ࡱ> 7 [bjbjUU V7|7|M lTTT ^ tR3334T4R266666;8.i8}8 giiiiii$š *878;888kIjj66;JkIkIkI8 jR66gkI8gkIkI1N "ۋ66 XzR.3mF:ۋ `0 mF ۋkIRRjjjjBioactivity of 1,8  cineole, camphor and carvacrol against rusty grain beetle (Cryptolestes ferrugineus Steph.) on stored wheat Vlatka Rozmana(, Irma Kalinovia, Anita Liakaa aFaculty of Agriculture in Osijek, Trg Sv. Trojstva 3, 31000 Osijek, Croatia Abstract The rusty grain beetle is the most prevalent pest in silos and mills in Croatia. These investigations aimed to determine bioactivity of 1,8-cineole, camphor, and carvacrol against rusty grain beetle under laboratory and storage conditions. Seven-day laboratory tests of fumigant activity in 5 dose rates (0.1; 1; 10; 50; 100 (l/720 ml vol.) obtained 100% mortality with 1,8-cineole and camphor even at the lowest dose, while carvacrol obtained 100% mortality at the dose of 10 (l/720 ml vol. Under storage conditions during 24-day exposure and dose of 200 (l/ 5 kg of wheat 100% mortality was obtained in16 days with 1,8-cineole, and with camphor in 24 days, while carvacrol obtained 65% mortality at the end of the testing. Tested isolates did not affect rheological properties of the flour. However, in treatment with carvacrol flour retained certain scent residues. Such investigations enable new alternatives to be found as substitutes for fumigants applied so far. Key words: Cryptolestes ferrugineus, 1,8-cineole, camphor, carvacrol, fumigant activity, stored wheat Introduction Cryptolestes ferrugineus (Stephens) is a secondary species of storage pests whose population in silos and mills in Croatia has been rising rapidly in the past five years and in comparison to the other main storage pests it takes first place (Kalinovi and Rozman, 2002). C. ferrugineus does not belong to the group of economically most harmful pests, such as pests of Sitophilus genus. However, their growing population and presence of all developmental stages of the species affect directly qualitative properties of the stored goods. Present measures of the pest control are mainly based on application of synthetic insecticides and fumigants. However, their non-selective and uncritical application in storage facilities brings up some serious issues, such as toxic effects to the wheat that is used in diet for people and livestock (Fishwick, 1988), and contamination of the environment (WMO, 1995). In practice in Croatia phosphine is presently in use in storage pests control. However, fumigation with phosphine should be also limited for evident increase in the resistance of some pest species to a phosphine compound, which has been observed in more than 45 countries (Bell and Wilson, 1995). The cause of the resistance is often non-professional application (short exposure, insufficient hermetization at raised temperature, lowered gas concentration) that support thesis that this above all efficient fumigant should be replaced in the near future. This means that phosphine in use can and has to be limited on the global level. Therefore, there is a necessity to find safe alternatives to the conventional insecticides and fumigants that would protect grain and its products. One alternative are monoterpenes isolated from essential oils of aromatic plants from the Mediterranean area of Croatia. The objective of these investigations is to detect bioactivity of 1,8-cineol, camphor and carvacrol isolated from essential oils of local aromatic plants of Lamiaceae and Lauraceae genus, as follows, Lavandula angustifolia Ch., Rosmarinus officinalis L., Thymus vulgaris L. and Laurus nobilis L., to control C. ferrugineus in stored wheat. 2. Materials and methods 2.1. Essential oils, compounds, gas chromatography The essential oils of the aromatic plants Lavandula angustifolia Ch., Rosmarinus officinalis L., Thymus vulgaris L. and Laurus nobilis L. were purchased from"Ireks aroma", Zagreb, Croatia. To determine the three main compounds present, we applied a method described by Andronikashvili and Reichmuth (2002) and Regnault-Roger et al. (2004). Oils were analyzed by gas chromatography, using a  Perkin-Elmer (USA) GC  type 8700, fitted with a  Supelco (USA) SP-2380 capillary column 30m, i.d. 0.32mm, 0.20 m film thickness. 2.2. Chemicals (Compounds) The three main compounds: 1,8-cineole - (eucalyptol, C10H18O, EC No: 207-431-5); camphor - ((1R)-(+) camphor 99%, C10H16O, EEC No: 207-355-2) and carvacrol (98% 5-isopropyl-2-methylphenol, C10H14O, EC No:207-889-6) discovered in oils of the aromatic plants analyzed were purchased from Sigma-Aldrich and used for tests in bioassay. Insects Culture of the rusty grain beetle C. ferrugineus was collected from the granaries in the eastern part of Croatia. They were reared in the laboratory on mixture of whole wheat flour and maize flour at the rate of 1:1 in glass containers containing 0.5 kg of the mixture with 12% m.c. The species was reared at 29 1oC and 70 5% r.h. in darkness to obtain the first generation (F1) for use in bioassays. Bioactivity of 1,8-cineole, camphor and carvacrol Bioactivity of 1,8 cineole, camphor and carvacrol was assessed by evaluation of fumigant activity against C. ferrugineus under laboratory and storage conditions. 2.3.1. Fumigant activity 7 days laboratory test To determine fumigant activity of the compounds tested a modified procedure described by Prates et al., 1998 was used. In silk mesh cages that were put into separate glass jars of 720 ml capacity containing 250 g of wheat of 12% m.c. four replicates of ten adults of C. ferrugineus species were placed. 1,8-cineole, camphor and carvacrol were tested at 5 dose rates (0.1; 1; 10; 50 and 100 l/720 ml volume) and over exposure periods of 1, 2, 3, 4, 5, 6 and 7 days under controlled conditions (temp. 291 oC, humidity 705%, in darkness). The compounds were applied with a  Hirschman digital micropipette on filter paper attached to the lids of the tightly sealed glass containers. To determine fumigant activity all the samples were observed daily over the exposure of 1-7 days or until 100% mortality of test insects, and compared to the untreated control samples. 2.3.2. Fumigant activity 24 days test in storage conditions Analyses under storage conditions were done at IPK Croatia d.o.o. Silos and Mill, Osijek. Tested market wheat was dried to 12-13% r.h. in the storage and cooled at the temperature of 10oC. All the samples each of them weighing 5 kg were packed into two-layered paper bags and infested with 10 C. ferrugineus adults that were previously placed into silk mesh cages. 1,8-cineol, camphor and carvacrol were applied at the dose of 200 ml to impregnated porous strips 10 cm in length and incorporated into the prepared wheat samples. Control was infested wheat samples with no treatment. Bags with samples were then sealed with adhesive tape and placed on wooden pallets under storage conditions (temp. 5 10C; r. h. 65-75%). Control and observation of the samples to 100% mortality were done every fourth day by lens on lighted, heated background during 24 hours storage. Bioassay was set in 4 replications. Qualitative analyses of tested wheat flour 2.4.1. Rheological analyses Rheological analyses of wheat flour were done at technological laboratory of IPK Croatiad.o.o. Silos and Mill, Osijek. Treated samples of wheat flour and control samples weighing 2 kg were analyzed by farinograph and extenzograph according to the standard methods. Analyses were done on 60% laboratory flour (after cleaning, wheat samples were prepared for grinding by soaking in two phases: I soaking at 13,5% m.c. and left aging for 24 h; II phase soaking at 14% m.c. h before grinding). Flour samples were then left 5 days for aging and tested for rheological properties. Dough properties from flour obtained in the grinding process (water absorption, dough progress, dough stability, resistention, soften level, quality number, and quality group) were determined by farinograph. Dough elasticity properties (energy, resistance, elasticity, and their relation) were determined by extenzograph. The samples were tested in 4 replications. The results obtained were compared to the control with no treatment. 2.4.2. Scent analyses To determine scent in the samples tested we used standard organoleptic method described by technologists at Ireks aroma d.o.o., Zagreb. The method implies scent evaporation by stirring wheat flour and hot water at the rate of 1: 1 with inhalation of the vapours of each sample during the first 30 seconds, and comparison to the scent vapours of the control with no treatment. Analyses were done at the Department for plant protection, Faculty of Agriculture in Osijek. 2.5. Statistical analysis Mortality data were subjected to a multi-factorial analysis of variance (ANOVA) for each day of the exposure period according to the GLM (general linear model). Significant differences were shown by LSD test (least significant difference) and entered in the tables. Data processing was conducted by the SAS System for Windows 98 and SPSS 11.0 for Windows. Results 3.1. Chemical compounds of the essential oils Chemical compounds of the essential oils of each aromatic plant were determined by gas chromatography. The nine compounds identified in the essential oil of L. angustifolia, R. officinalis, T. vulgaris and L. nobilis were entered in Table 1. In this paper 1,8-cineol, camphor and carvacrol were chosen to be presented in the bioactivity analysis. 3.2. Bioactivity of 1,8-cineole, camphor and carvacrol 3.2.1. Fumigant activity - 7 days laboratory test Fumigant activity (Table 2) of 1,8  cineole and camphor was observed to 100% mortality of C. ferrugineus after the 24 h exposure, at the dose levels of 10; 50 and 100 l/ 720 ml volume and 50 and 100 l/ 720 ml with carvacrol. However, 7 days laboratory test of fumigant activity in 5 dose rates (0.1; 1; 10; 50; 100 (l/720 ml vol.) obtained 100% mortality with 1,8-cineole and camphor even at the lowest dose (after 5 and 6 days, as following), while carvacrol obtained 100% mortality at the dose of 10 (l/720 ml vol. at the same time. 3.2.2. Fumigant activity 24 days test under storage conditions Results of fumigant activity of 1,8 cineole, camphor and carvacrol against C. ferrugineus and under storage conditions proved their bioactivity requiring significantly longer exposure period if comparing to the laboratory test. Significant differences were observed in the first four days in treatments with 1,8 cineole and camphor (57.5% mortality) in comparison to carvacrol and to the control (17.5% and 0% mortality, as follows) (Table 3). Under storage conditions, in general, applications of 1,8 cineole and camphor were similarly effective (Figure 1), without significant differences between them, while application of carvacrol showed as twice as weaker effect then the remaining two compounds. Under storage conditions during 24-day exposure and dose of 200 (l/ 5 kg of wheat 100% mortality was obtained in16 days with 1,8-cineole, and with camphor in 24 days, while carvacrol obtained 65% mortality at the end of the testing. In comparison to the control with no treatment all the three compounds showed significant differences in the mortality of C. ferrugineus. 3.3. Results of qualitative analyses of tested wheat flour 3.3.1. Rheological analyses Laboratory analyses of the wheat quality tested showed that tested isolates did not affect rheological properties of the flour. Results of the ekstenzograph analyses (Table 4) show that the treatments with 1,8 cineole, camphor and carvacrol did not exhibit negative effect on the dough quality during the elasticity process, and did not have negative effect on dough properties tested by farinograph. Such results were expected as wheat quality is a complex feature that is genetically inherited, and could be simultaneously exposed to the external factors, in our case, application of essential oil compounds. 3.3.2. Scent analyses Results of scent analyses show that in treatment with carvacrol flour retained certain scent residues (Table 5), while in the treatments with 1,8 cineole and camphor no scent residues remained in tested wheat flour. 4. Discussion C. ferrugineus as presently most prevalent pest species in Croatian silos and storages showed very high susceptibility to fumigant activity of 1,8 cineole, camphor and carvacrol isolated from essential oils of local lavender, laurel, rosemary and thyme. The species showed positive reaction to the treatment under storage conditions, particularly to the application of 1,8 cineole and camphor. Very few foreign authors investigated about the influence of terpenoids to the rusty grain beetle. Some authors emphasised that the species was susceptible to thymol (Suya et al., 1998).In our studies, as a whole, 9 compounds were isolated from essential oils of lavender, laurel, rosemary and thyme, and examined. However, for extensive data obtained, we were forced to present three most effective compounds. Our results placed thymol on the fifth place regarding its efficiency, but it does not mean that the compound is not effective against the pest species. By comparing the fumigant activities against C. ferrugineus of 3 essential oil compounds, 1,8-cineole was arguably the most effective, followed by camphor and carvacrol. Also, results of the analyses by using a gas chromatograph (Table 1) proved 1,8-cineole was present in all essential oils (lavender, rosemary, thyme and laurel) which strongly suggested that it was one of the principal active substances in these oils. On the basis of the positive results of the analyses, new preparation based on the particular compounds would be patented as a carrier of the toxic effect observed. In general, aromatic plants contain essential oils only in concentrations of 1 3% (akir, 1992). Large quantities of plant material would need to be processed to gain enough essential oil for commercial-scale tests. However, certain compounds in the oils exhibit much stronger activity than others. Plant varieties should be sought that produce these compounds in larger quantities, or synthetic production methods should be explored as an option to gain enough material for full-scale use. The preparation would be applicable in the practice of stored pests control, as well as in the ecologically acceptable plant protection. Acknowledgments This study was funded by Ministry of Science of Republic of Croatia. We are grateful to the employees of IPK Croatia d.o.o. Silos and Mill, Osijek for providing storage facilities, and to the laboratory stuff for quality analyses of tested wheat. We are grateful to chemical industry Saponia Osijek for GC analyses and also thank Ireks Aroma for their assistance in providing the necessary Dalmatian essential oil extracts. References Andronikashvili, M., Reichmuth, Ch., 2002. Repellency and toxicity of essential oils from Ocimum gratissimum (Lamiaceae) and Laurus nobilis (Lauraceae) from Georgia against the rust-red flour beetle Tribolium castaneum (Herbst.) (Coleoptera: Tenebrionidae). In: Credland, P.F., Armitage, D.M., Bell, C.H., Cogan, P.M., Highley, E. (Eds), Proceedings of the 8th International Working Conference of Stored Product Protection, York, UK, CAB International, pp. 749-762. Bell, C. H., Wilson, S. M., 1995. Phosphine tolerance and resistance in Trogoderma granarium Everts. (Coleoptera: Dermestidae). Journal of Stored Products Research, 31, 199-205. akir, C., 1992. Investigations on the fungitoxic potentials of some plants occuring in Antalya. M.Sc. Thesis, Akdeniz University, Antalya (in Turkish). Fishwick, F. B., 1988. Pesticide residues in grain arising from postharvest treatments. Aspects of Applied Biology, 17, 37-46. Kalinovi, I., Rozman, V., 2002. Entomofauna on stored wheat in republic of Croatia. In: Ioannidis , P., Savopoulou Soultani, M., Gazis, E. (Eds), Congress abstracts of the VIIth European Congress of Entomology, Thessaloniki, Greece, Helenic Entomological Society, pp. 30. Prates, H.T., Santos, J.P., Waquil, J.M., Fabris, J.D., Oliveira, A.B., Foster, J.E., 1998. Insecticidal activity of monoterpenes against Rhyzopertha dominica (F.) and Tribolium castaneum (Herbst). Journal of Stored Products Research 34, 243-249. Regnault-Roger, C., Ribodeau, M., Hamraoui, A., Bareau, I., Blanchard, P., Gil-Munoz, M.I., Barberan, F.T., 2004. Polyphenolic compounds of Mediterranean Lamiaceae and investigation of orientational effects on Acanthoscelides obtectus (Say). Journal of Stored Products Research 40, 395-408. Suya, W., Yongjia, J., Chunming, L., Yingjie, Z. 1998: Studies of population reproductive inhibiting effect on volatile oil from Elsholtiza sp. against stored-product insects. In: Zuxun, J., Quan, L., Yongsheng, L., Xianchang, T., Lianghua, G. (Eds), Proceedings of the 7th International Working Conference on Stored product Protection, Beijing, China, Sichuan Publishing House of Science & Technology, vol. 1, pp. 858-865. WMO, 1995. Scientific assessment of ozone depletion: World Meteorological Organization global ozone research and monitoring project. Report No. 37, WMO, Geneva, Switzerland. CompoundsLavandula angustifolia Chaix. lavenderRosmarinus officinalis Linn. rosemaryThymus vulgaris Linn. thymeLaurus nobilis Linn. laurel1,8-cineole camphor eugenol linalool carvacrol thymol borneol bornyl-acetate linalyl-acetate+ + + + + + ++ + + ++ + + + + + ++ + + Table 1. Main compounds identified in the essential oils Table 2. Results of fumigant activity in 7 days laboratory test on C. ferrugineus Exposure time  dose l/720ml)% mortality C.ferrugineus* Compounds1.8 cineolecamphorcarvacrol1 day0.175.0b22.5c0.0d195.0ab62.5b45.0c10100.0a100.0a82.5b50100.0a100.0a100.0a100100.0a100.0a100.0a2 day0.180.0b42.5c15.0d197.0a70.0b47.5c10--85.0b50---100---3 day0.185.0b70.0b55.0c1100.0a80.0b60.0c10--90.0b50---100---4 day0.192.5ab85.0b72.5b1-92.5ab75.0b10--97.5a50---100---5 day0.1100.0a97.5a80.0b1-100.0a85.0b10--100.0a50---100---6 day0.1-100.0a90.0b1--90.0b10---50---100--- 7 day0.1--95.0ab1--97.5a10---50---100---*means in the same column followed by the same letters are not significantly (P>0.05) different as determined by the LSD-test. Table 3. Results of fumigant activity in storage condition test on C. ferrugineus Exposure time  % mortality C.ferrugineus* Compounds1.8 cineolecamphorcarvacrolcontroldose 200 (l/ 5 kg of wheat EMBED Equation.3 4 day57.57.0c57.57.0c17.52.4e00.0f8 day656.0c62.52.9c252.7e00.0f12 day903.4b87.54.2b454.3d00.0f16 day1000.0a97.51.2a52.54.6d2.50.9f20 day1000.0a97.51.2a656.1c2.50.9f24 day1000.0a1000.0a656.1c2.50.9f*means in the same column and row followed by the same letters are not significantly (P>0.05) different as determined by the LSD-test. Figure 1. Fumigant activity of 1,8 cineole, camphor and carvacrol in storage conditions against C. ferrugineus  Table 4. Results of farinograph and ekstenzograph on tested stored wheat TreatmentsFARINOGRAMWater absorb%Dough progress min.Dough stability min.Resistention min.Soften level FJQuality No.Quality group1,8 - cineole62.71.70.21.99848.1B2camphor62.71.70.328353.3B2carvacrol631.80.32.18650.9B2control62.71.70.31.98051.6B2TreatmentsEKSTENZOGRAMenergy cm2resistence EJelasticity mmrelation EJ/mm1,8 - cineole55.13071222.52camphor61.43241282.53carvacrol66.43201302.46control61.53251352.44 Table 5. Results of scent analyses in tested wheat flour TreatmentsScent1,8 - cineole-camphor-carvacrol+control- ( Corresponding author. 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