found to be significantly different with respect to
control of late blight at comparative application rates and frequencies on any of the
cultivars and advanced breeding lines (20).

Decreased emphasis on regionalization within the US has resulted in increased
co-operation between potato research and extension programs with common geographic
and climatic characteristics. Such a program has come to be known as the Quad State
Program in which potato producing states within north central US share research findings

and recently has resulted in testing of common germplasm across locations.

22

An example of this co-operation has resulted in the inclusion of potato cultivars and
advanced breeding lines from four of the Land Grant University potato-breeding
programs in the USA: Michigan, Minnesota, North Dakota and Wisconsin.

It was recommended by Kirk et al. (21) that prior to release of new potato
varieties; trials should be conducted to determine appropriate crop protection
recommendations, particularly for potato late blight.

The objective of this study was to determine the susceptibility of different
cultivars and advanced breeding lines from the Quad State breeding programs and test the
efficacy of reduced rates of fluazinam applied at different frequencies on the foliage of
these cultivars and advanced breeding lines against the predominant genotype of the

pathogen P. infestans in north central US [US8, A2 mating type, metalaxyl insensitive

(17)]-

23

MATERIALS AND METHODS

Field experiments were conducted at the Muck Soils Research Farm in Bath,
Clinton County, Michigan in 2001 and 2002. After being plowed to 20 cm depth during
October following harvest of preceding crops, soils were prepared for planting with a
mechanical cultivator in early May and fertilizer applied during final bed preparation on
the day of planting. Cultivars and advanced breeding lines were planted in June 2001 and
2002 in two-row by 8 m plots (0.9 m row Spacing). Fertilizers were applied in
accordance with results from soil testing carried out in the spring of each year and about
250 kg N. ha'1 (total N) was applied in two equal doses at planting and billing. Additional
micronutrients were applied according to petiole sampling recommendations in both
years. Approximately 0.2, 0.3 and 0.2 kg. ha"l boron, manganese and magnesium,
respectively were applied as chelated formulations.

Cut and whole seed pieces (75-150g) of selected cultivars and advanced breeding
lines were used in all experiments. When percent relative humidity (RH) fell below 80%
(measured with RH sensors mounted within the canopy, described below), a mist
irrigation system (described below) was turned on to maintain RH at > 95% within the
plant canopy.

Plots were irrigated with turbine rotary garden sprinklers (Gihnour Group,
Somerset, PA, USA.) at 1055 1 H20 ha/hr and managed under standard potato
agronomic practices as necessary to maintain canopy and soil moisture conditions

conducive for development of foliar late blight (24).

24

Weeds were controlled by hilling and with metolachlor at 2.3 l.ha'1 10 days after
planting (DAP), bentazon salt at 2.3 l. ha"(20 and 40 DAP) and sethoxydim at 1.8 l.ha'1
(60 DAP). Insects were controlled with imidacloprid at 1.4 kg. ha’1(at planting), carbaryl
at 1.4 kg. ha'1 (31 and 55 DAP), endosulfan at 2.7 1. ha’1 (65 and 87 DAP) and permethrin

at 0.56 kg. ha" (48 DAP).

PATHOGEN PREPARATION AND INOCULATION

A single isolate of Phytophthora infestans (Pi 95-7; US8 genotype, metalaxyl
resistant; A2 mating type), the predominant biotype of the pathogen in North America (6)
was grown on rye agar plates (7) for 14 days in the dark at 15°C.

Sporulation media were made of modified rye B agar (4) consisting of the filtrate
of pre-rinsed rye (Secale cereale L.) seeds (100.0 g. l") boiled for 1 hour, de-ionized (di)
H20 added to a final volume of 1.0 1, glucose (7.5 g. l") , B-sitosterol (0.05 g. 1") and agar
(15.0 g. l").

The mycelial/sporangia] mat was rinsed in cold (4°C) sterile, distilled water and
scraped fi'om the agar plate surface with a rubber policeman. Sporangia were counted
with a hemacytometer and the final concentration was adjusted to 1x104 sporangia. ml'l.
Sporangia] cultures were incubated for 2-3 hours at 4°C to stimulate zoospore release.
Plots were inoculated by injecting a zoospore suspension of P. infestans into the
irrigation water feed pipeline under 0.5kg. cm‘2 C02 pressure and applied at a rate of

about 150ml of inoculum solution. m'2 trial area.

25

The amount and rate of inoculum applied was estimated from prior calibration of
the overhead irrigation system and was intended to expose all potato foliage to inoculum

of P. infestans.

PLANT MATERIAL

Ten potato cultivars advanced breeding lines, Jacqueline Lee, Snowden, Dakota
Pearl, Dakota Rose, Torridon, MN 19350, MN 1915, MNA 157-4, W 1355-1, and W
1386, previously identified with different responses to foliar late blight, were evaluated in
this study. Cultivars Jacqueline Lee and Snowden were used, respectively, as the resistant
and susceptible standards following five years of testing at Michigan State University (7,

8, 9, 10).

FUNGICIDE APPLICATIONS

Fluazinam 5SC (non-commercial formulation, ISK Biosciences Corporation, 5966
Heisley Road, PO Box 8000, Mentor, OH 44061-8000) a residual contact fungicide was
used for this experiment. The manufacturer’s recommended application rate [(MRAR)
100%] was 0.15kg atria“. application" (29).

Fungicide treatments were applied with an ATV rear-mounted spray boom (R&D
Sprayers, Opelousas, LA, USA.) that traveled at 1 m/s, delivering 230 L H20. ha'1 (3.5
kg. cm'2 pressure) with three XR11003VS nozzles per row positioned 30 cm apart and 45

cm above the canopy.

26

The first application of fungicide was made when plants were about 5 cm tall, about
30 days after planting. Potato plants were treated with fiingicide until complete
defoliation in non-treated plots of susceptible controls.
The fungicide was applied at a 5, 7, 10 and 14-day spray intervals at 33, 66 and 100%
MRAR in 2001 and at a 5, 10 and 15-day spray intervals at 50 and 100% MRAR in 2002.
The rate was adjusted to 50% MRAR after the fungicide omega (® Syngenta) was
introduced commercially in 2002. The 33% rate was below the legal recommended rate

of application.

EXPERIMENTAL DESIGN, DISEASE EVALUATION AND DATA ANALYSIS

The field trials were planted as described above into a completely randomized
plot design into plots with three replicates due to limitations Of seed supply.

Late blight symptoms were detected about 7 days after inoculation. Each plant
within each plot was visually rated at 3-5 day intervals for percent leaf and stem area with
late blight lesions. The mean percent blighted foliar area per treatment was calculated.

Evaluations continued until untreated plots of susceptible cultivars were 100% blighted.

27

For all plots and assessment dates, the area under the disease progress curve AUDPC (3)
was calculated and the relative area under the disease progress curve [RAUDPC (1)] was

used to estimate the relative severity of foliar late blight using the following formula:

20141" 71)*(_%§+_P;)

TTm,*100

0

RA UDPC =

 

Where T, was the 1th day after inoculation when an estimation of percent foliar late blight
was made, D, was the estimated percentage of area with blighted foliage at Ti and That
was the number of days after inoculation at which the final foliar assessment was
recorded.

Data were analyzed by ANOVA and all pair-wise comparison using Fisher’s least
significant differences (LSD) at or = 0.05 were calculated (SAS-Institute 2003. Statistical
Analysis Software v.8.2, Cary, NC .). Cultivars or advanced breeding lines with foliar late
blight severity not significantly higher than that of treated Jacqueline Lee were classified
as late blight resistant (R); susceptible (S) if the severity was significantly higher or not
significantly different from that of non-treated Snowden; and moderately resistant (M)
when foliar late blight severity was significantly higher than that of Jacqueline Lee but
significantly lower than that of non-treated Snowden.

A fungicide treatment on a cultivar that resulted in a RAUDPC value not
significantly higher than that of treated Snowden with a full rate of fluazinam at a 5-day

spray interval (2001 and 2002) was classified as effective late blight control (E).

28

Any fungicide treatment and cultivar combination in which the RAUDPC was
significantly higher than that of treated Snowden and significantly less than that of non-
treated Snowden was classified as a partially effective treatment (PE). If a fungicide
treatment on a cultivar resulted in a RAUDPC not significantly different from that of

non-treated Snowden it was classified as a non-effective treatment (NE).

MICROCLIMATE MEASUREMENTS

Climatic variables were measured with a Davis Weather Station (Spectrum
Groweather ET Station, Spectrum Technologies, Inc., 2389 W. Andrew Road, Plainfield,
IL 60544) equipped with air temperature and humidity sensors located within the potato
canopy on Site. Microclimate within the potato canopy was monitored beginning when
50% of the potato plants had emerged and ending when canopies of healthy plants
reached 100% senescence. The Wallin Late Blight Prediction Model (28) was developed
in the Eastern United States under conditions similar to those in Michigan and was
adapted to local conditions (1). Late blight disease severity values (DSV) were estimated
from the Wallin Late Blight Prediction Model and accumulated from inoculation to final

evaluation to estimate the conduciveness of the environment for late blight development.

29

RESULTS

2001 EXPERIMENT

Table 3. Environmental conditions for late blight development at Muck Soils Research
Farm in 2001.

 

 

 

Climatic variables Month
June July August September

Air temperature (°C)

- Maximum 32.4 35.1 30.0 30.7

- Minimum 17.4 23.1 16.2 15.6
Soil temperature (C)

- Maximum 29.8 29.6 26.5 24.7

- Minimum 22.6 24.1 21.6 17.8
Precipitation (mm) 27.90 14.73 6.35 3.05

 

 

From 50% emergence to harvest, 81 late blight disease severity values were
accumulated (base 80% ambient relative humidity). These conditions were conducive for
development of late blight.

Cultivars and advanced breeding lines are ranked in order of increasing RAUDPC
in untreated plots (Table 4). Application of fluazinam at full rate at a 5 or 7-day spray
interval resulted in an effective control in almost all cultivars and advanced breeding
lines (Table 4). The mean RAUDPC value for non-treated Jacqueline Lee and Torridon
was about 0.2, which were classified as resistant (Figure 1).

The thresholds used to determine the efficacy of the fungicide and variety
combination programs was a RAUDPC value of 2.7 (Snowden MRAR, 5 day interval)
and RAUDPC of 37.2 (Snowden, non-treated control); (Figure 1 and Table 4). There was
no significant effect of fungicide treatment on resistant cultivars Jacqueline Lee and

Torridon (Table 4).

30

Therefore fungicide treatment and variety combinations with an RAUDPC S 6.8 (not
significantly different from Snowden, 100% MRAR, 5-day application interval) were
defined as effective (E); combinations not significantly different from the non-treated
Snowden control (RAUDPC 2 33.1) were defined as non-effective (NE); and
combinations of RAUDPC values significantly different from both standards were
defined as partially effective (PE).

W 1355-1 was effectively protected by application of fluazinam on 5-day spray
interval at all rates, on 7-day spray intervals at 66% and 100% MRAR fluazinam and on
10-day and 14-day spray intervals at 100% MRAR fluazinam (Figure 2). The disease was
partially effectively controlled with 33% MRAR fluazinam applied at 7-day spray
interval and at 66% MRAR fluazinam applied at a 10 and 14-day spray intervals in the
foliage of this cultivar (Figure 2).

Snowden was effectively protected at 100% MRAR fluazinam applied at 5 and 7-
day spray intervals; late blight was partially effectively controlled at all other rates and
application frequencies in this cultivar (Figure 3).

MNA 157-4 was effectively protected by the fungicide at 100% MRAR fluazinam
at a 5—day spray interval; the disease was partially effectively controlled at all other rates
and application frequencies in this advanced breeding line except at 100% MRAR

fluazinam on 14-day spray interval where MNA 157-4 was not effectively protected

(Figure 4).

31

Dakota Pearl was partially protected by the fungicide at all rates and application
frequencies except at 33% MRAR fluazinam on 7, 10 and 14-day spray intervals. At 66%
of the MRAR on 10 and 14-day spray intervals and at 100% MRAR fluazinam on 14-day
spray interval, fluazinam did not provide sufficient protection in this cultivar (Figure 5).

Dakota Rose was effectively protected by the fungicide applied at 100% MRAR
fluazinarn on 5-day Spray interval; late blight was partially effectively protected at all
other rates and application frequencies in this cultivar (Figure 6).

MN 19350 was effectively protected at 100% MRAR fluazinam applied at a 5-
day spray interval and was partially protected at all other rates and application
frequencies except at 33% and 66% MRAR on 10 and 14-days application intervals and

at 100% MRAR fluazinam on 14-day spray interval (Figure 7).

32

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Figure l. Susceptibility expressed in terms of relative area under the disease progress
curve values of different cultivars and advanced breeding lines (untreated) to P. infestans
in 2001 growing season. Bars represent LSDp=0.05 = 4.60.

33

 

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5days 7days 10days

   

 

 

Appilcation interval

Figure 2. Range of RAUDPC values for W 1355-1 treated with different rates of
fluazinam in 2001 growing season. Bars represent LSDp=005 = 4.60.

34

 

50-
45j
40-
35.
30~
25J
20~

15]
10.

 

I Untreated control 33%MRAR 66%MRAR El 100%MRAR

 

 

 

RAUDPC (%)

 

 

 

 

7days 10days 14days

 

Application interval

Figure 3. Range of RAUDPC values for cultivar Snowden treated with different rates of
fluazinam in 2001 growing season. Bars represent LSDp=o.os = 4.60.

35

 

50 1i I Untreated control E133%MRAR I66%MRAR D100%MRAR

RAUDPC (°/o)

 

 

14days

Application interval

Figure 4. Range of RAUDPC values for MNA 157-4 treated with different rates of
fluazinam in 2001 growing season. Bars represent LSDp=o.05 = 4.60.

36

 

50‘ lUntieatedichntrol I330/iMRAR I66%MRAR D100%MRAfl

 

 

O

  
 

RAUDPC (%)
N N (A)
O 01

_l
01
I

10-

 

 

 

7days 1 Odays

Application interval

Figure 5. Range of RAUDPC values of cultivar Dakota Pearl treated with different rates
of fluazinam in 2001 growing season. Bars represent LSDP=Q05 = 4.60.

37

 

50 l {I Untreated control In 33% MRAR l66%MRAR [11009on

 

 

45 'l
40 i

  
  

00
01
PM

RAUDPC (%)
N N 0.)
0'1

 

7days

Application interval

Figure 6. Range of RAUDPC values for cultivar Dakota Rose treated with different rates
of fluazinam in 2001 growing season. Bars represent LSDp=o.05 = 4.60.

38

 

 

 

50 ,
lUntreated control E133%MRAR I66%MRAR D100%MRAFj

pal

5days 7days 10days 14days

RAUDPC (°/.)
-\ —¥ N N (J0 (JO -5 #-
O (I! 0 (J1 O 01 O 0'1

01

 

 

O

Application interval

Figure 7. Range of RAUDPC values for MN 19350 treated with different rates of
fluazinam in 2001 growing season. Bars represent LSDp=n05 = 4.60.

39

Table 4. Summary of efficacy results: fluazinam applied at reduced rates and frequencies on potato
cgfivars and advanced breeding lines from North Central US potato breeding programs, MSU 2001.
Cultivar/ABL Rate of Application frequency (days)

 

 

fluazinam'o 5 7 10 14
RAUDPC2
Jacqueline Lee 0 0.2 op3 RT
33 0.3 O’p’ E5 0.1 o’p’ E 0.06 p’ E 0.06 P’ E
66 0.2 O’p’ E 0.2 o’p’ E 0.06 P’ E 0.2 o’p’ E
100 0.06 p’ E 0.1 o’p’ E 0.06 P’ E 0.06 P’ E
Torridon 0 0.2 o’p’ R
33 0.2 o’p’ E 0.1 p’ E 0.1 p’ E 0.3 o’p’ E
66 0.2 o’p’ E 0.2 o’p’ E 0.1 o’p’ E 0.2 o’p’ E
100 0.2 o’p’ E 0.1 o’p’ E 0.0 p’ E 0.3 o’p’ E
MN19350 0 33.5 f-j S
33 27.1 n-r PE 31.9 g-l PE 33.2 f-k NE 36.4 c-f NE
66 25.0 p-t PE 27.3 m-r PE 34.7 e-h NE 34 f-i NE
100 4.6 l’-n’ E 10.4 g’-i’ PE 24.0 q-v PE 33.1 f-k NE
Snowden O 37.2 b-f S
33 24.7 q-t PE 21.6 s-x PE 20.3 u-y PE 21.4 t-x PE
66 8.9 h’-k’ PE 13.4 c’-g’ PE 16.6 y-c’ PE 18.5 x-b’ PE
100 2.7 l’-p’ E 6.0 j’-m’ E 20.7 t-y PE 19.3 w-a’ PE
Dakota Rose 0 38.6 b-e S
33 15.9 z-d’ PE 30.3 i-n PE 31.7 g-m PE 31.1 h-n PE
66 15.4 a’-e’ PE 23.8 r-v PE 20.1 v-z PE 29.0 k-p PE
100 6.6 i’-m’ E 10.1 g’-j’ PE 15.9 z-d’ PE 30.4 i-n PE
Dakota Pearl 0 40.5 a-c S
33 29.4 j-o PE 35.7 d-g NE36.7 c-f NE 35.4 d-g NE
66 10.7 P-i’ PE 25.7 o-s PE 36.7 c-f NE 39.5 b-d NE
100 11.9 d’-h’ PE 18.2 x-b’ PE 29.9 i-n PE 33.2 f-i NE
MNA157-4 0 41.3 a-b S
33 21.6 s-x PE 28.2 l-q PE31.1 h-n PE 32.2 g-l PE
66 9.4 g’-k’ PE l8.0 x-b’ PE 23.9 q-v PE 24.4 q-u PE
100 6.8 i’-l’ E 11.7 e’-h’ PE 22.8 s-w PE 34.6 e-h NE
W1355-1 O 43.9 a S
33 1.4 n’-p’ E 11.3 e’-h’ PE 11.8 d’-h’ PE 24.0 q-v PE
66 1.3 n’-p’ E 2.5 m’-p’ E 9.3 g’-k’ PE 14.8 b’-t‘ PE
100 0.6 n’-p’ E 5.7 k’-m’ E 4.3 l’-o’ E 4.2 1’-p’ E

 

1: Application rate of fluazinam as percent of manufacturer’s recommended rate 2: Relative area under the
disease progress curve from inoculation to 100% late blight in susceptible control (Snowden); max = 100.
3: Means followed by the same letter were not significantly different at p = 0.05; comparison between all
combinations of fungicide application rate and frequency of application in all cultivars/ABL.

4: Susceptibility of non-treated control to late blight; R = Resistant, not significantly different from
Jacqueline Lee (non-treated); S = Susceptible, not significantly different from Snowden (non-treated); M=
Moderately resistant, significantly different from both Jacqueline Lee and Snowden (non-treated).

5: Effectiveness of fungicide treatment in comparison to Snowden treated with a full application rate of
fluazinam at a 5-day interval or with non-treated Snowden control; B = RAUDPC not significantly different
from treated Snowden control; PE significantly different from treated Snowden control and non-treated
control; NE = not significantly different from Snowden non-treated control at p = 0.05.

40

2002 EXPERIMENT

Table 5. Environmental conditions for late blight development at Muck Soils Research
Farm in 2002.

 

 

 

Climatic variables Month
June July August September

Air temperature (°C)

- Maximum 33.4 33.6 31.5 32.9

- Minimum 18 22.5 20.3 18.2
Soil temperature (°C)

- Maximum 27.8 29.2 29.1 27.9

- Minimum 21.6 23.4 23.4 20.7
Precipitation (mm) 8.13 28 .96 10.42 0.0

 

 

From 50% emergence to harvest, 100 late blight disease severity values were
accumulated (base 80% ambient relative humidity). These conditions were conducive for
development of late blight.

Cultivars and advanced breeding lines were ranked in order of increasing
RAUDPC in untreated plots (Figure 8 and Table 6). There was no significant effect with
fungicide treatment on Jacqueline Lee and Torridon that were classified as late blight
resistant (R) with mean RAUDPC value = 0.2 (Table 6). Dakota Pearl, W 1335-1, Dakota
Rose and MN 19350 whose mean RAUDPC values were significantly higher than that of
Jacqueline Lee and not significantly lower than that of Snowden (non-treated) were
classified as moderately resistant (M); (Table 6). The mean RAUDPC values of cultivars
W 1386, MN 1915 and Snowden were higher than that of Snowden and were classified as

susceptible (S); (Table 6).

41

The thresholds used to determine the efficacy of the fungicide and cultivar
combination were RAUDPC = 1.7 and 344 Therefore, any fungicide treatment and
cultivar combination with RAUDPC value S 5.1 (not significantly different from
Snowden with a full application rate of fluazinam at a 5-day spray interval) were defined
as effective (E); any combinations with RAUDPC 3 29.6 (not significantly different from
the non-treated Snowden) were defined as non-effective (NE); and any combinations
with RAUDPC values significantly different from both standards were defined as
partially effective (PE).

All cultivars were effectively protected against late blight by full and half rate
applications of fluazinam on S-day spray interval and partially protected at all other rates
and application intervals except Dakota Pearl, which was only effectively protected at
full rate of fluazinam on 5-day spray interval (Figures 9-15). Advanced breeding lines W
13551-1 and W1386 were effectively protected by the fungicide application at both full
and half rate on 5-day spray interval and also at full rate on 10-day Spray interval (Figures

10, 13).

42

 

 

 

 

7////////////////x/////////////////

 

 

 

7////////////////////////////fl

 

 

 

 

7/////////////,///////////////.

 

 

 

 

7///////////////////////A

7///////////////////////2

 

 

4 a a a I
5 0 5 0 5 0 5 O
1

g oaon<m

mm
60
Mom
mW
coo mp.
to an
o .m)
r0 dd
bx mm
0V9 mm
Ls N am
6% n ms
.6 m. am
A“ m my.
0 W e.m2
be .m .mwm
oak hm:
xv d mbs
RV e fdo.
O C 60
e n 0c F
O a S
4.6 v mmw
ROI. M edL
0 I tat
N, 0 r ndn
66% n imm
\ o' d e
44 m mmw.
.0 C e.Vr.
r1
0% Pkm
0‘va awB
c to .Wancm
cos c mama
.0 max
warms
Q0 80.].
V usw
0 860
on {my
.6 88
060 fivm~
o co
S Wm.“
FC.1

43

 

50 i
I Untreated control 50% MRAR D100 MRAR

 

 

 

RAUDPC (%)

 

 

 

 

 

 

 

5days 1 Odays 1 5days

Application interval

Figure 9. Range of RAUDPC values for cultivar Dakota Pearl treated with different rates
of fluazinam in 2002 growing season. Bars represent LSDp=0_05 = 3.42.

44

 

50

 

 

45 J I Untreated control I50°/o MRAR El 100% MRAR
40 ~
35 ~

301

RAUDPC (%)
N
01

 

 

 

 

 

 

5days 10days 1 5days

Application interval

Figure 10. Range of RAUDPC values for W 1355-1 treated with different rates of
fluazinam in 2002 growing season. Bars represent LSDp=005 = 3.42.

45

 

50— l

45 I Untreted control 50% MRAR l:l100% MRAR :

 

40J
354
304
257
20-

RAUDPC (7.)

15p
10-

5‘ '7
OT ////A

5days 10days ’ 15days

 

 

 

 

Application interval

Figure 11. Range of RAUDPC values for cultivar Dakota Rose treated with different
rates of fluazinam in 2002 growing season. Bars represent LSDp=o.05 = 3.42.

46

 

50-

 

I Untreated control 50% MRAR D100% MRAR

 

 

45~

40~

RAUDPC (°/.)
N
01

 

   

 

 

 

 

5days 10days 15days

Application interval

Figure 12. Range of RAUDPC values for MN 19350 treated with different rates of
fluazinam in 2002 growing season. Bars represent LSDp=n_05 = 3.42.

47

 

I Untreated control 50% MRAR D100% MRAR

 

 

 

N 00 oo 4: J:- or
01 o or o 01 o
l l l l I 4

204

RAUDPC (°/o)

A
(TI
I

10-

 

 

 

 

5days 10days 15days

Application interval

Figure 13. Range of RAUDPC values for W 1386 treated with different rates of
fluazinam in 2002 growing season. Bars represent LSDp=o_o5 = 3.42.

48

01
O
1

 

I Untreated control 50% MRAR D100°/o MRAR

k
01
1

 

 

 

RAUDPC (%)
N (A) 00 -§
01 O 01 O

 

 

 

 

 

5days 1 Odays 1 5days

Application interval

Figure 14. Range of RAUDPC values for MN 1915 treated with different rates of
fluazinam in 2002 growing season. Bars represent LSDp=0_05 = 3.42.

49

 

50 —
45 I Untreated control 50% MRAR E1100% MRAR

 

RAUDPC ("/o)

 

 

5days 10days 15days

Application interval

Figure 15. Range of RAUDPC values for cultivar Snowden treated with different rates of
fluazinam in 2002 growing season. Bars represent LSDp=0.05 = 3.42.

50

All cultivars and advanced breeding lines tested in this study ranged from
resistant to susceptible to P. infestans but most were susceptible. Jacqueline Lee and
Torridon were classified as late blight resistant (R) with mean RAUDPC value = 0.2.
Dakota Pearl, W 1355-1, Dakota rose and MN 19350 were classified as moderately
resistant (M). W 1386, MN 1915, MNA 157-4 and cultivar Snowden were classified as
susceptible (S). W 1355-1, W 1386 and cultivar MN 19350 were very responsive to
fungicide applications. 'W 1355-1 was the most responsive of the susceptible cultivars
and advanced breeding lines during the two years of testing.

In 2001, application of fluazinam at 100% MRAR at a 5 or 7-day spray interval
resulted in an effective control in most cultivars and advanced breeding lines except those
from the North Dakota and Minnesota programs. All cultivars and advanced breeding
lines were partially effectively protected at all other rates and application intervals.

In 2002, all cultivars were at 50% and 100% MRAR of fluazinam effectively
protected against late blight on a 5-day spray interval and partially effectively protected

at all other rates and application intervals.

51

Table 6. Summary of efficacy results: fluazinam applied at reduced rates and frequencies on potato
cultivars and advanced breeding lines from North Central US potato breeding programs, MSU 2002.

 

 

 

Cultivar/ABL Application frequency (days)
Rate of fluazinaml
RAUDPC2
5 10 15

Jacqueline Lee 0 0.2 y3 R4

50 0.06 y E5 0.13 y E 0.13 y E

100 0.13 y E 0.06 y E 0.06 y E
Torridon O 0.2 y R

50 0.3 y E 0.2 y E 0.1 y E

100 0.2 y E 0.1 y E 0.1 y E
Dakota Pearl 0 28.0 b M

50 10.2 i-l PE 15.9 d-f PE 14.8 e-g PE

100 3.9 p-w E 17.7 c-e PE 18.9 cd PE
W 1355-1 0 28.4 b M

50 1.8 u-y E 6.1 m-s PE 7.1 l-q PE

100 1.0 w-y E 3.9 q-w E 6.3 m-s PE
Dakota Rose 0 29.1 b M

50 5.1 n-u E 15.5 d-g PE 18 c-e PE

100 2.5 t-y E 10.7 h-k PE 16.2 d-f PE
MN19350 0 29.6 b M ,

50 3.7 r-x E 7.3 l-p PE 10.1 H PE

100 2.9 S-y E 6.6 m-r PE 9.4 j-m PE
W 1386 0 33.3 a S

50 2.4 t-y E 5.4 n-t PE 8.2 k-n PE

100 1.7 v-y E 4.8 o-v E 6.2 m—s PE
MN 1915 0 33.7 a S

50 2.1 t-y E 14.2 fg PE 16.4 d-f PE

100 1.5 v-y E 12.5 g-j PE 14.1 f-h PE
Snowden 0 34.4 a S

50 4.0 p-w E 13.4 f-i PE 20.7 c PE

100 1.7 u-y E 8.0 k-o PE 9.0 k-m PE

 

1: Application rate of fluazinam as percent of manufacturer’s recommended rate.

2: Relative area under the disease progress curve from inoculation to 100% late blight in susceptible control
(Snowden); max = 100.

3: Means followed by the same letter were not significantly different at p = 0.05; comparison between all
combinations of fungicide application rate and frequency of application in all cultivars/ABL.

4: Susceptibility of non-treated control to late blight; R = Resistant, not significantly different from
Jacqueline Lee (non-treated); S = Susceptible, not significantly different from Snowden (non-treated); M=
Moderately resistant, significantly different from both Jacqueline Lee and Snowden (non-treated).

5: Effectiveness of fungicide treatment in comparison to Snowden treated with a full application rate of
fluazinam at a 5-day interval or with non-treated Snowden control; B = RAUDPC not significantly different
from treated Snowden control; PE Significantly different from treated Snowden control and non-treated
control; NE = not significantly different from Snowden non-treated control at p = 0.05.

52

DISCUSSION

Weather conditions were conducive for the development of late blight in both 2001
and 2002 at the Muck Soils Research Farm. The difference between 2001 and 2002 in
terms of accumulation of late blight disease severity values (DSV) was minimal. Late
blight is predicted to occur when inoculum is present, the host is in a susceptible state and
when climatic conditions are favorable for infection to occur; the Wallin model (28) and
the adapted Blightcast model (23) predict that infection will occur after the accumulation
of 18 DSV. The accumulated DSV used in these studies was adapted to a 80% RH
threshold, as recommended by Baker et al. (1). The traditional threshold of 90% RH
resulted in accumulations of 38 and 32 DSV in 2001 and 2002 over the same period,
respectively. Baker et a1. (1) reported that 80% RH measured 1.0 meter above the canopy
was equivalent to 90% RH within a mature potato canopy.

The results of this study were consistent with previous studies (12, 13, 21, 27) and
indicate that a combination of cultivar and advanced breeding line resistance and
managed application of protective fungicides will reduce foliar late blight to acceptable
levels in most situations. However, when environmental conditions were extremely
favorable for the development of late blight, lower application rates (33 and 66% MRAR)
provided unsatisfactory control for some moderately resistant and susceptible cultivars
and advanced breeding lines. In some cultivars and advanced breeding lines, 33% of the
MRAR of the fungicide was sufficient to achieve acceptable control, whereas other
cultivars and advanced breeding lines required 66% MRAR of fluazinam to control late

blight.

53

However, there was rarely a further reduction in disease in any cultivar and advanced
breeding lines when fluazinam was applied at the 100% MRAR of the fungicide.

On late blight susceptible cultivars, applications of fluazinam at either a 10-day
application interval or above were either partially or non- effective for controlling late
blight at any dose tested. In the resistant cultivars Torridon and Jacqueline Lee, the
fungicide did not reduce the RAUDPC in comparison with untreated plots of these
cultivars.

The opportunity to manage late blight by applying reduced rates of fungicides at
increased spray intervals to cultivars less susceptible to late blight was demonstrated in
this study. However, more critical dose response studies will be required before effective
rates of application can be established for new fungicides. In addition, the efficacy of
reduced rates and increased application intervals of fungicides against other potato
pathogens such as early blight has not been established and may prove to be a major
constraint in the adoption of managed fungicide applications.

The application rates and application frequencies of fungicides used in this study
were selected to cover the range of responses likely to be exhibited by the range of
cultivars and advanced breeding lines used over the period of the study. In addition, as
microclimatic conditions at the experimental site were likely to differ over the study
period it was important to have high and low level fungicide input treatments. The study
largely supported the dose rates recommended by the manufacturer but also showed that
less susceptible potato cultivars required lower levels of input for effective late blight

control.

54

As new cultivars with enhanced late blight resistance are developed and released
it will be important to provide growers with recommendations for the most effective and
economical chemical control of late blight in these new cultivars.

In the future, the type of information gathered in this study will be used to
develop models, based on cultivar resistance and response to fungicide application, to
advise and guide growers as to which fungicide, rate and frequency of application is
required to provide protection against late blight. Climatic conditions within the canopy
will also impact choice of fungicide and rate and frequency of application (1). Therefore,
new cultivars will need to be carefully screened in the manner described in this study,

over several seasons in order to develop accurate models for fungicide application.

55

LITERATURE CITED

10.

ll.

12.

. Baker, K.M, Andresen, J.A., Kirk, W.W. and Stein, J.M 2000. Crop disease

mitigation: Daily risk modeling for Michigan potato growers. 4th International
Conference on Integrating GIS and Environmental Modeling (GIS/EM4). Banff,
Alberta, Canada, 2-8 Sept, 2000. http://litllophytengdc.noaa.gov/cgi-
bin/subview3.cgi/. Paper 522.

 

Canada Pest Management Regulatory agency. 2003. Regulatory note REG2003.
Fluazinam. Health Canada, Ontario.

Campell, C. L. 1990. Introduction to plant disease epidemiology. New York, John
Wiley and Sons.

Caten, CE, and Jinks, J.L. 1968. Spontaneous variability of single isolates of
Phytophthora infestans. 1. Cultural variations. Canadian Journal of Botany 46:
329-348.

CIP, 1999. Global Initiative on Late Blight: Annual Report.

Deahl, CL. and Demuth.l995. Identification of mating types and metalaxyl
resistance in North-American pOpulations of Phytophthora infestans. American
Pot. J. 74(2):35-49. ‘

Douches, D.S., Chase R.W., Jastrzebski, K., Long, C., Hammerschmidt, R., Kirk,
W.W., Walters, K., and Coombs, J. 1997. Potato Variety Evaluations. Mich.
Potato Res. Rep.29: 5-32.

Douches, D.S., Chase R.W., Jastrzebski, K., Harnmerschmidt, R., Kirk, W.W.,
Long, C., Walters, K., Coombs, J. and Greyerbiehl, J. 1998. Potato Variety
Evaluations. Mich. Potato Res. Rep.30: 1 1-35.

Douches, D.S., Chase R.W., Jastrzebski, K., Harnmerschmidt, R., Kirk, W.W.,
Long, C., Walters, K.; Coombs, J. and Greyerbiehl, J. 1998. Potato Variety
Evaluations. Mich. Potato Res. Rep.31: 1-45.

Douches, D.S., Coombs, J., Long, C., Zarka, K., Walters, K., and Bisognin, D.
2000. Potato Variety Evaluations. Mich. Potato Res. Rep.32: 5-54.

Erwin, DC, and Ribereiro, OK. 1996. Phytophthora Diseases wordwide. St
Paul, USA: APS press.

Fry, WE. 1977a. Integrated effects of polygenic resistance and protective
fungicide on development of potato late blight. Phytopathology 65: 908-911.

56

13.

14.

15.

16.

17.

18.

19.

20.

21.

22.

23.

Fry, W.E. 1977b. Integrated control of potato late blight - effects of polygenic
resistance and techniques of timing fungicide applications. Phytopathology 67:
415-420.

Fry, W.E., and Goodwin, SB. 1997. Re-emergence of potato and tomato late
blight in the United States. Plant Disease. 81: 1349-1357.

Fry, W.E., Goodwin, S.B., Deyer, A.T., Matuszak, J.M., Drenth, A., Tooley ,
W.P., Sujkowski, L.S., Koh, Y. J., Cohen, B.A., Spiehnan, L.J., Deahl, K.L.,
Inglis, D.A. and Sandlan, KR 1993. Historical and recent migrations of
Phytophthora infestans. Chronology, Pathways, and implications. Plant Disease
77:653-661.

Goodwin, S.B., Schneider, RE. and Fry, W.E. 1995. Use of cellulose-acetate
electrophoresis for rapid identification of allozyme genotypes of Phytophthora
infestans. Plant Disease. 79:1 181—1 185.

Goodwin, S.B., Sujkowski, L.S., Fry, W.E., 1996. Widespread distribution and
probable origin of resistance to metalaxyl in clonal genotypes of Phytophthora
infestans in the United States and Western Canada. Phytopathology 86: 793-800.

Gray, J .I., and Whalon, ME. 1996. The assessment of pesticides at risk in the
future of Michigan food and feed plant agriculture: A process. East Lansing,
Michigan Agricultural Experiment Station. 15 pp.

Guenthner, J .F., Michael, KC. and Nolte P. 2001. The economic impact of potato
late blight on US growers. Potato Research 44: 121-125.

Kirk, W.W., Douches, D.S., Hammerschmidt, R., Stein, J .M., Felcher K.J., Baker
K.M., and Niemira, BA. 2000. Combining varietal resistance with managed
fungicide applications for the control of potato late blight. Afiican Potato
Association Conference Procedings.5: 329-335.

Kirk, W.W., Felcher, K.J., Douches D.S., Coombs, J ., Stein, J .M. Baker, K.M.,
and Hammerschmidt, R. 2001. Effect of host plant resistance and reduced rates
and frequencies of fungicide application to control potato late blight. Plant.
Disease. 85: 1113-1118.

Lambert, DH. and Currier, AL 1997. Differences in tuber rot development for
North American clones of Phytophthora infestans. American Potato Journal 74,
39-43.

MacKenzie, DR, 1981. Scheduling firngicide applications for potato late blight
with BLIGHTCAST. Plant Disease, 65 (5): 394-399.

57

24.

25.

26.

27.

28.

29.

Madden L.V. and Hughes, G. 1995. Plant disease incidence: distributions,
heterogeneity, and temporal analysis. Annual Review of Phytopathology 33:529-
564.

Syngenta web Site. 2003: wwwSyngentacropprotection-us.com

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Environmental documents. Volume 67, number 75.

Van Der Plank, J.E. 1968. Disease resistance in plants. New York Academic
press.

Wallin, J .R. 1953. The production and survival of sporangia of Phytophthora
infestans on tomato and potato plants in the field. Phytopathology 43:505-508.

Zeneca UK Website. 2001. http:/’/www.zencca-crop.co.uk/.

58

CHAPTER THREE

IMPACT OF PLANT AGE AND LEAF POSITION ON SUSCEPTIBILITY TO LATE
BLIGHT

INTRODUCTION

Breeding for late blight resistance in potatoes was initiated then intensified in
Europe and North America following serious epidemics that occurred in 1845-1847 (1,
12). Initially breeding for resistance was focused on horizontal resistance (12). Later
when race-specific resistance in the wild potato Solanum demissum was detected,
breeders turned their efforts to breed for vertical resistance by transferring major R-genes
from S. demissum into the domesticated potato (S. tuberosum); (12, 22). Since then,
vertical resistance was identified as being more effective than horizontal resistance,
resulting in very rapid selection and production of varieties reSistant to potato late blight.
A broad spectrum of acceptable qualities fi'om different Solanum spp. are necessary when
selecting for horizontal resistance in order to incorporate as many traits as possible from
different Species to achieve durable resistance to late blight in potatoes. Thus, this kind of
resistance becomes difficult to handle, labor intensive and time consuming.

Breeding for vertical resistance in potato plants resulted in the production of many
resistant cultivars until the late 1960’s when P. infestans overcame vertical resistance
(22). These varieties were no longer resistant and vertical resistance proved unreliable.

Currently, efforts are being redirected towards breeding for horizontal resistance.

59

Breeding for horizontal resistance has already resulted in the development of
some cultivars with intermediate resistance, known as partial resistance. However, fully
resistant varieties that can provide adequate protection without using fungicides have not
yet been achieved (23).

Results from some studies on factors related to resistance of potato foliage to late
blight such as plant age and leaf position have been published (2, 3, 4, 9, 10, 11, 13, 14,
15, 16, 17, 18, 19, 20, 21). Horizontal resistance to late blight is often observed in late
maturing cultivars rather than in early maturing ones (22). This has resulted in efforts to
search for factors affecting differences among these cultivars in terms of resistance.
Horizontal resistance is under polygenic control and expression of resistance is
quantitative resulting from an additive contribution of several genes. Different genes may
be expressed during the potato season. This was achieved in late maturing cultivars and
made early season screening difficult for horizontal resistance (22).

Results from some studies (3, 4, 18, 21) on the change in resistance to P. infestans
in potatoes allow some general conclusions to be drawn on late blight resistance in potato
foliage. In susceptible cultivars young plants are highly susceptible to infection; this
susceptibility decreases in resistant cultivars prior to flowering and may increase during
senescence (3, 18). At senescence, resistant cultivars become more resistant whereas
some other susceptible cultivars become more susceptible at equivalent maturities (3).
Apical leaves have been reported to be more resistant to potato late blight than the basal
leaves (3, 4, 21). The objective of this study was to determine the impact of plant age and
leaf position on susceptibility to late blight in some susceptible and resistant potato

cultivars.

60

MATERIALS AND METHODS

EXPERIMENTAL DESIGN AND PLANT MATERIAL

Three experiments, two in 2002 and one in 2003 were conducted. Three cultivars
were tested in each experiment except in one, experiment in 2003 where only two
cultivars were used. All cultivars tested were, in general, susceptible to potato late blight
except the cultivar Jacqueline Lee, which was previously classified as resistant (5, 6, 7,
8). In each of the three experiments, leaf positions 5, 10 and 15, counted from the base of
the main stem of the potato plant above the soil, were randomly harvested and tested for

susceptibility to P. infestans.

Experiment 1

Twenty potato tubers per cultivar of Russet Norkotah, Dakota Pearl and
Jacqueline Lee were grown at random in three field plots at MSU plant pathology farm
and used in experiment 1. Leaves to be inoculated with P. infestans were harvested from
each cultivar at plant age of 24, 39, 46, 59 and 78 days after emergence during this
experiment. Soil preparation, fertilizer application, insects and weed control were as

described above in chapter two.

61

Experiment 2

Cultivars F L1879, Dakota Rose and Jacqueline Lee were grown in a growth
chamber and were used to test for resistance to late blight. Ten potato plants per cultivar
were planted into individual pots (one tuber per pot) filled with potting soil. A total of 30

pots containing 30 potato tubers were placed in a climatic grth chamber.

The relative humidity was 80% in the climatic chamber and the illumination was
provided by 500 W Philips-HPIT lamps. Potato plants were grown under a constant
temperature of 24°C and a photoperiod of 16 hours day length. Leaves were harvested at
plant ages of 20, 33, 46, 60 and 72 days after emergence and inoculated with P. infestans

(described below).

Experiment 3

In the third experiment cultivars Snowden and Jacqueline Lee were used. In order
to minimize variation due to inoculation procedures, tubers were planted at intervals. The
experiment was conducted in similar conditions as in experiment 2. But three grth
chambers each containing ten pots per cultivar were used in this experiment at each of
three different planting times. Leaves to be inoculated with P. infestans were harvested
from the same position at plant age of 20, 32, 46, 60 and 74 days after planting. Leaves

harvested from the same position differed in age by seven days.

62

PATHOGEN PREPARATION AND INOCULATION

Inoculum was prepared from isolate Pi 95-7 of P. infestans (US-8 genotype; A2
mating type obtained from Dr William W. Kirk’s collection at MSU) and produced on
rye agar as described in Chapter Two. Sporangia were harvested from agar plates on the
day of inoculation. The suspension of sporangia was adjusted to a final concentration of 5
X 104 sporangia ml"1 and chilled at 4°C for 4 hours prior to inoculation to enhance the

release of zoospores.

Prior to inoculation, leaf discs were cut with a cork borer (12mm) from the
primary potato leaflet on each Side of the main vein, about 1 cm from the point of
attachment to the petiole. Five leaf discs were placed on rye B agar amended with
antibiotics (rifamycin 75 mg. 1", ampicillin 20mg. 1'1 and nystatin 75 mg. 1'1 dissolved in
1.0 m1 DMSO) to reduce contamination with bacteria.

A 50ul droplet of the sporangia/zoospore suspension was applied to each of five
leaf discs per plate. A total of 10 leaf discs, per cultivar per leaf position per plant age
were inoculated. Inoculated leaf discs were incubated at 21°C light/ 18 °C dark (12 hours
cycle) and were examined with a dissecting microscope 96 hours post-inoculation.
Symptoms and signs of infection by P. infestans, such as sporulation were noted.

Pathogen identity was confirmed with a compound microscope.

63

DISEASE EVALUATION AND DATA ANALYSIS

Late blight infection was estimated on the fifth day after inoculation by counting
Sporangia harvested from each leaf disc with late blight lesions. The leaf discs were
placed into a 1.5 ml micro-centrifuge tube with 1.0 ml of sterile de-ionized H20 (dinO)
and agitated to dislodge the sporangia. Samples were taken from each sporangium
suspension to count the final number of sporangia using a hemacytometer for a total of 6
counts per leaf disc. The number of sporangia produced per leaf disc per cultivar was
used to estimate susceptibility or resistance in different cultivars. Any plant age per leaf
position per cultivar treatment in which the average number of sporangia produced per
leaf disc was significantly different at p = 0.05 from that of the overall mean number of
sporangia produced per leaf disc for the resistant cultivar Jacqueline Lee was classified as
susceptible (S) to blight infection and resistant (R) to infectiOn when the mean number of
sporangia per leaf disc was not significantly different from that of the cultivar Jacqueline
Lee.

Data were analyzed by ANOVA and all pair-wise comparison calculated using
Fisher’s least significant differences (LSD) at or=0.05 (SAS-Institute 2003. Statistical

Analysis Software v.8.2, Cary, NC.).

64

RESULTS

EXPERIMENT 1

The average number of sporangia produced per inoculated leaf disc in the cultivar
Jacqueline Lee was 60. Any plant age or leaf position on a cultivar that resulted in an
average number of sporangia of P. infestans produced per leaf disc 5 60 (not significantly
different from that of the cultivar Jacqueline Lee) was defined as resistant to infection,
susceptible when the average number of Sporangia produced per leaf disc was > 60
(significantly different fiom that of the cultivar Jacqueline Lee). The average number of
sporangia of P. infestans produced per leaf disc for all cultivars tested was significantly
different from that of the cultivar Jacqueline Lee. The mean numbers of sporangia
produced per leaf disc for the susceptible cultivars Russet Norkotah and Dakota Pearl
were not significantly different from each other. The average number of sporangia
produced per leaf disc for the resistant cultivar Jacqueline Lee was significantly different

from both Russet Norkotah and Dakota Pearl (Table 7).

The analysis of variance indicated a significant effect of plant age on the average
number of sporangia of P. infestans produced per leaf disc (P<0.0001) at or=0.05 for all
cultivars. The effect of leaf position on the number of Sporangia of P. infestans produced

was also significant for all cultivars tested.

65

The numbers of sporangia of P infestans produced per leaf disc for the resistant
cultivar Jacqueline Lee were significantly different at plant ages of 22 and 39 days after
emergence (Table 7) where this cultivar was classified as susceptible to late blight
infection. Mean number of sporangia produced per leaf disc was not significantly
different at all leaf positions tested for this cultivar (Table 7). All leaf positions tested for
this cultivar were resistant to late blight infection, but resistance was not significantly

different from each other among these leaf positions (Table 7).

For the susceptible cultivar Russet Norkotah, the average numbers of sporangia of
P. infestans produced per leaf disc at all plant ages tested were significantly different
from that of the cultivar Jacqueline Lee except at the ages of 39 post-planting where the
mean number of sporangia of P. infestans produced was not significantly different from
that of the cultivar Jacqueline Lee (Table 7). The average numbers of sporangia produced
per leaf disc were also significantly different from that of the cultivar Jacqueline Lee at
all leaf positions tested for this cultivar (Table 7). All leaf positions tested for this cultivar
were susceptible to late blight infection, but susceptibility was significantly higher in leaf

position 5.

The numbers of sporangia of P. infestans produced per leaf disc for the cultivar
Dakota Pearl were significantly different from that of the cultivar Jacqueline Lee at all
plant ages tested except at age 24 post-emergence where the mean number of sporangia
of P. infestans was not significantly different from the number harvested from the
cultivar Jacqueline Lee (Table 7). Dakota Pearl was defined as resistant at plant age of 24

days after emergence and susceptible at all other plant ages tested.

66

Mean numbers of sporangia of P infestans produced per leaf disc were significantly
different from that of the cultivar Jacqueline Lee at all leaf positions tested for this
cultivar (Table 7). Dakota Pearl was also susceptible to infection at all leaf positions, but

susceptibility was Significantly higher in leaf positions 5 and 10 for this cultivar.

Experiment 2

The average number of sporangia produced per inoculated leaf disc in the cultivar
Jacqueline Lee was 70. Any plant age or leaf position on a cultivar that resulted in a
number of sporangia of P. infestans produced per leaf disc s 70 (not significantly
different from that of the cultivar Jacqueline Lee) was defined as resistant to blight
infection, susceptible when the mean number of sporangia produced per leaf disc was >
70 (Significantly different from that of the cultivar Jacqueline Lee). The average number
of sporangia of P. infestans produced per leaf disc for all cultivars tested was
significantly different from that of Jacqueline Lee. The average numbers of sporangia
produced per leaf disc for the susceptible cultivars FL1879 and Dakota Rose were not
significantly different from each other. The average number of sporangia produced per
leaf disc for the resistant cultivar Jacqueline Lee was significantly different from both FL

1879 and Dakota Rose (Table 7).

67

The analysis of variance showed a significant effect of plant age on the numbers
of Sporangia of P. infestans produced (P<0.0001) at or=0.05 for all cultivars. There was
no Significant effect of leaf position on susceptibility to blight infection in all cultivars
tested in experiment 2. For the susceptible cultivar FL1879, the average numbers of
sporangia produced per leaf disc were Significantly different from that of the cultivar
Jacqueline Lee at plant ages of 20 and 60 days after emergence. Mean numbers of
Sporangia produced per leaf disc were not significantly different from that of the cultivar
Jacqueline Lee at plant ages of 33, 46 and 72 days post-emergence (Table 7). FL1879
was classified as resistant to infection at plant ages of 33, 46 and 72 days and susceptible

at plant ages of 20 and 60 days after emergence

The average numbers of Sporangia produced per leaf disc for the other susceptible
cultivar Dakota Rose were significantly different from that of the cultivar Jacqueline Lee
at all plant ages tested except at plant age of 46 after emergence where the average
number of Sporangia produced was not significantly different from that of the cultivar
Jacqueline Lee (Table 7). Cultivar Dakota Rose was defined as susceptible to infection at

plant ages of 20, 33, 60 and 72 days and resistant at plant age of 46 days post-emergence.

For the resistant cultivar Jacqueline Lee, the average numbers of sporangia
produced per leaf disc were significantly different at plant ages of 20 and 33 days after
emergence (Table 7) where Jacqueline Lee was defined as susceptible to late blight

infection.

68

Experiment 3

The average number of sporangia produced per inoculated leaf disc in the cultivar
Jacqueline Lee was 59. Any plant age or leaf position on a cultivar that resulted in a
number of sporangia of P. infestans produced per leaf disc 5 59 (not significantly
different from that of the cultivar Jacqueline Lee) was defined as resistant to infection.
And treatments that resulted in the numbers of sporangia produced per leaf disc > 59
(Significantly different from that of the cultivar Jacqueline Lee) were defined as
susceptible to blight infection. The mean number of sporangia for the susceptible cultivar

Snowden was Significantly different from that of the cultivar Jacqueline Lee (Table 7).

The analysis of variance indicated a significant effect of plant age on the number
of sporangia of P. infestans produced per leaf disc at or=0.05. The effect of leaf position

on the number of sporangia of P. infestans produced was also significant.

For the susceptible cultivar Snowden, the average numbers of sporangia produced
per leaf disc were significantly different from that of the cultivar Jacqueline Lee at all
plant ages tested (Table 7). Mean numbers of sporangia produced were also significantly
different from that of the cultivar Jacqueline Lee for all leaf positions tested for this
cultivar (Table 7). All leaf positions tested for cultivar Snowden were susceptible to late
blight infection, but susceptibility was not significantly different from each other among

these leaf positions.

69

The average number of sporangia of P. infestans produced per leaf disc for the
resistant cultivar Jacqueline Lee was significantly different at plant ages of 20 and 32
days post-emergence (Table 7) where Jacqueline Lee was classified as susceptible to
blight infection. The average numbers of Sporangia of P. infestans produced per leaf disc

were significantly different at leaf position 5 (Table 7) where cultivar Jacqueline Lee was

classified as susceptible to infection.

70

 

Table 7. Summary of susceptibility results: average number of‘sporangia of P. infestans produced per leaf
disc per plant age per cultivar (all leaves together) and per leaf position (all plant ages together) for the six
cultivars tested in the three experiments

 

 

Plant age Number of sporangia per leaf disc

Experiment 1 Russet Dakota Jacqueline FL 1879 Dakota Snowden

Plant age Norkotah Pearl Lee Rose
- 24 days 220ab sc 40c R 110a s - - -
- 39 days 40b R 70bc S 80ab S - - -
- 46 days 190a S 250a S 50bc R - - -
- 59 days 250a 8 150b S 20c R - - -
- 78 days 120ab S 70bc S 20c R - - -
- LSDaLQOSa 140 90 50 -

Leaf position
- Leaf 5 250a S 190a S 603 R - - -
- LeaflO 13% S 150a S 503 R - - -
- Leaf15 110b S 90b S 50a R - - —
- LSDa=005 9O 60 30 - - -

Average for the cultivar 160 S 140 S 60 R - - -
Experiment 2

 

 

Plant age

- 20 days - - 250a S 130a S 310b S -

- 33 days - - 90b S 60b R 80bc S -

- 46 days - - 40b R 60b R 20c R -

- 60 days - - 40b R 190a S 990a S -

- 72 days - - 20 R 60b R 280bc S -

- LSDa=0,05 - - 80 80 250 -
Avege for the cultivar - - 70 R 90b S 310a S -
Experiment 3
Plant age

- 20 days - - 118 a S - - 520a S

- 32 days - - 86 ab S - - 210bc S

- 46 days - - 42 bc R - - 200bc S

- 60 days - - 38 c R - - 360ab S

- 74 days - - 39 c R - - l40c S

- LSDa=005 45 190
Leaf position

- Leaf 5 - - 72 a S - - 220a S

- Leaf 10 - - 56 a R - - 270a S

- Leaf 15 - - 48 a R - - 300a S

- 1,3130l =0.“ - - 31 - - 130
Average for the cultivar - - 59 R - - 268 S

 

a: LSD: least significant difference

b: Means followed by the same letter within a column in each experiment are not significantly different

c: S: Susceptible to infection, significantly different from the cultivar Jacqueline Lee; R = Resistant, not
significantly different from the cultivar Jacqueline Lee.

71

DISCUSSION

The effects of plant age and leaf position on susceptibility to potato late blight
infection were investigated in three different experiments conducted both in the field and
controlled environment using six different potato cultivars with a range on susceptibility
to late blight. The results of this study supported previous reports (5, 6, 7, 8) that
Jacqueline Lee is resistant to potato late blight.

Susceptibility to infection due to plant age in susceptible cultivars was variable
depending on cultivar tested. Resistant cultivar Jacqueline Lee was susceptible at young
plant ages in all experiments.

All leaf positions tested were susceptible to infection for all susceptible cultivars
in all experiments and resistant for the resistant cultivar Jacqueline Lee. Leaf position has
effect on susceptibility to infection for all cultivars tested in all experiments and leaf
position 5 was the most susceptible. V

Results from this research demonstrated the increase of resistance to blight
infection from leaf position 5 to leaf position 15 and supported those from previous
studies (2, 3, 11, 14, 16, 21) that there is gradual increase to blight susceptibility due to P.
infestans from apical leaves to basal ones and confirmed that apical leaves are the most
resistant to potato late blight infection.

Resistant cultivar Jacqueline Lee was susceptible for Short periods during the
early stages of leaf development and then became resistant at later stages. Plant age has
less effect on susceptible cultivars tested, which were susceptible at any stage of leaf

development.

72

This was in accordance with Carnegie and Calhoun (3) that young plants are very
susceptible to infection in the resistant cultivars, whereas susceptible cultivars are less
affected by plant age.

Plant age and leaf position were important factors that were affecting
susceptibility to blight infection in all cultivars tested but leaf position was the most
important factor during this study. I would therefore suggest breeders to consider both

plant age and leaf position in different tests and evaluations for resistance to potato late

blight.

73

LITERATURE CITED

10.

ll.

Bradshaw, J .E., Wastie, R.L., Stewart HE, and Mackay GR. 1995. Breeding for
resistance to late blight in Scotland. In Phytophthora infestans 150, edited by
Dowley, L.J., Bannon, E., Cooke, L.R., Keane, T., and O’Sullivan, Dublin
Ireland: Boole Press Ltd.

. Carnegie, SF, and Colhoun, J. 1980. Differential leaf susceptibility to

Phytophthora infestans on Potato plants of cv. King Edward, Phytopathol. Z. 98:
108-1 17.

Carnegie, SF, and Colhoun, J. 1982. Susceptibility of potato leaves to
Phytophthora infestans in relation to plant age and leaf position. Phytopathol. Z.
104: 157-167.

Dorrance, AB, and Inglis, D.A. 1997. Assessment of greenhouse and laboratory
screening methods for evaluating potato foliage for resistance to late blight. Plant
Dis. 81: 1206-1213.

Douches, D.S.; Chase R.W., J astrzebski, K., Long, C., Hammerschmidt, R., Kirk,
W.W., Walters, K., and Coombs, J. 1997. Potato Variety Evaluations. Mich.
Potato Res. Rep.29: 5-32.

Douches, D.S., Chase R.W., Jastrzebski, K., Hammerschmidt, R.; Kirk, W.W.,
Long, C., Walters, K., Coombs, J. and Greyerbiehl, J. 1998. Potato Variety
Evaluations. Mich. Potato Res. Rep.30: 11-35.

Douches, D.S., Chase R.W., Jastrzebski, K., Hammerschmidt, R., Kirk, W.W.,
Long, C., Walters, K.; Coombs, J. and Greyerbiehl, J. 1998. Potato Variety
Evaluations. Mich. Potato Res. Rep.31: 1-45.

Douches, D.S., Coombs, J ., Long, 0, Zarka, K., Walters, K., and Bisognin, D.
2000. Potato Variety Evaluations. Mich. Potato Res. Rep.32: 5-54.

Fry, W.E., and Apple, A.E.1986. Disease management implications of age-related
changes in susceptibility of potato foliage to Phytophthora infestans. Am. Potato
J. 63: 47-56.

Grainger, J. 1956. Host nutrition and attack by fungal parasite. Phytopathology
46: 445-456.

Hodgson, WA. 1961. Laboratory testing of the potato for partial resistance to
Phytophthora infestans. Am. Potato J. 38: 259-264.

74

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Landeo, J.A., Gastelo, M., Pinedo, H., and Flores F. 1995. Breeding for
Horizontal resistance to late blight in potato free of R genes. In Phytophthora
infestans 150 edited by Dowley, L.J., Bannon, E., Cooke, L.R., Keane, T., and
O’Sullivan, E. EAPR: Boole Press LtD.

Lapwood, D. H. 1961. Potato Haulm resistance to Phytophthora infestans II.
Lesion production and sporulation. Ann. Appl. Biol., 49, 316-330.

Lowings, RH, and Acha, LG. 1959. Some factors affecting growth of leaves.
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Malcolmson, J. F. 1969. Factors involved in resistance to blight (Phytophthora
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Mooi, J.C. 1965. Experiments on testing field resistance to Phytophthora
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Pflanzenkrankheiten und Pflanzenschutz. Journal of Plant Diseases and
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Stewart, HE. 1990. Effect of plant age and inoculum concentration on expression
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Stewart, H.E., Taylor, K., and Wastie, R. L. 1983. Resistance to late blight in
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75

 

CHAPTER FOUR
CONCLUSIONS AND SUMMARY

Plants need to be protected against diseases in order to reduce or avoid serious
epidemics, which can lead to severe farnines such as the Irish potato famine that occurred
in the 1840’s. The aim of controlling plant diseases is to reduce food losses and at the
same time to improve food quality (1).

Current strategies to protect potatoes against potato late blight disease include the
use disease free seed, growing resistant varieties, inoculum-suppressing cultural
practices, crop scouting, weather-based disease modeling and chemical control programs
(7).

Breeding for resistance has resulted in development of resistant varieties but high
levels of stable resistance have not been fully successful (2). Even resistant cultivars are
susceptible when immature and need to be protected with fungicide applications
especially at early stages of crop and canopy development to reduce the potential risk of
an epidemic to occur when inoculum becomes present.

In temperate regions such as Michigan, potatoes may be exposed to late blight
inoculum produced on infected volunteer potato plants and discarded piles of culled
potatoes or the disease may originate from infected seed tubers. Favorable conditions for

disease development will also favor disease spread throughout the canopy.

76

In warm regions such as the tropical highlands of Rwanda, inoculum is almost
continuously present due to continuous cropping and conditions for late blight occurrence
and spread. In most potato growing areas in Rwanda, average annual precipitation ranges
from 1200 mm to over 1600 mm. In general, rainfall is bimodal with a minor peak
occurring in October and a major peak in March/April. High elevations and low latitudes
combine to form an isothermal temperature regime with an average annual temperature of
about 16°C (3, 4). Moreover farmers do not have the financial capabilities to spray
fungicides regularly and they do not have enough knowledge either to manage the disease
well. In addition they use low quality seed due to inadequate seed selection and storage
from their harvested crop (farmers generally replant seed from their own fields).

Cultural management of the potato crop includes removal of cull piles and
volunteer potatoes to minimize the source of inoculum of P. infestans, irrigation
management and hilling of rows to reduce tuber contact with free water, planting and
harvesting time to reduce tuber damage and desiccation of potato foliage prior to harvest
to eliminate foliar-bome inoculum (7).

The basis of this thesis was to demonstrate the potential for using managed
fungicide application in combination with varieties ranging from marginal to full
tolerance of potato late blight. The reason for using potato varieties with a range of
tolerance to potato late blight was to determine if different amounts and frequencies of
application of contact/protectant fungicide are required for control of late blight in
individual varieties under late blight conducive conditions. Overall, the use of resistant
varieties would potentially reduce losses due to late blight and reduce the cost of crop

protection in potato production.

77

However, it could be argued that the major benefit would be the removal of a negative
impact on the environment and consequent positive impact on the safety and quality of a
globally important food source. Moreover, plant age has proved to be an important factor
when testing and evaluating potato late blight resistance.

The use of the modern fungicide, fluazinam in combination with cultivar
resistance was evaluated to be incorporated later into an integrated potato late blight
control program both in Michigan and Rwanda. Potatoes play an important role in the
Rwandan economy as a cash crop [estimates of Rwandan marketed potato production
range between 35% and 50% (6)] and the diet of Rwandan people. The country is able to
increase potato production especially in the northern parts (highland areas) of Rwanda if
late blight, the major constraint to crop production is better controlled. There is also a
potential potato market in the neighboring countries in the Democratic Republic of
Congo, Burundi, Uganda and Kenya that can be developed so that the grower can
generate more income and the country earn more foreign currencies (5).

The problem of potato late blight in Rwanda is associated with serious crop and
yield losses, costly fungicides for the Rwandan farmer with limited resources and few
resistant varieties (4). Thus the approach developed in this research would be applied in
Rwanda to reduce the cost of production and losses due to potato late blight. The
fungicide trials described here will be applied under the conditions of Rwanda on some
late blight tolerant/resistant varieties with improved potato yield that are available in

Rwanda.

78

The information gathered based on cultivar resistance and response to fungicide
application, will be used to advise and guide Rwandan growers as to the rate and
frequency of the fungicide application required to provide better protection against late
blight. Rwandan farmers with limited resources will be able to reduce fungicides input on
potato production, increase yield, generate more income and improve their life in a safer

environment.

79

LITERATURE CITED

1. Agrios, G.N. 1997. Plant Pathology. Forth Edition. San Diego: Harcourt Brace
and Company.

2. Douches, D.S., Chase R.W., Jastrzebski, K., Long, C., Hammerschmidt, R., Kirk,
W.W., Walters, K., and Coombs, J. 1997. Potato Variety Evaluations. Mich.
Potato Res. Rep.29: 5-32.

3. Durr, G. 1983. Potato Production and Utilization in Rwanda. Intemational Potato
Center. Lima, Peru.

4. Institut des Sciences Agronomiques du Rwanda (ISAR) 1983. Rapport Annuel.

5. Okoboi, G. 2001. The marketing potential of potatoes in Uganda and market
opportunities for Rwanda. International Institute of tropical Agriculture, Draft
Report.

6. Scott, G. 1988. Potatoes in Central Africa: A Study of Burundi, Rwanda, and
Zaire. International Potato Center. Lima, Peru.

7. Secor, G.A., and Gudmestad, NC. 1999. Managing fungal diseases of potato.
Can. J. Plant. Pathol. 21 no. 3: 213- 221.

80

   

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