Nusantara Bioscience vol. 4, no. 1, March 2012

Majorana hortensis photo by M. Martin Vicente

| Nus Biosci | vol. 4 | no. 1 | pp. 1‐44| March 2012 | | ISSN 2087‐3948| E‐ISSN 2087‐3956 |

| Nus Biosci | vol. 4 | no. 1 | pp. 1-44 | March 2012 | | ISSN 2087-3948 | E-ISSN 2087-3956 | I S E A

J o u r n a l

o f

B i o l o g i c a l

S c i e n c e s

EDITORIAL BOARD: Editor-in-Chief, Sugiyarto, Sebelas Maret University Surakarta, Indonesia (sugiyarto_ys@yahoo.com) Deputy Editor-in-Chief, Joko R. Witono, Bogor Botanical Garden, Indonesian Institute of Sciences, Bogor, Indonesia (jrwitono@yahoo.com) Editorial Advisory Boards: Agriculture, Muhammad Sarjan, Mataram University, Mataram, Indonesia (janung4@yahoo.com.au) Animal Sciences, Freddy Pattiselanno, State University of Papua, Manokwari, Indonesia (pattiselannofreddy@yahoo.com) Biochemistry and Pharmacology, Mahendra K. Rai, SGB Amravati University, Amravati, India (pmkrai@hotmail.com) Biomedical Sciences, Afiono Agung Prasetyo, Sebelas Maret University, Surakarta, Indonesia (afieagp@yahoo.com) Biophysics and Computational Biology: Iwan Yahya, Sebelas Maret University, Surakarta, Indonesia (iyahya@uns.ac.id) Ecology and Environmental Science, Cecep Kusmana, Bogor Agricultural University, Bogor, Indonesia (cecep_kusmana@ipb.ac.id) Ethnobiology, Luchman Hakim, University of Brawijaya, Malang, Indonesia (lufehakim@yahoo.com) Genetics and Evolutionary Biology, Sutarno, Sebelas Maret University, Surakarta, Indonesia (nnsutarno@yahoo.com) Hydrobiology, Gadis S. Handayani, Research Center for Limnology, Indonesian Institute of Sciences, Bogor, Indonesia (gadis@limnologi.lipi.go.id) Marine Science, Mohammed S.A. Ammar, National Institute of Oceanography, Suez, Egypt (shokry_1@yahoo.com) Microbiology, Charis Amarantini, Duta Wacana Christian University, Yogyakarta, Indonesia (charis@ukdw.ac.id) Molecular Biology, Ari Jamsari, Andalas University, Padang, Indonesia (ajamsari@yahoo.com) Physiology, Xiuyun Zhao, Huazhong Agricultural University, Wuhan, China (xiuyunzh@yahoo.com.cn) Plant Science: Pudji Widodo, General Soedirman University, Purwokerto, Indonesia (pudjiwi@yahoo.com) Management Boards: Managing Editor, Ahmad D. Setyawan, Sebelas Maret University Surakarta (unsjournals@gmail.com) Associated Editor (English Editor), Wiryono, State University of Bengkulu (wiryonogood@yahoo.com) Associated Editor (English Editor), Suranto, Sebelas Maret University Surakarta Technical Editor, Ari Pitoyo, Sebelas Maret University Surakarta (aripitoyo@yahoo.co.id) Business Manager, A. Widiastuti, Development Agency for Seed Quality Testing of Food and Horticulture Crops, Depok, Indonesia (nusbiosci@gmail.com) PUBLISHER: Society for Indonesian Biodiversity CO-PUBLISHER: School of Graduates, Sebelas Maret University Surakarta FIRST PUBLISHED: 2009 ADDRESS: Bioscience Program, School of Graduates, Sebelas Maret University Jl. Ir. Sutami 36A Surakarta 57126. Tel. & Fax.: +62-271-663375, Email: nusbiosci@gmail.com ONLINE: biosains.mipa.uns.ac.id/nusbioscience

Society for Indonesia Biodiversity

Sebelas Maret University Surakarta

ISSN: 2087-3948 E-ISSN: 2087-3956

Vol. 4, No. 1, Pp. 1-5 March 2012

The effect of zearalenone mycotoxins administration at late gestation days on the development and reproductive organs of mice YULIA IRNIDAYANTI♥ Department of Biology, Faculty of Mathematics and Natural Sciences, State University of Jakarta. Jl. Pemuda No. 10 Rawangun, Jakarta Timur 13220, Indonesia. Tel: +92-21-4894909. email: irnidayanti@yahoo.com Manuscript received: 30 December 2011. Revision accepted: 6 February 2012.

Abstract. Irnidayanti Y. 2012. The effect of zearalenone mycotoxins administration at late gestation days on the development and reproductive organs of mice. Nusantara Bioscience 4: 1-5. Zearalenone was injected subcutaneously with a dose of 30 mg/kg body weight to pregnant mice on the 13 to 16 days. Control was given only sesame oil. Control and treated mice were killed on day 18 of gestation by cervical dislocation. Observations of maternal body weight, reproductive performance, external and internal malformation were conducted. Histological analysis of fetal ovaries, uterus, and testes were also done. The results revealed that administration of zearalenone to mice at late gestation was not teratogenic. Zearalenone caused a tendency that the primordial follicles and follicular cells relatively decreased in number and the number of the degenerate primordial follicle relatively increased. Effects of zearalenone on the uterus caused a significant increase in the height of lumen epithelial cells and in the thickness of the uterine wall were significantly. The lamina propria and myometrium started to differentiate. In the male fetus, zearalenone caused a tendency deacrease in number of the Leydig cells.

Key words: zearalenone, primordial follicle, follicle cells, uterus, Leydig cells.

Abstrak. Irnidayanti Y. 2012. Pengaruh pemberian mikotoksin zearalenon pada umur kebuntingan lanjut terhadap perkembangan dan organ reproduksi mencit.Nusantara Bioscience 4: 1-5. Zearalenon diberikan pada induk mencit bunting pada umur kebuntingan 13 sampai dengan 16 hari secara subkutan. Mencit kontrol hanya diberi minyak wijen. Mencit kontrol dan perlakuan dibunuh pada umur kebuntingan 18 hari secara dislokasi leher. Pengamatan dilakukan terhadap berat badan induk, penampilan reproduksi, kelainan eksternal dan internal. Pengujian juga dilakukan terhadap histologis ovarium fetus, uterus fetus dan testis fetus. Hasil penelitian menunjukkan bahwa pemberian zearalenon kepada mencit pada umur kebuntingan lanjut, tidak bersifat teratogenik. Zearalenon cenderung menyebabkan folikel-folikel primordial dan sel-sel folikel primordial, relatif jumlahnya menurun dan jumlah folikel primordial yang berdegenerasi relatif meningkat. Pemberian zearalenon menyebabkan bertambah tingginya sel-sel epitel pada lumen uterus, secara signifikan dan bertambahnya ketebalan dinding uterus secara signifikan Lamina propria dan miometrium sudah mulai berdifferensiasi. Pada fetus jantan, zearalenon cenderung menyebabkan penurunan jumlah sel-sel Leydig.

Kata kunci: zearalenone, folikel primordial, sel-sel folikel, uterus, sel Leydig,

INTRODUCTION Zearalenone is a natural mycotoxin produced by Fusarium roseum and grows on grain stored in a very high humid (Stob et al. 1962; Christensen et al. 1965; Chang et al. 1979). It is a secondary metabolite produced by Fusarium, associated with hiperestrogenisme syndrome and bleeding in farm animals (Mirocha et al. 1976).

Mycotoxin has a trivial (Urry et al. 1966 ) name, zearalenone and its trade name, RAL (β-resorcylic acid lactone). Initial information about the chemical structure of zearalenone was expressed as enatiomorf of 6-(10hydroxy-6-oxo-trans-1-undecenyl)-β-resorcylic acid lactone lactone lactone,with a chemical formula of C18O5H22 (Urry et al. 1968). Zearalenone can absorb ultraviolet light with wavelengths of 314, 274, and 236 μm, has a melting point at 163-165°C, has a molecular weight of 318 and has the character of blue-green fluorescence (Mirocha et al. 1967).

Concern of toxic metabolites produced by fungus began when an investigation found evidence of an association between aflatoxin and carcinogenesis in humans (Shank et al. 1971). Hidy et al. (1977) and Hobson et al. (1977) reported that zearalenone in primates can cause keratinization in vaginal epithelium , inhibit ovulation, inhibit the occurrence of implantation and suppress gonadotropin secretion. Corn contaminated by mold is a type of grain most often found in hiperestrogenism cases in pigs. One to 17% of contaminated corn samples turned out to contain zearalenone (Bennett dan Shotwell 1979).

Reports from McNutt et al. 1928 showed that the occurrence of estrogenic syndromes such as vulvar and vaginal bleeding posterior part, associated with consumption of moldy feed. Although zearalenone does not have chemical structures such as steroids, but this substance has potent trophic activity on the uterus of some animals (Ueno et al. 1974). Unique chemical structure of zearalenone can interact directly with estrogen receptors in

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the body and cause biological and biochemical responses such as that caused by natural estrogen, estradiol (Katzenellenbogen et al. 1979).

Fusarium grows in humid conditions and optimal temperature for infection is 20-25°C, and cold temperatures (8-10°C) is required to produce an optimal zearalenone (Christensen and Kaufmann. 1969). Fusarium can contaminate grain stored in a very high humid room (Stob et al. 1962; Christensen et al. 1965). Corn contaminated by the fungus is a type of grain most often found in hiperestrogenism cases in pigs.Not only in corn seeds, zearalenone is also found in barley. Animal feed containing contaminated material by fungus can cause losses to farmers (Bannett and Shotwell. 1979), because it can cause some types of reproductive disorders, such as infertility, persistent estrus, pseudopregnancy, decreased fertility, reduced size of puppies, malformations, hipere-strogenism in young animals and the possibility of resorption of embryos (Chang et al. 1979). Therefore, the objective of this study was to investigate whether zearalenone affect fetal development of mice, differen-tiation and development of reproductive system, if the dams was given zearalenone subcutaneously at a dose of 30 mg/kg body weight on gestation 13 to 16 days MATERIALS AND METHODS Animals used in these experiments were mice (Mus musculus) Swiss Webster taken from Laboratory Animal Care, Department of Pharmacy, ITB. The animals were kept in cages, Department of Biology, ITB. Male and female mice were kept in separate cages. Each virgin female mice which was in a state of estrus, 11-12 weeks old, with a body weight of 23.5 to 29.5 grams was mated with a male mice of the same age. Matings of male mice with females were conducted at 17.00. The occurrence of vaginal plug in the next morning was a sign of copulation and that day was designated as gestation day zero. Then the female mice were weighed and separated from the males.

Zearalenone used in this study was made in Makor Chemical POB 6570, Jerusalem, Israel. Zearalenone crystals were dissolved in sesame oil. Zearalenone solution was injected daily, subcutaneously in mice at gestation of 13 to 16 days. The volume of injection for the control and treated mice was 0.1 ml/10 g body weight, with a dose of

30 mg/kg body weight. Control mice were only given sesame oil. Mice were killed by cervical dislocation at gestation 18 days, then observations was done to the parent body weight of mice, reproductive performance, external and internal abnormalities. To detect internal malformations, half of live fetuses were fixed in Bouin solution. Then, the mice were dissected and the cardiovascular, urogenital organs, lens, retina, nasal cavity and cerebrum were observed (Taylor 1986).

Histological observations were done with paraffin method (Sutasurya 1985). Fetal urogenital organs were fixed in bouin solution for 24 hours. Then, staining with Hematoxylin-Eosin was done and sliced with 8 μm thick.

In histological preparations of ovarian, the shape and differentiation of muscle layer of uterine epithelial cells were observed. The thickness of epithelium and the the uterine wall without epithelium was measured. Testicular histological observations were conducted by counting the number of seminiferous tubules, spermatogonia cells and Leydig cells. for each animal, the average number of slide readings was 15-20.

"Wilcoxon's rank sum test" was used to analyze nonparametric data, such as the percentage of intrauterine death, the percentage of live fetuses, the percentage of external and internal malformations. Parametric data, such as thickness of epithelium of the uterus, the uterine muscle wall thickness, number of seminiferous tubules, spermatogonia, and Leydig cell number were examined by analysis of variance at the level of 95% (Steel and Torrie 1989).

RESULTS AND DISCUSSION Observations on mice body weight were listed in Table 1. The injection of zearalenone with a dose of 30 mg/kg body weight on 13 to 16 days of gestation had no effect on body weight and the weight of the dams. It can be concluded that zearalenone given at a dose of 30 mg/kg body weight at late gestation was not toxic to mice. There were no external abnormalities, but there was bleeding in some fetuses. Similar result was also found in the study by Mirocha et al. (1976), that states the metabolites produced

by Fusarium zearalenone could cause bleeding in livestock.

Table 1. Weight state of mice that were given zearalenone with a dose of 30 mg/kg body weight at gestation days 13 to 16.

Gestation (days)

Dose of zearalenone (mg/kg bw)

Σ Dams observed

Body weight at GD-0 (g) x ± sem

Body weight at GD-18 (g) x ± sem

Increase of body weight at GD-18 (g) x ± sem

0

10

26.18 ± 0.44

44.04 ± 1.29

18.17 ± 0.66

30

10

27.86 ± 0.59

46.07 ± 1.35

18.21 ± 0.96

13 to 18

IRNIDA AYANTI – Zearalenone mycotoxins at late geestation days

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Figure 1. Rightt kidney is smaaller than the lefft kidney in fetaal mice 18 dayss of age from dams F d who was ggiven zearaleno one, a dose of 30 3 m / kg body weight mg w at gestatioon 13 to 16 dayys (Magnificatioon 8x). K: Conttrol, E: treatmennt Smaller rightt kidney (g).

Figure 2. Desceendency testis in F i fetal mice 188 days of age frrom dams who given zearalenoone with a dosee of 30 mg / kg g body weight on o 1 to 16 days (m 13 magnification 8x). C: Control E: E Treatment, Descendency D testis (dt), t (testiis).

Figure 3. Dilataation of the uteerus in fetal micce 18 days of age F a from dams, who was givenn zearalenone w with a dose of 30 mg / kg boddy w weight on gestaation 13 to 16 daays (magnificattion 8x). C: Conntrol E: Treatm ment Dilatation of o the uterus (duu), uterus (u).

Figure 4. Crosss section of fetaal ovaries mice 18 days of agee, from control dams (magnification 400x) Prrimary oocytes (OI), primordial F f follicle (fp), folllicular cells (f),, stroma (s). Deegenerating oocytes (od).

Figure 5. Crosss section of fetaal ovaries micee 18 days of agee from the dam F ms who was givven zearalenonee with a dose off 30 mg/kg boddy w weight on 13 too 16 days of gesstation (magniffication 400x). Primary P oocytees (OI), follicle primordial (fp)), follicular cellls (f), stroma (ss), o oocytes degenerrating (od).

Figure 6. Crosss-section of thhe uterine fetaal mice 18 dayys of age , fro F om the control dams, magniffication 200x. Epithelium E (epp), p prospective of myometrium m (bm m), primordium m perimetrium (bp) ( Figure 7. Crosss-section of the uterine fetal mice 18 days of age, F a from the parent who was given zearalenoone, with a dosee 30 mg/kg boddy w weight, magnifiication 200x. Eppithelium (ep), lamina propria (lm), circular muscle m (om), loongitudinal musscle (ol), perimeetrium (p).

F Figure 8. Crosss-section of fetaal testes of micee 18 days of agee from the contrrol dams, magnnification 400x. Leydig cells (ssL) Figure 9. Crosss-section of fetaal testes of mice 18 days of agge from the dam F ms who was givven zearalenonee with a dose of 30 mg/kg boddy w weight on gestaation 13 to 16 daays , magnificattion 400x. Leyddig cells (sL).

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Various abnormalities of the internal organ development in fetuses at the age of 18 days were found, such as the right kidney being smaller than the left one.

This abnormality was found in the fetus from the mice of treatment (Figure 1) as well as in control fetuses, and Statistically, there was no significant in percentage of this abnormality in between treatment and control. Therefore, we suspect that these incidents occured spontaneously.

Bilateral testicular descendency was only found in fetal treatment (Figure 2). Normal mice fetus has a pair of testicles that are located on the right and left of vesica urine (Taylor 1986). The failure of the testes to descend from the abdominal cavity to the scrotum was caused by the failure of migration of testes into the pelvic cavity. Descendency of bilateral testes was not found in control, and it was found only in 20% on treatment group. There was no significant difference between treatment and control.Nevertheless, zearalenone was likely to inhibit testicular descendences.

Dilatation of the uterus is a reproductive tract abnormalities, which was found in this study. The uterus is a major target organ of zearalenone in mice (James and Smith 1982). Dilatation of the uterus in this study was 27.50% and was not found in control. Dilatation of the uterus is caused by zearalenone, as supported by histological observation data (Figure 3).

The histological structure of fetal ovaries of treated mice showed a difference with that of control. Fetal ovaries of mice at the age of 18 days consisted of the medulla and cortex, but the boundary on the second part was not clear on the control fetuses (Figure 4). While in the fetal ovary slice of treated mice, the medulla and cortex boundary was already beginning to seem (Figure 5). In addition, primordial follicles were also found, but relatively fewer in number than of the control and degenerate primordial follicles were relatively more numerous than those in the controls (Figure 6 and 7). This is consistent with the results of research by Yasuda et al., (1985), which used ethinyl estradiol in mice. In normal fetal mice, a number of follicle cells surrounding the oocyte contribute to prevent the process of egg follicle atresia (Yasuda et al. 1986).

According to Rugh (1968), follicle cells begin to form on day 13 of gestation. At the time of follicle formation begins, then the secretion of estrogen begins (Yasuda et al.

1987). Therefore the activity of ethinyl estradiol same with activity zearalenone. The results was also supported by Abid et al., (2004) that zearalenone reduces cell viability and inhibits DNA synthesis and it induced DNA damage and increase MDA formation. Because of the maximal cell population in follicles are granulosa cells, which play an essential role in the development and maturation of follicle (Zhu et al., 2011), global suppression of oocytes transcriptional activity and the induction of oocytes meiotic and cytoplasmic maturation (Rodgers and Irving Rodgers, 2010; Sue et al., 2009). Moreover, granulosa cells are involved in ovarian local microenviroment control system, whereas apoptosis of granulosa cells may lead to follicular atresia.Therefore it can be concluded, that administration of zearalenone may interfere with interactions between

follicle cells with the oocyte, so that many of follicular cell atresia.

In cross sections of fetal uterine of control, the walls were composed of epithelial layer limiting cylindrical lumen, primordia myometrium and perimetrium which is the outermost layer (Figure 6). While on the cross-section of fetal uterine of treatment, the uterine wall consisted of a layer of cylindrical epithelium which were significantly higher than that of controls, lamina propria had already been taking shape; myometrium had already been differentiated into the circular muscle layer, longitudinal muscle layers was beginning to appear; new perimetrium showed a single layer of epithelium (Figure 7). The uterine wall thickness of fetuses of treated mice (98.53 μm) were significantly greater than that of controls (64.65 μm).

Similarly, a thick layer of the uterin without epithelium also significantly.Thus it can be concluded that administration of zearalenone can stimulate differentiation of the uterus lining fetal mice at the age of 18 days, as well as the lamina propria and circular muscle layer, which is beginning to look. The results of this study was also supported by the results of research by Ueno et al. (1974) that zearalenone stimulate cell proliferation and mitotic cells of the uterine muscle. Zearalenone has activity that also the same activity with of β-estradiol, its can bind estrogen receptors and involved in estrogen mediated event. Zearalenon has a potent estrogenic activity and it causes several physiological alteration of the reproductive tract (Hidy et al. 1977).

Histological structure of testes of treatment showed differences from that of the control. Testicular cross sections of control fetuses (Figure 8) consisted of interstitial tissue and seminiferous compressed tubules, without lumen . Whereas the seminiferous tubules in testes of treatment had started to form lumen (Figure 9). In control fetal testis interstitial tissue, Leydig cell group was composed of five to six cells. While in the testis of treatment, Leydig cell group was composed of two to three cells, which was significantly smaller amount than of control. This situation is supported by the results of Yasuda et al., (1986), that the target organ of ethinyil estradiol is Leydig cell nucleus, which can disrupt the function of DNA in the process of cell proliferation. From the results of this research, it can be concluded that zearalenone affects the number of Leydig cells. Zearalenone given at 13 to 16 days of gestation, possibly disrupts the function of DNA in the process of cell proliferation, because the process of mitosis of mesenchymal cells that differentiate into Leydig cells occur in fetuses at the age of 13 to 15 days and decreases at 18 days old fetuses.

CONCLUSION From this research it can be concluded that zearalenone given at late gestation, is non-teratogenic, but is more estrogenic in a way to accelerate the development of the uterus. Apparently, zearalenone disrupts ovarian development process. In male fetus zearalenone a relative decrease in the number of Leydig cells.

IRNIDAYANTI – Zearalenone mycotoxins at late gestation days

REFERENCES Abid-Essefi S, Ouanes Z, Hassen W, Baudrimont I, Creppy E, Bacha H. 2004. Cytotoxicity, inhibition of DNA and protein syntheses and oxidative damage in cultured cells exposed to zearalenone. Toxicol in Vitro 18 (4): 467-474.

Bannett GA, Shotwell OL. 1979 Zearalenone in cereal grains. J Amer Oil Chem 56: 812-819.

Chang K, Kurtz HJ, Mirocha CJ. 1979. Effects of the mycotoxin zearalenone on swine reproduction. Am J Vet Res 40: 1260 - 1267.

Christensen CM, Nelson GH, Mirocha CJ. 1965. Effect on the white rat uterus of a toxic substance isolated from Fusarium. Appl Microbiol 13: 653-659.

Christensen CM, Kaufmann HH. 1969. Grain storage: The role of fungi in quality loss. University of Minnesota Press, Minneapolis, Minnesota.

Hobson W, Bailey J, Fuller GB. 1977. Hormone effects of zearalenone in nonhuman primates. J Toxicol Environ Health 3: 43 Hidy PH, Baldwin RS, Greasham RL, Keith CL, McMullen JR. 1977.

Zearalenone and some derivatives: production and biological activities. Adv Appl Microbiol 22: 59-82 James LJ, Smith TK. 1982. Effect of dietary alfalfa on zearalenone toxicity and metabolism in rat and swine. J Anim Sci 55: 110-118.

Katzenellenbogen BS, Katzenellenbogen JA, Mordecai D. 1979.

Zearalenones: Characterization of the estrogenic potencies and receptor interactions of a series of fungal β-resorcylic acid lactones.

Endocrinology 105: 33-40.

Zhu L, Yuan H, Guo C, Lu Y, Deng S, Yang Y, Wei Q, Wen L, He Z. 2011. Zearalenone induces apoptosis and necrosis in porcine granulosa cells via a caspase-3- and caspase-9-dependent mitochondrial signaling pathway. J Cell Physiol, doi: 10.1002/jcp.22906.

Mc Nutt SH, Purwin P, Murray C. 1928. Vulvo vaginitis in swine, Preliminary report. J Amer Vet Med Assoc 73: 484.

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Mirocha CJ, Christensen CM, Nelson GH. 1967. Estrogenic metabolite produc ed by Fusarium graminearun in stored corn. Appl Microbiol 15: 497.

Mirocha CJ, Pathre SV, Schauerhamer B, Christensen CM. 1976. Natural occurrence of Fusarium toxins in feedstuff. Appl Environ Microbiol 32: 553-556.

Rodgers RJ, Irving-Rodgers HF. 2010. Morphological classification of bovine ovarian follicles. Reproduction 139 (2): 309-318.

Rugh R. 1968. The mouse - its reproduction and development. Burgess Publ. Co, Minneapolis.

Shank R.C, Bourgeois C.H, Keschamras N, Chandavimol P. 1971.

Aflatoxin and autopsy specimens from Thai children an acute disease of unknown aetiology. Food Cosmet Toxicol 9: 501-507.

Steel RGD, Toriie JH. 1981. Principles and procedures of statistics a biometrical approach. Mc Graw Hill Book Co. Singapore.

Stob M, Baldwin RS, Tuite J, Andrews FN, Gillette KG. 1962. Isolation of an anabolic, uterotrophic compound from corn infected with Gibberella zeae. Nature 196: 1318.

Su YQ, Sugiura K, Eppig JJ. 2009. Mouse oocyte control of granulosa cell development and function: paracrine regulation of cumulus cell metabolism. Semin Reprod Med 27 (1): 32-42.

Sutasurya LA. 1985. The guidance of making permanentpreparations.

Department of Biology ITB, Bandung. [Indonesia] Taylor P. 1986. Practical teratology. Academic Press. London.

Ueno Y, Shimida N, Yagasaki S, Enomoto M. 1974. Toxicological approach to the metabolites of Fusaria: effects of zearalenone on the uteri of mice and rats. Chem Pharm Bull 22: 219-227.

Urry WH, Wehrmeister HL, Hodge EB, Hidy PH. 1966. The structure of zearalenone. Tetrahedron Lett (27): 3109.

Yasuda Y, Konishi H, Tanimura T. 1985. Gonadal dysgenesis induced by prenatal exsposure to ethinyl estradiol in mice. Teratology 32: 219227.

Yasuda Y, Konishi H, Tanimura T. 1986. Leydig cell hyperplasia infetal mice treated transplacentally with ethinyl estradiol. Teratology 33: 281-288.

ISSN: 2087-3948 E-ISSN: 2087-3956

Vol. 4, No. 1, Pp. 6-10 March 2012

Toxicity response of Poecilia reticulata Peters 1859 (Cyprinodontiformes: Poeciliidae) to some agricultural pesticides 1

ALIAKBAR HEDAYATI1,♥, REZA TARKHANI1, AHMAD SHADI2 Department of Fishery, Faculty of Fisheries and Environment, Gorgan University of Agricultural Science and Natural Resources, Gorgan, Iran. Tel: +980131528572, Fax: +981712220320, ♥E-mail: hedayati@gau.ac.ir 2 Young Researchers Club, Gorgan Branch, Islamic Azad University, Gorgan, Iran.

Manuscript received: 31 March 2012. Revision accepted: 30 April 2012.

Abstract. Hedayati A, Tarkhani R, Shadi A. 2012. Mortality response of Poecilia reticulata Peters 1859 (Cyprinodontiformes: Poeciliidae) to some agricultural pesticides. Nusantara Bioscience 4: 6-10. This research was performed to determine and compare acute toxicity of diazinon and deltamethrin as potential dangerous organic pesticides to assess mortality effects of these chemicals to the freshwater guppy Poecilia reticulata. LC50 of 24 h, 48 h, 72 h, and 96 h was attained by probit analysis by Finney’s and using the maximum-likelihood procedure (SPSS). The 24-96 h LC50 for the diazinon were 40.9±0.98, 33.2±0.84, 23.2±0.74 and 16.8±0.57 ppm respectively. The 24-96 h LC50 for the deltamethrin were 0.297±0.13, 0.236±0.16, 0.204±0.47 and 0.195±0.06 ppm respectively. In the present study, LC50 values indicated that deltamethrin was more toxic than diazinon to this species. LC50 values obtained in the present study were different from the corresponding values that have been published in the literature for other species of fish..

Key words: fish, LC50, diazinon,deltamethrin, pollution, toxicity, guppy.

Abstrak. Hedayati A, Tarkhani R, Shadi A. 2012. Respon kematian Poecilia reticulata Peters 1859 (Cyprinodontiformes: Poeciliidae) terhadap beberapa pestisida pertanian. Nusantara Bioscience 4: 6-10. Penelitian ini dilakukan untuk menentukan dan membandingkan toksisitas akut diazinon dan deltametrin sebagai pestisida organik dengan potensi berbahaya untuk menilai efek kematian dari bahanbahan kimia ini pada guppy air tawar Poecilia reticulata. LC50 24 jam, 48 jam, 72 jam dan 96 jam dilakukan dengan analisis probit Finney dan menggunakan prosedur maximum-likelihood (SPSS). Nilai LC50 24-96 jam untuk diazinon adalah 40,9±0,98, 0,84±33,2, 23,2±0,74 dan 16,8±0,57 ppm. LC50 24-96 jam untuk deltametrin adalah 0.297±0,13, 0,236±0,16, 0,204±0,47 dan 0,195±0,06 ppm.

Dalam penelitian ini, nilai LC50 menunjukkan bahwa deltametrin lebih beracun dari diazinon untuk spesies ini. LC50 yang diperoleh dalam penelitian ini menunjukkan hasil yang berbeda dibandingkan dengan nilai LC50 pada spesies ikan lainnya.

Kata kunci: ikan, LC50, diazinon, deltametrin, polusi, toksisitas, guppy.

INTRODUCTION Increased use of pesticides results in contamination of natural ecosystems especially the aquatic ecosystem (Stalin et al. 2008). These toxic substances may accumulate in the food chain and cause serious ecological and health problems. Chemical pesticides with persistent molecules (long half-life periods) pose a threat to fish and also to the human population consuming the affected fish.

Presence of pesticide in surface waters was reported in Canada, North America and Europe since 50 years ago, and since then many documents have been demonstrated the toxic effects of these pollutants to aquatic environment (Tinoco-Ojanguren and Halperin 1998; Capel et al. 2001; Miller et al. 2002; Galloway and Handy 2003). Organophosphorus pesticides (OPs) are largely used in agriculture, and the aquatic environment near the fields is under influence of OPs such as diazinon [O,O-diethyl O- (2isopropyl-4-methyl-6-pyrimiinyl) phosphorothioate] (Tinoco-Ojanguren and Halperin 1998).

Diazinon is a contact organophosphorus pesticide extensively used in agriculture and possesses moderately

persistence constitution (Larkin and Tjeerdema 2000; Bazrafshan 2007). The toxicity of diazinon is due to the blocking of acetyl cholinesterase (AChE) activity, which causes deleterious impacts on non-target aquatic species close to agricultural fields (Larkin and Tjeerdema 2000).

The pyrethroids including deltamethrin are widely used as pediculicides and are among the most potent insecticides known (Smith and Stratton 1986; Viran et al. 2003).

Pyrethroids have been proved to be extremely toxic to fish and some aquatic arthropods, for example shrimp (Bradbury and Coats 1989; Srivastav 1997; Viran et al.

2003). The toxicity of pyrethroids on mammals, birds and amphibians have been reviewed by Bradbury and Coats (1989).

Acute toxicity of a pesticide refers to the chemical’s ability to cause injury to an animal from a single exposure, generally of short duration. The acute toxicity tests of pesticides to fish have been widely used to acquire rapid estimates of the concentrations that cause direct, irreversible harm to tested organisms (Parrish 1995; Pandey et al. 2005).

HEDIYATI et al. – Mortality of Poecilia reticulata to some pesticides

The acute toxicity data can provide useful information to identify the mode of action of a substance and also help to do comparison of dose response among different chemicals. The 96 h LC50 tests are conducted to measure the vulnerability and survival potential of organisms to particular toxic chemicals. Substances with lower LC50 values are more toxic because lower concentrations results 50% of mortality in organisms.

Guppies are from common fresh water fishes which are capable of tolerating a wide range of fluctuations in water quality and are good model fish for ecotoxicological studies. The present study was performed to determine and compare acute toxicity of diazinon and deltamethrin as potential dangerous organic pesticides to assess mortality effects of these chemicals to the freshwater guppy Poecilia reticulata.

MATERIALS AND METHODS Healthy, unsexed P. reticulata (guppy) were selected for the present study (Figure 1). Lethal experiments were conducted using 70 healthy guppy. Test chambers were glass aquaria of 120l. All samples were acclimated for a week in these aquaria before assays with continuous aeration. Water temperature was maintained at 27⁰C by using a heater. Fish were feed twice daily with formulated feed and dead fish were immediately removed to avoid possible water quality deterioration (Gooley et al. 2000).

Nominal concentrations of active ingredient tested were 0, 5, 15, 30, and 50 ppm of commercial dose (60%) for diazinon and 0, 0.03, 0.04, 0.06, 0.10, 0.20, 0.30 and 0.40 ppm of commercial dose (2.5%) for deltamethrin. Groups of seven guppies were exposed for 96 h in aerated glass aquaria with 120l of test medium. During acute toxicity experiment, the water in each aquarium was aerated and the temperature was 27⁰C. No food was provided to the specimens during the assay and test media were not renewed. Mortality rates were recorded at 0, 24, 48, 72 and 96 h. Acute toxicity tests were carried out according to Hotos and Vlahos (1998). The nominal concentration of diazinon and deltamethrin estimated to result in 50% mortality of guppy within 24 h (24-h LC50), 48 h, 72 h, and 96 h was attained by probit analysis by Finney’s (1971) method (Finney 1971) and using the maximum-likelihood procedure (SPSS 2002). The LC50 value is obtained by fitting a regression equation arithmically and also by graphical interpolation by taking logarithms of the diazinon and dentinol concentrations versus probit value of percentage mortality.

The 95% confidence limits for LC50 was estimated by using the formula LC50 (95% CL) = LC50±1.96 [SE (LC50)]. The SE of LC50 was calculated from the formula: SE ( LC 50) = 1 / b pnw Where: b=the slope of the chemical/probit response (regression) line; p=the number of chemical used, n = the number of animals in each group, w = the average weight of the observations (Hotos and Vlahos 1998). After the acute toxicity test, the LOEC (Lowest Observed Effect Concentration) and NOEC (No

7

Observed Effect Concentration) were determined for each measured endpoint.

RESULTS AND DISCUSSION A number of fish died during the acclimation period before exposure, and no control fish died during acute toxicity tests. The mortality of P.reticulata for diazinon doses, 5, 15, 30, and 50ppm for diazinon and 0.03, 0.04, 0.06, 0.10, 0.20, 0.30 and 0.40 ppm for deltamethrin were examined during the exposure times at 24, 48, 72 and 96 h (Table 1 and 2). Significantly increased mortality of P.reticulata was observed with increasing concentrations from 2 ppm to higher concentrations.

For diazinon there was 100% mortality at 30 and higher concentrations within the 96 h, whereas 100% mortality for 0.30 ppm deltamethrin was 72 h and for 0.40 ppm were 48 h after exposure (Table 2).

Table 1. Cumulative mortality of Guppy Fish (n=21, each concentration) exposed to acute commercial diazinon.

Concentration (ppm) 0 5 15 30 50

24 h 0 0 0

No. of mortality 48 h 72 h 0 0 0 0 5 0

96 h 0 0 6

6

11

16

21

15

18

21

21

Table 2. Cumulative mortality of Guppy Fish (n=21, each concentration) exposed to acute commercial deltamethrin.

Concentration (ppm) 0.00 0.03 0.04 0.06 0.10 0.20 0.30 0.40

24 h 0 0 0 0 0 0

No. of mortality 48 h 72 h 0 0 0 0 0 0 0 0 0 0

96 h 0 0 0 0 0

6

10

12

15

19

20

21

18

20

21

21

Median lethal concentrations of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% and 90% tests were presented in Table 3. Because mortality (or survival) data are collected for each exposure concentration in a toxicity test at various exposure durations (24, 48, 72, or 96 hours), data can be plotted in other ways; the straight line of best fit is then drawn through the points. These are time-mortality lines.

The LT50 (median lethal survival time) can be estimated for each concentration.

8

4 (1): 6-10, March 2012

A

B Figure 1.A. Wild-common guppy used in this research, B. Clumps of various ornamental guppies (Poecilia reticulata). (photo: from several sources)

Table 3. Lethal Concentrations (LC1-99) of commercial dose diazinon (mean±Standard Error) depending on time (24-96 h) for Guppy.

Point LC1 LC10 LC20 LC30 LC40 LC50 LC60 LC70 LC80 LC90 LC99

Concentration (ppm) (95% of confidence limits) 24 h 48 h 72 h 96 h 9.97±0.98 5.92±0.84 1.07±0.74 9.06±0.57 23.8±0.98 18.1±0.84 11.0±0.74 12.5±0.57 29.7±0.98 23.3±0.84 15.1±0.74 14.0±0.57 33.9±0.98 27.0±0.84 18.2±0.74 15.1±0.57 37.5±0.98 30.2±0.84 20.7±0.74 16.0±0.57 40.9±0.98 33.2±0.84 23.2±0.74 16.8±0.57 44.3±0.98 36.2±0.84 25.6±0.74 17.7±0.57 47.9±0.98 39.4±0.84 28.1±0.74 18.6±0.57 52.1±0.98 43.1±0.84 31.2±0.74 19.6±0.57 57.9±0.98 48.2±0.84 35.4±0.74 21.1±0.57 71.8±0.98 60.5±0.84 45.3±0.74 24.6±0.57

Table 4. Lethal Concentrations (LC1-99) of commercial dose deltamethrin (mean±Standard Error) depending on time (24-96 h) for Guppy fish.

Point LC1 LC10 LC20 LC30 LC40 LC50 LC60 LC70 LC80 LC90 LC99

Concentration (ppm) (95 % of confidence limits) 24 h 48 h 72 h 96 h 0.141±0.13 0.107±0.16 0.151±0.47 0.142±0.06 0.211±0.13 0.165±0.16 0.174±0.47 0.166±0.06 0.241±0.13 0.189±0.16 0.184±0.47 0.176±0.06 0.262±0.13 0.207±0.16 0.192±0.47 0.183±0.06 0.280±0.13 0.222±0.16 0.198±0.47 0.190±0.06 0.297±0.13 0.236±0.16 0.204±0.47 0.195±0.06 0.314±0.13 0.250±0.16 0.209±0.47 0.201±0.06 0.333±0.13 0.265±0.16 0.216±0.47 0.207±0.06 0.354±0.13 0.282±0.16 0.223±0.47 0.215±0.06 0.384±0.13 0.307±0.16 0.233±0.47 0.225±0.06 0.454±0.13 0.364±0.16 0.257±0.47 0.248±0.06

HEDIYATI et al. – Mortaliity of Poecilia reticulata r to some pesticides

Toxicity Testing T Statistical Endpointts are in two parts: p 11-Hypothesis Testing: is there a statisstically signifficant d difference betw ween the meaan response in the treatmentts and m mean responsse in control or referencee sample? LO OEC: L Lowest Obseerved Effectt Concentratiion; NOEC: No O Observed Effe fect Concentraation. 2- Poinnt Estimates: what t toxicant conceentration willl cause a specific effect on o the t test population? LC50: the median Lethhal Concentraation.

O result forr Toxicity Testing Statisticcal Endpoints is in Our F Figure 2 and 3.

3

Diazinon (ppm)

50 40 30 20 10 0 NOEC

LOEC

LC50

Accute toxicity tesst

Deltamethrin (PPM)

Figure 2. Acutte toxicity testting statistical endpoints in Guppy F G F exposed too crude Diazinoon in different times Fish t (24 h, 48 h, 72 h and 96 h respeectively).

9

ous concerns remains due to their poteential to causse serio adveerse effects on o human annd wildlife populations. p I In addittion we foundd that both Diiazinon and deeltamethrin arre lethaal substrates to P.reticulaata. The 96 h LC50 waas calcu ulated to bee 16.8±0.57pppm for com mmercial dosse diaziinon and 0.195±0.06 ppm ffor deltamethrrin and here we w report deltamethriin to be highlyy toxic to fish..

The T 96 h LC50 diazinon on different d fishees 5 values of d reported from tennths to severall tens of mg/L L (Tsuda et al. a 1997 7; Adedeji et al. 2008). Value of diazin non 96 h LC50 was 0.8 mg/L for guppy (Poecilia reticulata a) but for zebrra fish (Brachydanioo rerio) was 8 mg/L. Differrent factor havve been n suggested too cause selecttive toxicity of o diazinon on o diffeerent fishes: different detoxification, absorption a annd diffeerent inhibitioon of acetylchholinesterase (Adedeji et al. a 2008 8; Oh et al. 1991).

Previous P studdies indicatee the high h toxicity of o deltaamethrin to fish f species aand our resultts are in goood agreeement with these reportts. Boateng et al. (20066) reported that younng fish are moore susceptiblee, and differennt species respond differently d to cconcentrationss of chemicals: Mittaal et al. (19994) estimated deltamethrin toxicity to P. P reticculate to be LC L 50 = 0.0166 ppm. Viran n et al. (20033) report LC50 value of deltamethrrin in guppiess as 5.13 mg/L L. Mesttres and Mesttres (1992) fouund 96 h fish LC50 values as a follo ows: Salmo gairdneri, g 0.339 mg/L; Cyp yprinus carpioo, 1.84 mg/L; and Sarotherodonn mossambicca, 3.50 mg//L (Messtres and Messtres 1992). L LC50 value of deltamethrin d i in tilapia, Oreochrom mis niloticus as15.47 μg/L L was reporteed

by Boateng B et al. (2006). Althoough deltametthrin is thoughht to bee less toxic inn field conditiions due to its adsorption to t sedim ments, these data d are usefuul to assessmeent of potentiaal ecosystem risks (V Viran et al. 20003).

CONCLU USION

NOEC

LOEC

LC50

Acu ute toxicity tesst Figure 3. Acutte toxicity testting statistical endpoints in Guppy F G F exposed too crude deltameethrin in differeent times (24 h,, 48 h, Fish 7 h and 96 h reespectively).

72

Discussion D The resultss of present stuudy indicate that t both chem micals d diazinon and deltamethrin varied in theiir acute toxicity to P reticulata. The toxicity of P. o deltamethriin and diazinoon on P reticulata increased witth increasing concentrationn and P. e exposure tim me. Occurrennce of pessticides in high c concentrations s in agricultural wastewaterrs and their toxxicity t aquatic orrganisms espeecially fish species to s have been r reported by many m researcheers (Larkin andd Tjeerdema 2000; 2 C Capel et all. 2001; Galloway G andd Handy 2003).

2 C Contamination n of aquatic environment e w pesticidees via with r rainfall runofff is very poossible (Williis and McDoowell 1 1982).

Fishes are sensitivee to aquatic contaminationn,and

LC L 50 values inndicated that deltamethrin is more toxiic than diazinon to this t species. L LC50 obtained d in the presennt y were differrent from thee corresponding values thaat study havee been publishhed in the litterature for otther species of o fish.. Although the LC50 under a deefined set of o ul informationn, envirronmental connditions can provide usefu the numeric valuue could nott be used in n the field, so s subsequently wee used som me biomarkers for betteer undeerstanding of agricultural a peesticides toxiccity.

AC CKNOWLED DGEMENTS The T authors thhank the Aquaaculture Reseaarch Center annd Fisheery Group foor the supplyy of research material. This work k was suppported by thhe Gorgan University of o Agriicultural Sciennces and Natural Resourcess, Iran.

ENCES REFERE Adedeeji OB, Adedejii AO, Adeyemoo OK, Agbede SA. 2008. Acuute to oxicity of diazinnon to the Afrrican catfish (Clarias gariepinuus) African A J Biotechnnol 7 (5): 651-6544

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Bazrafshan ES, Naseri AH, Mahvi, M, Shayedhi M. 2007. Performance evaluation of electrocoagulation process for diazinon removal from aquaeous environments by using iron electrons, Iranian J Environ Health Sci Eng 4: 127-132.

Boateng JO, Nunoo FK, Dankwa HER, Ocran MH. 2006. Acute toxic effects of deltamethrin on Tilapia, Oreochromis niloticus (Linnaeus, 1758). West Africa J Appl Ecol 9: 1-5.

Bradbury SP, Coats JR. 1989.Comparative toxicology of the pyrethroid insecticides. Rev Environ Contamin Toxicol 108: 133-177.

Capel PD, Larson SJ, Winterstein TA. 2001. The behavior of thirty-nine pesticides in surface waters as a function of scale. Hydrol Process 15: 1251-1269.

Finney DJ. 1971. Probit Analysis. Cambridge University Press, Cambridge.

Galloway T, Handy R. 2003. Immunotoxicity of organophosphorous pesticides. Ecotoxicol 12: 345-63.

Gooley GJ, Gavine FM, Dalton W, De Silva SS, Bretherton M, Samblebe, M. 2000. Feasibility of aquaculture in dairy manufacturing wastewater to enhance environmental performance and offset costs.

Final Report DRDC Project No. MAF001. Marine and Freshwater Resources Institute, Snobs Creek.

Hotos GN, Vlahos N. 1998. Salinity tolerance of Mugil cephalus and Chelon labrosus, Pisces: Mugilidae/fry in experimental conditions.

Aquaculture 167: 329-338 Larkin DJ, Tjeerdema RS. 2000. Fate and effects of diazinon. Rev Environ Contam Toxicol 166: 49-82.

Mestres R, Mestres G. 1992. deltamethrin: uses and environmental safety.

Rev Environ Contamin Toxicol 124: 1-18.

Miller GG, Sweet LI, Adams JV, Omann GM, Passino-Reader DR, Meier PG. 2002. In vitro toxicity and interactions of environmental contaminants (Arochlor 1254 and mercury) and immunomodulatory agents (lipopolysaccharide and cortisol) on thymocytes from lake trout (Salvelinus namaycush). Fish Shellfish Immunol 13: 11-26.

Mittal PK, Adak T, Sharma VP. 1994. Comparative toxicity of certain mosquitocidal compounds to larvivorous fish. Poecilia reticulata. Ind J Malariol 31 (2): 43-47.

Oh HS, Lee SK, Kim YH, Roh JK. 1991. Mechanism of selective toxicity of diazinon to killifish (Oryzias latipes) and loach (Misgurnus anguillicaudatus). Aquat Toxicol Risk Assess 14: 343-353.

Pandey S, Kumar R, Sharma S, Nagpure NS, Srivastava SK, Verma MS. 2005. Acute toxicity bioassays of mercuric chloride and malathion on air-breathing fish Channa punctatus (Bloch). Ecotoxicol Environ Safety 61: 114-120 Parrish PR. 1995. Acute toxicity tests. In: Rand GM (ed) Fundamentals of Aquatic Toxicology: Effects, Environmental Fate, and Risk Assessment. 2nd. Taylor & Francis, Washington DC. Smith TM, Stratton GW. 1986. Effects of synthetic pyrethroid insecticides on nontarget organisms. Res Rev 97: 93-119.

SPSS. 2002. SPSS Inc., Chicago, Illinois, USA Srivastav AK. 1997. Impact of deltamethrin on serum calcium and inorganic phosphate of freshwater catfish, Heteropneustes fossilis.

Bull Environ Contam Toxicol 59: 841-846.

Stalin SI, Kiruba S, Manohar Das SS. 2008. A comparative study on the toxicity of a synthetic pyrethroid, deltamethrin and a neem based pesticide, azadirachtin to Poecilia reticulata Peters 1859 (Cyprinodontiformes: Poeciliidae). Turkish J Fish Aquat Sci 8: 1-5 Tinoco-Ojanguren R, Halperin DC. 1998. Poverty, production, and health: inhibition of erythrocyte cholinesterase via occupational exposure to organophosphate insecticides in Chiapas, Mexico. Arch Environ Health 53: 29-35.

Tsuda T, Kojima M, Harada H, Nakajima A, Aoki S. 1997. Acute toxicity, accumulation and excretion of organophosphorus insecticides and their oxidation products in killifish. Chemosphere 35: 939-949.

Viran R, Erkoc FU, Polat H. Kocak O. 2003. Investigation of acute toxicity of deltamethrin on guppies (Poecilia reticulata). Ecotoxicol Environ Safety 55: 82-85.

Willis GH, McDowell LL. 1982. Review: Pesticides in agricultural runoff and their effects on downstream water quality. Environ Toxicol Chem 1: 267-279.

ISSN: 2087-3948 E-ISSN: 2087-3956

Vol. 4, No. 1, Pp. 11-15 March 2012

Physiological effect of some antioxidant polyphenols on sweet marjoram (Majorana hortensis) plants ABDALLA EL-MOURSI, IMAN MAHMOUD TALAAT, LAILA KAMAL BALBAA

Department of Botany, National Research Centre, Dokki, Cairo 12622, Egypt. Tel. +202-3366-9948, +202-3366-9955, Fax: +202-3337-0931, e-mail: imannrc@yahoo.com Manuscript received: 19 January 2012. Revision accepted: 28 March 2012.

Abstract. El-Moursi A, Talaat IM, Balbaa LK. 2012. Physiological effect of some antioxidant polyphenols on sweet marjoram (Majorana hortensis) plants. Nusantara Bioscience 4: 11-15. Two pot experiments were conducted in the screen of the National Research Centre, Dokki, Cairo, Egypt to study the physiological effect of foliar application of some antioxidant polyphenols on growth and chemical constituents of sweet marjoram plants (Majorana hortensis L.). Plants were treated with curcuminoids, cinnamic acid and salicylic acid, each at 5 and 10 mg/L except the control plants.The results indicate that foliar application of curcuminoids increased growth parameters under study. Total sugars were also increased as a result of foliar application of curcuminoids. On the other hand, oil %, oil yield and nitrogen % were decreased as a result of curcuminoids treatments. Cinnamic acid at 5 mg/L resulted in the tallest plants in most cases. Application of cinnamic acid at 10 mg/L signicantly increased oil % and total oil yield/plant. Sugar content followed the same trend. Treatment of sweet marjoram plants with salicylic acid significantly increased oil % and oil yield, especially in plants treated with 10 mg/L SA. Total sugars % and total nitrogen % followed the same trend. The main constituents of the plant essential oil were also markedly affected.

Key words: sweet marjoram, antioxidant polyphenols, curcuminoids.

Abstract. El-Moursi A, Talaat IM, Balbaa LK. 2012. Pengaruh fisiologis beberapa polifenol antioksidan terhadap tanaman marjoram manis (Majorana hortensis). Nusantara Bioscience 4: 11-15. Dua percobaan pot telah dilakukan di rumah kaca Pusat Penelitian Nasional, Dokki, Kairo, Mesir untuk mempelajari pengaruh fisiologis aplikasi foliar beberapa polifenol antioksidan pada pertumbuhan dan kandungan kimia tanaman marjoram manis (Majorana hortensis L.). Tanaman diperlakukan dengan kurkuminoid, asam sinamat dan asam salisilat, masing-masing sebanyak 5 dan 10 mg/L, kecuali tanaman kontrol. Hasil yang diperoleh menunjukkan bahwa aplikasi foliar dari kurkuminoid meningkat parameter pertumbuhan tanaman yang diteliti. Total gula juga meningkat akibat aplikasi foliar kurkuminoid. Di sisi lain, persentase minyak, hasil minyak dan persentase nitrogen menurun akibat perlakuan kurkuminoid. Perlakuan asam sinamat pada 5 mg/L menghasilkan tanaman tertinggi dalam keseluruhan percobaan. Perlakuan asam sinamat pada 10 mg/L secara signifikan meningkat persentase minyak dan kandungan minyak total/tanaman. Kadar gula menunjukkan kecenderungan yang sama.

Perlakuan tanaman marjoram manis dengan asam salisilat secara signifikan meningkatkan persentase minyak dan kandungan minyak yang dihasilkan, terutama pada tanaman yang diperlakukan dengan asam salisilat sebanyak 10 mg/L. Total persentase gula dan total persentase nitrogen menunjukkan kecenderungan yang sama. Konstituen utama dari minyak atsiri tanaman juga sangat terpengaruh.

Key words: marjoram manis, polifenol antioksidan, kurkuminoid.

INTRODUCTION Marjoram (Majorana hortensis L) is an annual, sometimes biennial herb or sub-shrub, with an erect, square, slightly hairy stem. The greyish leaves are opposite, oval and short-stalked. The small, white or purplish twolipped flowers are arranged in roundish clusters (‘knots’) in the leaf axil. The fruit consists of four smooth nutlets, which ripen only in warm regions (Figure 1). All parts of the plant are pleasantly aromatic.

The flowering stems are the medicinal parts. Their constituents include 1-2% of an essential oil with a spicy fragrance containing terpinines and terpinol, plus tannins, bitter compounds, carotenes and vitamin C. These substances give sweet marjoram stomachic, carminative, choleretic, antispasmodic and weak sedative properties. In

herbalism it is used mainly for various gastrointestinal disorders and to aid digestion. It is also an ingredient of ointments and bath preparations used to alleviate rheumatism (Stodola and Volàk 1992).

Curcuminoids are antioxidant polyphenols and what is considered as a curcumin or a derivative of a curcumin with different chemical groups that have been formed to increase solubility of curcumins and make them suitable for drug formulation. These compounds are polyphenols and produce a pronounced yellow color. Many curcumin characters are unsuitable for use as drugs by themselves.

They have poor solubility in water at acidic and physiological pH, and also hydrolyze rapidly in alkaline solutions (Péret-Almeida et al. 2005). Therefore, curcumin derivatives are synthezised to increase their solubility and hence bioavailability (Tomren 2007). Curcuminoids are

12

4 (1): 11-15, March 2012

A

B

C

Figure 1. Sweet marjoram plant. A. general appearance, B. Spike, C. Flower (photos from several sources).

soluble in dimethyl sulfoxide (DMSO), acetone and ethanol (Tiyaboonchai 2007), but are poorly soluble in lipids. It is possible to increase curcuminoid solubility in aqueous phase with surfactants or co-surfactants (Jayaprakasha et al.

2006). Curcumin derivatives have been synthesized that could possibly be more potent than curcumin itself. Most common derivatives have different substituents on the phenyl groups (Tiyaboonchai 2007). There is currently an increasing demand for demethoxycurcumin and (curcuminoids) because of their recently discovered biological activity (Tønnesen et al. 2002).

The role of trans-cinnamic acid in stimulating growth and activating plants was studied by many investigators. It was reported that plants synthesize large amounts of phenylpropanoid acids, mainly hydroxycinnamic acids, which are often found in conjugated forms, such as glycosides or Glc esters. These conjugates have been identified in numerous plants (Molgaard and Ravn 1988 and Herrmann 1989). Glucosides may be bioactive by themselves as defense compounds or they may be storage forms (Dixon 2001). On the other hand, 1-O-acyl Glc esters may serve as activated intermediates analogous to CoA thioesters in plant secondary metabolism (Villegas, and Kojima 1986; Lehfeldt et al. 2000). Glycosylation of hydroxycinnamic acids to form both glycosides and Glc esters is catalyzed by a group of enzymes called glucosyltransferases (GTs), which transfer the Glc residue from mostly UDP-activated Glc (Mock and Strack 1993).

Related GTs are known that glycosylate other compounds, such as flavonoids (Cheng et al. 1994), alkaloids (Moehs et al. 1997 and Kita et al. 2000), terpenoids (Jones et al.

1999), cyanohydrins (Reed et al. 1993, thiohydroxymates,

and plant hormones, Jackson et al. 2001). Many glycosyltransferases are able to glycosylate more than one aglycon, and they appear to recognize only the part of the molecule where glycosylation takes place (Hoesel 1981).

Glycosylation normally takes place in the cytosol, but Glc conjugates are found in the vacuole (Vogt and Jones 2000).

Salicylic acid (SA) was reported to play a role of natural inductor of thermogenesis in Arum lily, induces flowering in a range of plants, controls ion uptake by roots and stomatal conductivity (Raskin 1992).

The aim of the present study was to investigate the effect of some antioxidant phenolic compounds (curcuminoids, cinnamic acid and salicylic acid) on the growth and chemical constituents of sweet marjoram plant.

MATERIAL AND METHODS Growth of sweet marjoram plant Two pot experiments were carried out during two successive seasons of (2007/2008- 2008/2009) at the screen of National Research Centre (NRC), Dokki, Cairo, Egypt.

Seeds of sweet marjoram were secured from Horticulture Research Institute, Agricultural Research Centre, Ministry of Agriculture, Egypt. The seeds were sown in the nursery on 21st Febreuary, 2007 and 2008, respectively. 45 days later, the seedlings were transferred into clay pots 30 cm in diameter, each pot contained 8 kg loamy clay soil. Fifteen days after sowing, the seedlings were thinned leaving two uniform plants. Each pot received equal and adequate amounts of water and fertilizers. Phosphorous as calcium superphosphate was mixed with the soil before sowing at

EL-MOURSI et al. – Effect of polyphenol on Majorana hortensis

the rate of 4.0 g/pot. Three grams of nitrogen as ammonium sulphate in three applications (one g for each) with two weeks intervals started 30 days after sowing, also, two grams of potassium sulphate were added as soil application. Other agricultural processes were performed according to normal practice. 30 days after planting, transplants were sprayed with different concentrations of curcuminoids, cinnamic acid or salicylic acid, each at (5, 10 mg/L) in addition to control plants which were sprayed with distilled water.

Chemical constituents of sweet marjoram plant Curcuminoids were extracted from ginger plants and were secured from Department of Natural Products, NRC, Dokki, Cairo, Egypt. Treatments were distributed in a completly randomized block design with three replications, each replicate comprising three pots. The plant herbage was harvested, by cutting 5 cm above the soil surface, and plant growth characters in terms of plant height, number of branches, and herbage fresh and dry weights were recorded. Total sugars percent were determined according to Dubois et al. (1956). Total nitrogen was determined using the modified Micro-Kjeldahl method according to Jackson (1973).

Chemical composition of essential oil Samples from the fresh herbage of each treatment were separately subjected to hydro-distillation in order to determine the percentage of essential oil according to the Egyptian Pharmacopoeia (1984). Qualitative and quantitative determination of the different main constituents of marjoram oil, obtained from the first cut from each treatment had been carried out in parallel with authentic samples of different oil components by GLC technique. The qualitative identification of the main oil fractions was carried out by comparing the relative retention time of different peaks with those of the pure authentic samples. The quantitative determination was achieved by the peak area percentage, which was measured for each fraction; to study the changes in the constituents of marjoram oil as a result of the effect of different treatments used.

For this purpose, gas liquid chromatographic apparatus (VARIAN-3700), equipped with FID, Hp 4270 Integrator, was used for the separation of marjoram oil fractions of the samples. The analysis conditions were as follows: The chromatography was fitted with (2m x 1/8``) columns, peaked with Diatomic G.Hb, (100-120) mesh, and coated with 10% DEGS. 12 Ft. S.S. The columns were operated, using a temperature program, a linear increase with rate of 4C/min, from (70C to 190C); with nitrogen at 30 mL/min, as a carrier gas. The flow rates for hydrogen and air were 30 and 300 mL/min, respectively. Detector temperature was 280C. Chart speed was 0.5 cm/min range: 32; sample size was about 2 mL. Sensitivity of the apparatus was 18-8 x32. The standard material was injected with the samples of marjoram oil under the same conditions.

13

Data analysis Data obtained were subjected to standard analysis of variance procedure. The values of LSD were obtained whenever F values were significant at 5% level as described by Snedecor and Cochran (1980).

RESULTS AND DISCUSSION Growth of sweet marjoram plant Data presented in Table 1 show that curcuminoids treatment at 5 mg/L significantly promoted plant height, number of branches, fresh and dry weights of herb in both cuttings. Fresh and dry weights of herb followed the same trend. Application of curcuminoids at 10 mg/L resulted in a marked decrease in the number of branches in the second cut. Cinnamic acid at 5 mg/L resulted in the tallest plants in most cases, number of branches, fresh and dry weights of herb followed the same trend. Salicylic acid treatments significantly increased plant height, number of branches, fresh and dry weights of herb, especially in plants treated with 5 mg/L SA (Table 1).

In this concern, Raskin (1992) reported that salicylic acid (SA) is an endogenous growth regulator of phenolic nature, which participates in the regulation of physiological processes in plants. SA, for example, plays a role as natural inductor of thermogenesis in Arum lily, induces flowering in a range of plants, controls ion uptake by roots and stomatal conductivity.

Talaat (2005) reported that foliar application of salicylic acid (50 or 100 µM) enhanced the vegetative growth of geranium plants, especially at 100 µM concentration.

Talaat and Balbaa (2010) also reported that exogenous application of trans-cinnamic acid on basil plants considerably increased plant growth at both the two cuttings. It was also recognized that the most promising results of vegetative growth criteria (i.e., plant height, number of branches, fresh and dry weights of herb) were obtained from plants treated with trans-cinnamic acid (250 mg/L).

Chemical constituents of sweet marjoram plant Data presented in Table 2 show that oil % and total oil yield/plant were significantly decreased as a result of foliar spray of curcuminoids at 5 mg/L and 10 mg/L. These results hold true for essential oil % and total oil yield/plant in both cuttings. Total nitrogen % followed the same trend.

On the other hand total sugars % was pronouncedly increased as a result of curcuminoids treatments.

Data also indicate that application of cinnamic acid, especially at 10 mg/L signicantly increased oil % and total oil yield/plant. Sugar content followed the same trend. On the other hand, total nitrogen % was markedly decreased as a result of cinnamic acid treatments.

Meanwhile, treatment of sweet marjoram plants with salicylic acid significantly increased oil % and oil yield, especially in plants treated with 10 mg/L SA. Total sugars % and total nitrogen % followed the same trend.

In this respect, Talaat and Balbaa, (2010) reported that chemical analysis of the leaves of sweet basil at both the

14 first and second cuts indicated that the contents of total essential oil % and oil yield in basil herb were significantly increased as a result of foliar application of transcinnamic acid. Similar results were obtained for total carbohydrates and total soluble sugars, Total nitrogen, total phosphorus and total potassium contents. Iron and zinc contents followed the same trend.

These findings were in agreement with those obtained by Tari et al. (2002) who reported that SA application resulted in a significant increase in total soluble sugar content in leaves of Camellia cuttings thus maintaining the carbohydrates pool in the chloroplasts at a high level. This increase may be implicated in osmotic adjustment as it has been described in tomato SA-treated plants (Tari et al. 2002). Talaat (2005) also reported that foliar application of salicylic acid (50 or 100 µM) increased total sugars %, total protein (μg/g FW), essential oil % and essential oil yield. Kaveh et al. (2004) reported that very low dose (50 µM) of SA considerably enhanced the growth and carbohydrate metabolism of tea cuttings.

Addition of 50 µM SA produced the most remarkable effects. There was a 2 fold significant increase in leaf area, leaf fresh weight and leaf dry weight. Leaf TSS was also doubled by this treatment. Invertase activity in SA treated cuttings was higher than in control with a significant increase for 50 µM SA. Chemical composition of essential oil To study the effect of different treatments on essential oil composition of sweet marjoram plants the oil of each treatment was separately subjected to gas liquid chromatography and the main compounds and their relative percentages are shown in (Table 3). Linalool ranged from 10.62% in plants treated with 5 mg/L salicylic to 32.36% in plants treated with 10 mg/L curcuminoids. The highest content of α-terpineol (38.11%) was observed in plants received 10 mg/L curcuminoids.

In this respect, Talaat (2005) reported that foliar treatment of pelargonium plants with salicylic acid at the rate of 50 μM/l resulted in the highest content of citronellol.

Gamal El-Din and Reda (2006) also reported that treatment of chamomile plants with salicylic acid, especially at 60 μM/l resulted in quantitative increases of some essential oil constituents.

4 (1): 11-15, March 2012 Table 1. Effect of some antioxidant polyphenols on vegetative growth of sweet marjoram plants Treatments (mg/L)

Number of branches 1st cut 2nd cut 1st cut 2nd cut 46.67 28.00 21.67 23.00 43.67 26.00 17.67 17.67 49.00 27.00 19.33 22.00 51.00 33.67 23.33 28.33 46.33 30.33 18.00 27.00 48.00 32.00 20.67 28.00 42.33 25.67 14.00 20.00 3.30 1.19 2.34 2.18 Plant Height

Curcuminoids 5 Curcuminoids 10 Cinnamic acid 5 Cinnamic acid 10 Salicylic acid 5 Salicylic acid 10 Control LSD (5%)

Fresh wt. of herb 1st cut 2nd cut 75.10 48.78 75.01 39.71 79.63 55.47 93.18 76.82 77.10 74.86 88.16 75.15 75.30 41.72 4.07 4.15

Dry wt. of herb 1st cut 2nd cut 31.20 17.08 30.53 13.61 27.47 28.11 32.78 30.27 25.98 22.15 32.87 29.83 24.27 13.73 4.32 3.60

Table 2. Effect of some antioxidant polyphenols on chemical constituents of sweet marjoram plants Treatments (mg/L) Curcuminoids 5 Curcuminoids 10 Cinnamic acid 5 Cinnamic acid 10 Salicylic acid 5 Salicylic acid 10 Control LSD (5%)

Total sugars % 1st cut 2nd cut 1st cut 2nd cut 1st cut 0.34 0.29 0.26 0.14 16.4 0.25 0.26 0.19 0.10 16.95 0.38 0.46 0.30 0.25 15.15 0.41 0.48 0.38 0.37 16.85 0.39 0.42 0.30 0.32 16.1 0.42 0.44 0.37 0.33 16.35 0.36 0.38 0.27 0.16 14.15 0.06 0.07 0.05 0.05 N.S. Oil %

Oil yield

Total Nitrogen % 1st cut 9.2 8.25 9.69 7.06 9.69 11.88 9.69 N.S.

Table 3. Effect of curcuminoids on essential oil constituents of sweet marjoram plants.

Treatments (mg/L) essential oil constituents α-pinene β-pinene camphene d-limonene cymene linalool α-terpineol geraniol carvone eugenol citronellol ethylcinnamate cavacrol thymole known unknown

Curcuma- Curcumi- Cinnamic Cinnamic Salicylic Salicylic noids 5 noids 10 acid 5 acid 10 acid 5 acid 10 Control (mg/L) (mg/L) (mg/L) (mg/L) (mg/L) (mg/L) 1.55 7.09 7.35 17.28 7.63 11.20 29.05 3.09 2.73 1.59 1.88 0.55 90.99 9.01

0.76 0.72 1.49 5.01 8.74 32.36 38.11 3.87 1.82 92.88 7.12

1.16 0.38 0.80 7.88 6.71 15.81 35.40 1.94 4.33 0.81 0.26 0.22 1.73 0.19 77.62 22.38

1.40 2.51 8.85 7.95 23.30 10.62 31.68 5.10 0.81 0.11 1.88 0.50 0.17 0.27 95.15 4.85

0.12 6.51 6.90 13.84 8.12 14.76 30.89 5.88 1.21 0.19 0.12 2.14 0.17 0.34 91.19 8.81

1.03 7.02 6.68 14.16 7.06 14.13 33.21 5.98 0.97 0.19 0.13 1.65 0.19 0.21 92.61 7.39

1.06 7.03 7.90 17.11 6.49 16.83 28.56 1.72 3.49 0.86 1.72 0.49 0.16 0.28 93.7 6.3

EL-MOURSI et al. – Effect of polyphenol on Majorana hortensis

CONCLUSION From the above mentioned data, it could be concluded that the antioxidant polyphenols (curcuminoids, cinnamic acid and salicylic acid) might play a role in plant phytochemical mechanisms through affecting the metabolism of terpenes, essential oil, carbohydrates and proteins, but further studies are needed to learn more about these mechanisms.

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ISSN: 2087-3948 E-ISSN: 2087-3956

Vol. 4, No. 1, Pp. 16-21 March 2012

Characterization of Carica pubescens in Dieng Plateau, Central Java based on morphological characters, antioxidant capacity, and protein banding pattern AINUN NIKMATI LAILY, SURANTO, SUGIYARTO

Bioscience Program, School of Graduates, Sebelas Maret University, Surakarta 57126, Central Java, Indonesia. Jl. Ir. Sutami 36A, Surakarta 57126, Central Java, Indonesia. Tel./Fax. +62-271-663375. email: sugiyarto_ys@yahoo.com Manuscript received: 2 February 2012. Revision accepted: 20 March 2012.

Abstract. Laily AN, Suranto, Sugiyarto. 2012. Characterization of Carica pubescens in Dieng Plateau, Central Java based on morphological characters, antioxidant capacity, and protein banding pattern. Nusantara Bioscience 4: 16-21. Carica pubescens Lenne & K. Koch is a species of fruit plant firstly cultivated in South America and has adapted to the highland environment, such as Dieng Plateau, Central Java (2000 m asl). It has a narrow habitat range and limited or unknown intraspecies variation. Therefore, important information about the characters of the plants at various altitudes is needed, so that it is possible to extend its distribution through transplantation to other areas. Characterization can be performed based on morphological characters, chemical content, and protein banding patterns. This study aimed to describe the morphological characters, the chemical content (antioxidant capacity), and the pattern of protein bands by staining, using coomassie brilliant blue on C. pubescens in the Dieng Plateau. The research was conducted in the villages of Kejajar (1400 m asl), Patak Banteng (1900 m asl), and Sembungan (2400 m asl). The observations of morphological characters were conducted in the field and continued in the laboratory. Morphological characters, the chemical content (antioxidant capacity), and the banding pattern of protein of C. pubescens were analyzed descriptively. The results showed that the morphological characters of C. pubescens in Dieng Plateau varried in stems, leaves and fruits. The antioxidant capacity decreased with decreasing habitat altitude, 2400 m asl altitude> 1900 m altitude> 1400 m asl. The Protein banding patterns did not vary, but the pattern in C. Papaya was different. The uniformity of the pattern of protein bands showed that genetic stability in C.pubescens was not affected by environmental factors.

Key words: Carica pubescens, morphological characters, antioxidant capacity, protein banding pattern.

Abstrak. Laily AN, Suranto, Sugiyarto. 2012. Karakterisasi Carica pubescens di Dataran Tinggi Dieng, Jawa Tengah berdasarkan sifat morfologi, kapasitas antioksidan, dan pola pita protein. Nusantara Bioscience 4: 16-21. Carica pubescens Lenne & K. Koch merupakan jenis tanaman buah yang pertamakali dibudidayakan di Amerika Selatan dan beradaptasi pada lingkungan dataran tinggi, misalnya Dataran Tinggi Dieng, Jawa Tengah (2000 m dpl). C. pubescens memiliki daerah persebaran sempit dan variasi intraspesies terbatas atau belum diketahui. Oleh karenanya, diperlukan informasi mengenai karakter tanaman pada berbagai ketinggian sehingga dimungkinkan untuk memperluas daerah penyebaran melalui transplantasi di daerah lain. Karakterisasi dapat dilakukan berdasarkan karakter morfologi, kandungan kimia, dan pola pita protein. Penelitian ini bertujuan untuk mendeskripsikan karakter morfologi, kandungan kimia (kapasitas antioksidan), dan pola pita protein dengan pewarnaan coomassie brilliant blue pada C. pubescens di Dataran Tinggi Dieng. Penelitian lapangan dilakukan di Desa Kejajar (1400 m dpl), Patak Banteng (1900 m dpl), dan Sembungan (2400 m dpl). Pengamatan karakter morfologi dilakukan di lapangan dan dilanjutkan di laboratorium. Karakter morfologi, kandungan kimia (kapasitas antioksidan), dan pola pita protein C. pubescens dianalisis secara deskriptif. Hasil penelitian menunjukkan bahwa karakter morfologi C. pubescens di Dataran Tinggi Dieng bervariasi pada batang, daun, dan buah. Kapasitas antioksidannya bervariasi dengan urutan dari ketinggian 2400 m dpl > 1900 m dpl > 1400 m dpl. Pola pita proteinnya tidak bervariasi antar ketinggian, namun berbeda dengan C. papaya. Keseragaman pola pita protein menunjukkan kestabilan genetik C. pubescens tidak dipengaruhi oleh perubahan lingkungan.

Kata kunci: Carica pubescens, karakter morfologi, kapasitas antioksidan, pola pita protein

INTRODUCTION The genus Carica of the family of Caricaceae has about 40 species, but only seven species are edible (Budiyanti et al., 2005). In Indonesia, one of the edible species is Carica pubescens Lenne & K. Koch which is cultivated only in the highland of Dieng, Central Java and locally known as karika or mountain papaya. C. pubescens is a species

introduced from the Andes, South America which grows at the altitude of 2000 meters above sea level (m asl), at low temperature and high rainfall.

Not all places in the Dieng Plateau are suitable for C. pubescens. C. pubescens does not grow well at the valley of Dieng at the altitude of ± 1400 m asl as in Kejajar village, but it grows very well at the top of the Dieng at the altitude of ± 2400 m asl, like in the village of Sembungan.

LAILY et al. – Carica pubescens of Dieng Plateau, Central Java

Thus, the higher the place in the Dieng Plateau the more C. pubescens will be found, hence it has a narrow distribution range.

Variations in karika are believed to be influenced by environmental and genetic factors. Sitompul and Guritno (1995) say that the appearance of plant forms is controlled by the genetic properties of plants under the influence of environmental factors. Environmental factors believed to influence the occurrence of morphological changes in plants are temperature, soil type, soil conditions, altitude, and humidity. If the environment factors are more powerful than the genetic factors, then the plants in different places with different environmental conditions will have different morphologies (Suranto 2001). But if the influence of environmental factors is weaker than that of the genetic factors then there will not be any morphological difference despite being planted in different places.

The problem faced today is the lack of information regarding the characterization of C. pubescens in terms of morphological features, chemical content, and protein banding pattern. Morphological features can be used to characterize patterns of genetic diversity, but the nature which can be described is limited and likely to be influenced by environmental factors, so that molecular genetic identification is required to overcome these limitations (Rahayu et al., 2006). Information about the molecular characters can be gathered by knowing the protein banding pattern of C. pubescens while the chemical character can be determined by measuring the antioxidant capacity of these plants. This study aimed to describe the morphological characters, the chemical content (antioxidant capacity), and the the pattern of protein bands by the staining of C. pubescens using coomassie brilliant blue in the Dieng Plateau, Central Java.

MATERIALS AND METHODS Time and places This study was conducted from July 2010 to February 2011. The field research on morphological characters of C. pubescens Lenne & K. Koch was conducted in the village of Kejajar (1400 ± 50 m asl), Patak Banteng (1900 ± 50 m asl), and Sembungan (2400 ± 50 m asl) in the Dieng Plateau, Wonosobo district, Central Java. The field research on morphological characters of superior C. papaya was conducted in Boyolali, Central Java (1500 ± 50 m asl).

Antioxidant capacity and protein banding pattern were analyzed at the Sebelas Maret University, Surakarta, Central Java.

Procedures Sampling Samples of C. pubescens in three different heights and those of C. papaya were taken for morphological observations in the laboratory, the analysis of antioxidant capacity, and the pattern of protein bands. Samples were required for the laboratory observations on the morphology of leaves, flowers, and fruits.

17

The observation on morphological characters Observations were conducted on 10 C. pubescens plants for the three different altitudes at the Dieng Plateau, with a comparison with the superior plant of C. papaya of Boyolali. Observation of morphological characters in the field was followed by observations in the laboratory. Parts of stems, leaves, flowers, fruits, and seeds of C. pubescens were observed and documented. The morphological characters of stems observed included the height, the diameter, the cross-sectional shape, the outer surface, the color, the branch, the trunk’s appearance.

The morphological characters of the leaves included color, the bone, the stalk’s length, the leaf’s diameter, and the leaf‘s blade. The morphological characters of the flowers were types of flowers, the basic form of flowers, the shape of the curve, the edge of the petals, the number of crowns, the number of stamens, the number of the ovule, the position of the stamens, the postion of the fruit in relation to the position of the base of the flower, and the shape of the flower. The morphological characters of the fruits observed were the color, the dominant of central cavity, the diameter, the length, and the length of stem, the shape of fruit, the lengthwise slice, and the crosswise slice. The characters of the morphology of seeds observed were the common form of the outside part of the seed, the engraving of the grain leather, and the color of the endosperm. The guideline for the observation of these morphological characters was taken from Tjitrosoepomo (1990), Muzayyinah (2008), and the Center for Plant Variety Protection of the Ministry of Agriculture of the Republic of Indonesia (2006).

Test of antioxidant capacity A total of 100 g of fruit extracts of C. pubescens and C. papaya were weighed and then dissolved in 1 mL methanol. The main liquor was taken using a micro pipette with multilevel dilution to obtain the test solution concentration of 10 ug / mL, 5 mg / mL, 2.5 microg / mL, and 1.25 microg / mL. One mL of each test solution was put into glass bottles and then added with 2 mL of DPPH (diphenyl picril hydrazil hydrate), then was left for 30 minutes. Methanol was used as a blank solution . DPPH absorbance was analyzed with a spectrophotometer of visible light at a wavelength of 517 nm.

The making of protein banding pattern The analysis of the protein band profile was carried out according to the methods of Coats et al. (1990), using SDSPAGE electrophoresis technique. The concentration of acrylamide for stacking gel was 3%, while for the gradient gel was 10%. Electrophoresis was run at a constant voltage of 110 VA, until the loading dye was near the bottom of the gel. The painting was done overnight using the solution of coomassie brilliant blue, followed by the laxative solution until the protein banding pattern emerged. Electrophoresis results were documented in a digital camera.

Data analyses Morphological character data, the chemical content (antioxidant capacity), and the banding pattern of proteins in C. pubescens and C. papaya were analyzed

18

4 (1): 16-21, March 2012

descriptively. The antioxidant capacities of the fruits of C. pubescens and C. papaya were analyzed based on the absorption percentage of DPPH. Antioxidant capability was measured as a decrease in DPPH solution absorbance due to the addition of the sample for 30 minutes. DPPH solution absorbance values before and after the addition of the extract were calculated as percent inhibition (% inhibition). Then the calculations was included in the regression equation with the extract concentration (mg/100 mL) as the abscissa (X axis) and the percentage of inhibition as the ordinate (Y axis). The value of IC50 derived from the calculation at the time of the percentage of inhibition was 50%. Y = ax + b (Cahyana 2002).

The banding patterns formed on the leaf organ samples of C. pubescens and C. papaya were analyzed based on whether or not the band appeared on the gel and also the thickness of the band which was formed, as has been done

by Suranto (1991, 2001, 2002) and Triawati (2005).

Banding patterns which were formed were drawn as zimograms. The diversity of banding pattern was determined by the value of Rf, which is the relative mobility values obtained from the comparison between the migration distance towards the migration of loading dye.

The data were obtained in the form of qualitative data, so the data analysis were done descriptively based on the results of electrophoresis of the leaf ’s organ of C. pubescens and C. papaya.

RESULTS AND DISCUSSION Morphological characters C. pubescens can be found in the Dieng Plateau, central Java at an altitude of 1400 m asl up to 2,400 m asl.

The word "pubescens" means hair (Center for Plant Variety protection of the Ministry of Agriculture of the Republic of Indonesia 2006). Morphological observations of C. pubescens found the presence of feathers in several organs of plants, among which was evident on the outer surface of the lower leaves (abaksial), leaf stalk, the outer surface of the flowers, both male flowers and female flowers. C. pubescens has more hair than another member of the genus Carica, namely C. papaya (Table 1).

100

100

100

90

90

90

80

80 In h ib is i %% inhibition

60 50 40 30

60

70

50 40 30

60 50 40 30

20

20

20

10

10

10

0

0

0

2

4

6

8

10

Extract conc. (mg/100 mL) K onse ntra si e kstra k (m g /100 m l)

A

12

14

y = 2.0346x + 48.003 R 2 = 0.9554

80

y = 4.7589x + 43.842 R 2 = 0.6405

70

In h ib is i %%inhibition

y = 5.2116x + 3.915 2 R = 0.7029

70 In h ib is i %%inhibition

Antioxidant capacity The leaf’s morphology of C. pubescens at different altitudes showed the presence of variations. The colors appeared thickher in plants that grew on the higher land. At the higher places, the green and the large size of the leaves increase the amount of chlorophyll and the area of crosssectional sliced leaf’s surface, so that the tree is able to harness the sun's rays that are not too high in terms of intensity for optimal photosynthetic activity.

Morphological features of flowers as the proliferation of generative organs of plants did not show any variation.

Plants at every altitude consistently showed all types of flowers, namely male, female, and hermaphrodite.

Morphological characters suggest that environmental factors, namely the air pressure and extreme temperatures on the higher altitude of the Dieng Plateau support the growth and development of C. pubescens.

Antioxidant capacity was measured by counting the amount of reduced DPPH purple color intensity that is proportional to the reduction of DPPH solution concentration (Figure 1).

The amount of the inhibition’s percentage of various concentrations of the extract that gave rise to IC 50 values indicated that the fruit of C. pubescens grown at different altitudes had different antioxidant activities. Of the three sample extracts derived from the three different altitudes, the extracts of C. pubescens at an altitude of 2400 m asl had the highest antioxidant capacity with IC 50 of 0.983 mg/100 mL, followed by C. pubescens grown at an altitude of 1900 m asl with IC50 of 1.2945 mg/100 mL, and C. pubescens grown at an altitude of 1400 m asl with IC50 of 8.843 mg/100 mL, while in C. papaya was 5.326 mg/100 ml. The order of antioxidant capacity from the largest to the smallest was as follows: C. pubescens at an altitude of 2400 m asl> C. pubescens at an altitude of 1900 m asl> C. papaya> C. pubescens at an altitude of 1400 m asl.

All three kinds of fruit extracts of C. pubescens from the three different altitudes had antioxidant capacity. The response of plants as a result of environmental factors can be seen in the morphology and physiology . Plants that normally live in areas of high elevation are the type that can adapt to the climatic conditions of low temperature, high humidity and low sun light intensity.

0

0

2

4

6

8

10

(mg/100 mL)m l) KExtract onse ntraconc. si e kstra k (m g /100

B

12

14

0

2

4 6 8 10 12 K onse ntra si e kstra k (m g /100 m l)

14

Extract conc. (mg/100 mL)

C

Figure 1. Linear regression curves for the determination of IC50 fruit extracts of C. pubescens growing at the altitude of: (a) 1400 m asl, (b) 1900 m asl, and (c) 2400 m asl.

LAILY et al. – Carica pubescens of Dieng Plateau, Central Java

19

Tabel 1. Morphological characteristics of C. pubescens in Dieng Plateau, Central Java Plant organs Stem Height (cm) Diameter (cm) Cross-sectional shape The outer surface Color Branch How the branches look Leaves Color Leaf vein Length of stalk (cm) Diameter (cm) Leaf blade Flowers Flowers type Shape of flowers receptacle Shape of curve edge of sepals Number of petals Number of stamens Number of ovule Position of stamens Position of ovary towards receptacle of flowers Shape of flowers

1400 m asl

Morphological characters 1900 m asl

2400 m asl

193,8 11,2 round silken to rough and has pustules brown to dark brown, green, greenish brown and white glossy no branches, 2-4

174,7 11,6 round rough, has pustules greenish brown

153,3 10,8 Round silken to rough dark brown, greenish brown

4

6-8

dark green, yellowish green, finger-like, reddish or yellowish 33,65 45,67

dark green, dark green finger-like, yelowish 44,45 47,8

dark green finger-like, reddish 44,54 54,2

male, female, hermaphrodite round spirostichous 5 5 5 above the ovary on the receptacle of the flower

male, female, hermaphrodite round spirostichous 5 5 5 above the ovary on the receptacle of the flower

male, female, hermaphrodite round Spirostichous 5 5 5 above the ovary on the receptacle of the flower

young-old green on young fruit, and yellow on ripe fruit pentagon 7,3 8,8 1,8

young-old green on young fruit, and yellow on ripe fruit pentagon 7,2 8,6 1,8

Fruits Color

bright-dark green on young fruits, and yellowish on ripe fruit Dominant shape of central space pentagon Diameter (cm) 5,4 Length (cm) 8,1 Length of stalk (cm) 2,95 Shape of fruits Cross-sectional shape Longitudinal-sectional shape

The production of flavonoids needs sugar as eritrosa phosphoenolpyruvate and eritrosa 4-phosphate that provide some carbon atoms required for the B- flavonoid ring as well as an acetate unit for the A flavanoid ring.

Sugars, especially sucrose, can be obtained from the decomposition of starch or fat in storage organs during development of the sprouts or photosynthesis in cells that contain chlorophyll. Light also affects the composition of the chloroplast.

The antioxidant capacity test shows that the higher concentration of the standard vitamin C means more

antioxidant activities. Using the regression equation, the obtained IC50 vitamin standard was -84.7875 C mg/100 mL. This value is lower than that in the IC50 fruit extract of C. pubescens and C. papaya. Fruit extracts of C. pubescens and C. papaya have a capacity of antioxidant because it contains flavonoids. Antioxidant capacity of Flavonoid is associated with the presence of phenolic hydroxyl group attached to the frame structure. Flavonoid compounds have been proven to be able to reduce free radical of DPPH. The activities are different, possibly because each extract that is believed to be flavonoid has a

20

4 (1): 16-21, March 2012

hydroxyl group with different number and location of flavonoid skeleton. Flavonoids with free hydroxyl group have a radical capturing activity and the presence of more than one hydroxy group on ring B in particular will increase the antioxidant activity.

Genetic differences and the environmental factors give the optimal growth of C. pubescens and C. papaya. The apparent variations in pattern of protein bands between C. pubescens and C. papaya showed the diversity of the synthesized protein, and it can be assumed there are differences in genetic makeup that encodes these proteins.

The diversity of banding pattern of each species showed an encoding genetic diversity, since protein is a direct product of the gene in the form of amino acids. Amino acids are encoded by the DNA specifically for each type of protein.

Resistance to damage may be caused by the pressure resistance of the protein molecule, being protected from damage by other molecules, a special structure, or a certain behavior patterns. Judged from the data pattern of protein bands, it appears that there is a striking difference between C. pubescens and C. papaya, on the level of molecular characteristics.

Protein banding patterns The protein banding patterns were analyzed in the form of zimogram that was the typical electrophoresis results, so it can be used as a characteristic feature of the leaf’s organ of C. pubescens. Data were analyzed qualitatively, based on whether or not the band appeared and whether thin and thick bands were found in the gel electrophoresis results.

The diversity of banding pattern was seen from the Rf values that were formed. Rf value is the value of relative mobility explained by Ferguson, obtained from the comparison of the migration distance of protein towards the migration distance of loading dye.

The zymogram gained from running along the karika leaves of C. pubescens at an altitude of 1400 m asl and the leaves of C. papaya is shown in Figure 3. These data show the similarity of protein banding pattern on the samples of C. pubescens from different altitudes. This suggests that the molecular basis of this plant is stable in response to various environmental factors. Environmental factors influence the morphology and physiology of the plant. The morphology of the plant is adapted to environmental conditions so that physiological processes can run optimally. Genetic variation is a key to optimal treatment towards the genetic resources. Morphological features can be used to characterize a species or an individual, but the nature described is only a small proportion of the genetic code.

Therefore, the molecular characterization of genetic variation should be made from the protein banding pattern because it produces more accurate data since the protein is a late gene expression, relatively simple, and not easily changed.

Rf

1

2

3

4

5

6

7

8

9

Rf

0 0,008

0 0,008

0,048 0,063 0,079

0,087

0,111

0,103

1

2

Rf

1

2

3

4

5

6

7

8

9

10

0 0,008 0,048 0,063 0,079 0,087 0,103 0,111

0,357

0,500 0,524 0,548 0,556 0,563 0,595 0,661 0,643 0,659 0,683 0,706

Figure 3. Zymogram protein banding pattern of the leaves of C. pubescens at 1400 m asl and C. papaya

3

4

5

6

7

0,357

0,500 0,524

0,548 0,563

0,556

0,595 0,661 0,643 0,659 0,683 0,706

0,706

A

B

Figure 2. Zimogram protein banding pattern on the same scale: (a) karika leaves of C. pubescens at an altitude of 1400 m asl, 1900 m asl and 2400 m asl, and (b) leaves of C. papaya. Note: 1: 2: 3: plants at an altitude of 1500 m asl, 4, 5, 6 plants at an altitude of 1900 m asl, and 7; 8; 9 plants at an altitude of 2400 m asl.

LAILY et al. – Carica pubescens of Dieng Plateau, Central Java

CONCLUSION Morphological characters of C. pubescens in Dieng Plateau showed a variation in the stems, leaves and fruits.

Antioxidant capacity of C. pubescens showed variations.

The antioxidant capacity increased with increasing altitude.

The banding patterns of protein of C. pubescens in Dieng Plateau did not show any variation. This suggests that the genetic stability is not affected by the environmental factors.

REFERENCES Budiyanti T, Purnomo S, Karsinah, Wahyudi A. 2005. Characterization of 88 papaya accession collected by Fruit-Crops Research Institute.

Buletin Plasma Nutfah. 11 (1): 21-27. [Indonesian] Coats SA, Wicker L. 1990. Protein variation among Fuller Rose case population (Coleoptra: Curculionidae). Ann Entomol 83 (6): 10541062.

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Muzayyinah. 2008. Plants terminology. Sebelas Maret University Press, Surakarta.

Center for Plant Variety Protection of the Ministry of Agriculture of the Republic of Indonesia. 2006. Guidance for testing the individual novelty, uniqueness, uniformity and stability. Center for Plant Variety Protection of the Ministry of Agriculture of the Republic of Indonesia, Jakarta.

Rahayu S, Sumitro SB, Susilawati T, Soemarno. 2006. Isoenzymic analysis to study genetic variation of Bali cattle in Province of Bali.

Hayati 12: 1-5.

Sitompul SM, Guritno B. 1995. Analysis of plant growth. Gadjah Mada University Press, Yogyakarta.

Suranto. 1991. Studies of population variation in species of Ranunculus.

[Thesis]. Departement of Plant Science, University of Tasmania, Hobart.

Suranto. 2001. Isozyme studies on the morphological variation of Ranunculus nanus populations. Agrivita 23 (2): 139-146.

Tjitrosoepomo G. 1990. Plants morphology. Gadjah Mada University Press, Yogyakarta.

Triawati RM. 2005. Study on the diversity of total protein banding pattern of leafhopper (Nephotettix virescens) of the endemic and nonendemic populations on rice tungro virus. [Honorary Thesis]. Faculty of Agriculture, Sebelas Maret University, Surakarta.

ISSN: 2087-3948 E-ISSN: 2087-3956

Vol. 4, No. 1, Pp. 22-26 March 2012

Community structure of parasitoids Hymenoptera associated with Brassicaceae and non-crop vegetation YAHERWANDI

Faculty of Agriculture, Andalas University, Limau Manis, Padang 25161, West Sumatra, Indonesia. Tel. +62-751-72774, Fax: +62-751-72702; email: yaherwandi_04@yahoo.com Manuscript received: 14 July 2011. Revision accepted: 16 February 2012.

Abstract. Yaherwandi. 2012. Community structure of parasitoids Hymenoptera associated with Brassicaceae and non-crop vegetation.

Nusantara Bioscience 4: 22-26. Parasitoids Hymenoptera have an important role in agroecosystem because of their ability in suppressing pest population. Their presence in the field is seen as the key to agricultural ecosystem. Their presence can be influenced by the availability of non-crop vegetation. Some adult parasitoids Hymenoptera require food in the form of pollen and nectar of wild flowers to ensure effective reproduction and longevity. The objective of this research was to study communities of parasitoid Hymenoptera in Brassicaceae field and non-crop vegetation around Brassicaceae fields. Samplings were conducted at two different landscape structures, i.e. Kayu Tanduak and Padang Laweh representing complex landscapes, whereas Alahan Panjang and Sungai Nanam representing simple landscapes. Insects were sampled by three trapping techniques (farmcop, sweep net, and yellow pan traps) in one line of transect for each landscape. A total of 84 species from 17 families of parasitoids Hymenoptera were collected in Bracicaceae field and in non-crop vegetation. Landscape structure, flowering vegetation, and pesticide application affected the species richness, diversity and evenness of parasitoids Hymenoptera in Brassicaceae fields and non-crop vegetation.

Key words: Brassicaceae, community structure, landscape, non-crop vegetation, parasitoid Hymenoptera.

Abstrak. Yaherwandi. 2012. Struktur komunitas Hymenoptera parasitoid yang berasosiasi dengan tanaman Brassicaceae dan tumbuhan liar. Nusantara Bioscience 4: 22-26. Hymenoptera parasitoid memiliki peran penting dalam agroekosistem karena kemampuannya dalam menekan populasi hama. Keanekaragaman Hymenoptera parasitoid dapat dipengaruhi oleh ketersediaan vegetasi liar berbunga, karena beberapa parasitoid dewasa Hymenoptera membutuhkan serbuk sari dan nektar untuk reproduksi dan kelangsungan hidupnya. Tujuan dari penelitian ini adalah untuk mempelajari keanekaragaman Hymenoptera parasitoid pada pertanaman Brassicaceae dan tumbuhan liar di sekitarnya. Pengambilan sampel serangga dilakukan pada dua lanskap pertanian yang berbeda, yaitu Kayu Tanduak dan Padang Laweh mewakili lanskap pertanian yang kompleks, sedangkan Alahan Panjang dan Sungai Nanam mewakili lanskap pertanian yang sederhana. Koleksi sampel serangga menggunakan tiga metode yaitu farmcop, jaring serangga, dan nampan kuning. Total Hymenoptera parasitoid yang telah dikoleksi pada pertanaman Brasicaceae dan tumbuhan liar adalah 84 spesies yang termasuk ke dalam 17 famili. Struktur lansekap pertanian, tumbuhan liar berbunga, dan aplikasi pestisida mempengaruhi kekayaan, keanekaragaman dan kemerataan spesies Hymenoptera parasitoid pada pertanaman Brassicaceae dan tumbuhan liar.

Kata kunci: Brassicaceae, struktur komunitas, lanskap, tumbuhan liar, Hymenoptera parasitoid.

INTRODUCTION Cabbage plants (Brassicaceae) such as broccoli, cabbage, cabbage flowers, petsai and caysin are vegetable commodities widely planted by farmers in Indonesia, including in West Sumatra. Vegetables commodity of West Sumatra not only meet the need in the province, but also support the need of the two neighboring provinces, namely Riau and Jambi. Brassicaceae fields in West Sumatra have a variety of problems, particularly pests and diseases.

Unfortunately, the use of pesticides in agricultural ecosystems has resulted in environmental pollution, decrease of arthropod diversity, the impoverishment of ecosystem and the emergence of pests resistant to these pesticides) We have several groups of farmers who have been producing organic vegetables in West Sumatra.

Organic farming is done in an effort to restore ecological functions (biorestoration) of various arthropods in agroecosystem. Therefore, it is necessary to find alternative controls without using pesticides, for example, by utilizing the natural enemies of insect herbivor or better known as biological control.

Biological control using parasitoids is an alternative pest control strategy that is currently being developed to replace the role of pesticides that tend to harm the environment and public health. Practical and more rational methods of Biological control have been introduced to enhance the role of parasitoid complex through habitat management.

The information on diversity, parasitization, distribution (dispersal rate) and ecological factors such as role of noncrop vegetation to influence ecology of parasitoids

YAHERWANDI – Parasitoid Hymenoptera on Brassicaceae

Hymenoptera in agroecosystem is important and very fundamental to the success of biological control..

Information about the Hymenoptera parasitoids in Indonesia, particularly of parasitoid complex associated with Brassicaceae and non-crop vegetation is still limited.

Therefore, this study aimed to study the diversity, distribution, and abundance of parasitoids Hymenoptera associated with Brassicaceae and non-crop vegetation in different types of agricultural landscape in West Sumatra.

The results of this study is expected to be used as a strong foundation for planning and development of integrated pest management (IPM) technologies in Indonesia.

MATERIALS AND METHODS Study sites Parasitoids Hymenoptera collection was conducted in different types of landscape of West Sumatra, Indonesia.

The villages of Sungai Nanam and Alahan Panjang, Solok district represent a simple structure of agricultural landscape or agricultural ecosystems dominated by red onions fields (95%). Kayu Tanduak village, Tanah Datar district and the village of Padang Laweh, Agam district represent a complex structure of agricultural landscape or agricultural ecosystems consisting of vegetables, rice and corn. Descriptions of research sites were presented in Table 1. Identification of insects material were conducted in the Laboratory of Insect Ecology, Department of Pest and Diseases Plant, Faculty of Agriculture, Andalas University, Padang. The research was conducted from March to November 2007.

Table 1. Description of research sites Sites

Altitude (m dpl)

Landscape type

Kayu Tanduk

800-850

Complex agricultural landscape mixed culture of vegetables (+60%), corn, and rice)

Padang Laweh

850-900

Complex agricultural landscape (mixed culture of vegetables (+60%), corn, and rice)

Alahan Panjang 850-1000

Simple agricultural landscape (monoculture of red onions (+95%), cabbage, and tomato)

Brassicaceae fields At each agricultural landscape a transect line approximately 1000 m in length was made along the existing fields. Sampling of Brassicaceae was done every 50 m along the transect. Collection Hymenoptera parasitoids at each sample point was conducted using three methods: sweep net, suction with farmcop, and yellow pan traps.

23

Method of sweep net. Netting was conducted at each sample point on the transect line. Netting which was ten times double swing that includes 50 plants per sample point. Insects were caught directly inserted into the vial containing of 70% alcohol.

Farmcop method. This method used a tool that consisted of a small electric vacuum cleaner which had been modified, 1.5-inch diameter plastic tube, a tool for insect traps consisting of 20 cm diameter bottles, a vial containing 70% alcohol, and 12-volt batteries 60 A. Sampling was done by direct suction on all plant parts of Brassicaceae.

Yellow pan trap method. Traps were made of yellow plastic container measuring 15 x 25 cm and 10 cm high.

Yellow pan traps were installed in the middle of fields.

Insects attracted to the yellow color would go into the traps. To kill the insects that perched on the traps, the traps were filled with soap water water solution to reduce surface tension, so the insects that entered would drown and die. A trap was placed in each sample point and left for 24 hours.

Insects caught were immediately cleaned and placed into the vial containing 70% alcohol.

Wild vegetation Collection of parasitoid Hymenoptera on wild plants was done by sweep net method and suction with farmcop.

Insects caught were directly placed into the vial containing 70% alcohol Identification of parasitoid Hymenoptera Identification was done on adult parasitoid Hymenoptera. All Imago of parasitoids Hymenoptera obtained from sweep net method, farmcop, and yellow pan traps were identified to family level using Gaulet and Huber (1993). Identification at the species level was based on morphological differences or morphospecies.

Data analysis Analyses of species diversity and abundance of parasitoid Hymenoptera were done using ShannonWienner Diversity Index, species richness and Simpson's evenness index (Magurran 1988; Ludwig and Reynolds 1988; Krebs 1999). To calculate species richness, ShannonWienner index, and Simpson's evenness index we used the program Primer for Windows version 5.

To create a smooth species accumulation curves, the number of species obtained at each sample point was randomized 50 times with the program EstimateS version 8:00. Randomization of parasitoid Hymenoptera species richness based on Jackknife-1 estimator (Cowell 2007).

Analysis of community similarity of parasitoid Hymenoptera in Brassicaceae, other vegetables, and wild vegetation was done using Sørensen similarity index. To obtain the Sørensen similarity index we used biodiv97 programs integrated in Microsoft Exel. Further analysis of community grouping with cluster analysis (UPGMA) was done using the program of Statistica 7 for Windows (StatSoft 2007).

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4 (1): 22-26, March 2012

Community of parasitoid Hymenoptera associated Brassicaceae field and non crops vegetation on different types of agricultural landscapes The total number of parasitoid Hymenoptera collected on Brassicaceae and non crops was 540 individuals consisting of 84 species and 17 families. The number of individuals, species, and families of parasitoids Hymenoptera associated Brasicaeae in the complex landscape was higher than that in the simple landscapes (Table 2). The high number of individuals, species, and the families of parasitoids Hymenoptera in complex agricultural landscapes was due to the flow of species from other habitats into Brassicaceae community.In other words, the parasitoid Hymenoptera community of Brassicaceae fields consisted of species of parasitoids Hymenoptera of rice fields, other vegetables, and non crop vegetation (Table 4). This result was similar to that found by Yaherwandi et al. (2007) on rice fields in the Cianjur watershed, West Java.

Table 2. Number of family, individual, and species of parasitoid Hymenoptera associated with Brassicaceae fields and non crops vegetation on different types of agricultural landscapes Simple landscape Complex landscape Brassica- Non BrassicaNon ceae crops ceae crops Betylidae 0 0 5 (1) 5 (1) Braconidae 127(12) 50 (9) 85 (14) 23 (6) Calcididae 3 (2) 1 (1) 0 0 Ceraphronidae 0 0 3 (1) 3 (1) Diapriidae 1 (1) 1 (1) 8 (3) 0 Encyrtidae 2 (1) 2 (1) 17 (3) 0 Eucoilidae 14 (5) 5 (1) 13 (3) 2 (1) Eulophidae 9 (4) 5 (3) 31 (7) 4 (1) Ichneumonidae 90 (15) 21 (8) 67 (11) 11 (4) Megaspilidae 3 (1) 3 (1) 4 (1) 4 (1) Mutillidae 0 0 2 (1) 0 Mymarommatidae 0 0 2 (1) 2 (1) Platigastridae 0 0 2 (1) 0 Pteromalidae 2 (2) 0 7 (2) 7 (2) Scelionidae 11 (4) 3 (3) 26 (7) 15 (4) Torymidae 1 (1) 0 0 0 Trichogrammatidae 0 0 3 (1) 0 Total 263 (48) 91 (28) 275 (57) 76 (22) Note: number in parentheses () is the number of species Family

However, the number of individuals, species, and family of parasitoid Hymenoptera collected onnon crop vegetation around Brassicaceae field was higher in simple landscapes than in complex landscapes (Table 2). These results indicate that the flow of species between Brassicaceae fields and non crop vegetation was quite high (Table 3). Alahan Panjang and Sungai Nanam are an agricultural area with simple landscape structure and application of pesticides is quite high (3 times per week), while the Padang Laweh and Kayu Tanduak are an

agricultural area with a complex landscape and pesticide application once a week. The use of pesticides is scheduled three times a week, causing conditions the agroecosystem of Alahan Panjang and Sungai Nanam less suitable for natural enemies, including parasitoid Hymenoptera. The same results has been reported by Yaherwandi et al.

(2008), especially at the time of pesticide application, many parasitoid took refuge in habitats of non crops around vegetables fields in Cianjur watershed West Java.

Table 3. Matrix similarity (Sørensen index) of parasitoids Hymenoptera on the Brassicaceae fields, red onions field, and non crops in a simple agricultural landscapes Crops

Red onions

Red onions

1.00

Brassicaceae

Brassicaceae

Non crops

0.31

0.38

1.00

0.57

Non crops

1.00

Table 4. Matrix similarity (Sørensen index) of parasitoids Hymenoptera on the Brassicaceae, other vegetables, rice fields, and non crops in a complex agricultural landscape Brassicaceae 1.00

Crops Brassicaceae

0.16

Other vegetbales 0.28

Non crops 0.20

1.00

0.36

0.18

1.00

0.29

Rice

Rice Other vegetables Non crops

1.00

Estimation of species richness of parasitoid Hymenoptera in the Brassicaceae fields Spesies accumulation curves of parasitoid Hymenoptera were still rising, but not too sharp in both landscape (Figure 1). The numbers of species collected in simple and complex landscapes were 48 and 57 species respectively (Table 3), while the estimation results with Jackknife-1 estimator for the simple and complex landscapes were 66 and 92 species respectively (Figure 2).

No. of species Jumlah spesies

RESULTS AND DISCUSSION

100 90 80 70 60 50 40 30 20 10 0

Lanskaplandscape Sedehana simple complex landscape Lanskap Kompleks

1

2

3

4

5

6

7

8

9

10

Sampel Sample

Figure 1. Accumulation curves of parasitoid Hymenoptera species on Brasicaecae fields based on data encryption of program of estimateS 8.00

No. of species

YAHERWANDI – Parasitoid Hymenoptera on Brassicaceae

Obs

Jack-1

Simple landscape

Obs

Jack-1

Complex landscape

Figure 2. Number of species of parasitoids Hymenoptera in Brassicaceae fileds based on observational data and Jackknife-1 estimator with program of EstimateS 8:00

This study has collected > 60% species of parasitoid Hymenoptera (Figure 2). This suggests that species richness collected was not maximal. According to Krebs (1999) the highest number of species estimated by the Jacknife estimator is twice the number of species obtained.

Furthermore, He said that Jacknife-1 estimatoris influenced by the total number of species, sample size and the number of unique species (rare species). Thus, the low number of species of parasitoid Hymenoptera collected was probably caused by the low number of samples (10 samples per landscape) and the ineffectiveness of tools used for collection of insects. Due to technical reasons Malaise traps were not used in this study, whereas the tool was effective enough to capture the active flying Hymenoptera (Noyes 1989; Pickering and Sharkey 1995). Many ecologists disagree with Jacknife estimator, because estimate of species richness in the community by the Jackknife estimator is biased positively or higher (over estimate) (Heltshe and Forrester 1983). However, Palmer (1990) states that the Jacknife estimator is more accurate than the eight other estimators.

Species richness, evenness, and diversity index of parasitoid Hymenoptera in Brassicaceae fields The diversity of habitats and structure of agricultural landscape affect species richness, evenness, and diversity of parasitoid Hymenoptera. Species diversity of parasitoid Hymenoptera was higher in the complex landscape than in a simple landscape. Species diversity index is the resultant of the value of species richness and evenness. It was obvious that the high diversity of species in complex landscapes, because the species richness and evenness were high (Table 5).

Kayu Tanduak and Padang Laweh consist of a variety of habitats (rice, Bracicaceae, other vegetables, and non crops) to form the structure of agricultural landscape more complex than vegetable ecosystem in the Alahan Panjang and Sungai Nanam (dominated by red onion (95%) and

25

cabbage 5%). Agricultural landscapes in Kayu Tanduak and Padang Laweh provide a variety of resources such as alternative host, food (pollen and nectar), and shelter for adult parasitoids Hymenoptera , when environmental conditions are not favorable. This agroecosystem can improve survival and diversity of parasitoid Hymenoptera.

(Dryer and Landis 1996; dryer and Landis 1997). Similar results have also been reported by Idris et al. (2002), Hooks and Johnson (2003), Menalled et al. (2003), Stephens et al.

(2006), Bianchi et al. (2006), and Yaherwandi et al. (2007).

The diversity of parasitoids is influenced by the type of agricultural landscape. The agricultural landscape with a complex structure has higher abundance, richness, and diversity of parasitoid species than the landscape with a simpler structure.

Table 5. Species richness, evenness, and diversity of parasitoid Hymenoptera associated with Brassicaceae crops and non crops on different types of agricultural landscapes Index Species richness Species evenness Species diversity

Simple 46 0.23 4.23

Landscape Complex 56 0.46 5.27

CONCLUSION The complex landscape had higher number of families, individuals, and species of parasitoid Hymenoptera than the simple landscape. The number of species collected in complex and simple landscapes has reached > 60% of existing species based on Jecknife-1 estimator. Species diversity of parasitoid Hymenoptera was higher in the complex landscape than in the simple landscape. Species similarities of communities of parasitoid Hymenoptera in cabbage fields and in non crop vegetation was > 40%.

ACKNOWLEDGEMENTS Our thanks goes to the Director of Research and Community Service Director General of Higher Education, Ministry of National Education who has funded this research. We also thank the Dean and the Chairman of the Department of Plant Pests and Diseases, Faculty of Agriculture Andalas University who have given permission to work at Laboratory of insect ecology. Our thanks were to all Wali Nagari of Kayu Tanduak, Padang Laweh, Alahan Panjang, and Sungai Nanam who gave permission to study in these four villages. Thanks were also conveyed to the students and all those who have helped this research.

REFERENCES Bianchi FJJA, Booij CJH, Tscharntke T. 2006. Sustainable pest regulation in agriculture landscape: a review on landscape composition, biodiversity and natural pest control. Proc R Soc B 273: 1715-1727.

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Colwell RK. 2007. EstimateS: Statistical estimate of spesies richness and shared spesies from samples. Version 6.0b1 [serial online]. http://www.viceroy.eeb.uconn.edu/estimates [16 Dsember 2003].

Dryer LE, Landis DA. 1996. Effect of habitat, temprature and sugar availability on longevity of Eriborus terebrans (Hym: Ichneumonidae). Environ Entomol 25: 1192-1201.

Dryer LE, Landis DA. 1997. influence of non-crop habitat on distribution of Eriborus terebrans (Hym: Ichneumonidae) in cornfields. Environ Entomol 26: 924-932 Goulet H, Huber JT. 1993. Hymenoptera of the world: An identification guide to families. Research Branch Agruculture Canada Publication, Ottawa.

Heltshe JE, Forrester NE. 1983. Estimating species richness using the jackknife procedure. Biometrics 39: 1-11 Hooks CR, Johnson MW. 2003. Impact of agriculture diversification on the insect community of cruciferous crops. Crop Protection 22: 223-238.

Idris AB, Nor SMd, Rohaida R. 2007. Study on diversity of insect community at different altitudes of Gunung Nuang in Selangor, Malaysia. J Biol Sci 2 (7): 505-507.

Krebs CJ. 1999. Ecological metodology. 2nd ed. Addison Wesley Longman, New York.

Ludwig, JA, Reynolds JF. 1988. Statistical Ecology. John Wiley & Sons, New York.

Magurran AE. 1988. Ecological diversity and its measurement. Chapman and Hall, London.

Menalled FD, Costamagna AC, Marino PC, Landis DA. 2003. Temporal variation in the response of parasitoids to agriculture landscape structure. Agric Ecosyst Environ 96: 29-35.

Noyes JS. 1989. A study of methods of sampling Hymenoptera (Insecta) in tropical rainforest, with special reference to the parasitica. J Nat Hist 23: 285-298 Palmer MW. 1990. The estimation of species richness by extrapolation.

Ecology 71: 1195-1199 Statsoft [Statistical Software]. 2007. Statistica 7.0 for Windows. Statsoft, Tulsa.

Stephens CJ, Schellhorn NA, Wood GM, Austin AD. 2006. Parasitic wasp assemblages assosiated with native and weedy plant species in an agriculture landscape. Austr J Entomol 45: 176-184.

Yaherwandi, Manuwoto S, Buchor D, Hidayat P, Prasetyo L. 2007.

Community diversity of Hymenoptera parasitoid on paddy ecosystem Jurnal HPT Tropika 7 (1): 10-20. [Indonesia] Yaherwandi, Manuwoto S, Buchor D, Hidayat P, Prasetyo L. 2008.

Community structure of Hymenoptera parasitoid on non-crop vegetations in paddy field in Cianjur watershed Jurnal HPT Tropika 8 (2): 90-101. [Indonesia]

ISSN: 2087-3948 E-ISSN: 2087-3956

Vol. 4, No. 1, Pp. 27-31 March 2012

Evaluation of the effectiveness of integrated management and mating disruption in controlling gypsy moth Lymantria dispar (Lepidoptera: Lymantriidae) populations GOODARZ HAJIZADEHď‚Š, MOHAMMAD REZA KAVOSI

Department of Forest Sciences, Gorgan University of Agricultural Sciences and Natural Resources, Beheshti St. 386, Gorgan, Golestan, Iran.

Tel./Fax. +98 171 2227867, ď‚Šemail: Goodarzhajizadeh@gmail.com Manuscript received: 29 February 2012. Revision accepted: 26 March 2012.

Abstract. Hajizadeh G, Kavosi MR. 2012. Evaluation of the effectiveness of integrated management and mating disruption in controlling gypsy moth Lymantria dispar (Lepidoptera: Lymantriidae) populations. Nusantara Bioscience 4: 27-31. This study was conducted during 2008 and 2009 in Daland National Park (north of Iran) to compare the effectiveness of mechanical control used in combination with mating disruption (integrated management) and only mating disruption in controlling gypsy moth, Lymantria dispar (L.) (Lepidoptera: Lymantriidae). Male moths and egg mass counts were taken before (2008) and after (2009) the two treatments were applied. In sites with integrated management and with mating disruption only, 1,828 and 1,793 egg masses/tree, and 412.75 and 207.75 male moths/trap were observed, respectively. Both the numbers of egg masses/tree and of male moths/trap were significantly lower in sites with integrated management than in sites with only mating disruption. This study shows that integrated management was more effective than mating disruption in reducing infestation levels in the study site.

Key words: egg masses, integrated management, Lymantria dispar, mating disruption, mechanical method, pheromone traps Abstrak. Hajizadeh G, Kavosi MR. 2012. Evaluasi tentang efektifitas manajemen terpadu dan gangguan perkawinan dalam mengontrol populasi ngengat gipsi Lymantria dispar (Lepidoptera: Lymantriidae). Nusantara Bioscience 4: 27-31. Penelitian ini dilakukan selama tahun 2008 dan 2009 di Taman Nasional Daland (bagian utara Iran) untuk membandingkan efektivitas pengendalian mekanis yang digunakan dikombinasi dengan gangguan perkawinan (manajemen terpadu) dan hanya gangguan perkawinan saja dalam pengendalian ngengat gipsi, Lymantria dispar (L.) (Lepidoptera: Lymantriidae). Penghitungan ngengat jantan dan jumlah massa telur dilakukan sebelum (2008) dan setelah (2009) dua perlakuan diterapkan. Di lokasi dengan manajemen terpadu dan dengan gangguan perkawinan saja, terdapat 1.828 dan 1.793 massa telur/pohon, serta 412,75 dan 207,75 ngengat jantan/perangkap. Jumlah massa telur/pohon dan ngengat jantan/perangkap secara signifikan jauh lebih rendah pada lokasi dengan manajemen terpadu daripada di lokasi dengan gangguan perkawinan saja. Studi ini menunjukkan bahwa manajemen terpadu lebih efektif daripada gangguan perkawinan dalam mengurangi tingkat serangan hama di lokasi penelitian.

Kata kunci: massa telur, manajemen terpadu, Lymantria dispar, gangguan perkawinan, cara mekanik, perangkap feromon

INTRODUCTION The gypsy moth, Lymantria dispar (L.) (Figure 1), is probably the most important forest defoliating pest in the northeastern United States. Defoliation and tree mortality associated with gypsy moth outbreaks can cause a multitude of ecological and economic effects (Twery 1991; Gottschalk 1993). In Iran, gypsy moth was observed for the first time in oak forests at Guilan state region, north Iran in 1937 (Kavosi 2008). The activity of this pest in central parts, western and south western forests of Iran has been admitted outside these regions (Hajizadeh and Kavosi 2011). The largest outbreaks of gypsy moth occurred in Talesh forest in Guilan state region in 1975 (Kavosi 2008).

In the 1960s and 1970s, most treatments for controlling this pest were conducted using conventional synthetic pesticides like carbaryl (sevin) and dylox (trichlorfon).

Since 1983, these have been increasingly replaced by biorational compounds like Bacillus thuringiensis variety

kurstaki (Berliner) and dimilin (Diflubenzuron) (Liebhold and McManus 1999). Starting in 1971, the management of gypsy moth using mating disruption has been the subject of considerable research efforts (Doane and McManus 1981; Reardon et al. 1998).

Application of the synthetic gypsy moth pheromone, disparlure, in a slow-release formulation interferes with the male mate-search behavior and subsequently decreases the number of fertilized eggs laid by females (Leonhardt et al.

1996; Reardon et al. 1998). Initially, this method applied on high-density gypsy moth populations got poor results (Cameron 1981). Later experiments in medium and lowdensity populations have proven that disparlure can substantially reduce gypsy moth abundance (Reardon et al.

1998). In recent years, the operational use of disparlure has increased (Sharov et al. 2002). Its effectiveness is inversely related to population density (Schwalbe et al. 1983; Webb et al. 1988, 1990).

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4 (1): 27-31, March 2012

A

C

B

D

Figure 1. The morphology of Lymantria dispar; A. larva, B. pupa, C. imago, D. antennae (photo: from several sources).

Historically, most treatments of gypsy moth populations have been conducted to prevent defoliation in the current year. Treatments are typically scheduled based on counts of overwintering egg mass populations, which can be used to predict defoliation (Gansner et al. 1985; Liebhold et al.

1993). Operational treatments of outbreak populations usually provide at least partial foliage protection, but they may have limited effects on densities in subsequent years or on the probability of defoliation in the future (Liebhold et al. 1996). The success of treatments targeted against outbreak populations of the gypsy moth is traditionally evaluated by the reduction in egg mass counts and defoliation in treated versus untreated blocks (Twardus and Machesky 1990; Liebhold et al. 1996). However, these methods are not applicable in low density populations because egg mass densities cannot be estimated with any

accuracy and populations are too low to cause noticeable defoliation. Thus, evaluation of preventive treatments has to be based on alternative methods. Larval counts under burlap bands are a sensitive sampling method at moderate population densities (Reardon et al. 1998; Wallner et al.

1990). And at extremely low densities, the only viable sampling method is the use of male moth counts in pheromone traps because they are most sensitive to variations at very low population levels. Another advantage of using pheromone traps for treatment evaluation is that they are less expensive and thus can be used on an operational basis rather than just in experiments. Liebhold et al. (1995) and Carter et al. (1992) found that the correlation between moths counts in pheromone traps and defoliation was weak in continuously infested areas of high density populations. However, at these densities many traps

HAJIZADEH & KAVOSI – Pest management of Lymantria dispar

29

become saturated and this may obscure correlations of trap counts with population density (Elkinton 1987). Granett (1974) avoided trap saturation by frequent moth removal and recorded a high correlation between trap catches and population numbers.

The objective of this study was to evaluate the effectiveness of integrated management and mating disruption in controlling gypsy moth, Lymantria dispar (L.) populations in Daland National Park (north of Iran).

instrument was so designed that enough gas could exit to burn the entire egg mass while keeping the bole of the trees undamaged. This was the first time gypsy moth mating disruption was carried out in Iran. The pheromone traps described previously for the evaluation gypsy moth population densities were used for the mating disruption.

This treatment was applied to the eastern part of study site.

MATERIALS AND METHODS

During the year the treatments were applied, 1.793 egg masses/tree and 207.75 males/trap, and 1.828 egg masses/tree and 412.75 males/trap were collected in sites with integrated management and in site with mating disruption only, respectively. The numbers of egg masses and male moths captured in the sites with integrated management were significantly lower than in sites with mating disruption only (Table 1 and 2). In the year following treatment application, the number of egg masses in the sites with integrated management was significantly different from that in the sites with mating disruption only, which were 0.93 egg masses/tree and 1.362 egg masses/tree, respectively (Table 3). Combining both treatments, the number of egg masses decreased significantly from the year of treatment application (2008) to the following year (2009)(Table 4).

Study site The experiment was conducted in Daland National Park, which is part of the larger Golestan forest in Hyrcanian, north Iran (latitude 36º2′ S-36º4′ S, longitude 36º3′ E-41º5′ E). This area is approximately 3750 m long and 2900 m wide and has a total area of 608 ha. The study region has an average temperature of 16.5°C, a total annual rainfall of 660 mm and an altitudinal range of 75-119 m above sea level. The park consists almost entirely of Parrotia persica, Quercus castanifolia, Zelkova carpinifolia, and Carpinus betulus, with a few small areas of other species such as Populus alba, Ficus carica, Morus alba, Cupressus Sempervirence horizentalis, Pinus eladerica, Thuja orientalis, and Acer insigne (Anon 2005).

The study site was newly infested with the gypsy moth.

This area was considered to be part of the eastern infestation front.

Description of treatments Integrated management and mating disruption only treatments were applied in 2008. For the evaluation of these treatmens egg masses and male moth counts were taken during the year treatment was applied (2008) and in the following year (2009). Egg masses counts were taken from burlap bands placed around the boles of trees in the study areas. This allows for evaluating gypsy moth population levels even at low densities and other egg masses sampling methods yield mostly zero counts (Bellinger et al. 1990).

The use of pheromone traps is one of the suitable methods for monitoring and control of L. dispar. Sampling was carried from early July to the end of August through the use of delta type traps (4 for each treatment-total of 8) installed at 1.5-2 m height with spacing of 100-200 m between each other. Adults captured were counted daily.

The delta trap did not contain any sticky material. A small piece of brown paper was placed inside to provide a surface on which the female could cling. The delta trap was suspended from a coat hanger stapled to the side of the tree bole.

Integrated management Integrated management consisted of burning of egg masses (mechanical method) combined with mating disruption. This treatment was applied to the western part of study site. A gas instrument was designed and used to mechanical control egg masses in defoliated trees. This

RESULTS AND DISCUSSION

Table 1. Number of egg masses collected during the year the control treatments were carried out in sites with integrated management and in sites with mating disruption only.

No. of egg masses Egg collected masses/tree Integrated management 631 1.828a Mating disruption only 443 1.793b Note: Treatments with the same letter are not significantly different at the 0.05 experiment-wise error rate.

Treatment

Table 2. Comparison of male counts in pheromone traps in sites with integrated management and in sites with mating disruption only (2008).

Treatment Integrated management Mating disruption only

4 4

Number of pheromone traps

Males/trap 207.75b 412.75a

Note: Treatments with the same letter are not significantly different at the 0.05 experiment-wise error rate.

Table 3. Comparison of number gypsy moth egg masses in sites with integrated management and in sites with mating disruption only, the year following the application of the control treatments (2009).

No. of egg masses Egg collected masses/tree Integrated management 15 0.930b Only pheromone traps 79 1.362a Treatments with the same letter are not significantly different at the 0.05 experiment-wise error rate.

Treatment

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4 (1): 27-31, March 2012

Table 4. Evaluation of the number egg masses collected during (2008)and after (2009) carrying out the control treatments.

No. of egg masses Egg collected masses/tree 2008 1074 1.81a 2009 94 1.30b Note: Treatments with the same letter are not significantly different at the 0.05 experiment-wise error rate.

Year

Discussion In this research we found that integrated management was more effective than mating disruption in controlling gypsy moth populations, as it can be attested by the significant lower numbers of egg masses and male moths.

Gypsy moth populations are mainly monitored using aerial maps of forest defoliation, counts of overwintering egg masses (Kolodny-Hirsch 1986), and counts of male moths in pheromone baited traps (Talerico 1981; Ravlin et al.

1987). Particularly, egg mass counts are the most reliable method for assessing decisions (Ravlin et al. 1987).

Methods such as collection and destruction of egg masses, use of sticky bands to prevent larvae from climbing trees, removal of larvae that congregate under burlap skirts wrapped around tree trunks, and pheromone traps are often recommended as alternative approaches to managing gypsy moth (Campbell 1983; Thorpe et al. 1995; Thorpe et al.

2007). However, Campbell (1983) and Thorpe et al. (1995) have shown that these tactics are not capable of protecting trees from defoliation during outbreaks, even when used in combination. Collection and destruction of egg masses is ineffective because most egg masses are well hidden or high in the tree where they are inaccessible. Even thorough searches by experts detect only a proportion of those present. Burlap bands wrapped around the lower trunk of trees can attract large numbers of gypsy moth larvae, which hide under them during the day when they are not feeding.

This tactic can be useful for detecting the presence of low gypsy moth populations, and may be useful for protecting small, isolated trees from defoliation. However, research and experience have demonstrated that trunk banding is ineffective in preventing defoliation of even moderate size trees. The use of pheromone traps to decrease gypsy moth populations is sometime recommended, but is also futile.

Only males are attracted to the traps, which are quickly saturated even when populations are very low (Herms 2003). Pheromone traps are very useful for delineating the distribution of gypsy moth populations, and are used effectively in monitoring programs. Application of gypsy moth sex pheromone over large areas has been used successfully to suppress populations through disruption of mating (Leonhardt et al. 1996). Wide spread application of pheromone (usually by aircraft) saturates the environment, preventing males from detecting pheromones produced by individual females. Mating disruption is most effective when gypsy moth populations are low but starting to increase. When Populations are high, the day-flying males can easily locate mates visually. In areas infested by gypsy moth for many years, there is little or no relationship between male moth counts and subsequent defoliation at the same location (Carter et al. 1992; Liebhold et al. 1995).

However, in the area along the expanding gypsy moth

front, the relationship among male moth counts, egg mass density, and defoliation may be quite different because of the strong population density gradient (Ravlin et al. 1991).

Egg mass counts are the most reliable cause method in medium and high density population, and thus they are widely used for making decision concerning aerial suppression of outbreak population (Schwalbe 1981; Ravlin et al. 1987). In the uninfected and transition zones, moth trapping remains the only reliable monitoring method. Thus, the analysis of the spatial distribution of moth counts is justified. Mating success may be the most important density dependent factor that affects sparse gypsy moth populations. Mating failure can cause instability in isolated populations because the proportion of non-mated females will increase as population density decreases. The relationship between pheromone trap catch and mating success has never been measured accurately.

Knowledge of this relationship will be useful for distinguishing between unstable and establish populations (Sharov et al. 1995a). There are several factors affecting the relationship between pheromone trap capture and female mating probability. One group of factors is related to pheromone source and trap design. Another group of factors affecting the mating trap capture relationship is associated with male moth behavior. The 3rd group of factors is associated with the female calling period (Sharov et al. 1995b).

CONCLUSION In this paper, we have developed a new method for evaluating treatments of low density, isolated gypsy moth populations that is based on male moth count and egg mass counts. It is recommended that field studies of contamination measure in other areas, especially in the northern forests, beconducted, so that we could use the knowledge in the management and population control programs. Also, the use of integrated methods to control pest gypsy moth areas is recommended. Finally, the methods of pest control training in gypsy moth to executive departments should be effective.

ACKNOWLEDGMENTS The authors thank everybody that helped in the field data collection. We also thank Ali Afshari, Gorgan University of Agricultural Sciences and Natural Resources, for reviewing an earlier draft of this paper.

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Gottschalk W, Twery MJ, Smith SI (eds.) Proceeding, U.S. Department of Agriculture Interagency gypsy moth research review 1990. USDA Forest Service General Technical Report NE-146, Radnor, PA. Reardon RC, Leonard DS, Mastro VC, Leonhardt BA, McLane W, Talley S, Thorpe K, Webb R. 1998. Using mating disruption to manage gypsy moth: a review. USDA Forest Service FHTET-98-01.

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Webb RE, Tatman KM, Leonhardt BA, Plimmer JR, Boyd VK, Bystrak PG, Schwalbe CP, Douglas LW. 1988. Effect of aerial application of racemic disparlure on male trap catch and female mating success of gypsy moth (Lepidoptera: Lymantriidae). Econ Entomol 81: 268-273.

ISSN: 2087-3948 E-ISSN: 2087-3956

Vol. 4, No. 1, Pp. 32-35 March 2012

The total protein band profile of the green leafhoppers (Nephotettix virescens) and the leaves of rice (Oryza sativa) infected by tungro virus ANI SULISTYARSI, SURANTO, SUPRIYADI

Bioscience Program, School of Graduates, Sebelas Maret University, Surakarta 57126, Central Java, Indonesia, Jl. Ir. Sutami 36A, Surakarta 57126, Central Java, Indonesia. Tel./Fax. +62-271-663375. email: priyadi_hpt@yahoo.co.id Manuscript received: 2 February 2012. Revision accepted: 28 March 2012.

Abstract. Sulistyarsi A, Suranto, Supriyadi. 2012. The total protein band pattern of the green leafhoppers (Nephotettix virescens) and the leaves of rice (Oryza sativa) infected by tungro virus. Nusantara Bioscience 4: 32-35. Tungro virus is one of most important diseases of rice plants caused by double infection with RTBV and RTSV which is transmitted by Nephotettix virescens Distant. The interaction between host and virus-vector are still quitted difficult to understand. The aims of this study were: (i) to know the character of the total protein band pattern of rice plants infected with tungro virus compared to the health one, (ii) to look at the different between the band profiles of total protein of N. virescens that consume the host rice plants infected by tungro virus and that of the healthy rice plants.

Total protein band profiles of rice plants were identified using SDS-PAGE. To extract the leaves, buffer merchapto-ethanol was used, while the sample extraction of green leafhoppers employed buffer PBS IX, and for staining the protein coomassie brilliant blue was used. Data were analyzed descriptively based on the score of the migration of the band (Rf). The results showed that the protein contains of every 0.5 g of healthy leaves and the infected by the virus were 0.567 g and 1.011 g respectively. Clear difference of the protein pattern was found in the healthy plant and the infected one. In general, the entire band in the infected plant was much thicker compared to the infected leaves. Protein bands with a higher quantity were expressed by the protein on the molecular weight of 108, and 117 kDa. These proteins are presumably from the group of β-galactosidase and bovine serum albumin. The function of such proteins is still unknown, but it may be related to the plant’s responses to virus infection, because the protein did not appear in the healthy plants. The total protein content of both N. virescens which acquired the healthy leaves and the infected one were 0.1395 g and 0.1546 g respectively. Qualitatively, there was no significant difference of the protein expression in those vectors, but slightly thicker band were observed in the infected leaves.

Key words: rice, tungro, Nephotettix virescens, protein banding.

Abstrak. Sulistyarsi A, Suranto, Supriyadi. 2012. Pola pita protein total wereng hijau (Nephotettix virescens) dan daun tanaman padi (Oryza sativa) yang terinfeksi virus tungro. Nusantara Bioscience 4: 32-35. Virus tungro merupakan salah satu penyakit penting tanaman padi disebabkan oleh infeksi ganda RTBV dan RTSV, yang disebarkan oleh Nephotettix virescens Distant. Interaksi antara inang dan vektor virus masih sulit dipahami. Penelitian ini bertujuan: (i) mengetahui karakter pola pita protein total tanaman padi yang terinfeksi virus tungro dan tanaman padi sehat, (ii) mengetahui karakter pola pita protein total N. virescens yang mengkonsumsi inang tanaman padi terinfeksi virus tungro dan tanaman padi sehat. Pola pita protein total tanaman padi diidentifikasi menggunakan metode elektroforesis dengan SDS-PAGE. Sampel daun diekstraks menggunakan buffer mercapto ethanol, sedangkan sampel wereng hijau diekstraks menggunakan buffer PBS IX; dan pewarnaan pita protein menggunakan coomassie brilliant blue. Data dianalisis secara deskriptif berdasarkan nilai migrasi pita (Rf). Hasil penelitian menunjukkan kadar protein 0,5 g daun tanaman padi sehat dan daun tanaman padi terinfeksi virus tungro masing-masing sebesar 0,567 μg dan 1,011 μg. Perbedaan pola pita protein secara jelas ditemukan di antara tanaman padi sehat dan terinfeksi. Pada umumnya, pola pita protein pada tanaman padi yang terinfeksi virus tungro lebih tebal dari pada tanaman padi sehat. Pita protein dengan kuantitas lebih tinggi diekspresikan pada protein dengan berat molekul 108, dan 117 kDa. Diduga protein ini dari kelompok β-galaktosidase dan bovine serum albumin. Fungsi protein tersebut belum diketahui, namun diduga berkaitan dengan respon tanaman terhadap infeksi virus, karena protein tersebut tidak muncul pada tanaman sehat. Kadar protein total wereng hijau yang mengkonsumsi daun tanaman padi sehat dan terinfeksi virus tungro masing-masing sebesar 0,1395 μg dan 0,1546 μg. Secara kualitatif, tidak terdapat perbedaan yang signifikan ekspresi protein pada inang, tetapi pada daun yang terinfeksi cenderung lebih tebal.

Kata kunci: padi, virus tungro, wereng hijau, Nephotettix virescens, pola protein

INTRODUCTION Tungro virus is one of the most important diseases of the rice plants in South and Southeast Asia causing significant economic looses. The disease is caused by double infection of rice virus tungro bacilliform virus (RTBV) and rice virus tungro spherical virus (RTSV),

which is transmitted by green leafhoppers (Nephotettix virescens Distant) (Muralidharan et al. 2003; Tyagi et al 2008). This vector is the most effective in transmitting the viruses on the rice plant and the very dominant species in the tropics (Abdul Rachim 2000). N. virescens is the most effective vector in transmitting the tungro virus (Supriyadi et al. 2004, 2008; Widiarta 2005) and have also been

SULISTYARSI et al. – Protein patterns of green leafhoppers and rice infected by tungro virus

recorded its population is more dominant than other vectors in the field (Himawati and Supriyadi 2003; Supriyadi et al.

2004; Widiarta 2005).

A healthy rice plant uninfected by tungro virus contains much chlorophyll and therefore can be used for photosynthesis and producing food for the plant. Accordingly, leaves of rice plants do not contain lots of protein so that the amino acid content of the plants is low. On the rice plants infected by the tungro virus, the DNA from the virus will then infect the plant cells and take over the functions of DNA of the plants in order to synthesize the protein, which is used by the viruses to replicate the viral DNA. Therefore, it is necessary to test the protein band profiles of healthy rice plants and infected ones. RTSV particles themselves do not play a role in transmitting the RTBV, but the one which has a role is the helper factor protein, that is a product of the interaction between RSV with the infected host plants (Hibino and Cabauatan 1986).

The process of tungro virus transmission by vectors has been known to involve the helper component which serves in binding virus particles on the mouth of the vector.

Helper components are thought to be specific proteins which are important for virus absorption at the vector stylet (Supriyadi et.al. 2004). According to Hibino (1996) protein as a helper component of the vector is produced in the body that is secreted into the mouth of the stylet. Helper proteins of the vector of N. virescens are produced by means of his own mouth (stylet) or the thorax which is secreted into the mouth of the tool, so it requires identification of the total protein.

Based on the above background the researcher raised several issues, namely the study of the total protein profile of host plants of the rice infected by tungro virus and total protein profiles of individuals of N. virescens. The research aimed: (i) to know the character of the total protein band profiles of rice plants infected with tungro virus and that of healthy rice plants, (ii) to know the character of the band profiles of total protein of N. virescens that consume the host rice plants infected by tungro virus and that of the healthy rice plants.

MATERIALS AND METHODS Place and time The observation on the rice plants infected by the tungro virus was done in the Laboratory of Science and Plant Diseases, Faculty of Agriculture, Sebelas Maret University (UNS) Surakarta, while the total protein band profile analysis of host plants infected by rice tungro virus and the total protein band profiles of green leafhopper N. virescens was conducted at the Laboratory of Microbiology, IUC, Gadjah Mada University, Yogyakarta. The study was conducted from February to July 2010.

Procedures The rice cultivar tested in this study was of Ciherang type. Rice plant samples were taken from endemic areas in Yogyakarta, Indonesia. The rice plants were planted in pots placed in a greenhouse. Samples of N. virescens from the

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field were grown in a rice box breeding nursery in our campus. Tungro virus transmission was conducted in a greenhouse. Samples for total protein electrophoresis profiles using the leaves of rice plants in both healthy and the rice infected by the tungro virus. As for the total protein profile analysis of green leafhoppers, samples were taken from the head and thorax of green leafhoppers that attacked both healthy rice plants and the plants infected by the tungro virus. Observation of the rice plant is described qualitatively to identify the healthy rice plants and the tungro virus- infected rice plants.

Total protein profile analysis was conducted to identify differences and/or total protein band profile similarity between the rice plants infected by tungro virus and healthy rice plants and also between green leafhoppers that consumed healthy rice plants and the plants infected by tungro virus. Profile analysis of total protein band was done with the technique of electrophoresis on SDS-PAGE with several stages, adopting the method of Wongsosupantio (1992), Coats et al. (1990), Cruz et al. (1998) and Takkara (2000). Leaf samples of rice plants employed merchaptoethanol extract buffer, while the green leafhoppers samples employed IX PBS buffer extract. Each procedure requires 0.5 g of sample. The concentration of acrylamide for stacking gel was 3%, while for gradient gels was 10%.

Electrophoresis was run at a constant voltage of 100 VA, until the bromphenol blue travelled near the bottom of the gel. Gels were fixed and then staining was done in one night with a solution of coomassie brilliant blue R-250.

Staining process was followed by distaining using a solution consisting of methanol, acetic acid, and water (40:40:20) which were all shaken until the protein bands appeared. Total protein electrophoresis results were documented in a digital photograph.

The data of the total protein band pattern of the rice and N. virescens were analyzed by zimogram to observe similarities or differences in the total protein band profiles between rice plants infected with tungro virus and healthy rice plants.

RESULTS AND DISCUSSION Description of the rice plants infected by tungro virus The rice plants uninfected by the tungro virus grew well the leaves were green and plants were relatively high. The rice plants infected by the tungro virus grew somewhat stunted; the young leaves turn yellowish from the tip, and the yellow leaves appear somewhat twisted; the older leaves look yellow to orange; the offsprings were few, and the heights of the plants were hardly even. In the breeding process, the tungro virus transmission appears on the third leave which looks somewhat twisted (Figure 1).

According to Gnanamanickam (2009), the general symptoms of rice plants infected by tungro virus are leaf discoloration that begins from leaf tip and extends to the blade or the lower leaf portion; infected leaf sometimes also show molted or stripped appearance and stunting. The presence of tungro virus can be confirmed by some serological tool based on protein characteristics (Suparyono et al. 2003).

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4 (1): 32-35, March 2012

A

B

C

D

Figure 1. Rice plants. A. Healthy plants, B. The breeding of the tungro virus-infected ones, C-D. Adult plants infected by tungro virus

A

B

Figure 2. The results of the total protein band profiles of rice leaf samples by SDS-PAGE electrophoresis. A. Protein banding pattern profiles, B. Zimogram, 1 = marker, 2 = leaves of healthy rice plants: 30 mg (12 mL), 3 = leaves of healthy rice plants: 40 mg (16 mL), 4 = healthy leaves of rice plants: 20 mg (8 mL), 5 = leaves of the rice plants infected by the tungro virus: 40 mg (6.6 mL).

Total protein band profiles of the healthy rice plants and the ones infected by the tungro virus The results of measurements with a spectrophotometer at a wavelength of (λ) = 595 nm indicated that the protein content of the healthy rice plants and the infected one were 0.567 μg and 1.011 μg respectively. Protein levels are used to determine the concentration of the sample used in the process of running electrophoresis.

The electrophoresis results of the protein band profiles of the healthy rice plants and the ones infected by green leafhoppers at a certain range of molecular weight showed a very thick gene expression, which was not expressed on the healthy rice plants (Figure 2). Profile of protein bands that appeared on the BM (16, 30, 47, 61, 89 and 200) kDa were expressed by the healthy rice plants and the rice plants infected by the tungro virus, which means both the healthy rice plants and the ones infected by the tungro virus

expressed the same protein band profile, which was based on markers consisting of protein glutamate dehydrogenase, ovalbumin, carbonic anhydrase, myoglobin, lysozyme, and aprotinin. On the tungro virus-infected plants, more quantity of expressed proteins was seen on the protein bands which looked thicker.

The tungro virus-infected rice plants showed a specific protein band profile which was in the range of protein bands close to 108 MW and 117 kDa which was displayed with a thick band. Protein bands on the range of molecular weight are typical proteins in the rice plants infected by the tungro virus. Based on the marker, both proteins were alleged to be β-galactosidase and bovine serum albumin expressed by the rice plants because of a virus infection.

The function of both types of proteins is not known with certainty, but it is allegedly associated with the plant responses to virus infection as a form of self defense, because both types of proteins were not expressed on the healthy rice plants, thus there is a need for further research.

According to Sereikaite et al. (2005) bovine serum albumin usually serves as a nutrient in microbial cells, and supports the growth of cells and is used for immunoblots and is correlated to the immunosorbent of the enzyme test.

In the total protein expression profile in rice plants infected by the tungro virus, it was difficult to distinguish between a viral protein and the protein of rice plants. With the addition of SDS in the electrophoresis, the protein of the plants and of the virus will be cut into polypeptide chains. It is suspected that the polypeptide of the virus protein has a molecular weight similar to that of the polypeptide of the plants so that they settle in the same location, or that the levels and types of viral polypeptides are relatively few compared to the polypeptide of plants so it is difficult to identify. This result was differ to Oluwafemi et al. (2007) which indicated that plants infected with maize streak virus (MSV) had protein patterns different from healthy plants. Perhaps, in this study, the isolated viral proteins had very little concentration.

SULISTYARSI et al. – Protein patterns of green leafhoppers and rice infected by tungro virus

The profile of protein bands of the green leafhoppers of the rice plants infected by the tungro virus No significant different of the profile of the protein bands (MW ≼ 200, 117, 89, 61, 48, 23, 29) was shown by N. virescens that also attacked the healthy rice plants and the rice plants infected by the tungro virus (Figure 3). On the green leafhoppers that ate rice plants infected by the tungro virus, the protein bands that appeared were slightly thicker which showed more protein concentration, which was the accumulation of the protein of tungro virusinfected plants that was also consumed. Whereas on the lower molecular weight, the protein banding patterns that emerged showed no much difference. The presence of viral proteins on vector species commonly observed, even several pathogenic virus can mediate manipulation of vector behavior may facilitate pathogen spread (Yamagishi and Yoshikawa 2009).

The above phenomenon could be explained that the more insect N. virescens feed the infected leaves, the more interaction between the protein of plant host and the protein of vector. This occurrence may stimulate the production of virus protein which expressed in the insect vector.

Therefore, if the insect tissue was extracted, more concentration of the protein on the gel occurred. It was interesting to look at, that the lower band both samples did not different. It could be one to the fact that small size of protein could not been stained intensively

A

B

Figure 3. The results of total protein band profiles of the green leafhoppers with SDS-PAGE electrophoresis. A. Protein banding pattern profiles, B. Zimogram, M = Marker, WS = green leafhoppers on the healthy plants, WT = green leafhoppers on the plants infected by the tungro virus

CONCLUSION The profile of the total banding patterns of the protein of the rice plants infected by the tungro virus was in contrast to the one of the healthy plants having a molecular

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weight of 108 and 117 kDa. The observation results on the banding pattern profiles of the protein of N. virescens that consumed the healthy rice plants and of the N. virescens that consumed the tungro virus-infected rice plants showed a difference in quantity as indicated by the thick-thin profile of protein bands at the molecular weight of 200, 117, 89, 61, 48, 23, 29 kDa. REFERENCES Coats SA, Wicker L. 1990. Protein variation among fuller rose case population (Coleoptera: Curculionidae). Ann Entomol 83 (6): 10541062.

Gnanamanickam SS. 2009. Biological Control of Rice Diseases. Progress in Biological Control 8. Springer, New York.

Hibino H, Cabunagan RC. 1986. Rice tungro associated viruses and their relation to host plants and vector leafhopper. Phytopatol 19: 173-182.

Hibino H. 1996. Biology and epidemiology of rice viruses. Ann Rev Phytopathol 34: 249-274.

Himawati, Supriyadi. 2003. Study the composition of green leafhoppers genus Nephotettix spp. (Hemiptera: Cicadellidae) in endemic and non-endemic areas of rice tungro disease. [Research Report]. DGHE, Department of Education, RI, Jakarta. [Indonesia] Muralidharan K, Krishnaveni D, Rajarajeswari NVL, Prasad ASR. 2003.

Tungro epidemics and yield losses in paddy fields in India. Current Sci 85 (8): 1143-1147.

Oluwafemi S, Thottappilly G, Alegbejo MD. 2007. Transmission, ELISA, and SDS-PAGE results of some maize streak virus isolates from different parts of Nigeria. J Plant Protection Res 47 (2): 197-212.

Rachim A. 2000. Efforts to reduce tungro in Bali. Adaptation Assessment Report of Tungro Resistant Rice in Bali. Agricultural Research and Development Center, Bogor. [Indonesia] Suparyono, Catindig JLA, Cabauatan PQ, Troung HX. 2003. Rice Doctor: Tungro. International Rice Research Institute (IRRI), Manila Supriyadi, Untung K, Trisyono A, Yuwono T. 2004. Character of the green leafhopper populations, Nephotettix virescens Distant (Hemiptera: Cicadellidae) in endemic and non-endemic areas of rice tungro disease. J Perlind Tanam Indon 10 (2): 112-120. [Indonesia] Supriyadi, Untung K, Trisyono A, Yuwono T. 2008. Population diversity of green leafhoppers, Nephotettix virescens Distant (Hemiptera: Cicadellidae) from endemic and non-endemic areas of rice tungro disease. National Seminar V. Entomology Society of Indonesia (PEI) Bogor Branch. Bogor, 18-19 Maret 2008. [Indonesia] Tyagi H, Rajasubramaniam S, Rajam MV, Dasgupta I. 2008. RNAinterference in rice against Rice tungro bacilliform virus results in its decreased accumulation in inoculated rice plants. Transgenic Res 17:897-904 Widiarta IN. 2005. Green leafhopper (Nephotettix virescens Distant): Population dynamics and control strategies for vector tungro disease.

J Litbang Pertan 24 (3): 85-92. [Indonesia] Wongsosupantio S. 1992. Protein Gel Electrophoresis. Inter-University Center for Biotechnology, Gadjah Mada University. Yogyakarta.

[Indonesia] Yamagishi N, Yoshikawa N (2009) Virus-induced gene silencing in soybean seeds and the emergence stage of soybean plants with Apple latent spherical virus vectors. Pl Mol Biol 71 (1-2): 15-24

ISSN: 2087-3948 E-ISSN: 2087-3956

Vol. 4, No. 1, Pp. 36-44 March 2012

Review: Soil solarization and its effects on medicinal and aromatic plants KHALID ALI KHALID

Department of Medicinal and Aromatic Plants, National Research Centre, El Buhouth St., 12311 Dokki, Cairo, Egypt. Tel. +202-3366-9948, +202-33669955, Fax: +202-3337-0931, e-mail: ahmed490@gmail.com Manuscript received: 1 March 2012. Revision accepted: 31 March 2012.

Abstract. Khalid KA. 2012. Review: Soil solarization and its effects on medicinal and aromatic plants. Nusantara Bioscience 4: 36-44.

Soil solarization or solar heating is a non-chemical disinfestation practice. Solarization effectively controls a wide range of soil borne pathogens, insects and weeds. Soil solarization is based on the exploitation of the solar energy for heating wet soil mulched with transparent plastic sheets to 40-55ºC in the upper soil layer. Thermal killing is the major factor involved in the pest control process, but chemical and biological mechanisms are also involved. The efficacy of the thermal killing is determined by the values of the maximum soil temperature and amount of heat accumulated (duration x temperature). The use of organic amendments (manure, crop residues) together with soil solarization (biofumigation) elevates the soil temperature by 1-3ºC, and improves pest control due to a generation and accumulation of toxic volatiles. Although cheaper than most chemicals used as soil fumigants, not all crops are worth the plastic prices, particularly in developing countries. Not all soil-borne pests and weeds are sufficiently controlled. Cheaper and more environmentally accepted mulching technologies are needed before expanding the range of the controlled pests by solarization.

Medicinal and aromatic plant production was affected by soil solarization.

Key words: solarization, soil borne diseases, disinfestation, mulches, plastic.

Abstrak. Khalid KA. 2012. Review: Solarisasi tanah dan dampaknya pada tanaman obat dan aromatik. Nusantara Bioscience 4: 36-44.

Solarisasi atau pemanasan tanah dengan matahari adalah praktek pembasmian hama dan penyakit secara non kimia. Solarisasi efektif mengendalikan berbagai patogen bawaan tanah, serangga dan gulma. Solarisasi tanah didasarkan pada pemanfaatan energi matahari untuk memanaskan tanah basah bermulsa dengan lembaran plastik transparan dengan suhu 40-55ºC pada bagian atas lapisan tanah. Pembasmian dengan panas merupakan faktor utama dalam proses pengendalian hama, tetapi mekanisme kimia dan biologi juga terlibat. Efektivitas pembasmian dengan panas tergantung oleh nilai-nilai suhu tanah maksimum dan jumlah panas yang terakumulasi (durasi x suhu). Penggunaan penutup organik (pupuk kandang, sisa tanaman) bersama dengan solarisasi tanah (biofumigation) meningkatkan suhu tanah 1-3ºC, dan meningkatkan pengendalian hama karena pembentukan dan akumulasi senyawa-senyawa volatil beracun. Meskipun lebih murah daripada kebanyakan bahan kimia yang digunakan sebagai fumigant tanah, tidak semua hasil panenan sepadan dengan biaya penyediaan plastik, terutama di negara berkembang. Tidak semua tanah yang mengandung hama dan gulma dapat dikendalikan sepenuhnya. Teknologi mulsa yang lebih murah dan lebih ramah lingkungan diperlukan sebelum memperluas jangkauan pengendalian hama dengan solarisasi. Produksi tanaman obat dan aromatik dipengaruhi solarisasi tanah.

Kata kunci: solarisasi, tanah penyakit bawaan, disinfestation, mulsa, plastik.

INTRODUCTION Soil borne plant pathogens survive in the soil and cause extensive damage to many crops. The most common approach for their control is by elimination before or after planting, by means of destructive methods of soil disinfestation. This should be done in such a manner as to reach the pathogens in all physical and biological niches in the soil. Chemical fumigants have proved to be of great advantage to agricultural production for many years. They are strong eradicators by nature, resulting in simultaneous control of a variety of pests. However, negative effects, i.e., eradication of beneficial organisms, and a negative shift in the biological equilibrium in the soil, are also possible during their use. Unfortunately, certain fumigants were found to possess limiting negative attributes, such as acute

and chronic health hazards, environmental pollution, and even potential atmospheric ozone depletion. The increased environmental concern due to these negative effects has been a major factor in triggering regulatory restriction on the use of soil fumigants. In many countries the use of fumigants, especially nematicides including 1, 2dibromochloropropane (DBCP), ethylene dibromide (EDB) and 1,3-dichloropropene, has been discontinued or suspended, and phase-out of methyl bromide, which is the most widely used soil fumigant, is currently underway.

Few soil disinfestation chemicals are still available, leaving the farmers in many cases without effective means to combat soilborne pests. None of the available methods used to control soilborne diseases is effective against all pathogens (including those caused by nematodes and bacteria, which are difficult to control), or can be used in

KHALID – Soil solarization of medicinal and aromatic plants

all instances. Thus, development of nonchemical methods for effective control of soil borne diseases is needed (Gamliel and Stapleton 1997, Pokharel and Larsen 2007; Pokharel et al. 2008).

Concern over environmental hazards and increased public awareness on human health issues caused by pesticides such as methyl bromide (MB) to the stratospheric ozone have directed much attention to alternative practices for chemical pest control (Katan 1999, 2000). Soil solarization or solar heating is a non-chemical disinfestation practice that has potential application as a component of a sustainable integrated pest management (IPM) approach. In addition, it also increases the availability of soil mineral nutrients, reduces crop fertilization requirements and results in improved plant growth and yield (Stapleton and DeVay 1986). Solarization was originally developed to control soil-borne pathogens as first reported by Katan et al. (1976), but it was soon found to be an effective treatment against a wide range of other soil-borne pests and weeds including more than 40 fungal plant pathogens, a few bacterial pathogens, 25 species of nematodes and many weeds (Stapleton 1997; Okharel and Hammon 2010). The virtues of solar energy are not new; however, the innovation in developing soil solarization is the use of a modern tool to this end, namely, plastic sheets.

Thus, implementation of this technology is easy to accomplish under a wide range of crop production systems.

Soil solarization is based on utilizing the solar energy for heating soil mulched with a transparent polyethylene (PE) sheet, reaching a level of 40-55ÂşC in the upper soil layer.

There is a gradient of temperatures from the upper to lower soil layer during the appropriate season. The temperature elevation is facilitated by wetting the soil before and/or during mulching with the PE sheet. The main factor involved in the pest control process is the physical mechanism of thermal killing. In addition, chemical and biological mechanisms are involved in the pest control process.

BASICS PRINCIPLE The basic principle of soil solarization is to elevate the temperature in a moist soil to a lethal level that directly affects the viability of certain organisms. The heating process also induces other environmental and biological changes in the soil that indirectly affect soil-borne pests as well as survival of beneficial organisms (Katan 1981). The values of the maximum soil temperature and amount of heat accumulated (duration x temperature) determine the potential of the thermal killing effect on soil-borne pests (Katan 1987) and weed seeds (Stapleton et al. 2000a, 2000b). Currently, the most common practice of soil solarization is based on mulching moistened soil with transparent PE. The duration of soil mulching that is required for successful effect is usually four to six weeks, depending on the pest, soil characteristics, climatic conditions and the PE properties (Katan 1981, 1987; Rubin and Benjamin 1984). Pest population and environmental conditions are unmanageable variables, while soil moisture

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and PE properties could be modified as needed. Soil pretreatment and appropriate PE technology may overcome unfavorable environmental conditions prevailing in some regions or in certain seasons, increasing weed (or pest) sensitivity and soil, shortening soil mulched duration (Stevens et al. 1991). Soil moisture improves temperature conductivity in soil and the sensitivity of microorganisms to toxic agents. Hence, pest control is better under wet heating than dry heating. This applies also to weed control, presumably because moist seeds are in a more advanced metabolic activity (Shlevin et al. 2004). Therefore, all soil pretreatments that improve water capacity, such as soil cultivation or drip irrigation during mulching, may improve soil solarization efficacy. Drip irrigation during the solarization process is essential for maintaining a wet soil surface, enabling the heat transfer to deeper layers.

Moreover, good soil preparation that leads to a smooth soil surface facilitates plastic mulching and prevents tearing (Figure 1).

Soil energy balance Mahrer (1991) discussed the mechanisms that affect the soil energy balance on bare and mulched soils. Soil energy balance can be mathematically described as follows: Rs + Rl-S-H-E = 0. Where Rs and Rl are the net fluxes of short and long wave radiation at the soil surface (radiative fluxes), S is conduction of heat in the soil (soil heat flux), H is the net heat exchange due to convection (sensible heat flux) and E is the net heat exchange due to evaporation and condensation of water (latent heat flux). These fluxes determine the temperature regime of the soil, and can be manipulated by covering the soil with appropriate mulches.

Radiative fluxes are determined by the photometric characteristics (transmission, absorption and reflection of electromagnetic radiation) of both the soil and the mulch.

Soils of darker colors tend to have higher temperatures due to increased light absorption. Mulches that are transparent to short-wave radiation and reflective to long-wave radiation increase the influx of heat into the soil by inducing a greenhouse effect.

Plastic mulches Mulches used for solarization are films of plastic polymers, usually polyethylene (PE), polyvinyl chloride (PVC), or ethylene-vinyl acetate (EVA). PE films are the most widely used. Among the desirable characteristics that make PE films popular are tensile strength, resistance to tearing when exposed to strong winds and low cost (Brown et al. 1991; Stevens et al. 1991). The optical properties of PVC and EVA are more desirable than those of PE for soil solarization, but their manufacture is more complicated and therefore, they are more expensive (Lamberti and Basile 1991). Gutkowski and Terranova (1991) observed that temperatures in soils mulched with EVA films are higher than in soils mulched with PE films. Noto (1994) found that temperatures for PVC film were slightly higher than those for PE. Cascone and D’Emilio (2000) compared the performance of EVA and co-extruded EVA-EVA and EVA-PE films on the effectiveness of greenhouse soil solarization for controlling soil-borne pathogens, but since

4 (1): 36-44, March 2012

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A

C

B

D

Figure 1. The four steps to solarize soil. A. cultivate and remove plant matter, B. level and smooth the soil, C. irrigate, D. and lay a clear tarp on the soil surface for 4 to 8 weeks, depending on local conditions (Stapleton 2008).

the mulches were of different color and thickness, an identification of the polymer effects could not be done.

Plastic films can contain additives that improve their properties for use in solarization. Additives include pigments, heat-retaining substances, wetting agents, ultraviolet stabilizers and photodegradable or biodegradable additives (Brown et al. 1991; Stevens et al. 1991).

Pigmentation of the plastic influences the efficiency of the mulch in soil energy management. Alkayssy and Alkaraghouli (1991) tested the performance of different color plastic mulches for soil solarization and reported that soil temperatures decreased for the colors in the following order: red, transparent, green, blue, yellow and black.

Traditionally, soil solarization has been implemented using either transparent or black mulches. Black PE films are usually pigmented with carbon black fillers, while transparent films have no pigment at all. Chase et al.

(1999b) and Campiglia et al. (2000) observed that soil temperatures under transparent film were higher than under black mulch, while Ham et al. (1993) reported the opposite.

Rieger et al. (2001) found black and clear mulches were equally effective for increasing soil temperatures.

Heat-retaining substances and wetting agents also influence the photometric characteristics of mulch. Mineral additives such as aluminum silicates can be added to PE films to increase their opacity to long-wave radiation and enhance the greenhouse effect in the soil (Chase et al.

1999b). Wetting agents in the film allow humidity to condense in a thin, continuous layer that also traps heat without significantly reducing the light transmittance of the plastic (Lamberti and Basile 1991).

Plastic films degrade when exposed to ultraviolet (UV) radiation, one of the components of natural light. This degradative process compromises film integrity, which is required in order to minimize heat losses from the soil.

Plastic degradation due to exposure to natural radiation has been slowed down by the addition of UV stabilizers, such as benzophenones, nickel compounds and hindered amines.

Carbon black, a common pigment for black films, also acts as a UV stabilizer: as a general rule, black films last longer

KHALID – Soil solarization of medicinal and aromatic plants

than films of other color (Abu-Irmaileh 1991; Brown et al.

1991; Stevens et al. 1991).

The durability of plastic films can be further controlled by the addition of other substances that increase the rate of degradative processes. Photodegradable PE films contain substances that accelerate the degradation of plastic exposed to light (for example, ferric ion complexes or calcium carbonate). Biodegradable plastics include substances in the polymer matrix that can be metabolized by microorganisms in the soil, accelerating the disintegration of the film into small particles. Film degradation has been considered as an alternative to inconvenient and costly removal and disposal procedures traditionally used for plastic mulches (Brown et al. 1991a; Stevens et al. 1991).

Physical and chemical changes In addition to direct physical destruction of soilborne pest inoculums, other changes in the physical soil environment occur during solarization. Among the most striking of these is the increase in concentration of soluble mineral nutrients commonly observed following treatment.

For example, the concentrations of ammonium-and nitratenitrogen are consistently increased across a range of soil types after solarization. Results of a study in California showed that in soil types ranging from loamy sand to silty clay, NH4-N and NO3-N concentration in the top 15cm soil depth increased 26-177 kg/ha (Katan 1987; Stapleton and DeVay 1995). Concentrations of other soluble mineral nutrients, including calcium, magnesium, phosphorus, potassium, and others also sometimes increased, but less consistently. Increases in available mineral nutrients in soil can play a major role in the effect of solarization, leading to increased plant health and growth, and reduced fertilization requirements. Increases in some of the mineral nutrient concentrations can be attributed to decomposition of organic components of soil during treatment, while other minerals, such as potassium, may be virtually cooked on the mineral soil particles undergoing solarization.

Improved mineral nutrition is also often associated with chemical soil fumigation (Chen et al. 1991). According to Stapleton et al. (1985) summer solarization of six wet field soils of four different textures raised soil temperatures by l0-12°C at 15 cm depth. Soil solarization increased concentrations of NO3--N and NH4+-N up to six times those in nontreated soils. Concentrations of P, Ca2+, Mg2+ and electrical conductivity (EC) increased in some of the solarized soils. Solarization did not consistently affect available K+, Fe3+, Mn2+, Zn2+, Cu2+ Cl-concentrations, soil pH or total organic matter. Concentrations of mineral nutrients in wet soil covered by transparent polyethylene film, but insulated against solar heating, were the same as those in nontreated soil. Increases in NO3--N plus NH4+-N were no longer detected in fallowed soils 9 months after solarization.

Biological changes In addition to direct physical and chemical effects, solarization causes important biological changes in treated soils. The destruction of many mesophilic microorganisms during solarization creates a partial (biological vacuum) in

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which substrate and nutrients in soil are made available for recolonization following treatment (Katan 1987; Stapleton and DeVay 1995). Solarization has been used successfully to reduce various plant pathogenic fungi (Katan 1981). The reduction of pathogens during solarization has been ascribed not only to high temperatures but also to the production of some volatiles such as carbon dioxide, ethylene and other substances which are toxic to fungi (Rubin and Benjamin 1984). Many soil borne plant parasites and pathogens are not able to compete as successfully for those resources as other microorganisms which are adapted to surviving in the soil environment.

This latter group, which includes many antagonists of plant pests, is more likely to survive solarization, or to rapidly colonize the soil substrate made available following treatment. Bacteria including Bacillus and Pseudomonas spp., fungi such as Trichoderma, and some free-living nematodes have been shown to be present in higher numbers that kill pathogens following solarization. Their enhanced presence may provide a short-or long-term shift in the biological equilibrium in solarized soils which prevents recolonization by pests, and provides a healthier environment for root and overall plant productivity (Katan 1987; Gamliel and Stapleton 1993a; Stapleton and DeVay 1995).

Restrictions The major constraints that limit the adoption of soil solarization in practice are the relatively long duration of the process and the climatic dependency. The cost of solarization is relatively low compared with other available alternatives; however, it can be a limiting factor depending on the country, the crop type, the production system (e.g. organic versus conventional farming) and the cost and availability of alternatives. Soil solarization as a nonchemical tool for weed management was proven to be more cost-effective and profitable than MB (Stapleton et al.

2005) or some other treatments (Boz 2004), especially in highly-valued crops (Abdul-Razik et al. 1988; Vizantinpoulos and Katranis 1993). Technological innovations, such as mulching the soil with sprayable polymers or using a variety of PE sheets or other mulch techniques (Gamliel and Becker 1996; Al-Kayssi and Karaghouli 2002), will facilitate the application and use of soil solarization in agriculture. These facilitations should result in reduced mulch duration, an increased geographical range of usage, a broader range of controlled weeds, improved persistency of the PE sheets, decreased PE pollution and a significant decrease in the total economical cost of mulching.

However, in addition to the favorable effects of soil solarization, there are also unfavorable ones: (i) there are geographical limitations on where the method can be used in terms of solar radiation availability; (ii) the soil is occupied for at least one month with the mulch; (iii) although cheaper than most chemicals used for soil fumigation, not all crops are worth the PE prices ; (iv) it is difficult to protect the PE sheets from damage caused by wind and animals; (v) there is no fully environmentallyaccepted solution for the used PE; and (vi) not all soilborne pests and weeds are sufficiently controlled.

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4 (1): 36-44, March 2012

Perfection Under conducive conditions and proper use, solarization can provide excellent control of soilborne pathogens in the field, greenhouse, nursery, and home garden.

However, under marginal environmental conditions, with thermo tolerant pest organisms or those distributed deeply in soil, or to minimize treatment duration, it is often desirable to combine solarization with other appropriate pest management techniques in an integrated pest management approach to improve the overall reliability of treatment (Stapleton 1997). Solarization is compatible with numerous other methods of physical, chemical, and biological pest management.

This is not to say that solarization is always improved by combining with other methods. Many field trials have shown that, under the prevailing conditions, pesticidal efficacy of solarization or another management strategy alone could not be improved by combining the treatments (Stapleton and DeVay 1995). However, even in such cases, combination of solarization with a low dose of an appropriate pesticide may provide the benefit of a more predictable treatment which is sought by commercial users.

For example, although combining solarization with a partial dose of 1, 3-dichloropropene did not statistically improve control of northern root knot nematode (Meloidogyne hapla) over either treatment alone; it did reduce recoverable numbers of the pest to near undetectable levels to a soil depth of 46 cm (Stapleton and DeVay 1983).

Solarization can also be combined with a wide range of organic amendments, such as composts, crop residues, green manures, and animal manures to sometimes increase the pesticidal effect of the combined treatments (RamirezVillapudua and Munnecke 1987; Gamliel and Stapleton 1993a,b; Chellemi et al. 1997). Incorporation of these organic materials by themselves may act to reduce numbers of soilborne pests in soil by altering the composition of the resident microbiota, or of the soil physical environment (biofumigation). Combining these materials with solarization can sometimes greatly increase the biocidal activity of the amendments. However, this appears to be an inconsistent phenomenon, and such effects should not be generalized without conducting confirmatory research. The concentrations of many volatile compounds emanating from decomposing organic materials into the soil atmosphere have been shown to be significantly higher when solarized (Gamliel and Stapleton 1993b).

The successful addition of biological control agents to soil before, during, or after the solarization process in order to obtain increased and persistent pesticidal efficacy has long been sought after by researchers. There have been great hopes of adding specific antagonistic and/or plant growth promoting microorganisms to solarized soil, either by inundating release or with transplants or other propagative material, to establish a long-term diseasesuppressive effect to subsequently planted crops (Katan 1987; Stapleton and DeVay 1995). Although no consistent advantage has been shown by this method to date, there have been a few instances of demonstrated benefit. For example, Tjamos and Fravel (1995) showed that the fungus Talaromyces yavus, when added to solarized soil which

was heated only to sublethal levels, was detrimental to the survival of Verticillium dahliae microsclerotia. In most studies, however, it appears that re-colonization of solarized soil by the native biota is just as beneficial to subsequent crops as the addition of specific microorganisms (Stapleton and DeVay 1995). This area is likely to remain to be a topic of interest and experimentation for many researchers.

EFFECT OF SOLARIZATION ON MEDICINAL AND AROMATIC PLANTS Stapleton et al. (1985) indicated that fresh and dry weights of radish, pepper and Chinese cabbage plants usually were greater when grown in solarized soils than in nontreated soils. Fertilization of solarized soils sometimes resulted in greater plant growth responses than observed in solarized than non-fertilized soils. Solarization of soil within plastic bags for 4 weeks also increased availability of nutrients such as NH4+, N, PO4+ and K+ for gerbera (Gerbera jamesonii L.) plants (Kaewruang et al. 1989).

The long-term effect of solarization on the control of pink root disease and on onion yields was studied during four successive years. No disease symptoms were noted in the first year. However, total and quality yields were increased by 29% and 57%, respectively, denoting an increased growth response phenomenon. In the following years, disease incidence increased substantially in the untreated plots, but solarization had a long-term effect in reducing disease incidence. Soil solarization has a great potential for increasing onion yield in the Mediterranean region (Satour et al. 1989).

Solarization of soil was found beneficial for plant growth in cowpea under field conditions. Root nodulation, infection by mycorrhizal fungi and yield were higher in plants grown in solarized soil. These increases were to the extent of 104.7, 20.0 and 23.7 per cent respectively when compared to control treatment without solarization (Nair 1990). Solarization of soil within plastic bags for four weeks increased availability of nutrients; solarization also significantly controlled annual weed and increased strawberry yield 12% over the yield of nontreated plots (Hartz et al. 1993). Two field experiments resulted in the reduction to undetectable levels of Sclerotium cepivorum in the upper 20 cm layer of soil, even in heavily infested soils, after solarization for 8-11 weeks. White rot progress curves in subsequent crops of garlic indicated disease onset ~ 4 months after planting. Rates of disease progress and final incidence of dead plants were greatly reduced in solarized plots, with yield increments of 40.6-155.5% over the unsolarized control plots. However, a garlic crop in the second year after solarization had increased disease levels and yield reductions that was unacceptable to the growers; this is, apparently, attributable to the high incidence of white rot of garlic that can be induced by low inoculum densities in the soil. Disease progress curves in the unsolarized plots suggested that secondary infections occur.

KHALID – Soil solarization of medicinal and aromatic plants

The effect of soil solarization on the quality of garlic was beneficial because of the increased growth response observed. Soil solarization, during the summer before the susceptible crop is planted, provides a reliable and practical method of control of white rot of garlic (Basallote-Ureba and Melero-Vara 1993). Soil solarization on raised Strawberry beds was complicated by weed growth on the top edges and sides of the bed. Soil solarization is a useful alternative for flat bed culture, but is practically limited on raised beds due to insufficient weed control (Himelrick 1995).

Combining organic amendments with soil solarization is a nonchemical approach to improvement of the control of soil borne plant diseases. Pathogen control in solarizedamended soil is attributed to a combination of thermal killing and enhanced generation of biotoxic volatile compounds. Apparently, pathogen sensitivity to biotoxic volatile compounds is enhanced with an increase of soil temperature and acts in combination with antagonistic microbial activity. Enhanced biocontrol may also be involved in some amendments. Toxic volatile compounds including alcohols, aldehydes, sulfides, isothiocyanates, and others were detected in soil amended with cruciferous residues during heating so field solarization of soil amended with composted chicken manure gave better control of pathogens and higher yield of lettuce and tomato than either treatment alone (Gamliel and Stapleton 1997).

Marketable Tomato yields in plots using soil solarization and similar to yields obtained in plots fumigated with methyl bromide + chloropicrin (Chellemi et al. 1997).

According to GrĂźnzweig et al. (1999) the effect of solarization on plant nutrients and their role in the IGR (increased growth response) was studied with tomato plants grown in solarized or non-solarized (control) sandy soil, under controlled conditions. Solarization considerably increased the soil concentrations of water extractable N, K, Ca, Mg and Na at most sites, whereas Cl and diethylenetriaminepentaacetic acid (DTPA) extractable Mn, Zn, Fe and Cu were decreased by the treatment. Plant growth and specific leaf area were enhanced in solarized as well as in N-supplemented control soil. In tomato plants grown in solarized soil, concentrations of most nutrients in the xylem sap, including N, were increased compared to the control, whereas Cl and SO4 levels decreased. The most significant increase in leaf nutrient concentration caused by soil solarization was recorded for N. Furthermore, leaf N concentration was highly and positively correlated with shoot growth. The concentration of Cu increased in leaves from the solarization, whereas that of SO4 and Cl decreased, the latter presumably below the critical toxicity level. The correlation between shoot growth and leaf concentration was positive for Cu and inverse for Cl and SO4. In conclusion, we found that soil solarization significantly affects nutrient composition in tomato plants, and provided strong evidence that N, and eventually also Cl, play a major role in IGR. Soil solarization experiments were completed in three commercial olive orchards in southern Spain; soil-solarized plots remained free of weeds, but tress in solarized plots did not show significant growth increase measured by trunk

41

perimeter (Lopez-Escudero and Blanco-Lopez 2001).

Raising the cuttings in solarized mixture fortified with Trichoderma harzianum and VAM is reported to produce robust disease-free rooted black pepper cuttings (Sarma 2000; Anandaraj et al. 2001). Solarization mediated favorable effects were observed in bhindi (Bawazir et al.

1995), onion (Adelunji 1994), coriander (Herrera and Ramirez 1996), lime (Stapleton and Devay 1986), chilies (Haripriya and Manivannan 2000) and black pepper (Sainamol et al. 2003).

Solarized soil with different levels of cattle manure resulted in a significant increase in growth and yield characters, i.e. plant height, branch number (plant-1), flower-head number (plant-1), fresh and dry weights of flower heads (g plant-1), fresh and dry weights of vegetative parts (g plant-1) and seed yield (g plant-1) as well as increase the chemical composition (essential oil, total flavonoides , total crotenoides, N, P, K, Fe, Zn and Mn) compared with the treatments of cattle manure only (Khalid et al. 2006).

According to Thankaman (2008) solarized potting mixture in combination with nutrients and biocontrol agents was evaluated for production of vigorous diseasefree rooted cuttings of black pepper. Plants raised in solarized potting mixture had better growth than plants rose in nonsolarized potting mixture (soil, sand, and farm yard manure 2: 1: 1 proportion). Among the various treatments, plants raised in solarized potting mixture with recommended nutrients (urea, superphosphate, potash and magnesium sulphate 4: 3: 2: 1) showed significant increase in number of leaves (5.3), length of roots (20 cm), leaf area (177 cm2), nutrient contents and biomass (3.7 g pl-1). The results indicated the superiority of solarized potting mixture for reducing the incidence of diseases besides yielding vigorous planting material. Cost of production of rooted cuttings with biocontrol agents was found to be cheaper in the case of rooted black pepper cuttings raised in solarized potting mixture. Bio control agents or bio fertilizers can be mixed with solarized potting mixture. The tomato yield and nutrient contents (N, P, K, Ca, Mg, Mn, Zn and Cu) were increased in leaves by soil solarization (Cimen et al. 2010).

FUTURE EXPECTATION Having user-friendly mathematical models for predicting treatment duration and efficacy (i.e. when a solarization treatment is done) available to end-users would greatly aid the adoption of solarization, but these generally have not been successfully implemented as agricultural production tools because of the passive and complex mode of action of the process over a broad range of target organisms. Nevertheless, because of the potential utility of such predictive models, they continue to be a focus of development (Katan 1987; Stapleton 1997). Also, though solarization can be an effective soil disinfestant in numerous geographic areas for certain agricultural and horticultural applications, there are inherent limitations, and situations are presented where it may be desirable to

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increase the efficacy and/or predictability of solarization through combination with other methods of soil disinfestations. Since solarization is a passive process with biocidal activity dependent to a great extent upon local climate and weather.There are occasions when even during optimal periods of the year, cool air temperatures, extensive cloud cover, frequent or persistent precipitation events, or other factors may not permit effective soil treatment. In these cases, integration of solarization with other disinfestation methods may be essential in order to increase treatment efficacy and predictability. As methyl bromide is phased out, many current users will turn to other pesticides for soil disinfestation. Combining these pesticides (perhaps at lower dosages) with solarization (perhaps for a shorter treatment period) may prove to be the most popular option for users who wish to continue using chemical soil disinfestants (Stapleton 2000a). In any case, as global environmental quality considerations grow in importance along with the increasing human population in the 21st century and beyond, evolving concepts such as solarization and other uses of solar energy in agriculture will likely to become increasingly important (Stapleton 2000b). Known limitations of soil solarization are high implementation costs for developing countries, and the requirement of special logistics and managerial abilities.

Because of these limitations, solarization is used primarily for highly-valued crops (Chen et al. 2000).

Acording to Barakat and Al-Masri (2012a), soil solarization tests against Fusarium oxysporum f. sp. lycopersici, the causal agent of tomato Fusarium wilt, were conducted for seven weeks through July and August 2008 and 2009 in the climatic conditions of Al-Aroub Agricultural Experimental Station, located in the southern mountains of the West Bank, Palestine. Double polyethylene (DPE) sheets, regular polyethylene (PE) sheets, and virtually impermeable films (VIF) were compared to examine their effects on soil temperature, disease severity, and plant growth. Results showed that in comparison to the control, PE, DPE, and VIF treatments increased the mean maximum soil temperatures by 10.2, 14.1, and 8.8ยบC, respectively, in 2008 and by 10.2, 12.6, and 8.3ยบC respectively, in 2009. The longest length of time recorded for temperature above 45ยบC under DPE sheets were 220 hours in 2008 and 218 hours in 2009. The treatments reduced the pathogen population by 86% and the disease by 43% under the DPE treatment in 2009 and to a lesser extent by the other treatments. Increases of up to 94% in fresh plant weight and up to 60% in plant dry weight were evident under the same treatment. The treatments also increased soil organic matter, both nitrogen forms, and major cations.

Acording to Lombardo et al. (2012), the relative efficacy of soil solarization and fumigation with chloropicrin and 1, 3 - dichloropropene (CP+1, 3-D) was evaluated in greenhouse-grown tomatoes. Experiments were conducted over two seasons in southern Italy, aimed at evaluating the effects of soil treatment on soil-borne pest control, and the vegetative growth and fruit production of tomato. Solarization provided a better level of control over the major fungal pathogens (Fusarium oxysporum f. sp.

lycopersici and f. sp. radicis lycopersici, as well as Pyrenochaeta lycopersici) than CP+1, 3-D fumigation.

Solarization was also more effective in reducing the population of Meloidogyne spp. in the soil, and was particularly valuable for the suppression of the parasitic plant branched broomrape Phelipanche ramosa (syn.

Orobanche ramosa). In both seasons, solarization was more beneficial than CP+1, 3-D fumigation in terms of plant growth and crop productivity. In conclusion, solarization provided a good level of control over some important tomato pests and weeds, while at the same time improving the productivity in an environmentally friendly manner. It should therefore represent a viable alternative to methyl bromide fumigation for the greenhouse production of tomato.

According to Barakat and Al-Masri (2012b), the use of integrated pest management is a valid alternative to conventional chemical treatments. This study was carried out to evaluate the effects of Brassica carinata seed meals amendment, combined with solarization, on soil activity and lettuce cultivation. B. carinata seed meals pellets are biofumigant plant tissues originated as byproducts of the biodiesel industry. Microbiological data combined with lettuce production results suggested that, after biofumigation, soil microbial communities changed toward a new equilibrium that creates better root plant conditions to improve high lettuce yields. Moreover, Brassica seed meals, combined with solarization, preserved soil microflora against detrimental effects of heating, as revealed by enzymatic and functional analysis.

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References Author-year citations are required. In the text give the authors name followed by the year of publication and arrange from oldest to newest and from A to Z. In citing an article written by two authors, both of them should be mentioned, however, for three and more authors only the first author is mentioned followed by et al., for example: Saharjo and Nurhayati (2006) or (Boonkerd 2003a, b, c; Sugiyarto 2004; El-Bana and

Nijs 2005; Balagadde et al. 2008; Webb et al. 2008). Extent citation as shown with word “cit” should be avoided. Reference to unpublished data and personal communication should not appear in the list but should be cited in the text only (e.g., Rifai MA 2007, personal communication; Setyawan AD 2007, unpublished data). In the reference list, the references should be listed in an alphabetical order. Names of journals should be abbreviated. Always use the standard abbreviation of a journal’s name according to the ISSN List of Title Word Abbreviations (www.issn.org/222661-LTWA-online.php). The following examples are for guidance.

Journal: Saharjo BH, Nurhayati AD. 2006. Domination and composition structure change at hemic peat natural regeneration following burning; a case study in Pelalawan, Riau Province. Biodiversitas 7: 154-158.

The usage of “et al” in long author lists will also be accepted: Smith J, Jones M Jr, Houghton L et al. 1999. Future of health insurance. N Engl J Med 965: 325–329 Article by DOI: Slifka MK, Whitton JL. 2000. Clinical implications of dysregulated cytokine production. J Mol Med. Doi:10.1007/s001090000086 Book: Rai MK, Carpinella C. 2006. Naturally occurring bioactive compounds.

Elsevier, Amsterdam.

Book Chapter: Webb CO, Cannon CH, Davies SJ. 2008. Ecological organization, biogeography, and the phylogenetic structure of rainforest tree communities. In: Carson W, Schnitzer S (eds) Tropical forest community ecology. Wiley-Blackwell, New York.

Abstract: Assaeed AM. 2007. Seed production and dispersal of Rhazya stricta. 50th annual symposium of the International Association for Vegetation Science, Swansea, UK, 23-27 July 2007.

Proceeding: Alikodra HS. 2000. Biodiversity for development of local autonomous government. In: Setyawan AD, Sutarno (eds) Toward mount Lawu national park; proceeding of national seminary and workshop on biodiversity conservation to protect and save germplasm in Java island. Sebelas Maret University, Surakarta, 17-20 July 2000.

[Indonesia] Thesis, Dissertation: Sugiyarto. 2004. Soil macro-invertebrates diversity and inter-cropping plants productivity in agroforestry system based on sengon.

[Dissertation]. Brawijaya University, Malang. [Indonesia] Online document: Balagadde FK, Song H, Ozaki J, Collins CH, Barnet M, Arnold FH, Q u a k e S R , Y o u L . 2 0 0 8 . A s yn t h e t i c E s c h e r i c h i a c o l i p r e d a t o r - p r e y e c o s y s t e m . M o l S ys t B i o l 4 : 1 8 7 . www.molecularsystemsbiology.com Tables should be numbered consecutively and accompanied by a title at the top. Illustrations Do not use figures that duplicate matter in tables.

Figures can be supplied in digital format, or photographs and drawings, which can be ready for reproduction. Label each figure with figure number consecutively.

Uncorrection proofs will be sent to the corresponding author by e-mail as .doc or .docx files for checking and correcting of typographical errors. To avoid delay in publication, proofs should be returned in 7 days.

A charge The cost of each manuscript is IDR 250,000,- plus postal cost or IDR 150,000,- for SIB members. There is free of charge for non Indonesian author(s), but need to pay postal cost for hardcopy.

Reprints Two copies of journal will be supplied to authors; reprint is only available with special request. Additional copies may be purchased by order when sending back the uncorrection proofs by e-mail.

Disclaimer No responsibility is assumed by publisher and co-publishers, nor by the editors for any injury and/or damage to persons or property as a result of any actual or alleged libelous statements, infringement of intellectual property or privacy rights, or products liability, whether resulting from negligence or otherwise, or from any use or operation of any ideas, instructions, procedures, products or methods contained in the material therein.

NOTIFICATION: All communications are strongly recommended to be undertaken through email.

| Nus Bioscci | vol. 4 | no. 1 | pp. 1‐ 44 | M March 2012 | | ISSN 2087 N 2087‐3956 | 7‐3948 | E‐ISSN I S E A J o u r n a l o f B i o l o g i c a l S c i e n c e s

The effect of zearaleno one mycotoxiins administrration at late e gestation daays on the deevelopment and reprod ductive organ ns of mice YULIA IRN NIDAYANTI

1‐5

Toxicity response of Po oecilia reticullata Peters 18 859 (Cyprino odontiformess: Poeciliidaee) to some agriculturaal pesticides ALIAKBAR R HEDAYATI, REZA TARKH HANI, AHMA AD SHADI

6‐10

Physiologiccal effect of ssome antioxiidant polyph henols on swe eet marjoram m (Majorana a hortensis) plants ABDALLA EL‐MOURSI,, IMAN MAH HMOUD TALA AAT, LAILA K KAMAL BALB BAA

11‐15

Characterization of Carrica pubescen ns in Dieng P Plateau, Centrral Java base ed on morpho ological characterss, antioxidantt capacity, an nd protein baanding patterrn AINUN NIK KMATI LAILY Y, SURANTO,, SUGIYARTO O

16‐21

Communitty structure o of parasitoidss Hymenopte era associate ed with Brasssicaceae and non‐crop vegetation n YAHERWA ANDI

22‐26

Evaluation n of the effecttiveness of in ntegrated maanagement aand mating d disruption in ccontrolling gypsy motth Lymantria dispar (Lepid doptera: Lym mantriidae) populations GOODARZZ HAJIZADEH H, MOHAMM MAD REZA KA AVOSI

27‐31

The total protein band profile of the green leafhoppers (Nephotettix virescens) and of the rice (Oryza sativa) infected by tungro virus ANI SULISTYARSI, SURANTO, SUPRIYADI

Review: Soil solarization and its effects on medicinal and aromatic plants

KHALID ALI KHALID

32‐35 43 36-44

Societty for Indon nesian Biodive ersity

Sebellas Maret Univversity Surakkarta

ISSN 2087-3948

Published three timess in one yearr PRINTED IN INDONESIIA

E-ISSN 2087-3956 2