Screening of some cucumber accessions to two-spotted spider mite (Tetranychus urticae)

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1 International Research Journal of Applied and Basic Sciences. Vol., 3 (8), , 2012 Available online at www. irjabs.com ISSN X 2012 Screening of some cucumber accessions to two-spotted spider mite (Tetranychus urticae) Marie Shoorooei 1, Mostafa Nasertorabi 1, Amanollah Soleimani 2, Ecehagh Moghbeli 3, Esmaeil Madadkhah 1, Hamid Moghbeli 2 1- Department of Horticulture, College of Aboureihan, University of Tehran, Tehran, Iran. 2- Department of Plant Science, Faculty of Agriculture, University of Jiroft, Jiroft, Iran. 3- Department of Horticulture, Faculty of Agriculture, Ferdowsi University of Mashhad, Mashhad, Iran. *Corresponding Author mshoorooei@gmail.com Abstract The two-spotted spider mite, Tetranychus urticae (Koch) is one of the most important pests of cucurbits, that can infests more than 200 species. In order to finding resistance sources in cucumber, ten accessions from Gene Bank were selected for further studying. Ten seeds of each accession were cultivated in seed trays (one seed per pot). At six leaf stage, one leaf (about 4 cm 2 ) from each plant was detached and placed around a circle with a diameter of 100 cm. In order to determine the antixenosis, ten adult females were released at the center of circle for each leaf. Experimental plots were arranged as a randomized complete block design with three replications. The number of mites feeding on each leaf was recorded after 24 and 48 hours. Statistical analysis showed significant differences between accession and indicated antixenosis resistance (p<0.05). Maximum number of mites were observed at C104 (12.79±0.53) and C118 (12.3±0.4) and the minimums were observed in C90 (5.58±0.65) and C39 (5.82±0.46). Therefore, the mentioned accessions are supposed to be typically susceptible and resistance to T.urticae respectively. The content of cucurbitcin B, D, E and I in extreme susceptible and resistant genotypes was also by HPLC to declare the relation between pest attraction or and secondary metabolites. Keywords: Antixenosis, Cucumis sativus, cucurbitacins, HPLC, Tetranychus urticae, resistance. Introduction Tetranychus urticae Koch (TSSM) is one of the most important pests of greenhouse cucumbers, especially under hot and dry conditions (Hussey and Scopes 1985). This species is adapted to various environmental conditions and the greenhouses are ideal areas for that, which can complete a generation in one week (Düzgünes and Çobanolu. 1983). The active stages of this pest are sap feeders that produce irregular patterns of small light colored spots on the leaf. Yield losses in cucurbites are primarily due to feeding on the lower leaf surfaces. It is particularly dominant in intensive, high-yield cropping systems, and affects crops by direct feeding, thereby reducing the area of photosynthetic activity and causing leaf abscission in severe infestations (Davidison and Lyon. 1979, Shih and Wang.1996, Gorman et al.2001, Ragkou et al., 2004). The control of T. urtica has relied on application of acaricides, with frequent chemical sprays needed to achieve effective control. However, this is not a sustainable measure against that when considering its capacity of developing resistance to acaricides by high reproductive potential and short life cycle (Hoyt et al. 1985). Genetic resistance to spider mites has been reported in many field and vegetable crops (De Ponti 1985, Flexner et al.1995, Gracen et al. 1997, Grazzini et al. 1997). Host-plant resistance to TSSM has been reported in different crops such as strawberry (Gimenez-Ferrer et al. 1994), raspberry (Wilde et al. 1991) and tomato (Saeidi and Mallik 2006). Within the genus Cucumis mite resistance research has focused mostly on the cucumber (Cucumis sativus L), because of the injury caused in greenhouse production (Tulisalo 1972). Plant resistance to a pest can be caused by antixenosis, antibiosis, tolerance, or some combinations of these mechanisms (Pain, Smith 2005). Therefore,

2 conducted choice tests to establish antixenosis (or nonpreference), in which the plant is a poor host to the insect (Smith 2005). Many authors have reported on the involvement of secondary metabolites in the interaction between plants and herbivore insects (Gershenzon, 1994). Of all possible secondary metabolites, terpenoids in particular have been shown to be involved in both indirect and direct plant defense. An important example of these direct defense compounds is the cucurbitacins. Cucurbitacins are bitter triterpenoid compounds that are toxic to most organisms, including mammals such as man, and occur widely in wild and cultivated Cucurbitaceae (Mir`o, 1995). Many different forms of cucurbitacins have been identified, all with the general cucurbitane carbon skeleton: 19-(10->9)-abeo-10-lanost- 5-ene (Chao Chen 2005). Resistance was initially linked to the presence of some cucurbitacins in the foliage, but results are inconsistent (Soans et al. 1973, Gould. 1978, Balkema-Boomstra. 2003). The objectives of this work were to identify additional sources of resistance to T. urticae on several cucumber genotypes, and detemined relation of some cucurbitacinse to mite resistance. Materials and Methods Experimental design The experimental design was Randomized Complete Block with three replications and 10 pots per ecotype in each replication. Mean comparisons were performed using Duncan's multiple range test. Spider mite colony A colony of two spotted spider mites was collected on rose from Pakdasht greenhouses in 2008 and cultured on bean (Phaseolus vulgaris L.) were grown in pots (15 cm diameter, three plants per pot) for tree generation in insect proof cages in climatic chamber (mass rearing, ºC, 60-65% RH and photoperiod of 16:8 h (L:D) (Ay et al, 2005). Plant material 10 cucumber accessions collected from different parts of Iran (the National Gene Bank, Karaj). Ten seeds of each ecotype cultivated in pots with 12 cm diameter (one seed per pot) in greenhouses (with mean annual temperature of 24 ºC, RH of 70% and 16h day/8h night, via natural lighting plus high pressure Sodium Lamps to supply an average lighting level of Lux) college of Aboureihan, University of Tehran. Chemicals Cucurbitacin E, D and I (purchased from Extrasynthese SA, Genay, France) were prepared by dissolving 5 mg of them in 5mL methanol (HPLC grade, Merck, Germany). Cucurbitacin B (purchased from Bionaturis, Netherlands) was prepared by dissolving 10 mg in 10mL acetonitrile (HPLC grade Merck, Germany). The solutions were stored at -20ºC and calibration standards were prepared by serial dilution of working solutions. Antixenosis tests The antixenosis of 10 accessions was evaluated in greenhouse. Laboratory tests were conducted to evaluate the preference of T. urtica for different cucumber ecotypes. Laboratory trials were carried out in climatic chambers at 21-25ºC, 55-80% RH, and a photoperiod of 16:8 (L:D). When plants at six leaf stage, one leaf (about 4 cm 2 ) from each plant was placed randomized arranged in a circle around a platform (100 cm diameter with a distance of 4 cm between plants). Ten adult females were released in a Petri dish with 9 cm diameter at the center of circle for each leaf. The number of mites feeding on each leaf was recorded after 24 and 48 hours (Bernal and Setamou, 2003). Measure of cucurbitacins Cucurbitacins content was determined on another disk of the same leaf that was used for the spider mite resistance test in the first experiment. HPLC (Analytical, Knauer, Germany, equipped with quaternary pump (K-1001), an auto sampler and a variable UV visible detector K-2600) analysis of cucurbitacins was carried out essentially as described by Gorski et al. (1986). cucurbitacin B, D, E and I. from leaf tissue were performed using MeOH.The HPLC was equipped with a C18 (125nm 4nm, Nucleosil-100,5µm) column using 30% acetonitrile in NANO pure water as the fluent at 0.7 ml/min and a column temperature of 25±C. cucurbitacins were determined by using external standards.

3 Results and Discussion Among 10 genotypes for antixenosis test, the positional arrangement of plants in the no-choice test influenced the feeding preference of T. urticae, as was shown in research of Costamagna et al (2005). The choice test demonstrated a significant effect of cucumber accession on the number of T. urticae feeding host. The number of mites on a given cucumber accessions did vary over different after insect release. Statistical analysis showed significant differences between accessions and indicated antixenosis resistance (p<0.05). The mean of spider mites on cucumber accessions are shown in Figure 1. Figure 1. Mean Number of mites present on different cucumber accessions leaf genotypes in the antixenosis test. Means associated with different letters differ significantly at P<0.05 by Duncan s test. Maximum number of mites were observed at C104 (12.79±0.53) and C118 (12.3±0.4) and the minimums were observed in C90 (5.58±0.65) and C39 (5.82±0.46). Therefore, the above mentioned accession are supposed to be typically susceptible and resistance to T.urticae respectively. The number of mites on cucumbers leaves reflected preferential choices of T.urticae for different genotypes after 24 and 48 h in this test. When the pooled mean percentages of mites recorded on the ten cucumber accessions were compared. Gradient HPLC separation of the cucurbitacins standards revealed UV-absorbing peaks at 235 nm is indicated in Figure 2. Figure 2. Gradient separation of the cucurbitacins standards revealed UV-absorbing peaks. Cucurbitacins content, determined by using HPLC in the first experiment with a disk from the same leaf as used for the spider mite test, varied considerably between the different cucumber accessions. Data analysis of cucurbitacin showed that there is no significant relation between cucurbitacins content of accessions to resistance to mite. Balkema-Bomestra et al (2003) concentration of cucurbitacin C in the leaves is an important parameter in spider mite resistance in cucumber. Dinan et al. (1997) demonstrated that cucurbitacins B and D, isolated from seeds of Iberis umbellata, act as antagonists at the ecdysteroid

4 receptor in a Drosophila melanogaster cell line. In rare cases, plants respond to insect feeding by releasing some cucurbitacins to limit the insects damage (Gry et al., 2006). According to Chuihua et al. (2004), the highest amount of cucurbitacins in plants are normally found in roots, therefore resistant plant should have a rapid translocation of cucurbiacin from root to leaf to defend against the feeding. Table 1. Concentration of different cucurbitacins in cucumber accessions. Accession cod Cucurbitacins (µg/g FW) D I B E C C C C C C C C C C Cucurbitacins (tetracyclic triterpenoids and their glycosides) occur commonly in this family and impart a bitter flavor, discouraging or even poisoning herbivores. Cucurbitacins among the bitterest substances known have been investigated as anticancer agents, insecticides and beetle attractants. Most cultivated Cucurbitaceae species have been selected to produce low levels of cucurbitacins, although the bitterness of cultivated species may increase in response to environmental stress such as heat or shortage of water. The concentration of cucurbitacins is generally highest in roots and leaves. Asian bitter melons are grown especially for this bitter flavour. References Ay R, Sökeli E, Gürkan MO, Response to some acaricides of two-spotted spider mite (Tetranychus urticae Koch) from protected vegetables in Isparta. Turk. J. Agric. For. 29: Balkema-Boomstra A, Zijlstra S, Verstappen F, Inggamer H, Mercke P, Jongsma M, Bouwmeester H, Role of cucurbitacin c in resistance to spider mite (Tetranychus urticae) in cucumber: Jornal of chemical ecology, Vol. 29, No.1. Bernal J, Setamou M, Fortuitous antixenosis in transgenic sugarcane: antibiosis-expressing cultivar is refractory to ovipositing herbivore pests. Environ. Entomol. 32(4): Chao Chen J, Hua Chiu M, Lin Nie R, cordell GA, Qiu SX, Ccurbitacins and cucurbitan glycosides: structures and biological activities. The Royal Society of Chemistry 22: Chuihua K, Wenju L, Xiao Y, Maoxin Z, Fei H, Mechanism of aulacophora femoralis chinensis weise feeding behavior and chemical response of host Cucumis sativus L. Chinese Science Bulletin 49: Constamagna AC, Lenicov R, Zanelli M, Maize and oat antixenosis and antibiosis against delphacodes kuscheli (Homoptera: Delphacidae), Vector of Mal de Rio Cuarto of Maize in Argentina. J. Econ. Entomol 98(4): Davidison RH, Lyon WF, Insect pests of farm, garden and orchard (7th edition). Wiley & Sons. New York. De Ponti OMB, Host plant resistance and its manipulation through plant breeding. in: Helle, W. and Sabelis, M.W. [Eds.] Spider Mites, Their Biology, Natural Enemies and Control. Volume 1B, pp Elsevier, New York, NY. Dinan L, Harmatha J, Lafont R, Chromatographic procedures for the isolation of plant steroids. J. Chromatogr. A 935: Düzgünes Z, Çobanolu S, The life history and tables Tetranychus urticae Koch and Tetranychus cinnabarinus (Boisduval) (Acarina: Tetyranychidae) under the various temperatures and humidities. Plant Protection Bulletin 23(4): Flexner JL, Westigard PH, Hilton R, Croft BA, Experimental evaluation of resistance management for two spotted spider mite (Acari: Tetranychidae) on southern Oregon pear: J. Econ. Entomol 88: Gershenzon J, The cost of plant chemical defense against herbivory: a biochemical perspective. pp , in E. A. Bernays (ed.). Insect-Plant Interactions, Vol. V. CRC Press, Boca Raton, Florida.

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