The study assessed the risk of resistance developing in the oomycete fungus Pseudoperonospora cubensis, which causes cucumber downy mildew, to the fungicide flumorph. Researchers compared flumorph to dimethomorph and azoxystrobin in terms of the ease of isolating resistant mutants, the level of resistance induced, and the fitness of resistant mutants. Attempts to induce resistance to flumorph and dimethomorph by adapting sporangia on treated leaves were unsuccessful. Moderately resistant mutants were isolated using UV mutagenesis for flumorph and dimethomorph, but resistance levels were much lower than for azoxystrobin-resistant mutants.
Climate extremes likely to drive land mammal extinction during next supercont...
Assessing the Risk of Resistance in Pseudoperonospora cubensis to the Fungicide Flumorph in vitro (2006)
1. Pest Management Science Pest Manag Sci 64:255–261 (2008)
Assessing the risk of resistance
in Pseudoperonospora cubensis to the
fungicide flumorph in vitro
Shusheng Zhu,1,2 Pengfei Liu,1 Xili Liu,1∗ Jianqiang Li,1 Shankui Yuan3 and
Naiguo Si4
1Department of Plant Pathology, China Agricultural University, Beijing 100094, China
2Key Laboratory of Agriculture Biodiversity for Plant Disease Management, Ministry of Education, Key Laboratory of Plant Pathology,
Yunnan Agricultural University, Kunming 650201, China
3Centre of Agrochemicals for Biological and Environmental Technology Institute for the Control of Agrochemicals, Ministry of Agriculture,
Beijing 100026, China
4China Shenyang Research Institute of the Chemical Industry, Shenyang 110021, China
Abstract
BACKGROUND: The oomycete fungicide flumorph is a recently introduced carboxylic acid amide (CAA)
fungicide. In order to evaluate the risk of developing field resistance to flumorph, the authors compared it with
dimethomorph and azoxystrobin with respect to the ease of obtaining resistant isolates to these fungicides, the
level of resistance and their fitness in the laboratory.
RESULTS: Mutants with a high level of resistance to azoxystrobin were isolated readily by adaptation and
UV irradiation, and their fitness was as good as that of the parent isolates. Attempts to generate mutants
of Pseudoperonospora cubensis (Burk. & MA Curtis) Rostovsev resistant to flumorph and dimethomorph by
sporangia adaptation on fungicide-treated leaves were unsuccessful. However, moderately resistant mutants were
isolated using UV mutagenesis, but their resistance level [maximum resistance factor (MRF) < 100] was much
lower than that of the azoxystrobin-resistant mutant (MRF = 733). With the exception of stability of resistance, all
mutants showed low pathogenicity and sporulation compared with wild-type isolates and azoxystrobin-resistant
mutants. There is cross-resistance between flumorph and dimethomorph, suggesting that they have the same
resistance mechanism.
CONCLUSION: The above results suggest that the resistance risk of flumorph may be similar to that of
dimethomorph but lower than that of azoxystrobin and can be classified as moderate. Thus, it can be managed by
appropriate product use strategies.
2007 Society of Chemical Industry
Keywords: cucumber downy mildew; fungicide resistance; flumorph; dimethomorph; azoxystrobin
1 INTRODUCTION
Flumorph, a recently introduced carboxylic acid
amide (CAA) fungicide, was developed by Shenyang
Research Institute of Chemical Industry of China
for the control of oomycete pathogens and has
been patented in China (ZL.96115551.5), the USA
(US6020332) and Europe (0 860 438B1).1 It exhibits
a very high level of protective and curative activity
against members of the family Peronosporaceae and
the genus Phytophthora but not Pythium.2 In China,
field resistance to a major systemic fungicide class,
such as the phenylamides, has occurred widely in
some species of plant pathogenic oomycetes.3–5 Thus,
the development of flumorph was expected to replace
the phenylamides for resistance management.
Flumorph has a similar chemical structure and anti-fungal
activity to the CAA fungicide dimethomorph,6
which has been widely used for oomycete disease con-trol,
and the resistance risk of different pathogens
to dimethomorph is diverse. For some Phytophthora
pathogens, such as P. infestans (Mont.) de Bary,7–9
P. capsici Leonian and P. parasitica Dastur,8,10,11 the
resistance risk to dimethomorph may be low, based on
laboratory studies and field monitoring.12 However,
in populations of the grape downy mildew pathogen,
Plasmopara viticola Berliner & de Toni, less sensi-tive
isolates have been found in certain regions of
Europe.13 The risk of Phytophthora spp. being resistant
to flumorph is also considered to be low to moder-ate,
in spite of resistant mutants of P. infestans and
∗ Correspondence to: Xili Liu, Department of Plant Pathology, China Agricultural University, Beijing 100094, China
E-mail: seedling@cau.edu.cn
(Received 3 September 2006; revised version received 9 July 2007; accepted 9 August 2007)
Published online 20 December 2007; DOI: 10.1002/ps.1515
2007 Society of Chemical Industry. Pest Manag Sci 1526–498X/2007/$30.00
2. S Zhu et al.
P. capsici being selected after ultraviolet treatment.4,14
The field performance of flumorph-based fungicides
has remained excellent for control of potato light blight
in China.4,5,15 However, the resistance risk of downy
mildew causal agents to flumorph in the field is still
unclear.
In order to define the risk of resistance develop-ment
of cucumber downy mildew, Pseudoperonospora
cubensis (Berk. & MA Curtis) Rostovsev, in the field
to flumorph, laboratory studies were conducted. In
this study, flumorph was compared with two com-mercial
systemic oomycete fungicides, dimethomorph
and azoxystrobin, both with well-understood resis-tance
potential,7–12,16 to determine (i) the ease of
isolating resistant mutants using ultraviolet (UV) light
mutagenesis and sporangia adaptation, (ii) the level of
resistance that can be induced and (iii) the fitness of
mutants on cucumber leaves. Based on these data, the
resistance risk of P. cubensis to flumorph was defined.
2 MATERIALS AND METHODS
2.1 Isolates
Four P. cubensis isolates were collected from different
geographical districts (Table 1) where no CAA fungi-cides
had been applied, and these were maintained
by weekly transfers to detached leaves on wet filter
paper in petri dishes at 20 ◦C with a 12:12 h light:dark
photoperiod.
2.2 Chemicals
Technical-grade flumorph (96%), dimethomorph
(97%), azoxystrobin (95%), cymoxanil (98%) and
metalaxyl (98%) were kindly supplied by Shenyang
Research Institute of Chemical Industry of China
(Shengyang, China), Genyun Co., Ltd (Jiangsu,
China), Syngenta China Ltd (Beijing, China), Wan-quanCo.,
Ltd (Hebei,China) and Agrolex P. Ltd (Bei-jing,
China) respectively. Stock solutions of 10 gL−1
of active ingredient of each fungicide were made in
methanol and stored at 4 ◦C in darkness. For sensi-tivity
testing, stocks of fungicides were serially diluted
with double-distilled water containing 0.05mL L−1
Tween 20. The maximum concentration of methanol
used in treatment solutions was less than 1mL L−1.
2.3 Sensitivity assays
Fungicide sensitivity was determined as described
previously.17 Briefly, leaf discs (15mm diameter) were
cut with a cork borer from healthy leaves (the second
from the tip) of four-true-leaf stage greenhouse-grown
cucumber plants (cv. Changchunmici). All leaf discs
were randomized and placed into containers to which
50mL of each fungicide solution was added. Control
discs were treated with distilled water containing
1ml L−1 methanol and 0.05mL L−1 Tween 20.
After 30min soaking, the leaf discs were removed
from the container and blotted dry with paper
towel. There were 30 leaf discs in three replicates
for every concentration of each fungicide. Fresh
sporangia of P. cubensis were harvested from diseased
cucumber leaves into cold water (4 ◦C). Leaf discs
were inoculated by placing one drop (10 μL) of
inoculum (1 × 104 sporangiamL−1) on the middle of
each disc. Dishes containing leaf discs were incubated
at 20 ◦C for 20 h in a humid chamber in darkness to
allow infection, and then maintained at 20 ◦C with a
12 h photoperiod for disease development. Six days
after inoculation, the mean percentage of sporulating
surface area on the leaf discs was determined. The
median effective concentration value (EC50) for each
isolate was calculated by regressing the percentage of
growth inhibition against the logarithm value of the
fungicide concentration using the software Microsoft
Excel 2003. The tests were replicated 3 times for each
isolate.
2.4 Induction of resistant isolates
2.4.1 Sporangia selection by adaptation
Sporangia suspensions (1 × 104 mL−1) were prepared
for each of the four wild-type isolates (Table 1)
and were sprayed on cucumber leaves treated with
flumorph (0.70mg L−1), dimethomorph (0.50mg
L−1) or azoxystrobin (0.07mg L−1), and then
incubated for 6 days as described above. The fungicide
concentration used for selection had previously
been found to be highly inhibitory yet sublethal
for all isolates of P. cubensis. The same sporangia
suspensions were also sprayed onto fungicide-free
leaves as control. Newly produced sporangia from
each treatment were used to subculture the isolate
onto new healthy cucumber leaves, treated with the
same concentration of flumorph, dimethomorph or
azoxystrobin, for a total of ten generations. After the
final transfer on fungicide-treated leaves, sporangia
were cycled once on fungicide-free leaves and the
EC50 was then calculated for each of the fungicide-exposed
and control isolates according to the above
method.
Table 1. Pseudoperonospora cubensis isolates collected from different geographical districts
EC50 (±SE) (mg L−1)
Isolate Year isolated Origin Flumorph Dimethomorph Azoxystrobin
K17 2002 Peking 0.17(±0.010) 0.14(±0.023) 0.012(±0.001)
T3 2003 Tianjin 0.24(±0.006) 0.19(±0.015) 0.017(±0.001)
LP2 2002 Hebei 0.13(±0.013) 0.11(±0.020) 0.015(±0.003)
M5 2002 Inner Mongolia 0.19(±0.011) 0.18(±0.011) 0.021(±0.001)
256 Pest Manag Sci 64:255–261 (2008)
DOI: 10.1002/ps
3. Risk of resistance in P. cubensis to the fungicide flumorph
2.4.2 Induction by UV mutagenesis
The method utilized to isolate mutants from UV-mutated
sporangia was based on procedures previously
described.10 Four wild-type isolates (Table 1) for
mutagenesis were grown on healthy cucumber
leaves until new sporangia were produced. UV
mutagenesis was performed with a UV lamp at
a wavelength of 254nm by irradiating suspensions
of sporangia (1 × 105 sporangiamL−1) in open petri
dishes (9cm diameter) for 1min at a distance of
30 cm. After irradiation, they were kept for 30 min
in the dark to minimize photorepair of radiation
damage. Sporangia suspensions were then sprayed
onto flumorph-treated (10mg L−1), dimethomorph-treated
(10mg L−1) or azoxystrobin-treated (1mg
L−1) cucumber leaves which did not support the
growth of wild-type isolates, as well as onto fungicide-free
leaves. Wild-type sporangia suspensions that
had not been exposed to UV were also included
in each experiment. All leaves were incubated
overnight in a humid chamber in darkness to
allow infection, and then maintained at 20 ◦C with
a 12:12 h light:dark photoperiod. After 6 days,
the number of lesions was examined, and two
lesions with the most vigorously growing sporangia
on fungicide-treated leaves were cycled once on
fungicide-free leaves. Subsequently, the resistance
level was determined by calculating the EC50 value
for each UV-mutant isolate compared with the parent
isolate.
2.5 Characteristics of resistant mutants
2.5.1 Stability of resistant isolates
After the initial sensitivities of mutants to fungicides
had been determined, all mutants were maintained
by weekly transfers to fungicide-free leaves. After the
tenth generation, sensitivity to flumorph was estimated
again using the method in Section 2.3 and compared
with the initial EC50 to give an indication of the
stability of the acquired resistance trait.
2.5.2 Pathogenicity and sporulation
Considering the low stability recorded for fungicide-adapted
isolates, pathogenicity and sporulation were
determined for UV-induced mutants only and were
compared with those of the parent isolates on
cucumber leaf discs. A sporangial suspension (1 ×
104 sporangiamL−1) of each sensitive or resistant
isolate was inoculated onto 30 leaf discs and incubated
according to the method in Section 2.3, and the
lesion area on each leaf disc was determined. Each
of the three sets of ten discs was placed in a 15mL
centrifuge tube containing 10mL distilled water and
mechanically agitated for 15 s. The sporangia released
were quantified with a haemocytometer, and the mean
number of sporangia cm−2 of lesion was calculated.
2.5.3 Cross-resistance
The sensitivity of flumorph-resistant and flumorph-sensitive
isolates was tested on a series of concen-trations
of metalaxyl-, dimethomorph-, cymoxanil-or
azoxystrobin-treated leaf discs in petri dishes
(15 cm diameter) by the method described in Sec-tion
2.3. Six days after inoculation, the mean
percentage of sporulating surface area on the
leaf discs at each of the different concentra-tions
was determined for the calculation of EC50.
The sensitivities of isolates to flumorph, dimetho-morph,
azoxystrobin and metalaxyl were compared
and cross-resistance was analysed using regression
analysis.18
3 RESULTS
3.1 Selection by adaptation on
fungicide-treated leaves
The lesion areas of all isolates on leaves treated
with sublethal doses of fungicide were significantly
smaller than those of the control, even if the
lesion area increased with subculture number for
all isolates. The isolates grown on azoxystrobin-treated
leaves demonstrated greatly reduced sen-sitivity,
whereas isolates that were grown on
flumorph- or dimethomorph-treated leaves showed
relatively little or no change in sensitivity (Table 2).
The average resistance factors (RFs) of isolates
grown on either flumorph- or dimethomorph-treated
leaves were all <5 and were significantly
lower than those for azoxystrobin, where average
RF values were >20 (Table 2). All azoxystrobin-resistant
isolates were stable, whereas two of
four flumorph-resistant isolates and one of four
dimethomorph-resistant isolates lost their resistance
(Table 2).
3.2 Isolation of resistant mutants by UV
irradiation and selection on fungicide-treated
leaves
After UVmutagenesis, approximately 20–30% of spo-rangia
could still infect fungicide-free leaves compared
with untreated controls. On fungicide-treated leaves,
however, only UV-mutated sporangia could cause
symptoms, but lesion areas were small. The average
number of lesions on flumorph- and dimethomorph-treated
leaves was 1.5 and 2 respectively. This
was significantly lower than the average number of
lesions on azoxystrobin-treated leaves, which was 5
(P = 0.05). The sensitivity of all mutants grown on
azoxystrobin-treated leaves had greatly decreased, with
RF values of >100 (Table 3). For flumorph and
dimethomorph, all mutants also showed reduced sen-sitivity,
but there were only two flumorph-resistant
mutants and two dimethomorph-resistant mutants
showing RF values above 50, and their mean RF
value was much lower than that of the azoxystrobin-resistant
mutants (Table 3). Although the resistance
levels of flumorph, dimethomorph and azoxystrobin
mutants were diverse, the EC50 values of all mutants
were not significantly changed compared with the
initial EC50 after ten generations on fungicide-free
leaves.
Pest Manag Sci 64:255–261 (2008) 257
DOI: 10.1002/ps
4. S Zhu et al.
Table 2. Resistance characteristics of Pseudoperonospora cubensis isolates obtained by adaptation on detached fungicide-treated cucumber
leaves for ten generations (R10) and their stability after ten successive generations on fungicide-free leaves (S10)
EC50 (mg L−1)bc RFd
Flumorph Dimethomorph Azoxystrobin
Isolatea R10 S10 R10 S10 R10 S10 FA DA AA
Ke17(C) 0.17b 0.15a 0.13b 0.14a 0.012b 0.011b – – –
Ke17-1(A) 0.34a 0.16a 0.32a 0.13a 0.360a 0.451a 2 2 30
T3(C) 0.26b 0.23b 0.24b 0.24b 0.019b 0.021b – – –
T3-1(A) 0.55a 0.31a 0.79a 0.88a 0.443a 0.424a 2 3 23
LP2(C) 0.10b 0.11a 0.12b 0.14b 0.014b 0.015b – – –
LP2-1(A) 0.16a 0.14a 0.49a 0.53a 0.231a 0.313a 3 4 16
M5(C) 0.20b 0.17b 0.18b 0.14b 0.023b 0.019b – – –
M5-1(A) 0.41a 0.24a 0.30a 0.27a 0.440a 0.413a 2 2 19
Mean – – – – – – 2b 3b 22a
a (C), wild-type isolates grown on fungicide-free leaves; (A), adapted isolates after ten generations on fungicide-treated leaves. b The first column
(R10) for each compound is the initial EC50 value of mutants to the fungicide, and the second column (S10) is the EC50 value of mutants after ten
successive subcultures on fungicide-free leaves. c Figures followed by the same letter were not significantly different between adapted isolates and
their parent isolate using Fisher’s LSD (P = 0.05). d RF (resistance factor) = EC50 value for fungitoxicity towards the adapted isolate divided by the
EC50 value for fungitoxicity towards the parent isolate; FA, DA and AA represent the isolates subcultured 10 times on flumorph-, dimethomorph-and
azoxystrobin-treated leaves, respectively.
Table 3. Resistance characteristics of UV-induced mutants of Pseudoperonspora.cubensis isolated after one generation (R1) on fungicide-treated
leaves and their stability after ten successive generations on fungicide-free leaves (S10)
EC50 (mg L−1)bc RFd
Flumorph Dimethomorph Azoxystrobin
Isolatea R1 S10 R1 S10 R1 S10 FU DU AU
Ke1(C) 0.17b 0.15b 0.13b 0.14b 0.012c 0.011c – – –
Ke17(UV1) 0.38a 0.41a 0.82a 0.79a 0.66b 0.57b 2 6 55
Ke17(UV2) 0.71a 0.77a 0.71a 0.80a 3.01a 3.09a 4 5 251
T3(C) 0.26b 0.23c 0.24c 0.24c 0.019c 0.021c – – –
T3(UV1) 1.30a 1.21b 1.96a 2.14a 5.36b 5.74b 5 8 282
T3(UV2) 1.84a 2.09a 1.34b 1.58b 13.93a 14.15a 7 6 733
LP2(C) 0.10c 0.11c 0.12c 0.14c 0.014c 0.015c – – –
LP2(UV1) 1.29b 1.22b 1.18b 1.03b 2.62b 2.89b 13 10 187
LP2(UV2) 6.49a 7.38a 10.6a 11.48a 4.93a 5.41a 65 88 352
M5(C) 0.21c 0.17c 0.18c 0.14c 0.023c 0.019c – – –
M5(UV1) 3.86b 3.72b 2.43b 2.08b 13.42a 11.58a 19 14 583
M5(UV2) 10.57a 11.04a 12.9a 12.19a 4.89b 5.14b 53 72 213
Mean – – – – – – 21b 28b 332a
a (C), wild-type isolates grown on fungicide-free leaves; (UV1) and (UV2), UV-induced mutants. b The first column (R1) for each compound is the initial
EC50 value of mutants to the fungicide, and the second column (S10) is the EC50 value of mutants after ten successive subcultures on fungicide-free
leaves. c Figures followed by the same letter were not significantly different between adapted isolates and their parent isolate using Fisher’s LSD
(P = 0.05). d RF (resistance factor) = EC50 value for fungitoxicity towards the adapted isolate divided by the EC50 value for fungitoxicity towards the
parent isolate; FU, DU and AU represent the mutants grown on flumorph-, dimethomorph- and azoxystrobin-treated leaves after mutation with UV
respectively.
3.3 Pathogenicity and sporulation
Compared with their parent isolates, the pathogenicity
and sporulation of all flumorph- and dimethomorph-resistant
mutants were significantly decreased. How-ever,
all azoxystrobin mutants retained the pathogenic-ity
and sporulation characteristics of the wild-type
parent isolates (Table 4).
3.4 Cross-resistance
All resistant mutants and their parent isolates were
chosen for cross-resistance studies. Cross-resistance
was present between flumorph and dimethomorph,
with a correlation coefficient of 0.95 (Fig. 1A).
However, a low correlation coefficient was found
between flumorph and azoxystrobin (Fig. 1B), cymox-anil
(Fig. 1C) and metalaxyl (Fig. 1D), indicating no
cross-resistance between them.
4 DISCUSSION
Isolating a mutant with a high level of resistance
to flumorph or dimethomorph was more difficult to
258 Pest Manag Sci 64:255–261 (2008)
DOI: 10.1002/ps
5. Risk of resistance in P. cubensis to the fungicide flumorph
Table 4. Pathogenicity and sporulation characteristics on detached healthy leaves of UV-induced mutants of Pseudoperonospora cubensis
previously grown in the presence of flumorph (F), DMM (D) or azoxystrobin (A) compared with the parent isolates
Pathogenicitybc (mm2) (± SE) Sporulationc (×103 sporangia cm−2)
Isolatea F D A F D A
Ke17(C) 120 (±27)a 120 (±27)a 120 (±27)a 22.41a 25.41a 25.41a
Ke17(UV1) 10 (±2)b 12 (±3)b 128 (±54)a 16.65b 17.88b 24.61a
Ke17(UV2) 13 (±5)b 10 (±3)b 142 (±38)a 17.24b 14.74b 21.27a
T3(C) 97 (±14)a 97 (±14)a 97 (±14)a 20.16a 20.16a 20.16a
T3(UV1) 21 (±7)b 16 (±7)b 108 (±24)a 12.28c 13.70b 21.02a
T3(UV2) 31 (±11)b 27 (±8)b 150 (±53)a 16.34b 14.57b 22.71a
LP2(C) 105 (±17)a 105 (±17)a 105 (±17)a 23.96a 23.96a 23.96a
LP2(UV1) 38 (±5)c 27 (±5)c 118 (±32)a 16.55b 16.42c 21.64a
LP2(UV2) 72 (±4)b 68 (±13)b 147 (±70)a 16.75b 19.50b 23.72a
M5(C) 102 (±34)a 102 (±34)a 102 (±34)a 26.29a 26.29a 26.29a
M5(UV1) 65 (±7)b 54 (±11)b 116 (±28)a 20.35b 21.91b 25.81a
M5(UV2) 83 (±12)b 73 (±17)b 126 (±47)a 19.77b 20.86b 27.38a
a (C), wild-type isolates grown on fungicide-free leaves; (UV1) and (UV2), UV-induced mutants. b The pathogenicity of isolates was assessed by their
lesion area on leaf discs. c Figures followed by the same letter within a column were not significantly different using Fisher’s LSD (P = 0.05).
Figure 1. Cross-resistance between flumorph and (A) dimethomorph, (B) azoxystrobin, (C) cymoxanil and (D) metalaxyl.
achieve by adaptation on fungicide-treated leaves than
it was for azoxystrobin. After repeated subculturing of
P. cubensis on cucumber leaves treated with a sublethal
concentration of flumorph or dimethomorph, the
sensitivity of all isolates slightly decreased, with
resistance factors of <4. The resistance levels of
some isolates to flumorph or dimethomorph were
significantly decreased after ten generations on
fungicide-free leaves, whichmay reflect a physiological
adaptation but not mutation. Although resistance
was stable for other isolates, their sensitivities were
only slightly reduced. This may reflect reduced
uptake, detoxification or overproduction of the target
protein.19 Previous data also showed that attempts
to generate mutants of P. infestans and P. capsici
resistant to dimethomorph and flumorph by mycelial
adaptation on fungicide-amended media failed.4,8,9,14
However, obtaining isolates resistant to azoxystrobin
by adaptation on fungicide-treated leaves was easier
than obtaining isolates resistant to flumorph and
dimethomorph. The resistant isolates obtained from
a sublethal concentration of azoxystrobin-treated
cucumber leaves were approximately 20 times less
sensitive than the wild-type isolates, but their
resistance was inhibited by the addition of 10mg L−1
salicylhydroxamate (SHAM) (unpublished data). This
result suggested that the occurrence of resistance was
the result of induction of an alternative oxidase (AOX)
but not the mutation of target in mutants.20,21
The development of resistance to flumorph,
dimethomorph and azoxystrobin in P. cubensis fol-lowing
UV exposure of sporangia was easier than after
repeated selection on sublethal treated leaves, but
there were differences in the ease with which mutants
were obtained as well as in the levels of resistance
and fitness among the flumorph-, dimethomorph- and
Pest Manag Sci 64:255–261 (2008) 259
DOI: 10.1002/ps
6. S Zhu et al.
azoxystrobin-resistant mutants. Attempts to obtain
mutants with a high level of resistance to dimetho-morph
were unsuccessful, with resistance factors of
<100 being recorded, in sharp contrast to experiments
with azoxystrobin, all of which yielded highly resistant
mutants with resistance factors of >150. Consistent
with this low level of resistance, all dimethomorph-resistant
mutants showed weaker fitness compared
with wild-type and azoxystrobin mutants. These
results are consistent with those of other researchers.
Field resistance to azoxystrobin has been well docu-mented,
and various studies have shown that resistant
mutants with a high level of resistance can be isolated
readily in the laboratory.16 However, only moder-ately
resistant mutants of P. parasitica and P. cap-sici
to dimethomorph were isolated using ultraviolet
and chemical mutagenesis respectively.10,11,14 For flu-morph,
attempts to isolate mutants with a high level
of resistance by UV mutagenesis were also unsuc-cessful.
Studies of the fitness of flumorph mutants
showed that, with the exception of stability of resis-tance,
the mutation(s) appeared to have low resistance
level, pathogenicity and sporulation compared with
wild-type isolates. The resistance level and fitness of
flumorph-resistant mutants were very similar to those
of dimethomorph-resistant mutants but lower than
those of azoxystrobin-resistant mutants.
The difference in resistance risk of P. cubensis to
CAA fungicides and QoI or phenylamides in vitro may
be due to their genetic difference. The resistance of
pathogen to phenylamides and QoI is controlled by
one semi-dominant nuclear gene and a mitochondrial
gene respectively, and thus the resistance risks of
pathogen to phenylamides and QoI fungicides are
high.22,23 A recent study with P. viticola showed that
resistance to CAA fungicides is controlled by recessive
nuclear genes, and hence resistance is expressed
only in homozygous offspring, which may require
several cycles of sexual reproduction to become fixed
and expressed in phenotypically aggressive isolates.13
Thus, the resistance risk of P. cubensis to flumorph and
dimethomorph is lower than to azoxystrob in vitro.
Under field conditions, however, the resistance
risk of P. cubensis to flumorph is high. After 6–8
successive applications of flumorph alone, resistant
isolates with a high level of resistance and good fitness
were easily detected.24 The difference in resistance
risk of P. cubensis to flumorph between laboratory and
field conditions may be due to the diploid nature
of oomycetes. For P. viticola and P. cubensis, the
occurrence of a sexual generation is unlikely when
only a single isolate is cultured in vitro, but they can
reproduce sexually in the field,25–27 and therefore
the chance of producing recessive resistance gene
homozygous mutants by sexual reproduction is higher
under field conditions and their resistance risk to CAA
fungicides is also higher than in the laboratory.
On the basis of the above data the intrinsic risk
and extent of resistance to flumorph in P. cubensis are
postulated to be moderate and considerably lower for
CAAs than for phenylamides and QoIs. Therefore, it
is expected that CAA resistance in P. cubensis can be
managed under field conditions by using appropriate
strategies such as a restricted number of applications
and the use of mixtures with non-cross-resistant fungi-cides.
The present cross-resistance results suggested
that there is cross-resistance between flumorph and
dimethomorph, but not with azoxystrobin, cymox-anil
or metalaxyl. This result was consistent with
previous reports and supported the hypothesis that
flumorph and dimethomorph have the same mode of
action.4 In addition, flumorph-resistant isolates also
show decreased sensitivity to another CAA fungicide,
iprovalicarb.24 Previous reports showed that popula-tions
of P. viticola can be found in certain regions that
are simultaneously resistant to dimethomorph, benthi-avalicarb,
iprovalicarb and mandipropamid.13 These
data indicated that there is cross-resistance between
flumorph and other CAA fungicides. Thus, flumorph
and other CAA fungicides could replace other fungi-cides
to manage resistance, with coapplication with
other fungicides to avoid or delay the occurrence of
resistance, but simultaneous usage of each should be
avoided owing to their cross-resistance.
ACKNOWLEDGEMENTS
This study was supported by the Shenyang Research
Institute of the Chemical Industry of China and the
National Science Foundation (Grant No. 30400294).
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