Toxicity evaluation of botanical insecticides and their mixtureagainst the spiraling whitefly，Aleurodicus dispersus

Jian-ming WANG，Bing LING，Xi CAO，Shu-lin BAO，Mao-xin ZHANG

Laboratory of Insect Ecology，South China Agriculture University，Guangzhou，Guangdong 510642，China

Introduction

The spiraling whitefly(Aleurodicus dispersus Russell)is a destructive invasive pest.This is native to the Caribbean islands and Central America(Russell，1965)，but has spread very fast in African and Asian countries besides several Pacific islands and poses serious problem(Martin ＆ Lucas，1984;Neuenschwander，1994).A.dispersus is a highly polyphagous pest(Lambkin，1999;Srinivasa，2000).Its extensive host range covers 182 plants belonging to 146 genera and 57 families in Hainan(Yu，2011).The major host plants of economic concern are Psidium guajava L.，Carica papaya L.，Musa sapientum L.，Annona squamosa L.，Euphoria longana Lam，Solanum melongena L.，Phaseolus vulgaris L.， and Manihot glaziovii Muell.，in addition to several species of avenue trees such as Pterocarpus indicus Willd.，Terminalia catappa L.，Canna indica L.，and Cercis chinensis Bunee in the urban environment(Han et al.，2008).Both adults and nymphs of A.dispersus suck sap from the leaf surfaces and can cause leaf wilting and dropping.Honeydew and wax excreted by the nymphs and adults on leaf surfaces can decrease the photosynthetic capacity of the host plants(Ramani et al.，2002).

A.dispersus was first found in Hainan，China，in April 2006(Yu，2011;Yu et al.，2007).Within a short period of time，the species has spread to the whole island，and damage to both economic crops and street trees have been documented(Han et al.，2008;Zheng et al.，2008).

Some synthetic insecticides are proven effective in spiraling whitefly control(Kambrekar et al.，2003;Lin et al.，2007;Liu et al.，2007).However，they are hazardous to human health because of the pest's widespread distribution and its presence in densely populated areas.In addition，indiscriminate use of synthetic insecticides kills beneficial insects and leads to the development of pest resistance to synthetic insecticides.Thus，there is an urgent need for alternative approaches to pest management that can completely or partially replace current chemically-based pest management practices(Dubey ＆ Sundararaj，2004).Botanical insecticides can contribute towards this goal as attractive alternatives to synthetic chemical insecticides for pest management because of their high potency，efficacy and more eco-friendly nature(Isman ，2006).With this in mind，nine botanically-based insecticide products and their mixtures were evaluated for the suppression of A.dispersus.We found several promising alternatives，and identified a combination with a synergistic effect，holding out promise for a more enviornmentally friendly control of this invasive species.Institute，Chinese Academy of Tropical Agriculture，Danzhou，Hainan Province，where they were grown free from any insecticides.A.dispersus adults，at least 24 h old，were collected randomly from populations of several hundred individuals reared on T.catappa for use in the bioassays.

Botanical insecticides

Technical grade of Matrine［98%active ingredient(AI)］，Stemonine(2%AI)，Sanguinarine(15%AI)and Celangulin(6% AI)，were purchased from Shanxi Fangsheng Biological Development Co.Ltd.，China.Osthole technical(98%AI)was purchased from Nanjing Zelang Medical Technology Co.Ltd.，China.Rotenone(40%AI)and pyrethrum(50%AI)were obtained from Yunnan Nanbao Plant Chemicals Co.Ltd.，China.Azadirachtin(20%AI)was purchased from Luxi Light Neem Industry Development Co.Ltd.，China.

The chloroform extract of bitter melon(Momordica charantia L.，BM)leaves were obtained and isolated following Abo＆ Matsuda(2000).Fresh leaves for extraction were collected from mature M.charantia(after the fruiting stage).The leaves were cut into pieces and extracted with methanol at room temperature(29～31℃，24 h).The extract was filtered and subsequently concentrated to dryness under reduced pressure with arotary evaporator.Themethanol extract was dissolved in water and successively partitioned with hexane and chloroform.A portion of the chloroform extract was dried under reduced pressure.

Bioassays of botanical insecticides

All bioassays were conducted in the laboratory at 30±1℃ and 16 h∶8 h(L∶D)regime.

Activity of botanical insecticides to adults of A.dispersus by spraying

A 10 mL sample of each test solution(concentration 500 mg·L－1with 50%acetone in water)was prepared.Bioassays were carried out on freshly cut leaves of young T.catappa，which had not been subjected to any prior chemical treatment.Single leaves were placed onto moistened filter paper in Petri dishes(9 cm diameter)，with the undersides of the leaves facing upward.After anaesthetizing the A.dispersus adults(＞ 24 h old)on the T.catappa with ether，thirty anaesthetized adults of A.dispersus were gently

Materials and methods

Insects

The various developental stages of A.dispersus used in assays were obtained from a natural population of A.dispersus in Terminalia catappa L.trees growing on the grounds of the Environment and Plant Protection introduced onto each leaf in the Petri dishes with a fine brush.When most of the insects had recovered and resumed activity，dead and weak insects were removed before starting the exposure.One mL of the 500 mg·L－1test solution was sprayed onto each leaf，by means of a spray tower(Potter，Burkard Company).The solvent was quickly removed by air-drying and the lid of Petri dish was covered by a plastic wrap.Control dishes were treated similarly with 50%acetone in water.Each test was replicated three times.The treated adults were examined for mortality at 12 h intervals after treatment.Adults that did not show any responses when probed with a small brush were considered dead.In the case of pyrethrum，rotenone，azadirachtin and BM extracts，A.dispersus adults were examined for mortality at 24 h after treatment of five concentrations gradient，respectively.

Log-probit regression equations were established for pyrethrum，rotenone，azadirachtin and BM extracts and their 24 h median lethal concentrations(LC50)were calculated，respectively.

Activity of botanical insecticides applied as dry films to adults

Bioassays were carried out on freshly cut leaves of young T.catappa which had not been subjected to any chemical treatment.The leaves were placed individually on moistened filter paper in Petri dishes(9 cm diameter)with the underside of the leaf facing upward.One mL of the 500 mg·L－1test solution was sprayed onto each leaf using a spray tower，and the solvent was quickly removed by air-drying.Thirty adults of A.dispersus(＞24 h old)were anaesthetized and gently introduced onto the treated leaves in the Petri dishes.When most of the adults were awake and active，inactive individuals were eliminated，and the Petri dish lid was closed.Controls were treated similarly with 50%acetone in water.Each test was replicated three times.Insects were examined for mortality at 12 h intervals after treatment.Adults that did not show any activity when probed with a small brush were considered dead.Mortalities were compared statistically.

Activity of pyrethrum to the eggs and nymphs of A.dispersus by spraying

Ten mL samples of pyrethrum dissolved in 50%acetone in water were diluted to concentrations of 12.5，25，50，100，200 and 400 mg·L－1.The assays were performed on fresh P.guajava leaves heavily infested with a natural population of eggs and various stages of nymphs of A.dispersus.The leaves were placed individually on moistened filter paper in Petri dishes(9 cm diameter)with the eggs and nymphs on the undersides of the leaves facing upward.The number of eggs and nymphs in each Petri dish were counted with a stereo dissecting microscope.A 1 mL aliquot of the 500 mg·L－1test solution was applied to the eggs and nymphs on the leaf in each Petri dish using a spray tower.The solvent was quickly removed by airdrying and the lid of Petri dish was then covered.Control leaves were treated similarly with 50%acetone in water.Each test was replicated six times.The eggs and nymphs of A.dispersus were examined for mortality after five days.Eggs that exhibited shrinkage and darkening were considered dead.Nymphs that showed activity when probed with a small brush were considered to be survivors.

Tests with mixed extracts

The synergistic effect of the mixtures of azadirachtin or rotenone with pyrethrum on adults of A.dispersus were determined by the co-toxicity coefficient(CTC)method in the laboratory(Sun ＆ Johnson，1960;Zhang et al.，2008).Five concentrations of two different mixtures were applied by a spray tower to treat thirty A.dispersus adults incubated in Petri dishes.The control was distilled water.Each treatment was replicated five times.The number of mortality was recorded at 24 h after treatment.CTC of the mixtures were calculated as(Zhang et al.，2008):

concentration of pesticide A，B and mixture，respectively.PAand PBmean percentage of pesticide A and B in the active constituent of mixture respectively.When CTC is 100，it indicates a probability of similar action.With CTC ＞＞100，a synergistic action can be assumed，while at CTC ＜＜100 indicates antagonism.

Statistical analysis

Mean values were given with the standard error(SE).Differences in mortalities among treatments were analyzed by one-way ANOVA of arcsine square root transformed values，using Duncan's Multiple Range Test for post-hoc comparison of means.LC50values of the tested botanical insecticides and confidence limits were calculated for A.dispersus with Log dosage-mortality probit regression equations.All statistical analyses were performed using SAS 9.0 software(SAS Institute Inc.2005).

Results

Toxicity of botanical insecticides to adults of A.dispersus by spraying

Mortalities of A.dispersus adults on T.catappa leaves，sprayed with different botanical insecticides under laboratory conditions showed significant differences(Table 1).The maximum adult mortality was recorded for pyrethrum and rotenone(mortality of 98.33%and 90.98%at 12 h and 100%and 98.89%at 24 h，respectively.Celangulin，azadirachtin and BM extracts were also relatively effective with mortality at 36 h between 58.8%and 71.5%.There were no significant differences between control and matrine，sanguinarine，osthole and stemonine(Table 1).

Table 1 Toxicity of the nine tested botanical insecticides to adults of A.dispersus by spraying

Based on the Log-probit regression equations(Table 2)A.dispersus adults were the most sensitive to pyrethrum and the least sensitive to BM extracts.LC50of pyrethrum was 1.56 mg·L－1，which was significantly lower than that of rotenone，azadirachtin and BM extracts.

Table 2 Comparative toxicity of nine botanical insecticides to A.dispersus adults after 24 h

The effects of sprays of azadirachtin or rotenone mixed with pyrethrum on adult A.dispersus

The effects of two different mixtures on the adults of A.dispersus were similarly synergistic.Bioassay results revealed that the synergism was significant，and their CTC were 193.11 and 224.35，respectively(Table 2).

Toxicity of botanical insecticides applied as dry films to adults of A.dispersus

When adult A.dispersus residing on T.catappa leaves were treated with dry films of different botanical insecticides under laboratory conditions(Table 3)，pyrethrum caused the highest mortalities througout the experiment，followed by azadirachtin(34.8%mortality at 24 h，increasing to 81.8%at 36 h).Matrine，rotenone and sanguinarine were also effective，but they exhibited significantly lower mortalities than the first two(Table 3).None of the other botanical insecticides caused significantly greater mortality than the control.Rotenone，BM extracts and celangulin，when applied as dry films against adults of A.dispersus had reduced effects compared to the effects of spraying.

Toxicity of pyrethrum to the eggs and nymphs of A.dispersus by spraying

Log-probit regression equations of pyrethrum to eggs and nymphs of A.dispersus were shown in Table 4.LC50of pyrethrum to the eggs and nymphs were 77.39 mg·L－1and 61.42 mg·L－1，respectively.Toxicity of pyrethrum to A.dispersus adults was higher than that to nymphs or eggs.The fourth instar nymph was the least sensitive to pyrethrum.

Table 3 Toxicity of the nine tested botanical insecticides applied as dry films to adults of A.dispersus

Table 4 Toxicity of pyrethrum to eggs and nymphs of A.dispersus by spraying

Discussion

Chemicals derived from pyrethrum(Tanacetum cinerariifolium Trev.)， neem (Azadirachta indica Juss)，and other plant species are traditionally used in many crop pest control operations(Atkinson et al.，2004;Liang et al.，2003).Neem products，tobacco extracts and rosin soap had been found effective against A.dispersus in several countries(Dubey ＆Sundararaj，2004;Kambrekar et al.，2003;Lüet al.，2009;Singh et al.，2005).Zhong et al.(2009)observed that ethanol extracts of Celosia argentea L.and Eupatorium odoratum L.had high bioactivities against A.dispersus with LC50being 753.40 mg·L－1and 999.81 mg·L－1，respectively.Our results indicated that pyrethrum，rotenone，azadirachtin，BM extracts，and celangulin at 500 mg·L－1concentration exhibited significant insecticidal action on A.dispersus adults with mortality above 57.5% ，while matrine，sanguinarine，osthole and stemonine showed little insecticidal action on A.dispersus adults by spraying.Among the nine botanical insecticides，the maximum mortality of adults was recorded for pyrethrum，whether applied by spraying or as a dry film(Tables 1，2 and 3).Acute toxicity of pyrethrum to A.dispersus adults was the highest and the most rapid than that to the eggs and nymphs(Tables 2 and 4).This may be releated to the thicker waxy layer covering the insect body at these stages.Consequently，we recommend that pyrethrum should be applied during the peak occurrence of adult A.dispersus.

The second effective botanical insecticide spray was rotenone.Although the toxicity of azadirachtin to A.dispersus adults was lower than that of pyrethrum and rotenone，its toxicity when applied as a dry film was higher with a longer duration of toxicity(Table 3).Dubey＆ Sundararaj(2004)reported that fortnightly and three-weekly applications of neem effectively controlled nymphal populations of A.dispersus，causing 62.2%mortality even at 21 days after treatment.Formulatoin does matter:Neemark retained its toxicity(from an initial mortality of 55%to 45%on day 15)better than Neemazal(45%to 25%mortality，Kambrekar et al.，2003).The different results of toxicity tests might be due to the formulation effect and the response of A.dispersus to other toxic components of the neem.

The results of this study showed that the synergistic effects of pyrethrum mixed with azadirachtin or rotenone to the adults of A.dispersus were significant(Table 2).There are different mechanisms of action on A.dispersus between each insecticide.The pyrethrum is to block voltage-gated sodium channels in nerve axons(Xu，2001).Rotenone is a mitochondrial poison，which blocks the electron transport chain and prevents energy production(Goyal ＆ Srivastava，1990).In contrast to action mechanisms of pyrethrum and rotenone，azadirachtin has diverse biological effects on insects，demonstrating antifeedant，oviposition deterrent，repellent，insect growth inhibitor，mating disruptor，and toxic properties(Xu，2001).Mixed use of insecticides with different action mechanisms can enhance the synergism and also delay the development of resistance in A.dispersus.

Extracts of M.charantia leaves were evaluated not only for oviposition deterrence，antifeedant effect and inhibition of development but also for toxic action against many phytophagous insect pests(Li et al.，2001;Ling et al.，2008，2009;Mekuria et al.，2005).Our study confirmed that chloroform extracts of M.charantia leaves had insecticidal effect to A.dispersus adults.The acetone extracts of fresh leaves of M.charantia are toxic to the adults of Liriomyza sativae Blanchard，causing up to 50.48%mortality(Li et al.，2001).The methanol extracts of the leaves of M.charantia had insecticidal activity on larvae of Culex quinquefasciatus Say with LC50of 465.85 mg · L－1(Prabakar＆ Jebanesan，2004).Methanol extracts of M.charantia have ovicidal action in Leucoptera coffeella(Guérin-Mèneville)(Alves et al.，2011).The extracts of M.charantia leaves contain a variety of cucurbitane triterpenoid compounds，such as momordicin Ⅰ，momordicin Ⅱ，23-dihydroxy-3-O-malonycucurbita-5，24-dien-19-al，(19S，23E)-5β，19-epoxy-19-methoxy-cucurbita-6，23-diene-3β，and 25-diol，(19R，23E)-5β，19-epoxy-19-methoxycucurbita-6，23-diene-3β，25-diol，which deter oviposition of Liriomyza trifolii(Burgess)and L.sativae adults，and inhibit feeding and development of Plutella xylostella L.(Ling et al.，2008，2009;Mekuria et al.，2005).The study have established the insecticidal potential of M.charantia extracts on A.dispersus，further research is required to determine the active components and formulate them.

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