Fungicide resistance in cucurbit downy mildew – methodological, biological and population aspects

RESEARCH ARTICLE

Fungicide resistance in cucurbit downymildew – methodological, biological and population aspectsJ. Urban & A. Lebeda

Department of Botany, Faculty of Science, Palacky University in Olomouc, Olomouc-Holice, Czech Republic

Keywords

Biochemical aspects; cucurbits; fungicide

resistance/tolerance; metalaxyl; methodology;

mode of action; fosetyl-Al; Pseudoper-

onospora cubensis; spatial and temporal

changes; strobilurin.

Correspondence

A. Lebeda, Department of Botany, Faculty of

Science, Palacky University in Olomouc,

Slechtitelu 11, CZ-783 71 Olomouc-Holice,

Czech Republic

Email: [email protected]

Received: 18 February 2006; revised version

accepted: 16 May 2006.

doi:10.1111/j.1744-7348.2006.00070.x

Abstract

Cucurbit downy mildew, caused by Pseudoperonospora cubensis, is among the

most devastating diseases of cucurbitaceous plants. In spite of improved

cultural practices and breeding for resistant cultivars, chemical control is still

a very important tool to manage the disease. During the last several decades,

many fungicides from various chemical classes have been developed. The

occurrence of strains of P. cubensis resistant/tolerant to some fungicides

encouraged research of this phenomenon. The first part of this article summa-

rises the many different methodological approaches such as field trials, in vitro

testing on active plant tissues or molecular diagnoses developed for the detec-

tion of resistant/tolerant strains of P. cubensis, as well as methods to collect and

maintain pathogen isolates. The second part outlines the commonly used fun-

gicides to control P. cubensis and their features like systemicity, biological and

biochemical mode of action and translocation behaviour within plants. The

last part deals with geographical aspects such as first appearance of resistance

problems, distribution of resistance, temporal development of resistance

under selection pressure by a fungicide, fitness of resistant subpopulations in

competition with sensitive ones in the absence of a fungicide, as well as

genetic and molecular sources of resistance.

Introduction

The use of fungicides has been the preferred method for

control of plant pathogens (De Waard et al., 1993; Knight

et al., 1997), including Oomycetes (Cohen & Coffey,

1986; Gisi, 2002), despite many attempts to improve crop

resistance and to identify promising natural antagonists of

the downy mildews for their biological control (Lebeda &

Schwinn, 1994). The discovery and introduction of sys-

temic fungicides with specific activity against Oomycete

plant pathogens improved considerably the control of

foliar downy mildews (Schwinn & Staub, 1995).

Cucurbit downy mildew, caused by Pseudoperonospora

cubensis (Berk. et Curt.) Rost., is one of the most impor-

tant diseases of cucurbitaceous crops. This disease has

a worldwide distribution and probably occurs wherever

cucurbits are grown (Holmes et al., 2004). There are

more than 50 cucurbit species known as hosts of

P. cubensis. The most important crop hosts are considered

Cucumis sativus, Cucumis melo, Cucurbita pepo, Cucurbita max-

ima, Citrullus lanatus, Benincasa hispida, Luffa cylindrica and

Lagenaria siceraria (Lebeda & Widrlechner, 2003). The

fungus is highly variable in its pathogenicity (Lebeda &

Widrlechner, 2003, 2004), and disease control by growing

resistant cultivars of cucumber (C. sativus) has not, as yet,

been effective (Lebeda, 1992, 1999; Lebeda & Prasil, 1994;

Lebeda & Widrlechner, 2003; Lebeda & Urban, 2004).

On the contrary, effective sources of resistance based on

race specificity are known in C. melo and Cucurbita spp.

(Lebeda, 1991; Lebeda & Krıstkova, 1993, 2000; Lebeda &

Widrlechner, 2004). However, according to the terminol-

ogy of McDonald & Linde (2002), P. cubensis belongs to

the group of ‘the highest risk pathogens’ with high evolu-

tionary potential (Lebeda & Urban, 2004; Urban & Lebeda,

2004a,b), and thus an employment of resistance genes

has to be combined with other practices of an integrated

management system to minimise the high risk of over-

coming such genes by the pathogen.

Annals of Applied Biology ISSN 0003-4746

Ann Appl Biol 149 (2006) 63–75 ª 2006 The AuthorsJournal compilation ª 2006 Association of Applied Biologists

63

In this situation, the rational use of fungicides is a first

step towards integrated control. According to Gisi (2002),

the sales value of fungicides against downy mildews

amounted to 1.2 billion SFr in 1996, of which 10% were

used to fight mainly P. cubensis on cucurbit crops. For

many decades, contact fungicides such as copper for-

mulations were the only fungicides available for the

control of P. cubensis. During the last several decades,

new and mostly systemic fungicides have been devel-

oped and widely accepted for disease control (for exam-

ple, fosetyl-Al in 1977, phenylamides 1977–1983,

propamocarb 1978, dimethomorph 1988, cyazofamid

2001). Systemic fungicides have a specific, single-site

mode of action, which means they are active at one

point in one metabolic pathway of the pathogen. There-

fore, the risk is high that resistance to them will develop.

There are several reports (Samoucha & Cohen, 1985;

O’Brien & Weinert, 1995; Ishii et al., 2002) about

appearance and increase of subpopulations resistant to

single-site inhibitors shortly after their market introduc-

tion and continuous, exclusive use under high disease

pressure. On the other hand, contact fungicides are par-

ticular multisite inhibitors and, therefore, the risk that

resistance to them will develop is far lower than with

systemic fungicides. For this reason, an integrated dis-

ease management programme should involve the use of

a diversity of fungicides, more disease-resistant cultivars,

together with weather forecasts, epidemiological studies,

disease monitoring, computer simulations etc. All these

combined and coordinated will lead to a more precise

prediction of critical periods and, consequently,

a reduced number of fungicide applications (Lebeda &

Schwinn, 1994). In the case of powdery mildew of

cucurbits (Podosphaera xanthii), two types of resistance of

the pathogen to fungicides were reported: ‘qualitative’

and ‘quantitative’ (McGrath, 2001). Qualitative resis-

tance results from modification of a single major gene.

Pathogens are then either resistant or sensitive, and

resistance is shown as a complete failure of a fungicide.

If resistance resulted from the modification of several

interacting genes, it is referred to as quantitative resis-

tance, and pathogens exhibit a range in sensitivity.

Effective control of the pathogen population can be

achieved by higher rates of a fungicide or by more fre-

quent applications. However, other mechanisms causing

range in sensitivity levels, such as mutations at different

codons in one gene or different substitutions at the

same codon, have been reported (Albertini et al., 1999;

Kim et al., 2003; Ma et al., 2003).

The aim of this study is to summarise the recent knowl-

edge about fungicide resistance in P. cubensis, to show

methodological approaches that have been used for

research of this phenomenon, summarise the types of

efficient fungicides and outline the geographical and

temporal aspects of this phenomenon.

Methodological approaches for research of

resistance to fungicides in Pseudoperonospora

cubensis

For research of resistance/tolerance of P. cubensis to fun-

gicides and for the detection of resistant strains in popu-

lations of the fungus, it is possible to employ different

methodological approaches. These can be grouped into

either field or laboratory experiments. In view of the

highly complex interactions between an obligate parasite

and its host (Lebeda & Schwinn, 1994), it seems to be

preferable to use different organs or plant tissues (e.g.

leaf discs, cotyledons, leaves or whole plants) rather

than various culture media in laboratory experiments.

The properties of a fungicide under study, such as sys-

temicity, translocation movement within plants or mode

of action in the life cycle of the pathogen, also determine

or influence the method appropriate for its application.

In the following part of this review, the basic methods

for research into these features are summarised.

Sampling and establishment of

Pseudoperonospora cubensis cultures

Details of the methodological aspects of this step were

described in our previous studies (Lebeda, 1986; Lebeda &

Widrlechner, 2003). The samples of infected leaves are

put on wet filter paper in plastic containers, with the

adaxial leaf surfaces facing up. After some time of incuba-

tion, the inoculum is prepared by shaking in distilled

water a piece with a single lesion and full-grown spores of

an infected leaf. The spore suspension is atomised over the

adaxial surface of a leaf of a highly susceptible genotype of

a host placed in a Petri dish on wet filter paper. Inoculated

leaves are incubated in a growth chamber with constant

conditions; first, they have to be kept in dark for 24 h

(Lebeda & Urban, 2004), followed by 12-h light/dark peri-

ods and a temperature of 18�C during light/15�C during

darkness. The pathogen produces conidiosporangiophores

with spores, usually 7–8 days after inoculation.

Maintenance of Pseudoperonospora

cubensis isolates

There are several ways to maintain P. cubensis isolates.

The most suitable methods for obligate parasites of plants

were summarised by Lebeda & Bartos (1988). Pure cul-

tures of P. cubensis can be stored in Petri dishes at 220�Cand/or 280�C, which keeps spores viable for about

6 months. After this period, it is necessary to renew the

Fungicide resistance in cucurbit downy mildew J. Urban & A. Lebeda

64 Ann Appl Biol 149 (2006) 63–75 ª 2006 The Authors

Journal compilation ª 2006 Association of Applied Biologists

cultures by fresh inoculations (Urban & Lebeda,

2004a,b). Isolates can also be maintained by weekly

transfers to detached cotyledons or leaves on wet filter

paper (Lebeda, 1986, 1991) or water agar in Petri dishes

(O’Brien & Weinert, 1995; Ishii et al., 2002) or by trans-

ferring them to detached whole plants in separate growth

chambers (Samoucha & Cohen, 1984; Ishii et al., 2001).

Field trials

In field trials, the effectiveness of fungicides is tested in

a natural environment and while using normal cultural

practices such as spray timing, use of mixtures, type of

application etc. However, field trials have not yet been

used often because they do not provide any exact informa-

tion about the structure of the pathogen population as far as

resistance to a fungicide is concerned.With this approach it

is impossible to work with a defined collection of P. cubensis

isolates and repeat the tests. Such trials were used, for

example by Mitani et al. (2003) to examine the efficacy of

the novel systemic fungicide cyazofamid. The field tests

were arranged as randomised plots (two replicates) with

5–7 plants per plot. Plants were sprayed with the fungi-

cide and infected by naturally occurring spores without

any artificial inoculation. Each leaf was later visually eval-

uated by estimating the lesion area. The disease intensity

on leaves was expressed in terms of a disease index (0–4

scale), where 0: no disease, 1: 1–5% infected, 2: 6–25%,

3: 26–49%, 4: more than 50%. The disease severity of the

plot was expressed by the following formula:

Disease severity ¼ 100� ðBþ 2C þ 3Dþ 4EÞ=4ðAþ Bþ C þ Dþ EÞ;

where A, B, C, D and E represent the number of leaves

rated at disease index 0, 1, 2, 3 and 4, respectively.

Laboratory tests

Various methods are available to assess the resistance/

tolerance of strains of cucurbit downy mildew against

fungicides under in vitro conditions.

Testing on leaf discs

Floating leaf disc bioassay (Anonymous, 1982)

This method employs leaf discs cut from leaves of a highly

susceptible genotype of a cucurbitaceous host plant. Discs

are floated, abaxial surface up, on fungicide solutions in

Corning multiwell plates. The method was recently used

by Urban & Lebeda (2004a,b) for efficacy screening of

metalaxyl, propamocarb and fosetyl-Al. There were four

leaf discs in three replicates for every concentration of

each fungicide. After 24 h, each disc was inoculated with

a spore suspension (1 � 105 spores mL21) using a glass

sprayer. Inoculum was prepared from 2- to 3-day-old

spores of P. cubensis. Subsequently, leaf discs were placed

in darkness (18/15�C) for 24 h and then incubated in

a growth chamber (12-h photoperiod, 18�C in the light,

15�C in the dark). Evaluation was carried out 6–14 days

after inoculation by using a 0–4 scale (percentage of disc

area covered by sporangiophores with spores): 0, no sporu-

lation; 1, �25%; 2, >25–50%; 3, >50–75%, 4, >75%.

The total degree of infection was expressed as a percent-

age of the maximum scores according to Towsend &

Heuberger (1943). Three types of reactions were as-

signed: (a) sensitive [degree of infection (DI) = 0–10%];

(b) tolerant (DI = 10.1–34.9%) and (c) resistant (DI

� 35%). Values of effective dose (ED) 50 (fungicide con-

centration that inhibits fungal growth by 50%) were

determined for each screened isolate and expressed in

ranges of fungicide concentrations.

A similar approach to test the efficacy of metalaxyl had

also been used by O’Brien & Weinert (1995), but details

of the tests differed from those described above. Leaf

discs cut from cucumber cotyledons were inoculated by

placing a single 10-ll droplet of sporangial suspension

(1 � 104 mL21) in the centre of each disc. Discs were

incubated at 20�C for 12 days, then rated on a 0–3 scale

for sporulation intensity: 0, no symptoms; 1, reaction

spot with no sporulation; 2, sporulation, low moderate

covering <50% of the disc surface and 3, sporulation

covering >50% of the disc.

A modified leaf disc bioassay (Anonymous, 1982)

This method has been especially used for efficacy screen-

ing of several fungicides against cucurbit powdery mildew

(McGrath, 2001; Sedlakova & Lebeda, 2004). Yet,

O’Brien & Weinert (1995) employed this method for

screening the efficacy of metalaxyl against P. cubensis.

Cucumber plants grown in a glasshouse were sprayed

at weekly intervals with metalaxyl at various concen-

trations. Leaf discs were cut from the youngest fully

expanded leaves 24 h after the second of two metalaxyl

applications. The discs were then placed inverted on

water agar in Petri dishes and inoculated by single 10-lldroplets (1 � 104 mL21). There were three replicate

discs per treatment. Five days later, the discs were exam-

ined and rated on a 0–3 scale: 0, no symptoms; 1, chlo-

rotic spot with either no sporulation or very few spores;

2, moderate sporulation and 3, intense sporulation.

Testing on cotyledons

Using fungicide as a soil drench treatment

This method is appropriate for testing fungicides that have

a high level of systemic behaviour within plants. Cohen

& Samoucha (1984) and Samoucha & Cohen (1985)

J. Urban & A. Lebeda Fungicide resistance in cucurbit downy mildew


65

used the method to test metalaxyl, propamocarb, cypro-

furam + folpet, SAN 371 F and fosetyl-Al. Cucumbers

were used about 10 days after sowing when cotyledons

were fully expanded. Fungicides dissolved in water were

used as a soil drench 24–72 h before inoculation. Treated

plants were kept in a greenhouse until inoculation. Coty-

ledons were inoculated by placing a droplet of 10-ll inocu-lum (10 ± 1 sporangia per droplet) on the adaxial surface

of each cotyledon. These were incubated for 20 h at 17�Cin the dark and then transferred to growth chambers for

7 days [20�C, 60–70% relative humidity (RH), illuminated

12 h day21 with very high output (VHO) fluorescent

lamps]. Disease data were recorded 7 days after inocula-

tion by counting the number of infected leaves.

Using fungicide in sprayed application

Thismethod is appropriate for all types of fungicides (com-

pletely, partially and non-systemic). Samoucha & Cohen

(1984) used it for testing mancozeb efficacy. It was simi-

lar to the method described above but the fungicide was

applied as a spray with a glass atomizer. Spray droplets

were allowed to dry for about 2 h and then the plants

were inoculated.

Testing on whole plants

Using fungicide as a soil drench treatment

This method is appropriate for fungicides with a high level

of systemicity within plants. Samoucha & Cohen (1985)

used it for testing of metalaxyl. Plants in their two-leaf

stage (3 weeks old) were treated by metalaxyl as soil

drench 1 day before inoculation. The frequency of iso-

lates resistant to metalaxyl was calculated according to

the ratio between disease severity in treated and

untreated plants. O‘Brien & Weinert (1995), also work-

ing with metalaxyl, evaluated two leaves per plant by

a 0–6 scale (percent leaf area affected): 0, no symptoms;

1, 1–3 chlorotic spots (<1%); 2, 1–10%; 3, >10–25%; 4,

>25–50%; 5, >50–75% and 6, >75%.

Using fungicide in sprayed application

This method is appropriate for all types of fungicides.

Samoucha & Cohen (1984) used it to test the efficacy of

mancozeb. It was similar to the method described above

but the fungicide was applied as a spray with a glass

atomizer (all surfaces of leaves). Spray droplets were

allowed to dry for about 2 h and then the plants were

inoculated. Disease data were recorded 7 days after inocu-

lation. The leaf area showing downy mildew symptoms

on two inoculated leaves was measured by tracing on

a transparent paper and weighing the cut-outs. O’Brien

& Weinert (1995) employed this method for metalaxyl

testing, and two leaves on a plant were evaluated by

a 0–6 scale as described above or, in a different experi-

ment, by a 0–3 scale (percent leaf area affected): 0, no

symptoms; 1, <10%; 2, 10–50%; 3, >50%. Cohen et al.

(1995) used the method also for dimethomorph testing

and Ishii et al. (2002) to test the efficacy of azoxystrobin.

Disease development on 2- to 3-week-old cucumber

plants was evaluated 7 days after inoculation. Each leaf

was assessed by using the following scale: 0, no visible

symptoms; 1, 0–5% of the leaf area infected; 2, 6–25%;

3, 26–50%; 4, 51–75% and 5, more than 76%. Disease

severity was calculated as follows: ([5A + 4B + 3C +

2D + E]/5F) � 100, where A, B, C, D and E are the

number of leaves corresponding to the scales, 5, 4, 3, 2

and 1, respectively. Mitani et al. (2001) used this method

also for determining biological properties such as sys-

temic behaviour, preventative and curative activity of

cyazofamid.

Molecular diagnosis

Conventional methods for detecting fungicide resistance

are generally labour intensive and time consuming if large

numbers of isolates are to be tested. Advances in molecu-

lar biology have provided new opportunities for rapidly

detecting fungicide-resistant genotype once the mech-

anisms of resistance have been elucidated at a molecular

level. However, such methods are not so easily applicable

and conventional techniques have to be used in cases

when resistance mechanisms are complex, as was, for

example, concluded in resistance of some plant pathogens

to dicarboximide fungicides (Ma & Michailides, 2005).

Several molecular techniques, such as PCR, PCR-restric-

tion fragment length polymorphism (PCR-RFLP), allele-

specific PCR and allele-specific real-time PCR, have been

used successfully to detect fungicide-resistant genotypes

of several plant pathogens (Ma & Michailides, 2005). In

P. cubensis, Ishii (2001) and Ishii et al. (2002) reported

screening for resistance to strobilurins using the PCR-

RFLP method. A leaf disc bearing P. cubensis spores was

used for DNA extraction with mortar and pestle. After

purifying the pathogen DNA by a commercially available

kit, a fragment of the cytochrome b gene was success-

fully amplified by PCR. Subsequently, products were

treated with the restriction enzyme ItaI, which should

specifically recognise the mutated sequence in the

fragment.

Stochastic variation

A lot of experimental data are of random nature and dif-

ferent methods of statistical analysis are used to process

them. There are experiments with an implicit form of




randomness and unobvious stochastic variation is not

taken into account. The simple analysis based on average

numbersmay lead to deviation from reliable estimates and

inaccurate conclusions and interpretations. Kosman

(2002) reported methodological aspects of stochastic vari-

ation on example of bioassay for quantitative estimation

of fungicide resistance in Phytophthora infestans mixed

populations (Kadish & Cohen, 1988) and proposed the

corresponding probabilistic model for measuring fre-

quency of fungicide-resistant phenotype in a pathogen

population. Application of the model may improve the

estimates and raise reliability of the results.

Types of fungicides and their efficacy

Fungicides active against P. cubensis are distinguished

either by their biological mode of action in the disease

cycle or according to their biochemical mode of action

(Table 1). They can simultaneously control a broad spec-

trum of diseases or only one or several diseases (narrow

spectrum).

According to their systemicity, there are non-systemic

(contact) or systemic fungicides. Contact fungicides form

a protective barrier on the plant surface and exhibit only

a preventative activity (inhibition of pathogen develop-

ment prior to penetration into the tissue). With the

exceptions of fluazinam and zoxamide, they are multisite

inhibitors and interact mostly unspecifically with many

biochemical steps in the metabolism pathway of the

pathogen.

Therefore, the risk is low that resistance to these fungi-

cides will develop. There are only a few reports about the

infection of P. cubensis on parts of a plant other than

leaves (Van Haltern, 1933; D’Ercole, 1975). Thus, the

control of P. cubensis is focused on the protection of

foliage, and the effective usage of contact fungicides on

extensive field crops of cucurbitaceous plants is practi-

cally impossible because of the difficulty of applying it

evenly, particularly to the abaxial leaf surfaces.

Systemic fungicides exhibit various levels of systemicity

within plants. They can be fully systemic (e.g. metalaxyl)

or partially systemic. Partially systemic fungicides are

either locally systemic (e.g. dimethomorph) or mesoste-

mic (trifloxystrobin; Margot et al., 1998). A meso-systemic

fungicide has a high affinity with the plant surface, is

absorbed by the waxy layers of the plant and is redis-

tributed at the plant surface by superficial vapour

movement and redeposition. It penetrates plant tissue,

exhibits translaminar activity but no transport within

the vascular system of the plant. The term ‘quasi-

systemic’ or ‘surface systemic’ has been used for kresoxim-

methyl (Ypema & Gold, 1999). Systemic fungicides can

exhibit preventative, curative or eradicative activity.

Curative compounds affect pathogen stages between the

penetration and the appearance of the first symptoms,

eradicative compounds in later stages of pathogen devel-

opment (hyphal growth within plant tissues and the

production of spores).

There are differences in modes of translocation of

various systemic fungicides within the plant. It can be

apoplastic (acropetal) mobility, i.e. translocation within

the free space, cell walls and xylem elements of the plant

tissue governed by diffusion and the rate of transpiration,

or the mobility is symplastic (basipetal), i.e. translocation

through plasmodesmata from cell to cell, involving uptake

and distribution from source to sink via the phloem

(Neumann & Jacob, 1995). Systemic fungicides recently

used against P. cubensis are single-site inhibitors and,

therefore, they come with a high risk that resistance to

them may develop.

Fungicides active against P. cubensis and their basic

characteristics are summarised in Table 1.

Resistance of Pseudoperonospora cubensis to

fungicides; mechanisms, spatial and temporal

aspects of resistance

Mechanisms of resistance

Fungicide resistance can be conferred by various mecha-

nisms (Gisi et al., 2000; Ma & Michailides, 2005), includ-

ing an altered target site, which reduces the binding of

the fungicide; the synthesis of an alternative enzyme

capable of substituting the target enzyme; the over-

production of the fungicide target; an active efflux or

reduced uptake of the fungicide and a metabolic break-

down of the fungicide. In addition, some unrecognised

mechanisms could also be responsible for fungicide

resistance.

Since resistance of P. cubensis to the phenylamide fun-

gicide metalaxyl first appeared, there have been many

reports of failure of metalaxyl to control other Oomycete

pathogens such as Phytophthora infestans (e.g. Fisher &

Hayes, 1984; Deahl et al., 1993; Daayf & Platt, 1999;

Shattock, 2002; Stein & Kirk, 2003), Plasmopara viticola

(Clerjeau & Simone, 1982; Moreau et al., 1987) and

Bremia lactucae (Crute, 1987). Genetics of metalaxyl

resistance is not known in P. cubensis; however, inheri-

tance studies of Phytophthora infestans on metalaxyl

resistance suggested that a single locus exhibiting incom-

plete resistance was controlling resistance to phenyl-

amides (Shattock, 1988; Shaw & Shattock, 1991).

Resistance to metalaxyl was reported to be controlled by

a single incompletely dominant gene also in Phytophthora

capsici (Lucas et al., 1990) and Phytophthora megasperma

var. sojae (Bhat et al., 1993). Some authors have



67

Table

1Li

sto

fch

em

ica

lco

mp

ou

nd

sa

ctiv

ea

ga

inst

Pseudoperonospora

cubensis

an

dso

me

oth

er

Oo

myc

ete

sa

Leve

lo

f

Sys

tem

icit

y

Cro

ss-r

esi

sta

nce

Gro

up

Co

mm

on

Na

me

of

Co

mp

ou

nd

Typ

eo

fA

ctiv

ity/

Tra

nsl

oca

tio

n

Be

ha

vio

ur

Wit

hin

Pla

nts

Bio

che

mic

al

Mo

de

of

Act

ion

Re

fere

nce

sb

Fu

llysy

ste

mic

Ph

en

yla

mid

es

Me

tala

xy

l,m

eta

lax

yl-M

,

be

na

lax

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ofu

race

Pre

ven

tati

ve(?

),cu

rati

ve,

era

dic

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ve/a

po

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stic

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sym

pla

stic

,tr

an

sla

min

ar

Inh

ibit

ion

of

rRN

A

syn

the

sis

Reuvenietal.(1980),Georgopoulos&Grigoriu

(1981),

Wyn

n&

Cru

te(1

98

3),Cohen&Samoucha

(1984),

Fis

he

r&

Ha

yes

(19

84

),D’Ercole

&Nipoti

(1985),Samoucha&Cohen(1985),Moss

(1987),

Da

vid

se(1

98

8,

19

95

),Ackerm

ann(1990),

Gri

n‘k

o

(19

92

),O’Brien&Weinert

(1995),

Sch

win

n&

Sta

ub

(19

95

),G

isi

(20

02

),S

ha

tto

ck(2

00

2),Urban&Le

beda

(2004a,b),Kuzm

a(2005)

Am

ino

aci

da

mid

e

carb

am

ate

s

Ipro

valic

arb

Pre

ven

tati

ve,

cura

tive

,

era

dic

ati

ve/a

po

pla

stic

,

sym

pla

stic

Aff

ect

am

ino

aci

d

me

tab

olis

m(?

)

Ste

nze

letal.

(19

98

),G

isi

(20

02

)

Ph

osp

ho

na

tes

fose

tyl-A

lP

reve

nta

tive

,cu

rati

ve/a

po

pla

stic

,

sym

pla

stic

(?),

***

Bo

mp

eixetal.

(19

80

),Fa

rihetal.

(19

81

),La

ng

cake

(19

81

),Cohen&

Samouch

a(1984),

Gu

est

(19

84

),D

erc

ks&

Cre

asy

(19

89

),

Sm

illieetal.

(19

89

),D

ust

inetal.

(19

90

),N

em

est

oth

y&

Gu

est

(19

90

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ros

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P.cubensis.




identified molecular markers, by using bulk segregant

analysis, linked to loci controlling resistance in Phytoph-

thora infestans (Fabritius et al., 1997; Judelson & Roberts,

1999). So far, six random amplified polymorphic DNA

markers have been identified, and mapping assays indi-

cate that one or two semi-dominant loci, MEX, are

involved. The data further suggested that not all alleles

are functionally equivalent and, in addition, that epi-

static minor genes interacted with the MEX loci. Lee

et al. (1999) reported that one dominant gene plus minor

genes may be involved in phenylamide resistance. In

biochemical studies, endogenous RNA polymerase activ-

ity of isolated nuclei of Phytophthora megasperma and

Phytophthora infestans was highly sensitive to metalaxyl

in sensitive isolates but insensitive in resistant isolates,

suggesting that a target site mutation is responsible for

resistance (Davidse, 1988).

The mechanism of resistance of P. cubensis and some

other phytopathogens against strobilurin fungicides

azoxystrobin and kresoxim-methyl is well known. In

strobilurin-resistant isolates of P. cubensis, a single point

mutation leading to an amino acid change from glycine

to alanine at codon 143 (G143A) of the mitochondrial

cytochrome b gene conferred strobilurin resistance

(Heaney et al., 2000; Ishii et al., 2000, 2001, 2002). The

G143A mutation has been correlated with strobilurin

resistance in a wide variety of plant pathogens

from many hosts, for example Mycosphaerella fijiensis

(Sierotzki et al., 2000a), Blumeria graminis f. sp. tritici

(Sierotzki et al., 2000b) and Plasmopara viticola (Gisi et al.,

2002). In addition, mutation at different codon of cyto-

chrome b gene (Kim et al., 2003) and various

substitutions at codon 143 (Avila-Adame & Koller, 2003)

were reported to be responsible for strobilurin resistance.

Miguez et al. (2004) reported an additional resistance

mechanism of Mycosphaerella graminicola to azoxystrobin

involving activation of an alternative oxidase, which

increased flexibility in respiration and allowed resistant

strains to survive in the presence of the fungicide.

Spatial and temporal aspects of

fungicide resistance

Fungicides are an important tool for managing cucurbit

downy mildew. Unfortunately, fungicides that are

single-site inhibitors come with a high risk of resistance

developing against them (Gisi, 2002) and, furthermore,

P. cubensis has a high potential for resistance develop-

ment. While contact fungicides as multisite inhibitors

have a considerably lower risk of resistance developing

against them (McGrath, 2001), their usage in crops is

less effective because of the necessity to protect in partic-

ular the abaxial leaf surfaces of the hosts.

Metalaxyl resistance

There are several reports about failures of various systemic

fungicides and build up of resistant subpopulations of

P. cubensis (Table 2). The best-known example is the phe-

nylamide fungicide metalaxyl. The first failure of meta-

laxyl was observed in the winter of 1979 in plastic

houses with cucumbers in the Hadera district in Israel.

Isolates of the pathogen collected from these green-

houses could not be controlled with a rate of metalaxyl

300 times higher than the rate recommended for the

control of sensitive strains (Reuveni et al., 1980). After

this breakdown, an extensive collection of P. cubensis isol-

ates from many sites in Israel during 1980–84 was tested

(Samoucha & Cohen, 1985). The results showed a rapid

build up of resistant strains under selection pressure of

metalaxyl and a high fitness of these strains in

Table 2 Occurrence of Pseudoperonospora cubensis strains resistant/tolerant to fungicides

Chemical Group/

Chemical Class Common Name

Countries where Resistant/

Tolerant Strains Occurred References

Phenylamides Metalaxyl Israel Reuveni et al. (1980), Samoucha & Cohen (1985)

Greece Georgopoulos and Grigoriu (1981)

Italy D‘Ercole & Nipoti (1985)

USA Moss (1987)

Russia Grin’ko (1992)

Australia O’Brien & Weinert (1995)

Czech Republic Ackermann (1990), Urban & Lebeda (2004a,b)

Strobilurins Azoxystrobin,

kresoxim-methyl

Japan Takeda et al. (1999), Heaney et al. (2000), Ishii (2001),

Ishii et al. (2002)

Taiwan Ishii (2001)

Phosphonates fosetyl-Al Israel Cohen & Samoucha (1984)

Czech Republic Urban & Lebeda (2004a,b)

Carbamates Propamocarb Israel Cohen & Samoucha (1984)

Phthalimides Folpet Israel Cohen & Samoucha (1984)

Dithiocarbamates Mancozeb Israel Samoucha & Cohen (1984)



69

competition with sensitive ones in the absence of the

fungicide. The worldwide distribution of metalaxyl-resis-

tant strains of P. cubensis has since been confirmed by

several reports from other countries, for example Greece

(Georgopoulos & Grigoriu, 1981), Italy (D’Ercole &

Nipoti, 1985), USA (Moss, 1987) and Russia (Grin‘ko,

1992). In Australia, O’Brien & Weinert (1995) reported

three levels of sensitivity to metalaxyl of P. cubensis

isolates collected in the Murrumbidgee Irrigation Area

(MIA), New South Wales, and the Burdekin district,

Queensland. In floating disc experiments, the ED 50

values of sensitive, intermediate and resistant isolates

were in the ranges >0.01–<0.1, >1–<10 and >100 lgmetalaxyl mL21, respectively. They postulated that the

reduced (intermediate) sensitivity of P. cubensis isolates

from MIA represents probably a transitory stage and

later collections will show the presence of resistant

types.

The phenomenon of occurrence of metalaxyl-resistant/

tolerant strains of P. cubensis and its historical develop-

ment has been explored also in the Czech Republic

(Urban & Lebeda, 2004a,b; J. Urban & A. Lebeda,

unpublished data). A large number of isolates were

collected in the Czech Republic, especially during 2001–

03, but also used were 15 isolates collected in the

former Czechoslovakia during 1985–2000 (Lebeda, 1992;

Lebeda & Gadasova, 2002); all were screened for reaction

to metalaxyl (Table 3). The results showed a continuous

selection of highly resistant strains in Czech populations

of P. cubensis. Until 2000, strains sensitive to metalaxyl

probably predominated, although the existence of some

resistant strains was proved already for 1995. Since 2000,

resistant strains have become predominant, and a gradual

selection for higher resistance in Czech populations of

P. cubensis was observed during the years 2000–03 (Urban

& Lebeda, 2004a,b).

Strobilurin resistance

The rapid decline in efficacy of strobilurin fungicides, i.e.

azoxystrobin and kresoxim-methyl, against P. cubensis is

well known from Japan. Field resistance to strobilurin

fungicides was first reported in populations of wheat

powdery mildew (B. graminis f. sp. tritici) in northern

Germany in 1998 (Erichsen, 1999). Subsequently, the

resistance problem has also occurred in downy mildew

on cucumber in Japan and Taiwan (Ishii, 2001). In

Japan, kresoxim-methyl and azoxystrobin were officially

registered in December 1997 and April 1998, respect-

ively. Most cucumber growers followed the manu-

facturers’ recommended rate and applied their products

only a couple of times per crop in alternation with other

fungicides with different modes of action. However,

a reduced efficacy of these fungicides and build up of

highly resistant subpopulations of P. cubensis were already

observed in 1999 (Takeda et al., 1999). Heaney et al.

(2000) briefly mentioned that resistant subpopulations of

P. cubensis were present at almost all locations sampled in

Japan. Ishii et al. (2002) reported about persistence of

resistant P. cubensis strains in the absence of strobilurin

fungicides in several greenhouses in Tsukuba and Akeno,

Ibaraki. Although these fungicides were removed from

practice in 1999, a high proportion of the strains were still

resistant in 2000 and 2001. It suggests that resistant

isolates are not necessarily less fit than sensitive isolates

in the environment. In case of Magnaporthe grisea, several

parameters tested to measure fitness penalties inherent

to the mutational changes in cytochrome b gene and

responsible for QoI resistance revealed that the G143A

mutant was not compromised (Avila-Adame & Koller,

2003). Also, cross-resistance between azoxystrobin and

kresoxim-methyl was confirmed in P. cubensis isolates.

Resistance to fosetyl-Al

and other types of fungicides

Despite good effectiveness of fosetyl-Al, there are reports

about the appearance of a potential development of highly

resistant strains of P. cubensis in the Czech Republic and

Israel. Cohen & Samoucha (1984) detected metalaxyl-

resistant isolates of P. cubensis collected in 1979 and 1982

in Israel, which were also resistant to a soil drench of

fosetyl-Al at the concentration 3200 lg a.i. mL21. They

considered cross-resistance between metalaxyl and

fosetyl-Al, but this is questionable because the two

Table 3 Temporal shift in occurrence of metalaxyl-resistant isolates of

Pseudoperonospora cubensis collected in the Czech Republic in the

years 1985–2002 (Urban & Lebeda, 2004a,b; J. Urban & A. Lebeda,

unpublished data)

Isolatea (year)

Metalaxyl Concentration (lg a.i. mL21)b

0 50.4 100.0 200.0c 400.0 800.0

3/85 (1985) + 2 2 2 2 2

1/88 (1988) + 2 2 2 2 2

4/95 (1995) + 2 2 2 2 2

1/98 (1998) + 2 2 2 2 2

35/01 (2001) + + (2) (2) 2 2

28/01 (2001) + + + (2) 2 2

60/02 (2002) + + + + (2) 2

43/02 (2002) + + + + + +

Reaction (Urban & Lebeda, 2004a,b) of P. cubensis isolates to metalaxyl:

2, sensitive (no sporulation); (2), tolerant (limited sporulation); +,

resistant (profuse sporulation).aFor origin and pathogenicity characteristics see Lebeda (1991, 1992),

Lebeda & Gadasova (2002), Lebeda & Urban (2004), Lebeda & Widr-

lechner (2003, 2004).bSource of Ridomil Plus 48 WP (AgroBio Opava Ltd., Opava, Czech Republic).cThe concentration recommended by the producer (Kuzma, 2001, 2005).




fungicides are members of different cross-resistance/

chemical groups (Table 1). In the Czech Republic, the

efficacy of fosetyl-Al against P. cubensis was monitored

in detail during 2001–03 (Urban & Lebeda, 2004a,b;

J. Urban & A. Lebeda, unpublished data). Only in 2001,

a high proportion of isolates with a certain level of resis-

tance/tolerance to this fungicide was detected, and while

this could signal a selection of more resistant strains,

such responses did not recur in 2002 and 2003. This

could relate to the nature of the Czech population,

which is characterised by epidemic occurrence caused

by the annual airflow transport of inoculum from the

southern and south-eastern areas of Europe (Lebeda,

1990) and annual restoration and extinction with no or

only rare overwintering through oospores, especially in

combination with using other fungicides, could be

responsible for the loss of locally developed resistance

to fosetyl-Al. In addition, the more resistant sub-

populations to the fungicide could exhibit less fitness

than original wild-type populations and decreased fre-

quency in relation to weaker selection pressure of the

fungicide to subpopulations having high sensitivity.

Cohen & Samoucha (1984) also described prop-

amocarb resistance of metalaxyl-resistant P. cubensis

isolates. That fungicide gave no control as a soil drench

at a concentration of 420 g a.i. mL21. The authors

considered cross-resistance between propamocarb and

metalaxyl but again this is questionable.

Multisite inhibitorswithpreventative activity usedmostly

inmixtureswith systemic fungicides bear a lower risk for the

development of resistance. However, in Israel, metalaxyl-

resistant strains of cucurbit downy mildew that also ex-

hibited a certain level of resistance against the contact

fungicide mancozeb were detected (Samoucha & Cohen,

1984). In the same country, resistance against the mixture

of cyprofuram + folpet (Cohen & Samoucha, 1984) by

metalaxyl-resistant strains of P. cubensis was also re-

ported; nevertheless, from the viewpoint of used testing

methodology (soil drench treatment), folpet should not

be considered as an active substance in this case.

Conclusions and future prospects

The preceding sections described various methodological

approaches to study the resistance of P. cubensis to fungi-

cides. When choosing an appropriate method, it is espe-

cially important to consider the specific features of

a fungicide so that objective results will be obtained.

Recently, some molecular methods have been developed

(Ishii et al., 2002) to rapidly screen for sensitive or resist-

ant strains. These methods will be particularly useful for

future research into the genetic background of fungicide

resistance.

During the last several decades, many new fungicides

effective against cucurbit downy mildew have been intro-

duced to the market (Table 1), thereby replacing the

contact fungicides used earlier, such as the copper for-

mulations. They exhibit various levels of systemicity, from

non-systemic to partially and fully systemic and also differ

in translocation behaviour within plants, as well as in their

biological and biochemical modes of action (Table 1).

These newer fungicides are mostly single-site inhibitors

in the metabolism pathway of the pathogen and thus bear

a high risk of development of resistance to them. There

are reports about failures in their control of P. cubensis

(Table 2) and other downy mildews (Gisi, 2002). In the

case of P. cubensis, the mechanisms of resistance are not

well known, with the exception of the strobilurin fungi-

cides such as azoxystrobin and kresoxim-methyl (Takeda

et al., 1999; Ishii et al., 2002). Further research in this

field is necessary to gain a better understanding of resist-

ance mechanisms, knowledge that would be part of the

basis for effective disease management.

There are numerous reports about a rapid increase of

fungicide-resistant populations and subpopulations of P.

cubensis under selection pressure from different fungi-

cides (Tables 2 and 3). Resistance to the phenylamide

fungicide metalaxyl has been reported frequently from

many countries around the world since its first appear-

ance in Israel in the 1980s. It has recently become obvi-

ous that resistant strains of P. cubensis predominate in

the pathogen populations of many countries and geo-

graphic regions (Table 2). Failures of other fungicides to

be effective against P. cubensis were only detected in

a few countries, but they are likely to occur in other

areas as well. It was clearly demonstrated that fungicide-

resistant strains persist in pathogen populations even

after withdrawal of a fungicide. More detailed research

should focus on the geographic distribution and the

dynamics of spatial and temporal aspects of fungicide

resistance in P. cubensis. Intensive international collab-

oration in this area is required.

Current recommendations for dealing with fungicide

resistance include the use of diverse fungicides and plant

defence inducers as well within an integrated disease

management programme that also utilises a facet of

non-chemical practices, such as more resistant cultivars,

weather forecasts, epidemiological studies, disease moni-

toring etc (Lebeda & Widrlechner, 2003). It is also advis-

able not to use fungicides with a proven loss of efficacy

such as metalaxyl.

Acknowledgements

The authors thank Dr J. Nielsen (Winnipeg, Canada) for

reading and remarks on the first draft of the manuscript.



71

This research was supported by grants: (a) QD 1357 and

(b) ‘National Programme of Genepool Conservation of

Microorganisms and Small Animals of Economic Impor-

tance’ (Ministry of Agriculture of the Czech Republic); (c)

MSM 6198959215 (Ministry of Education, Youth and

Sports of the Czech Republic).

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Fungicide resistance in cucurbit downy mildew – methodological, biological and population aspects