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Keywords:

  • flumorph;
  • fungicide resistance;
  • inheritance;
  • Phytophthora capsici

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Baseline sensitivity to flumorph, a carboxylic acid amide (CAA) fungicide used to control some oomycetes, was examined using 83 Phytophthora capsici isolates, resulting in a unimodal distribution of effective concentration for 50% inhibition of mycelial growth ranging from 0·716 to 1·363, with a mean of 1·033 ± 0·129 μg mL−1. To assess the potential risk of developing flumorph resistance, 13 flumorph-resistant mutants of P. capsici were obtained using ultraviolet irradiation. Most of these mutants and their progeny had high levels of fitness, including mycelial growth, sporulation and virulence. The resistance to flumorph changed slightly, either increasing or decreasing, after 10 transfers on agar media. Cross-resistance was found between flumorph and other CAA fungicides (dimethomorph and iprovalicarb), but not between flumorph and non-CAA fungicides (cymoxanil, metalaxyl, azoxystrobin and cyazofamid). To investigate the genetics of the flumorph resistance, 619 progeny were obtained by self-crossing and sexual hybridization. Segregation of sensitivity to fungicide was measured as a ratio of sensitive (S) to resistant (R) isolates. Segregation of the progeny, from self-crossed isolate PCAS1 (flumorph resistant), was 1:15 in the first generation; and 0:1 or 1:15 in the second generation. In sexual hybridization, segregation of progeny was 0:1 and 1:7 for R × R hybridization; and 1:3 for R × S hybridization. Therefore, the resistance of P. capsici against flumorph was controlled by two dominant genes.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Phytophthora capsici is a destructive oomycete pathogen to many vegetable crops, especially those in the Solanaceae and Cucurbitaceae families. The pathogen has been found everywhere in the world (Leonian, 1922; Erwin & Ribeiro, 1996; Hausbeck & Lamour, 2004). Phytophthora capsici can infect all parts of the plant, including the roots, crown, leaves and fruits. The pathogen is a soil inhabitant which is disseminated through soil and irrigation water (Granke et al., 2009). The disease cycle can be completed in a month or days if weather conditions are favourable. Controlling the disease is highly dependent on fungicides because there are not many effective cultural methods or resistant varieties available (Oelke et al., 2003; Hausbeck & Lamour, 2004).

Although fungicides are effective, their frequent applications can increase the possibility of fungicide resistance in the pathogen, especially for site-specific fungicides. For example, mefenoxam and metalaxyl have been widely used for managing oomycete diseases, but resistance to these fungicides has developed quickly in many oomycetes (Reuveni et al., 1980; Lambert & Salas, 1994; Gisi & Cohen, 1996), resulting in the fungicide treatment being less effective. Furthermore, because of cross-resistance occurring among similar fungicides, resistance to a specific fungicide can be developed even before that particular fungicide is used in an area.

Flumorph is another chemical that has been used for controlling Phytophthora spp. (Yuan et al., 2006). This systemic fungicide belongs to the carboxylic acid amides (CAA) group, which includes dimethomorph, flumorph, benthiavallicarb, iprovalicarb, valiphenal and mandipropamid (Russell, 2006), with high levels of protective and curative activities against some oomycete pathogens (Miyake et al., 2005; Cohen et al., 2007; Gisi et al., 2007). Knowing the risk for flumorph resistance development will help delay its development in the field, optimize application strategies and anticipate the risk of resistance to other CAA fungicides (Keinath, 2007; Zhu et al., 2008). However, the risk of resistance in P. capsici to this fungicide is not well understood, although it has been reported in Pseudoperonospora cubensis (Zhu et al., 2007, 2008) and P. infestans (Yuan et al., 2006).

Some genetic mechanisms of fungicide resistance in several oomycetes have been determined. For example, resistance to phenylamide fungicides in Phythophthora spp. is controlled by a single, semidominant gene (Shattock, 1988; Bhat et al., 1993; Gisi & Cohen, 1996; Knapova & Gisi, 2002), which resulted in phenylamide resistance developing quickly. The resistance of Plasmopara viticola to CAA fungicides is controlled by two recessive genes (Gisi et al., 2007). No such information appears to be available for P. capsici. Thus, it is of interest to investigate the genetics of P. capsici resistance to flumorph to evaluate the resistance risk.

In this study, the objectives were to (i) establish baseline sensitivity of P. capsici to flumorph; (ii) obtain mutants of P. capsici resistant to flumorph; (iii) study the biological characteristics of the mutants and their progeny; (iv) examine cross-resistance between flumorph and other fungicides in the mutants; and (v) investigate the inheritance of P. capsici resistance to flumorph. A preliminary report of this work has been published (Meng et al., 2008).

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Isolates of P. capsici and fungicides

All isolates used in this study were maintained on PDA medium (200 g potato, 18 g dextrose, 14 g agar and distilled water to 1 L) (Fang, 1977) in 9-cm Petri dishes at 25°C. Phytophthora capsici was isolated from infected pepper (Capsicum annuum) plants in six cities or provinces in China: Anhui, Beijing, Fujian, Hebei, Inner Mongolia and Shannxi, where no CAA fungicides had been applied. To generate flumorph-resistant mutants, 16 isolates were randomly chosen from these collections, and two standard P. capsici isolates, PCAS1 (P1314, mating type A1) and PCAS2 (P1319, mating type A2), originally collected from diseased plants, were kindly provided by Professor Michael Coffey (University of California, Riverside, USA). These isolates were single-spore cultured and kept on PDA immersed in mineral oil in the dark at room temperature for long-term storage. To transfer P. capsici cultures in the study, mycelial plugs (5 mm in diameter) were cut from agar plates with a cork borer and transferred with a needle. Mycelial growth was determined by measuring diameters of cultures with a ruler. Values given are the average of two diameter measurements taken perpendicularly. The above methods and materials were used throughout the experiments, unless otherwise noted.

Technical-grade flumorph (96% purity) was provided by the Research Institute of Chemical Industry (Shenyang, China). Dimethomorph (95%; Frey Agrochemicals Ltd), iprovalicarb (98%; Sigma-Aldrich Shanghai Trading Co. Ltd), cymoxanil (98%; Xinyi Agrochemicals Company), metalaxyl (97%; Agrolex P. Ltd), azoxystrobin (96%; Syngenta (China) Investment Co. Ltd), cyazofamid (96%; Sigma-Aldrich Shanghai Trading Co. Ltd) were all obtained from indicated manufacturers or distributors. All these chemicals were dissolved in acetone to make stock solutions (10 mg mL−1) and stored at 4°C in the dark until use. All were used within 3 months.

Baseline sensitivity of P. capsici to flumorph in vitro

A series of concentrations of flumorph were incorporated into PDA plates (non-fungicide-amended plates were used as a control), which contained the same concentration of acetone (0·1% by volume). Mycelial plugs (5 mm in diameter) of P. capsici from the leading edge of an actively growing colony were transferred onto flumorph-amended PDA plates. Each isolate was tested on triplicate plates and incubated at 25°C for 4 days in darkness. Colony diameter (with subtraction of the plug diameter) was measured perpendicularly and averaged for each treatment and expressed as the percentage of growth inhibition. Median effective concentration value (EC50) was calculated by regressing the percentage of growth inhibition against the logarithmic values of fungicide concentration. Baseline sensitivity was determined according to the EC50 values of all isolates.

Generation of P. capsici mutants and their sensitivity to flumorph

Mutagenesis

Both mycelial plugs and zoospores (see next paragraph) of P. capsici isolates, including PCAS1, PCAS2 and 16 other isolates from China, were used to produce mutants, following Rubin et al. (2008), with slight modification. Based on the baseline sensitivity of P. capsici to flumorph, PDA amended with 5 μg mL−1 flumorph (PDA-F5) was used as a standard selective medium for screening for fungicide-resistant mutants. For mycelial mutation, a 5-day-old culture was placed under an ultraviolet (UV) (Reuveni et al., 1980) light (20 W, 254 nm) at a distance of 20 cm for 1 min, followed by 30 min of incubation in the dark at 25°C to avoid light repairing the DNA. The UV-treated culture was cut randomly into 2-mm (diameter) mycelial plugs using a cork borer and then placed on PDA-F5, with mycelia facing to the medium (Fig. 1).

image

Figure 1.  Procedure for selecting flumorph-resistant mutants and investigating segregation of resistance in single-spore isolates of Phytophthora capsici. Isolate identifiers are preceded by their sensitivity to flumorph in brackets (S, sensitive; R, resistant). ST, sensitivity test; RM, resistant mutants; UV, ultraviolet; MTD, mating type determination; CR, crossing; SCR, self-crossing; S0, S1, S2: parental isolates and first generation and second generation of progeny of self-crossing hybridization, respectively; F0, F1: parental isolates and progeny of sexual hybridization, respectively.

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For zoospore mutation, 5-day-old P. capsici cultures grown on carrot agar medium (CAM, juice of 200 g ground carrots filtered with a cheese cloth, 14 g agar and distilled water up to 1 L) was incubated in the light for 48 h at 25°C for sporangia formation. Sterile water (10 mL) was added and the plates were placed in a refrigerator (4°C) for 15–30 min. Zoospores were normally released after 30 min of incubation at 25°C following the refrigeration. With the aid of a haemocytometer, the zoospore suspension was adjusted to 106–107 zoospores mL−1 in a Petri dish by adding sterile water. Zoospore suspensions were exposed to UV light (20 W, 254 nm) at a distance of 20 cm for 10 min, followed by 30 min of incubation in the dark. The treated zoospores in suspension were spread on PDA-F5 plates (100 μL per plate) with a sterile cell spreader.

The treated isolates, either mycelia or zoospores, were incubated for 5 days in the dark at 25°C. Colonies appearing on the media were transferred to plates and incubated for 5 days, then transferred onto PDA-F5 to confirm fungicide resistance. Colonies that grew on the PDA-F5 media were considered resistant mutants (phenotype). Mutation frequency of flumorph resistance was calculated as the number of mutants out of the total number of zoospores or number of plugs from a treated culture. Once the flumorph-resistant mutants were selected, they were purified from a single zoospore produced as described above.

Sensitivity of mutants to flumorph

PDA was made amended with various concentrations of flumorph: 1, 1·5, 2, 2·5 and 3 μg mL−1 for PCAS1; 0·5, 0·75, 1, 1·25 and 1·5 μg mL−1 for PCAS2; 5, 10, 15, 20 and 25 μg mL−1 for all mutants except U-3 and U-4; 75, 100, 125, 150, 175 and 200 μg mL−1 for mutant U-3; and 15, 20, 25, 30, 35 and 40 μg mL−1 for mutant U-4. This adjustment of flumorph concentrations was to optimize the EC50 calculation. Mycelial plugs of 3- to 5-day-old cultures from PDA plates were transferred onto fungicide-amended PDA at various concentrations. There were three replicates (plates) for each concentration. The plates were incubated in the dark at 25°C. Mycelial growth was measured on the fourth day of incubation. Inhibition rate (%) was calculated as (diameter of culture on fungicide-free PDA − diameter of culture on flumorph-amended PDA) × 100/(diameter of culture on fungicide-free PDA). Values of effective concentration for 50% mycelial growth inhibition (EC50) were determined for each fungicide. Based on the baseline sensitivity to flumorph, the isolates were divided into two groups: sensitive (S, EC50≤1·6 μg mL−1), and resistant (R, EC50 >1·6 μg mL−1). This experiment was conducted twice.

Cross-resistance

The sensitivity of all P. capsici isolates was tested on a series of concentrations of fungicide-amended PDA plates. The fungicides included dimethomorph (50, 100, 150 and 200 μg mL−1 for U-3; and 1, 2, 3 and 4 μg mL−1 for all other isolates), metalaxyl (0·2, 0·4, 0·6, 0·8 and 1 μg mL−1), azoxystrobin (0·05, 0·1, 0·2 and 0·3 μg mL−1), iprovalicarb (50, 75, 100, 125 and 150 μg mL−1), cyazofamid (5, 10, 15, 20 and 30 μg mL−1) and cymoxanil (5, 10, 10, 20, 30 and 40 μg mL−1). There were four replicates for each treatment. Mycelial growth of P. capsici was measured on the fourth day. Correlation was calculated with the EC50 values between flumorph and one of the other tested fungicides. This experiment was conducted twice.

Characterization of flumorph-resistant P. capsici mutants and their sexual progeny

Resistance stability

Cultures of P. capsici mutants were grown on PDA. When mycelia covered three-quarters of the plate, the culture was transferred onto a new PDA plate. The same operation was repeated for 10 transfers. Cultures from the first and the tenth transfer were used for examining stability of resistance. Sensitivity of P. capsici mutants to flumorph was determined by measuring EC50 on flumorph-amended PDA, as described above. There were three replicates for each isolate.

Mycelial growth and sporulation in vitro

Mycelial growth of P. capsici mutants and their parental isolates were compared on PDA plates incubated at 25°C for 5 days in the dark by measuring colony diameter every 12 h. Only measurement at 72 h was used for data analysis, when good growth was visible, but before plates were completely covered. For sporangial formation, all isolates were grown on CAM at 25°C for 5 days in the dark, followed by 48 h of incubation in the light. After incubation at 4°C for 30 min the culture was placed at room temperature to release zoospores, which were counted under a microscope with a haemocytometer.

Sporulation in vivo and virulence

Mycelial plugs of 5-day-old P. capsici cultures, with different resistance levels, and their parental isolates were transferred onto the surface of detached pepper leaves, which were rinsed with sterile water and placed in a Petri dish on a piece of wet filter paper (Whatman No.3, Whatman International Ltd) and incubated at 20°C in a 16-h light/8-h dark cycle, with 80% relative humidity, for 4 days. The necrotic lesions on the leaves were measured by calculating the oval shape area, which was used as an estimate of virulence of P. capsici. Five leaves, as a composite sample, were placed in a 50-mL centrifuge tube containing 15 mL distilled water, and agitated with a vortex (Denville Scientific, Inc.) for 15 s. The released sporangia were quantified with a haemocytometer under a microscope. This experiment was repeated once.

Oospore production by mutants

Mutants with different levels of fungicide sensitivity were co-cultured with an isolate of the opposite mating type on 10% V8 medium (100 mL V8 juice, 0·2 g CaCO3, 14 g agar and distilled water to 1 L) in the dark. Isolates or mutants with unknown mating types were co-cultured both with PCAS1 (A1 mating type) and PCAS2 (A2 mating type), which determined mating types for those mutants (Chang & Ko, 1990). Oospores were produced after 15 days of incubation. The culture (with medium) was ground using a blender to make an oospore suspension. Oospores were counted with a haemocytometer under a microscope. This was also done on media at various concentrations of flumorph to determine the inhibition of oospore production by flumorph.

Characterization of sexual progeny

Isolates of P. capsici with different sensitivities to flumorph were used as parents. Two cultures with opposite mating types were hybridized by co-culturing as described above. Growth characteristics of the progeny were examined, including mycelial growth, zoospore production and virulence, using the methods described above.

Segregation of resistance in P. capsici progeny

Mating type determination

Mating types of flumorph-resistant mutants and sexual progeny were determined by co-inoculation with reference isolates PCAS1 (A1 mating type) and PCAS2 (A2 mating type) as described by Chang & Ko (1990), and the number of isolates for each mating type was recorded (Fig. 1).

Segregation of phenotypic resistance in self-crossed progeny

Isolates with different resistance levels to flumorph were selected for self-crossing, after their mating types were determined. A mycelial disc (35 mm in diameter) of 5-day-old culture was cut from V8 medium and transferred onto the centre of a Petri dish (100 mm in diameter) containing 1% water agar, with the side of aerial mycelia facing up, and then covered with a sterile polycarbonate membrane (47 mm in diameter, Whatman Inc.). The membrane allows exchange of molecules, such as hormones required for sexual reproduction, but not mycelial penetration. A mycelial disc of the same size from a second isolate with an opposite mating type was transferred onto the top of the membrane, with mycelia facing the membrane. Therefore, the two cultures were separated by the membrane. The plate was sealed with Parafilm (Pechiney Plastic Packaging) and incubated in the dark for 2 months at 25°C for oospore formation and maturation. The culture and attached media from each side of the membrane were ground separately in distilled water in a blender. The homogenized culture was vacuum-filtered through a sieve with 54-μm openings to remove media and other residues, and then through a sieve with 31-μm openings to remove mycelia and sporangia or sporangiophores in the filtration. The material remaining on the sieves was rinsed twice with sterile distilled water, which was collected and included. The flow-through aliquot was passed through a 15-μm sieve. Oospores retained on the 15-μm sieve were washed three times and resuspended in sterile distilled water. After mixing with a 0·25% KMnO4 solution for 20 min in a shaker incubator at 50 rpm at room temperature, the oospore suspension was washed free of KMnO4 on a 15-μm sieve and resuspended with 2 mL distilled water. The concentration of the oospore suspension was determined with a haemocytometer (Fig. 1).

Segregation of flumorph resistance in self-crossing progeny

The oospore suspension was spread (about 100 oospores per plate) onto S + L medium (Ann & Ko, 1988) supplemented with 50 μg ampicillin and 50 μg penicillin mL−1 and incubated at 25°C for 24–72 h, illuminated with black light (Nanjing Ziguang company, China) with a 16-h light/8-h dark cycle each day. Germinated oospores were transferred individually to PDA at 12-h intervals, starting from the second day after plating, until all germinated oospores were transferred. The single-oospore culture was the first generation of self-crossing progeny (S1). From the S1 progeny, isolates with different levels of resistance, determined as described above, were randomly picked to generate the second generation (S2) of progeny by self-crossing (Fig. 1).

Segregation of flumorph resistance in sexual hybridization progeny

Hybridization was conducted by direct crossing between two isolates of P. capsici with opposite mating types. Isolate PCAS1 was crossed with several PCAS2-derived mutants. Mycelial plugs of the two isolates were cut from just behind the edge of actively growing cultures aged 3–5 days, and transferred onto a V8 medium plate with 30 mm of space between the two cultures. The plate was sealed with Parafilm and incubated in the dark for 14 days at 25°C for oospore formation, followed by incubation for at least 45 days to allow oospores to mature. Mycelia mixed in media were ground in a blender in order to release oospores. An oospore suspension was prepared as described for self-crossing. Oospore germination was determined and each isolate was tested for flumorph sensitivity as described above (Fig. 1).

Statistical analysis

For fungicide sensitivity tests, the fungicide concentration was transformed to logarithmic form (X) and the inhibition rate was expressed as a probability (Y). Regression was analysed between X and Y. A resistance factor (RF) was calculated as the ratio of the EC50 of tested isolates (resistant mutants in this case) over EC50 of control isolates (parents). Data were analysed using sas (version 9, SAS Inc.). proc glm was used for analysis of variance, and Duncan’s multiple comparisons were performed for separation of means. proc reg was used for linear regression. Segregation was analysed by the chi-square test.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Baseline sensitivity of P. capsici to flumorph in vitro

The frequency distribution of the EC50 values for 83 P. capsici isolates showed a unimodal curve (Fig. 2), ranging from 0·716 to 1·363 μg mL−1. The mean EC50 value was 1·033 ± 0·1287 μg mL−1. The low EC50 values indicated that there was no resistant subpopulation among the isolates.

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Figure 2.  Sensitivity of 83 field isolates of Phytophthora capsici to flumorph. EC50 = effective concentration for 50% mycelial growth inhibition. = total number of isolates obtained from the field.

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Generation of P. capsici mutants and their sensitivity to flumorph

Based on the results of baseline sensitivity, parental isolate PCAS1 (EC50 = 2·37 μg mL−1) was determined to be resistant, while isolate PCAS2 was sensitive (EC50 = 0·93 μg mL−1) (Table 1). All 13 flumorph-resistant mutants of P. capsici were obtained from isolate PCAS2 with UV mutagenesis, including U2-1 to U2-4 from mycelia, and U2-11 to U2-19 from zoospores. The mutation frequency was 7 × 10−9 using zoospores, and 4 × 10−4 using mycelial plugs. Among the 13 P. capsici mutants, 12 showed resistance to flumorph with RF values ranged from 11·64 to 36·46, while U2-3 had a high resistance level (RF = 216·67, Table 1). No mutant with a higher EC50 was obtained from PCAS1. Additionally, from the 16 isolates collected from China, one mutant from a zoospore of isolate DZ16 and three mutants from mycelial plugs of the same isolate N7 were obtained. However, the four mutants and their parents only produced a very few oospores via self-crossing or hybridization (data not shown), which is not good for investigation of segregation. Thus, mutants from DZ167 and N7 were not used in this study.

Table 1.   Sensitivity of Phytophthora capsici isolates to flumorph, and their resistance stability, measured by mycelial growth on PDA plates
IsolateaFirst transferTenth transferChange of EC50d
EC50 (μg mL−1)bRFcEC50 (μg mL−1)RF
  1. aPCAS1 and PCAS2 are wild-types. PCAS2 was used to generate mutant isolates U2-1 to U2-19 with UV mutagenesis.

  2. bEC50 is effective concentration for 50% mycelial growth inhibition.

  3. cRF (resistance factor) = (EC50 of test isolates)/(EC50 of isolate PCAS2).

  4. dChange of EC50 =  (EC50 of first transfer − EC50 of tenth transfer)/EC50 of first transfer. t-test was performed by comparing RFs between the first and tenth transfers; * and ** indicate significant difference at α = 0·05 and 0·01, respectively.

PCAS12·372·01
PCAS20·9310·9010
U2-115·4716·3510·4111·170·32**
U2-211·6112·4610·4511·220·10*
U2-3201·92216·67282·91303·58−0·40*
U2-433·9836·4624·4126·190·28*
U2-1114·9416·0413·0514·000·13
U2-1215·8116·9613·5514·540·14*
U2-1310·8511·6411·2312·05−0·04*
U2-1413·4914·4811·1411·950·17**
U2-1514·1115·1411·9212·790·16*
U2-1614·8815·9710·7111·500·28**
U2-1715·6316·779·6910·400·38*
U2-1815·5616·6910·9911·800·29**
U2-1916·2217·4012·4613·370·23*

Characterization of flumorph-resistant mutants of P. capsici and their sexual progeny

Resistance stability

Most mutants decreased (from 9% to 31%) in resistance after 10 transfers. The resistance of U2-3 (< 0·01) and U2-13 (< 0·05) increased by 40·1% and 3·5%, respectively, compared with the parental isolate (Table 1). The resistance decrease was significant for the rest of the isolates except U2-11 (Table 1).

Mycelial growth, sporulation in vivo and in vitro, and virulence

In the period of 72 h, the mycelial growth ranged from 4·85 to 5·73 cm. Isolates U2-2, U2-11 and U2-19 grew faster than the parental isolate PCAS2 (52·8 mm), U2-18 was not significantly different, and U2-1 and U2-3 grew slower than parental isolate PCAS2 (Table 2). All mutants produced significantly more zoospores (1·1–2·5 × 107 zoospores per plate) than PCAS2 (4·9 × 106 zoospores per plate) on CAM (Table 2). However, zoospore production on leaves varied among these mutants (6·9–11·0 × 103 zoospores per leaf), with one producing significantly more and two significantly less zoospores than PCAS2 (9·9 × 103 zoospores per leaf). Isolate U2-1 had significantly lower oospore production than the parental isolate, but the rest of the isolates did not differ from it. In addition, the resistant mutants were not significantly different from the parental isolate in virulence (Table 2).

Table 2.   Mycelial growth (colony diameter) on PDA plates at 72 h, zoospore production on carrot agar plates (*), zoospore production on pepper leaves (†), oospore production on V8 plates and symptom development (lesion area) on detached pepper leaves of Phytophthora capsici isolates resistant to flumorph
IsolateaHyphal growth (cm)Zoospores* (×107 per plate)Zoospores† (×104 per leaf)Oosporesb(×105 per plate)Lesion area (cm2)
  1. Multiple comparisons were performed using Duncan’s test at significance level α = 0·05. Values in the same column followed by the same letter are not significantly different.

  2. aPCAS2 is the wild-type (sensitive); the remaining isolates are mutants derived from PCAS2.

  3. bOospores were produced by co-culturing the isolate in question and a second isolate of a compatible mating type.

PCAS25·28 c0·49 g0·99 bc8·2 a4·35
U2-15·15 d1·1 f1·06 ab6·8 b3·80
U2-25·73 a1·8 c0·69 d7·6 ab3·61
U2-34·85 e2·5 a1·10 a8·5 a3·70
U2-115·52 b1·3 e0·94 c7·6 ab3·87
U2-185·38 c1·6 d0·68 d8·5 a2·91
U2-195·56 b1·9 b0·90 c8·8 a3·63
Oospore production by mutants

Oospores were produced at the rate of 105 oospores per plate for all progeny from the hybridization of mutants × PCAS1, which was similar to the hybridization of PCAS2 × PCAS1 (8·2 × 105 per plate), although U2-1 × PCAS1 produced fewer (6·8 × 105 per plate) (Table 2). As the concentration of flumorph increased on agar media, oospore production decreased, the cross PCAS1 (R) × PCAS2 (S) being more affected than PCAS1 (R) × U2-3 (R) (Fig. 3).

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Figure 3.  Inhibitory effects of flumorph at various concentrations on oospore production of Phytophthora capsici. Isolates were co-cultured as PCAS1 (R) × PCAS2 (S), and PCAS1 (R) × U2-3 (R), where R, resistant and S, sensitive to flumorph. In the regression equation, = log(oospores)/plate and = concentration of flumorph (μg mL−1).

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Characterization of sexual progeny

Progeny of the cross PCAS1 × U2-3 had a lower rate of mycelial growth than progeny of PCAS1 × PCAS2 or PCAS1 × U2-1. There was no significant difference in zoospore production between different groups of progeny. Progeny of PCAS1 × U2-1 had larger lesions than the other two groups (Table 3).

Table 3.   Biological characteristics of hybrids of Phytophthora capsici isolates
Parental isolates crossedaSensitivity of parental isolatesbMycelial growthZoospore productionVirulence
Number of tested progenyColony diameter (cm)Number of tested progenyZoospores (×105 mL−1)Number of tested progenyLesion area (cm2)
  1. aIsolates PCAS1 and PCAS2 were wild-types. U2-1 and U2-3 were mutants derived from PCAS2 using ultraviolet light.

  2. bSensitivity was measured by effective concentration of flumorph for 50% mycelial growth inhibition of the isolate, where S, sensitive and R, resistant.

  3. Multiple comparisons were performed in the same column, using Duncan’s test at significance level α = 0·05. Values followed by the same letter are not significantly different.

PCAS1 × PCAS2R × S156·14 a151·80104·25 b
PCAS1 × U2-1R × R155·93 a120·62145·66 a
PCAS1 × U2-3R × R144·80 b151·40104·07 b
Cross-resistance

There was cross-resistance among CAA fungicides. Phytophthora capsici mutants resistant to flumorph were also resistant to other CAAs, including dimethomorph and iprovalicarb (Fig. 4a,b). However, there was no correlation of resistance between flumorph and non-CAAs, such as metalaxyl (phenylamide fungicides), cymoxanil (cyanoacetamideoxime fungicide) or cyazofamid (Qi inhibitors) (Fig. 5a–c). There was an exception in that azoxystrobin (Qo-inhibiting fungicides) showed possible negative cross-resistance with flumorph (Fig. 5d).

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Figure 4.  Correlation tests for cross-resistance in Phytophthora capsici between flumorph and the carboxylic acid amide fungicides dimethomorph (a) and iprovalicarb (b). In the regression equations, x is log(EC50) of flumorph and y is log(EC50) of one of the other test fungicides.

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Figure 5.  Correlation tests for cross-resistance in Phytophthora capsici between flumorph and the non-carboxylic acid amide fungicides metalaxyl (a), cymoxanil (b), cyazofamid (c) and azoxystrobin (d). In the regression equations, x is log(EC50) of flumorph and y is log(EC50) of one of the other test fungicides.

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Segregation of phenotypic traits of resistance in P. capsici progeny

Segregation of flumorph resistance in self-crossed progeny

A total of 269 self-crossed progeny were obtained from PCAS1, combined for two generations (Table 4). Offspring were not obtained from PCAS2. The observed segregation ratio of the first generation was 8:137 (S:R, where S = sensitive and R = resistant), which fitted a hypothetical ratio of 1:15 (S:R) (Table 4). From S1 progeny, 16 isolates with different levels of sensitivity to flumorph were chosen for a second self-crossing generation (S2), but only three isolates successfully produced progeny, including S1-83, S1-85 and S1-22, which all showed resistant characteristics. Groups S2-83 and S2-85 had 49 and 43 progeny, respectively. There were no sensitive isolates detected from these two groups, which fitted a 0:1 ratio (S:R). Group S2-22 had 33 progeny and segregated at 2:31(S:R), which fitted a 1:15 (S:R) ratio (Table 4). All the results passed the chi-square test (Table 4).

Table 4.   Analysis of resistance inheritance of Phytophthora capsici to flumorph via self-crossing using the chi-square test
Self progenyNumber of isolates tested (n)Sensitivity of parentsHypothetical segregationaHypothetical genotypebExpected segregationaObserved segregationaPcNull hypothesis
  1. aSegregation ratio of sensitive:resistant isolates. Wild-type isolate PCAS1 was self-crossed to generate the first generation, including 145 progeny. Sixteen isolates were randomly selected from the first generation and further self-crossed to generate the second generation. Three isolates were successfully germinated to establish groups of S2-83, S2-85 and S2-22. Isolates were sensitive if effective concentration of flumorph for 50% mycelial growth inhibition (EC50) <1·6 μg mL−1, and resistant if EC50 >1·6 μg mL−1.

  2. bEach letter designates a gene, with dominant (upper case) and/or recessive (lower case) alleles.

  3. c> 0·05 indicates that the null hypothesis (i.e. no difference between hypothetical and observed segregations) was passed by the chi-square test.

PCAS1145R1:15AaBb9·1:135·98:1370·716Accepted
S2-8349R0:1AaBB (AABb)0:490:491Accepted
S2-8543R0:1AaBB (AABb)0:430:431Accepted
S2-2233R1:15AaBb2·1:30·92:310·964Accepted
Segregation of flumorph resistance in sexual hybridization progeny

Hybridization of P. capsici isolates yielded a total of 350 offspring from all combinations of parental isolates that had different levels of sensitivity to flumorph (Table 5). Progeny in group H-1 were derived from PCAS1 (Ra: EC50 = 2·366 μg mL−1) × PCAS2 (S: EC50 = 0·93 μg mL−1) and segregated at a ratio of 31:77 (S:R), which fitted the hypothetical ratio of 1:3 (S:R). This ratio would be expected if two genes are involved with both alleles heterozygous in PCAS1 but recessive homozygous in PCAS2. In group H-4, a total of 180 progeny were derived from PCAS1 (Ra: EC50 = 2·366 μg mL−1) × U2-3 (Rb: EC50 = 201·922 μg mL−1), and the observed ratio was 8:172 (S:R). Progeny of H-2-A, derived from PCAS1 (Ra: EC50 = 2·366 μg mL−1) × U2-1 (Rc: EC50 = 15·24 μg mL−1), segregated at 6:56 (S:R), which passed the chi-square test of hypothetical segregation 1:7 (S:R) (Table 5).

Table 5.   Analysis of resistance inheritance of Phytophthora capsici to flumorph via hybridization using the chi-square test
Filial generationNumber of isolates tested (n)Sensitivity of parentsHypothetical segregationaHypothetical genotypebExpected segregationaObserved segregationaPcNull hypothesis
  1. aSegregation ratio of sensitive:resistant isolates. Isolates were sensitive if effective concentration of flumorph for 50% mycelial growth inhibition (EC50) <1·6 μg mL−1, and resistant if EC50 >1·6 μg mL−1. Ra: EC50 = 2·366 μg mL−1, Rb: EC50 = 201·922 μg mL−1, Rc: EC50 = 15·240 μg mL−1.

  2. bEach letter designates a gene, with dominant (upper case) and/or recessive (lower case) alleles.

  3. c> 0·05 indicates that the null hypothesis (i.e. no difference between hypothetical and observed segregations) was passed by the chi-square test.

H-1108Ra × S1:3AaBb × aabb27:8131:770·3741Accepted
H-4180Ra × Rb0:1AaBb × AABB0:1808:1720·5510Accepted
H-2-A62Ra × Rc1:7AaBb × aaBb (Aabb)7·8:54·36:560·5016Accepted

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

It was demonstrated that all 83 Phytophthora capsici isolates had low EC50 values, indicating that no resistant population had become established where the samples were collected. These results can be used as baseline sensitivity of P. capsici to flumorph for field resistance monitoring. Phytophthora capsici mutants resistant to flumorph were obtained by UV mutagenesis and the resistance was stable through as many as 10 transfers.

Stable resistance to flumorph has not been seen in mutants of other oomycetes. For example, mutants of Phytophthora infestans showed a low level of or unstable resistance to CAAs in several studies (Bagirova et al., 2001; Stein & Kirk, 2004; Rubin et al., 2008), resulting in the conclusion that the resistance of P. infestans to dimethomorph is negligible in nature (Stein & Kirk, 2004). This could be the reason why no resistant isolates of P. infestans have been found in the field (Stein & Kirk, 2004; Yuan et al., 2006; Cohen et al., 2007; FRAC CAA working group reports). In comparison, it seems easier to obtain mutants of P. capsici resistant to fungicides such as flumorph (this study), zoxamide (Young et al., 2001) and iprovalicarb (Lu et al., 2010). Although some mutants showed significantly greater or less growth and virulence after many transfers or via heritage, most of them had a similar level of high fitness and aggressiveness to the wild-type isolate that they were derived from. This showed that the resistance is independent from growth characteristics, which is required for potential resistance development of P. capsici.

Phytophthora capsici mutants showed cross-resistance among CAA fungicides, including flumorph, dimethomorph and iprovalicarb, but not with most non-CAAs. Similar results were found for other oomycetes with CAA fungicides (Yuan et al., 2006; Zhu et al., 2008). This means a mutant could occur even if a particular fungicide is not applied. The fact that wild-type isolate PCAS1 had natural resistance to flumorph is evidence that mutants may exist stably in nature.

Sexual reproduction plays an important role in fungicide resistance because it leads to a much higher possibility of resistance development by gene recombination. For instance, resistant isolates of P. infestans were rare both in the field (Stein & Kirk, 2004; Yuan et al., 2006; Cohen et al., 2007) and the laboratory (Bagirova et al., 2001; Yuan et al., 2006; Rubin et al., 2008), whereas CAA-resistant isolates of Plasmopara viticola were found frequently, with a high resistance factor (RF >300) and stable resistance inheritance (Gisi et al., 2007). Correspondingly, only the A1 mating type of P. infestans is found in most regions of the world (Goodwin et al., 1994, 1995), but both A1 and A2 mating types of P. viticola have been detected in the field with a ratio of 1:1 (Gobbin et al., 2003; Scherer & Gisi, 2006). This correspondence may help to predict fungicide resistance development in P. capsici, which has been shown to have a 1:1 distribution of mating types in many places, such as New Jersey (Papavizas et al., 1981), North Carolina (Ristaino, 1990), Michigan (Lamour & Hausbeck, 2000) and many other states in the USA (Hausbeck & Lamour, 2004). Both mating types A1 and A2 were found in the same field in China, with a ratio of around 3:1 (Cui, China Agricultural University). This trend seems to be increasing, which is favourable for sexual reproduction.

A segregation ratio of 1:15 (S:R) from S1-progeny by self-crossing showed that the genotype of test isolates with natural flumorph resistance was heterozygous, and that the fungicide resistance is controlled by two dominant genes. This was further confirmed by segregation of S2-progeny and sexual hybridization offspring. The different ratios of segregation resulting from three groups of the second generation of self-crossed P. capsici indicated that the genotypes of the three isolates selected from S1-progeny were not identical, but they all contained the dominant gene(s). The segregation ratio of 1:3 (S:R) from hybridization between resistant and sensitive isolates also illustrated that the gene(s) controlling the resistance was/were dominant. In hybridization group H-4, eight sensitive isolates were detected, which was not expected based on hypothetical genotype models. This could be attributed to self-crossing by PCAS1 carrying heterozygous genes on two loci during the hybridization. A simple sequence repeat method was used to separate the selfing progeny from the sexual hybridization populations, but this was not successful (data not shown). This should be attempted in the future as it would provide good supporting data (Donahoo & Lamour, 2008). Other workers have reported that the proportion of selfed progeny is very low in the whole cross offspring (Shattock, 1988). This appears to contradict the present study, where a large number of selfed progeny was obtained using a polycarbonate membrane, maintaining unique genetic backgrounds and ensuring the accuracy of genetic analysis. This method should be recommended in similar studies.

The genetic background of fungicide resistance affects resistance development. Resistance controlled by dominant gene(s) will develop much more quickly than that controlled by recessive gene(s), causing a decline in efficacy of fungicides in a short time. The results of the present study indicated that the flumorph resistance in P. capsici is controlled by two dominant genes, implying that it is easy for the resistance to spread once it has occurred, via sexual and asexual reproduction in nature. However, the percentage of highly resistant isolates is relatively low because the theoretical expression probability of completely homozygous dominant genes (AABB) is low.

In conclusion, flumorph-resistant mutants of P. capsici were successfully obtained in laboratory conditions and may occur in nature, if they possess a high level of fitness or survival ability; resistance to flumorph is controlled by two dominant genes in P. capsici; and cross-resistance was found between flumorph and other CAA fungicides. Considering that both mating types coexist in the field, the risk of flumorph resistance is therefore intermediate to high. Appropriate management strategies should be taken to avoid or delay the development of fungicide resistance. These include detecting sensitivity shifts over time in P. capsici populations, and applying CAAs combined with non-CAAs, especially using multi-site protective fungicides.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

This work was supported by the Key Research Project Foundation of the State Ministry of Education of China and the National Science Foundation of China (grant no. 30671390) and the United States Department of Agriculture Specialty Crop Research Initiative program (NIFA grant no. 2008-51180-04881). We thank Dr Linda Hanson for a critical review of this manuscript.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References