Assessing the Risk of Resistance in Pseudoperonospora cubensis to the Fungicide Flumorph in vitro (2006)

Pest Management Science Pest Manag Sci 64:255–261 (2008)

Assessing the risk of resistancein Pseudoperonospora cubensis to thefungicide flumorph in vitroShusheng Zhu,1,2 Pengfei Liu,1 Xili Liu,1∗ Jianqiang Li,1 Shankui Yuan3 andNaiguo Si41Department of Plant Pathology, China Agricultural University, Beijing 100094, China2Key Laboratory of Agriculture Biodiversity for Plant Disease Management, Ministry of Education, Key Laboratory of Plant Pathology,Yunnan Agricultural University, Kunming 650201, China3Centre of Agrochemicals for Biological and Environmental Technology Institute for the Control of Agrochemicals, Ministry of Agriculture,Beijing 100026, China4China 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 withdimethomorph and azoxystrobin with respect to the ease of obtaining resistant isolates to these fungicides, thelevel of resistance and their fitness in the laboratory.

RESULTS: Mutants with a high level of resistance to azoxystrobin were isolated readily by adaptation andUV irradiation, and their fitness was as good as that of the parent isolates. Attempts to generate mutantsof Pseudoperonospora cubensis (Burk. & MA Curtis) Rostovsev resistant to flumorph and dimethomorph bysporangia adaptation on fungicide-treated leaves were unsuccessful. However, moderately resistant mutants wereisolated using UV mutagenesis, but their resistance level [maximum resistance factor (MRF) < 100] was muchlower than that of the azoxystrobin-resistant mutant (MRF = 733). With the exception of stability of resistance, allmutants showed low pathogenicity and sporulation compared with wild-type isolates and azoxystrobin-resistantmutants. There is cross-resistance between flumorph and dimethomorph, suggesting that they have the sameresistance mechanism.

CONCLUSION: The above results suggest that the resistance risk of flumorph may be similar to that ofdimethomorph but lower than that of azoxystrobin and can be classified as moderate. Thus, it can be managed byappropriate product use strategies. 2007 Society of Chemical Industry

Keywords: cucumber downy mildew; fungicide resistance; flumorph; dimethomorph; azoxystrobin

1 INTRODUCTIONFlumorph, a recently introduced carboxylic acidamide (CAA) fungicide, was developed by ShenyangResearch Institute of Chemical Industry of Chinafor the control of oomycete pathogens and hasbeen patented in China (ZL.96115551.5), the USA(US6020332) and Europe (0 860 438B1).1 It exhibitsa very high level of protective and curative activityagainst members of the family Peronosporaceae andthe genus Phytophthora but not Pythium.2 In China,field resistance to a major systemic fungicide class,such as the phenylamides, has occurred widely insome species of plant pathogenic oomycetes.3–5 Thus,the development of flumorph was expected to replacethe 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 pathogensto dimethomorph is diverse. For some Phytophthorapathogens, such as P. infestans (Mont.) de Bary,7–9

P. capsici Leonian and P. parasitica Dastur,8,10,11 theresistance risk to dimethomorph may be low, based onlaboratory 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 ofEurope.13 The risk of Phytophthora spp. being resistantto 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, ChinaE-mail: [email protected](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

S Zhu et al.

P. capsici being selected after ultraviolet treatment.4,14

The field performance of flumorph-based fungicideshas remained excellent for control of potato light blightin China.4,5,15 However, the resistance risk of downymildew causal agents to flumorph in the field is stillunclear.

In order to define the risk of resistance develop-ment of cucumber downy mildew, Pseudoperonosporacubensis (Berk. & MA Curtis) Rostovsev, in the fieldto flumorph, laboratory studies were conducted. Inthis study, flumorph was compared with two com-mercial systemic oomycete fungicides, dimethomorphand azoxystrobin, both with well-understood resis-tance potential,7–12,16 to determine (i) the ease ofisolating resistant mutants using ultraviolet (UV) lightmutagenesis and sporangia adaptation, (ii) the level ofresistance that can be induced and (iii) the fitness ofmutants on cucumber leaves. Based on these data, theresistance risk of P. cubensis to flumorph was defined.

2 MATERIALS AND METHODS2.1 IsolatesFour P. cubensis isolates were collected from differentgeographical districts (Table 1) where no CAA fungi-cides had been applied, and these were maintainedby weekly transfers to detached leaves on wet filterpaper in petri dishes at 20 ◦C with a 12:12 h light:darkphotoperiod.

2.2 ChemicalsTechnical-grade flumorph (96%), dimethomorph(97%), azoxystrobin (95%), cymoxanil (98%) andmetalaxyl (98%) were kindly supplied by ShenyangResearch Institute of Chemical Industry of China(Shengyang, China), Genyun Co., Ltd (Jiangsu,China), Syngenta China Ltd (Beijing, China), Wan-quan Co., Ltd (Hebei, China) and Agrolex P. Ltd (Bei-jing, China) respectively. Stock solutions of 10 g L−1

of active ingredient of each fungicide were made inmethanol and stored at 4 ◦C in darkness. For sensi-tivity testing, stocks of fungicides were serially dilutedwith double-distilled water containing 0.05 mL L−1

Tween 20. The maximum concentration of methanolused in treatment solutions was less than 1 mL L−1.

2.3 Sensitivity assaysFungicide sensitivity was determined as describedpreviously.17 Briefly, leaf discs (15 mm diameter) werecut with a cork borer from healthy leaves (the second

from the tip) of four-true-leaf stage greenhouse-growncucumber plants (cv. Changchunmici). All leaf discswere randomized and placed into containers to which50 mL of each fungicide solution was added. Controldiscs were treated with distilled water containing1 ml L−1 methanol and 0.05 mL L−1 Tween 20.After 30 min soaking, the leaf discs were removedfrom the container and blotted dry with papertowel. There were 30 leaf discs in three replicatesfor every concentration of each fungicide. Freshsporangia of P. cubensis were harvested from diseasedcucumber leaves into cold water (4 ◦C). Leaf discswere inoculated by placing one drop (10 µL) ofinoculum (1 × 104 sporangia mL−1) on the middle ofeach disc. Dishes containing leaf discs were incubatedat 20 ◦C for 20 h in a humid chamber in darkness toallow infection, and then maintained at 20 ◦C with a12 h photoperiod for disease development. Six daysafter inoculation, the mean percentage of sporulatingsurface area on the leaf discs was determined. Themedian effective concentration value (EC50) for eachisolate was calculated by regressing the percentage ofgrowth inhibition against the logarithm value of thefungicide concentration using the software MicrosoftExcel 2003. The tests were replicated 3 times for eachisolate.

2.4 Induction of resistant isolates2.4.1 Sporangia selection by adaptationSporangia suspensions (1 × 104 mL−1) were preparedfor each of the four wild-type isolates (Table 1)and were sprayed on cucumber leaves treated withflumorph (0.70 mg L−1), dimethomorph (0.50 mgL−1) or azoxystrobin (0.07 mg L−1), and thenincubated for 6 days as described above. The fungicideconcentration used for selection had previouslybeen found to be highly inhibitory yet sublethalfor all isolates of P. cubensis. The same sporangiasuspensions were also sprayed onto fungicide-freeleaves as control. Newly produced sporangia fromeach treatment were used to subculture the isolateonto new healthy cucumber leaves, treated with thesame concentration of flumorph, dimethomorph orazoxystrobin, for a total of ten generations. After thefinal transfer on fungicide-treated leaves, sporangiawere cycled once on fungicide-free leaves and theEC50 was then calculated for each of the fungicide-exposed and control isolates according to the abovemethod.

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

Risk of resistance in P. cubensis to the fungicide flumorph

2.4.2 Induction by UV mutagenesisThe method utilized to isolate mutants from UV-mutated sporangia was based on procedures previouslydescribed.10 Four wild-type isolates (Table 1) formutagenesis were grown on healthy cucumberleaves until new sporangia were produced. UVmutagenesis was performed with a UV lamp ata wavelength of 254 nm by irradiating suspensionsof sporangia (1 × 105 sporangia mL−1) in open petridishes (9 cm diameter) for 1 min at a distance of30 cm. After irradiation, they were kept for 30 minin the dark to minimize photorepair of radiationdamage. Sporangia suspensions were then sprayedonto flumorph-treated (10 mg L−1), dimethomorph-treated (10 mg L−1) or azoxystrobin-treated (1 mgL−1) cucumber leaves which did not support thegrowth of wild-type isolates, as well as onto fungicide-free leaves. Wild-type sporangia suspensions thathad not been exposed to UV were also includedin each experiment. All leaves were incubatedovernight in a humid chamber in darkness toallow infection, and then maintained at 20 ◦C witha 12:12 h light:dark photoperiod. After 6 days,the number of lesions was examined, and twolesions with the most vigorously growing sporangiaon fungicide-treated leaves were cycled once onfungicide-free leaves. Subsequently, the resistancelevel was determined by calculating the EC50 valuefor each UV-mutant isolate compared with the parentisolate.

2.5 Characteristics of resistant mutants2.5.1 Stability of resistant isolatesAfter the initial sensitivities of mutants to fungicideshad been determined, all mutants were maintainedby weekly transfers to fungicide-free leaves. After thetenth generation, sensitivity to flumorph was estimatedagain using the method in Section 2.3 and comparedwith the initial EC50 to give an indication of thestability of the acquired resistance trait.

2.5.2 Pathogenicity and sporulationConsidering the low stability recorded for fungicide-adapted isolates, pathogenicity and sporulation weredetermined for UV-induced mutants only and werecompared with those of the parent isolates oncucumber leaf discs. A sporangial suspension (1 ×104 sporangia mL−1) of each sensitive or resistantisolate was inoculated onto 30 leaf discs and incubatedaccording to the method in Section 2.3, and thelesion area on each leaf disc was determined. Eachof the three sets of ten discs was placed in a 15 mLcentrifuge tube containing 10 mL distilled water andmechanically agitated for 15 s. The sporangia releasedwere quantified with a haemocytometer, and the meannumber of sporangia cm−2 of lesion was calculated.

2.5.3 Cross-resistanceThe 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 meanpercentage of sporulating surface area on theleaf 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 comparedand cross-resistance was analysed using regressionanalysis.18

3 RESULTS3.1 Selection by adaptation onfungicide-treated leavesThe lesion areas of all isolates on leaves treatedwith sublethal doses of fungicide were significantlysmaller than those of the control, even if thelesion area increased with subculture number forall isolates. The isolates grown on azoxystrobin-treated leaves demonstrated greatly reduced sen-sitivity, whereas isolates that were grown onflumorph- or dimethomorph-treated leaves showedrelatively little or no change in sensitivity (Table 2).The average resistance factors (RFs) of isolatesgrown on either flumorph- or dimethomorph-treated leaves were all <5 and were significantlylower than those for azoxystrobin, where averageRF values were >20 (Table 2). All azoxystrobin-resistant isolates were stable, whereas two offour flumorph-resistant isolates and one of fourdimethomorph-resistant isolates lost their resistance(Table 2).

3.2 Isolation of resistant mutants by UVirradiation and selection on fungicide-treatedleavesAfter UV mutagenesis, approximately 20–30% of spo-rangia could still infect fungicide-free leaves comparedwith untreated controls. On fungicide-treated leaves,however, only UV-mutated sporangia could causesymptoms, but lesion areas were small. The averagenumber of lesions on flumorph- and dimethomorph-treated leaves was 1.5 and 2 respectively. Thiswas significantly lower than the average number oflesions on azoxystrobin-treated leaves, which was 5(P = 0.05). The sensitivity of all mutants grown onazoxystrobin-treated leaves had greatly decreased, withRF values of >100 (Table 3). For flumorph anddimethomorph, all mutants also showed reduced sen-sitivity, but there were only two flumorph-resistantmutants and two dimethomorph-resistant mutantsshowing RF values above 50, and their mean RFvalue was much lower than that of the azoxystrobin-resistant mutants (Table 3). Although the resistancelevels of flumorph, dimethomorph and azoxystrobinmutants were diverse, the EC50 values of all mutantswere not significantly changed compared with theinitial EC50 after ten generations on fungicide-freeleaves.

Pest Manag Sci 64:255–261 (2008) 257DOI: 10.1002/ps

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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 30T3(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 23LP2(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 16M5(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 19Mean – – – – – – 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 tensuccessive subcultures on fungicide-free leaves. c Figures followed by the same letter were not significantly different between adapted isolates andtheir parent isolate using Fisher’s LSD (P = 0.05). d RF (resistance factor) = EC50 value for fungitoxicity towards the adapted isolate divided by theEC50 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 55Ke17(UV2) 0.71a 0.77a 0.71a 0.80a 3.01a 3.09a 4 5 251T3(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 282T3(UV2) 1.84a 2.09a 1.34b 1.58b 13.93a 14.15a 7 6 733LP2(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 187LP2(UV2) 6.49a 7.38a 10.6a 11.48a 4.93a 5.41a 65 88 352M5(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 583M5(UV2) 10.57a 11.04a 12.9a 12.19a 4.89b 5.14b 53 72 213Mean – – – – – – 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 initialEC50 value of mutants to the fungicide, and the second column (S10) is the EC50 value of mutants after ten successive subcultures on fungicide-freeleaves. 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 theparent isolate; FU, DU and AU represent the mutants grown on flumorph-, dimethomorph- and azoxystrobin-treated leaves after mutation with UVrespectively.

3.3 Pathogenicity and sporulationCompared with their parent isolates, the pathogenicityand 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-typeparent isolates (Table 4).

3.4 Cross-resistanceAll resistant mutants and their parent isolates werechosen 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 foundbetween flumorph and azoxystrobin (Fig. 1B), cymox-anil (Fig. 1C) and metalaxyl (Fig. 1D), indicating nocross-resistance between them.

4 DISCUSSIONIsolating a mutant with a high level of resistanceto flumorph or dimethomorph was more difficult to



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 25.41aKe17(UV1) 10 (±2)b 12 (±3)b 128 (±54)a 16.65b 17.88b 24.61aKe17(UV2) 13 (±5)b 10 (±3)b 142 (±38)a 17.24b 14.74b 21.27aT3(C) 97 (±14)a 97 (±14)a 97 (±14)a 20.16a 20.16a 20.16aT3(UV1) 21 (±7)b 16 (±7)b 108 (±24)a 12.28c 13.70b 21.02aT3(UV2) 31 (±11)b 27 (±8)b 150 (±53)a 16.34b 14.57b 22.71aLP2(C) 105 (±17)a 105 (±17)a 105 (±17)a 23.96a 23.96a 23.96aLP2(UV1) 38 (±5)c 27 (±5)c 118 (±32)a 16.55b 16.42c 21.64aLP2(UV2) 72 (±4)b 68 (±13)b 147 (±70)a 16.75b 19.50b 23.72aM5(C) 102 (±34)a 102 (±34)a 102 (±34)a 26.29a 26.29a 26.29aM5(UV1) 65 (±7)b 54 (±11)b 116 (±28)a 20.35b 21.91b 25.81aM5(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 theirlesion 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 thanit was for azoxystrobin. After repeated subculturing ofP. cubensis on cucumber leaves treated with a sublethalconcentration of flumorph or dimethomorph, thesensitivity of all isolates slightly decreased, withresistance factors of <4. The resistance levels ofsome isolates to flumorph or dimethomorph weresignificantly decreased after ten generations onfungicide-free leaves, which may reflect a physiologicaladaptation but not mutation. Although resistancewas stable for other isolates, their sensitivities wereonly slightly reduced. This may reflect reduceduptake, detoxification or overproduction of the targetprotein.19 Previous data also showed that attemptsto generate mutants of P. infestans and P. capsiciresistant to dimethomorph and flumorph by mycelialadaptation on fungicide-amended media failed.4,8,9,14

However, obtaining isolates resistant to azoxystrobin

by adaptation on fungicide-treated leaves was easierthan obtaining isolates resistant to flumorph anddimethomorph. The resistant isolates obtained froma sublethal concentration of azoxystrobin-treatedcucumber leaves were approximately 20 times lesssensitive than the wild-type isolates, but theirresistance was inhibited by the addition of 10 mg L−1

salicylhydroxamate (SHAM) (unpublished data). Thisresult suggested that the occurrence of resistance wasthe 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 afterrepeated selection on sublethal treated leaves, butthere were differences in the ease with which mutantswere obtained as well as in the levels of resistanceand fitness among the flumorph-, dimethomorph- and


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azoxystrobin-resistant mutants. Attempts to obtainmutants with a high level of resistance to dimetho-morph were unsuccessful, with resistance factors of<100 being recorded, in sharp contrast to experimentswith azoxystrobin, all of which yielded highly resistantmutants with resistance factors of >150. Consistentwith this low level of resistance, all dimethomorph-resistant mutants showed weaker fitness comparedwith wild-type and azoxystrobin mutants. Theseresults are consistent with those of other researchers.Field resistance to azoxystrobin has been well docu-mented, and various studies have shown that resistantmutants with a high level of resistance can be isolatedreadily in the laboratory.16 However, only moder-ately resistant mutants of P. parasitica and P. cap-sici to dimethomorph were isolated using ultravioletand chemical mutagenesis respectively.10,11,14 For flu-morph, attempts to isolate mutants with a high levelof resistance by UV mutagenesis were also unsuc-cessful. Studies of the fitness of flumorph mutantsshowed that, with the exception of stability of resis-tance, the mutation(s) appeared to have low resistancelevel, pathogenicity and sporulation compared withwild-type isolates. The resistance level and fitness offlumorph-resistant mutants were very similar to thoseof dimethomorph-resistant mutants but lower thanthose of azoxystrobin-resistant mutants.

The difference in resistance risk of P. cubensis toCAA fungicides and QoI or phenylamides in vitro maybe due to their genetic difference. The resistance ofpathogen to phenylamides and QoI is controlled byone semi-dominant nuclear gene and a mitochondrialgene respectively, and thus the resistance risks ofpathogen to phenylamides and QoI fungicides arehigh.22,23 A recent study with P. viticola showed thatresistance to CAA fungicides is controlled by recessivenuclear genes, and hence resistance is expressedonly in homozygous offspring, which may requireseveral cycles of sexual reproduction to become fixedand expressed in phenotypically aggressive isolates.13

Thus, the resistance risk of P. cubensis to flumorph anddimethomorph is lower than to azoxystrob in vitro.

Under field conditions, however, the resistancerisk of P. cubensis to flumorph is high. After 6–8successive applications of flumorph alone, resistantisolates with a high level of resistance and good fitnesswere easily detected.24 The difference in resistancerisk of P. cubensis to flumorph between laboratory andfield conditions may be due to the diploid natureof oomycetes. For P. viticola and P. cubensis, theoccurrence of a sexual generation is unlikely whenonly a single isolate is cultured in vitro, but they canreproduce sexually in the field,25–27 and thereforethe chance of producing recessive resistance genehomozygous mutants by sexual reproduction is higherunder field conditions and their resistance risk to CAAfungicides is also higher than in the laboratory.

On the basis of the above data the intrinsic riskand extent of resistance to flumorph in P. cubensis arepostulated to be moderate and considerably lower for

CAAs than for phenylamides and QoIs. Therefore, itis expected that CAA resistance in P. cubensis can bemanaged under field conditions by using appropriatestrategies such as a restricted number of applicationsand the use of mixtures with non-cross-resistant fungi-cides. The present cross-resistance results suggestedthat there is cross-resistance between flumorph anddimethomorph, but not with azoxystrobin, cymox-anil or metalaxyl. This result was consistent withprevious reports and supported the hypothesis thatflumorph and dimethomorph have the same mode ofaction.4 In addition, flumorph-resistant isolates alsoshow decreased sensitivity to another CAA fungicide,iprovalicarb.24 Previous reports showed that popula-tions of P. viticola can be found in certain regions thatare simultaneously resistant to dimethomorph, benthi-avalicarb, iprovalicarb and mandipropamid.13 Thesedata indicated that there is cross-resistance betweenflumorph and other CAA fungicides. Thus, flumorphand other CAA fungicides could replace other fungi-cides to manage resistance, with coapplication withother fungicides to avoid or delay the occurrence ofresistance, but simultaneous usage of each should beavoided owing to their cross-resistance.

ACKNOWLEDGEMENTSThis study was supported by the Shenyang ResearchInstitute of the Chemical Industry of China and theNational Science Foundation (Grant No. 30400294).

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