Toxicity of Naturally Occurring Compounds of Lamiaceae and Lauraceae to Three Stored-Product Insects

ARTICLE IN PRESS

Journal of Stored Products Research 43 (2007) 349–355 www.elsevier.com/locate/jspr

Toxicity of naturally occurring compounds of Lamiaceae and Lauraceae to three stored-product insects

V. Rozmana,Ã, I. Kalinovica, Z. Korunicb

aFaculty of Agriculture in Osijek, University of J. J. Strossmayer in Osijek, Trg Sv. Trojstva 3, 31000 Osijek, Croatia bDiatom Research and Consulting Inc., 14 Greenwich Dr., Guelph, Ont., Canada N1H 8B8

Accepted 18 September 2006

Abstract

The compounds 1,8-cineole, camphor, eugenol, linalool, carvacrol, thymol, borneol, bornyl acetate and linalyl acetate occur naturally in the essential oils of the aromatic plants Lavandula angustifolia, Rosmarinus ofﬁcinalis, Thymus vulgaris and Laurus nobilis. These compounds were evaluated for fumigant activity against adults of Sitophilus oryzae, Rhyzopertha dominica and Tribolium castaneum. The insecticidal activities varied with insect species, compound and the exposure time. The most sensitive species was S. oryzae, followed by Rhyzopertha dominica. Tribolium castaneum was highly tolerant of the tested compounds. 1,8-Cineole, borneol and thymol were highly effective against S. oryzae when applied for 24 h at the lowest dose (0.1 ml/720 ml volume). For Rhyzopertha dominica camphor and linalool were highly effective and produced 100% mortality in the same conditions. Against Tribolium castaneum no oil compounds achieved more than 20% mortality after exposure for 24 h, even with the highest dose (100 ml/720 ml volume). However, after 7 days exposure 1,8-cineole produced 92.5% mortality, followed by camphor (77.5%) and linalool (70.0%). These compounds may be suitable as fumigants because of their high volatility, effectiveness, and their safety. r 2006 Elsevier Ltd. All rights reserved.

Keywords: 1; 8-cineole; Camphor; Eugenol; Linalool; Carvacrol; Thymol; Borneol; Bornyl acetate; Linalyl acetate; Fumigant toxicity; Sitophilus oryzae; Rhyzopertha dominica; Tribolium castaneum; Wheat

1. Introduction organisms. Botanicals with low human toxicity currently available include rotenone and pyrethrum. Others now During the past few decades, application of synthetic available include neem, used as insect repellent, feeding pesticides to control agricultural pests has been a standard deterrent and growth regulator; eugenol, the component of practice. However, with growing evidence that many clover oil that is applied to cockroaches, ants, dust mites, conventional pesticides can adversely affect the environ- flies, wasp, spiders, crickets and fleas; and D-limonene ment, requirements for safer means of pest management isolated from citrus oils and citrus fruit peel and used as a have become crucial. Therefore, the use of safe, low- contact insecticide against ants, roaches, fleas, silverfish toxicity botanical pesticides is now emerging as one of the and palmetto bugs. prime means to protect crops, their products and the The insecticidal activity of a large number of essential environment from pesticide pollution, a global problem oils and other plant extracts has been assessed against (Prakash and Rao, 1997). Generally, they should cause less several major agricultural pests (Regnault-Roger et al., damage to human and environmental health than conven- 1993; Regnault-Roger and Hamraoui, 1993; Golob et al., tional insecticides. Many of them degrade rapidly and do 1999; Weaver and Subramanyam, 2000; Keíta et al., 2001; not accumulate in the body and environment while some Lee et al., 2001; Andronikashvili, and Reichmuth, 2002; are very pest specific and do little or no damage to other Kalinovic et al., 2002; Papachristos and Stamopoulos, 2002; Kim et al., 2003; Park I.K. et al., 2003). Prakash and ÃCorresponding author. Tel.: +385 31 224 244; fax: +385 31 207 017. Rao (1997) described 866 different plant species that E-mail address: [email protected] (V. Rozman). produce chemicals useful against insects and listed their

256 biologically active chemical components. However, in 2.2. Chemicals (compounds) spite of the wide recognition that many plants possess insecticidal properties, only a handful of pest control The main compounds present in oils of the aromatic products directly obtained from plants are presently in use plants (Table 1) were purchased from ‘‘Sigma-Aldrich’’ because the commercialization of new botanicals can be (Export Division Gru¨nwalder Weg 30 D-82041 Deisenho- hindered by a number of issues (Isman, 1997). Botanicals fen, Germany) and ‘‘Fluka’’ (Industriestrasse 25, CH-9471 used as insecticides presently constitute 1% of the world Buchs, SG Switzerland). These compounds were tested by insecticide market. bioassay. The focus over the last few years has been on the determination of the insecticidal activity of isolated 2.3. Insects chemical compounds from plant extracts in order to find out the most biologically active chemical compo- Cultures of the rice weevil—Sitophilus oryzae, the lesser nents (Regnault-Roger and Hamraoui, 1995; Clemente grain borer—Rhyzopertha dominica and the red flour et al., 2000; Huang et al., 2002; Lee et al., 2003; Park I.K. beetle—Tribolium castaneum (Herbst), were collected from et al., 2003; Park C. et al., 2003; Regnault-Roger et al., granaries in the eastern part of Croatia. They were reared 2004). in the laboratory to obtain the first generation (F1) for use The purpose of the present study was to investigate the in bioassays. Adults used in the experiments were 1–3 fumigant activity of naturally occurring compounds 1,8- weeks old and of mixed sex. Sitophilus oryzae and cineole, camphor, eugenol, linalool, carvacrol, thymol, Rhyzopertha dominica were reared on whole wheat grain borneol, bornyl acetate and linalyl acetate of the essential in glass containers containing 0.5 kg of clean wheat with oils of Lavandula angustifolia Chaix, Rosmarinus officinalis 13% moisture content (m.c.) while Tribolium castaneum L., Thymus vulgaris L. and Laurus nobilis L. against three was reared on a mixture of whole wheat flour and maize stored-product beetles. This paper describes for the first flour at the ratio of 1:1 in glass containers containing 0.5 kg time the fumigation activity of thymol, borneol, eugenol of the mixture with 12% m.c. All species were reared at and linalyl acetate against Sitophilus oryzae (L.) and 2971 1Cand7075% relative humidity (r.h.) in darkness. eugenol, borneole, bornyl acetate, carvacrol, linalool and linalyl acetate against Rhyzopertha dominica (F.). 2.4. Fumigant activity

2. Materials and methods The method used to determine the fumigant activity of tested compounds was based on that described by Prates et 2.1. Essential oils, compounds, gas chromatography al. (1998a). Four replicates of 10 adults of each of the three test insect species were placed in silk mesh cages that were The essential oils of Lavandula angustifolia (cultivar from put into separate glass jars of 720 ml capacity containing island Hvar, common name ‘‘budrika’’), Rosmarinus 250 g of wheat of 12% m.c. Nine compounds identified by officinalis (‘‘Miss Jessopp’s Upright’’ cultivar), Thymus analyzing the essential oils of Lavandula angustifolia, vulgaris (‘‘Argenteus’’ cultivar) and Laurus nobilis (‘‘Angu- Rosmarinus officinalis, Thymus vulgaris and Laurus nobilis stifolia’’ cultivar) were purchased from ‘‘Ireks aroma’’, and purchased from ‘‘Sigma’’, ‘‘Aldrich’’ and ‘‘Fluka’’ Zagreb, Croatia. Oils were analyzed by gas chromatogra- were tested at 5 dose rates (0.1, 1, 10, 50 and 100 ml/720 ml phy, using a ‘‘Perkin-Elmer’’ (USA) GC—type 8700, fitted volume) and over exposure periods of 1, 2, 3, 4, 5, 6 and 7 with a ‘‘Supelco’’ (USA) SP-2380 capillary column 30 m, days under controlled conditions (2971 1C, 7075% r.h., i.d. 0.32 mm, 0.20 mm film thickness. A similar method is in darkness). The substances were applied with a ‘‘Hirsch- described by Andronikashvili and Reichmuth (2002) and man’’ digital micropipette on to filter paper attached to the Regnault-Roger et al. (2004). lids of the glass containers, which were then tightly sealed.

Table 1 List of the compounds purchased from Sigma, Aldrich and Fluka

Compounds EC number Produced by

1,8-cineole ( ¼ eucalyptol) 207-431-5 Sigma Camphor (1R)-(+) camphor 99% 207-355-2 Sigma Eugenol (2-methoxy-4-[2-prophenyl]phenol) 202-589-1 Sigma Linalool 97% linalool 201-134-4 Aldrich Carvacrol 98% 5-isopropyl-2-methylphenol 207-889-6 Aldrich Thymol 99.5% 5-methyl-2-isopropylphenol 201-944-8 Sigma Borneol 87% borneol, 12% isoborneol 208-080-0 Sigma Bornyl acetate (-)-bornyl acetate, 97% — Aldrich Linalyl acetate linalyl acetate 95% — Fluka ARTICLE IN PRESS V. Rozman et al. / Journal of Stored Products Research 43 (2007) 349–355 351

All the samples for determination of fumigant activity were 100% mortality was obtained with the highest dose of observed daily over the exposure period of 1–7 days, or 100 ml/720 ml volume after 2 days exposure. After 7 days until 100% mortality of the test insects, and compared to exposure only with the lowest dose of linalyl acetate (0.1 ml/ the untreated control samples. 720 ml volume) was there any survival of S. oryzae (mortality 97.5%). 2.5. Statistical analysis For Rhyzopertha dominica (Table 4), besides camphor and linalool, eugenol, linalyl acetate, 1,8-cineole, bornyl Mortality data were subjected to a multi-factorial acetate, borneol and thymol all gave complete control at analysis of variance (ANOVA) for each day of the the dose rates of 1 ml/720 ml volume and above within 24 h. exposure period according to the general linear model For carvacrol, the highest doses (50 and 100 ml/720 ml (GLM). Significant differences were identified by least volume) were required for 100% mortality after 24 h. After significant difference (LSD) tests and entered in the tables. 7-days exposure survival occurred only at the lowest Data processing was conducted by the SAS System for dose (0.1 ml/720 ml volume), mortality with carvacrol or Windows 98 and SPSS 11.0 for Windows. thymol being 82.5% and with eugenol, linalyl acetate and borneol, 95%. 3. Results Fumigant efficacy tests of the compounds against Tribolium castaneum showed very low susceptibility in In general, fumigant activity for all compounds was almost all treatments. After the 24 h exposure no oil observed after the 24-h exposure, at the lowest dose level achieved more than 20% mortality even with 100 ml/720 ml of 0.1 ml/720 ml volume. Sitophilus oryzae and Rhyzopertha (Table 5). Most of the isolates achieved higher mortality as dominica showed very high susceptibility to all com- the dose was increased to 100 ml/720 ml volume, and the pounds. 1,8-Cineole, borneol and thymol were highly exposure lengthened to 7 days, especially 1,8-cineole effective against S. oryzae, while for Rhyzopertha (92.5% mortality after 7 days) followed by linalool and dominica camphor and linalool were highly effective, these camphor (70.0–77.5% mortality after 7 days). The other six oils giving 100% mortality at the lowest dose level. compounds were less effective, causing up to 60% In contrast, Tribolium castaneum was highly tolerant at mortality after 7 days exposure. Mortality in untreated the lowest dose level, with only eugenol achieving control samples was nil for all insect species, at the end of significantly higher mortality (12.5%) than the control the 7-day test period. (Table 2). Complete control was obtained after the 7-day exposure 4. Discussion at a dose of 1 ml/720 ml volume, or above with all compounds for S. oryzae and Rhyzopertha dominica, but In general, aromatic plants contain essential oils only in not for Tribolium castaneum (Tables 3–5). For S. oryzae concentrations of 1–3% w/w (C- akir, 1992). Large quan- (Table 3), in addition to 1,8-cineole, borneol and thymol, tities of plant material would need to be processed to gain good results were obtained with eugenol, camphor, bornyl enough essential oil for commercial-scale tests. However, acetate, carvacrol and linalyl acetate (100% mortality at certain compounds in the oils exhibit much stronger doses of 10 ml/720 ml volume or above after 24 h). Treat- activity than others. Plant varieties should be sought that ment with linalool was less effective against S. oryzae. produce these compounds in larger quantities, or synthetic

Table 2 Toxicity of nine compounds against three test insect species exposed for 24 h

Compounds Mortality after 24 h (%) Dose (0.1 ml/720 ml) Sitophilus oryzae Rhyzopertha dominica Tribolium castaneum

Mean7SE* Mean7SE* Mean7SE*

Eugenol 85.078.6ab 80.0712.2a 12.576.29a Linalyl acetate 82.5711.8ab 87.5712.5a 0.070.0b 1,8-cineole 100.070.0a 97.572.5a 0.070.0b Carvacrol 72.578.5b 82.5710.3a 0.070.0b Camphor 85.076.4ab 100.070.0a 0.070.0b Linalool 85.076.4ab 100.070.0a 0.070.0b Bornyl acetate 85.078.6ab 92.574.78a 0.070.0b Borneol 100.070.0a 92.574.78a 0.070.0b Thymol 100.070.0a 77.5722.5a 0.070.0b Untreated 0.070.0c 0.070.0b 0.070.0b

*Means in the same column followed by the same letters are not signiﬁcantly different (P40.05) as determined by the LSD-test. ARTICLE IN PRESS 352 V. Rozman et al. / Journal of Stored Products Research 43 (2007) 349–355

Table 3 Fumigant activity of the compounds against Sitophilus oryzae

Exposure time Dose (ml/720 ml) % Mortality of Sitophilus oryzae*

Eugenol Linalyl acetate 1,8 cineole Carvacrol Camphor Linalool Bornyl acetate Borneol Thymol

1 day 0.1 85.0b 82.5b 100.0a 72.5b 85.0b 85.0b 85.0b 100.0a 100.0a 1 92.5ab 90.0a 100.0a 87.5b 95.0a 87.5ab 95.0a 100.0a 100.0a 10 100.0a 100.0a 100.0a 100.0a 100.0a 90.0ab 100.0a 100.0a 100.0a 50 100.0a 100.0a 100.0a 100.0a 100.0a 97.5a 100.0a 100.0a 100.0a 100 100.0a 100.0a 100.0a 100.0a 100.0a 97.5a 100.0a 100.0a 100.0a 2 days 0.1 90.0a 82.5b — 72.5b 87.5b 87.5b 90.0a —— 1 95.0a 92.5ab — 92.5ab 97.5a 90.0ab 95.0a —— 10 — — — — — 92.5ab ——— 50 — — — — — 97.5ab ——— 100 — — — — — 100.0a ——— 3 days 0.1 100.0a 92.5a — 85.0b 90.0a 90.0a 97.5a —— 1 100.0a 97.5a — 97.5a 100.0a 95.0a 100.0a —— 10 — — — — — 95.0a ——— 50 — — — — — 100.0a ——— 4 days 0.1 — 95.0a — 92.5a 95.0a 95.0a 97.5a —— 1 — 100.0a — 100.0a — 97.5a ——— 10 — — — — — 97.5a ——— 5 days 0.1 — 95.0a — 97.5a 97.5a 97.5a 97.5a —— 1 — — — — — 97.5a ——— 10 — — — — — 97.5a ——— 6 days 0.1 — 97.5a — 100.0a 100.0a 100.0a 100.0a —— 1 — — — — — 100.0a ——— 10 — — — — — 100.0a ——— 7 days 0.1 — 97.5a ————— —— 1—————————

*Means in the same column followed by the same letters are not significantly different (P40.05) as determined by the LSD-test. — ¼ 100% kill already recorded. production methods should be explored as an option to We extended the range of active oil substances of gain enough material for full-scale use. aromatic plants tested by four more compounds (thymol, Positive results for contact and fumigant activity of borneol, eugenol and linalyl acetate) in the control of monoterpenes were obtained against S. oryzae. Shaaya et S. oryzae, all of which proved capable of achieving al. (1991) and Weaver et al. (1994) have demonstrated the complete control within 24 h at doses ranging from 0.1 to susceptibility of rice weevil to terpenoids and determined 10 ml/720 ml. camphor, 1,8-cineole and carvacrol as compounds showing Singh and Upadhyay (1993) showed that Rhyzopertha fumigant toxicity. Prates et al. (1998b) found terpenoids to dominica was susceptible to plant extracts, particularly to be effective for rice weevil control by isolating 1,8-cineole the essential oil extracted from rhizomes of Acorus calamus and limonene from Brazilian flora. They reported that L. Xu et al. (1994) tested the toxicity of estragol isolated these compounds had lethal effects on rice weevil caused by from the essential oil of Clausena dunniana L. and Dunkel their influence on the respiratory and digestive systems. Lee and Sears (1998) determined toxicity of essential oil vapors et al. (2001) showed that menthone, limonene and linalool from Artemisia tridenata Nutt. ssp. vaseyana (Rydb.) isolated from Mentha arvensis L. had toxic activity against against Rhyzopertha dominica. By gas chromatography rice weevil. Lee et al. (2003) showed that the plant Dunkel and Sears (1998) isolated 1,8-cineole and camphor monoterpenes cineole, fenchrone and pulegone in pre- as the major agents of toxicity. Shaaya et al. (1991) and liminary fumigant analyses successfully controlled the pest, Prates et al. (1998a) obtained similar results by evaluating and recommended these monoterpenes as suitable fumi- essential oil compounds from Eucalyptus spp., isolating gants because of their high volatility, fumigant efficacy and 1,8-cineole and limonene as the principal compounds with safety. Apart from these monoterpenes, bornyl acetate, insecticidal activity. These authors proved there was limonene, mircen, felandren, pinen, sabinen and terpinen contact toxicity through the insect cuticle, and fumigant isolated from Korean native plants showed very good toxicity through the respiratory and digestive systems. repellent activity (Park I.K. et al., 2003; Park C. et al., Azadirachtin proved effective even against multi-resistant 2003). strains of Rhyzopertha dominica by inhibiting the growth of ARTICLE IN PRESS V. Rozman et al. / Journal of Stored Products Research 43 (2007) 349–355 353

Table 4 Fumigant activity of the compounds against Rhyzopertha dominica

Exposure time Dose (ml/720 ml) % Mortality of Rhyzopertha dominica*

Eugenol Linalyl acetate 1,8 cineole Carvacrol Camphor Linalool Bornyl acetate Borneol Thymol

1 day 0.1 80.0b 87.5ab 97.5a 82.5b 100.0a 100.0a 92.5b 92.5b 77.5b 1 100.0a 100.0a 100.0a 87.5ab 100.0a 100.0a 100.0a 100.0a 100.0a 10 100.0a 100.0a 100.0a 87.5ab 100.0a 100.0a 100.0a 100.0a 100.0a 50 100.0a 100.0a 100.0a 100.0a 100.0a 100.0a 100.0a 100.0a 100.0a 100 100.0a 100.0a 100.0a 100.0a 100.0a 100.0a 100.0a 100.0a 100.0a 2 days 0.1 82.5b 90.0a 97.5a 82.5b — — 92.5b 92.5b 77.5b 1 — — — 87.5ab ——— —— 10 — — — 100.0a ——— ——

3 days 0.1 85.0b 90.0a 97.5a 82.5b — — 92.5b 92.5b 77.5b 1 — — — 92.5ab ——— ——

4 days 0.1 85.0b 95.0a 100.0a 82.5b — — 97.5a 95.0b 80.0b 1 — — — 100.0a ——— —— 5 days 0.1 90.0a 95.0a — 82.5b — — 97.5a 95.0b 80.0b 1————————— 6 days 0.1 95.0a 95.0a — 82.5b — — 97.5a 95.0b 80.0b 7 days 0.1 95.0a 95.0a — 82.5b — — 100.0a 95.0b 82.5b

*Means in the same column followed by the same letters are not significantly different (P40.05) as determined by the LSD-test. — ¼ 100% kill already recorded. subsequent generations (Rahim, 1998). Suya et al. (1998) Tribolium castaneum with the essential oil of Lavandula showed the fumigant activity of thymol against Rhyzo- spica L. that contained 40% of 1,8-cineole. The laboratory pertha dominica after its isolation from the essential oil of studies of Lee et al. (2003) proved that Tribolium castaneum Elsholtzia spp. could be controlled by 1,8-cineole, 1-fechone and pulegone, We extended the range of active oil substances of but only at the highest doses. aromatic plants tested against Rhyzopertha dominica by The results of our study are mostly in the agreement with six more compounds: eugenol, borneol, bornyl acetate, the results of other investigators. By comparing the carvacrol, linalool and linalyl acetate, which, with the fumigant activities of nine essential oil compounds against exception of carvacrol, achieved complete control within the three insect species, 1,8-cineole was arguably the most 24 h at a dose of 1 ml/720 ml. effective, followed by camphor and linalool. In our results, Tribolium castaneum was highly resistant to the essential oils tested. Only 1,8-cineole applied at the 5. Conclusions highest concentration (100 ml/720 ml volume) showed acceptable efficacy against Tribolium castaneum after 7 The results of fumigant activity of some compounds days exposure. Prates et al. (1998a) assessed 1,8-cineole and from laurel, lavender, rosemary and thyme essential oils limonene, monoterpenes from Eucaliptus and Citrus spp. against S. oryzae, Rhyzopertha dominica and Tribolium against Tribolium castaneum and found that although castaneum, serious pests of Croatian silos and storages, limonene was more effective than 1,8-cineole, neither identified some possible alternatives for fumigants cur- compound showed a high level of efficacy. Qiantai and rently in use. Yongcheng (1998) assessed the contact efficacy of camphor If the problem of cost-effective commercial production as the major isolate from essential oils of Cinnamomum can be solved, some of the compounds tested could find a cassia Blume, Illicium verum Hook.f. and Cinnamomum place in IPM strategies, especially where the emphasis is on camphora (L.), against Rhyzopertha dominica, S. zeamais environmental and food safety and on replacing the more and Tribolium castaneum, and found camphor effective dangerous and toxic fumigants and insecticides. against S. zeamais and Rhyzopertha dominica, whereas the isolate against Tribolium castaneum showed repellent, but Acknowledgments not insecticidal activity. Xiaoqing et al. (1998) suggested that the pest could be controlled by compounds from We are grateful to ‘‘Saponia’’ Osijek for GC analyses Citrus spp. (particularly limonene) after evaluating seven and also thank ‘‘Ireks Aroma’’ for their assistance in plant extracts for contact, repellent and fumigant efficacy. providing the necessary Dalmatian essential oil extracts. Clemente et al. (2000) obtained positive results by treating We thank Dr. Paul Fields of Cereal Research Centre, ARTICLE IN PRESS 354 V. Rozman et al. / Journal of Stored Products Research 43 (2007) 349–355

Table 5 Fumigant activity of the compounds against Tribolium castaneum

Exposure time Dose (ml/720 ml) % Mortality of Tribolium castaneum*

Eugenol Linalyl acetate 1,8 cineole Carvacrol Camphor Linalool Bornyl acetate Borneol Thymol

1 day 0.1 12.5b 0.0b 0.0b 0.0b 0.0b 0.0b 0.0a 0.0a 0.0a 1 12.5b 0.0b 0.0b 7.5ab 0.0b 0.0b 0.0a 0.0a 0.0a 10 13.0b 0.0b 0.0b 12.5ab 0.0b 2.5b 0.0a 0.0a 0.0a 50 13.0b 0.0b 2.5b 15.0ab 2.5b 2.5b 2.5a 0.0a 2.5a 100 17.5a 12.5a 17.5a 20.0a 12.5a 12.5a 5.0a 0.0a 5.0a 2 days 0.1 12.5b 0.0c 0.0b 5.0b 0.0b 0.0b 0.0b 0.0b 0.0b 1 12.5b 0.0c 0.0b 12.5ab 0.0b 0.0b 0.0b 0.0b 0.0b 10 13.0b 7.5abc 0.0b 17.5ab 0.0b 5.0b 0.0b 0.0b 0.0b 50 13.0b 10.0ab 5.0b 20.0a 5.0ab 5.0b 2.5b 0.0b 2.5ab 100 20.0a 12.5a 27.5a 20.0a 12.5a 25.0a 17.5a 15.0a 7.5a 3 days 0.1 12.5b 0.0b 0.0b 5.0b 0.0b 0.0b 0.0b 0.0b 0.0b 1 17.5ab 12.5ab 0.0b 12.5ab 0.0b 2.5b 2.5b 0.0b 0.0b 10 17.5ab 12.5ab 2.5b 20.0ab 0.0b 5.0b 2.5b 0.0b 0.0b 50 22.5ab 15.0ab 5.0b 25.0a 5.0b 5.0b 5.0b 2.5b 5.0ab 100 25.0a 17.5a 37.5a 25.0a 25.0a 32.5a 17.5a 15.0a 7.5ab 4 days 0.1 15.0b 0.0b 0.0b 5.0b 0.0b 0.0b 2.5b 0.0b 0.0c 1 20.0ab 12.5ab 0.0b 17.5ab 0.0b 5.0b 2.5b 0.0b 0.0c 10 20.0ab 12.5ab 2.5b 25.0a 2.5b 5.0b 7.5b 2.5b 0.0c 50 25.0a 22.5a 10.0b 27.5a 10.0b 7.5b 7.5b 7.5b 12.5b 100 27.5a 30.0a 42.5a 30.0a 40.0a 52.5a 32.5a 20.0a 22.5a 5 days 0.1 17.5b 0.0b 0.0b 7.5c 0.0b 0.0b 5.0b 0.0b 0.0c 1 25.0ab 12.5ab 0.0b 17.5b 0.0b 5.0b 7.5b 0.0b 0.0c 10 25.0ab 22.5a 2.5b 25.0ab 2.5b 5.0b 7.5b 2.5b 0.0c 50 35.0a 25.0a 10.0b 32.5a 10.0b 7.5b 15.0b 7.5b 12.5b 100 37.5a 30.0a 50.0a 37.5a 45.0a 57.5a 40.0a 25.0a 35.0a 6 days 0.1 20.0b 0.0b 0.0b 7.5c 0.0b 0.0b 5.0b 0.0b 0.0c 1 30.0ab 17.5ab 0.0b 22.5b 0.0b 5.0b 7.5b 0.0b 0.0c 10 30.0ab 27.5a 2.5b 30.0ab 5.0b 5.0b 10.0b 2.5b 0.0c 50 45.0a 30.0a 10.0b 40.0a 10.0b 7.5b 20.0b 7.5b 12.5b 100 50.0a 30.0a 57.5a 50.0a 57.5a 72.5a 52.5a 30.0a 40.0a 7 days 0.1 20.0b 0.0b 0.0c 7.5c 0.0c 0.0b 5.0b 0.0b 0.0c 1 30.0ab 17.5ab 0.0c 27.5b 0.0c 5.0b 7.5b 0.0b 0.0c 10 30.0ab 27.5a 2.5c 30.0ab 5.0bc 7.5b 10.0b 2.5b 0.0c 50 52.5a 30.0a 12.5b 47.5a 10.0b 7.5b 20.0b 7.5b 12.5b 100 57.5a 37.5a 92.5a 55.0a 70.0a 77.5a 57.5a 30.0a 50.0a

*Means in the same column followed by the same letters are not signiﬁcantly different (P40.05) as determined by the LSD-test.

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