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Keywords:

  • cyanoacrylate fungicide;
  • Fusarium graminearum;
  • Triticum aestivum;
  • wheat head blight

Abstract

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

The fungicide JS399-19 is a novel cyanoacrylate fungicide active against Gibberella zeae, and has been marketed in China for control of fusarium head blight (FHB) on wheat. Forty-three isolates sensitive to fungicide JS399-19 were collected from three commercial wheat fields in China. Forty-five isolates resistant to JS399-19, obtained from five sensitive isolates by selection for resistance to JS399-19, were selected. Three sensitivity levels were identified: sensitive (S), moderately resistant (MR) and highly resistant (HR) to JS399-19, based on a previous study. Eight isolates representing the three sensitivity-level phenotypes were randomly selected for a study on the inheritance of JS399-19 resistance by analysing the sensitivity of hybrid F1 progeny. A nitrate-non-utilizing mutant (nit) was used as a genetic marker to confirm that individual perithecia were the result of outcrossing. Five crosses were assessed: S × S, S × HR, MR × HR, HR × HR and MR × S. In crosses between parents with different sensitivity levels, such as S × HR, MR × HR and MR × S, the progeny fitted a 1:1 segregation ratio of the two parental phenotypes. No segregation was observed in the crosses S × S and HR × HR. It was concluded that the MR and HR phenotypes in G. zeae were conferred by different allelic mutations within the same locus. In these isolates, resistance to JS399-19 was not affected by modifying genes or cytoplasmic components.


Introduction

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

Gibberella zeae (anamorph = Fusarium graminearum) is one of the main pathogens causing fusarium head blight (FHB) on wheat and barley. FHB is the most economically important disease worldwide, especially in the USA (McMullen et al., 1997) and China (Chen et al., 2000), and is responsible for extensive damage to wheat in humid and semi-humid regions of the world (Bai & Shaner, 1994; McMullen et al., 1997). In China, FHB generally occurs in the middle and lower reaches of the Yangtze River and Huaihe River valleys and the eastern coastal region. However, an increase in the disease in the north and west wheat-growing areas of China has occurred in the last decade. FHB not only resulted in reported losses of 5–15% of grain yield in moderate epidemic years and up to 40% in severe epidemic years, but also reduced grain quality as a result of the presence of mycotoxins such as deoxynivalenol (DON, vomitoxin) (Snijders, 1990; Proctor et al., 1995). These mycotoxins inhibited amino acid incorporation and protein production in plant tissues (Casale & Hart, 1988), and also caused emesis and feed refusal in animals (Vesonder et al., 1976; Forsyth et al., 1977). Application of fungicides for control of FHB has been relied on over the past few decades, because few cultivars with effective resistance are available. The benzimidazole fungicides, particularly carbendazim, have been extensively used one or more times during each period of wheat heading and flowering in areas with warm and moist weather since the early 1970s. However, carbendazim faces the threat of resistance development in the field (Ye & Zhou, 1985; Zhou & Wang, 2001). It was reported that a single gene controlled resistance to carbendazim in G. zeae in China, and was generally expressed as a high degree of resistance to the fungicide (Yuan & Zhou, 2005). As a result, the frequency of resistant populations has increased dramatically and FHB control decreased markedly in some areas (Wang et al., 2002). China now is facing a challenge to find alternative fungicides for control of FHB on wheat.

JS399-19 (2-cyano-3-amino-3-phenylancryic acetate), which is a new cyanoacrylate fungicide and exhibits specific activity inhibiting mycelial growth of plant pathogenic Fusarium spp., was developed by the Jiangsu Branch of the National Pesticide Research & Development South Center (NPRDSC) in 1998 and introduced into the market recently (Li et al., 2008). The efficacy of JS399-19 in controlling FHB at a treatment concentration of 562·5 g a.i. ha−1 was better than that of carbendazim at 750 g a.i. ha−1 in a field experiment where carbendazim had been extensively used for decades and control failures of carbendazim had occurred (Li et al., 2008). However, mutants resistant to JS399-19 were easily obtained under the selection pressure of the fungicide in a previous study (Chen et al., 2008). Although the new fungicide has just been marketed in China, no information on the inheritance of resistance to this fungicide has been available until now. Moreover, strategies to manage the resistance risk should be developed and implemented to avoid unexpected control failures and sustain the usefulness of the new product. Outcrossing of G. zeae was recently confirmed by using the non-nitrate-utilizing (nit) mutation as a genetic marker (Bowden & Leslie, 1999), providing the possibility of studying the genetics of resistance to JS399-19 in this species. The objectives of this study were to determine whether resistant laboratory isolates could be divided into different resistance levels based on genetics, to assess the mode of inheritance of JS399-19 resistance, and to estimate the number of genes that control the different resistance phenotypes in order to understand the resistance risk of the chemical.

Materials and methods

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

Fungicide and media

Technical-grade JS399-19 (95·5%) was provided by NPRDSC and dissolved in methanol to 10 mg mL−1 for stock solution, which was diluted into media for testing inhibition of mycelia growth.

Potato sucrose agar (PSA) was used for routine culture and sensitivity tests to JS399-19. Minimal medium (MM) was made by adding 2 g NaNO3 to 1 L basal medium (Correll et al., 1987). This was amended with 30 g potassium chlorate and 1·6 g l-asparagine L−1 to make potassium chlorate medium (MMC), which was used to isolate nit mutants. MM, MO2 (MM amended with 0·2% NaNO2), MH (MM amended with 0·2% hypoxanthine) and MA (MM amended with 1·0% ammonium tartrate) were used to determine the types of nit mutation. Mung bean broth (MBB; 1 L H2O amended with 50 g mung beans) was used to produce conidia. Carrot agar was used for producing perithecia (Klittich & Leslie, 1988). Water agar (WA) medium (1·8% agar) was used to establish single-spore isolates.

Pathogen

Monoconidial isolates of 43 sensitive isolates and 45 JS399-19-resistant isolates, previously characterized at 10 µg JS399-19 mL−1 (resistant isolates grew normally on PSA amended with 10 µg JS399-19 mL−1, while sensitive ones could not), were used in this study. The sensitive isolates were collected from three commercial wheat fields: Tongzhou (Jiangsu Province), Jiaxing (Zhejiang Province) and Shanghai, where JS399-19 had never been used. These regions have a temperate-humid climate. The samples were collected during the harvest season of 2006. Each isolate originated from separate diseased head of wheat. The JS399-19-resistant isolates were obtained from five field isolates (2021, NT, NT14, S3 and S7) by selection for resistance to the fungicide based on a previous study (Chen et al., 2008). Briefly, this was done by taking fresh mycelial plugs from colony margins of the five selected sensitive field isolates, transferring them to PSA plates amended with 10 µg JS399-19 mL−1 and incubating in a growth chamber for 2 weeks. Any spontaneous and fast-growing sectors from the otherwise restricted colonies on these PSA plates were selected for resistance identification on PSA plates amended with 10 µg JS399-19 mL−1. For the stable JS399-19-resistant mutants, single-spore isolates were established. In total, 45 JS399-19-resistant isolates were randomly selected (18 from 30 resistant isolates recovered from 2021, five from 12 resistant isolates recovered from NT, 10 from 19 resistant isolates recovered from NT14, six from 12 resistant isolates recovered from S3, and six from 12 resistant isolates recovered from S7). All isolates were grown on PSA at 25°C for 3–5 days and stored on PSA slants at 4°C. The EC50 values of JS399-19 against all these isolates were determined later in this study (see details below).

Phenotypic characterization of JS399-19 resistance

For mycelial growth studies, autoclaved PSA was amended with JS399-19 stock solution before solidification to obtain final concentrations of 0·025, 0·05, 0·1, 0·2, 0·4, 0·8 and 1·6 µg mL−1. The 43 sensitive isolates of G. zeae were cultured on PSA Petri dishes (9 cm in diameter). Mycelia plugs, 5 mm in diameter, were cut from the margin of actively growing colonies of 3-day-old cultures of the fungus. One plug was placed in the centre of each 9-cm Petri dish with the mycelium in contact with the medium amended with JS399-19. Mycelial growth was measured using the sensitivity assay technique of a previous study (Karaoglanidis et al., 2002), in which fungicide-amended and fungicide-free Petri dishes were incubated at 25°C. Radial growth was measured (minus the diameter of the inoculation plug) after 3 days of incubation by calculating the mean of two perpendicular colony diameters. Tests were replicated twice, with three Petri dishes per concentration of the fungicide. The EC50 for each isolate, defined as the concentration of the fungicide that caused a reduction in colony diameter by 50%, was estimated by linear regression of the log of colony diameter versus fungicide concentration as described previously (Li et al., 2008), and mean EC50 value was calculated with the EC50 values from the two replicated tests. The 45 resistant isolates were also assessed for JS399-19 sensitivity by transferring 5-mm-diameter plugs from the leading edge of an actively growing colony to a series of PSA dishes amended with 0, 12·5, 25, 50, 100, 200 and 400 µg JS399-19 mL−1. Each isolate was tested in triplicate and incubated at 25°C for 72 h. Then, the mean EC50 values inhibiting the growth of the resistant isolates were calculated in the same way. In all cases (including non-amended control plates), the final methanol concentrations were the same for all media. According to mean EC50 values (see Results), isolates were classified as sensitive (S), moderately resistant (MR) or highly resistant (HR).

Genetic marker

For each of the sensitivity phenotypes, several representative isolates were selected to study the genetic control of resistance. For each parental isolate nit mutants were obtained as fast-growing sectors on MMC. The physiological types of the nit mutants were determined on media amended with different nitrogen sources, as described above (Correll et al., 1987). Genetic terminology for these mutants followed the guidelines for plant pathogenic fungi (Yoder et al., 1986). The mutant designations were appended to the original isolate number. Thus, Y2021Anit1 and Y2021Anit3 were the nit1 and nit3 mutants, respectively, of isolate Y2021A. Sensitivity to chlorate and JS399-19 were compared between each nit mutant and its parental isolate. Vegetative compatibility of the parental isolates was tested on MM (Correll et al., 1987).

Inheritance of JS399-19 resistance in asexual and selfed sexual progeny

For each fungicide-sensitivity phenotype, mycelial plugs of each isolate were grown in liquid culture (200 mL of 5% mung bean broth in a 250-mL Erlenmeyer flask) to produce macroconidia. The flask was incubated at 25°C on a rotary shaker at 200 r.p.m. (Throw = 3 cm) with a 12-h photoperiod for 7 days. These isolates were also grown on carrot agar in 9-cm-diameter dishes at 25°C with a 12-h photoperiod for 7 days to produce perithecia. After 7 days for conidia or 3 weeks for ascospores, the spores were smeared on WA plates and incubated at 25°C for 10 h to allow germination. Individual germlings were taken and cultured separately. Fifty conidia and ascospore progeny were recovered, and their sensitivity phenotypes were determined by measuring mean EC50 values as described above.

Crosses and preparation of hybrid progeny

Eight isolates, representing the three sensitivity-level phenotypes, were randomly selected for a study on the inheritance of JS399-19 resistance. Each parent in all crosses was marked with a different class of nit mutation. Small (1-mm3) mycelia plugs from cultures of each parent were placed on opposite sides of 9-cm-diameter dishes of carrot agar, 5 mm apart. The cultures were incubated at 25°C with a 12-h photoperiod for 7 days in a growth chamber, which allowed sufficient time for the cultures to cross and produce perithecia (Bowden & Leslie, 1999).

Approximately 50 putative heterozygous perithecia for each cross were sampled along the interface of colonies of the two parents under a dissecting microscope. The perithecia were crushed with a sterile needle on a concave slide and the ascospores were washed into a plastic tube with sterile water. The mixed ascospore suspensions were diluted to 1 × 103 spores mL−1 and smeared onto WA. Germlings were transferred individually to MM plates amended with streptomycin to minimize contamination with bacteria. For each cross, over 100 single-ascospore isolates were collected. After 3–5 days of incubation at 25°C, the sensitivity phenotype of each single-ascospore culture was evaluated. The presence of both nit mutants and wild-type progeny on MM indicated outcrossing between the two parental isolates had occurred.

Once outcrossing of the parents was confirmed, single perithecia were sampled along the interface and washed individually with sterile water three to five times to remove possibly contaminating conidia. Perithecia were then crushed on a concave slide, and the ascospores were washed with 0·2 mL sterile water and smeared onto MM plates amended with streptomycin. Over 100 germinating single ascospores of each perithecium were individually removed to MM plates and incubated at 25°C for 5 days. Only progeny from a perithecium where the nit:wild-type ratio was 3:1 were examined further, based on the results of a previous study in which perithecia that yielded approximately 25% wild-type and 75%nit mutant colonies were considered to be heterozygous, while perithecia that yielded only nit mutant progeny or less than 25% wild type with P < 0·01 for 3:1 segregation were considered to be homozygous (Bowden & Leslie, 1999). All of the progeny of each perithecium were transferred to PSA slants, cultures of the F1 hybrid progeny were established, and the sensitivity phenotype of each culture was determined.

Results

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

Phenotypic expression of JS399-19 resistance

Three fungicide-sensitivity levels were identified among the isolates examined (Table 1). Based on the baseline sensitivity of G. zeae in the previous study (Chen et al., 2008), 43 of these field isolates were determined to be sensitive (S). The resistant mutants were divided into moderate-level resistance (MR) and high-level resistance (HR) (Table 1), determined by EC50, values based on the previous study in which resistant mutants were tentatively divided into MR and HR phenotypes by EC50 values of 15·1–75·0 µg mL−1 and >75·0 µg mL−1, respectively. (Chen et al., 2008). Of all the 45 resistant mutants, 17 were MR and 28 were HR (Table 1). The mean EC50 values of the 43 S isolates, 17 MR isolates and 28 HR isolates were also calculated (Table 1).

Table 1.  Classification of phenotypes of Gibberella zeae isolates
PhenotypeaNumber of isolatesEC50 values (µg mL−1)b
  • a

    S: sensitive; MR: moderately resistant; HR: highly resistant.

  • b

    EC50 values of JS399-19 against the isolates were determined by mycelial growth assays and calculated using linear regression models. The range of values is given, with the mean value in parentheses.

S430·075–0·228 (0·141)
MR1727·470–61·497 (44·427)
HR28145·046–197·350 (169·979)

Three isolates (2043, JT24, NT14), one isolate (Y2021B) and four isolates (Y2021A, Y2021C, Y2021F, YNT) representing the S, MR and HR fungicide sensitivity phenotypes, respectively, were selected for the analysis of JS399-19 resistance (Table 2).

Table 2.  Parental isolates used in genetic crosses of Gibberella zeae
Genetic crossParental isolates, with nit phenotypeaParental JS399-19 phenotypeb
  • a

    2043-1, JT24-1 and NT14-1 were nit mutants recovered from field isolates 2043 (Jiaxing, Zhejiang Province), JT24-1 (Tongzhou, Jiangsu Province) and NT14 (Shanghai), respectively; Y2021F-1, Y2021B-5, YNT-1, Y2021C-4 and Y2021A-2 were nit mutants recovered from JS399-19-resistant isolates Y2021F, Y2021B, YNT, Y2021C and Y2021A, respectively [where Y2021F, Y2021B, Y2021C and Y2021A were recovered from field isolate 2021 (Tongzhou, Jiangsu Province) and YNT was recovered from the field isolate NT (Shanghai) through selection for resistance to the fungicide JS399-19].

  • b

    S: sensitive; MR: moderately resistant; HR: highly resistant.

A2043-1, nit3 × JT24-1, nit1S × S
B2043-1, nit3 × Y2021F-1, nit1S × HR
CY2021B-5, nit3 × YNT-1, nit1MR × HR
DY2021C-4, nit3 × YNT-1, nit1HR × HR
EY2021B-5, nit3 × JT24-1, nit1MR × S
FY2021A-2, nit3 × NT14-1, nit1HR × S

Genetic markers

Four classes (nit1, nit3, NitM and nnu) of nit mutants were identified from the fast-growing sectors of the eight selected isolates. All nit mutants showed similar levels of fungicide sensitivity to their respective parents (data not shown). The nit mutant complementation tests also showed that Y2021A, Y2021B, Y2021C, Y2021F and 2021, which were originally recovered from the field sensitive isolate 2021, were in the same vegetative compatibility group (VCG), whereas 2043, JT24, NT14 and YNT represented different VCGs. Consequently, each pair of isolates used in crossing tests (Table 2) represented two different VCGs (Table 2).

Resistance stability in asexual and self-crossed reproduction

All the conidia and ascospore progeny of three representative isolates (2043, Y2021B and YNT, representing S, MR and HR, respectively) exhibited unchanged resistance levels to JS399-19 based on their EC50 values (data not shown), indicating resistance stability in asexual and self-crossed reproduction.

Crosses and determination of progeny phenotypes

A total of six crosses were performed (Table 2), and all were found to be fertile. Individual perithecia were sampled along the interface line for each cross, and single-ascospore progeny of each perithecium were cultured. Generally, nearly 25% of progeny from hybrid perithecia exhibited wild-type growth on MM. The ratio was not significantly different inline image from the expected 3:1 segregation ratio for nit mutants and wild-type recombinants (Tables 3 and 4). However, the progeny of most inbred perithecia of either parental isolate exhibited nit growth on MM.

Table 3.  Segregation of JS399-19-resistant phenotypes among the ascospore progeny of different Gibberella zeae perithecia from the same genetic cross
Genetic crossPerithecia numberAscospore viability on WAa (%)Total progeny observedbNumber of progenyχ2 (3:1)cNumber of each phenotypedχ2 (1:1)ce
nitWild-typeSMRHR
  • a

    WA: water agar medium (1·8% agar).

  • b

    Germinated single ascospores (progeny) were transferred individually to minimal medium plates and over 100 single-ascospore cultures were established to determine nit physiological phenotypes and sensitity (S, MR or HR) to fungicide JS399-19.

  • c

    Only progeny from a perithecium where the nit:wild-type ratio was 3:1 were determined to be from outcrossing and available for further examination inline image.

  • d

    S: sensitive; MR: moderately resistant; HR: highly resistant.

  • e

    –: no segregation.

BB-198144101431·56770670·56
B-299133105280·90620710·48
B-398124101360·27570670·33
DD-19710482220·6300104
D-29712285371·5700122
D-398136107290·7900136
Table 4.  Chi-squared (χ2) test of the progeny in six genetic crosses of Gibberella zeae
Genetic crossAscospore viability on WA (%)Total progeny observedNumber of progenyχ2 (3:1)aNumber of each phenotypebχ2 (1:1)ac
nitWild-typeSMRHR
  • a

    inline image.

  • b

    S: sensitive; MR: moderately resistant; HR: highly resistant.

  • c

    –: no segregation.

A99108 77310·60108 0  0
B99146117291·79 71 0 750·06
C98137101360·06  067 700·03
D99120 97231·88  0 0120
E97126 89371·06 6165  00·07
F98132104280·82 62 0 700·37

Genetic studies of the fungicide sensitivity of all crosses showed that the viability of ascospores was never lower than 97% and, therefore, it could be expected that none of the genotypes could be missing in large samples (Tables 3 and 4). Segregation of JS399-19-resistant phenotypes of three hybrid perithecia of both cross B and cross D were examined (Table 3). Segregation among progeny of different perithecia from the same parents was similar (Table 3), so for all the six crosses, only one hybrid perithecium was tested.

In cross A, all progeny of 2043-1, nit3 × JT24-1, nit1 were sensitive and similar to both parents (Table 4). All progeny of cross D were HR; none of the progeny were differentiated as S or MR, indicating that the HR phenotype was controlled by a single locus in these isolates.

In crosses B and F (between HR and S isolates) and in cross E (between MR and S isolates), segregation fitted a 1:1 ratio, indicating that in each MR and HR isolate the resistance was controlled by a single Mendelian gene. In these crosses, the resistant progeny were phenotypically similar to their respective resistant parent.

In cross C, in which parents had different resistant phenotypes, the progeny also had only the parental phenotypes, segregated in a 1:1 ratio. Therefore, both MR and HR were controlled by the same locus in the isolates characterized.

There was no linkage observed between the genes controlling nit mutations and resistance. Thus, the nit characters and JS399-19-resistant phenotypes segregated independently among the hybrid progeny.

Discussion

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

Benzimidazole fungicides are site-specific fungicides that interfere with cell division. Continuous carbendazim-resistance monitoring showed that the frequency of resistant isolates in some regions of China increased gradually, and the efficacy of carbendazim against G. zeae decreased dramatically after 1998 (Wang & Zhou, 2002). Resistant isolates could not be controlled by increasing the application rate or by shortening the spray interval and were fit and competitive in nature, even without selective pressure (Wang et al., 2002). The efficacy of carbendazim was in direct relation to the resistant pathogen population in the field (Zhou & Wang, 2001). JS399-19 is a novel fungicide which strongly inhibited the mycelial growth of G. zeae and exhibited an excellent efficacy in controlling FHB in field trials where control failures of carbendazim had occurred (Li et al., 2008). Furthermore, it also showed the ability to delay the development of carbendazim resistance in field trials where control failures of carbendazim occurred (Li et al., 2008). The absence of cross-resistance between JS399-19 and other well-known fungicides, such as carbendazim, tebuconazole, prochloraz and azoxystrobin, suggested that the mode of action and resistance mechanism of JS399-19 are different from those of these fungicides (Chen et al., 2008; Li et al., 2008). Management of benzimidazole resistance relies on reducing selection pressure by limiting exposure to fungicides with the same mode of action, and using tank mixtures or replacing benzimidazole fungicides with others having different biochemical modes of action. Thus, JS399-19 might become the major fungicide instead of carbendazim, or a good companion fungicide in controlling FHB in China.

In the present study, JS399-19-resistant isolates of G. zeae, previously obtained by selection for the resistance to the fungicide, could all be classified as either MR or HR on the basis of their mean EC50 values. This indicated that at least two different mutations were involved in JS399-19 resistance in G. zeae. Genetic analysis of the crosses indicated that the resistance to JS399-19 was governed by a single major gene in G. zeae and that the two levels of resistance could be conferred by mutations at two sites or one site with two different mutations in the gene. There was a consistent 1:1 ratio of segregation in the crosses between parents with different resistance levels, and there was no indication that cytoplasmic components or modifying genes were involved in the resistance. The segregation pattern for the two levels of resistance to JS399-19 in G. zeae provides an indication of polymorphism in a single Mendelian gene.

It is concluded that a major gene controlled resistance to JS399-19 in G. zeae, and was generally expressed as a high degree of resistance to the fungicide, which might lead to a site-specific mode of action and make the resistance appear quickly under high selection pressure of the fungicide. Thus, the resistance of G. zeae isolates to the new fungicide JS399-19 in vitro might presumably be at high risk and appropriate precautions against resistance development should be taken. However, all the resistant isolates were recovered from the laboratory, which might not mean that JS399-19-resistant field isolates would exhibit the same level of resistance. Large scale monitoring for fungicide resistance to this product should be performed, since field JS399-19 resistance would help to clarify this point.

Considering there was no positive cross-resistance between JS399-19 and fungicides belonging to other chemical classes, such as ergosterol biosynthesis inhibitors (tebuconazole and prochloraz) and strobilurins (azoxystrobin), combining or alternating application of JS399-19 with tebuconazole, prochloraz or azoxystrobin could be a valuable tool to limit fungicide resistance development (Chen et al., 2008; Li et al., 2008). Continuous JS399-19 resistance monitoring should be carried out, since the new fungicide has been marketed and used in China.

Acknowledgements

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

This study was sponsored by: (i) the State ‘973’ Programs from the Ministry of Science and Technology of China (no. 2006CB101907); (ii) the State ‘863’ Programs from the Ministry of Science and Technology of China (no. 2008AA10Z414); (iii) the National Natural Science Foundation of China (nos 30671048 and 30671384); (iv) the Key Technology R&D programme from the Ministry of Science and Technology of China (no. 2006BAE01A04-08); and (v) doctorate funding of the Chinese Education Department (20050307028).

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