Pest Management Science Pest Manag Sci 64:255–261 (2008) 
Assessing the risk of resistance 
in Pseudoperonospora cubensis ...
S Zhu et al. 
P. capsici being selected after ultraviolet treatment.4,14 
The field performance of flumorph-based fungicid...
Risk of resistance in P. cubensis to the fungicide flumorph 
2.4.2 Induction by UV mutagenesis 
The method utilized to iso...
S Zhu et al. 
Table 2. Resistance characteristics of Pseudoperonospora cubensis isolates obtained by adaptation on detache...
Risk of resistance in P. cubensis to the fungicide flumorph 
Table 4. Pathogenicity and sporulation characteristics on det...
S Zhu et al. 
azoxystrobin-resistant mutants. Attempts to obtain 
mutants with a high level of resistance to dimetho-morph...
Risk of resistance in P. cubensis to the fungicide flumorph 
11 Chabane K, Leroux P andMaia N, Resistance toDMMin labo-rat...
Nächste SlideShare
Wird geladen in …5
×

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

408 Aufrufe

Veröffentlicht am

DOI 10.1111/j.1365-3059.2005.01338.x

Veröffentlicht in: Wissenschaft
0 Kommentare
0 Gefällt mir
Statistik
Notizen
  • Als Erste(r) kommentieren

  • Gehören Sie zu den Ersten, denen das gefällt!

Keine Downloads
Aufrufe
Aufrufe insgesamt
408
Auf SlideShare
0
Aus Einbettungen
0
Anzahl an Einbettungen
2
Aktionen
Geteilt
0
Downloads
4
Kommentare
0
Gefällt mir
0
Einbettungen 0
Keine Einbettungen

Keine Notizen für die Folie

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

  1. 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. 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. 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. 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. 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. 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). REFERENCES 1 Liu WC, Li ZL, Zhang ZJ and Liu CL, Antifungal activity and prospect of flumorph and its mixtures. Zhejiang Chemical Industry 31:(Suppl.): 87–88 (2000). 2 Liu CW and Liu CL, New high efficiency fungicide flumorph. Journal of Pesticides 41:8–11 (2001). 3 Zhang ZM, Li YQ, Zhu JH and Tian SM, Preliminary study on metalaxyl-resistant strains of Phytophthora infestans in China. Proc Int Workshop on Potato Late Blight, Pyongchang, Ganwon, Korea, pp. 101–105 (2001). 4 Yuan SK, Liu XL, Si NG, Dong J, Gu BG and Jiang H, Sensitivity of Phytophthora infestans to flumorph: in vitro determination of baseline sensitivity and the risk of resistance. Plant Pathol 55:258–263 (2006). 5 Zhu XQ, Che XB, Guo LY and Wang YH, Mating type of Phytophthora infestans from six provinces (cities) in China and their sensitivity to several fungicides. Plant Prot 30:20–23 (2004). 6 Zhu SS, Yuan SK, Fan JR, Wang Y, Liu M and Liu XL, The effect of six fungicides on different developmental stages in the life cycle of Pseudoperonospora cubensis. Chinese J Pestic Sci 7:119–125 (2005). 7 Bagirova SF, Li AZ, Dolgova AV, Elansky SN, Shaw DS and Dyakov YT, Mutants of Phytophthora infestans resistant to DMM fungicide. J Russian Phytopathol Soc 2:9–18 (2001). 8 Young DH, Spiewak SL and Slawecki RA, Laboratory studies to assess the risk of development of resistance to zoxamide. Pest Manag Sci 57:1081–1087 (2001). 9 Stein JM and Kirk WW, The generation and quantification of resistance to dimethomorph in Phytophthora infestans. Plant Dis 88:930–934 (2004). 10 Chabane K, Leroux P and Bompeix G, Selection and charac-terization of Phytophthora parasitica mutants with ultraviolet-induced resistance to dimethomorph or metalaxyl. Pestic Sci 39:325–329 (1993). 260 Pest Manag Sci 64:255–261 (2008) DOI: 10.1002/ps
  7. 7. Risk of resistance in P. cubensis to the fungicide flumorph 11 Chabane K, Leroux P andMaia N, Resistance toDMMin labo-ratory mutants of Phytophthora parasitica, in Modern Fungicides and Antifungal Compounds, ed. by Lyr H, Russell PE and Sisler HD. Intercept Limited, Andover, UK, pp. 387–391 (1996). 12 Dereviagina MK, Elanskij SN and Dyakov YT, Resistance of Phytophthora infestans to the DMM fungicides. Mikologiya Fitopatologiya 33:208–213 (1999). 13 Gisi U, Waldner M, Kraus N, Dubuis PH and Sierotzki H, Inheritance of resistance to carboxylic acid amide (CAA) fungicides in Plasmopara viticola. Plant Pathol 56:199–208 (2007). 14 Yuan SK, Liu XL, Gu BG, Dong J, Jiang H and Si NG, Induction and characterization of laboratory mutants of Phytophthora capsici resistant to DMM and flumorph. Agricultural Sciences in China 4:101–105 (2005). 15 Wang YH, Guo LY, Liang DL and Zhu XQ, Mating type dis-tribution and sensitivity to several chemicals of Phytophthora infestans from Inner Mongolia and Gansu province, China. J China Agricultural University 8:78–82 (2003). 16 Bartlett DW, Clough JM, Godwin JR, Hall AA, Hamer M and Parr-Dobrzanski B, The strobilurin fungicides. Pest Manag Sci 58:649–662 (2002). 17 Wong FP and Wilcox WF, Distribution of baseline sensitivities to azoxystrobin among isolates of Plasmopara viticola. Plant Dis 84:275–281 (2000). 18 Wong FP and Wilcox WF, Sensitivity to azoxystrobin among isolates of Uncinula necator: baseline distribution and relationship to myclobutanil sensitivity. Plant Dis 86:394–404 (2002). 19 Dekker J, Development of resistance to modern fungicides and strategies for its avoidance, in Modern Selective Fungicides: Properties, Applications, Mechanisms of Action, ed. by LyrH Gustav Fischer Verlag, Jena, Germany, pp. 23–38 (1995). 20 Ziogas BN, Baldwin BC and Young JE, Alternative respiration: a biochemical mechanism of resistance to azoxystrobin (ICIA5504) in Septoria tritici. Pestic Sci 50:28–34 (1997). 21 Wood PM and Hollomon D, A critical evaluation of the role of alternative oxidase in the performance of strobilurin and related fungicides acting at the Qo site of complex III. Pest Manag Sci 59:499–511 (2003). 22 Fabritius A-L, Shattock RC and Judelson HS, Genetic analysis of metalaxyl insensitivity loci in Phytophthora infestans using linked DNA markers. Phytopathology 87:1034–1340 (1997). 23 Ziogas BN, Markoglou AN and Tzima A, A non-Mendelian inheritance of resistance to strobilurin fungicides in Ustilago maydis. Pest Manag Sci 58:908–916 (2002). 24 Zhu SS, Liu XL, Liu PF, Li JQ, Yuan SK and Si NG, Resis-tance risk of Pseudoperonospora cubensis to flumorph in cucum-ber plastic houses. Plant Pathol 56:967–975 (2007). 25 Tran Manh Sung C, Strizyk S and Clerjeau M, Simulation of the date of maturity of Plasmopara viticola oospores to predict the severity of primary infections in grapevine. Plant Dis 74:120–124 (1990). 26 Wong FP, Burr HN and Wilcox WF, Heterothallism in Plas-mopara viticola. Plant Pathol 50:427–432 (2001). 27 Guo MH and Li H, Overwintering survival and influencing factors of oospores of Plasmopara viticola. Chinese Agricultural Science Bulletin 21:358–361 (2005). Pest Manag Sci 64:255–261 (2008) 261 DOI: 10.1002/ps

×