Aphicidal Activity of an Ageraphorone Extract From Eupatorium adenophorum Against Pseudoregma bambucicola (Homoptera: Aphididae, Takahashi).

The bamboo aphid, Pseudoregma bambucicola, is an important insect pest of bamboo that affects normal bamboo growth and induces sooty molds. The control of P. bambucicola involves the application of chemicals, such as imidacloprid, to which many species are resistant. In this study, we isolate a novel botanical pesticide (9-oxo-10,11-dehydro-ageraphorone) from an Eupatorium adenophorum(Asteraceae: Compositae) petroleum ether extract and test the aphicidal activity of this compound against P. bambucicola in laboratory bioassay and field-based experiments. This ageraphorone compound at a concentration of 2 mg/ml caused 73.33% mortality (corrected mortality [Subtracted the mortality of the negative control]: 70%) of P. bambucicola by laboratory bioassay within 6 h. Even at lower concentrations, this compound caused greater 33% mortality (corrected mortality: 30%) of aphids. Field experiments with naturally infested bamboo plants showed that two applications of 2 mg/ml ageraphorone to infested plants completely cleared infestations within 30 d. These effects were similar to those of the positive control (imidacloprid). These results reveal that 9-oxo-10,11-dehydro-ageraphorone exhibits significant aphicidal activity against bamboo aphids. We suggest that future research be directed at developing this ageraphorone compound from E. adenophorum as an aphicidal agent for biocontrol.

Aphids, or plant lice, are small, sap-sucking insects (Paik 1972). They are among the most important insect pests of many crops worldwide and cause damage that lowers plant quality and results in reduced crop production (Kim et al. 2011, Baek et al. 2013). Pseudoregma bambucicola (Takahashi, 1921) is a parthenogenic aphid species (holocyclic) that is widely distributed throughout the warmer regions of eastern Asia. This species infests Bambusa bamboo stems, branches, twigs, leaves, and shoots, developing into large and high-density colonies that impair normal bamboo growth (Fukatsu et al. 2001, Ijichi et al. 2004). The delicate branches of infested bamboo turned brown and die, while the aphid infestation also induces sooty molds, the growth of which inhibits photosynthesis. P. bambucicola has a very high reproductive potential and can cause substantial injury and even death to young plants (Petitt and Smilowitz 1982).

The prevention and control of aphids involves two commercially available management tools: insecticidal seed treatments and treatments that induce host plant resistance. The control of P. bambucicola, in particular, involves the application of chemicals, such as imidacloprid (Kim et al. 2011). Such chemicals pollute the environment (Park et al. 2011). Moreover, as most aphid species have become resistant to many aphicidal agents (Rashid et al. 2013), managing these pests in greenhouses and in the field is becoming problematic (Pavela 2006, Dang et al. 2010, Kim et al. 2011). Therefore, efficient and environmentally friendly pest control alternatives must be developed to replace synthetic pesticides.

Botanical aphicidal agents are safer than synthetic pesticides, biodegrade naturally, and are not likely to cause insecticide resistance among pests. A growing number of studies have attempted to develop plant-derived aphicidal agents, and many biologically active compounds have been identified that are toxic to insect pests. For example, studies have revealed aphicidal activity in crude extracts and phospholipids from Chenopodium ficifolium, oil from Jatropha curcas, alkaloids from Corydalis turtschaninovii tubers, rhamnolipids from Pseudomonas spp., and alkaloids from Macleaya cordata seeds (Dang et al. 2010, Habou et al. 2011, Park et al. 2011, Kim et al. 2011, Rashid et al. 2013).

Eupatorium adenophorum (Crofton weed) (Asteraceae: Compositae) (Auld and Martins 1975) is a harmful, perennial herbaceous weed, recognized as the most important invasive plant species in China (Zhao et al. 2012). E. adenophorum is a global pest of crops and forests that has caused environmental and ecological damages in at least 30 countries (Lei et al. 2012). Previous research has revealed the potential of this weed in the development of plant-derived pesticides. In this study, we isolate a novel compound (9-oxo-10,11-dehydro-ageraphorone) from an E. adenophorum petroleum ether extract and test the aphicidal activity of this compound against the bamboo aphid P. bambucicola with laboratory bioassay and field-based experiments.

Materials and Methods

Ethics Statement

No specific permissions were required for the activities conducted in this study. The location is neither privately owned nor protected. The experiments did not involve endangered or protected species.

Plants and Aphids

E. adenophorum was collected in Xichang City, Sichuan province, China. The aerial parts of E. adenophorum were air-dried and then crushed in a knife mill. Morphological identification of the plant was based on a taxonomic key (Li 1998). Aphids were collected from a bamboo tree naturally infested with P. bambucicola. The laboratory bioassay experiments used wingless parthenogenetic P. bambucicola obtained from the bamboo plant. The field experiments involved the direct treatment of insects on the naturally infested bamboo plant.

Isolation and Identification of Ageraphorone

The aerial parts of E. adenophorum were dried and crushed in a knife mill. Subsequently, 8 kg of E. adenophorum plant material was soaked in 95% ethanol (600 ml of 95% ethanol per 100 g plant material) for 30 min at room temperature. The material was then extracted twice with boiling ethanol under reflux, this step was 1 h per cycle. Then, the extracts were combined and concentrated by evaporation using a rotary evaporation apparatus. Finally, the ethanol extract was obtained. Then, we extracted ethanol extract with petroleum ether and obtained the petroleum ether extract of E. adenophorum (Nong et al. 2014a).The petroleum ether extract was then chromatographed on a silica gel column using a gradient of ratios of petroleum ether to acetone (50:1 → 5:1) as the eluents to obtain the compound. We used 1H NMR and 13C NMR (Wang et al. 2007) to identify the structures of the compound obtained from the E. adenophorum petroleum ether extract. The compound was subsequently identified as 9-oxo-10,11-dehydro-ageraphorone by nuclear magnetic resonance (NMR). NMR spectra were measured with a Bruker DRX-400 instrument with tetramethylsilane as the internal standard (Nong et al. 2014b).

Aphicidal Activity of Ageraphorone to P. bambucicola With Laboratory Bioassay

This was performed according to the methods of Nong et al. (2012, 2014a) and Chermenskaya et al. (2012) with some modifications. Glycerin and distilled water (1:1) were used for solvent preparation. The ageraphorone compound was used at four different concentrations (2.0, 1.0, 0.5, and 0.25 mg/ml). We added 2 ml of each concentration to a Petri dish (10 cm in diameter) containing filter paper to absorb the liquid. Ten P. bambucicola aphids were then placed on the filter paper in each Petri dish and incubated at 20 ± 2°C and 80 ± 10% relative humidity (Chermenskaya et al. 2012). Three replicates were performed for each concentration. The viability of the P. bambucicola insects was checked regularly by needle stimulation and aphids that displayed no reaction were recorded as dead. Imidacloprid was used as the positive control, whereas glycerin and distilled water (1:1) were used as negative controls.

Aphicidal Activity of Ageraphorone to P. bambucicola in vivo (Field Experiment)

The field experiment was performed in a bamboo grove where severe infestation by P. bambucicola was observed. Each treatment was repeated thrice. We selected nine bamboo trees showing P. bambucicola infestation, these nine infested plants were chosen that appeared to have comparable infestations were chosen and then these were randomly assigned to treatments. These nine bamboo plants were randomly divided into three groups (A, B, and C). Plants in group A were treated with 2 mg/ml of 9-oxo-10,11-dehydro-ageraphorone (treatment group), whereas plants in group B were treated with imidacloprid (positive control group), and plants in group C were administered with glycerin and distilled water (1:1) (negative control group). All nine bamboo plants were treated twice on days 0 and 4. We used a small sprayer (especially used for thin-layer chromatography) for the application of ageraphorone, imidacloprid, and glycerin. Spraying approximately 20 ml of each substance on each bamboo tree appeared to distribute the chemicals on the plant and cover the aphids. Observations were conducted on day 0 (prior to spraying) and on days 4, 8, 12, and 30, using either a three-point or one-point sampling strategy. On days 0 and 4, we sampled three points on the plant (the upper, middle, and lower parts of each aphid gathering area). On days 8, 12, and 30, we only sampled one of those three areas. Sampling aphids involved collected the insects from a 1 cm2 area/per point on the plant to calculate the mean aphid number. The aphid reduction rate and field treatment effect were calculated using the following formulas (Xu et al. 2009):

Aphid reduction rate (ARR, %) = (number of aphid before spraying – number of aphid after spraying)/number aphid before spraying × 100%.

Field treatment effect (%) = (ARR of treated group – ARR of control group)/(100 – ARR of control group) × 100%.

Statistical Analyses

Analysis of variance tests were conducted using SAS software (SAS Institute 2002) to assess the significance of differences in insect mortality rates under the different ageraphorone concentrations. The median lethal time (LT50) was calculated using a complementary log–log model. We also assessed the statistical significance of field experiment results, mainly the field treatment effect (%) of different concentrations of extracts and different treatment times were considered statistically significant when P < 0.05. The significance values were corrected with Duncan’s multiple comparisons test.

Results

Identification of the Aphicidal Compound

The structure of the active compound was identified by comparing its NMR data with data from literature. The NMR spectra of the compound showed the presence of one C = CH group (δH 6.23, 1H, br s; δC 135.8 s, 146.3 d), four methyl groups (δH 2.03, 1.87, 1.71, each 3H, s; δH 0.96 3H, d, J = 6.8 Hz), and two carbonyl groups (δC 197.7 s, 202.8 s) (Nong et al. 2014b). The spectral data were in agreement with published data (Shi et al. 2012) and this compound was identified as 9-oxo-10,11-dehydro-ageraphorone (molecular formula C15H20O2; Fig. 1).

Fig. 1.

The structure of 9-oxo-10,11-dehydro-ageraphorone.

Aphicidal Activity of Ageraphorone Against P. bambucicola With Laboratory Bioassay

We found that 9-oxo-10,11-dehydro-ageraphorone was highly toxic to P. bambucicola (Table 1). The 2 mg/ml concentration of the compound caused 73.33% (corrected mortality [subtracted the mortality of the negative control]: 70%) mortality among aphids within 6 h. Even concentrations of 1.0 and 0.5 mg/ml of the ageraphorone compound caused 60% and 33.33% (corrected mortality: 57% and 30%) aphid mortality, respectively (Table 1).

Table 1.

The aphicidal activity of 9-oxo-10,11-dehydro-ageraphorone against P. bambucicola with laboratory bioassay

Different concentration of ageraphorone	Mean mortality (%) ± SE of each observation time (min)
	120 min	180 min	300 min	360 min
2 mg/ml (ageraphorone)	16.67 ± 3.33c(b)	23.33 ± 3.33c(b)	50.00 ± 10.00b(a)	73.33 ± 6.67a(ab)
1 mg/ml (ageraphorone)	13.33 ± 3.33c(bc)	26.67 ± 3.33bc(b)	40.00 ± 5.77b(ab)	60.00 ± 5.77a(b)
0.5 mg/ml (ageraphorone)	3.33 ± 3.33c(cd)	13.33 ± 6.67bc(bc)	23.33 ± 3.33ab(b)	33.33 ± 6.67a(c)
0.25 mg/ml (ageraphorone)	3.33 ± 3.33c(cd)	13.33 ± 6.67bc(bc)	23.33 ± 3.33ab(b)	33.33 ± 6.67a(c)
Positive (imidacloprid)	40.00 ± 5.77b(a)	46.67 ± 3.33b(a)	56.67 ± 6.37b(a)	83.33 ± 3.33a(a)
Untreated (glycerin and distilled water 1:1)	0.00 ± 0.00a(d)	3.33 ± 3.33a(c)	3.33 ± 3.33a(c)	3.33 ± 3.33a(d)

Ten P. bambucicola aphids and three replicates were performed for each concentration. SE: standard error of means. Different lower case letters within a row denote significant differences between different times (P < 0.05). Different lower case letters in brackets within a column denote significant differences between different concentrations (P < 0.05).

Toxicity Analysis of Ageraphorone With Laboratory Bioassay (Median Lethal Time, LT50)

The toxicity of ageraphorone extract against P. bambucicola was tested with laboratory bioassay using a complementary log–log model. The data demonstrate that the extract of ageraphorone from E. adenophorum has a strong toxic effect against P. bambucicola. The probit regression analysis by regression line of different concentration of ageraphorone show that the toxicity of ageraphorone has to be time- and concentration dependent. The LT50 values of the 2.0, 1.0, and 0.5 mg/ml concentrations of ageraphorone were 4.5, 5.4, and 8.9  h, respectively. The laboratory bioassay aphicidal activity of 9-oxo-10,11-dehydro-ageraphorone showed dose and time dependence (Table 2).

Table 2.

The probit regression analysis of toxicity (LT50) of ageraphorone against aphids with laboratory bioassay

Different concentration of ageraphorone	Regression line	LT50/(h) (95% FL)
2 mg/ml	Y = 3.256X–2.117	4.5 (3.8–5.6)
1 mg/ml	Y = 2.609X–1.907	5.4 (4.3–8.4)
0.5 mg/ml	Y = 2.614X–2.486	8.9 (6.3–34.9)

Regression line: the equation reflect the relationship between the toxicity and the concentration of ageraphorone; LT50, median lethal time value; 95% FL, the overall parameter is 95% in this range.

Field Experiment

Prior to the field experiment, infested plants that appeared to have comparable infestations were chosen and then these were randomly assigned to treatments. Aphid density area was greatly reduced in group A (ageraphorone group) on days 4 and 8 postspraying (Fig. 2A2 and A3), and by day 12, nearly all of the infested plants in this group had completely recovered. Moreover, no recurrence of infestation was observed on day 30 in the plants in this group.

Fig. 2.

Observations of the field treatment effects on infected bamboos. Plants in group A were treated with 2 mg/ml of the ageraphorone extract from E. adenophorum. Plants in group B were treated with imidacloprid, whereas plants in group C were untreated (treated with glycerine and distilled water 1:1). The photos A1, B1, and C1 show aphid infestations prior to “treatment.” The photos A2, B2, and C2 show observations of typical infestations on day 4 posttreatment, whereas photos A3, B3, and C3 show observations of typical infestations on day 8 posttreatment.

Infested bamboos in the positive control group (group B, treated with imidacloprid) also exhibited improvement during the experimental period with enhanced recovery after the second treatment (Fig. 2B2 and B3). As expected, bamboo plants in the negative control group (group C, treated with glycerol and water) showed no signs of recovery (i.e., no reduction in aphid infestation; Fig. 2C2 and C3) and actually increased over the course of the experiment (at day 8). Although the ageraphorone compound acted slower than did the imidacloprid, by 30 d postspraying, no aphids were present on any of the bamboo plants in either group (A or B), with both compounds exhibiting a 100% field efficacy rate (Table 3). Thus, the ageraphorone compound is ultimately, as effective as the standard currently used aphicidal agent.

Table 3.

The results of field trial-field treatment effect (%) score of different groups (mean ± SE) against P. bambucicola

Treatment groups	Field treatment effect (%) score of different groups (mean ± SE)
	0 d	4 d	8 d	12 d	30 d
A: 2 mg/ml ageraphorone	0.00 ± 0.00d(a)	31.33 ± 6.57c(ab)	76.67 ± 1.67b(a)	96.33 ± 0.88a(a)	100.00 ± 0.00a(a)
B: Positive control (imidacloprid)	0.00 ± 0.00d(a)	51.00 ± 2.31c(a)	90.00 ± 5.29b(a)	100.00 ± 0.00a(a)	100.00 ± 0.00a(a)
C: Untreated control (glycerin and distilled water 1:1)	0.00 ± 0.00a(a)	17.00 ± 9.54a(b)	12.33 ± 7.88a(b)	7.17 ± 6.43a(b)	6.00 ± 6.00a(b)

The field experiment was performed in a bamboo grove where severe infestation by P. bambucicola. Nine bamboo plants were randomly divided into three groups (A, B, and C). Plants in group A were treated with 2 mg/ml of 9-oxo-10,11-dehydro-ageraphorone, whereas group B were treated with imidacloprid, and plants in group C were administered with glycerin and distilled water (1:1). SE, standard error. Field treatment effect (%) = (aphid reduction rate of treated group – aphid reduction rate of control group)/(100 – aphid reduction rate of control group) × 100%. Different lower case letters within a row denote significant differences between different times (P < 0.05). Different lower case letters in brackets within a column denote significant differences between different concentrations (P < 0.05).

Discussion

To discover new natural products requires screening candidate plants, obtaining active ingredients, and isolating and identifying the active plant constituents (Chermenskaya et al. 2012). We applied this procedure to obtain the compound 9-oxo-10,11-dehydro-ageraphorone from E. adenophorum petroleum ether extract. Previous studies have shown that oral administration of 9-oxo-10,11-dehydro-ageraphorone can cause hepatotoxicity in mice and antifeedant activity in Helicoverpa armigera (Bhardwaj et al. 2001, Shi et al. 2012). However, few studies have investigated the effect of compound 9-oxo-10,11-dehydro-ageraphorone against aphids. In this study, we used the methods of laboratory bioassay and field experiments, and the results were shown that this compound exhibits significant aphicidal activity against bamboo aphids.

Studies have shown that the main active ingredients of E. adenophorum include alkaloids, monoterpenoids, flavonoids, sesquiterpenes, and phenols, and these compounds are mainly concentrated in the leaves (He et al. 2006, Yan et al. 2006). Of these compounds, alkaloids of E. adenophorum showed strong insecticidal activity against Aphis gossypii (Sun et al. 2004). The sesquiterpene compound 2-acetoxy-3,4,6,11-tetrahydrocadinen-7-one, a pesticidal chemical constituent of E. adenophorum, exhibits antifeedant properties against Piers rapae larvae (Zhou et al. 2003). In addition, studies have shown that extracts from E. adenophorum can control Panonychus citri and several species of weevils (Li et al. 1995).

The results of our bioassay clearly demonstrated the potent aphicidal activity of this natural product. In particular, our field results for 9-oxo-10,11-dehydro-ageraphorone showed that this compound is more effective against aphids than were 5 mg/ml of n-hexane and dichloromethane extracts from C. ficifolium against A. gossypii (Dang et al. 2010). Moreover, the effect of 9-Oxo-10,11-dehydro-ageraphorone on aphids in our field experiment was also similar to that of alkaloids from the seeds of M. cordata against cotton aphid, A. gossypii (Baek et al. 2013). Hence, the identification of this novel compound provides a basis for further in-depth research on E. adenophorum as a medicinal plant. However, further studies should first verify the toxicity of ageraphorone to beneficial insects (natural enemies) prior to the development of ageraphorone for medicinal purposes. Integrated insect control methods using plant extracts combined with conventional pesticides present a sustainable means of controlling pest species (Xu et al. 2009). E. adenophorum extracts offer a natural pesticide that can be used as an alternative to chemical pesticides. As this weedy species is readily available, it can potentially reduce the costs of pest control. With further research, increasing numbers of botanical pesticides will be identified for use in biological control.