Wu Peng, Qin Zhi-wei*, Zhou Xiu-yan, Wu Tao, Xin Ming, and Guo Qian-qian

1College of Life Science, Agriculture and Forest, Qiqihar University, Qiqihar 161006, Heilongjiang, China

2College of Horticulture, Northeast Agricultural University, Harbin 150030, China

Study on Relationship Between Cucumber Germplasm and Propamocarb Residue Using Subjective Rating Technique

Wu Peng1,2, Qin Zhi-wei2*, Zhou Xiu-yan2, Wu Tao2, Xin Ming2, and Guo Qian-qian1

1College of Life Science, Agriculture and Forest, Qiqihar University, Qiqihar 161006, Heilongjiang, China

2College of Horticulture, Northeast Agricultural University, Harbin 150030, China

Propamocarb (PM) residue in cucumber (Cucumis sativus L.) receives little attention. As is well known to all, high PM residue of cucumber could lead to increase in the violation rates of maximum residue limits and ultimately cause harm to human health. Knowledge of PM residue could help cucumber breeders in developing cultivars with low PM residue and improving cucumber quality. In this study, 32 representative cucumber accessions (26 breeding lines and six cultivars) from different regions of China were evaluated for their PM residue in fruit and leaf to provide meaning to the subjective rating, which was highly correlated with PM residual content of fruit (r=0.97) and leaf (r=0.94). In addition, PM residual content of North China ecotype was the highest and Pickling ecotype was the lowest in fruit and leaf of cucumber. The leaf had significantly higher (P＜0.01) PM residual content than the fruit, and poor correlation between leaf and fruit was represented. This study verified PM residual relationship between fruit and leaf, and laid the foundation for further identification of germplasm resources and breeding of new varieties for low PM residue of cucumber.

cucumber, ecotype, gas chromatography, germplasm, propamocarb residue

Introduction

Cucumber (Cucumis sativus L.) is thought to originate from India or China (Harlan, 1975). Statistically, China is the world's largest cucumber producer, with 1.5 million hm2of harvested area and 25 million metric tons in 2004 (FAO, 2005). Cucumber is one of the most important species that people like eating fresh. However, its growth process is often affected by many diseases and then demands a mass of pesticides. Cucumber is one of the main vegetable varieties with increases in the violation rates of maximum residue limits (MRLs) at present. Pesticide residue has been one of the most important problems in the quality of cucumber fruits (Adachi and Okano, 2006). In recent years, cucumber downy mildew is the important disease in cucumber. Propamocarb (PM) has been a kind of pesticide that mainly controls downy mildew of cucumber in China. Then, the residue of PM in cucumber is a consequent problem. Propamocarb has a human toxicity, with ADI of 0.4 mg • kg-1bw/day and ARfD of 2 mg • kg-1bw set by the Codex Alimentarius.

Gas chromatography (GC) is a major scientific and technological achievement from the 1950s, and has been widely used in the qualitative and quantitativeanalyses of mixtures. The analyses of pesticides are normally carried out by means of GC column in combination with a group-selective or elementselective detector, such as an electron capture or nitrogen phosphorous system (Mondello, 2007). FID detector was used in this study to detect PM residue in fruits and leaves of cucumber, and could meet the requirement of quantitative detection of PM residue. Specific germplasm collections are valuable to plant breeders as a source of new genes or desired traits to improve cultivated crops (Wada and Read., 2011). The study of Sarangthem et al. (2013) assessed and prioritized the wild Hedychium resources of Manipur, thus established a desirable potential germplasm for breeding and further biotechnological interventions. Santalla et al. (2004) reported the evaluation of runner bean cultivars from Spain for morphological, agronomical and seed quality traits in different environments. They found some valuable germplasms that could be used either in production or breeding, including interspecific hybridization with common bean. In our research, the identification of potential germplasms with low PM residue after PM treatment might be of considerable value in the resistant improvement of cucumber cultivars. To date, no studies had been undertaken to screen the cucumber germplasm resources of low and high PM residue. Therefore, the present study was undertaken to screen the cucumber germplasm resources of low and high PM residue using a subjective rating technique.

In addition, PM residual content between leaves and fruits was compared, and the residual correlation was verified. Ultimately, the accessions with low or high PM residue of cucumber were used in further identification of germplasm resources and breeding of new varieties for improving the cucumber germplasm quality. The objectives of this research were: (i) to identify potential cucumber germplasm with low PM residue after PM treatment and (ii) to verify correlation between fruits and leaves for PM residue in cucumber.

Materials and Methods

Plant materials and growing conditions

A total of 32 cucumber accessions from China, which were comprised of North China ecotype (NC), South China ecotype (SC), European greenhouse ecotype (EG) and Pickling ecotype (PE), were used to determine PM residual content in fruits and leaves. These genetic materials were maintained in the germplasm collection at the Cucumber Research Group of Northeast Agricultural University, Harbin, China. The seeds used in the experiment were obtained from plants grown in the same environment in 2010. Seeds of 32 cucumber accessions were sown on 18; March 2011, with the seedlings grown in a mixture of 1 peat: 1 vermiculite (v/v) on the research greenhouse. At the three-leaf stage, seedling plants were transplanted gently into a growth chamber containing the same water and fertility conditions, which grew under controlled environmental conditions. Plants were trained on a trellis of 1.8 m high, and the branches were pruned. Greenhouse temperatures averaged 30℃(days) and 22℃ (nights).

Experiments were carried out with all the fruits and their corresponding leaves of the mixed sample of each cucumber accession plant, and samples were collected on 55 day after colonization (DAC). At the same time, samples were weighed, immediately frozen in liquid nitrogen, and then were stored at –80℃ until required for determination of PM residue.

Experimental design

The experiment was conducted in a randomized complete block design and three replications was used, with 1-plant plots. A total of 32 cucumber accessions plants were sprayed by 400 times PM solution, which was higher than the recommended dose (800 times) in order to analyze it better. PM solution was sprayed firstly at 34 DAC (first harvest period), and then sprayed once every 5 days, with 3 times continuously. The root fruits and connected leaves of 32 cucumberaccessions were collected at 55 DAC. Each cucumber accession had 40 plants from which 5-10 plants were randomly selected to determine the content of PM residue.

PM residual content of 32 cucumber accessions in fruits and leaves was subjectively rated on a scale of 1 to 9 (1=extra small, 2=very small, 3=small, 4 =medium small, 5=medium, 6=medium large, 7=large, 8=very large, and 9=extra large), modified from Walters and Wehner (1994). The higher scale stood for high PM residual content while the lower scale stood for low PM residual content.

Determination of PM residue

The quantitative determination of PM residue of 32 cucumber accessions was employed with three replicates by gas chromatography (GC) analysis. GC analyses were carried out on a Shimadzu GC-2010 gas chromatography operated with a split/splitless injector and a Shimadzu autosampler AOC-20s and autoinjector AOC-20i (Shimadzu, Japan); column: Rtx-1, 30 m× 0.25 mm I.D., 0.25 μm film thickness; temperature program: 70 to 230℃ at 30℃/min; injection volume: 1.0 μL; inlet pressure: 100 kPa; carrier gas: N2, linear velocity u: 31.8 cm • s-1; column flow: 1.25 mL • min-1; splitless detection: FID, at 290℃. H2flow: 40.0 mL• min-1; air flow: 400.0 mL• min-1. Data were acquired by a GC solution software (Shimadzu). The analyte was extracted with acetone-water and cleaned up by liquid-liquid partition into diethyl, ether. Recoveries of PM were 85%-94% and the relative standard deviation was 1%-2%. This analytical method could meet the requirements of quantitative detection of PM residue in fruits and leaves.

Statistical analysis

The experiments were setup in a completely randomized design. Each replicate contained mixed sample randomly collected from 5 to 10 plants after treatment. Three replications per accession were used with measurement date as a block. Data means and standard errors (S.E.) which were then averaged from three repeated experiments. The data were analyzed by an analysis of variance (ANOVA) and correlation (CORR) procedures using SPSS (version 13.0), and significant differences were calculated by Duncan's multiple range test (P＜0.05). Pearson correlation coefficients were computed between fruits and leaves for PM residue measured (Steel et al., 1997).

Results

PM residue of 32 cucumber accessions in fruit

PM residual content of 32 cucumber accessions in fruit is shown in Table 1. Significant differences existed among fruits of 32 cucumber accessions for PM residue, and the content was a continuous change trend with a range from 0.148 to 2.983 mg • kg-1. However, PM residual content of most accessions fell into the 0.557 to 1.595 mg • kg-1(18% or 56%), and the average content of PM residue was 1.017 mg • kg-1in fruit. The complete data set would be available from Cucumber Research Group of Northeast Agricultural University, Harbin, China. Jinyan 4 had the largest PM residual content of all accessions evaluated (2.983 mg • kg-1PM residue). Meanwhile, it was significantly different from 31 other accessions. The accession with the smallest PM residual content was D0351 (0.148 mg • kg-1PM residue). PM residual content of Jinyan No. 4 and D9320 exceeded MRLs (2.0 mg • kg-1), and other 30 cucumber accessions were not more than MRLs. According to our measurements, low PM residues in fruit was a scale of ＜0.557 mg • kg-1in residual content, most had medium PM residues ＞0.557 and ＜1.595 mg • kg-1in residual content, and high PM residues＞1.595 mg • kg-1in residual content. PM residue in fruit and our subjective rating were positive correlation (r= 0.97), indicating that PM residue of cucumber in fruit could be accurately predicted from a subjective rating. Rating increased as PM residual content increased and was best described by the linear relationship, y=0.3482 (rating)–0.5035, R2=0.9418 (Fig. 1).

Total 32 cucumber accessions were comprised of 13 NC, 6 SC, 8 EG and 5 PE. The average PM residualcontent in fruit of NC was 1.451 mg • kg-1, SC was 1.033 mg • kg-1, EG was 0.694 mg • kg-1and PE was 0.387 mg • kg-1. PM residue of four different cucumber ecotypes had a large difference, and NC had the highest PM residue and PE had the lowest. The average PM residual content of NC was significantly higher (P＜0.01) than PE and EG. In addition, amplitude of variation of NC was 0.743 -2.983 mg • kg-1, SC was 0.333-2.368 mg • kg-1, EG was 0.233-1.499 mg • kg-1and PE was 0.148-0.751 mg • kg-1. Meanwhile, NC had smaller coefficient of variation (0.407) than SC (0.711), EG (0.579) and PE (0.651) (Table 1).

Fig. 1 Relationship between PM residue in fruits and rating

Table 1 PM residue of fruits with four different cucumber ecotypes of 32 cucumber accessions

Continued

PM residue of 32 cucumber accessions in leaves

PM residue of 32 cucumber accessions in leaves is shown in Table 2. Data for one cucumber accession (D0327-4-2) was not collected due to poor plant growth. Total 31 cucumber accessions were significant differenct in PM residue of leaves, with a range of 0.944 mg • kg-1to 101.113 mg • kg-1and the highest difference of nearly 100 times. The average PM residual content was 32.131 mg • kg-1. Jinyan 4 and D0422 had the highest and the lowest PM residual contents of all evaluated cucumber accessions (101.113 and 0.944 mg • kg-1PM residue). In addition, PM residual content of most accessions exceeded MRLs (2.0 mg • kg-1) in leaves except for D0422, D0406-15 and 129-1. According to our results, PM residue in leaves had very large amplitude of variation of 32 cucumber accessions. Low PM residues in leaves was a scale of ＜6.252 mg • kg-1in residual content, most had medium PM residues ＞6.252 and ＜46.471 mg • kg-1in residual content, and high PM residues were ＞46.471 mg • kg-1in residual content. PM residue in leaves and our subjective rating had highly positive correlation (r=0.94), indicating that PM residue of cucumber in leaves can be also accurately predicted from a subjective rating. PM residual content and rating were best described by the linear relationship: y=10.206 (rating)–15.362, R2=0.9423 (Fig. 2).

The average PM residual content of NC, SC, EG and PE in leaves were 37.591, 26.469, 38.11 and 16.741 mg • kg-1, respectively. NC had the highest PM residual content and PE had the lowest, but there were no significant difference (P＞0.05) of four different cucumber ecotypes. In addition, NC had the largest amplitude of variation (1.494-101.113 mg • kg-1) of four cucumber ecotypes, and PE had the smallest amplitude of variation (1.379-46.471 mg • kg-1) and the largest coefficient of variation (1.121) (Table 2).

Table 2 PM residue of fruits with four different cucumber ecotypes of 32 cucumber accessions

Correlation for PM residue between fruits and leaves

The average PM residual content of fruits was 1.017 mg • kg-1. However, the average residual content of leaves reached up to 32.131 mg • kg-1. The leaves had a significantly higher (P＜0.01) PM residual content than the fruits. Minimum and maximum contents of leaves were significantly higher (P＜0.01) than minimum and maximum content of fruits. Furthermore, correlation analysis was carried out between fruits and leaves with PM residue, and the results showed that correlation coefficient of fruits and leaves was 0.228, which had low positively correlation. Regression equation of PM residue between leaf and fruit: Y=0.770+0.028X–0.001X2(Table 3).

Fig. 2 Relationship between PM residue in leaves and rating

Table 3 Statistics of PM residue of fruits and leaves in 32 cucumber accessions

Discussion

Different ecotypes of plant have been shown to differ substantially in their composition of secondary metabolites (Huang et al., 2010), so we investigated the four different ecotypes of cucumber for PM residues. The results showed that PM residues of four different cucumber ecotypes had a large difference in fruits and leaves, and NC had the highest PM residues and PE had the lowest. This might be related to PE having less nap, papilloma, smaller fruit size and blade surface tension than NC (data not shown). Meanwhile, amplitude of variation of PE was the smallest. This suggested that PE has better stability and repeatability of results. In this report, we thought that different cucumber ecotypes had different PM residual contents and cucumber germplasm resources of low PM residue should be chosen in PE. A total 32 cucumber accessions from Northeast Agricultural University including NC, SC, EG and PE were used as they would provide basic idea about extent of diversity, and had very good representative of cucumber germplasm resources. In this study, significant differences among 32 cucumber cultivars were noted for PM residues in fruits and leaves evaluated, indicating the existence of variation in the studied germplasm. D0351 had the lowest PM residue in fruits, and Jinyan No. 4 had the highest PM residual content in fruits and leaves. These results showed that the fruit of D0351 had stronger ability against PM-induced stress than other 31 accessions, and Jinyan No. 4 had the weakest ability.

Jinyan No. 4 belongs to NC, and D0351 belongs to PE, which were parallel to the results formerly,and showed that NC had the highest PM residue and PE had the lowest. This might be due to different ecological types of cucumber had different structures and metabolic pathways. Leaves are the source organ, with functions of complete production, temporary save, output and immune reactions, and the fruits are the library organ and the main function is to reprocess the output of the source material and storage; functional specificity is stronger. Our results displayed that the leaves had a significantly higher (P＜0.01) PM residual content than the fruits. This might be due to leaves having more stoma, water stoma and larger surface area than fruits. In addition, this result showed that the fruits had stronger ability against PM-induced stress than leaves. This was parallel to the research of Azuma et al. (2010), which showed that fruit growth had greater ability to stand salinity stress compared to leaf growth. D0351 of PE and Jinyan No. 4 of NC should be chosen as significant germplasm resources of cucumber for low and high PM residues, and studied with PM detoxification system against PM-induced stress of cucumber.

Low residual plants (D0351) were characterized by shorter internodes, higher ration of female to male flowers, and smaller fruits than high residual plants (D9320). D0351 plants had smaller leaf area (219.00± 21.3 cm2vs. 550.39±38.8 cm2) compared with Jinyan No. 4 of NC. Correlation coefficient between leaf area and PM residue of fruits was 0.92, and reached a significant (P＜0.01) level of positive correlation. This result showed that PM residual content might be affected by morphological traits of plants. In this study, we found that correlation between fruits and leaves for PM residue was poor, but could be found that leaves had a dramatically higher (P＜0.01) PM residual content than the fruits. This might be due to exit transport path from leaves to fruits. Therefore, transportation mechanism of PM residue between leaves and fruits would be studied further.

The subjective rating technique provided a simple, accurate and rapid method to estimate PM residue of cucumber fruits and leaves. Rating is a subjective measurement, and the person rating must first understand how to accurately rate the size of PM residue. Before rating can be used as an accurate measurement of PM residue, the rater must determine which PM residue goes into each of the nine categories; to accomplish this task, the rater should lay out PM residual systems, one for each of the nine categories, so the rating scale can be referred to when a judgment is made. The subjective rating and PM residue in fruits and leaves were highly correlated (r=0.97 and 0.94), indicating that PM residue of cucumber in fruit and leaf can be accurately predicted from a subjective rating. This method of evaluating content of PM residue showed that significant genotypic differences for PM residue existed among the accessions tested. Knowledge of PM residue will allow cucumber breeders to develop cultivars that have lower PM residual content. That might help decreasing PM residues, thus leading to improve cucumber quality and market competitiveness.

Conclusions

Considerable diversity supported with PM residue of cucumber pointed out certain strategic issues for germplasm explorers. In this study, the identification of cucumber accessions with PM residue of fruits and leaves had initiated additional studies using these accessions having the low or high PM residue from this screening research. For example, Jinyan 4 had the highest PM residual content no matter what fruits and leaves, while D0351 had the lowest PM residue in fruits and D0422 had the lowest residue in leaves. Additional work was needed to select the accessions of low or high PM residue, stabilize the best selections using self pollination, and used the selections for interspecific hybridization with the common cucumber as a source for low or high PM residue. More importantly, the inheritance and molecular marker of PM residue in cucumber were necessary for economizing germplasm collections and conservation programmes.

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S642.2

A

1006-8104(2014)-01-0001-09

Received 17 September 2013

Supported by the National Natural Science Foundation of China (31272158); 863 Program of the National Science and Technology of China (2012AA100105)

Wu Peng (1983-), male, Ph. D, lecturer, engaged in the research of vegetable physiology and molecular biology. E-mail: wupeng216@126.com

* Corresponding author. Qin Zhi-wei, professor, supervisor of Ph. D student, engaged in the research of cucumber genetics and breeding. E-mail: qzw303@126.com