Abstract: We were aimed to confirm the oviposition preference of P. rapae against C. religiosa (Capparidaceae plant) and cabbage (Brassicaceae plant) and then analyzed the AITC and sinigrin contents and myrosinase activity in these plants to clarify the attractant and oviposition stimulant properties of C. religiosa and cabbage to P. rapae. We analyzed the attractant and oviposition stimulant of a Brassicaceae plant, Brassica oleracea var. capitata L. (cabbage) and a Capparidaceae plant, Crataeva religiosa to Pieri srapae crucivora Boisduval by the oviposition preference test. The oviposition preference of P. rapae was the highest in cabbage, followed by C. religiosa, but the lettuce (control plant) was rarely selected. Allylisothiocyanate (AITC), which is assumed to be one of the attractant and sinigrin as oviposition stimulants were detected in cabbage using GC-MS. Their compounds were not detected in C. religiosa, but the higher content of methylisothiocyanate (MITC) that is analogous to AITC was found. Myrosinase activity was analyzed by HPLC and detected in cabbage and C. religiosa, but not in lettuce. Glucosinolate of sinigrin analogous in C. religiosa fractionated by HPLC was decreased by myrosinase treatment and then produced MITC. We identified that this compound was glucocapparin as a precursor of MITC by LC-MS. These results revealed that C. religiosa have a glucosinolate-myrosinase metabolic system and pathway.
INTRODUCTION
Recently, considering environmental pollution and the safety of food, consumers are strengthening the concern for organic product and chemical-free vegetables and producers are doing more organic farming. Alternative biological pesticides are thus being used to replace agricultural chemicals.
Brassicaceae plants, including many types of important vegetable crops, are extensively damaged by the P. rapae crucivora Boisduval. P. rapae specifically search for Brassicaceae plants and oviposits on the leaves of these plants (Sato et al., 1999). It is known that P. rapae utilize sinigrin as an oviposition stimulant in Brassicaceae plants (Huang and Renwick, 1994; Traynier and Truscott, 1991). Most butterflies have an oviposition preference and choose suitable plants upon which to lay eggs; it is thought that they respond to chemical compounds in host plants as a cue to search for a place to lay eggs (Honda, 1995). Glucosinolates (sinigrin, etc.) are plant secondary products as particular components of the family Brassicaceae, which have a glucosinolate-myrosinase system. When tissue disruption occurs due to pest or pathogen, glucosinolates are hydrolyzed with myrosinase and produce a volatile compound, (AITC: allylisothiocyanate), playing a role as defense to other pest (Griffiths et al., 2001; Tsao et al., 2002; Bridges et al., 2002; Tolra et al., 2000; Kushad et al., 1999). Bridges et al. (2002), Finiguerra et al. (2001) and Tsao et al. (2002) reported that AITC was one of a factor that P. rapae search for Brassicaceae plants.
In our previous study (Huang and Renwick, 1993), it was recognized that P. rapae was attracted to C. religiosa in the Capparidaceae plant, C. religiosa is the same order and the different family from Brassicaceae plants. Also, Fahey et al. (2001) reported that a sinigrin analogue contained in the subfamily Cleomoideae in the Capparidaceae plant. In this study, we confirmed the oviposition preference of P. rapae against C. religiosa (Capparidaceae plant) and cabbage (Brassicaceae plant). In addition, we analyzed the AITC and sinigrin contents and myrosinase activity in these plants to clarify the attractant and oviposition stimulant properties of C. religiosa and cabbage to P. rapae.
MATERIALS AND METHODS
This study was conducted at the Meiji University greenhouse from July 1st to December 25th 2006.
Plant materials: Seedings of cabbage (Brassicacea oleracea var. capitata L.) of the Brassicaceae plant and lettuce (Lactuca sativa L.) of the Asteraceae plant were grown in the Meiji University greenhouse from July 6th to September 24th 2006 according to cultural guidelines approved by the systematized techniques of agriculture (Iwama, 1974). Crataeva religiosa Forst. (Brassicales: Capparidaceae) were obtained from the Museum of Insects in Hiroshima City Forest Park.
Insect: Gravid adult females of Pieris rapae crucivora Boisduval were collected at cabbage patches in Kanagawa prefecture. They were released in a cage (1.6x1.6x1.8 m) covered by a white shading net in the greenhouse and for 24 h oviposited on potted cabbages. An approximately 10% sucrose solution was provided for feeding. The potted cabbages with deposited eggs were placed in natural conditions in the greenhouse until hatching. The hatched larvae were reared on cabbage in a plastic case (10x20x7 cm) at 25°C under a 16L: 8D photoperiod until adult emergence. The adults were kept in a cage in the greenhouse until use in the experiments.
Oviposition preference of P. rapae: The first experiment was conducted in the cage of the greenhouse from September 25 to November 25, 2006. Each of three combinations of lettuce (negative control), C. religiosa and cabbage of nearly the same size were placed in the cage. Ten butterflies were then released in the cage for 24 h and the number of eggs counted at each plant. Oviposition (%) was calculated using the equation below. The experiment was carried out using three replications.
Quantitative and identification of glucosinolates: The intact glucosinolate was extracted according to Tolra et al. (2000). The glucosinolate was extracted from 5 g of C. religiosa with 70% aqueous methanol in a boiling water bath for 10 min. After cooling and centrifugation, the proteins of the supernatant were precipitated with a solution containing lead acetate and barium acetate. The tube was stopped and allowed to stand on the water-bath for 10 min. During this time the tube was vigorously shaken twice. The tube was centrifuged and the supernatant transferred to a sample tube. This solution was used for desulfatedglucosinolate extraction and HPLC-MS analysis.
Desulfatedglucosinolates were analyzed according to Kushad et al. (1999). Desulfation of the glucosinolate was performed on DEAE anion exchange columns. A 1 mL of the intact glucosinolate solution was layered on a DEAE Sephadex A-25 column. Glucosinolates were desulfated with arylsulfatase while on the column by 10 units of sulfatase suspended in 500 μL of distilled water to the column and then capped for 18 h. The desulfatedglucosinolate were eluted from the column with 2 mL of water. The eluted solution was used on HPLC-MS analysis. The experiment was carried out using three replications.
Liquid chromatograph-mass spectrometry analysis: HPLC-MS was performed with a Shimadzu LC-MS-2010EV/A. The HPLC-UV analysis was carried out on a Shimadzu CBM-20Alite HPLC with a UV detector using a Wako WS5C18 column (4 mm i.d. x200 mm) . The column was maintained at 40°C. The detection wavelength was 228 nm. The flow rate was 1 mL min-1. The HPLC-MS condition for glucosinolate analysis and the gradient condition are shown in Table 1 and 2.
Myrosinase activity: All procedures were undertaken at 4°C. Five grams of C. religiosa leaf sample, all of which were stored at -80°C, were homogenized in buffer A (50 mM Mops-NaOH [pH 7.5], 1 mM Na2EDTA, 2.5 mM DTT and 0.05% (v/v) Triton X-100) and then centrifuged at 12000 rpm for 20 min. The supernatant liquid was passed through a PD-10 column and eluted with buffer B (50 mM Mops-NaOH [pH 7.5], 0.5 mg mL-1 BSA, 2.5 mM DTT and 0.05 % (v/v) Triton X-100). The eluate was an enzyme solution. Enzyme activity was determined using sinigrin as the substrate. Fifty microliters of enzyme solution were reacted in a mixture including 0.5 mL of 0.2 M phosphoric acid buffer solution (pH 7.0), 0.1 mL of 10 mM ascorbic acid, 0.15 mL of distilled water and 0.2 mL of 1 mM sinigrin at 37°C for 10 min. The reaction was stopped by heating at 100°C for 10 min. The mixture was passed through a 0.45 μm chromatodisc (Millipore Co., Ltd. Japan). The HPLC analysis was performed under the same conditions as the quantitative analysis of glucosinolate. The experiment was carried out using three replications.
| Table 1: | LC-MS condition for glucosinolate analysis |
| Table 2: | Composition of the mobile phase used in the HPLC for separation of glucosinolates |
ZAcetonitlile | |
Quantitative analysis and identification of Isothiocyanate (ITC): Solid-phase micro extraction (SPME) was performed with a 100 μm SPME fiber (Supelco Inc., Bellefonte, PA) coated with poly dimethylsiloxane (DVB/PDMS). For headspace SPME sampling, 1.01 mL of mixture including 0.5 mL of 0.2 M phosphoric acid buffer solution (pH 7.0), 0.1 mL of 10 mM ascorbic acid, 0.15 mL of distilled water, 0.2 mL of glucosinolate fraction solution, 0.01 mL of 1000 ppm cyclohexanol (internal standard), 0.1 mL of myrosinase solution or distilled water (control) at 37°C for 10 min after myrosinase hydrolyze was put into a 4 mL vial and closed with a hole cap faced with PTFE/white silicone; the septum was then equilibrated at 40°C for 10 min. The fiber was exposed to headspace volatiles for 40°C at 10 min. The fiber was then pulled into the sample vial, which was transferred directly to the GC injector. The analyses were desorbed from the SPME coating at 250°C for 3 min in the injection port of the GC operated in the splitless mode.
A Shimadzu GC-MS QP2010 (Shimadzu Co., Ltd., Kyoto, Japan) was used to identify the isothiocyanate in C. religiosa. The injector, interface and ion source temperatures were 230°C. The column was a DB-Wax fused silica capillary column (60 mx0.25 mm i.d.; J and W Scientific, Folsom, CA) and the oven temperature was held at 40°C for 10 min and then increased by 3°C min-1 to 220°C, at which it was held for 30 min. The resulting GC-MS data for the volatile compounds were identified by comparison of their mass spectra with those of a corresponding reference standard and those listed in databases NIST 27 and 147 (Shimadzu Co., Ltd., Kyoto, Japan). The experiment was carried out using three replications.
Statistical analysis: Analysis of variance was performed on data between and within the groups. The number of eggs laid on each plant was statistically analyzed by a chi-square test under the null hypothesis that the probabilities of P. rapaeovipositing on either plant were equal:
Mean separation of contents of glucosinolate and isothiocyanate and myrosinase activity was determined by Turkey-Kramer test at p = 0.05 and standard division of the mean (SD).
RESULTS AND DISCUSSION
The oviposition preference of P. rapae to cabbage, C. religiosa and lettuce are shown in Fig. 1. P. rapae significantly preferred cabbage (99%, χ2 test, χ2 = 106.11, p<0.001) and C. religiosa (93%, χ2 = 21.56, p<0.001) compared with lettuce. Although P. rapae predominantly oviposited on cabbage (80%) rather than on C. religiosa (20%), it was clearly indicated that P. rapae oviposited on C. religiosa. These results supported our previous result (Ikeura et al., 2008).
Next, we measured AITC, sinigrin and myrosinase activity in used plants (Fig. 2a-c). The AITC and sinigrin contents and myrosinase activity in cabbage highly selected by P. rapae were 2.61 μmol g-1, 15.51 μmol g-1 and 2.51 μmol/m/gFW, respectively. The myrosinase activity of C. religiosa selected by P. rapae was 3.41 μmol/m/gFW, but AITC and sinigrin were not detected at all. However, we found that MITC, an analogue of AITC, was abundantly contained at 12.61 μmol g-1 in C. religiosa. In lettuce, these compounds and myrosinase activity were not detected at all. These results demonstrated that the glucosinolate-myrosinase system exists in C. religiosa, therefore we presumed its glucosinolate-myrosinase pathway (Fig. 3). The pathway is initiated by the conversion of alanine to aldoxime. Biosynthetic steps after aldoxime formation are believed to involve conversion to a thiohydroximate, introduction of thioglucoside sulfur from cysteine and S-glycosyl transfer from UDP-glucose. S-glycosylation of thiohydroximate is catalyzed by thiohydroximateglucosyltransferase and results in a desulfoglucosinolate.
| Fig. 1: | Oviposition preference of P. rapae to Cabbage, C. religiosa and lettuce. (a) cabbage vs. lettuce, (b) C. religiosa vs. lettuce and (c) cabbage vs. C. religiosa. Numbers in parentheses indicate the average number of oviposited eggs |
| Fig. 2: | Contents of both of isothiocyanate and sinigrin and myrosinase activity in cabbage, C. religiosa and lettuce. (a) AITC and MITC, (b) sinigrin, (c) myrosinase activity. Parallel bars represent the standard deviation of the mean. The same letters are not significantly different at p<0.001 by Turkey-Kramer test |
| Fig. 3: | Pathway for biosynthesis of glucocapparin and MITC |
The final step in glucosinolates formation is the sulfation of desulfoglucosinolates catalyzed by desulfoglucosinolatesulfotransferase. Then, the glucosinolate was hydrolyzed by myrosinase and methylisothiocyanate (MITC) was produced (Fahey et al., 2001). To confirm the glucosinolate-myrosinase system in C. religiosa, the glucosinolate fraction extracted from C. religiosa using HPLC was treated with myrosinase enzyme and then the glucosinolate and MITC contents were measured (Table 3). The content of glucosinolate decreased rapidly from 37-13 μmol g-1 with myrosinase treatment and we found MITC in this fraction, these data indicated to be the glucosinolate-myrosinase system in C. religiosa, which the glucosinolate was hydrolyzed by myrosinase and MITC was produced. These results were supported by the report that MITC was contained in Boscia senegalensis Pers. of the Capparideae plants (Seck et al., 1993).
Next, the mass spectrum of glucosinolate which was analogous to sinigrin in C. religiosa and obtained by HPLC/ESI-MS is shown in Fig. 4.
| Table 3: | The contents of glucocapparin and mitc after myrosinase treatment |
zValues within a row with different letters are significantly different (Turkey-Kramer test, p<0.05) | |
Negative ion ESI in the full scan mode indicated that the glucosinolate had mass spectrum of 333 (ions at m/z 332, [M-H]-) (Fig. 4a). Supporting these data, desulfatedglucosinolate was obtained by mass spectrum of [C6H11O5-] (m/z 163), [C6H9O4-] (m/z 145) and [CH3CN-] (m/z 41) ion in the selected ion mode (Fig. 4b). These mass spectrum corresponded to the mass spectrum of fragment ion (m/z 254, 163 and 145) reported by Kiddle et al. (2001). From these results, we identified the glucosinolate as glucocapparin (methylglucosinolate). There has been no report of the oviposition preference of P. rapae and glucocapparin and MITC in C. religiosa, however Fahey et al. (2001) reported that glucocapparin contained in the subfamily Cleomoideae in the Capparidaceae plant.
| Fig. 4: | Negative ion electrospray mass spectra of intact (a) glucocapparin and (b) desulfatedglucocapparin |
Hovanitz and Chang (1963) observed that P. rapaeoviposited on Cleome. These reports supported our results that glucocapparin presented in C. religiosa, in the Capparidaceae plant. Traynier and Truscott (1991) indicated that oviposition stimulants of P. rapae were sinigrin, gluconasturtiin and glucobrassicin. Additionally, our results demonstrated that glucocapparin may be a new oviposition stimulant.
Present results elucidated that P. rapaeoviposited on C. religiosa of the Capparideae family and that the glucosinolate-myrosinase system (glucocapparin was hydrolyzed by myrosinase and produced MITC) is presented in C. religiosa. It is a strong possibility that MITC is an attractant and glucocapparin is an oviposition stimulant to P. rapae. In the future, we will attempt to prove these hypotheses.
ACKNOWLEDGMENT
We would like to thank the Museum of Insects in Hiroshima City Forest Park for supplying C. religiosa.