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Insecticidal activity and fungitoxicity of plant extracts and components of horseradish (Armoracia rusticana) and garlic (Allium sativum) a
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Paola Tedeschi , Marilena Leis , Marco Pezzi , Stefano Civolani , Annalisa Maietti & Vincenzo Brandolini
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Department of Pharmaceutical Science, University of Ferrara, Ferrara, Italy
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Department of Biology and Evolution, University of Ferrara, Ferrara, Italy
Available online: 04 Jul 2011
To cite this article: Paola Tedeschi, Marilena Leis, Marco Pezzi, Stefano Civolani, Annalisa Maietti & Vincenzo Brandolini (2011): Insecticidal activity and fungitoxicity of plant extracts and components of horseradish (Armoracia rusticana) and garlic (Allium sativum), Journal of Environmental Science and Health, Part B, 46:6, 486-490 To link to this article: http://dx.doi.org/10.1080/03601234.2011.583868
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Journal of Environmental Science and Health, Part B (2011) 46, 486–490 C Taylor & Francis Group, LLC Copyright ISSN: 0360-1234 (Print); 1532-4109 (Online) DOI: 10.1080/03601234.2011.583868
Insecticidal activity and fungitoxicity of plant extracts and components of horseradish (Armoracia rusticana) and garlic (Allium sativum)
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PAOLA TEDESCHI1, MARILENA LEIS2, MARCO PEZZI2, STEFANO CIVOLANI2, ANNALISA MAIETTI1 and VINCENZO BRANDOLINI1 1
Department of Pharmaceutical Science, University of Ferrara, Ferrara, Italy Department of Biology and Evolution, University of Ferrara, Ferrara, Italy
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To avoid environmental pollution and health problems caused by the use of traditional synthetic pesticides, there is a trend to search for naturally occurring toxicants from plants. Among the compounds discussed for anti-fungal and insecticidal activity, the natural extracts from garlic and horseradish have attracted considerable attention. The objective of this study is to determine the insecticidal and anti-fungal activity of Armoracia rusticana and Allium sativum L. extracts against larvae of Aedes albopictus (Skuse) and some pathogenic fungi. For the insecticidal test, horseradish and garlic extracts were prepared from fresh plants (cultivated in Emilia Romagna region) in a solution of ethanol 80 % and the two different solutions were used at different concentrations (for the determination of the lethal dose) against the fourth instar mosquito’s larvae. The fungicidal test was carried out by the agar plates technique using garlic and horseradish extracts in a 10 % ethanol solution against the following organisms: Sclerotium rolfsii Sacc., Trichoderma longibrachiatum, Botrytis cinerea Pers., Fusarium oxysporum Schlecht. and Fusarium culmorum (Wm. G. Sm.) Sacc. The first results demonstrated that the horseradish ethanol extracts present only a fungistatic activity against Sclerotium rolfsii Sacc., Fusarium oxysporum Schlecht. and F. culmorum (Wm.G. Sm) Sacc. while garlic extracts at the same concentration provided a good fungicidal activity above all against Botrytis cinerea Pers. and S. rolfsii. A. rusticana and A. sativum preparations showed also an interesting and significant insecticidal activity against larvae of A. albopictus, even if horseradish presented a higher efficacy (LC50 value of 2.34 g/L), approximately two times higher than garlic one (LC50 value of 4.48 g/L). Keywords: Garlic, horseradish, mosquito, pathogenic fungi.
Introduction Horseradish (Armoracia rusticana Gaertner, Meyer et Scherb), a perennial herb, is an important Brassica crop in United States and North Europe. Most of the commercial crop is crushed fresh into sauces or used as a food additive for its pungent flavour.[1–3] It has been noticed to release a strong pungent and lachrymatory odour when cut, shredded, or grated or when in contact with water. The odour comes from compounds including isothiocyanates enzymatically degraded from sinigrin and thioglucosides that are naturally present in the plant. Allyl isothiocyanate, a member of the isothiocyanates family, is also present in many other plant species in the Cruciferae family, such as mustard, wasabi, Brassica nigra Koch and B. juncea
Address correspondence to Vincenzo Brandolini, Department of Pharmaceutical Science, University of Ferrara, Ferrara, Via Fossato di Mortara 17/19-44121, Italy; E-mail:
[email protected]
Czem.[4] In fact Brassica vegetables are rich in sulphurcontaining glucosides called glucosinolates[2] which provide the bitter flavour and sulphurous aroma characteristic of Brassica and other Cruciferae as a result of their breakdown into isothiocyanates.[2,5,6] In recent years, research has been focused on a number of aspects of the biological activity of isothiocyanates.[4] Glucosinolates and their breakdown products were reported to suppress soil-borne organisms such as bacteria, fungi, viruses, nematodes and weeds.[2,7,8] Among many plant products, the bulbs or cloves of common garlic (Allium sativum L., Liliaceae) are widely used as food as well as dressing for foods.[9] Garlic has been shown to inhibit the growth of a variety of microorganisms, not only bacteria but also fungi and viruses. The anti-microbial activity of garlic is believed to be due to the effect of allicin, the main ingredient of garlic, generated by phosphopyridoxal enzyme allinase.[10] The past three decades have seen a dramatic global geographic spreading of Aedes albopictus (Skuse) that
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Insecticidal effects of horseradish and garlic continues today.[11] In Italy A. albopictus is currently recognized as the most dangerous mosquito and currently applied conventional control methods gave unsatisfactory results.[12] This is particularly relevant if control is intended to interrupt pathogen transmission in cases of possible arbovirus epidemics, such as the Chikungunya outbreak that occurred in Ravenna, Italy in 2007.[13] To avoid environmental pollution and health problems caused by the use of traditional synthetic pesticides, there is a trend to search for naturally occurring toxicants from plants. Furthermore, a lot of phytochemicals are safer to the environment or humans than traditional chemical insecticides and fungicides. In our study we focused the attention on larvicidal and fungicidal or fungistatic activity of some natural plant extracts against larvae of A. albopictus and against some pathogenic fungi (which caused severe and constant diseases in agriculture) such as Sclerotium rolfsii Sacc., Trichoderma longibrachiatum, Botrytis cinerea Pers., Fusarium oxysporum Schlecht. and Fusarium culmorum (Wm. G. Sm.) Sacc., using garlic and horseradish extracts.
Materials and methods Insecticidal test Garlic and horseradish extracts were prepared from fresh plants (cultivated in Emilia Romagna region) in a solution of ethanol 80 %. The solutions were prepared from fresh garlic and horseradish (moisture was between 62 and 65 % for garlic and 64 and 66 % for horseradish). The ethanol solutions were obtained by the following procedure: about 25 g of thinly ground garlic and horseradish were placed in 100 mL of ethanol at 80 % for 12 hours and then filtered through Whatman cellulose membrane filter. For the bioassay tests, tiger mosquito larvae of Rimini F36 strain (Centro Agricoltura Ambiente “Giorgio Nicoli” Crevalcore, Bologna – Italy) were used. The eggs, laid on strips of tissue paper and placed in a glass containing approximately 700 mL of drinking water decanted with 0.25 g of bacterial broth (Nutrient broth, Oxoid LTD, Basingstoke and Hampshire, England) were maintained in an incubator (ISCO, Milan, Italy) at 27 ◦ C under a 16:8 h Light:Night photoperiod till the hatching. The first instar larvae were placed in a plastic tray containing 3 litres of water, adding daily a pinch of fish food (Tetra, Germany). Five days after the eggs hatching, the fourth instar larvae were used for bioassays. The bioassay was realized in 500 mL plastic cups, added with the two extracts at different concentrations for the determination of lethal dose. Each glass filled with 100 mL of the decanted water was added garlic or horseradish extract at different concentra-
tions and 25 fourth instar larvae. An ethanol solution at 80 % (1 mL) alone was used for the control test. Larvae were considered dead if they were unable to move when disturbed. Statistical analysis All data were analyzed by the POLO-PC program,[14] which yields the biological response curve in semi-log graph reporting the relationship between applied dose (g/L) and mortality. The program allows to insert mortality “raw” data and applies the Abbott’s formula (Equation 1).[15] CM (%) n.i. dead after treatment − n.i. dead in the Co ∗ 100 = 100 − n.i. dead in the Co (1) where CM = corrected mortality, n.i. = number of insects, Co = control To obtain this mortality curve, data on fourth instar larvae of A. albopictus were transformed into logarithmic values (x variable) and mortality expressed in probits (y variable). Based on the output of the program we obtained the LC50 , for the extracts examined and the related confidence intervals (95 % CI). The software can also provide a theoretical model of biological response, which may not coincide with the real answer. To evaluate this situation, the program performs the Goodness-of-Fit test that uses the calculation of X 2 (with significance level P < 0.05, Finney et al.[16]). Antifungal test Microrganisms. The pathogenic fungi used in the tests were: Sclerotium rolfsii, Trichoderma longibrachiatum, Botrytis cinerea, Fusarium oxysporum and F. culmorum. The cultures were obtained from the Central Bureau voor Schimmelcultures, Baarn, The Netherlands. All the organisms were maintained on potato dextrose agar (PDA, Difco Laboratories, Detroit, USA). In vitro tests. The in vitro tests were carried out using garlic and horseradish extracts in a 10 % ethanol solution. The solutions were prepared using fresh garlic and horseradish (moisture was between 62 and 65 % for garlic and 64 and 66 % for horseradish). The ethanol solutions of garlic or horseradish were obtained by the following procedure: 20 g and 50 g of thinly ground garlic and horseradish were placed in 100 mL of 10 % ethanol solution for 12 hours and then filtered through Whatman cellulose membrane filter. The extracts obtained were added to autoclaved PDA, 1mL of extract in 10mL of PDA and then poured into Petri dishes (8.5 cm in diameter). An ethanol solution at 10 % alone was added to PDA for the control plates.
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Table 1. Lethal concentration for Aedes albopictus exposed to Allium sativum and Armoracia rusticana extracts. Extract Allium sativum Armoracia rusticana
N. larvae
Slope ± SE
Intercept ± SE
675 625
8.67 ± 0.67 3.02 ± 0.23
−5.65 ± 0.46 −1.11 ± 0.11
LC50 (95%CI) 4.48 (4.29–4.65) 2.34 (2.03–2.68)
χ 2(n) 21.8(21) 29.5 (19)
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SE = standard error LC50 = concentration necessary to kill 50% of insects; expressed in g/L CI 95% = 95% confidence interval n = degrees of freedom
Fungi tests. The pathogenic fungi were inoculated immediately after the preparation of the Petri dishes by placing a disk of mycelium (0.6 cm in diameter, cut with a sterile cork borer) from the rim of 7 day old cultures on PDA plates, in the centre of the Petri dishes. Petri dishes were scaled with Parafilm and kept at a temperature of 22 ± 1◦ C. The diametral development of cultures on the solid medium was calculated at 3, 6, 9 and 12 days measuring the growth of the fungal colonies along the two preset diametral lines. During the experiments the growth conditions and the medium sterility were checked for each strain. The incubation conditions were the same as those in potato dextrose test. Fungitoxicity was expressed in terms of percentage of mycelian growth inhibition and calculated according to the formula of Zygadlo et al. (Equation 2).[17] I = 100 (C − T)C−1 ,
(2)
where I = inhibition; C = average diameter of fungal grown in PDA + ethanol solution at 10 % and T = average diameter of fungi cultivated in PDA + garlic or horseradish extract.
All the experiments were performed in triplicate and the standard deviation was calculated. Statistical analysis The analysis of variance (ANOVA) was carried out on all data by StatMost program.
Results and discussion Insecticidal activity Larvae of A. albopictus showed significant mortality following exposure to the two different compounds. The two plant extracts tested varied in toxicity to larvae of A. albopictus (Table 1). Of these, the extracts produced more than 80 % of mortality at 4.50 and 5.55 g/L respectively for horseradish and garlic. Good insecticidal activity against larvae was achieved with A. rusticana compound with an LC50 value (Fig. 1, part B) of 2.34 g/L (ranged from 2.03 to 2.68).
Fig. 1. Biological response curve on the larvae of A. albopictus, relative to treatment with the extract of Allium sativum (part A) and Armoracia rusticana (part B). (Chart in semi-logarithmic scale). The % of mortality was expressed in g/L (Abbott’s formula).
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Insecticidal effects of horseradish and garlic
Fig. 2. Activity as inhibition percentage of garlic and horseradish extracts against S. rolfsii, F. oxysporum, T. longibrachiatum, F. culmorum, R. solani and B. cinerea.
The results indicated that garlic extract (Fig. 1, part A) which presented an LC50 of 4.48 (ranged between 4.29 and 4.65 g/L) was less toxic than horseradish. The comparison of LC50 of the two plant compounds shows that the extract of A. rusticana has an insecticidal efficacy two times higher than the A. sativum one (2.34 g/L against 4.48 g/L; Table 1). Fungicidal activity The garlic ethanol extracts demonstrated a good antifungal activity against the pathogenic fungi tested even if with same difference between the different organisms (Fig. 2). A good efficacy was shown at 20 g/100 mL of extract which totally inhibited the growth of S. rolfsii, R. solani and B. cinerea. The same garlic extract was less active against F. oxysporum and T. longibrachiatum, which were not completely inhibited even at the higher extract concentration (50 g/100 mL). The mycelian growth of the fungi tested was only partially inhibited with horseradish ethanol extracts. The results of the screening given in Figure 2 showed a very little effect of A. rusticana against S. rolfsii, F. culmorum and F. oxysporum at both concentrations used (20 g/100 mL and 50 g/100 mL); while T. longibrachiatum, R. solani and B. cinerea appeared not inhibited by horseradish solutions.
Conclusion A. rusticana and A. sativum preparations showed interesting and significant insecticidal activity against larvae of A. albopictus, even if horseradish presented a higher efficacy, approximately two times higher than the garlic one. The first results of antifungal tests demonstrated that horseradish ethanol extracts present only a fungistatic ac-
tivity against S. rolfsi, F. oxysporum and F. culmorum while garlic extracts at the same concentration provided a good fungicidal activity against all the phytopathogenic fungi tested and above all against B. cinerea and S. rolfsii. Although higher concentrations of these natural anti-fungal and insecticidal products should be used, their intrinsically low toxicity towards humans makes them an interesting alternative to current chemicals. This study has demonstrated that garlic and horseradish extracts could be promising alternatives to synthetic pesticides and work is still in progress to detect the active compounds with potential larvicidal and fungicidal effects and quantify each substance.
References [1] Weil, M.J.; Zhang, Y.; Nair, M.G. Tumor cell proliferation and cyclooxygenase inhibitory constituents in horseradish (Armoracia rusticana) and Wasabi (Wasabia japonica). J. Agric. Food Chem. 2005, 53, 1440–1444. [2] Li, X.; Kushad, M. Correlation of glucosinolate content to myrosinase activity in horseradish (Armoracia rusticana). J. Agric. Food Chem. 2004, 52, 6950–6955. [3] Kushad, M.M.; Brown, A.F.; Kurilich, A.C.; Julik, J.A.; Klein, P.; Walling, M.A.; Jeffery, E.H. Variation of glucosinolates in vegetable crops of Brassica oleracea. J. Agric. Food Chem. 1999, 47, 1541–1548. [4] Wu, H.; Zhang, G.A.; Zeng, S.; Lin, K.C. Extraction of allyl isothiocyanate from horseradish (Armoracia rusticana) and its fumigant insecticidal activity on four stored-product pests of paddy. Pest Manag Sci. 2009. 65, 1003–1008. [5] Bennett, R.N.; Mellon, F.A.; Botting, N.P.; Eagles, L.; Rosa, E.A.; Williamson, G. Identification of the major glucosinate (4mercaptobutyl glucosinates) in leaves of Eruca sativa L. (salad rocket). Phytochemistry. 2002, 61, 25–30. [6] Engel, E.; Baty, D.; Le Corre, I.; Souchon, N.; Martin, N. Flavor-active compounds potentially implicated in cooked cauliflower acceptance. J. Agric. Food Chem. 2002, 50, 6459– 6467.
Downloaded by [Universita Di Ferrara], [Vincenzo Brandolini] at 01:44 06 July 2011
490 [7] Mayton, H.S.; Olivier, C.; Vaughn, S.F.; Loria, R. Correlation of fungicidal activity of Brassica species with allyl isothiocyanate production in macerated leaf tissue. Phytophatol. 1996, 86, 267–271. [8] Spak, J.; Kolarvoa, J.; Fenwick, G.R. The effect of glicosinolates (mustard oil glucosides) and products of their enzymatic degradation on the infectivity of turnip mosaic virus. Biol. Plant. 1993, 35, 73–80. [9] Brandolini, V.; Tedeschi, P.; Cereti, E.; Maietti, A.; Barile, D.; Coisson, J.D.; Mazzotta, D.; Arlorio, M.; Martelli, A. Chemical and genomic approach applied to the characterization and identification of Italian Allium sativum L. J. Agric. Food Chem. 2005, 53, 678–683. [10] Tedeschi, P.; Maietti, A.; Boggian, M.; Vecchiati, G.; Brandolini V. Fungitoxicity of lyophilized and spray-dried garlic extract. J. of Environmental Science and Health Part. B. 2007, 42, 795–799. [11] Lambrechts, L.; Scott, T.W.; Gubler, D.J. Consequences of the expanding global distribution of Aedes albopticus for Dengue virus transmission. Plos neglected tropical diseases, 2010, 4, 646–655.
Tedeschi et al. [12] Balestrino, F.; Medici, A.; Candini, G.; Carrieri, M.; Maccagnani, B.; Calvitti, M.; Maini, S.; Bellini, R. Gamma ray dosimetry and mating capacity studies in the laboratory on Aedes albopictus males. J. Med. Entomol. 2010, 47, 581–591. [13] Rezza, G.; Nicoletti, L.; Angelici, R.; Romi, R.; Finarelli, A.C.; Panning, M.; Cordioli, P.; Fortuna, C.; Boros, S.; Magurano, F. Infection with Chikungunya virus in Italy: an outbreak in a temperate region. Lancet. 2007, 370, 1840–1846. [14] LeOraSoftware. POLO-PC: a use guide to probit and logit Analysis. User’s manual, LeOra Software: Berkeley, California, 2002, 28 pp. [15] Abbott, W.S. A method of computing the effectiveness of an insecticide. J. Econ. Entomol. 1925, 18, 265–267. [16] Finney, D.J. Probit Analysis, 3rd Edition, Cambridge University Press: Cambridge, U.K. ISBN 0521080041, 1971. [17] Zygadlo, J.A.; Guzman, C.A.; Grosso, N.R. Antifungal properties of the leaf oils of Tagetes minuta L:, T. filifolia Lag. J. Essent. Oil Res. 1994, 6, 617–621.
