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

EDITORIAL BOARD:

Editor-in-Chief, Sugiyarto, Sebelas Maret University Surakarta, Indonesia ([email protected])Deputy Editor-in-Chief, Joko R. Witono, Bogor Botanical Garden, Indonesian Institute of Sciences, Bogor, Indonesia([email protected])

Editorial Advisory Boards:Agriculture, Muhammad Sarjan, Mataram University, Mataram, Indonesia ([email protected])Animal Sciences, Freddy Pattiselanno, State University of Papua, Manokwari, Indonesia([email protected])Biochemistry and Pharmacology, Mahendra K. Rai, SGB Amravati University, Amravati, India ([email protected])Biomedical Sciences, Afiono Agung Prasetyo, Sebelas Maret University, Surakarta, Indonesia ([email protected])Biophysics and Computational Biology: Iwan Yahya, Sebelas Maret University, Surakarta, Indonesia ([email protected])Ecology and Environmental Science, Cecep Kusmana, Bogor Agricultural University, Bogor, Indonesia([email protected])Ethnobiology, Luchman Hakim, University of Brawijaya, Malang, Indonesia ([email protected])Genetics and Evolutionary Biology, Sutarno, Sebelas Maret University, Surakarta, Indonesia ([email protected])Hydrobiology, Gadis S. Handayani, Research Center for Limnology, Indonesian Institute of Sciences, Bogor, Indonesia([email protected])Marine Science, Mohammed S.A. Ammar, National Institute of Oceanography, Suez, Egypt ([email protected])Microbiology, Charis Amarantini, Duta Wacana Christian University, Yogyakarta, Indonesia ([email protected])Molecular Biology, Ari Jamsari, Andalas University, Padang, Indonesia ([email protected])Physiology, Xiuyun Zhao, Huazhong Agricultural University, Wuhan, China ([email protected])Plant Science: Pudji Widodo, General Soedirman University, Purwokerto, Indonesia ([email protected])

Management Boards:Managing Editor, Ahmad D. Setyawan, Sebelas Maret University Surakarta ([email protected])Associated Editor (English Editor), Wiryono, State University of Bengkulu ([email protected])Associated Editor (English Editor), Suranto, Sebelas Maret University SurakartaTechnical Editor, Ari Pitoyo, Sebelas Maret University Surakarta ([email protected])Business Manager, A. Widiastuti, Development Agency for Seed Quality Testing of Food and Horticulture Crops, Depok,Indonesia ([email protected])

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 UniversityJl. Ir. Sutami 36A Surakarta 57126. Tel. & Fax.: +62-271-663375, Email: [email protected]

ONLINE:biosains.mipa.uns.ac.id/nusbioscience

Society for Indonesia Biodiversity Sebelas Maret University Surakarta

| 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

ISSN: 2087-3948 Vol. 4, No. 1, Pp. 1-5 E-ISSN: 2087-3956 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: [email protected]

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-(10-hydroxy-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

4 (1): 1-5, March 2012

2

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 cardio-vascular, 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 non-parametric 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)

Body weight at GD-18

(g)

Increase of body weight at GD-18

(g) x ± sem x ± sem x ± sem

13 to 18

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

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4 (1): 1-5, March 2012

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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.Never- theless, 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 adminis-tration 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 develop-ment process. In male fetus zearalenone a relative decrease in the number of Leydig cells.

IRNIDAYANTI – Zearalenone mycotoxins at late gestation days 5

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.

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: 219-227.

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

ISSN: 2087-3948 Vol. 4, No. 1, Pp. 6-10 E-ISSN: 2087-3956 March 2012

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

ALIAKBAR HEDAYATI1,♥, REZA TARKHANI1, AHMAD SHADI2 1Department of Fishery, Faculty of Fisheries and Environment, Gorgan University of Agricultural Science and Natural Resources, Gorgan, Iran. Tel:

+980131528572, Fax: +981712220320, ♥E-mail: [email protected] 2Young 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 bahan-bahan 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). Organo-phosphorus 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- (2-isopropyl-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 7

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.


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:

pnwbLCSE /1)50( = 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

Observed Effect Concentration) were determined for each measured endpoint.


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)

No. of mortality 24 h 48 h 72 h 96 h

0 0 0 0 0 5 0 0 0 0

15 0 0 5 6 30 6 11 16 21 50 15 18 21 21

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

Concentration (ppm)

No. of mortality 24 h 48 h 72 h 96 h

0.00 0 0 0 0 0.03 0 0 0 0 0.04 0 0 0 0 0.06 0 0 0 0 0.10 0 0 0 0 0.20 0 6 10 12 0.30 15 19 20 21 0.40 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.

4 (1): 6-10, March 2012

8

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 Concentration (ppm) (95% of confidence limits) 24 h 48 h 72 h 96 h

LC1 9.97±0.98 5.92±0.84 1.07±0.74 9.06±0.57 LC10 23.8±0.98 18.1±0.84 11.0±0.74 12.5±0.57 LC20 29.7±0.98 23.3±0.84 15.1±0.74 14.0±0.57 LC30 33.9±0.98 27.0±0.84 18.2±0.74 15.1±0.57 LC40 37.5±0.98 30.2±0.84 20.7±0.74 16.0±0.57 LC50 40.9±0.98 33.2±0.84 23.2±0.74 16.8±0.57 LC60 44.3±0.98 36.2±0.84 25.6±0.74 17.7±0.57 LC70 47.9±0.98 39.4±0.84 28.1±0.74 18.6±0.57 LC80 52.1±0.98 43.1±0.84 31.2±0.74 19.6±0.57 LC90 57.9±0.98 48.2±0.84 35.4±0.74 21.1±0.57 LC99 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 Concentration (ppm) (95 % of confidence limits) 24 h 48 h 72 h 96 h

LC1 0.141±0.13 0.107±0.16 0.151±0.47 0.142±0.06 LC10 0.211±0.13 0.165±0.16 0.174±0.47 0.166±0.06 LC20 0.241±0.13 0.189±0.16 0.184±0.47 0.176±0.06 LC30 0.262±0.13 0.207±0.16 0.192±0.47 0.183±0.06 LC40 0.280±0.13 0.222±0.16 0.198±0.47 0.190±0.06 LC50 0.297±0.13 0.236±0.16 0.204±0.47 0.195±0.06 LC60 0.314±0.13 0.250±0.16 0.209±0.47 0.201±0.06 LC70 0.333±0.13 0.265±0.16 0.216±0.47 0.207±0.06 LC80 0.354±0.13 0.282±0.16 0.223±0.47 0.215±0.06 LC90 0.384±0.13 0.307±0.16 0.233±0.47 0.225±0.06 LC99 0.454±0.13 0.364±0.16 0.257±0.47 0.248±0.06

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to their potend wildlife piazinon and deata. The 96

ppm for comfor deltamethry toxic to fish.diazinon on dl tens of mg/Lalue of diazinilia reticulata mg/L. Differtive toxicity ooxification, aholinesterase

e the highand our resultts. Boateng ore susceptibleconcentrationsdeltamethrin

6 ppm. Viranrin in guppiesund 96 h fish 39 mg/L; Cypn mossambic

LC50 value of das15.47 μg/L

ough deltametions due to itul to assessme003).

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t be used inme biomarkeesticides toxic

DGEMENTS

aculture Reseay of research he Gorgan ral Resources

ENCES

o OK, Agbede rican catfish (C4

ential to causpopulations. Ieltamethrin ar h LC50 wa

mmercial dosrin and here w. different fisheL (Tsuda et anon 96 h LCa) but for zebrrent factor havof diazinon oabsorption an(Adedeji et a

h toxicity ots are in gooet al. (2006

e, and differens of chemical toxicity to P

n et al. (2003s as 5.13 mg/LLC50 values a

yprinus carpioca, 3.50 mg/deltamethrin iL was reportethrin is thoughs adsorption tent of potentia

is more toxid in the presenng values thather species oefined set oul information

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city.

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se In re as se

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nd is of

ute us)

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

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Hotos GN, Vlahos N. 1998. Salinity tolerance of Mugil cephalus and Chelon labrosus, Pisces: Mugilidae/fry in experimental conditions. Aquaculture 167: 329-338

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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

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Smith TM, Stratton GW. 1986. Effects of synthetic pyrethroid insecticides on nontarget organisms. Res Rev 97: 93-119.

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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.

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ISSN: 2087-3948Vol. 4, No. 1, Pp. 11-15 E-ISSN: 2087-3956March 2012

Physiological effect of some antioxidant polyphenols on sweet marjoram(Majorana hortensis) plants

ABDALLA EL-MOURSI, IMAN MAHMOUD TALAAT, LAILA KAMAL BALBAADepartment of Botany, National Research Centre, Dokki, Cairo 12622, Egypt. Tel. +202-3366-9948, +202-3366-9955, Fax: +202-3337-0931, e-mail:

[email protected]

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 NationalResearch Centre, Dokki, Cairo, Egypt to study the physiological effect of foliar application of some antioxidant polyphenols on growthand chemical constituents of sweet marjoram plants (Majorana hortensis L.). Plants were treated with curcuminoids, cinnamic acid andsalicylic acid, each at 5 and 10 mg/L except the control plants.The results indicate that foliar application of curcuminoids increasedgrowth 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 plantsin most cases. Application of cinnamic acid at 10 mg/L signicantly increased oil % and total oil yield/plant. Sugar content followed thesame trend. Treatment of sweet marjoram plants with salicylic acid significantly increased oil % and oil yield, especially in plantstreated with 10 mg/L SA. Total sugars % and total nitrogen % followed the same trend. The main constituents of the plant essential oilwere 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 marjorammanis (Majorana hortensis). Nusantara Bioscience 4: 11-15. Dua percobaan pot telah dilakukan di rumah kaca Pusat PenelitianNasional, Dokki, Kairo, Mesir untuk mempelajari pengaruh fisiologis aplikasi foliar beberapa polifenol antioksidan pada pertumbuhandan kandungan kimia tanaman marjoram manis (Majorana hortensis L.). Tanaman diperlakukan dengan kurkuminoid, asam sinamatdan asam salisilat, masing-masing sebanyak 5 dan 10 mg/L, kecuali tanaman kontrol. Hasil yang diperoleh menunjukkan bahwa aplikasifoliar dari kurkuminoid meningkat parameter pertumbuhan tanaman yang diteliti. Total gula juga meningkat akibat aplikasi foliarkurkuminoid. Di sisi lain, persentase minyak, hasil minyak dan persentase nitrogen menurun akibat perlakuan kurkuminoid. Perlakuanasam sinamat pada 5 mg/L menghasilkan tanaman tertinggi dalam keseluruhan percobaan. Perlakuan asam sinamat pada 10 mg/L secarasignifikan 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 minyakyang dihasilkan, terutama pada tanaman yang diperlakukan dengan asam salisilat sebanyak 10 mg/L. Total persentase gula dan totalpersentase 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 two-lipped flowers are arranged in roundish clusters (‘knots’) inthe leaf axil. The fruit consists of four smooth nutlets,which ripen only in warm regions (Figure 1). All parts ofthe plant are pleasantly aromatic.

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

herbalism it is used mainly for various gastrointestinaldisorders and to aid digestion. It is also an ingredient ofointments and bath preparations used to alleviaterheumatism (Stodola and Volàk 1992).

Curcuminoids are antioxidant polyphenols and what isconsidered as a curcumin or a derivative of a curcuminwith different chemical groups that have been formed toincrease solubility of curcumins and make them suitable fordrug formulation. These compounds are polyphenols andproduce a pronounced yellow color. Many curcumincharacters are unsuitable for use as drugs by themselves.They have poor solubility in water at acidic andphysiological pH, and also hydrolyze rapidly in alkalinesolutions (Péret-Almeida et al. 2005). Therefore, curcuminderivatives are synthezised to increase their solubility andhence bioavailability (Tomren 2007). Curcuminoids are

4 (1): 11-15, March 201212

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 ispossible to increase curcuminoid solubility in aqueousphase with surfactants or co-surfactants (Jayaprakasha et al.2006). Curcumin derivatives have been synthesized thatcould possibly be more potent than curcumin itself. Mostcommon derivatives have different substituents on thephenyl groups (Tiyaboonchai 2007). There is currently anincreasing demand for demethoxycurcumin and (curcumi-noids) because of their recently discovered biologicalactivity (Tønnesen et al. 2002).

The role of trans-cinnamic acid in stimulating growthand activating plants was studied by many investigators. Itwas reported that plants synthesize large amounts ofphenylpropanoid acids, mainly hydroxycinnamic acids,which are often found in conjugated forms, such asglycosides or Glc esters. These conjugates have beenidentified in numerous plants (Molgaard and Ravn 1988and Herrmann 1989). Glucosides may be bioactive bythemselves as defense compounds or they may be storageforms (Dixon 2001). On the other hand, 1-O-acyl Glcesters may serve as activated intermediates analogous toCoA thioesters in plant secondary metabolism (Villegas,and Kojima 1986; Lehfeldt et al. 2000). Glycosylation ofhydroxycinnamic acids to form both glycosides and Glcesters is catalyzed by a group of enzymes calledglucosyltransferases (GTs), which transfer the Glc residuefrom 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 etal. 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 glycosyl-transferases are able to glycosylate more than one aglycon,and they appear to recognize only the part of the moleculewhere glycosylation takes place (Hoesel 1981).Glycosylation normally takes place in the cytosol, but Glcconjugates are found in the vacuole (Vogt and Jones 2000).

Salicylic acid (SA) was reported to play a role ofnatural inductor of thermogenesis in Arum lily, inducesflowering in a range of plants, controls ion uptake by rootsand stomatal conductivity (Raskin 1992).

The aim of the present study was to investigate theeffect of some antioxidant phenolic compounds (curcumi-noids, cinnamic acid and salicylic acid) on the growth andchemical constituents of sweet marjoram plant.

MATERIAL AND METHODS

Growth of sweet marjoram plantTwo pot experiments were carried out during two

successive seasons of (2007/2008- 2008/2009) at the screenof National Research Centre (NRC), Dokki, Cairo, Egypt.Seeds of sweet marjoram were secured from HorticultureResearch Institute, Agricultural Research Centre, Ministryof Agriculture, Egypt. The seeds were sown in the nurseryon 21st Febreuary, 2007 and 2008, respectively. 45 dayslater, the seedlings were transferred into clay pots 30 cm indiameter, each pot contained 8 kg loamy clay soil. Fifteendays after sowing, the seedlings were thinned leaving twouniform plants. Each pot received equal and adequateamounts of water and fertilizers. Phosphorous as calciumsuperphosphate was mixed with the soil before sowing at

A B C

4 (1): 11-15, March 201212










A B C

4 (1): 11-15, March 201212










A B C

EL-MOURSI et al. – Effect of polyphenol on Majorana hortensis 13

the rate of 4.0 g/pot. Three grams of nitrogen as ammoniumsulphate in three applications (one g for each) with twoweeks intervals started 30 days after sowing, also, twograms of potassium sulphate were added as soilapplication. Other agricultural processes were performedaccording to normal practice. 30 days after planting,transplants were sprayed with different concentrations ofcurcuminoids, cinnamic acid or salicylic acid, each at (5,10 mg/L) in addition to control plants which were sprayedwith distilled water.

Chemical constituents of sweet marjoram plantCurcuminoids were extracted from ginger plants and

were secured from Department of Natural Products, NRC,Dokki, Cairo, Egypt. Treatments were distributed in acompletly randomized block design with three replications,each replicate comprising three pots. The plant herbagewas harvested, by cutting 5 cm above the soil surface, andplant growth characters in terms of plant height, number ofbranches, and herbage fresh and dry weights wererecorded. Total sugars percent were determined accordingto Dubois et al. (1956). Total nitrogen was determinedusing the modified Micro-Kjeldahl method according toJackson (1973).

Chemical composition of essential oilSamples from the fresh herbage of each treatment were

separately subjected to hydro-distillation in order todetermine the percentage of essential oil according to theEgyptian Pharmacopoeia (1984). Qualitative andquantitative determination of the different mainconstituents of marjoram oil, obtained from the first cutfrom each treatment had been carried out in parallel withauthentic samples of different oil components by GLCtechnique. The qualitative identification of the main oilfractions was carried out by comparing the relativeretention time of different peaks with those of the pureauthentic samples. The quantitative determination wasachieved by the peak area percentage, which was measuredfor each fraction; to study the changes in the constituents ofmarjoram oil as a result of the effect of different treatmentsused.

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 thesamples. The analysis conditions were as follows: Thechromatography was fitted with (2m x 1/8``) columns,peaked with Diatomic G.Hb, (100-120) mesh, and coatedwith 10% DEGS. 12 Ft. S.S. The columns were operated,using a temperature program, a linear increase with rate of4C/min, from (70C to 190C); with nitrogen at 30mL/min, as a carrier gas. The flow rates for hydrogen andair were 30 and 300 mL/min, respectively. Detectortemperature was 280C. Chart speed was 0.5 cm/minrange: 32; sample size was about 2 mL. Sensitivity of theapparatus was 18-8 x32. The standard material was injectedwith the samples of marjoram oil under the sameconditions.

Data analysisData obtained were subjected to standard analysis of

variance procedure. The values of LSD were obtainedwhenever F values were significant at 5% level asdescribed by Snedecor and Cochran (1980).


Growth of sweet marjoram plantData 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 bothcuttings. Fresh and dry weights of herb followed the sametrend. Application of curcuminoids at 10 mg/L resulted in amarked decrease in the number of branches in the secondcut. Cinnamic acid at 5 mg/L resulted in the tallest plants inmost cases, number of branches, fresh and dry weights ofherb followed the same trend. Salicylic acid treatmentssignificantly increased plant height, number of branches,fresh and dry weights of herb, especially in plants treatedwith 5 mg/L SA (Table 1).

In this concern, Raskin (1992) reported that salicylicacid (SA) is an endogenous growth regulator of phenolicnature, which participates in the regulation of physiologicalprocesses in plants. SA, for example, plays a role as naturalinductor of thermogenesis in Arum lily, induces floweringin a range of plants, controls ion uptake by roots andstomatal conductivity.

Talaat (2005) reported that foliar application of salicylicacid (50 or 100 µM) enhanced the vegetative growth ofgeranium plants, especially at 100 µM concentration.Talaat and Balbaa (2010) also reported that exogenousapplication of trans-cinnamic acid on basil plantsconsiderably increased plant growth at both the twocuttings. It was also recognized that the most promisingresults of vegetative growth criteria (i.e., plant height,number of branches, fresh and dry weights of herb) wereobtained from plants treated with trans-cinnamic acid (250mg/L).

Chemical constituents of sweet marjoram plantData presented in Table 2 show that oil % and total oil

yield/plant were significantly decreased as a result of foliarspray of curcuminoids at 5 mg/L and 10 mg/L. Theseresults hold true for essential oil % and total oil yield/plantin both cuttings. Total nitrogen % followed the same trend.On the other hand total sugars % was pronouncedlyincreased as a result of curcuminoids treatments.

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

Meanwhile, treatment of sweet marjoram plants withsalicylic 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 thatchemical analysis of the leaves of sweet basil at both the

4 (1): 11-15, March 201214

first and second cuts indicated that thecontents of total essential oil % and oil yieldin basil herb were significantly increased asa result of foliar application of trans-cinnamic acid. Similar results were obtainedfor total carbohydrates and total solublesugars, Total nitrogen, total phosphorus andtotal potassium contents. Iron and zinccontents followed the same trend.

These findings were in agreement withthose obtained by Tari et al. (2002) whoreported that SA application resulted in asignificant increase in total soluble sugarcontent in leaves of Camellia cuttings thusmaintaining the carbohydrates pool in thechloroplasts at a high level. This increasemay be implicated in osmotic adjustment asit has been described in tomato SA-treatedplants (Tari et al. 2002). Talaat (2005) alsoreported that foliar application of salicylicacid (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 SAconsiderably enhanced the growth andcarbohydrate metabolism of tea cuttings.Addition of 50 µM SA produced the mostremarkable effects. There was a 2 foldsignificant increase in leaf area, leaf freshweight and leaf dry weight. Leaf TSS wasalso doubled by this treatment. Invertaseactivity in SA treated cuttings was higherthan in control with a significant increasefor 50 µM SA.

Chemical composition of essential oilTo study the effect of different

treatments on essential oil composition ofsweet marjoram plants the oil of eachtreatment was separately subjected to gasliquid chromatography and the maincompounds and their relative percentagesare shown in (Table 3). Linalool rangedfrom 10.62% in plants treated with 5 mg/Lsalicylic to 32.36% in plants treated with 10mg/L curcuminoids. The highest content ofα-terpineol (38.11%) was observed in plantsreceived 10 mg/L curcuminoids.

In this respect, Talaat (2005) reportedthat foliar treatment of pelargonium plantswith salicylic acid at the rate of 50 μM/lresulted in the highest content of citronellol.Gamal El-Din and Reda (2006) alsoreported that treatment of chamomile plantswith salicylic acid, especially at 60 μM/lresulted in quantitative increases of someessential oil constituents.

Table 1. Effect of some antioxidant polyphenols on vegetative growth of sweetmarjoram plants

Treatments(mg/L)

Plant Height Number ofbranches

Fresh wt. ofherb

Dry wt. ofherb

1st cut 2nd cut 1st cut 2nd cut 1st cut 2nd cut 1st cut 2nd cutCurcuminoids 5 46.67 28.00 21.67 23.00 75.10 48.78 31.20 17.08Curcuminoids 10 43.67 26.00 17.67 17.67 75.01 39.71 30.53 13.61Cinnamic acid 5 49.00 27.00 19.33 22.00 79.63 55.47 27.47 28.11Cinnamic acid 10 51.00 33.67 23.33 28.33 93.18 76.82 32.78 30.27Salicylic acid 5 46.33 30.33 18.00 27.00 77.10 74.86 25.98 22.15Salicylic acid 10 48.00 32.00 20.67 28.00 88.16 75.15 32.87 29.83Control 42.33 25.67 14.00 20.00 75.30 41.72 24.27 13.73LSD (5%) 3.30 1.19 2.34 2.18 4.07 4.15 4.32 3.60

Table 2. Effect of some antioxidant polyphenols on chemical constituents ofsweet marjoram plants

Treatments(mg/L)

Oil % Oil yield Totalsugars %

TotalNitrogen %

1st cut 2nd cut 1st cut 2nd cut 1st cut 1st cutCurcuminoids 5 0.34 0.29 0.26 0.14 16.4 9.2Curcuminoids 10 0.25 0.26 0.19 0.10 16.95 8.25Cinnamic acid 5 0.38 0.46 0.30 0.25 15.15 9.69Cinnamic acid 10 0.41 0.48 0.38 0.37 16.85 7.06Salicylic acid 5 0.39 0.42 0.30 0.32 16.1 9.69Salicylic acid 10 0.42 0.44 0.37 0.33 16.35 11.88Control 0.36 0.38 0.27 0.16 14.15 9.69LSD (5%) 0.06 0.07 0.05 0.05 N.S. N.S.

Table 3. Effect of curcuminoids on essential oil constituents of sweet marjoramplants.

Treatments(mg/L)

essential oilconstituents

Curcuma-noids 5(mg/L)

Curcumi-noids 10(mg/L)

Cinnamicacid 5(mg/L)

Cinnamicacid 10(mg/L)

Salicylicacid 5(mg/L)

Salicylicacid 10(mg/L)

Control

α-pinene 1.55 0.76 1.16 1.40 0.12 1.03 1.06β-pinene 7.09 0.72 0.38 2.51 6.51 7.02 7.03camphene 7.35 1.49 0.80 8.85 6.90 6.68 7.90d-limonene 17.28 5.01 7.88 7.95 13.84 14.16 17.11cymene 7.63 8.74 6.71 23.30 8.12 7.06 6.49linalool 11.20 32.36 15.81 10.62 14.76 14.13 16.83α-terpineol 29.05 38.11 35.40 31.68 30.89 33.21 28.56geraniol 3.09 - 1.94 5.10 5.88 5.98 1.72carvone 2.73 - 4.33 0.81 1.21 0.97 3.49eugenol 1.59 - 0.81 0.11 0.19 0.19 0.86citronellol - - 0.26 1.88 0.12 0.13 1.72ethylcinnamate - - 0.22 0.50 2.14 1.65 0.49cavacrol 1.88 3.87 1.73 0.17 0.17 0.19 0.16thymole 0.55 1.82 0.19 0.27 0.34 0.21 0.28known 90.99 92.88 77.62 95.15 91.19 92.61 93.7unknown 9.01 7.12 22.38 4.85 8.81 7.39 6.3

EL-MOURSI et al. – Effect of polyphenol on Majorana hortensis 15

CONCLUSION

From the above mentioned data, it could be concludedthat the antioxidant polyphenols (curcuminoids, cinnamicacid and salicylic acid) might play a role in plantphytochemical mechanisms through affecting themetabolism of terpenes, essential oil, carbohydrates andproteins, but further studies are needed to learn more aboutthese mechanisms.

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Comment (10): deletethe yellow highlightedword and add the greyhighlighted wordpelargonium


Characterization of Carica pubescens in Dieng Plateau, Central Javabased on morphological characters, antioxidant capacity, and protein

banding pattern

AINUN NIKMATI LAILY, SURANTO, SUGIYARTOBioscience 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: [email protected]

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 onmorphological 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 DiengPlateau, Central Java (2000 m asl). It has a narrow habitat range and limited or unknown intraspecies variation. Therefore, importantinformation about the characters of the plants at various altitudes is needed, so that it is possible to extend its distribution throughtransplantation to other areas. Characterization can be performed based on morphological characters, chemical content, and proteinbanding patterns. This study aimed to describe the morphological characters, the chemical content (antioxidant capacity), and the patternof protein bands by staining, using coomassie brilliant blue on C. pubescens in the Dieng Plateau. The research was conducted in thevillages of Kejajar (1400 m asl), Patak Banteng (1900 m asl), and Sembungan (2400 m asl). The observations of morphologicalcharacters were conducted in the field and continued in the laboratory. Morphological characters, the chemical content (antioxidantcapacity), and the banding pattern of protein of C. pubescens were analyzed descriptively. The results showed that the morphologicalcharacters of C. pubescens in Dieng Plateau varried in stems, leaves and fruits. The antioxidant capacity decreased with decreasinghabitat 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 byenvironmental 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 sifatmorfologi, kapasitas antioksidan, dan pola pita protein. Nusantara Bioscience 4: 16-21. Carica pubescens Lenne & K. Kochmerupakan 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 intraspesiesterbatas atau belum diketahui. Oleh karenanya, diperlukan informasi mengenai karakter tanaman pada berbagai ketinggian sehinggadimungkinkan untuk memperluas daerah penyebaran melalui transplantasi di daerah lain. Karakterisasi dapat dilakukan berdasarkankarakter 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 diDataran Tinggi Dieng. Penelitian lapangan dilakukan di Desa Kejajar (1400 m dpl), Patak Banteng (1900 m dpl), dan Sembungan (2400m 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 karaktermorfologi C. pubescens di Dataran Tinggi Dieng bervariasi pada batang, daun, dan buah. Kapasitas antioksidannya bervariasi denganurutan dari ketinggian 2400 m dpl > 1900 m dpl > 1400 m dpl. Pola pita proteinnya tidak bervariasi antar ketinggian, namun berbedadengan C. papaya. Keseragaman pola pita protein menunjukkan kestabilan genetik C. pubescens tidak dipengaruhi oleh perubahanlingkungan.

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

INTRODUCTION

The genus Carica of the family of Caricaceae has about40 species, but only seven species are edible (Budiyanti etal., 2005). In Indonesia, one of the edible species is Caricapubescens Lenne & K. Koch which is cultivated only in thehighland of Dieng, Central Java and locally known askarika or mountain papaya. C. pubescens is a species

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

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

LAILY et al. – Carica pubescens of Dieng Plateau, Central Java 17

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

Variations in karika are believed to be influenced byenvironmental and genetic factors. Sitompul and Guritno(1995) say that the appearance of plant forms is controlledby the genetic properties of plants under the influence ofenvironmental factors. Environmental factors believed toinfluence the occurrence of morphological changes inplants are temperature, soil type, soil conditions, altitude,and humidity. If the environment factors are more powerfulthan the genetic factors, then the plants in different placeswith different environmental conditions will have differentmorphologies (Suranto 2001). But if the influence ofenvironmental factors is weaker than that of the geneticfactors then there will not be any morphological differencedespite being planted in different places.

The problem faced today is the lack of informationregarding the characterization of C. pubescens in terms ofmorphological features, chemical content, and proteinbanding pattern. Morphological features can be used tocharacterize patterns of genetic diversity, but the naturewhich can be described is limited and likely to beinfluenced by environmental factors, so that moleculargenetic identification is required to overcome theselimitations (Rahayu et al., 2006). Information about themolecular characters can be gathered by knowing theprotein banding pattern of C. pubescens while the chemicalcharacter can be determined by measuring the antioxidantcapacity of these plants. This study aimed to describe themorphological characters, the chemical content(antioxidant capacity), and the the pattern of protein bandsby the staining of C. pubescens using coomassie brilliantblue in the Dieng Plateau, Central Java.


Time and placesThis 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 villageof Kejajar (1400 ± 50 m asl), Patak Banteng (1900 ± 50 masl), and Sembungan (2400 ± 50 m asl) in the DiengPlateau, Wonosobo district, Central Java. The fieldresearch on morphological characters of superior C. papayawas conducted in Boyolali, Central Java (1500 ± 50 m asl).Antioxidant capacity and protein banding pattern wereanalyzed at the Sebelas Maret University, Surakarta,Central Java.

Procedures

SamplingSamples of C. pubescens in three different heights and

those of C. papaya were taken for morphologicalobservations in the laboratory, the analysis of antioxidantcapacity, and the pattern of protein bands. Samples wererequired for the laboratory observations on the morphologyof leaves, flowers, and fruits.

The observation on morphological charactersObservations were conducted on 10 C. pubescens plants

for the three different altitudes at the Dieng Plateau, with acomparison with the superior plant of C. papaya ofBoyolali. Observation of morphological characters in thefield was followed by observations in the laboratory. Partsof stems, leaves, flowers, fruits, and seeds of C. pubescenswere observed and documented. The morphologicalcharacters of stems observed included the height, thediameter, the cross-sectional shape, the outer surface, thecolor, the branch, the trunk’s appearance. Themorphological characters of the leaves included color, thebone, the stalk’s length, the leaf’s diameter, and the leaf‘sblade. The morphological characters of the flowers weretypes of flowers, the basic form of flowers, the shape of thecurve, the edge of the petals, the number of crowns, thenumber of stamens, the number of the ovule, the position ofthe stamens, the postion of the fruit in relation to theposition of the base of the flower, and the shape of theflower. The morphological characters of the fruits observedwere the color, the dominant of central cavity, the diameter,the length, and the length of stem, the shape of fruit, thelengthwise slice, and the crosswise slice. The characters ofthe morphology of seeds observed were the common formof the outside part of the seed, the engraving of the grainleather, and the color of the endosperm. The guideline forthe observation of these morphological characters wastaken from Tjitrosoepomo (1990), Muzayyinah (2008), andthe Center for Plant Variety Protection of the Ministry ofAgriculture of the Republic of Indonesia (2006).

Test of antioxidant capacityA total of 100 g of fruit extracts of C. pubescens and C.

papaya were weighed and then dissolved in 1 mLmethanol. The main liquor was taken using a micro pipettewith multilevel dilution to obtain the test solutionconcentration of 10 ug / mL, 5 mg / mL, 2.5 microg / mL,and 1.25 microg / mL. One mL of each test solution wasput into glass bottles and then added with 2 mL of DPPH(diphenyl picril hydrazil hydrate), then was left for 30minutes. Methanol was used as a blank solution . DPPHabsorbance was analyzed with a spectrophotometer ofvisible light at a wavelength of 517 nm.

The making of protein banding patternThe analysis of the protein band profile was carried out

according to the methods of Coats et al. (1990), using SDS-PAGE electrophoresis technique. The concentration ofacrylamide for stacking gel was 3%, while for the gradientgel was 10%. Electrophoresis was run at a constant voltageof 110 VA, until the loading dye was near the bottom of thegel. The painting was done overnight using the solution ofcoomassie brilliant blue, followed by the laxative solutionuntil the protein banding pattern emerged. Electrophoresisresults were documented in a digital camera.

Data analysesMorphological character data, the chemical content

(antioxidant capacity), and the banding pattern of proteinsin C. pubescens and C. papaya were analyzed

4 (1): 16-21, March 201218

descriptively. The antioxidant capacities of the fruits of C.pubescens and C. papaya were analyzed based on theabsorption percentage of DPPH. Antioxidant capabilitywas measured as a decrease in DPPH solution absorbancedue to the addition of the sample for 30 minutes. DPPHsolution absorbance values before and after the addition ofthe extract were calculated as percent inhibition (%inhibition). Then the calculations was included in theregression equation with the extract concentration (mg/100mL) as the abscissa (X axis) and the percentage ofinhibition as the ordinate (Y axis). The value of IC50derived from the calculation at the time of the percentageof inhibition was 50%. Y = ax + b (Cahyana 2002).

The banding patterns formed on the leaf organ samplesof C. pubescens and C. papaya were analyzed based onwhether or not the band appeared on the gel and also thethickness of the band which was formed, as has been doneby Suranto (1991, 2001, 2002) and Triawati (2005).Banding patterns which were formed were drawn aszimograms. The diversity of banding pattern wasdetermined by the value of Rf, which is the relativemobility values obtained from the comparison between themigration distance towards the migration of loading dye.The data were obtained in the form of qualitative data, sothe data analysis were done descriptively based on theresults of electrophoresis of the leaf ’s organ of C.pubescens and C. papaya.


Morphological charactersC. 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 Varietyprotection of the Ministry of Agriculture of the Republic ofIndonesia 2006). Morphological observations of C.pubescens found the presence of feathers in several organsof plants, among which was evident on the outer surface ofthe lower leaves (abaksial), leaf stalk, the outer surface ofthe flowers, both male flowers and female flowers. C.pubescens has more hair than another member of the genusCarica, namely C. papaya (Table 1).

Antioxidant capacityThe leaf’s morphology of C. pubescens at different

altitudes showed the presence of variations. The colorsappeared thickher in plants that grew on the higher land. Atthe higher places, the green and the large size of the leavesincrease the amount of chlorophyll and the area of cross-sectional sliced leaf’s surface, so that the tree is able toharness the sun's rays that are not too high in terms ofintensity for optimal photosynthetic activity.Morphological features of flowers as the proliferation ofgenerative organs of plants did not show any variation.Plants at every altitude consistently showed all types offlowers, namely male, female, and hermaphrodite.Morphological characters suggest that environmentalfactors, namely the air pressure and extreme temperatureson the higher altitude of the Dieng Plateau support thegrowth and development of C. pubescens.

Antioxidant capacity was measured by counting theamount of reduced DPPH purple color intensity that isproportional to the reduction of DPPH solutionconcentration (Figure 1).

The amount of the inhibition’s percentage of variousconcentrations of the extract that gave rise to IC 50 valuesindicated that the fruit of C. pubescens grown at differentaltitudes had different antioxidant activities. Of the threesample extracts derived from the three different altitudes,the extracts of C. pubescens at an altitude of 2400 m aslhad the highest antioxidant capacity with IC 50 of 0.983mg/100 mL, followed by C. pubescens grown at an altitudeof 1900 m asl with IC50 of 1.2945 mg/100 mL, and C.pubescens grown at an altitude of 1400 m asl with IC50 of8.843 mg/100 mL, while in C. papaya was 5.326 mg/100ml. The order of antioxidant capacity from the largest tothe smallest was as follows: C. pubescens at an altitude of2400 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 fromthe three different altitudes had antioxidant capacity. Theresponse of plants as a result of environmental factors canbe seen in the morphology and physiology . Plants thatnormally live in areas of high elevation are the type thatcan adapt to the climatic conditions of low temperature,high humidity and low sun light intensity.

A B 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.

y = 5.2116x + 3.915

R 2 = 0.7029

0

10

20

30

40

50

60

70

80

90

100

0 2 4 6 8 10 12 14

K on se n tra si e kstra k (m g /100 m l)

% I

nh

ibis

i

y = 4.7589x + 43.842

R 2 = 0.6405

0

10

20

30

40

50

60

70

80

90

100

0 2 4 6 8 10 12 14

K on se n tra si e kstra k (m g /100 m l)

% I

nh

ibis

i

y = 2.0346x + 48.003

R 2 = 0.9554

0

10

20

30

40

50

60

70

80

90

100

0 2 4 6 8 10 12 14K on se n tra si e kstra k (m g /100 m l)

% I

nh

ibis

i

Extract conc. (mg/100 mL) Extract conc. (mg/100 mL) Extract conc. (mg/100 mL)

% in

hibi

tion

% in

hibi

tion

% in

hibi

tion


Tabel 1. Morphological characteristics of C. pubescens in Dieng Plateau, Central Java

Plant organs Morphological characters1400 m asl 1900 m asl 2400 m asl

StemHeight (cm) 193,8 174,7 153,3Diameter (cm) 11,2 11,6 10,8Cross-sectional shape round round RoundThe outer surface silken to rough and has pustules rough, has pustules silken to roughColor brown to dark brown, green,

greenish brown and white glossygreenish brown dark brown, greenish brown

Branch no branches, 2-4 4 6-8How the branches look

LeavesColor dark green, yellowish green, dark green, dark green dark greenLeaf vein finger-like, reddish or yellowish finger-like, yelowish finger-like, reddishLength of stalk (cm) 33,65 44,45 44,54Diameter (cm) 45,67 47,8 54,2Leaf blade

FlowersFlowers type male, female, hermaphrodite male, female, hermaphrodite male, female, hermaphroditeShape of flowers receptacle round round roundShape of curve edge of sepals spirostichous spirostichous SpirostichousNumber of petals 5 5 5Number of stamens 5 5 5Number of ovule 5 5 5Position of stamens above the ovary above the ovary above the ovaryPosition of ovary towardsreceptacle of flowers

on the receptacle of the flower on the receptacle of the flower on the receptacle of the flower

Shape of flowers

FruitsColor bright-dark green on young fruits,

and yellowish on ripe fruityoung-old green on young

fruit, and yellow on ripe fruityoung-old green on young fruit,and yellow on ripe fruit

Dominant shape of central space pentagon pentagon pentagonDiameter (cm) 5,4 7,3 7,2Length (cm) 8,1 8,8 8,6Length of stalk (cm) 2,95 1,8 1,8Shape of fruits

Cross-sectional shape

Longitudinal-sectional shape

The production of flavonoids needs sugar as eritrosaphosphoenolpyruvate and eritrosa 4-phosphate thatprovide some carbon atoms required for the B- flavonoidring as well as an acetate unit for the A flavanoid ring.Sugars, especially sucrose, can be obtained from thedecomposition of starch or fat in storage organs duringdevelopment of the sprouts or photosynthesis in cells thatcontain chlorophyll. Light also affects the composition ofthe chloroplast.

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

antioxidant activities. Using the regression equation, theobtained IC50 vitamin standard was -84.7875 C mg/100mL. This value is lower than that in the IC50 fruit extractof C. pubescens and C. papaya. Fruit extracts of C.pubescens and C. papaya have a capacity of antioxidantbecause it contains flavonoids. Antioxidant capacity ofFlavonoid is associated with the presence of phenolichydroxyl group attached to the frame structure. Flavonoidcompounds have been proven to be able to reduce freeradical of DPPH. The activities are different, possiblybecause each extract that is believed to be flavonoid has a

38

4 (1): 16-21, March 201220

hydroxyl group with different number and location offlavonoid skeleton. Flavonoids with free hydroxyl grouphave a radical capturing activity and the presence of morethan one hydroxy group on ring B in particular willincrease the antioxidant activity.

Protein banding patternsThe protein banding patterns were analyzed in the form

of zimogram that was the typical electrophoresis results, soit can be used as a characteristic feature of the leaf’s organof C. pubescens. Data were analyzed qualitatively, basedon whether or not the band appeared and whether thin andthick bands were found in the gel electrophoresis results.The diversity of banding pattern was seen from the Rfvalues that were formed. Rf value is the value of relativemobility explained by Ferguson, obtained from thecomparison of the migration distance of protein towards themigration distance of loading dye.

The zymogram gained from running along the karikaleaves of C. pubescens at an altitude of 1400 m asl and theleaves of C. papaya is shown in Figure 3. These data showthe similarity of protein banding pattern on the samples ofC. pubescens from different altitudes. This suggests that themolecular basis of this plant is stable in response to variousenvironmental factors. Environmental factors influence themorphology and physiology of the plant. The morphologyof the plant is adapted to environmental conditions so thatphysiological processes can run optimally. Geneticvariation is a key to optimal treatment towards the geneticresources. Morphological features can be used tocharacterize a species or an individual, but the naturedescribed is only a small proportion of the genetic code.Therefore, the molecular characterization of geneticvariation should be made from the protein banding patternbecause it produces more accurate data since the protein isa late gene expression, relatively simple, and not easilychanged.

Genetic differences and the environmental factors givethe optimal growth of C. pubescens and C. papaya. Theapparent variations in pattern of protein bands between C.pubescens and C. papaya showed the diversity of thesynthesized protein, and it can be assumed there aredifferences in genetic makeup that encodes these proteins.The diversity of banding pattern of each species showed anencoding genetic diversity, since protein is a direct productof the gene in the form of amino acids. Amino acids areencoded by the DNA specifically for each type of protein.Resistance to damage may be caused by the pressureresistance of the protein molecule, being protected fromdamage by other molecules, a special structure, or a certainbehavior patterns. Judged from the data pattern of proteinbands, it appears that there is a striking difference betweenC. pubescens and C. papaya, on the level of molecularcharacteristics.

1Rf

2 3 4 5 6 7 8 9 10

0,0480,063

0,556

0,079

0,111

0,7060,683

0,524

0

0,6590,6430,661

0,500

0,008

0,0870,103

0,357

0,563

0,548

0,595

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

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 masl 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 masl, and 7; 8; 9 plants at an altitude of 2400 m asl.

0,087

0,563

0,103

0,706

1

0,595

0,548

Rf2 3 4 5 6 7

0

0,357

0,008

0,0480,063

0,556

0,079

0,111

0,706

1

0,683

0,524

Rf2 3 4 5 6 7 8 9

0

0,6590,6430,661

0,500

0,008


CONCLUSION

Morphological characters of C. pubescens in DiengPlateau 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 DiengPlateau did not show any variation. This suggests that thegenetic stability is not affected by the environmental factors.

REFERENCES

Budiyanti T, Purnomo S, Karsinah, Wahyudi A. 2005. Characterization of88 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 casepopulation (Coleoptra: Curculionidae). Ann Entomol 83 (6): 1054-1062.

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

Center for Plant Variety Protection of the Ministry of Agriculture of theRepublic of Indonesia. 2006. Guidance for testing the individualnovelty, uniqueness, uniformity and stability. Center for Plant VarietyProtection of the Ministry of Agriculture of the Republic ofIndonesia, Jakarta.

Rahayu S, Sumitro SB, Susilawati T, Soemarno. 2006. Isoenzymicanalysis 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 MadaUniversity 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 ofRanunculus nanus populations. Agrivita 23 (2): 139-146.

Tjitrosoepomo G. 1990. Plants morphology. Gadjah Mada UniversityPress, Yogyakarta.

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


Community structure of parasitoids Hymenoptera associated withBrassicaceae and non-crop vegetation

YAHERWANDIFaculty of Agriculture, Andalas University, Limau Manis, Padang 25161, West Sumatra, Indonesia. Tel. +62-751-72774, Fax: +62-751-72702; email:

[email protected]

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 insuppressing pest population. Their presence in the field is seen as the key to agricultural ecosystem. Their presence can be influenced bythe availability of non-crop vegetation. Some adult parasitoids Hymenoptera require food in the form of pollen and nectar of wildflowers to ensure effective reproduction and longevity. The objective of this research was to study communities of parasitoidHymenoptera in Brassicaceae field and non-crop vegetation around Brassicaceae fields. Samplings were conducted at two differentlandscape structures, i.e. Kayu Tanduak and Padang Laweh representing complex landscapes, whereas Alahan Panjang and SungaiNanam representing simple landscapes. Insects were sampled by three trapping techniques (farmcop, sweep net, and yellow pan traps) inone line of transect for each landscape. A total of 84 species from 17 families of parasitoids Hymenoptera were collected in Bracicaceaefield 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 dantumbuhan liar. Nusantara Bioscience 4: 22-26. Hymenoptera parasitoid memiliki peran penting dalam agroekosistem karenakemampuannya dalam menekan populasi hama. Keanekaragaman Hymenoptera parasitoid dapat dipengaruhi oleh ketersediaan vegetasiliar berbunga, karena beberapa parasitoid dewasa Hymenoptera membutuhkan serbuk sari dan nektar untuk reproduksi dankelangsungan hidupnya. Tujuan dari penelitian ini adalah untuk mempelajari keanekaragaman Hymenoptera parasitoid pada pertanamanBrassicaceae dan tumbuhan liar di sekitarnya. Pengambilan sampel serangga dilakukan pada dua lanskap pertanian yang berbeda, yaituKayu Tanduak dan Padang Laweh mewakili lanskap pertanian yang kompleks, sedangkan Alahan Panjang dan Sungai Nanam mewakililanskap pertanian yang sederhana. Koleksi sampel serangga menggunakan tiga metode yaitu farmcop, jaring serangga, dan nampankuning. Total Hymenoptera parasitoid yang telah dikoleksi pada pertanaman Brasicaceae dan tumbuhan liar adalah 84 spesies yangtermasuk 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 vegetablecommodities widely planted by farmers in Indonesia,including in West Sumatra. Vegetables commodity of WestSumatra not only meet the need in the province, but alsosupport the need of the two neighboring provinces, namelyRiau and Jambi. Brassicaceae fields in West Sumatra havea variety of problems, particularly pests and diseases.

Unfortunately, the use of pesticides in agriculturalecosystems has resulted in environmental pollution,decrease of arthropod diversity, the impoverishment ofecosystem and the emergence of pests resistant to thesepesticides) We have several groups of farmers who havebeen producing organic vegetables in West Sumatra.

Organic farming is done in an effort to restore ecologicalfunctions (biorestoration) of various arthropods inagroecosystem. Therefore, it is necessary to find alternativecontrols without using pesticides, for example, by utilizingthe natural enemies of insect herbivor or better known asbiological control.

Biological control using parasitoids is an alternativepest control strategy that is currently being developed toreplace the role of pesticides that tend to harm theenvironment and public health. Practical and more rationalmethods of Biological control have been introduced toenhance the role of parasitoid complex through habitatmanagement.

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

YAHERWANDI – Parasitoid Hymenoptera on Brassicaceae 23

Hymenoptera in agroecosystem is important and veryfundamental to the success of biological control..Information about the Hymenoptera parasitoids inIndonesia, particularly of parasitoid complex associatedwith Brassicaceae and non-crop vegetation is still limited.

Therefore, this study aimed to study the diversity,distribution, and abundance of parasitoids Hymenopteraassociated with Brassicaceae and non-crop vegetation indifferent types of agricultural landscape in West Sumatra.The results of this study is expected to be used as a strongfoundation for planning and development of integrated pestmanagement (IPM) technologies in Indonesia.


Study sitesParasitoids Hymenoptera collection was conducted in

different types of landscape of West Sumatra, Indonesia.The villages of Sungai Nanam and Alahan Panjang, Solokdistrict represent a simple structure of agriculturallandscape or agricultural ecosystems dominated by redonions fields (95%). Kayu Tanduak village, Tanah Datardistrict and the village of Padang Laweh, Agam districtrepresent a complex structure of agricultural landscape oragricultural ecosystems consisting of vegetables, rice andcorn. Descriptions of research sites were presented in Table1. Identification of insects material were conducted in theLaboratory of Insect Ecology, Department of Pest andDiseases Plant, Faculty of Agriculture, Andalas University,Padang. The research was conducted from March toNovember 2007.

Table 1. Description of research sites

Sites Altitude(m dpl) Landscape type

Kayu Tanduk 800-850 Complex agricultural landscapemixed 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 fieldsAt each agricultural landscape a transect line

approximately 1000 m in length was made along theexisting fields. Sampling of Brassicaceae was done every50 m along the transect. Collection Hymenopteraparasitoids at each sample point was conducted using threemethods: sweep net, suction with farmcop, and yellow pantraps.

Method of sweep net. Netting was conducted at eachsample point on the transect line. Netting which was tentimes double swing that includes 50 plants per samplepoint. Insects were caught directly inserted into the vialcontaining of 70% alcohol.

Farmcop method. This method used a tool thatconsisted of a small electric vacuum cleaner which hadbeen modified, 1.5-inch diameter plastic tube, a tool forinsect traps consisting of 20 cm diameter bottles, a vialcontaining 70% alcohol, and 12-volt batteries 60 A.Sampling was done by direct suction on all plant parts ofBrassicaceae.

Yellow pan trap method. Traps were made of yellowplastic 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 thetraps. To kill the insects that perched on the traps, the trapswere filled with soap water water solution to reduce surfacetension, so the insects that entered would drown and die. Atrap was placed in each sample point and left for 24 hours.Insects caught were immediately cleaned and placed intothe vial containing 70% alcohol.

Wild vegetationCollection of parasitoid Hymenoptera on wild plants

was done by sweep net method and suction with farmcop.Insects caught were directly placed into the vial containing70% alcohol

Identification of parasitoid HymenopteraIdentification was done on adult parasitoid

Hymenoptera. All Imago of parasitoids Hymenopteraobtained from sweep net method, farmcop, and yellow pantraps were identified to family level using Gaulet andHuber (1993). Identification at the species level was basedon morphological differences or morphospecies.

Data analysisAnalyses of species diversity and abundance of

parasitoid Hymenoptera were done using Shannon-Wienner Diversity Index, species richness and Simpson'sevenness index (Magurran 1988; Ludwig and Reynolds1988; Krebs 1999). To calculate species richness, Shannon-Wienner index, and Simpson's evenness index we used theprogram Primer for Windows version 5.

To create a smooth species accumulation curves, thenumber of species obtained at each sample point wasrandomized 50 times with the program EstimateS version8:00. Randomization of parasitoid Hymenoptera speciesrichness based on Jackknife-1 estimator (Cowell 2007).

Analysis of community similarity of parasitoidHymenoptera in Brassicaceae, other vegetables, and wildvegetation was done using Sørensen similarity index. Toobtain the Sørensen similarity index we used biodiv97programs integrated in Microsoft Exel. Further analysis ofcommunity grouping with cluster analysis (UPGMA) wasdone using the program of Statistica 7 for Windows(StatSoft 2007).

4 (1): 22-26, March 201224


Community of parasitoid Hymenoptera associatedBrassicaceae field and non crops vegetation on differenttypes of agricultural landscapes

The total number of parasitoid Hymenoptera collectedon Brassicaceae and non crops was 540 individualsconsisting of 84 species and 17 families. The number ofindividuals, species, and families of parasitoidsHymenoptera associated Brasicaeae in the complexlandscape was higher than that in the simple landscapes(Table 2). The high number of individuals, species, and thefamilies of parasitoids Hymenoptera in complexagricultural landscapes was due to the flow of species fromother habitats into Brassicaceae community.In other words,the parasitoid Hymenoptera community of Brassicaceaefields consisted of species of parasitoids Hymenoptera ofrice fields, other vegetables, and non crop vegetation(Table 4). This result was similar to that found byYaherwandi et al. (2007) on rice fields in the Cianjurwatershed, West Java.

Table 2. Number of family, individual, and species of parasitoidHymenoptera associated with Brassicaceae fields and non cropsvegetation on different types of agricultural landscapes

FamilySimple landscape Complex landscapeBrassica-

ceaeNon

cropsBrassica-

ceaeNon

cropsBetylidae 0 0 5 (1) 5 (1)Braconidae 127(12) 50 (9) 85 (14) 23 (6)Calcididae 3 (2) 1 (1) 0 0Ceraphronidae 0 0 3 (1) 3 (1)Diapriidae 1 (1) 1 (1) 8 (3) 0Encyrtidae 2 (1) 2 (1) 17 (3) 0Eucoilidae 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) 0Mymarommatidae 0 0 2 (1) 2 (1)Platigastridae 0 0 2 (1) 0Pteromalidae 2 (2) 0 7 (2) 7 (2)Scelionidae 11 (4) 3 (3) 26 (7) 15 (4)Torymidae 1 (1) 0 0 0Trichogrammatidae 0 0 3 (1) 0Total 263 (48) 91 (28) 275 (57) 76 (22)Note: number in parentheses () is the number of species

However, the number of individuals, species, andfamily of parasitoid Hymenoptera collected onnon cropvegetation around Brassicaceae field was higher in simplelandscapes than in complex landscapes (Table 2). Theseresults indicate that the flow of species betweenBrassicaceae fields and non crop vegetation was quite high(Table 3). Alahan Panjang and Sungai Nanam are anagricultural area with simple landscape structure andapplication 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 pesticideapplication once a week. The use of pesticides is scheduledthree times a week, causing conditions the agroecosystemof Alahan Panjang and Sungai Nanam less suitable fornatural enemies, including parasitoid Hymenoptera. Thesame results has been reported by Yaherwandi et al.(2008), especially at the time of pesticide application, manyparasitoid took refuge in habitats of non crops aroundvegetables fields in Cianjur watershed West Java.

Table 3. Matrix similarity (Sørensen index) of parasitoidsHymenoptera on the Brassicaceae fields, red onions field, and noncrops in a simple agricultural landscapes

Crops Red onions Brassicaceae Non crops

Red onions 1.00 0.31 0.38Brassicaceae 1.00 0.57Non crops 1.00

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

Crops Brassica-ceae Rice Other

vegetbalesNon

cropsBrassicaceae 1.00 0.16 0.28 0.20Rice 1.00 0.36 0.18Other vegetables 1.00 0.29Non crops 1.00

Estimation of species richness of parasitoidHymenoptera in the Brassicaceae fields

Spesies accumulation curves of parasitoid Hymenopterawere still rising, but not too sharp in both landscape (Figure1). The numbers of species collected in simple andcomplex landscapes were 48 and 57 species respectively(Table 3), while the estimation results with Jackknife-1estimator for the simple and complex landscapes were 66and 92 species respectively (Figure 2).

Figure 1. Accumulation curves of parasitoid Hymenopteraspecies on Brasicaecae fields based on data encryption of programof estimateS 8.00

0102030405060708090

100

1 2 3 4 5 6 7 8 9 10

Sampel

Jum

lah

sp

esie

s

Lanskap SedehanaLanskap Komplekssimple landscapecomplex landscape

Sample

No.

of s

peci

es

YAHERWANDI – Parasitoid Hymenoptera on Brassicaceae 25

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

This study has collected > 60% species of parasitoidHymenoptera (Figure 2). This suggests that speciesrichness collected was not maximal. According to Krebs(1999) the highest number of species estimated by theJacknife estimator is twice the number of species obtained.Furthermore, He said that Jacknife-1 estimatoris influencedby the total number of species, sample size and the numberof unique species (rare species). Thus, the low number ofspecies of parasitoid Hymenoptera collected was probablycaused by the low number of samples (10 samples perlandscape) and the ineffectiveness of tools used forcollection of insects. Due to technical reasons Malaise trapswere not used in this study, whereas the tool was effectiveenough to capture the active flying Hymenoptera (Noyes1989; Pickering and Sharkey 1995). Many ecologistsdisagree with Jacknife estimator, because estimate ofspecies richness in the community by the Jackknifeestimator is biased positively or higher (over estimate)(Heltshe and Forrester 1983). However, Palmer (1990)states that the Jacknife estimator is more accurate than theeight other estimators.

Species richness, evenness, and diversity index ofparasitoid Hymenoptera in Brassicaceae fields

The diversity of habitats and structure of agriculturallandscape affect species richness, evenness, and diversityof parasitoid Hymenoptera. Species diversity of parasitoidHymenoptera was higher in the complex landscape than ina simple landscape. Species diversity index is the resultantof the value of species richness and evenness. It wasobvious that the high diversity of species in complexlandscapes, because the species richness and evenness werehigh (Table 5).

Kayu Tanduak and Padang Laweh consist of a varietyof habitats (rice, Bracicaceae, other vegetables, and noncrops) to form the structure of agricultural landscape morecomplex than vegetable ecosystem in the Alahan Panjangand Sungai Nanam (dominated by red onion (95%) and

cabbage 5%). Agricultural landscapes in Kayu Tanduakand Padang Laweh provide a variety of resources such asalternative host, food (pollen and nectar), and shelter foradult parasitoids Hymenoptera , when environmentalconditions are not favorable. This agroecosystem canimprove survival and diversity of parasitoid Hymenoptera.(Dryer and Landis 1996; dryer and Landis 1997). Similarresults have also been reported by Idris et al. (2002), Hooksand 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 ofagricultural landscape. The agricultural landscape with acomplex structure has higher abundance, richness, anddiversity of parasitoid species than the landscape with asimpler structure.

Table 5. Species richness, evenness, and diversity of parasitoidHymenoptera associated with Brassicaceae crops and non cropson different types of agricultural landscapes

Index LandscapeSimple Complex

Species richness 46 56Species evenness 0.23 0.46Species diversity 4.23 5.27

CONCLUSION

The complex landscape had higher number of families,individuals, and species of parasitoid Hymenoptera than thesimple landscape. The number of species collected incomplex and simple landscapes has reached > 60% ofexisting species based on Jecknife-1 estimator. Speciesdiversity of parasitoid Hymenoptera was higher in thecomplex landscape than in the simple landscape. Speciessimilarities of communities of parasitoid Hymenoptera incabbage fields and in non crop vegetation was > 40%.

ACKNOWLEDGEMENTS

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

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No.

of s

peci

es

Obs Jack-1Complex landscape

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Heltshe JE, Forrester NE. 1983. Estimating species richness using thejackknife procedure. Biometrics 39: 1-11

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Yaherwandi, Manuwoto S, Buchor D, Hidayat P, Prasetyo L. 2007.Community diversity of Hymenoptera parasitoid on paddy ecosystemJurnal HPT Tropika 7 (1): 10-20. [Indonesia]

Yaherwandi, Manuwoto S, Buchor D, Hidayat P, Prasetyo L. 2008.Community structure of Hymenoptera parasitoid on non-cropvegetations in paddy field in Cianjur watershed Jurnal HPT Tropika 8(2): 90-101. [Indonesia]


Evaluation of the effectiveness of integrated management and matingdisruption in controlling gypsy moth Lymantria dispar (Lepidoptera:

Lymantriidae) populations

GOODARZ HAJIZADEH, MOHAMMAD REZA KAVOSIDepartment of Forest Sciences, Gorgan University of Agricultural Sciences and Natural Resources, Beheshti St. 386, Gorgan, Golestan, Iran.

Tel./Fax. +98 171 2227867, email: [email protected]


Abstract. Hajizadeh G, Kavosi MR. 2012. Evaluation of the effectiveness of integrated management and mating disruption incontrolling gypsy moth Lymantria dispar (Lepidoptera: Lymantriidae) populations. Nusantara Bioscience 4: 27-31. This study wasconducted during 2008 and 2009 in Daland National Park (north of Iran) to compare the effectiveness of mechanical control used incombination 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 wereapplied. In sites with integrated management and with mating disruption only, 1,828 and 1,793 egg masses/tree, and 412.75 and 207.75male moths/trap were observed, respectively. Both the numbers of egg masses/tree and of male moths/trap were significantly lower insites with integrated management than in sites with only mating disruption. This study shows that integrated management was moreeffective 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 mengontrolpopulasi ngengat gipsi Lymantria dispar (Lepidoptera: Lymantriidae). Nusantara Bioscience 4: 27-31. Penelitian ini dilakukan selamatahun 2008 dan 2009 di Taman Nasional Daland (bagian utara Iran) untuk membandingkan efektivitas pengendalian mekanis yangdigunakan dikombinasi dengan gangguan perkawinan (manajemen terpadu) dan hanya gangguan perkawinan saja dalam pengendalianngengat gipsi, Lymantria dispar (L.) (Lepidoptera: Lymantriidae). Penghitungan ngengat jantan dan jumlah massa telur dilakukansebelum (2008) dan setelah (2009) dua perlakuan diterapkan. Di lokasi dengan manajemen terpadu dan dengan gangguan perkawinansaja, terdapat 1.828 dan 1.793 massa telur/pohon, serta 412,75 dan 207,75 ngengat jantan/perangkap. Jumlah massa telur/pohon danngengat jantan/perangkap secara signifikan jauh lebih rendah pada lokasi dengan manajemen terpadu daripada di lokasi dengangangguan perkawinan saja. Studi ini menunjukkan bahwa manajemen terpadu lebih efektif daripada gangguan perkawinan dalammengurangi 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), isprobably the most important forest defoliating pest in thenortheastern United States. Defoliation and tree mortalityassociated with gypsy moth outbreaks can cause amultitude of ecological and economic effects (Twery 1991;Gottschalk 1993). In Iran, gypsy moth was observed for thefirst time in oak forests at Guilan state region, north Iran in1937 (Kavosi 2008). The activity of this pest in centralparts, western and south western forests of Iran has beenadmitted outside these regions (Hajizadeh and Kavosi2011). The largest outbreaks of gypsy moth occurred inTalesh forest in Guilan state region in 1975 (Kavosi 2008).

In the 1960s and 1970s, most treatments for controllingthis pest were conducted using conventional syntheticpesticides like carbaryl (sevin) and dylox (trichlorfon).Since 1983, these have been increasingly replaced bybiorational compounds like Bacillus thuringiensis variety

kurstaki (Berliner) and dimilin (Diflubenzuron) (Liebholdand McManus 1999). Starting in 1971, the management ofgypsy moth using mating disruption has been the subject ofconsiderable 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 themale mate-search behavior and subsequently decreases thenumber of fertilized eggs laid by females (Leonhardt et al.1996; Reardon et al. 1998). Initially, this method appliedon high-density gypsy moth populations got poor results(Cameron 1981). Later experiments in medium and low-density populations have proven that disparlure cansubstantially reduce gypsy moth abundance (Reardon et al.1998). In recent years, the operational use of disparlure hasincreased (Sharov et al. 2002). Its effectiveness is inverselyrelated to population density (Schwalbe et al. 1983; Webbet al. 1988, 1990).

4 (1): 27-31, March 201228

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 populationshave been conducted to prevent defoliation in the currentyear. Treatments are typically scheduled based on counts ofoverwintering egg mass populations, which can be used topredict defoliation (Gansner et al. 1985; Liebhold et al.1993). Operational treatments of outbreak populationsusually provide at least partial foliage protection, but theymay have limited effects on densities in subsequent yearsor on the probability of defoliation in the future (Liebholdet al. 1996). The success of treatments targeted againstoutbreak populations of the gypsy moth is traditionallyevaluated by the reduction in egg mass counts anddefoliation in treated versus untreated blocks (Twardus andMachesky 1990; Liebhold et al. 1996). However, thesemethods are not applicable in low density populationsbecause egg mass densities cannot be estimated with any

accuracy and populations are too low to cause noticeabledefoliation. Thus, evaluation of preventive treatments hasto be based on alternative methods. Larval counts underburlap bands are a sensitive sampling method at moderatepopulation densities (Reardon et al. 1998; Wallner et al.1990). And at extremely low densities, the only viablesampling method is the use of male moth counts inpheromone traps because they are most sensitive tovariations at very low population levels. Another advantageof using pheromone traps for treatment evaluation is thatthey are less expensive and thus can be used on anoperational basis rather than just in experiments. Liebholdet al. (1995) and Carter et al. (1992) found that thecorrelation between moths counts in pheromone traps anddefoliation was weak in continuously infested areas of highdensity populations. However, at these densities many traps

A B

C D

HAJIZADEH & KAVOSI – Pest management of Lymantria dispar 29

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

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


Study siteThe experiment was conducted in Daland National

Park, which is part of the larger Golestan forest inHyrcanian, north Iran (latitude 36º2′ S-36º4′ S, longitude36º3′ E-41º5′ E). This area is approximately 3750 m longand 2900 m wide and has a total area of 608 ha. The studyregion has an average temperature of 16.5°C, a total annualrainfall of 660 mm and an altitudinal range of 75-119 mabove sea level. The park consists almost entirely ofParrotia persica, Quercus castanifolia, Zelkovacarpinifolia, and Carpinus betulus, with a few small areasof other species such as Populus alba, Ficus carica, Morusalba, Cupressus Sempervirence horizentalis, Pinuseladerica, 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 easterninfestation front.

Description of treatmentsIntegrated management and mating disruption only

treatments were applied in 2008. For the evaluation ofthese treatmens egg masses and male moth counts weretaken during the year treatment was applied (2008) and inthe following year (2009). Egg masses counts were takenfrom burlap bands placed around the boles of trees in thestudy areas. This allows for evaluating gypsy mothpopulation levels even at low densities and other eggmasses sampling methods yield mostly zero counts(Bellinger et al. 1990).

The use of pheromone traps is one of the suitablemethods for monitoring and control of L. dispar. Samplingwas carried from early July to the end of August throughthe use of delta type traps (4 for each treatment-total of 8)installed at 1.5-2 m height with spacing of 100-200 mbetween each other. Adults captured were counted daily.The delta trap did not contain any sticky material. A smallpiece of brown paper was placed inside to provide a surfaceon which the female could cling. The delta trap wassuspended from a coat hanger stapled to the side of the treebole.

Integrated managementIntegrated management consisted of burning of egg

masses (mechanical method) combined with matingdisruption. This treatment was applied to the western partof study site. A gas instrument was designed and used tomechanical control egg masses in defoliated trees. This

instrument was so designed that enough gas could exit toburn the entire egg mass while keeping the bole of the treesundamaged. This was the first time gypsy moth matingdisruption was carried out in Iran. The pheromone trapsdescribed previously for the evaluation gypsy mothpopulation densities were used for the mating disruption.This treatment was applied to the eastern part of study site.


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

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

Treatment No. of egg massescollected

Eggmasses/tree

Integrated management 631 1.828a

Mating disruption only 443 1.793b

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

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

Treatment Number ofpheromone traps Males/trap

Integrated management 4 207.75b

Mating disruption only 4 412.75a


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

Treatment No. of egg massescollected

Eggmasses/tree

Integrated management 15 0.930b

Only pheromone traps 79 1.362a

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

4 (1): 27-31, March 201230

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

Year No. of egg massescollected

Eggmasses/tree

2008 1074 1.81a

2009 94 1.30b


DiscussionIn this research we found that integrated management

was more effective than mating disruption in controllinggypsy moth populations, as it can be attested by thesignificant lower numbers of egg masses and male moths.Gypsy moth populations are mainly monitored using aerialmaps of forest defoliation, counts of overwintering eggmasses (Kolodny-Hirsch 1986), and counts of male mothsin pheromone baited traps (Talerico 1981; Ravlin et al.1987). Particularly, egg mass counts are the most reliablemethod 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 skirtswrapped around tree trunks, and pheromone traps are oftenrecommended as alternative approaches to managing gypsymoth (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 protectingtrees from defoliation during outbreaks, even when used incombination. Collection and destruction of egg masses isineffective because most egg masses are well hidden orhigh in the tree where they are inaccessible. Even thoroughsearches by experts detect only a proportion of thosepresent. Burlap bands wrapped around the lower trunk oftrees can attract large numbers of gypsy moth larvae, whichhide under them during the day when they are not feeding.This tactic can be useful for detecting the presence of lowgypsy moth populations, and may be useful for protectingsmall, isolated trees from defoliation. However, researchand experience have demonstrated that trunk banding isineffective in preventing defoliation of even moderate sizetrees. The use of pheromone traps to decrease gypsy mothpopulations is sometime recommended, but is also futile.Only males are attracted to the traps, which are quicklysaturated even when populations are very low (Herms2003). Pheromone traps are very useful for delineating thedistribution of gypsy moth populations, and are usedeffectively in monitoring programs. Application of gypsymoth sex pheromone over large areas has been usedsuccessfully to suppress populations through disruption ofmating (Leonhardt et al. 1996). Wide spread application ofpheromone (usually by aircraft) saturates the environment,preventing males from detecting pheromones produced byindividual females. Mating disruption is most effectivewhen gypsy moth populations are low but starting toincrease. When Populations are high, the day-flying malescan easily locate mates visually. In areas infested by gypsymoth for many years, there is little or no relationshipbetween male moth counts and subsequent defoliation atthe 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 massdensity, and defoliation may be quite different because ofthe strong population density gradient (Ravlin et al. 1991).Egg mass counts are the most reliable cause method inmedium and high density population, and thus they arewidely used for making decision concerning aerialsuppression of outbreak population (Schwalbe 1981;Ravlin et al. 1987). In the uninfected and transition zones,moth trapping remains the only reliable monitoringmethod. Thus, the analysis of the spatial distribution ofmoth counts is justified. Mating success may be the mostimportant density dependent factor that affects sparsegypsy moth populations. Mating failure can causeinstability in isolated populations because the proportion ofnon-mated females will increase as population densitydecreases. The relationship between pheromone trap catchand mating success has never been measured accurately.Knowledge of this relationship will be useful fordistinguishing between unstable and establish populations(Sharov et al. 1995a). There are several factors affectingthe relationship between pheromone trap capture andfemale mating probability. One group of factors is relatedto pheromone source and trap design. Another group offactors affecting the mating trap capture relationship isassociated with male moth behavior. The 3rd group offactors is associated with the female calling period (Sharovet al. 1995b).

CONCLUSION

In this paper, we have developed a new method forevaluating treatments of low density, isolated gypsy mothpopulations that is based on male moth count and egg masscounts. It is recommended that field studies ofcontamination measure in other areas, especially in thenorthern forests, beconducted, so that we could use theknowledge in the management and population controlprograms. Also, the use of integrated methods to controlpest gypsy moth areas is recommended. Finally, themethods of pest control training in gypsy moth to executivedepartments should be effective.

ACKNOWLEDGMENTS

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

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The total protein band profile of the green leafhoppers (Nephotettixvirescens) and the leaves of rice (Oryza sativa) infected by tungro virus

ANI SULISTYARSI, SURANTO, SUPRIYADIBioscience 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: [email protected]


Abstract. Sulistyarsi A, Suranto, Supriyadi. 2012. The total protein band pattern of the green leafhoppers (Nephotettix virescens) andthe leaves of rice (Oryza sativa) infected by tungro virus. Nusantara Bioscience 4: 32-35. Tungro virus is one of most important diseasesof rice plants caused by double infection with RTBV and RTSV which is transmitted by Nephotettix virescens Distant. The interactionbetween host and virus-vector are still quitted difficult to understand. The aims of this study were: (i) to know the character of the totalprotein band pattern of rice plants infected with tungro virus compared to the health one, (ii) to look at the different between the bandprofiles 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 wasused. Data were analyzed descriptively based on the score of the migration of the band (Rf). The results showed that the protein containsof 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 proteinpattern was found in the healthy plant and the infected one. In general, the entire band in the infected plant was much thicker comparedto 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 stillunknown, but it may be related to the plant’s responses to virus infection, because the protein did not appear in the healthy plants. Thetotal protein content of both N. virescens which acquired the healthy leaves and the infected one were 0.1395 g and 0.1546 grespectively. Qualitatively, there was no significant difference of the protein expression in those vectors, but slightly thicker band wereobserved 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 pentingtanaman padi disebabkan oleh infeksi ganda RTBV dan RTSV, yang disebarkan oleh Nephotettix virescens Distant. Interaksi antarainang dan vektor virus masih sulit dipahami. Penelitian ini bertujuan: (i) mengetahui karakter pola pita protein total tanaman padi yangterinfeksi virus tungro dan tanaman padi sehat, (ii) mengetahui karakter pola pita protein total N. virescens yang mengkonsumsi inangtanaman padi terinfeksi virus tungro dan tanaman padi sehat. Pola pita protein total tanaman padi diidentifikasi menggunakan metodeelektroforesis dengan SDS-PAGE. Sampel daun diekstraks menggunakan buffer mercapto ethanol, sedangkan sampel wereng hijaudiekstraks menggunakan buffer PBS IX; dan pewarnaan pita protein menggunakan coomassie brilliant blue. Data dianalisis secaradeskriptif berdasarkan nilai migrasi pita (Rf). Hasil penelitian menunjukkan kadar protein 0,5 g daun tanaman padi sehat dan dauntanaman padi terinfeksi virus tungro masing-masing sebesar 0,567 μg dan 1,011 μg. Perbedaan pola pita protein secara jelas ditemukan diantara tanaman padi sehat dan terinfeksi. Pada umumnya, pola pita protein pada tanaman padi yang terinfeksi virus tungro lebih tebal daripada 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 didugaberkaitan dengan respon tanaman terhadap infeksi virus, karena protein tersebut tidak muncul pada tanaman sehat. Kadar protein totalwereng hijau yang mengkonsumsi daun tanaman padi sehat dan terinfeksi virus tungro masing-masing sebesar 0,1395 μg dan 0,1546 μg. Secarakualitatif, 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 ofthe rice plants in South and Southeast Asia causingsignificant economic looses. The disease is caused bydouble infection of rice virus tungro bacilliform virus(RTBV) and rice virus tungro spherical virus (RTSV),

which is transmitted by green leafhoppers (Nephotettixvirescens Distant) (Muralidharan et al. 2003; Tyagi et al2008). This vector is the most effective in transmitting theviruses on the rice plant and the very dominant species inthe tropics (Abdul Rachim 2000). N. virescens is the mosteffective vector in transmitting the tungro virus (Supriyadiet al. 2004, 2008; Widiarta 2005) and have also been

SULISTYARSI et al. – Protein patterns of green leafhoppers and rice infected by tungro virus 33

recorded its population is more dominant than other vectorsin the field (Himawati and Supriyadi 2003; Supriyadi et al.2004; Widiarta 2005).

A healthy rice plant uninfected by tungro virus containsmuch chlorophyll and therefore can be used for photo-synthesis and producing food for the plant. Accordingly,leaves of rice plants do not contain lots of protein so thatthe amino acid content of the plants is low. On the riceplants infected by the tungro virus, the DNA from the viruswill then infect the plant cells and take over the functionsof 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 ofhealthy rice plants and infected ones. RTSV particlesthemselves 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 theinfected host plants (Hibino and Cabauatan 1986).

The process of tungro virus transmission by vectors hasbeen known to involve the helper component which servesin binding virus particles on the mouth of the vector.Helper components are thought to be specific proteinswhich are important for virus absorption at the vector stylet(Supriyadi et.al. 2004). According to Hibino (1996) proteinas a helper component of the vector is produced in the bodythat is secreted into the mouth of the stylet. Helper proteinsof the vector of N. virescens are produced by means of hisown mouth (stylet) or the thorax which is secreted into themouth of the tool, so it requires identification of the totalprotein.

Based on the above background the researcher raisedseveral issues, namely the study of the total protein profileof host plants of the rice infected by tungro virus and totalprotein profiles of individuals of N. virescens. The researchaimed: (i) to know the character of the total protein bandprofiles of rice plants infected with tungro virus and that ofhealthy rice plants, (ii) to know the character of the bandprofiles of total protein of N. virescens that consume thehost rice plants infected by tungro virus and that of thehealthy rice plants.


Place and timeThe observation on the rice plants infected by the

tungro virus was done in the Laboratory of Science andPlant Diseases, Faculty of Agriculture, Sebelas MaretUniversity (UNS) Surakarta, while the total protein bandprofile analysis of host plants infected by rice tungro virusand the total protein band profiles of green leafhopper N.virescens was conducted at the Laboratory of Micro-biology, IUC, Gadjah Mada University, Yogyakarta. Thestudy was conducted from February to July 2010.

ProceduresThe rice cultivar tested in this study was of Ciherang

type. Rice plant samples were taken from endemic areas inYogyakarta, Indonesia. The rice plants were planted in potsplaced in a greenhouse. Samples of N. virescens from the

field were grown in a rice box breeding nursery in ourcampus. Tungro virus transmission was conducted in agreenhouse. Samples for total protein electrophoresisprofiles using the leaves of rice plants in both healthy andthe rice infected by the tungro virus. As for the total proteinprofile analysis of green leafhoppers, samples were takenfrom the head and thorax of green leafhoppers that attackedboth healthy rice plants and the plants infected by thetungro virus. Observation of the rice plant is describedqualitatively to identify the healthy rice plants and thetungro virus- infected rice plants.

Total protein profile analysis was conducted to identifydifferences and/or total protein band profile similaritybetween the rice plants infected by tungro virus and healthyrice plants and also between green leafhoppers thatconsumed healthy rice plants and the plants infected bytungro virus. Profile analysis of total protein band was donewith the technique of electrophoresis on SDS-PAGE withseveral stages, adopting the method of Wongsosupantio(1992), Coats et al. (1990), Cruz et al. (1998) and Takkara(2000). Leaf samples of rice plants employed merchapto-ethanol extract buffer, while the green leafhoppers samplesemployed IX PBS buffer extract. Each procedure requires0.5 g of sample. The concentration of acrylamide forstacking 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 thegel. Gels were fixed and then staining was done in onenight with a solution of coomassie brilliant blue R-250.Staining process was followed by distaining using asolution consisting of methanol, acetic acid, and water(40:40:20) which were all shaken until the protein bandsappeared. Total protein electrophoresis results weredocumented in a digital photograph.

The data of the total protein band pattern of the rice andN. virescens were analyzed by zimogram to observesimilarities or differences in the total protein band profilesbetween rice plants infected with tungro virus and healthyrice plants.


Description of the rice plants infected by tungro virusThe rice plants uninfected by the tungro virus grew well

the leaves were green and plants were relatively high. Therice plants infected by the tungro virus grew somewhatstunted; the young leaves turn yellowish from the tip, andthe yellow leaves appear somewhat twisted; the olderleaves look yellow to orange; the offsprings were few, andthe heights of the plants were hardly even. In the breedingprocess, the tungro virus transmission appears on the thirdleave which looks somewhat twisted (Figure 1).

According to Gnanamanickam (2009), the generalsymptoms of rice plants infected by tungro virus are leafdiscoloration that begins from leaf tip and extends to theblade or the lower leaf portion; infected leaf sometimes alsoshow molted or stripped appearance and stunting. The presenceof tungro virus can be confirmed by some serological toolbased on protein characteristics (Suparyono et al. 2003).

4 (1): 32-35, March 201234

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 leafsamples by SDS-PAGE electrophoresis. A. Protein bandingpattern profiles, B. Zimogram, 1 = marker, 2 = leaves of healthyrice plants: 30 mg (12 mL), 3 = leaves of healthy rice plants: 40mg (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 (6.6mL).

Total protein band profiles of the healthy rice plantsand the ones infected by the tungro virus

The results of measurements with a spectrophotometerat a wavelength of (λ) = 595 nm indicated that the proteincontent of the healthy rice plants and the infected one were0.567 μg and 1.011 μg respectively. Protein levels are usedto determine the concentration of the sample used in theprocess of running electrophoresis.

The electrophoresis results of the protein band profilesof the healthy rice plants and the ones infected by greenleafhoppers at a certain range of molecular weight showeda very thick gene expression, which was not expressed onthe healthy rice plants (Figure 2). Profile of protein bandsthat appeared on the BM (16, 30, 47, 61, 89 and 200) kDawere expressed by the healthy rice plants and the riceplants infected by the tungro virus, which means both thehealthy rice plants and the ones infected by the tungro virus

expressed the same protein band profile, which was basedon markers consisting of protein glutamate dehydrogenase,ovalbumin, carbonic anhydrase, myoglobin, lysozyme, andaprotinin. On the tungro virus-infected plants, morequantity of expressed proteins was seen on the proteinbands which looked thicker.

The tungro virus-infected rice plants showed a specificprotein band profile which was in the range of proteinbands close to 108 MW and 117 kDa which was displayedwith a thick band. Protein bands on the range of molecularweight are typical proteins in the rice plants infected by thetungro virus. Based on the marker, both proteins werealleged to be β-galactosidase and bovine serum albuminexpressed by the rice plants because of a virus infection.The function of both types of proteins is not known withcertainty, but it is allegedly associated with the plantresponses to virus infection as a form of self defense,because both types of proteins were not expressed on thehealthy rice plants, thus there is a need for further research.According to Sereikaite et al. (2005) bovine serum albuminusually serves as a nutrient in microbial cells, and supportsthe growth of cells and is used for immunoblots and iscorrelated to the immunosorbent of the enzyme test.

In the total protein expression profile in rice plantsinfected by the tungro virus, it was difficult to distinguishbetween a viral protein and the protein of rice plants. Withthe addition of SDS in the electrophoresis, the protein ofthe plants and of the virus will be cut into polypeptidechains. It is suspected that the polypeptide of the virusprotein has a molecular weight similar to that of thepolypeptide of the plants so that they settle in the samelocation, or that the levels and types of viral polypeptidesare relatively few compared to the polypeptide of plants soit is difficult to identify. This result was differ toOluwafemi et al. (2007) which indicated that plantsinfected with maize streak virus (MSV) had proteinpatterns different from healthy plants. Perhaps, in thisstudy, the isolated viral proteins had very littleconcentration.

A B C D

SULISTYARSI et al. – Protein patterns of green leafhoppers and rice infected by tungro virus 35

The profile of protein bands of the green leafhoppers ofthe rice plants infected by the tungro virus

No significant different of the profile of the proteinbands (MW ≥ 200, 117, 89, 61, 48, 23, 29) was shown byN. virescens that also attacked the healthy rice plants andthe rice plants infected by the tungro virus (Figure 3). Onthe green leafhoppers that ate rice plants infected by thetungro virus, the protein bands that appeared were slightlythicker which showed more protein concentration, whichwas the accumulation of the protein of tungro virus-infected plants that was also consumed. Whereas on thelower molecular weight, the protein banding patterns thatemerged showed no much difference. The presence of viralproteins on vector species commonly observed, evenseveral pathogenic virus can mediate manipulation ofvector behavior may facilitate pathogen spread (Yamagishiand Yoshikawa 2009).

The above phenomenon could be explained that themore insect N. virescens feed the infected leaves, the moreinteraction between the protein of plant host and the proteinof vector. This occurrence may stimulate the production ofvirus protein which expressed in the insect vector.Therefore, if the insect tissue was extracted, moreconcentration of the protein on the gel occurred. It wasinteresting to look at, that the lower band both samples didnot different. It could be one to the fact that small size ofprotein could not been stained intensively

A B

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

CONCLUSION

The profile of the total banding patterns of the proteinof the rice plants infected by the tungro virus was incontrast to the one of the healthy plants having a molecular

weight of 108 and 117 kDa. The observation results on thebanding pattern profiles of the protein of N. virescens thatconsumed the healthy rice plants and of the N. virescensthat consumed the tungro virus-infected rice plants showeda difference in quantity as indicated by the thick-thinprofile 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 casepopulation (Coleoptera: Curculionidae). Ann Entomol 83 (6): 1054-1062.

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

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

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

Himawati, Supriyadi. 2003. Study the composition of green leafhoppersgenus Nephotettix spp. (Hemiptera: Cicadellidae) in endemic andnon-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. CurrentSci 85 (8): 1143-1147.

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

Rachim A. 2000. Efforts to reduce tungro in Bali. Adaptation AssessmentReport of Tungro Resistant Rice in Bali. Agricultural Research andDevelopment 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 thegreen leafhopper populations, Nephotettix virescens Distant(Hemiptera: Cicadellidae) in endemic and non-endemic areas of ricetungro disease. J Perlind Tanam Indon 10 (2): 112-120. [Indonesia]

Supriyadi, Untung K, Trisyono A, Yuwono T. 2008. Population diversityof green leafhoppers, Nephotettix virescens Distant (Hemiptera:Cicadellidae) from endemic and non-endemic areas of rice tungrodisease. 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. RNA-interference in rice against Rice tungro bacilliform virus results in itsdecreased accumulation in inoculated rice plants. Transgenic Res17:897-904

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-UniversityCenter for Biotechnology, Gadjah Mada University. Yogyakarta.[Indonesia]

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

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

KHALID ALI KHALIDDepartment of Medicinal and Aromatic Plants, National Research Centre, El Buhouth St., 12311 Dokki, Cairo, Egypt. Tel. +202-3366-9948, +202-3366-

9955, Fax: +202-3337-0931, e-mail: [email protected]

Manuscript received: 1 March 2012. Revision accepted: 31 March 2012.

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

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

INTRODUCTION

Soil borne plant pathogens survive in the soil and causeextensive damage to many crops. The most commonapproach for their control is by elimination before or afterplanting, by means of destructive methods of soildisinfestation. This should be done in such a manner as toreach the pathogens in all physical and biological niches inthe soil. Chemical fumigants have proved to be of greatadvantage to agricultural production for many years. Theyare strong eradicators by nature, resulting in simultaneouscontrol of a variety of pests. However, negative effects, i.e.,eradication of beneficial organisms, and a negative shift inthe biological equilibrium in the soil, are also possibleduring their use. Unfortunately, certain fumigants werefound to possess limiting negative attributes, such as acute

and chronic health hazards, environmental pollution, andeven potential atmospheric ozone depletion. The increasedenvironmental concern due to these negative effects hasbeen a major factor in triggering regulatory restriction onthe use of soil fumigants. In many countries the use offumigants, especially nematicides including 1, 2-dibromochloropropane (DBCP), ethylene dibromide (EDB)and 1,3-dichloropropene, has been discontinued orsuspended, and phase-out of methyl bromide, which is themost widely used soil fumigant, is currently underway.Few soil disinfestation chemicals are still available, leavingthe farmers in many cases without effective means tocombat soilborne pests. None of the available methodsused to control soilborne diseases is effective against allpathogens (including those caused by nematodes andbacteria, which are difficult to control), or can be used in

ISSN: 2087-3948Vol. 4, No. 1, Pp. 36-44 E-ISSN: 2087-3956 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 bornepathogens, 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 andaccumulation 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 aromaticplant production was affected by soil solarization.

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 efektifmengendalikan 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.

all instances. Thus, development of nonchemical methodsfor 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 increasedpublic awareness on human health issues caused bypesticides such as methyl bromide (MB) to thestratospheric ozone have directed much attention toalternative practices for chemical pest control (Katan 1999,2000). Soil solarization or solar heating is a non-chemicaldisinfestation practice that has potential application as acomponent of a sustainable integrated pest management(IPM) approach. In addition, it also increases theavailability of soil mineral nutrients, reduces cropfertilization requirements and results in improved plantgrowth and yield (Stapleton and DeVay 1986). Solarizationwas originally developed to control soil-borne pathogens asfirst reported by Katan et al. (1976), but it was soon foundto be an effective treatment against a wide range of othersoil-borne pests and weeds including more than 40 fungalplant pathogens, a few bacterial pathogens, 25 species ofnematodes and many weeds (Stapleton 1997; Okharel andHammon 2010). The virtues of solar energy are not new;however, the innovation in developing soil solarization isthe use of a modern tool to this end, namely, plastic sheets.Thus, implementation of this technology is easy toaccomplish under a wide range of crop production systems.Soil solarization is based on utilizing the solar energy forheating soil mulched with a transparent polyethylene (PE)sheet, reaching a level of 40-55ºC in the upper soil layer.There is a gradient of temperatures from the upper to lowersoil layer during the appropriate season. The temperatureelevation is facilitated by wetting the soil before and/orduring mulching with the PE sheet. The main factorinvolved in the pest control process is the physicalmechanism of thermal killing. In addition, chemical andbiological mechanisms are involved in the pest controlprocess.

BASICS PRINCIPLE

The basic principle of soil solarization is to elevate thetemperature in a moist soil to a lethal level that directlyaffects the viability of certain organisms. The heatingprocess also induces other environmental and biologicalchanges in the soil that indirectly affect soil-borne pests aswell as survival of beneficial organisms (Katan 1981). Thevalues of the maximum soil temperature and amount ofheat accumulated (duration x temperature) determine thepotential 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 soilsolarization is based on mulching moistened soil withtransparent PE. The duration of soil mulching that isrequired for successful effect is usually four to six weeks,depending on the pest, soil characteristics, climaticconditions and the PE properties (Katan 1981, 1987; Rubinand Benjamin 1984). Pest population and environmentalconditions are unmanageable variables, while soil moisture

and PE properties could be modified as needed. Soil pre-treatment and appropriate PE technology may overcomeunfavorable environmental conditions prevailing in someregions or in certain seasons, increasing weed (or pest)sensitivity and soil, shortening soil mulched duration(Stevens et al. 1991). Soil moisture improves temperatureconductivity in soil and the sensitivity of microorganismsto toxic agents. Hence, pest control is better under wetheating than dry heating. This applies also to weed control,presumably because moist seeds are in a more advancedmetabolic activity (Shlevin et al. 2004). Therefore, all soilpretreatments that improve water capacity, such as soilcultivation or drip irrigation during mulching, may improvesoil solarization efficacy. Drip irrigation during thesolarization process is essential for maintaining a wet soilsurface, enabling the heat transfer to deeper layers.Moreover, good soil preparation that leads to a smooth soilsurface facilitates plastic mulching and prevents tearing(Figure 1).

Soil energy balanceMahrer (1991) discussed the mechanisms that affect the

soil energy balance on bare and mulched soils. Soil energybalance can be mathematically described as follows: Rs +Rl-S-H-E = 0. Where Rs and Rl are the net fluxes of shortand long wave radiation at the soil surface (radiativefluxes), S is conduction of heat in the soil (soil heat flux),H is the net heat exchange due to convection (sensible heatflux) and E is the net heat exchange due to evaporation andcondensation of water (latent heat flux). These fluxesdetermine the temperature regime of the soil, and can bemanipulated by covering the soil with appropriate mulches.Radiative fluxes are determined by the photometriccharacteristics (transmission, absorption and reflection ofelectromagnetic radiation) of both the soil and the mulch.Soils of darker colors tend to have higher temperatures dueto increased light absorption. Mulches that are transparentto short-wave radiation and reflective to long-waveradiation increase the influx of heat into the soil byinducing a greenhouse effect.

Plastic mulchesMulches used for solarization are films of plastic

polymers, usually polyethylene (PE), polyvinyl chloride(PVC), or ethylene-vinyl acetate (EVA). PE films are themost widely used. Among the desirable characteristics thatmake PE films popular are tensile strength, resistance totearing when exposed to strong winds and low cost (Brownet al. 1991; Stevens et al. 1991). The optical properties ofPVC and EVA are more desirable than those of PE for soilsolarization, but their manufacture is more complicated andtherefore, they are more expensive (Lamberti and Basile1991). Gutkowski and Terranova (1991) observed thattemperatures in soils mulched with EVA films are higherthan in soils mulched with PE films. Noto (1994) foundthat temperatures for PVC film were slightly higher thanthose for PE. Cascone and D’Emilio (2000) compared theperformance of EVA and co-extruded EVA-EVA andEVA-PE films on the effectiveness of greenhouse soilsolarization for controlling soil-borne pathogens, but since

KHALID – Soil solarization of medicinal and aromatic plants 37

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 cleartarp on the soil surface for 4 to 8 weeks, depending on local conditions (Stapleton 2008).

the mulches were of different color and thickness, anidentification of the polymer effects could not be done.Plastic films can contain additives that improve their pro-perties for use in solarization. Additives include pigments,heat-retaining substances, wetting agents, ultravioletstabilizers and photodegradable or biodegradable additives(Brown et al. 1991; Stevens et al. 1991).

Pigmentation of the plastic influences the efficiencyof the mulch in soil energy management. Alkayssy andAlkaraghouli (1991) tested the performance of differentcolor plastic mulches for soil solarization and reported thatsoil temperatures decreased for the colors in the followingorder: red, transparent, green, blue, yellow and black.Traditionally, soil solarization has been implemented usingeither transparent or black mulches. Black PE films areusually pigmented with carbon black fillers, whiletransparent films have no pigment at all. Chase et al.(1999b) and Campiglia et al. (2000) observed that soiltemperatures under transparent film were higher than underblack mulch, while Ham et al. (1993) reported the opposite.

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

Heat-retaining substances and wetting agents alsoinfluence the photometric characteristics of mulch. Mineraladditives such as aluminum silicates can be added to PEfilms to increase their opacity to long-wave radiation andenhance the greenhouse effect in the soil (Chase et al.1999b). Wetting agents in the film allow humidity tocondense in a thin, continuous layer that also traps heatwithout significantly reducing the light transmittance of theplastic (Lamberti and Basile 1991).

Plastic films degrade when exposed to ultraviolet (UV)radiation, one of the components of natural light. Thisdegradative process compromises film integrity, which isrequired in order to minimize heat losses from the soil.Plastic degradation due to exposure to natural radiation hasbeen slowed down by the addition of UV stabilizers, suchas benzophenones, nickel compounds and hindered amines.Carbon black, a common pigment for black films, also actsas a UV stabilizer: as a general rule, black films last longer

A B

C D

4 (1): 36-44, March 201238

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

The durability of plastic films can be further controlledby the addition of other substances that increase the rate ofdegradative processes. Photodegradable PE films containsubstances that accelerate the degradation of plastic expo-sed to light (for example, ferric ion complexes or calciumcarbonate). Biodegradable plastics include substances inthe polymer matrix that can be metabolized bymicroorganisms in the soil, accelerating the disintegrationof the film into small particles. Film degradation has beenconsidered as an alternative to inconvenient and costlyremoval and disposal procedures traditionally used forplastic mulches (Brown et al. 1991a; Stevens et al. 1991).

Physical and chemical changesIn addition to direct physical destruction of soilborne

pest inoculums, other changes in the physical soilenvironment occur during solarization. Among the moststriking of these is the increase in concentration of solublemineral nutrients commonly observed following treatment.For example, the concentrations of ammonium-and nitrate-nitrogen are consistently increased across a range of soiltypes after solarization. Results of a study in Californiashowed that in soil types ranging from loamy sand to siltyclay, NH4-N and NO3-N concentration in the top 15cm soildepth increased 26-177 kg/ha (Katan 1987; Stapleton andDeVay 1995). Concentrations of other soluble mineralnutrients, including calcium, magnesium, phosphorus,potassium, and others also sometimes increased, but lessconsistently. Increases in available mineral nutrients in soilcan play a major role in the effect of solarization, leading toincreased plant health and growth, and reduced fertilizationrequirements. Increases in some of the mineral nutrientconcentrations can be attributed to decomposition oforganic components of soil during treatment, while otherminerals, such as potassium, may be virtually cooked onthe mineral soil particles undergoing solarization.Improved mineral nutrition is also often associated withchemical soil fumigation (Chen et al. 1991). According toStapleton et al. (1985) summer solarization of six wet fieldsoils of four different textures raised soil temperatures byl0-12°C at 15 cm depth. Soil solarization increasedconcentrations of NO3

--N and NH4

+-N up to six times thosein nontreated soils. Concentrations of P, Ca2+, Mg2+ andelectrical conductivity (EC) increased in some of thesolarized soils. Solarization did not consistently affectavailable K+, Fe3+, Mn2+, Zn2+, Cu2+ Cl-concentrations, soilpH or total organic matter. Concentrations of mineralnutrients in wet soil covered by transparent polyethylenefilm, but insulated against solar heating, were the same asthose in nontreated soil. Increases in NO3

--N plus NH4+-N

were no longer detected in fallowed soils 9 months aftersolarization.

Biological changesIn addition to direct physical and chemical effects,

solarization causes important biological changes in treatedsoils. The destruction of many mesophilic microorganismsduring solarization creates a partial (biological vacuum) in

which substrate and nutrients in soil are made available forrecolonization following treatment (Katan 1987; Stapletonand DeVay 1995). Solarization has been used successfullyto reduce various plant pathogenic fungi (Katan 1981). Thereduction of pathogens during solarization has beenascribed not only to high temperatures but also to theproduction of some volatiles such as carbon dioxide,ethylene and other substances which are toxic to fungi(Rubin and Benjamin 1984). Many soil borne plantparasites and pathogens are not able to compete assuccessfully for those resources as other microorganismswhich are adapted to surviving in the soil environment.This latter group, which includes many antagonists of plantpests, is more likely to survive solarization, or to rapidlycolonize the soil substrate made available followingtreatment. Bacteria including Bacillus and Pseudomonasspp., fungi such as Trichoderma, and some free-livingnematodes have been shown to be present in highernumbers that kill pathogens following solarization. Theirenhanced presence may provide a short-or long-term shiftin the biological equilibrium in solarized soils whichprevents recolonization by pests, and provides a healthierenvironment for root and overall plant productivity (Katan1987; Gamliel and Stapleton 1993a; Stapleton and DeVay1995).

RestrictionsThe major constraints that limit the adoption of soil

solarization in practice are the relatively long duration ofthe process and the climatic dependency. The cost ofsolarization is relatively low compared with other availablealternatives; however, it can be a limiting factor dependingon the country, the crop type, the production system (e.g.organic versus conventional farming) and the cost andavailability of alternatives. Soil solarization as a non-chemical tool for weed management was proven to be morecost-effective and profitable than MB (Stapleton et al.2005) or some other treatments (Boz 2004), especially inhighly-valued crops (Abdul-Razik et al. 1988; Vizantin-poulos and Katranis 1993). Technological innovations,such as mulching the soil with sprayable polymers or usinga variety of PE sheets or other mulch techniques (Gamlieland Becker 1996; Al-Kayssi and Karaghouli 2002), willfacilitate the application and use of soil solarization inagriculture. These facilitations should result in reducedmulch duration, an increased geographical range of usage,a broader range of controlled weeds, improved persistencyof the PE sheets, decreased PE pollution and a significantdecrease in the total economical cost of mulching.However, in addition to the favorable effects of soilsolarization, there are also unfavorable ones: (i) there aregeographical limitations on where the method can be usedin terms of solar radiation availability; (ii) the soil isoccupied for at least one month with the mulch; (iii)although cheaper than most chemicals used for soilfumigation, not all crops are worth the PE prices ; (iv) it isdifficult to protect the PE sheets from damage caused bywind and animals; (v) there is no fully environmentally-accepted solution for the used PE; and (vi) not all soil-borne pests and weeds are sufficiently controlled.


PerfectionUnder conducive conditions and proper use, solari-

zation can provide excellent control of soilborne pathogensin the field, greenhouse, nursery, and home garden.However, under marginal environmental conditions, withthermo tolerant pest organisms or those distributed deeplyin soil, or to minimize treatment duration, it is oftendesirable to combine solarization with other appropriatepest management techniques in an integrated pestmanagement approach to improve the overall reliability oftreatment (Stapleton 1997). Solarization is compatible withnumerous other methods of physical, chemical, andbiological pest management.

This is not to say that solarization is always improvedby combining with other methods. Many field trials haveshown that, under the prevailing conditions, pesticidalefficacy of solarization or another management strategyalone 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 anappropriate pesticide may provide the benefit of a morepredictable treatment which is sought by commercial users.For example, although combining solarization with apartial dose of 1, 3-dichloropropene did not statistically im-prove control of northern root knot nematode (Meloidogynehapla) over either treatment alone; it did reducerecoverable numbers of the pest to near undetectable levelsto a soil depth of 46 cm (Stapleton and DeVay 1983).

Solarization can also be combined with a wide range oforganic amendments, such as composts, crop residues,green manures, and animal manures to sometimes increasethe pesticidal effect of the combined treatments (Ramirez-Villapudua and Munnecke 1987; Gamliel and Stapleton1993a,b; Chellemi et al. 1997). Incorporation of theseorganic materials by themselves may act to reduce numbersof soilborne pests in soil by altering the composition of theresident microbiota, or of the soil physical environment(biofumigation). Combining these materials with solari-zation can sometimes greatly increase the biocidal activityof the amendments. However, this appears to be aninconsistent phenomenon, and such effects should not begeneralized without conducting confirmatory research. Theconcentrations of many volatile compounds emanatingfrom decomposing organic materials into the soilatmosphere have been shown to be significantly higherwhen solarized (Gamliel and Stapleton 1993b).

The successful addition of biological control agents tosoil before, during, or after the solarization process in orderto obtain increased and persistent pesticidal efficacy haslong been sought after by researchers. There have beengreat hopes of adding specific antagonistic and/or plantgrowth promoting microorganisms to solarized soil, eitherby inundating release or with transplants or otherpropagative material, to establish a long-term disease-suppressive effect to subsequently planted crops (Katan1987; Stapleton and DeVay 1995). Although no consistentadvantage has been shown by this method to date, therehave been a few instances of demonstrated benefit. Forexample, Tjamos and Fravel (1995) showed that the fungusTalaromyces yavus, when added to solarized soil which

was heated only to sublethal levels, was detrimental to thesurvival of Verticillium dahliae microsclerotia. In moststudies, however, it appears that re-colonization ofsolarized soil by the native biota is just as beneficial tosubsequent crops as the addition of specific micro-organisms (Stapleton and DeVay 1995). This area is likelyto remain to be a topic of interest and experimentation formany researchers.

EFFECT OF SOLARIZATION ON MEDICINALAND AROMATIC PLANTS

Stapleton et al. (1985) indicated that fresh and dryweights of radish, pepper and Chinese cabbage plantsusually were greater when grown in solarized soils than innontreated soils. Fertilization of solarized soils sometimesresulted in greater plant growth responses than observed insolarized than non-fertilized soils. Solarization of soilwithin plastic bags for 4 weeks also increased availabilityof 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 pinkroot disease and on onion yields was studied during foursuccessive years. No disease symptoms were noted in thefirst year. However, total and quality yields were increasedby 29% and 57%, respectively, denoting an increasedgrowth response phenomenon. In the following years,disease incidence increased substantially in the untreatedplots, but solarization had a long-term effect in reducingdisease incidence. Soil solarization has a great potential forincreasing onion yield in the Mediterranean region (Satouret al. 1989).

Solarization of soil was found beneficial for plantgrowth in cowpea under field conditions. Root nodulation,infection by mycorrhizal fungi and yield were higher inplants grown in solarized soil. These increases were to theextent of 104.7, 20.0 and 23.7 per cent respectively whencompared to control treatment without solarization (Nair1990). Solarization of soil within plastic bags for fourweeks increased availability of nutrients; solarization alsosignificantly controlled annual weed and increasedstrawberry yield 12% over the yield of nontreated plots(Hartz et al. 1993). Two field experiments resulted in thereduction to undetectable levels of Sclerotium cepivorum inthe upper 20 cm layer of soil, even in heavily infested soils,after solarization for 8-11 weeks. White rot progress curvesin subsequent crops of garlic indicated disease onset ~ 4months after planting. Rates of disease progress and finalincidence of dead plants were greatly reduced in solarizedplots, with yield increments of 40.6-155.5% over theunsolarized control plots. However, a garlic crop in thesecond year after solarization had increased disease levelsand yield reductions that was unacceptable to the growers;this is, apparently, attributable to the high incidence ofwhite rot of garlic that can be induced by low inoculumdensities in the soil. Disease progress curves in theunsolarized plots suggested that secondary infectionsoccur.

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The effect of soil solarization on the quality of garlicwas beneficial because of the increased growth responseobserved. Soil solarization, during the summer before thesusceptible crop is planted, provides a reliable and practicalmethod of control of white rot of garlic (Basallote-Urebaand Melero-Vara 1993). Soil solarization on raisedStrawberry beds was complicated by weed growth on thetop edges and sides of the bed. Soil solarization is a usefulalternative for flat bed culture, but is practically limited onraised beds due to insufficient weed control (Himelrick1995).

Combining organic amendments with soil solarizationis a nonchemical approach to improvement of the controlof soil borne plant diseases. Pathogen control in solarized-amended soil is attributed to a combination of thermalkilling and enhanced generation of biotoxic volatilecompounds. Apparently, pathogen sensitivity to biotoxicvolatile compounds is enhanced with an increase of soiltemperature and acts in combination with antagonisticmicrobial activity. Enhanced biocontrol may also beinvolved in some amendments. Toxic volatile compoundsincluding alcohols, aldehydes, sulfides, isothiocyanates,and others were detected in soil amended with cruciferousresidues during heating so field solarization of soilamended with composted chicken manure gave bettercontrol of pathogens and higher yield of lettuce and tomatothan either treatment alone (Gamliel and Stapleton 1997).Marketable Tomato yields in plots using soil solarizationand similar to yields obtained in plots fumigated withmethyl bromide + chloropicrin (Chellemi et al. 1997).

According to Grünzweig et al. (1999) the effect ofsolarization on plant nutrients and their role in the IGR(increased growth response) was studied with tomato plantsgrown in solarized or non-solarized (control) sandy soil,under controlled conditions. Solarization considerablyincreased the soil concentrations of water extractable N, K,Ca, Mg and Na at most sites, whereas Cl anddiethylenetriaminepentaacetic acid (DTPA) extractableMn, Zn, Fe and Cu were decreased by the treatment. Plantgrowth and specific leaf area were enhanced in solarized aswell as in N-supplemented control soil. In tomato plantsgrown in solarized soil, concentrations of most nutrients inthe xylem sap, including N, were increased compared tothe control, whereas Cl and SO4 levels decreased. The mostsignificant increase in leaf nutrient concentration caused bysoil solarization was recorded for N. Furthermore, leaf Nconcentration was highly and positively correlated withshoot growth. The concentration of Cu increased in leavesfrom the solarization, whereas that of SO4 and Cldecreased, the latter presumably below the critical toxicitylevel. The correlation between shoot growth and leafconcentration was positive for Cu and inverse for Cl andSO4. In conclusion, we found that soil solarizationsignificantly affects nutrient composition in tomato plants,and provided strong evidence that N, and eventually alsoCl, play a major role in IGR.

Soil solarization experiments were completed in threecommercial olive orchards in southern Spain; soil-solarizedplots remained free of weeds, but tress in solarized plotsdid not show significant growth increase measured by trunk

perimeter (Lopez-Escudero and Blanco-Lopez 2001).Raising the cuttings in solarized mixture fortified withTrichoderma harzianum and VAM is reported to producerobust disease-free rooted black pepper cuttings (Sarma2000; Anandaraj et al. 2001). Solarization mediatedfavorable effects were observed in bhindi (Bawazir et al.1995), onion (Adelunji 1994), coriander (Herrera andRamirez 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 manureresulted in a significant increase in growth and yieldcharacters, i.e. plant height, branch number (plant-1),flower-head number (plant-1), fresh and dry weights offlower heads (g plant-1), fresh and dry weights of vegetativeparts (g plant-1) and seed yield (g plant-1) as well asincrease the chemical composition (essential oil, totalflavonoides , total crotenoides, N, P, K, Fe, Zn and Mn)compared with the treatments of cattle manure only (Khalidet al. 2006).

According to Thankaman (2008) solarized pottingmixture in combination with nutrients and biocontrolagents was evaluated for production of vigorous disease-free rooted cuttings of black pepper. Plants raised insolarized potting mixture had better growth than plants rosein nonsolarized potting mixture (soil, sand, and farm yardmanure 2: 1: 1 proportion). Among the various treatments,plants raised in solarized potting mixture withrecommended nutrients (urea, superphosphate, potash andmagnesium sulphate 4: 3: 2: 1) showed significant increasein number of leaves (5.3), length of roots (20 cm), leaf area(177 cm2), nutrient contents and biomass (3.7 g pl-1). Theresults indicated the superiority of solarized pottingmixture for reducing the incidence of diseases besidesyielding vigorous planting material. Cost of production ofrooted cuttings with biocontrol agents was found to becheaper in the case of rooted black pepper cuttings raised insolarized potting mixture. Bio control agents or biofertilizers can be mixed with solarized potting mixture. Thetomato 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 forpredicting treatment duration and efficacy (i.e. when asolarization treatment is done) available to end-users wouldgreatly aid the adoption of solarization, but these generallyhave not been successfully implemented as agriculturalproduction tools because of the passive and complex modeof action of the process over a broad range of targetorganisms. Nevertheless, because of the potential utility ofsuch predictive models, they continue to be a focus ofdevelopment (Katan 1987; Stapleton 1997). Also, thoughsolarization can be an effective soil disinfestant innumerous geographic areas for certain agricultural andhorticultural applications, there are inherent limitations,and situations are presented where it may be desirable to


increase the efficacy and/or predictability of solarizationthrough combination with other methods of soildisinfestations. Since solarization is a passive process withbiocidal activity dependent to a great extent upon localclimate and weather.There are occasions when even duringoptimal periods of the year, cool air temperatures,extensive cloud cover, frequent or persistent precipitationevents, or other factors may not permit effective soiltreatment. In these cases, integration of solarization withother disinfestation methods may be essential in order toincrease treatment efficacy and predictability. As methylbromide is phased out, many current users will turn to otherpesticides for soil disinfestation. Combining thesepesticides (perhaps at lower dosages) with solarization(perhaps for a shorter treatment period) may prove to be themost popular option for users who wish to continue usingchemical soil disinfestants (Stapleton 2000a). In any case,as global environmental quality considerations grow inimportance along with the increasing human population inthe 21st century and beyond, evolving concepts such assolarization and other uses of solar energy in agriculturewill likely to become increasingly important (Stapleton2000b). Known limitations of soil solarization are highimplementation costs for developing countries, and therequirement of special logistics and managerial abilities.Because of these limitations, solarization is used primarilyfor highly-valued crops (Chen et al. 2000).

Acording to Barakat and Al-Masri (2012a), soilsolarization tests against Fusarium oxysporum f. sp.lycopersici, the causal agent of tomato Fusarium wilt, wereconducted for seven weeks through July and August 2008and 2009 in the climatic conditions of Al-AroubAgricultural Experimental Station, located in the southernmountains of the West Bank, Palestine. Doublepolyethylene (DPE) sheets, regular polyethylene (PE)sheets, and virtually impermeable films (VIF) werecompared to examine their effects on soil temperature,disease severity, and plant growth. Results showed that incomparison to the control, PE, DPE, and VIF treatmentsincreased the mean maximum soil temperatures by 10.2,14.1, and 8.8ºC, respectively, in 2008 and by 10.2, 12.6,and 8.3ºC respectively, in 2009. The longest length of timerecorded for temperature above 45ºC under DPE sheetswere 220 hours in 2008 and 218 hours in 2009. Thetreatments reduced the pathogen population by 86% andthe disease by 43% under the DPE treatment in 2009 and toa lesser extent by the other treatments. Increases of up to94% in fresh plant weight and up to 60% in plant dryweight were evident under the same treatment. Thetreatments also increased soil organic matter, both nitrogenforms, and major cations.

Acording to Lombardo et al. (2012), the relativeefficacy of soil solarization and fumigation withchloropicrin and 1, 3 - dichloropropene (CP+1, 3-D) wasevaluated in greenhouse-grown tomatoes. Experimentswere conducted over two seasons in southern Italy, aimedat evaluating the effects of soil treatment on soil-borne pestcontrol, and the vegetative growth and fruit production oftomato. Solarization provided a better level of control overthe major fungal pathogens (Fusarium oxysporum f. sp.

lycopersici and f. sp. radicis lycopersici, as well asPyrenochaeta lycopersici) than CP+1, 3-D fumigation.Solarization was also more effective in reducing thepopulation of Meloidogyne spp. in the soil, and wasparticularly valuable for the suppression of the parasiticplant branched broomrape Phelipanche ramosa (syn.Orobanche ramosa). In both seasons, solarization wasmore beneficial than CP+1, 3-D fumigation in terms ofplant growth and crop productivity. In conclusion,solarization provided a good level of control over someimportant tomato pests and weeds, while at the same timeimproving the productivity in an environmentally friendlymanner. It should therefore represent a viable alternative tomethyl bromide fumigation for the greenhouse productionof tomato.

According to Barakat and Al-Masri (2012b), the use ofintegrated pest management is a valid alternative toconventional chemical treatments. This study was carriedout to evaluate the effects of Brassica carinata seed mealsamendment, combined with solarization, on soil activityand lettuce cultivation. B. carinata seed meals pellets arebiofumigant plant tissues originated as byproducts of thebiodiesel industry. Microbiological data combined withlettuce production results suggested that, afterbiofumigation, soil microbial communities changed towarda new equilibrium that creates better root plant conditionsto improve high lettuce yields. Moreover, Brassica seedmeals, combined with solarization, preserved soilmicroflora against detrimental effects of heating, asrevealed by enzymatic and functional analysis.

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cytokine production. J Mol Med. Doi:10.1007/s001090000086Book: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 treecommunities. In: Carson W, Schnitzer S (eds) Tropical forestcommunity ecology. Wiley-Blackwell, New York.

Abstract:Assaeed AM. 2007. Seed production and dispersal of Rhazya stricta. 50th

annual symposium of the International Association for VegetationScience, Swansea, UK, 23-27 July 2007.

Proceeding:Alikodra HS. 2000. Biodiversity for development of local autonomous

government. In: Setyawan AD, Sutarno (eds) Toward mount Lawunational park; proceeding of national seminary and workshop onbiodiversity conservation to protect and save germplasm in Javaisland. 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,

Qu ak e SR , You L. 2 0 0 8 . A s yn th e t i c Esch er i ch ia co l ip red a to r -p rey ec os ys t em. Mol S ys t B i o l 4 : 1 8 7 .www.molecularsystemsbiology.com

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Review: Soil solarization and its effects on medicinal and aromatic plants 36-44KHALID ALI KHALID

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ANI SULISTYARSI, SURANTO, SUPRIYADI

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