Open Access
Translator Disclaimer
1 September 2015 Toxicity of an Acetogenin-Based Bioinsecticide Against Diaphorina citri (Hemiptera: Liviidae) and its Parasitoid Tamarixia radiata (Hymenoptera: Eulophidae)
Leandro Do Prado Ribeiro, Mônica Silva Santos, Gabriel Luiz Padoan Gonçalves, José Djair Vendramim
Author Affiliations +

The Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Liviidae), is currently the most important insect pest affecting citrus worldwide due to its relation (as insect vector) with huanglongbing (greening) disease. To determine an alternative tool for D. citri control, this study evaluated the insecticidal activity of ethanolic extract from Annona mucosa Jacq. (Magnoliales: Annonaceae) seeds (ESAM), which has the acetogenin rolliniastatin-1 as its major compound, against D. citri. ESAM caused high mortality in both 3rd instar nymphs (LC50 = 429.43, 247.95, 148.16, 96.89, and 57.76 mg/L after 24, 48, 72, 96, and 120 h of exposure, respectively) and adults (LC50 = 5,359.00, 2,464.00, 1,507.00, and 795.51 mg/L after 48, 72, 96, and 120 h of exposure, respectively), showing higher effectiveness than Azamax® 1.2 EC (azadirachtin 3-tigloylazadirachtol, positive control) at the recommended concentration, which showed insecticidal effects only on nymphs. At a sublethal concentration (LC25), ESAM caused significant reductions in feeding and oviposition of D. citri adults. However, the adult emergence of the ectoparasitoid Tamarixia radiata (Waterston) (Hymenoptera: Eulophidae) was reduced when exposed (by contact) to ESAM in its larval stage. In a greenhouse trial (seedlings cultivated in vases), the insecticidal activity of formulated ESAM was superior to that of Azamax® 1.2 EC, showing a residual effect of approximately 6 d (effectiveness > 80%). The effectiveness of ESAM (> 99%) for D. citri control also was confirmed in a commercial sweet orange farm (field trial). In light of these results, ESAM can constitute a useful component in the framework of D. citri integrated pest management, mainly in domestic orchards and organic systems.

Huanglongbing (HLB), also known as citrus greening disease, is currently the main phytosanitary problem of the citrus industry worldwide (Alemán et al. 2007; Grafton-Cardwell et al. 2013). In Brazil (the largest citrus producer worldwide), the disease is caused by Candidatus Liberibacter asiaticus and Candidatus Liberibacter americanus (Coletta-Filho et al. 2004; Teixeira et al. 2005). These phloem-limited bacteria are transmitted in a persistent manner by the Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Liviidae) (Hall et al. 2012). Although this insect has occurred naturally within Brazil's citrus groves for more than 60 yr without causing economic damage (Costa Lima 1942), in 2004, its relationship with HLB transmission was discovered in orchards of São Paulo State (Leal et al. 2010). The disease has since spread rapidly to other citrus producing regions (Belasque Jr et al. 2010), requiring significant multi-institutional research efforts to contain its advance and economic impact.

As all citrus species farmed are HLB susceptible and varieties tolerant to the disease's expression are still unavailable (Grafton-Cardwell et al. 2013), control of the insect vector is considered essential to reduce disease spread in orchards (Hall et al. 2012; Boina & Bloomquist 2015). In this context, the use of botanical derivatives is an important tool in integrated D. citri management in organic orchards, a market niche with huge potential for expansion in Brazil (Turra et al. 2014). Although a strategy still poorly exploited and used, studies have demonstrated the potential of botanical derivatives for D. citri control, including extracts from Azadirachta indica A. Juss (Sapindales: Meliaceae) (Shivankar et al. 2000; Borad et al. 2001; Shivankar et al. 2003; Weathersbee III & McKenzie 2005; Khan et al. 2012), Vitex negundo L. (Lamiales: Lamiaceae), and Acorus calamus L. (Acorales: Acoraceae) (Shivankar et al. 2000), essential oils of aromatic plants (Mann et al. 2011, 2012), and, more recently, a matrine-based biopesticide (Zanardi et al. 2015). However, it is still necessary to perform studies aiming to detect new sources of botanical insecticides that can be used in managing this pest species, especially plant species with great abundance under neotropical conditions.

In our current research program aiming to detect new sources of insecticides/acaricides, compounds derived from Annona mucosa Jacq. (Magnoliales: Annonaceae) were chosen as this plant is observed to cause pronounced lethal and sublethal effects in some pest species of agricultural importance, such as Sitophilus zeamais Motschulsky (Coleoptera: Curculionidae) (Ribeiro et al. 2013), Panonychus citri (McGregor) (Prostigmata: Tetranychidae) (Ribeiro et al. 2014c), Trichoplusia ni Hübner (Lepidoptera: Noctuidae), and Myzus persicae (Sulzer) (Aphidomorpha: Aphididae) (Ribeiro et al. 2014a). Based on the promising initial results, biomonitored fractionation was conducted, indicating that the toxicity of this A. mucosa derivate is due to synergy of structurally diverse acetogenins, the majority being the bis-tetrahydrofuranic acetogenin rolliniastatin-1 (Ribeiro 2014). Despite the potential of derivatives from Annonaceae in arthropod pest management, little is known regarding their action against sucking insects that transmit phytopathogens.

Due to its importance in establishing a natural biological balance, preservation of the control exercised by natural enemies (entomophagous and entomopathogenic) should be considered in a screening program of new insecticide compounds, thus allowing for reduced economic costs and environmental impacts from applying these xenobiotics. Under the citrus growing conditions in Brazil, Tamarixia radiata (Waterston) (Hymenoptera: Eulophidae) is the main parasitoid of D. citri (Gómez-Torres et al. 2012) because of its high parasitism efficiency, large dispersal capacity, and good field adaptation (Étienne et al. 2001). In addition, this ectoparasitoid has been used in classical biological control programs for D. citri in Brazil (Diniz et al. 2012; Gómez-Torres et al. 2012) and in other citrus producing countries (Étienne et al. 2001; Williams et al. 2013). A previous study (Ribeiro et al. 2014b) found that derivatives from A. mucosa are compatible with 3 species of entomopathogenic fungi, but to our knowledge, no previously published study has evaluated the impact of derivatives from these promising Annonaceae on entomophagous insects.

Given these considerations, the present study aimed to compare the toxicity of A. mucosa ethanolic seed extract (ESAM) and a limonoidbased commercial bioinsecticide on D. citri in laboratory, semi-field (greenhouse), and field experiments. In addition, we evaluated the effects of ESAM on oviposition and feeding behavior of D. citri adults and its impact on T. radiata ectoparasitoid emergence in the laboratory.

Materials and Methods


The D. citri (nymphs and adults) and T. radiata specimens used in the bioassays were obtained from a population reared in the laboratory under controlled conditions (26 ± 2 °C, 70 ± 10% RH, and 14:10 h L:D photoperiod). Seedlings (approx. 15 cm) of orange jessamine (Murraya paniculata [L.] Jacq.; Sapindales: Rutaceae), considered as one of the most suitable host species for D. citri (Michaud & Olsen 2004), were used for rearing purpose. For multiplication of T. radiata, orange jessamine seedlings were used and were infested with 4th and 5th instar nymphs of D. citri (host for the immature stage) and honey to feed the adults as described by Gómez-Torres et al. (2012).


The A. mucosa seeds used to prepare the crude extract were obtained from mature fruit collected on 17 Mar 2011 from specimens grown on the “Luiz de Queiroz” College of Agriculture campus, Piracicaba, São Paulo, Brazil (22°42′28.5″S, 47°37′59.6″W; altitude: 534 m). A voucher specimen, previously identified by Dr. Heimo Rainer (Department of Systematics and Evolution of Higher Plants, University of Vienna, Vienna, Austria), was deposited in the ESA herbarium of the Department of Biological Sciences at ESALQ/USP in Piracicaba, São Paulo, Brazil, under registration number 120985.

To prepare the extracts, the seeds were dried in an oven at 40 °C for 48 to 72 h and subsequently ground in a knife mill. The powder obtained was stored in sealed glass and kept refrigerated (approx. -10 °C) until use. Organic extract was obtained using the ethanol (99.5%) soaking technique (in a 1:5 ratio, w/v) as previously described (Ribeiro 2014).


Laboratory Tests

All laboratory trials were conducted in a climate-controlled room (26 ± 2 °C, 70 ± 10% RH, and 14:10 h L:D photoperiod) under a completely randomized design.

Toxicity of the Extract Obtained Compared with a Limonoidbased Bioinsecticide

To conduct the bioassays, lime (Citrus limona L. Osbeck var. ‘Cravo’; Sapindales: Rutaceae) seedlings grown in plastic tubes were previously pruned, and after shoots were emitted (2–3 cm long), they were used as experimental units.

Effect on Nymphs. The insecticidal action (via residual contact) of the A. mucosa ethanolic seed extract (ESAM) on D. citri 3rd instars was compared with that of a limonoid-based bioinsecticide (azadirachtin [6,220.15 g/L] + 3-tigloylazadirachtol [2,596.60 mg/L]; Azamax® 1.2 EC, UPL Brasil Ltda., Campinas, São Paulo, Brazil), commercialized in Brazil to manage D. citri on citrus (Agrofit 2014). For this goal, ‘Cravo’ lime seedlings were sprayed with the treatments via a micro-atomizer (Arprex® model 5A, Mogi das Cruzes, São Paulo, Brazil) coupled to a pneumatic pump adjusted to provide a pressure of 0.5 kgf/cm2. For all treatments, the spray volume used was 2 mL of solution per seedling, stipulated based on preliminary tests to obtain complete and uniform coverage (point of runoff).

The concentration used in both treatments (extract and positive control) was 2,500 mg/L, which is recommended for Azamax® 1.2 EC to control D. citri in Brazil (Agrofit 2014). Due to its rapid degradation, the limonoid levels (azadirachtin + 3-tigloylazadirachtol) in the formulation was quantified using the analytical method described by Forim et al. (2010) at the moment of product usage. The negative controls consisted of the solvents used to solubilize the extract and the commercial bioinsecticide (acetone:deionized water [1:3, v/v] and deionized water, respectively).

After applying the treatments, the seedlings were kept in a climatecontrolled room for 2 h to dry the residue. Next, the seedlings were arranged in cages (2 L) constructed as described by Zanardi et al. (2015). Subsequently, ten 3rd instars were transferred from the rearing stock to each seedling using a fine brush and a stereoscopic microscope. Six replicates were used for each treatment level (n = 60). Nymph mortality was evaluated every 24 h for 5 d using a stereoscopic microscope. Dead nymphs were considered to be those that were dried and did not react to the touch of a fine brush.

Effect on Adults. The same procedures and experimental units used in the test with nymphs were employed to evaluate the insecticidal action on D. citri adults. However, the concentration of ESAM and commercial bioinsecticide used was 10,000 mg/L (4 times the concentration recommended for Azamax® 1.2 EC in controlling nymphs), defined based on previous tests. After applying the treatments and drying the residue, each seedling was infested with 10 adults (non-sexed) from the rearing stock aged between 5 and 8 d old. Similar to the previous test, 6 seedlings (replicates) per treatment (n = 60) were used, and the mortality of the exposed insects was evaluated daily for 5 d.

Concentration—Response Curve

To estimate LC50 and LC90, corresponding to the levels necessary to cause 50 and 90% mortality, respectively, in the exposed insect population (separately by stage), preliminary tests were performed to determine the concentrations that caused a 95% insect mortality and a mortality level similar to that obtained in the control. Based on these results, 6 concentrations were established for testing (intervals: 31.25 – 1,000 mg/L for nymphs and 250 – 10,000 mg/L for adults) based on the procedures described by Finney (1971). The same experimental procedures described for the previous test were adopted for these estimates, and the mortality evaluations were performed daily for 5 d.

Estimated Mean Lethal Time (LT50)

LT50 (time necessary to kill 50% of the population) values of ESAM for D. citri nymphs and adults were estimated at different concentrations (125; 250; 500; and 1,000 mg/L [nymphs] and 1,000; 2,000; 3,981; 6,309; and 10,000 mg/L [adults]). For this purpose, the same aforementioned experimental procedures were adopted, and mortality was evaluated daily for 5 d.

Evaluating Deterrent Effects of ESAM on Oviposition and Feeding

Deterred Oviposition. The deterrent effect of ESAM on D. citri adult oviposition was evaluated at the previously estimated LC25 (exposure time = 120 h) using a test without opportunity to choose (confinement). The same experimental units and procedures afore mentioned were adopted for this bioassay. The acetone:deionized water solution (1:3, v/v) used to solubilize the extract served as the negative control.

After drying the residues, the seedlings were isolated into cages (2 L) and infested with 5 D. citri adult couples per seedling, and already fertilized females were selected (expanded abdomen with yellow-orange color; Skelley & Hoy 2004; Wenninger & Hall 2007). Ten plants were used for each treatment, totaling 50 couples per treatment. After 48 h of infestation, the number of eggs oviposited on each plant was counted using a stereoscopic microscope.

Deterred Feeding. Discs of leaves from sweet orange (Citrus sinensis [L.] Osbeck var. ‘Pêra’; Sapindales: Rutaceae) (3.5 cm wide) were submerged in an extract solution (in the LC25 previously estimated for D. citri adults [exposure time = 120 h]) for 5 s and resuspended in acetone:deionized water (1:3, v/v). After applying the treatment, the discs were kept in a climate-controlled room on a paper towel for 2 h to dry the residue. Next, the discs were placed on Petri dishes (3.5 cm wide) containing solidified agar:deionized water solution (2.5% [w/v]).

In each Petri dish, 10 non-sexed adults aged between 5 and 8 d old were released with 10 replicates per treatment level (n = 100). A filter paper disc was added to the top of each Petri dish. The paper discs were thus kept face down in the Petri dishes to collect the honeydew excreted by the confined insects according to the procedure described by Boina et al. (2009). After 48 h, the filter paper discs were removed and submerged into ninhydrin:acetone solution (1% [v/v]) for 3 min. After 24 h, the discs were then scanned, and the honeydew drop area was estimated using Quant software version 1.0.1 (Vale et al. 2001).

Effect of ESAM on the Ectoparasitoid Tamarixia radiata

Orange jessamine branches infested with 4th and 5th instars of D. citri from the rearing stock were placed on buds of orange jessamine seedlings reared in plastic tubes (50 mL) for spontaneous migration of the nymphs. After 24 h (the period necessary for attachment and natural distribution of the nymphs on seedlings), nymphs were counted using a stereoscopic microscope. Next, the seedlings infested with the nymphs were placed into cages (2 L) that were infested with 10 T. radiata females for each D. citri nymph. The parasitoid females remained in contact with the D. citri nymphs for 48 h for the occurrence of parasitism. After this period, the parasitoid females were removed, and the plants containing the nymphs were kept in the respective cages in a climate-controlled room.

Four days after removing the parasitoid, the parasitized D. citri nymphs (mummified) were counted and sprayed with the extract at the LC90 previously estimated for D. citri adults (exposure time = 120 h), adopting the same aforementioned equipment and procedures. Acetone:deionized water (1:3, v/v) was used as a control, and 10 replicates (seedlings) were used for each treatment. Evaluation was performed after 9 d of applying the treatments, counting the number of parasitoids that emerged in each experimental unit.


Sweet orange seedlings (approx. 80 cm) kept in pots (10 L) were sprayed with an aqueous emulsion of ESAM containing 5 g/L of Tween 80® (Ribeiro et al. 2014a) until the point of runoff using a Guarany® sprayer backpack equipped with a constant-flow full cone nozzle (FullJet®). The concentration of the extract used corresponded to the LC90 previously estimated for D. citri adults (exposure time = 120 h). Azamax® 1.2 EC bioinsecticide was used as a positive control, and the solvents employed to solubilize the formulated extract (methanol:water [1:10, v/v] + Tween 80® [0.5%, v/v]) and commercial bioinsecticide (deionized water) were used as negative controls.

At 3 h (time 0 = period necessary for drying the residues) and 1, 3, 6, 12, and 24 d after spraying, leaves from the apical portion of the treated seedlings were covered with acrylic cages (5 × 4 × 2 cm) and infested with 10 non-sexed adults aged between 5 and 8 d old, with 5 replicates per treatment level (n = 50). After this, the infested seedlings were kept in a greenhouse, and after 5 d, the mortality of the adults exposed in each treatment was evaluated.


The efficacy of the formulated ESAM compared with a limonoidbased bioinsecticide (Azamax® 1.2 EC) was evaluated in a commercial sweet orange orchard (C. sinensis var. ‘Valência’; approximately 4 yr old) with plants grown 4 × 6 m apart, as implemented in Piracicaba, São Paulo, Brazil (22°42′30″S, 47°38′0″W). In this orchard, no pesticides were applied for 12 mo before the onset of the experiment.

Branches from the apical portion of the plants of the plot were selected randomly and marked. Next, the plants were sprayed, using the same equipment described previously until the point of runoff. The concentration of the formulated ESAM used corresponded to the LC90 previously estimated for D. citri adults (exposure time = 120 h). The bioinsecticide Azamax® 1.2 EC (10,000 mg/L) was used as a positive control, and the solvents employed to solubilize the formulated extract (methanol:water [1:10, v/v] + Tween 80® [0.5%, v/v]) and commercial bioinsecticide (deionized water) were used as negative controls. Five replicates (plants) were used for each treatment.

After applying and drying the residue, selected branches were covered with a cage of voile fine tissue (20 × 15 cm) and infested with 20 non-sexed adults aged between 5 and 8 d old (n = 100). The mortality of the exposed insects was evaluated 5 d after infestation.


Generalized linear models (GLM) (Nelder & Wedderburn 1972) with quasi-binomial, quasi-Poisson, and Gaussian distributions were used for data analysis of mortality ratios, D. citri egg counts, and honeydew drop area, respectively. In all cases, the goodness of fit was tested using half-normal plots of probabilities with simulated envelope (Hinde & Demétrio 1998). When the treatments differed significantly, multiple comparisons (Tukey test, α = 0.05) were performed using the glht function of the multicomp package, with the P values adjusted for the treatments with qualitative levels, whereas non-linear regressions were used to compare the treatments with quantitative levels. All analyses were performed using R statistical software version 2.15.1 (R Core Team 2012). The mortality data obtained in the semi-field and field tests were corrected using the formula proposed by Schneider-Orelli (1947).

To estimate the lethal concentrations (LC50 and LC90), a binomial model with complementary log-log link function (gompit model) was used, using the Probit Procedure (SAS version 9.2; SAS Institute 2011). In turn, to estimate mean lethal time (LT50), the method proposed by Throne et al. (1995) was used for probit correlated data analysis.


The yield of the obtained extract from the maceration process of A. mucosa seeds in ethanol at a 1:5 (w/v) ratio was 18.79% (g of extract/g of seed powder). Regardless of the D. citri stage (nymphs or adults), the insecticidal action of ESAM was higher than that of the limonoid-based bioinsecticide (Azamax® 1.2 EC) used as a positive control, which was only effective in controlling nymphs (Table 1). ESAM caused complete mortality of exposed insects when tested at concentrations of 2,500 mg/L and 10,000 mg/L, respectively, for nymphs and adults.

Depending on the exposure time, ESAM caused high mortality in D. citri nymphs (LC50 = 429.43, 247.95, 148.16, 96.89, and 57.76 mg/L after 24, 48, 72, 96, and 120 h of exposure, respectively; Table 2) and D. citri adults (LC50 = 5,359.00, 2,464.00, 1,507.00, and 795.51 mg/L, after 48, 72, 96, and 120 h of exposure, respectively; Table 2). Similarly, the mean lethal time (LT50) estimated was concentration dependent with significantly increased mortality throughout the exposure time (LT50 [nymphs] = 76.41, 41.99, 17.50, and 10.31 h at concentrations of 125, 250, 500, and 1,000 mg/L, respectively; LT50 [adults] = 91.90, 84.35, 47.93, 41.43, and 31.23 h at concentrations of 1,000, 2,000, 3,981, 6,309, and 10,000 mg/L, respectively; Table 3).

Table 1.

Mortality (mean ± SE) of 3rd instar nymphs and adults of Diaphorina citri exposed to residual ethanolic extract from Annona mucosa seeds (ESAM) or commercial limonoid-based bioinsecticide (Azamax® 1.2 EC, positive control). Note: The treatments were tested at concentrations of 2,500 mg/L and 10,000 mg/L for nymphs and adults, respectively.


Regarding sublethal effects, ESAM (at the LC25 estimated for adults [exposure time = 120 h]) significantly reduced the number of deposited eggs and the feeding of D. citri adults, which indicates that this product has deterrent action on oviposition and feeding (Figs. 1 and 2).

Despite the potential bioactive effects on D. citri, ESAM reduced the emergence of ectoparasitoid T. radiata adults (Fig. 3) when its larval stage (2nd instar) was exposed (by contact) to the extract applied at the LC90 estimated for controlling D. citri adults (exposure time = 120 h).

The results obtained in the greenhouse test (semi-field) also confirmed the higher efficacy of ESAM compared with the limonoid-based bioinsecticide (Azamax® 1.2 EC) used as a positive control (Fig. 4). Under the same conditions, ESAM exhibited persistence of approx. 6 d (efficacy > 80%), showing a pronounced decrease in its insecticidal action after this period (Fig. 4). In contrast, Azamax® 1.2 EC caused higher adult mortality between 3 and 6 d after application, corroborating its systemic action and demonstrating reduced translocation speed in the citrus plants within the first days after leaf application.

In the test conducted in the Valencia sweet orange orchard, the formulated ESAM, when tested at the LC90 estimated under laboratory conditions (4,463.00 mg/L), exhibited high efficacy (> 90%) at controlling D. citri adults (Table 4). Despite the lower concentration used, the level of activity caused by this extract was significantly higher than that of the Azamax® 1.2 EC insecticide used as a positive control, which was tested at a concentration of 10,000 mg/L.


Our results, obtained under laboratory, semi-field, and field conditions, indicate promising insecticidal action of ESAM for D. citri, and its efficacy levels were higher than those of a commercial limonoid-based bioinsecticide (Azamax® 1.2 EC, positive control) used to manage this pest species in Brazilian citrus orchards. Corroborating the toxicity of ESAM for sucking insects, our previous study (Ribeiro et al. 2014a) demonstrated that this extract has superior aphidicidal action (against M.s persicae) compared with commercial acetogenin-based (Anosom® 1 EC) and pyrethrinbased (Insect Spray®) bioinsecticides in laboratory and semi-field tests, without having any phytotoxic effects on the plant species (cabbage and citrus) used in the study.

Table 2.

Estimated LC50 and LC90 (in mg/L) and confidence interval of ethanolic extract from Annona mucosa seeds (ESAM) for 3rd instar nymphs and adults of Diaphorina citri at different exposure times.


In addition to the lethal toxicity of ESAM, our laboratory results also showed that at sublethal levels, it has pronounced deterrent effects on feeding and oviposition, effects that can affect the demography and population dynamics of D. citri, a hypothesis to be tested in the field in further studies. Moreover, compounds that inhibit feeding or alter feeding behavior can affect a phytopathogen's ability to transmit via insect vectors (Halbert & Manjunath 2004) because acquisition of the bacteria associated with HLB by D. citri makes salivation and ingestion of the phloem sap necessary (Bonani et al. 2010). The phloem is where the bacteria are found inside citrus plants (Batool et al. 2007). Given the perspective of applying phagodeterrent compounds in managing phytopathogen-transmitting insect pests, the interference of ESAM in D. citri feeding behavior will be the subject of a future study using the electrical penetration graph technique.

Table 3.

Estimated mean lethal time (LT50, in h) and confidence interval of ethanolic extract from Annona mucosa seeds (ESAM) for 3rd instar nymphs and adults of Diaphorina citri at different levels.


Table 4.

Percentage mortality (mean ± SE) of Diaphorina citri adults 120 h after applying an aqueous emulsion of ethanolic extract from Annona mucosa seeds (ESAM) or a commercial limonoid-based bioinsecticide (Azamax® 1.2 EC, positive control) in a test conducted in a commercial ‘Valência’ sweet orange orchard.


Typically, the bioactivity of ESAM is due to the synergy of compounds from different chemical classes (especially acetogenins, alkaloids, and triglycerides) and/or of different polarities (Ribeiro et al. 2013), where the bis-tetrahydrofuranic acetogenin rolliniastatin-1 is the major active component (Ribeiro 2014). Acetogenins are considered potent complex I inhibitors (NADH: ubiquinone oxidoreductase) of the mitochondrial electron transport system and NADH: oxidase of the plasma membrane, which induces cellular apoptosis (programmed cell death), perhaps as a result of ATP deprivation (Tormo et al. 1999). Lately, acetogenins have attracted much interest due to their promising insecticidal action (Alali et al. 1999; Ribeiro et al. 2013) and repellence/deterrence of feeding and oviposition (Blessing et al. 2010). Studies on the structure-activity relationship have proven that acetogenins with adjacent bis-tetrahydrofuranic rings and 3 hydroxyl groups (e.g., rolliniastatin-1) have more pronounced entomotoxicity compared with acetogenins containing other distributions of functional groups in their structure (He et al. 1997).

Fig. 1.

Effect of ethanolic extract from Annona mucosa seeds (ESAM) on oviposition preference of copulated Diaphorina citri females (A) and on feeding in non-sexed D. citri adults (B). Both tests were conducted without opportunity to choose, and the concentration of the extract used was equivalent to the LC25 (224.92 mg/L) previously estimated for D. citri adults (120 h of exposure).


Fig. 2.

Filter paper discs (experimental units) of the control and treatment with ethanolic extract from Annona mucosa seeds (ESAM). Dark points inside the discs comprise honeydew drop areas excreted by Diaphorina citri adults exposed to the referred treatments, which were stained with a ninhydrin:acetone solution (1%, v/v).


Although a deeper evaluation of the possible chronic effects of ESAM on the ectoparasitoid T. radiata is necessary, both under laboratory and field conditions, our results showed a negative impact of the extract on the emergence of this natural enemy. However, Leatemia & Isman (2004), using direct spraying and residual contact tests, found variation in the susceptibility of generalist predators to an extract from Annona squamosa L. (Magnoliales: Annonaceae) seeds, wherein larvae of Chrysoperla carnea (Stephens) (Neuroptera: Chrysopidae) were less susceptible than adults of Orius insidiosus (Say) (Hemiptera: Anthocoridae). However, the compatibility of derivatives from A. mucosa with entomopathogenic fungi (Beauveria bassiana Bals.-Criv. Vuill. [Hypocreales: Cordycipitaceae] isolate ESALQ-PL63; Isaria fumosorosea Wize [Hypocreales: Cordycipitaceae] isolate ESALQ-1296; and Metarhizium anisopliae [Metschn.] Sorokin [Hypocreales: Clavicipitaceae] isolate ESALQ-E9) (Ribeiro et al. 2014b) is a positive aspect for including it in integrated D. citri management programs. Several studies have demonstrated the potential of entomopathogens in managing this insect vector (Hoy et al. 2010; Avery et al. 2011; Guizar-Guzman & Sanchez-Peña 2013), and commercial formulations of mycoinsecticides aiming to control D. citri are under development for the Brazilian market (Ribeiro et al. 2014b).

Fig. 3.

Percentage of emergence of Tamarixia radiata adults exposed to ethanolic extract from Annona mucosa seeds (ESAM) during their larval stage. The concentration of extract used was equivalent to the LC90 (4,463.00 mg/L) estimated for D. citri adults (120 h of exposure).


In addition to aspects related to the agronomic efficacy of these natural derivatives, it is necessary to carefully evaluate their possible effects on non-target organisms (especially mammals) and their behavior in the environment. In a preliminary approach, González-Coloma et al. (2002) found that cells from the ovary of a mammal (Chinese hamster) were less sensitive (approx. 400 times) to the acetogenin rolliniastatin-1 than cells from Spodoptera frugiperda Smith & Abbot (Lepidoptera: Noctuidae) (Sf9), although enzymatic and immunochemical studies have revealed high similarity between the enzymes involved in cellular respiration in insects, mammals, and fungi (Lümmen 1998). However, membrane factors dependent on the structure and metabolic capacity of inactivating acetogenins in different groups can provide different levels of sensitivity (Ribeiro 2014), an aspect that should be investigated further.

Fig. 4.

Corrected mortality (Schneider-Orelli 1947) of Diaphorina citri adults exposed to residue on ‘Valência’ sweet orange seedlings treated with an aqueous emulsion of ethanolic extract from Annona mucosa seeds - ESAM (•) - or the commercial limonoid-based bioinsecticide Azamax® 1.2 EC (○) used as a positive control, at different periods after application in a semi-field trial (greenhouse). The mortality levels were lower than 7.5% in the controls.


Based on the results obtained, we conclude that ESAM has promising bioactivity for D. citri and may constitute a useful component for managing this pest species in Brazil and in other citrus producing countries, especially in domestic orchards and organic citrus production systems. Given this perspective, studies on optimizing extraction processes and formulations should be conducted, especially to enable controlled-release nanoformulations that provide increased residual effects of bioinsecticides developed based on A. mucosa seed extract.


The authors thank the Research Foundation of São Paulo State (FAPESP — Grant 2010/52638-0), Brazil, for financial support.

References Cited


Agrofit. 2014. Phytosanitary Agrochemicals System [online]. Ministry of Agriculture, Livestock and Food Supply, Brazil. accessed 14 Dec 2014). Google Scholar


FQ Alali , XX Liu , EJL McLaughlin. 1999. Annonaceous acetogenins: recent progress. Journal of Natural Products 62: 504–540. Google Scholar


JH Alemán , H Baños , J Ravelo. 2007. Diaphorina citri y la enfermedad huanglongbing: una combinación destructiva para la producción citrícola. Revista de Protección Vegetal 22: 154–165. (In Spanish) Google Scholar


PB Avery , VW Wekesa , WB Hunter , DG Hall , CL McKenzie , LS Osborne , CA Powell , ME Rogers. 2011. Effects of the fungus Isaria fumosorosea (Hypocreales: Cordycipitaceae) on reduced feeding and mortality of the Asian citrus psyllid, Diaphorina citri (Hemiptera: Psyllidae). Biocontrol Science and Technology 21: 1065–1078. Google Scholar


A Batool , Y Iftikhar , SM Mughal , MM Khan , MJ Jaskani , M Abbas , IA Khan. 2007. Citrus greening disease - a major cause of citrus decline in the world - a review. Horticultural Science 34: 159–166. Google Scholar


J Belasque Jr , JC Barbosa , CA Massari , AJ Ayres. 2010. Incidência e distribuição do huanglongbing no estado de São Paulo, Brasil. Citrus Research and Technology 31: 1–9. (In Portuguese) Google Scholar


LT Blessing , OA Colom , S Popich , A Neske , A Bardón. 2010. Antifeedant and toxic effects of acetogenins from Annona montana on Spodoptera frugiperda. Journal of Pest Science 83: 307–310. Google Scholar


DR Boina , JR Bloomquist . 2015. Chemical control of the Asian citrus psyllid and of huanglongbing disease in citrus. Pest Management Science (in press). Google Scholar


DR Boina , EO Onagbola , M Salyani , LL Stelinski. 2009. Antifeedant and sublethal effects of imidacloprid on Asian citrus psyllid, Diaphorina citri. Pest Management Science 65: 870–877. Google Scholar


JP Bonani , A Fereres , E Garzo , MP Miranda , B Appezzato-Da-Gloria , JRS Lopes. 2010. Characterization of electrical penetration graphs of the Asian citrus psyllid, Diaphorina citri, in sweet orange seedlings. Entomologia Experimentalis et Applicata 134: 35–49. Google Scholar


PK Borad , MJ Patel , NM Vaghela , BH Patel , MG Patel , HP Babubhai , JR Patel. 2001. Evaluation of some botanicals against citrus leafminer (Phyllocnistis citrella) and psylla (Diaphorina citri) on kagzilime (Citrus aurantifolia). Indian Journal of Agricultural Sciences 71: 177–179. Google Scholar


HD Coletta-Filho , MLPN Targon , MA Takita , JD De Negri , J Pompeu-Junior , MA Machado. 2004. First report of the causal agent of huanglongbing (‘Candidatus Liberibacter asiaticus’) in Brazil. Plant Disease 88: 1382. Google Scholar


AM Costa Lima. 1942. Insetos do Brasil, Homoptera. Escola Nacional de Agronomia, Rio de Janeiro, Brazil. (In Portuguese) Google Scholar


AJF Diniz , GR Alves , JRP Parra , PT Yamamoto . 2012. Tamarixia radiata: uma alternativa para controle do psilídeo dos citros. Citricultura Atual 1: 12–14. (In Portuguese) Google Scholar


J Étienne , S Quilici , D Marinal , A Franck. 2001. Biological control of Diaphorina citri (Hemiptera: Psyllidae) in Guadeloupe by imported Tamarixia radiata (Hymenoptera: Eulophidae). Fruits 56: 307–315. Google Scholar


DJ Finney. 1971. Probit Analysis. Cambridge University Press, Cambridge, United Kingdom. Google Scholar


MR Forim , MFG Fernandes , QB Cass , JB Fernandes , PC Vieira. 2010. Simultaneous quantification of azadirachtin and 3-tigloylazadirachtol in Brazilian seeds and oil of Azadirachta indica: application to quality control and marketing. Analytical Methods 2: 860–869. Google Scholar


ML Gómez-Torrez , DE Nava , JRP Parra. 2012. Life table of Tamarixia radiata (Hymenoptera: Eulophidae) on Diaphorina citri (Hemiptera: Psyllidae) at different temperatures. Journal of Economic Entomology 105: 338–343. Google Scholar


A González-Coloma , A Guadaño , C Inés , R Martínez-Díaz , D Cortes. 2002. Selective action of acetogenin mitochondrial complex I inhibitors. Zeitschrift für Naturforschung C 57: 1028–1034. Google Scholar


EE Grafton-Cardwell , LL Stelinski , PA Stansly. 2013. Biology and management of Asian citrus psyllid, vector of the huanglongbing pathogens. Annual Review of Entomology 58: 413–432. Google Scholar


L Guizar-Guzman , R Sanchez-Peña. 2013. Infection by Entomophthora sensu stricto (Entomophthoromycota: Entomophthorales) in Diaphorina citri (Hemiptera: Liviidae) in Veracruz, Mexico. Florida Entomologist 96: 624–627. Google Scholar


SE Halbert , KL Manjunath. 2004. Asian citrus psyllid (Sternorryncha: Psyllidae) and greening disease of citrus: a literature review and assessment of risk in Florida. Florida Entomologist 87: 3330–3353. Google Scholar


DG Hall , ML Richardson , ED Ammar , SE Halbert. 2012. Asian citrus psyllid, Diaphorina citri, vector of citrus huanglongbing disease. Entomologia Experimentalis et Applicata 146: 207–223. Google Scholar


H He , L Zeng , Q Ye , G Shi , N Oberlines , G Zhao , C Njoku , J McLaughlin. 1997. Comparative SAR evaluations of annonaceous acetogenins for pesticidal acitivity. Journal of Pesticide Science 49: 372–378. Google Scholar


J Hinde , CGB Demétrio. 1998. Overdispersion: models and estimation. Computational Statistics and Data Analysis 27: 151–170. Google Scholar


MA Hoy , S Singh , ME Roger. 2010. Evaluations of a novel isolate of Isaria fumosorosea for control of the Asian citrus psyllid, Diaphorina citri (Hemiptera: Psyllidae). Florida Entomologist 93: 24–32. Google Scholar


I Khan , M Zahid , GZ Khan. 2012. Toxicity of botanic and synthetic pesticide residues to citrus psyllid Diaphorina citri Kuwayama and Chrysoperla carnea (Stephens). Pakistan Journal of Zoology 44: 197–201. Google Scholar


RM Leal , JC Barbosa , MG Costa , JBJ Junior , PT Yamamoto , J Dragone. 2010. Distribuição espacial de huanglongbing (greening) em citros utilizando a geoestatística. Revista Brasileira de Fruticultura 32: 808–818. (In Portuguese) Google Scholar


JA Leatemia , MB Isman. 2004. Toxicity and antifeedant activity of crude seed extracts of Annona squamosa (Annonaceae) against lepidopteran pests and natural enemies. International Journal of Tropical Insect Science 24: 150–158. Google Scholar


P Lümmen. 1998. Complex I inhibitors as insecticides and acaricides. Biochimica et Biophysica Acta 1364: 287–296. Google Scholar


RS Mann , RL Rouseff , JM Smoot , WS Castle , LL Stelinski. 2011. Sulfur volatiles from Allium spp. affect Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Psyllidae), response to citrus volatiles. Bulletin of Entomological Research 1: 89–97. Google Scholar


RS Mann , S Tiwari , JM Smoot , RL Rouseff , LL Stelinski. 2012. Repellency and toxicity of plant-based essential oils and their constituents against Diaphorina citri Kuwayama (Hemiptera: Psyllidae). Journal of Applied Entomology 136: 87–96. Google Scholar


JP Michaud , LE Olsen. 2004. Suitability of Asian citrus psyllid, Diaphorina citri, as prey for ladybeetles. BioControl 49: 417–431. Google Scholar


JA Nelder , RWM Wedderburn. 1972. Generalized linear models. Journal of the Royal Statistical Society A 135: 370–384. Google Scholar


R Core Team. 2012. R: a language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. Google Scholar


LP Ribeiro. 2014. Exploring genetic biodiversity: secondary metabolites from Neotropical Annonaceae as a potential source of new pesticides. Ph.D. thesis, College of Agriculture, University of São Paulo, Piracicaba, Brazil. Google Scholar


LP Ribeiro , JD Vendramim , KU Bicalho , MS Andrade , JB Fernandes , RA Moral , CGB Demétrio. 2013. Annona mucosa Jacq. (Annonaceae): a promising source of bioactive compounds against Sitophilus zeamais Mots. (Coleoptera: Curculionidae). Journal of Stored Products Research 55: 6–14. Google Scholar


LP Ribeiro , Y Akhtar , JD Vendramim , MB Isman. 2014a. Comparative bioactivity of selected seed extracts from Brazilian Annona species and an acetogeninbased commercial bioinsecticide against Trichoplusia ni and Myzus persicae. Crop Protection 62: 100–106. Google Scholar


LP Ribeiro , LHC Mota , CP D'Alessandro , JD Vendramim , I Delalibera Jr. 2014b. In vitro compatibility of an acetogenin-based bioinsecticide with three species of entomopathogenic fungi. Florida Entomologist 94: 1395–1403. Google Scholar


LP Ribeiro , OZ Zanardi , JD Vendramim , PT Yamamoto. 2014c. Comparative toxicity of an acetogenin-based extract and commercial pesticides against citrus red mite. Experimental and Applied Acarology 64: 87–98. Google Scholar


SAS Institute. 2011. Statistical analysis system: getting started with the SAS learning. Version 9.2. SAS Institute Inc., Cary, North Carolina, USA. Google Scholar


O Schneider-Orelli. 1947. Entomologisches Praktikum. Sauerlander, Aarau, Switzerland. Google Scholar


VJ Shivankar , CN Rao , S Singh . 2000. Studies on citrus psylla, Diaphorina citri Kuwayama: a review. Agricultural Reviews 21: 199–204. Google Scholar


VJ Shivankar , CN Rao , S Singh. 2003. Evaluation of plant based biopesticides against psylla, Diaphorina citri Kuwayama and Mallada boninensis Okamoto against insect pests of citrus In O Koul , GS Dhaliwal , SS Marwaha , JK Arora [eds.], Biopesticides and Pest Management. Campus Books International, New Delhi, India. Google Scholar


LH Skelley , MA Hoy. 2004. A synchronous rearing method for the Asian citrus psyllid and its parasitoids in quarantine. Biological Control 29: 14–23. Google Scholar


DC Teixeira , C Saillard , S Eveillard , JL Danett , PI Costa , AJ Ayres , J Bové. 2005. ‘Candidatus Liberibacter americanus’, associated with citrus huanglongbing (greening disease) in São Paulo State, Brazil. International Journal of Systematic and Evolutionary Microbiology 55: 1857–1862. Google Scholar


JE Throne , DK Weaver , VB Chew , E James. 1995. Probit analysis of correlated data: multiple observations over time at one concentration. Journal of Economic Entomology 88: 1510–1512. Google Scholar


JR Tormo , T Gallardo , R Aragón , D Cortes , E Estornell. 1999. Annonaceous acetogenins as inhibitors of mitochondrial complex I. Current Topics in Phytochemistry 2: 69–90. Google Scholar


C Turra , CEF Vian , FAG Nielsen , PS Santos , LFF Penteado. 2014. Overview of the Brazilian citriculture certification. Journal of Agricultural and Environmental Ethics 27: 663–679. Google Scholar


FXR Vale , E Fernandes-Filho , JR Liberato. 2001. Quant - Quantificação de doenças: versão 1.0.1. Universidade Federal de Viçosa, Viçosa, Brazil. (In Portuguese) Google Scholar


III AA Weathersbee , CL McKenzie. 2005. Effect of neem biopesticide on repellency, mortality, oviposition and development of Diaphorina citri (Homoptera: Psyllidae). Florida Entomologist 88: 401–407. Google Scholar


EJ Wenninger , DG Hall. 2007. Daily timing of mating and age at reproductive maturity in Diaphorina citri (Hemiptera: Psyllidae). Florida Entomologist 90: 715–722. Google Scholar


T Williams , HC Arredondo-Bernal , LA Rodríguez-del-Bosque. 2013. Biological pest control in Mexico. Annual Review of Entomology 58: 119–140. Google Scholar


OZ Zanardi , LP Ribeiro , TF Ansante , MS Santos , GP Bordini , PT Yamamoto , JD Vendramim. 2015. Bioactivity of a matrine-based biopesticide against four pest species of agricultural importance. Crop Protection 67: 160–167. Google Scholar
Leandro Do Prado Ribeiro, Mônica Silva Santos, Gabriel Luiz Padoan Gonçalves, and José Djair Vendramim "Toxicity of an Acetogenin-Based Bioinsecticide Against Diaphorina citri (Hemiptera: Liviidae) and its Parasitoid Tamarixia radiata (Hymenoptera: Eulophidae)," Florida Entomologist 98(3), 835-842, (1 September 2015).
Published: 1 September 2015

Annona mucosa
botanical insecticide
inseticida botânico
Get copyright permission
Back to Top