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Acaricidal Activity of Boenninghausenia sessilicarpa Against Panonychus citri
Agricultural Sciences in China
September 2009
2009, 8(9): 1097-1102
Acaricidal Activity of Boenninghausenia sessilicarpa Against Panonychus citri YANG Hui-zhi1, 2, 3, HU Jun-hua1, LI Qing3, LI Hong-jun1, LIU Hao-qiang1, YAO Ting-shan1, RAN Chun1 and LEI Hui-de1 Institute of Citrus, Chinese Academy of Agricultural Sciences, Chongqing 400716, P.R.China Changdu Vocational & Technical School, Changdu 854000, P.R.China 3 College of Agriculture, Sichuan Agricultural University, Ya’an 625014, P.R.China 1 2
Abstract Acaricidal activity of Boenninghausenia sessilicarpa against Panonychus citri was tested in the laboratory. Four solvents were used to prepare crude extracts, petroleum ether, chloroform, ethyl acetate, and ethanol, among which ethanol was the most effective one. The results suggested that ethanol extracts of B. sessilicarpa had eminent acaricidal and ovicidal activities. Concentrated extracts were prepared using petroleum ether, chloroform, ethyl acetate, or distilled water as solvent. Mite mortality rates in the concentrated extracts by petroleum ether, chloroform, or distilled water were significantly lower than those by ethyl acetate. The LC50 values of eggs and female mites were 0.7639 and 1.1033 mg mL-1, respectively. After liquid chromatography and thin layer chromatography, the concentrated extracts were separated into 14 groups of fractions and further tests for their acaricidal and ovicidal activities were conducted. Fraction 2 was found to possess higher acaricidal and ovicidal activities. The mortality of eggs and adult mites were 85.83 and 63.07%, respectively. Moreover, fraction 2 showed moderate oviposition inhibition effect (0.8795) against P. citri when the used dose was higher than 2.5 mg mL-1. Key words: Boenninghausenia sessilicarpa, Panonychus citri, acaricidal activity, ovicidal activity
INTRODUCTION Panonychus citri (McGregor) is one of the most serious citrus pest mites in the USA, England, Japan, Korea, and most sub-tropical areas of the world. Apart from citrus, the mite inhabits many other plants, such as deciduous fruit trees, ornamental trees, vegetables, and grasses (Bowman and Bartlett 1978; Uchida 1982; Osakabe 1987). It causes serious damage to citrus, rapidly developing injurious populations in early spring, late spring, and mid fall in southern China. Furthermore, recent study has demonstrated that P. citri could not only cause asthma and rhinitis in citrus farmers, but also increase bronchial responsiveness of the citrus red
mite-sensitive farmer and children in Korea (Kim et al. 1999, 2001). Chemical acaricides have been used routinely to control the mite pest in Chinese orchards. However, the long-term use of chemicals has caused resistant development in the mite population (Gerson and Cohen 1989; Meng et al. 2000) and became more toxic to natural enemies. Application of synthetic acaricides has led to environmental and human health concerns. Therefore, it is urgent to search for an acaricide that is effective against the spider mite but nontoxic to the benificials. Phytogenous acaricides are well suitable for the strategy of integrated mite management because they are the secondary metabolites that can be rapidly degraded (Misra et al. 1996) and generally less persistence in the environment than the synthetic
Received 5 December, 2008 Accepted 23 March, 2009 Correspondence HU Jun-hua, Associate Professor, Tel: +86-23-68349005, E-mail: [email protected]
© 2009, CAAS. All rights reserved. Published by Elsevier Ltd. doi:10.1016/S1671-2927(08)60317-X
1098
products. In addition, these naturally occurring products may delay the resistant development (Feng and Isman 1995). Products with short-term residual activity are also compatible with the use of biological control agents (van Lenteren and Woets 1988). There are a few reports on phytogenous acaricides. Neem (Sundaram and Sloane 1995; Venzon et al. 2008), tansy, and wormwood extracts (Chiasson et al. 2001) were found to be effective against two-spotted spider mite adults and Polyphagotarsonemus latus. Kochia scoparia (Hou 2004), Juglans regia leaf (Wang et al. 2007) and Capsicum frutescens extracts (Antonious et al. 2007) possess toxicity to Tetranychus viennensis and T. cinnabarinus. Ginkgolic acid (Pan et al. 2006), plumbagin (Han 2004), and Lisea cubeba (Mo et al. 2008) were recently evaluated for their acaricidal effects on P. citri. Boenninghausenia sessilicarpa is a perennial herbaceous plant with a widespread distribution and often grown on the marginal area of forest. It contains seboehausine, rutin, scopolean, umbelliferon, daphnoretin, and essential oil. However, no study on the acaricidal or insecticidal activities of B. sessilicarpa has been reported. This paper is implemented to evaluate the potential acaricidal activity of B. sessilicarpa against P. citri in the laboratory conditions.
YANG Hui-zhi et al.
vents were analytical grade (Chongqing Chemical Reagent Company, China). The plant powder was continuously soaked in solvent for 1 d.
Solvent partition of the B. sessilicarpa crude extracts Based on preliminary results of the acaricidal activities, the crude extract from ethanol was selected to prepare crude extracts for further concentration and partitioning. 50 g of the crude extract was diluted in 500 mL of distilled water in a separation funnel. 250 mL of partitioning solvent (petroleum ether, chloroform, or ethyl acetate) was added into the diluted extract. The extracts were concentrated using a rotary evaporator and weighed.
Isolation of active constitutes from the concentrated extracts of B. sessilicarpa
Boenninghausenia sessilicarpa was collected from Baihua Mountain (25°22´N, 102°42´E) in Wuding County, Yunnan Province, China. Panonychus citri were collected from Poncirus trifoliate of experimental citrus orchard of Institute of Citrus, Chinese Academy of Agricultural Sciences, which had not been treated with any acaricides before sampling.
A 1 000-mm glass column was used to isolate the ethyl acetate extracts. A mixture of 500 g of silica gel and 600 mL of petroleum ether was loaded into the column, and then 5 g of the extract was added onto the top of the mixture. Combinations of petroleum ether:ethyl acetate (1:1), ethyl acetate:ethanol (10:1), ethyl acetate: ethanol (9:1), ethyl acetate:ethanol (5:1), and ethyl: acetate:ethanol (1:1) were slowly added to the column sequentially at the rate of 3-4 drops per second, each for 150 mL. These mobile phases were continuously added to the column until all the components of the concentrated extract were eluted from the silica gel. The fractions (each 150 mL) were collected at the bottom of the column with collection bottles. The fractions were separated into different components according to polarity by thin layer chromatography (TLC). In total, 25 components were identified. Based on band appearance in TLC, the 25 fractions were combined into 14 groups. Each group was then evaporated to obtain a concentrated fraction of B. sessilicarp.
Solvents for B. sessilicarpa extract
Contact efficacy bioassay
The oven-dried plants (60°C, 48 h) were ground to fine powder. Four solvents, petroleum ether, chloroform, ethyl acetate, and ethanol, were used to extract active components of B. sessilicarpa. All sol-
The slide dip method was used to evaluate the contact efficacy of the extracts (Anonymous 1974). Forty female adult mites were placed dorsally on a piece of 1-cm2 double-coated tape glued on a glass microscope
MATERIALS AND METHODS Insects and plants
© 2009, CAAS. All rights reserved. Published by Elsevier Ltd.
Acaricidal Activity of Boenninghausenia sessilicarpa Against Panonychus citri
1099
slide. Three slides were prepared for each treatment with a total of 120 mites per treatment. The end of the slide with mites was dipped into the extract dilution or the control for 5 s. The slides were taken out and the extra solution was removed with filter paper. After dipping, the slides containing mites were maintained at room temperature, (25 ± 1)°C, and a photoperiod of 14 h in light and 10 h in dark. Mortality was assessed under a binocular microscope after 24 h. The mites were considered dead if their movement was imperceptible after repeated gentle probing with a fine artist’s brush. For regression analysis of toxicity, based on the primary screen, five concentrations were selected as a serial of test solutions.
by different solvents revealed that all solvents had significant (P < 0.05) effects on the crude extraction of B. sessilicarpa. Ethanol was the most effective solvent and petroleum ether was the least effective solvent (Table 1). The ethanol extracts caused a higher mortality rate than other three solvents and had a strong toxicity to the eggs of P. citri (Table 2). In the tests of the crude ethanol extracts, the mites and eggs responded in a concentration-dependent manner. The LC50 of the ethanol extracts against adults and eggs were 0.9241 and 1.7032 mg mL-1, respectively.
Ovicidal activity bioassay
The result indicates that the ethyl acetate extracts of B. sessilicarpa caused a higher mortality rate against adult female mites than the other three extracts, petroleum ether, chloroform, and water (Table 3). The ethyl acetate extracts caused a mean mite mortality rate
P. citri adult females were transferred to 2-cm diameter citrus leaf disks and left for 24 h for oviposition. Mites were removed when at least 100 eggs per disk were laid. Leaf disks were kept on moist soft cotton swabs placed in 4-cm diameter plastic petri dishes. Three leaf disks were prepared for each treatment. Leaf disks were dipped into the treatment solution for 5 s and Petri dishes were weighed before and after treatment, as described for the slides in the bioassay with adults. The effect of the treatment on egg viability was assessed daily by counting eggs remaining on the leaf disks for 10 d. Hatched eggs and live or dead nymphs were also counted. Percentage of the hatched eggs was estimated indirectly by counting live nymphs. Nymphs were considered dead if no movement was observed after repeated gentle probing with a small writing brush. All nymphs (alive and dead) were removed daily from the leaf disks. Percentages of the hatched eggs (number of live nymphs divided by the total number of the eggs on leaf disk) were obtained and subjected to an ANOVA statistical analysis by using SPSS 12.0.
RESULTS Comparison of acaricidal activities of the crude extracts using the different solvents Comparison of acaricidal activities of the crude extracts
Acaricidal activities of the ethanol extracts of B. sessilicarpa against P. citri
Table 1 The mortalities of the extracts from B. sessilicarpa against P. citri adults by using the contact bioassay Solvents
Extraction rate (%)
Corrected mortality (%)
12.16 8.00 7.95 5.05
31.67 c 51.67 b 54.17 b 91.67 a
Petroleum ether Chloroform Ethyl acetate Ethanol
Adjusted mortality (%) 31.51 51.33 54.19 90.99
c b b a
The mean values within each of the columns followed by same letters indicate no significant difference at a 0.05 level. The same as below. The extract concentration used in the assay was at 4 mg mL -1.
Table 2 The mortality of P. citri contacted with the ethanol extracts of B. sessilicarpa Treatment Eggs Female adults
LC 50 (mg mL-1)
Regressive equation
Correlation coefficient
95% confidence limit
1.7032 0.9241
y = -0.555 + 1.719x y = -1.599 + 2.225x
0.9710 0.9982
1.6315-1.8124 0.9024-0.9621
Table 3 The mortality of P. citri treated with the extracts of B. sessilicarpa at 2 mg mL-1 Treatment Eggs
Female adults
Extracts Petroleum ether Chloroform Ethyl acetate Water Petroleum ether Chloroform Ethyl acetate Water
Corrected mortality (%) 18.67 44.33 77.00 54.67 30.00 54.17 81.67 70.17
d c a b c b a a
Adjusted mortality (%) 18.61 44.12 76.84 54.58 25.01 53.88 81.42 70.01
d c a b c b a a
© 2009, CAAS. All rights reserved. Published by Elsevier Ltd.
1100
YANG Hui-zhi et al.
(81.67%). Moreover, egg hatch was reduced to some degree by all treatments, with the ethyl acetate extracts being the most significant. The LC50 of the ethyl acetate extracts against P. citri adult female mites and eggs were 0.7639 and 1.1033 mg mL -1, respectively (Table 4). The mean mite mortality and ovicidal activity of the ethyl acetate extracts were significant different from the other extracts.
Toxicity fraction of B. sessilicarpa extracts to P. citri Based on the band appearance in the TLC, 25 initial fractions were grouped into 14 final fractions (Table 5). The bioassays results indicated that acaricidal compounds were present in fractions 3-4 and 910 against adult mite, but ovicidal compounds appeared in fractions 3-4 and 6-8. Fractions 3-4 had the highest overall percentages of adult mite mortality (85.83%) and ovicidal activity (63.07%), which were significant
Table 4 The mortality of P. citri treated with the ethyl acetate extracts of B. sessilicarpa Treatment Eggs Female adults
LC 50 (mg mL-1)
Regressive equation
Correlation coefficient
95% confidence limit
1.1033 0.7639
y = -1.751 + 2.219x y = -1.983 + 2.422x
0.9929 0.9912
1.0924-1.1327 0.7238-0.7961
different from the other 13 fractions. Discontinuity in the biological activity was evident. For example, almost no adult mite mortality and ovicidal activity were associated with fraction 14 (Table 6). The fraction 2 showed fine oviposition inhibition effect on P. citri, especially when the concentration was at 2.5 mg mL -1, the oviposition inhibition rate was 0.8795. With concentration decreased, the oviposition inhibition rates declined. At 0.5 mg mL-1, the inhibition action disappeared.
DISCUSSION All the results together indicate that the extracts of B. sessilicarpa have acaricidal activity against adult females and eggs of citrus red mite. This is the first report showing acarcidal activity of B. sessilicarpa, although the mode of action of B. sessilicarpa acaricides is unknown. Fraction 2 was found to possess higher acaricidal and ovicidal activities (85.83 and 63.07%). Moreover, the fraction 2 showed moderate oviposition inhibition effect (0.8795) against P. citri when the used dose was higher than 2.5 mg mL-1. The identification of the active components and repellent toxicity analysis in this study should facilitate future investigations on the action mechanism of the active components of the B. sessilicarpa extracts. There are no previous reports
Table 5 Mortality of P. citri treated with the liquid ethyl ethanol fractions of B. sessilicarpa at 1 mg mL-1 Fraction 1 3 5 7 9 11 13 Rutin 1) 1)
Corrected mortality (mean ± SE, %) Eggs 9.10 ± 1.10 e 29.93 ± 2.02 c 6.71 ± 0.90 f 31.84 ± 2.80 bc 13.01 ± 0.42 e 7.09 ± 0.35 f 6.28 ± 0.58 fg 2.56 ± 0.02 h
Corrected mortality (mean ± SE, %)
Fraction
Adult female mite 66.67 ± 2.20 b 18.33 ± 1.00 fg 70.00 ± 1.44 b 47.50 ± 2.50 d 22.50 ± 1.20 f 10.00 ± 1.40 h 12.50 ± 0.50 g 4.67 ± 0.06 l
Eggs
2 4 6 8 10 12 14 Control
Adult female mite
63.07 ± 0.60 a 36.83 ± 1.50 b 14.44 ± 2.10 de 3.85 ± 0.20 g 8.24 ± 0.34 df 20.46 ± 1.30 d 1.11 ± 0.62 h 1.21 ± 0.02
85.83 ± 1.50 a 42.50 ± 2.50 d 59.17 ± 2.20 c 29.17 ± 0.70 f 45.83 ± 2.20 d 5.83 ± 0.20 hi 11.67 ± 0.56 gh 4.17 ± 0.28
Rutin was reported to have acaricidal activity against Tetranychus truncates (Xu and Guan 1993).
Table 6 The oviposition inhibition effect of the fraction 2 on P. citri Concentration (mg mL-1) 2.5 2 1.5 1 0.5 0
12 h
24 h
No. of egg laid per female
Oviposition inhibition index
Oviposition inhibition rate
No. of egg laid per female
Oviposition inhibition index
Oviposition inhibition rate
0.3793 0.6552 0.8644 1.5330 2.1695 3.1500
0.7851 0.6556 0.5693 0.3452 0.1821 -
0.8795 0.7920 0.7256 0.5132 0.3112 -
0.8181 1.3928 2.1428 3.1403 4.6035 4.6101
0.6816 0.5359 0.3653 0.1896 0 -
0.8225 0.6979 0.5352 0.3188 0 -
© 2009, CAAS. All rights reserved. Published by Elsevier Ltd.
Acaricidal Activity of Boenninghausenia sessilicarpa Against Panonychus citri
on insecticidal activity of the B. sessilicarpa extracts against various pest insects. B. sessilicarpa contains rutin, which was reported to have insecticidal and acaricidal activities (Xu and Guan 1993), but was not toxic to P. citri in this study. It is obvious that active components to P. citri are not rutin. Many phytogenous components have been demonstrated to have insecticidal activity, but few plants have acaricidal activities. Although B. sessilicarpa as herbal medicine has been studied for a long time, insecticidal and acaricidal activities of B. sessilicarpa have not been reported. This study indicates that B. sessilicarpa- and B. sessilicarpa-derived materials can be one of potential P. citri control agents. Applying active components from B. sessilicarpa into a miticide preparation could potentially increase efficiency of the miticide and reduce the potential for resistance development in mite populations. The use of B. sessilicarpa extract to control broad mite P. citri could also provide an alternative tool for mite management that fulfils the requirements of organic farmers. It also has the advantage of being non-toxic to mammals. However, field experiments are still necessary to confirm its efficacy in reducing the P. citri population under natural conditions.
Acknowledgements
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Tanacetum vulgare (Asteraceae) essential oils obtained by three methods of extraction. Journal of Economic Entomology, 94, 167-171. Feng R, Isman M B. 1995. Selection for resistance to azadirachtin in the green peach aphid, Myzus persicae. Cellular and Molecular Life Sciences, 51, 831-833. Gerson U, Cohen E. 1989. Resurgences of spider mites (Acari: Tetranychidae) induced by synthetic pyrethroids. Experimental and Applied Acarology, 6, 29-46. Han J Y, Zeng X N, Du L X. 2004. Acaricidal activity of root extracts of Plumbago zeylanica L. Acta Phytophylacica Sinica, 31, 85-90. (in Chinese) Hou H, Zhao L L, Shi G L, Li Z H. 2004. Studies on the acaricidic activity of the extracts of Kochia scoparia. Plant Protection, 30, 42-45. (in Chinese) Kim Y K, Park H S, Kim H Y, Jee Y K, Son J W, Bae J M, Lee M H, Cho S H, Min K U, Kim Y Y. 2001. Citrus red mite (Panonychus citri) may be an important allergen in the development of asthma among exposed children. Clinical and Experimental Allergy, 31, 582-589. Kim Y K, Son J W, Kim H Y, Park H S, Lee M H, Cho S H, Min K U, Kim Y Y. 1999. Citrus red mite (Panonychus citri) is the most common sensitizing allergen of asthma and rhinitis in citrus farmers. Clinical and Experimental Allergy, 29, 11021109. van Lenteren J C, Woets J. 1988. Biological and integrated pest control in greenhouses. Annual Review of Entomology, 33, 239-269.
This work was supported by the Special Public Sector Research of China (nyhyzx07-057) and the National K e y Te c h n o l o g y R & D P r o g r a m o f C h i n a (2007BAD47B04, 2008BAD92B08).
Meng H S, Wang K Y, Jiang X Y, Yi M Q. 2000. Studies on the resistance of Panonychus citri to several acaricides. Pesticides,
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Anonymous. 1974. Recommended methods for detection measurement of resistance of agricultural pests to pesticides. Tentative methods for spider mites and their eggs, Tetranychus spp. and Panonychus ulmi (Koch). FAO Plant Protection Bulletin, 22, 103-107. Antonious G F, Meyer J E, Rogers J A, Hu Y H. 2007. Growing hot pepper for cabbage looper, Trichopulsia ni (Hubner) and spider mite, Tetranychus urticae (Koch) control. Journal of Environmental Science and Health (Part B: Pesticides, Food Contaminants and Agricultural Wastes), 42, 559-567. Bowman C E, Bartlett P W. 1978. Panonyhus citri (McGregor) (Acari: Tetranychidae) infesting imported citrus mites in England. Plant Pathology, 27, 201. Chiasson H, Bélanger A, Bostanian N, Vincent C, Poliquin A. 2001. Acaricidal properties of Artemisia absinthium and
39, 26-28. (in Chinese) Misra G, Pavlostathis S G, Perdue E M, Araujo R. 1996. Aerobic biodegradation of selected monoterpenes. Applied Microbiology and Biotechnology, 45, 831-838.
red mite Panonychus citri McGregor. Journal of Environmental Entomology, 30, 44-49. (in Chinese) Osakabe M. 1987. Esterase activities and developmental success of the citrus red mite, Panonychus citri (McGregor) (Acarina: Tetranychidae), on several plants. Applied Entomology and Zoology, 22, 35-44. Pan W G, Luo P, Fu R B, Gao P, Long Z F, Xu F Y, Xiao H B, Liu S G. 2006. Acaricidal activity against Panonychus citri of a ginkgolic acid from the external seed coat of Ginkgo biloba. Pest Management Science, 62, 283-287. Sundaram K M S, Sloane L. 1995. Effects of pure and formulated azadirachtin, a neem-based biopesticide, on the phytophagous spider mite, Tetranychus urticae Koch. Journal of Environmental Science and Health, B30, 801-814.
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Uchida M. 1982. Ecological Studies on the Abundance and Diapause of Spider Mites and the Damage Caused by the
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Xu M J, Guan Z H. 1993. Studies on flavonoids from tomato plant: their bioactivities to cabbageworm and toxicity to
Polyphagotarsonemus latus (Banks) (Acari: Tarsonemidae). Crop Protection, 27, 869-872.
truncated spider mites. Acta Agriculturae Universitatis Pekinensis, 19, 55-61. (in Chinese)
Wang Y N, Shi G L, Zhao L L, Liu S Q, Yu T Q, Clarke S R, Sun (Edited by ZHANG Juan)
© 2009, CAAS. All rights reserved. Published by Elsevier Ltd.
September 2009
2009, 8(9): 1097-1102
Acaricidal Activity of Boenninghausenia sessilicarpa Against Panonychus citri YANG Hui-zhi1, 2, 3, HU Jun-hua1, LI Qing3, LI Hong-jun1, LIU Hao-qiang1, YAO Ting-shan1, RAN Chun1 and LEI Hui-de1 Institute of Citrus, Chinese Academy of Agricultural Sciences, Chongqing 400716, P.R.China Changdu Vocational & Technical School, Changdu 854000, P.R.China 3 College of Agriculture, Sichuan Agricultural University, Ya’an 625014, P.R.China 1 2
Abstract Acaricidal activity of Boenninghausenia sessilicarpa against Panonychus citri was tested in the laboratory. Four solvents were used to prepare crude extracts, petroleum ether, chloroform, ethyl acetate, and ethanol, among which ethanol was the most effective one. The results suggested that ethanol extracts of B. sessilicarpa had eminent acaricidal and ovicidal activities. Concentrated extracts were prepared using petroleum ether, chloroform, ethyl acetate, or distilled water as solvent. Mite mortality rates in the concentrated extracts by petroleum ether, chloroform, or distilled water were significantly lower than those by ethyl acetate. The LC50 values of eggs and female mites were 0.7639 and 1.1033 mg mL-1, respectively. After liquid chromatography and thin layer chromatography, the concentrated extracts were separated into 14 groups of fractions and further tests for their acaricidal and ovicidal activities were conducted. Fraction 2 was found to possess higher acaricidal and ovicidal activities. The mortality of eggs and adult mites were 85.83 and 63.07%, respectively. Moreover, fraction 2 showed moderate oviposition inhibition effect (0.8795) against P. citri when the used dose was higher than 2.5 mg mL-1. Key words: Boenninghausenia sessilicarpa, Panonychus citri, acaricidal activity, ovicidal activity
INTRODUCTION Panonychus citri (McGregor) is one of the most serious citrus pest mites in the USA, England, Japan, Korea, and most sub-tropical areas of the world. Apart from citrus, the mite inhabits many other plants, such as deciduous fruit trees, ornamental trees, vegetables, and grasses (Bowman and Bartlett 1978; Uchida 1982; Osakabe 1987). It causes serious damage to citrus, rapidly developing injurious populations in early spring, late spring, and mid fall in southern China. Furthermore, recent study has demonstrated that P. citri could not only cause asthma and rhinitis in citrus farmers, but also increase bronchial responsiveness of the citrus red
mite-sensitive farmer and children in Korea (Kim et al. 1999, 2001). Chemical acaricides have been used routinely to control the mite pest in Chinese orchards. However, the long-term use of chemicals has caused resistant development in the mite population (Gerson and Cohen 1989; Meng et al. 2000) and became more toxic to natural enemies. Application of synthetic acaricides has led to environmental and human health concerns. Therefore, it is urgent to search for an acaricide that is effective against the spider mite but nontoxic to the benificials. Phytogenous acaricides are well suitable for the strategy of integrated mite management because they are the secondary metabolites that can be rapidly degraded (Misra et al. 1996) and generally less persistence in the environment than the synthetic
Received 5 December, 2008 Accepted 23 March, 2009 Correspondence HU Jun-hua, Associate Professor, Tel: +86-23-68349005, E-mail: [email protected]
© 2009, CAAS. All rights reserved. Published by Elsevier Ltd. doi:10.1016/S1671-2927(08)60317-X
1098
products. In addition, these naturally occurring products may delay the resistant development (Feng and Isman 1995). Products with short-term residual activity are also compatible with the use of biological control agents (van Lenteren and Woets 1988). There are a few reports on phytogenous acaricides. Neem (Sundaram and Sloane 1995; Venzon et al. 2008), tansy, and wormwood extracts (Chiasson et al. 2001) were found to be effective against two-spotted spider mite adults and Polyphagotarsonemus latus. Kochia scoparia (Hou 2004), Juglans regia leaf (Wang et al. 2007) and Capsicum frutescens extracts (Antonious et al. 2007) possess toxicity to Tetranychus viennensis and T. cinnabarinus. Ginkgolic acid (Pan et al. 2006), plumbagin (Han 2004), and Lisea cubeba (Mo et al. 2008) were recently evaluated for their acaricidal effects on P. citri. Boenninghausenia sessilicarpa is a perennial herbaceous plant with a widespread distribution and often grown on the marginal area of forest. It contains seboehausine, rutin, scopolean, umbelliferon, daphnoretin, and essential oil. However, no study on the acaricidal or insecticidal activities of B. sessilicarpa has been reported. This paper is implemented to evaluate the potential acaricidal activity of B. sessilicarpa against P. citri in the laboratory conditions.
YANG Hui-zhi et al.
vents were analytical grade (Chongqing Chemical Reagent Company, China). The plant powder was continuously soaked in solvent for 1 d.
Solvent partition of the B. sessilicarpa crude extracts Based on preliminary results of the acaricidal activities, the crude extract from ethanol was selected to prepare crude extracts for further concentration and partitioning. 50 g of the crude extract was diluted in 500 mL of distilled water in a separation funnel. 250 mL of partitioning solvent (petroleum ether, chloroform, or ethyl acetate) was added into the diluted extract. The extracts were concentrated using a rotary evaporator and weighed.
Isolation of active constitutes from the concentrated extracts of B. sessilicarpa
Boenninghausenia sessilicarpa was collected from Baihua Mountain (25°22´N, 102°42´E) in Wuding County, Yunnan Province, China. Panonychus citri were collected from Poncirus trifoliate of experimental citrus orchard of Institute of Citrus, Chinese Academy of Agricultural Sciences, which had not been treated with any acaricides before sampling.
A 1 000-mm glass column was used to isolate the ethyl acetate extracts. A mixture of 500 g of silica gel and 600 mL of petroleum ether was loaded into the column, and then 5 g of the extract was added onto the top of the mixture. Combinations of petroleum ether:ethyl acetate (1:1), ethyl acetate:ethanol (10:1), ethyl acetate: ethanol (9:1), ethyl acetate:ethanol (5:1), and ethyl: acetate:ethanol (1:1) were slowly added to the column sequentially at the rate of 3-4 drops per second, each for 150 mL. These mobile phases were continuously added to the column until all the components of the concentrated extract were eluted from the silica gel. The fractions (each 150 mL) were collected at the bottom of the column with collection bottles. The fractions were separated into different components according to polarity by thin layer chromatography (TLC). In total, 25 components were identified. Based on band appearance in TLC, the 25 fractions were combined into 14 groups. Each group was then evaporated to obtain a concentrated fraction of B. sessilicarp.
Solvents for B. sessilicarpa extract
Contact efficacy bioassay
The oven-dried plants (60°C, 48 h) were ground to fine powder. Four solvents, petroleum ether, chloroform, ethyl acetate, and ethanol, were used to extract active components of B. sessilicarpa. All sol-
The slide dip method was used to evaluate the contact efficacy of the extracts (Anonymous 1974). Forty female adult mites were placed dorsally on a piece of 1-cm2 double-coated tape glued on a glass microscope
MATERIALS AND METHODS Insects and plants
© 2009, CAAS. All rights reserved. Published by Elsevier Ltd.
Acaricidal Activity of Boenninghausenia sessilicarpa Against Panonychus citri
1099
slide. Three slides were prepared for each treatment with a total of 120 mites per treatment. The end of the slide with mites was dipped into the extract dilution or the control for 5 s. The slides were taken out and the extra solution was removed with filter paper. After dipping, the slides containing mites were maintained at room temperature, (25 ± 1)°C, and a photoperiod of 14 h in light and 10 h in dark. Mortality was assessed under a binocular microscope after 24 h. The mites were considered dead if their movement was imperceptible after repeated gentle probing with a fine artist’s brush. For regression analysis of toxicity, based on the primary screen, five concentrations were selected as a serial of test solutions.
by different solvents revealed that all solvents had significant (P < 0.05) effects on the crude extraction of B. sessilicarpa. Ethanol was the most effective solvent and petroleum ether was the least effective solvent (Table 1). The ethanol extracts caused a higher mortality rate than other three solvents and had a strong toxicity to the eggs of P. citri (Table 2). In the tests of the crude ethanol extracts, the mites and eggs responded in a concentration-dependent manner. The LC50 of the ethanol extracts against adults and eggs were 0.9241 and 1.7032 mg mL-1, respectively.
Ovicidal activity bioassay
The result indicates that the ethyl acetate extracts of B. sessilicarpa caused a higher mortality rate against adult female mites than the other three extracts, petroleum ether, chloroform, and water (Table 3). The ethyl acetate extracts caused a mean mite mortality rate
P. citri adult females were transferred to 2-cm diameter citrus leaf disks and left for 24 h for oviposition. Mites were removed when at least 100 eggs per disk were laid. Leaf disks were kept on moist soft cotton swabs placed in 4-cm diameter plastic petri dishes. Three leaf disks were prepared for each treatment. Leaf disks were dipped into the treatment solution for 5 s and Petri dishes were weighed before and after treatment, as described for the slides in the bioassay with adults. The effect of the treatment on egg viability was assessed daily by counting eggs remaining on the leaf disks for 10 d. Hatched eggs and live or dead nymphs were also counted. Percentage of the hatched eggs was estimated indirectly by counting live nymphs. Nymphs were considered dead if no movement was observed after repeated gentle probing with a small writing brush. All nymphs (alive and dead) were removed daily from the leaf disks. Percentages of the hatched eggs (number of live nymphs divided by the total number of the eggs on leaf disk) were obtained and subjected to an ANOVA statistical analysis by using SPSS 12.0.
RESULTS Comparison of acaricidal activities of the crude extracts using the different solvents Comparison of acaricidal activities of the crude extracts
Acaricidal activities of the ethanol extracts of B. sessilicarpa against P. citri
Table 1 The mortalities of the extracts from B. sessilicarpa against P. citri adults by using the contact bioassay Solvents
Extraction rate (%)
Corrected mortality (%)
12.16 8.00 7.95 5.05
31.67 c 51.67 b 54.17 b 91.67 a
Petroleum ether Chloroform Ethyl acetate Ethanol
Adjusted mortality (%) 31.51 51.33 54.19 90.99
c b b a
The mean values within each of the columns followed by same letters indicate no significant difference at a 0.05 level. The same as below. The extract concentration used in the assay was at 4 mg mL -1.
Table 2 The mortality of P. citri contacted with the ethanol extracts of B. sessilicarpa Treatment Eggs Female adults
LC 50 (mg mL-1)
Regressive equation
Correlation coefficient
95% confidence limit
1.7032 0.9241
y = -0.555 + 1.719x y = -1.599 + 2.225x
0.9710 0.9982
1.6315-1.8124 0.9024-0.9621
Table 3 The mortality of P. citri treated with the extracts of B. sessilicarpa at 2 mg mL-1 Treatment Eggs
Female adults
Extracts Petroleum ether Chloroform Ethyl acetate Water Petroleum ether Chloroform Ethyl acetate Water
Corrected mortality (%) 18.67 44.33 77.00 54.67 30.00 54.17 81.67 70.17
d c a b c b a a
Adjusted mortality (%) 18.61 44.12 76.84 54.58 25.01 53.88 81.42 70.01
d c a b c b a a
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(81.67%). Moreover, egg hatch was reduced to some degree by all treatments, with the ethyl acetate extracts being the most significant. The LC50 of the ethyl acetate extracts against P. citri adult female mites and eggs were 0.7639 and 1.1033 mg mL -1, respectively (Table 4). The mean mite mortality and ovicidal activity of the ethyl acetate extracts were significant different from the other extracts.
Toxicity fraction of B. sessilicarpa extracts to P. citri Based on the band appearance in the TLC, 25 initial fractions were grouped into 14 final fractions (Table 5). The bioassays results indicated that acaricidal compounds were present in fractions 3-4 and 910 against adult mite, but ovicidal compounds appeared in fractions 3-4 and 6-8. Fractions 3-4 had the highest overall percentages of adult mite mortality (85.83%) and ovicidal activity (63.07%), which were significant
Table 4 The mortality of P. citri treated with the ethyl acetate extracts of B. sessilicarpa Treatment Eggs Female adults
LC 50 (mg mL-1)
Regressive equation
Correlation coefficient
95% confidence limit
1.1033 0.7639
y = -1.751 + 2.219x y = -1.983 + 2.422x
0.9929 0.9912
1.0924-1.1327 0.7238-0.7961
different from the other 13 fractions. Discontinuity in the biological activity was evident. For example, almost no adult mite mortality and ovicidal activity were associated with fraction 14 (Table 6). The fraction 2 showed fine oviposition inhibition effect on P. citri, especially when the concentration was at 2.5 mg mL -1, the oviposition inhibition rate was 0.8795. With concentration decreased, the oviposition inhibition rates declined. At 0.5 mg mL-1, the inhibition action disappeared.
DISCUSSION All the results together indicate that the extracts of B. sessilicarpa have acaricidal activity against adult females and eggs of citrus red mite. This is the first report showing acarcidal activity of B. sessilicarpa, although the mode of action of B. sessilicarpa acaricides is unknown. Fraction 2 was found to possess higher acaricidal and ovicidal activities (85.83 and 63.07%). Moreover, the fraction 2 showed moderate oviposition inhibition effect (0.8795) against P. citri when the used dose was higher than 2.5 mg mL-1. The identification of the active components and repellent toxicity analysis in this study should facilitate future investigations on the action mechanism of the active components of the B. sessilicarpa extracts. There are no previous reports
Table 5 Mortality of P. citri treated with the liquid ethyl ethanol fractions of B. sessilicarpa at 1 mg mL-1 Fraction 1 3 5 7 9 11 13 Rutin 1) 1)
Corrected mortality (mean ± SE, %) Eggs 9.10 ± 1.10 e 29.93 ± 2.02 c 6.71 ± 0.90 f 31.84 ± 2.80 bc 13.01 ± 0.42 e 7.09 ± 0.35 f 6.28 ± 0.58 fg 2.56 ± 0.02 h
Corrected mortality (mean ± SE, %)
Fraction
Adult female mite 66.67 ± 2.20 b 18.33 ± 1.00 fg 70.00 ± 1.44 b 47.50 ± 2.50 d 22.50 ± 1.20 f 10.00 ± 1.40 h 12.50 ± 0.50 g 4.67 ± 0.06 l
Eggs
2 4 6 8 10 12 14 Control
Adult female mite
63.07 ± 0.60 a 36.83 ± 1.50 b 14.44 ± 2.10 de 3.85 ± 0.20 g 8.24 ± 0.34 df 20.46 ± 1.30 d 1.11 ± 0.62 h 1.21 ± 0.02
85.83 ± 1.50 a 42.50 ± 2.50 d 59.17 ± 2.20 c 29.17 ± 0.70 f 45.83 ± 2.20 d 5.83 ± 0.20 hi 11.67 ± 0.56 gh 4.17 ± 0.28
Rutin was reported to have acaricidal activity against Tetranychus truncates (Xu and Guan 1993).
Table 6 The oviposition inhibition effect of the fraction 2 on P. citri Concentration (mg mL-1) 2.5 2 1.5 1 0.5 0
12 h
24 h
No. of egg laid per female
Oviposition inhibition index
Oviposition inhibition rate
No. of egg laid per female
Oviposition inhibition index
Oviposition inhibition rate
0.3793 0.6552 0.8644 1.5330 2.1695 3.1500
0.7851 0.6556 0.5693 0.3452 0.1821 -
0.8795 0.7920 0.7256 0.5132 0.3112 -
0.8181 1.3928 2.1428 3.1403 4.6035 4.6101
0.6816 0.5359 0.3653 0.1896 0 -
0.8225 0.6979 0.5352 0.3188 0 -
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Acaricidal Activity of Boenninghausenia sessilicarpa Against Panonychus citri
on insecticidal activity of the B. sessilicarpa extracts against various pest insects. B. sessilicarpa contains rutin, which was reported to have insecticidal and acaricidal activities (Xu and Guan 1993), but was not toxic to P. citri in this study. It is obvious that active components to P. citri are not rutin. Many phytogenous components have been demonstrated to have insecticidal activity, but few plants have acaricidal activities. Although B. sessilicarpa as herbal medicine has been studied for a long time, insecticidal and acaricidal activities of B. sessilicarpa have not been reported. This study indicates that B. sessilicarpa- and B. sessilicarpa-derived materials can be one of potential P. citri control agents. Applying active components from B. sessilicarpa into a miticide preparation could potentially increase efficiency of the miticide and reduce the potential for resistance development in mite populations. The use of B. sessilicarpa extract to control broad mite P. citri could also provide an alternative tool for mite management that fulfils the requirements of organic farmers. It also has the advantage of being non-toxic to mammals. However, field experiments are still necessary to confirm its efficacy in reducing the P. citri population under natural conditions.
Acknowledgements
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This work was supported by the Special Public Sector Research of China (nyhyzx07-057) and the National K e y Te c h n o l o g y R & D P r o g r a m o f C h i n a (2007BAD47B04, 2008BAD92B08).
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