Toxicity and Antifeedant Activity of Calotropis gigantea L. Leaf Extract Against Plutella xylostella L. (Lepidoptera: Plutellidae)

Toxicity and Antifeedant Activity of Calotropis gigantea L. Leaf Extract Against Plutella xylostella L. (Lepidoptera: Plutellidae)

Nur KhasanahEdhi Martono Y Andi Trisyono Arman Wijonarko 

Faculty of Agriculture, Tadulako University, Palu, Central Sulawesi 94118, Indonesia

Faculty of Agriculture, Gadjah Mada University, Yogyakarta 55281, Indonesia

Corresponding Author Email: 
khasanah@untad.ac.id
Page: 
677-682
|
DOI: 
https://doi.org/10.18280/ijdne.160609
Received: 
7 November 2021
|
Revised: 
2 December 2021
|
Accepted: 
8 December 2021
|
Available online: 
21 December 2021
| Citation

© 2021 IIETA. This article is published by IIETA and is licensed under the CC BY 4.0 license (http://creativecommons.org/licenses/by/4.0/).

OPEN ACCESS

Abstract: 

Many parts of the Calotropis gigantea plant are known to contain bioactive compounds, but leaves contain the most. This study aimed to determine the toxicity and antifeedant activity of C. gigantea leaves against Plutella xylostella. The study was carried out from November 2019 to July 2020. Toxicity was tested using the leaf dipping and spraying methods. Antifeedant activity was tested using a no-choice test and a choice test. Identification of the compound composition of the leaf extract of C. gigantea was carried out at the Integrated Research and Testing Laboratory, Gadjah Mada University. Extract toxicity data obtained were analyzed by Probit analysis. The results showed that the antifeedant activity of C. gigantea leaf extract a no-choice and with choice at each concentration had a significant effect on the consumption of P. xylostella larvae rations. The toxicity (LC50) of the leaf extract of C. gigantea to P. xylostella by the dipping method was 2,958 µgl-1 while the spraying application was 3.944 µgl-1. The composition of chemical compounds contained in the leaf extract of C. gigantea is saponins, alkaloids, flavonoids, tannins, phenols, terpenoids. With the composition of these chemical compounds, the leaf extract of C. gigantea has the potential as a source of vegetable insecticide compounds against P. xylostella.

Keywords: 

antifeedant, compound composition, leaf dipping, spraying

1. Introduction

Plants and insects have a long history of interacting with each other, especially phytophagous insects that consume plant tissues. Plants, when interacting with herbivores, produce secondary metabolites to protect themselves from herbivores or pathogens [1]. Plants are sources of various chemical compounds with rich bioactivities [2]. Plant-based chemical compounds are produced by above and below-ground plant tissues, while both support the defenses mechanism of plants against insects [3]. Chemical compounds related to plant defense (secondary metabolites) include alkaloid, saponin, tannin, phenol and terpenoids. Secondary metabolites act as toxics, repellence, or anti-nutritious against herbivores and are a source of promising biopesticides for pest management [1].

The use of secondary metabolites for pest management is an effective alternative solution. According to Campos et al. [4], secondary metabolite compounds have several mechanisms of action against targeted insects and have low toxicity on non-target organisms. Secondary metabolites act as toxics, deterrents, repellents and reduce nutritional content [5]. Toxic compounds from plant secondary metabolites can be used as botanical insecticides [1]. Many research has shown plant extracts as potential bio-insecticides. Several plant families that have been recognized and formulated into commercial botanical insecticides, include Amaranthaceae, Asteraceae, Fabaceae, Meliaceae, Piperaceae, Salicaceae, Solanaceae [6].

Calotropis gigantea belongs to Asclepiadaceae. C. gigantea grows across Southeast Asia, Pacific Island, Australia, South America and Africa [7]. C. gigantea is known as a weed that produces white sap from its whole structure and has potential as a medicinal plant and its activity as insecticide. C. gigantea belongs to Asclepiadaceae that includes 280 genera and approximately 2,000 species. Calotropis gigantea (Linn) R.Br. and C. procera (Ait) R.Br. are two interrelated species. Both have similar botanical appearances and pharmacological effects [8]. Plant parts of C. procera and C. gigantea are toxic [9]. Compounds contained in C. gigantea leaf extract consist of alkaloids, steroids, terpenoid, flavonoid, tannin, and phenol [7, 8].

C. gigantea leaves are parts that contain the most phytochemicals compared to other plant parts. Saps contain secondary metabolites that are used for defenses against herbivores and pathogens [10]. Bioactive compounds within C. gigantea cause it to resistant against insect and phytopathogen [11]. Toxicity test C. gigantea leaf against Helicoperva armigera showed that leaves had highest larvicidal activity compared to other plant parts and had antifeedant activity against H. armigera [12]. Based on these activities, C. gigantea leaf extracts have potential as a source of bioactive compounds with insecticidal properties against Plutella xylostella. This research aims to determine toxicity and antifeedant activity of C. gigantea leaf extract against P. xylostella. Considering the importance of food safety for consumers and the environment, finding sources of bioinsecticide is very important. This research hopes to provide information for the use of potential of active ingredients in C. gigantea as a bioinsecticide, especially as a toxin and antifeedant against P. xylostella.

2. Materials and Methods

2.1 Insect collection and rearing

P. xylostella was collected from Cangkringan Village, Yogyakarta Province and kept in a greenhouse. Maintenance includes planting seeds of bok choy (Brassica rapa var. chinensis) as food and as a place to lay P. xylostella eggs. Maintenance of bak choy plants by giving organic fertilizer which aims to obtain good plant growth [13].

Four plants were placed in four rearing boxes measuring 25 x 40 cm along with twelve pairs of P. xylostella imago which were fed 10% honey solution. the. Every three days, the plants were placed in an oviposition box measuring 40 x 60 cm. Plants are replaced until the imago dies. Thus, it was obtained that the larval needs of 4,400 individuals would be met through the insect collection and rearing.

2.2 Calotropis gigantea leaf extract

Extraction was done using a maceration method [14]. As much as 350 g of macerated C. gigantea leaf was added into a container, 1.4 L of ethanol was added and stirred to prevent sedimentation. The product was then sealed and stored in room temperature and reduced exposure to direct sunlight. Immersion was done for 48 hours, filtered using filter paper and Buchner funnel. Solution and solvent were separated using a Vacum Rotary evaporator (Heidolph; Weirtheim and Waterbath Cole-Parmer type 7049-05) at temperatures 40 - 180℃ and 160 - 280 rpm for 3 hours.

2.3 Toxicity test

P. xylostella larvae were tested using a feeding assay. The bok choy leaves were washed and made into circular leaf discs with a diameter of 2.5 cm. Petri dishes (diameter 9 cm height 2 cm) were used as bioassay arena. Concentration used were 15, 7.5, 3.75, 1.875, 0.937 µgl-1 and control. Five bok choy leaf disc was immersed into solutions for 1 minute and air dried on a petri dish. Twenty larvae were placed on to the petri dish according to designated treatment. Mortality percentage was observed every 24, 48, and 72 hours. Contact assay was done using a spraying assay. Concentration used were 20, 10, 5, 2.5, 1.250 µgl-1. Twenty larvae were placed on a petri dish and each concentration was replicated 10 times. As much as 2 ml of C. gigantea leaf extract were sprayed using Potter Precision Laboratory Spray Tower (Burkard Manufacturing Co. Ltd. Rickmansworth Herts. UK) with aperture nozzle of 0.6985 m, pressure at 1.12 kg cm2. Percentage of mortality was observed every 24, 48, and 72 hours.

2.4 Antifeedant test

Antifeedant bioassay was done using a no-choice antifeedant test and choice test [15]. Bok choy leaves were washed and made into 2.5 cm disc using a cork borer and placed on a petri dish. Concentration used were 15, 7.5, 3.75, 1.875, 0.937 µg l-1 and control. In the no-choice test, six-leaf discs were immersed in C. gigantea leaf extract for 1 minute according to designated concentration and air-dried. Twenty larvae were placed into petri dishes according to designated treatments. Choice test was done using 3 bok choy leaf that were immersed in C. gigantea according to designated concentration and 3 leaf discs immersed in distilled water for 1 minute. Treated and untreated leaves were place alternated within the petri dish in a circular pattern with the middle part empty for placing larvae. Twenty larvae were placed in the space and allowed to choose leaf disc. Each treatment was replicated 10 times. Area of consumed leaves were calculated using previous stated method. Observations were done every 3, 6 and 9 hours after treatment.

2.5 Identification of chemical compounds within C. gigantea

Two grams of C. gigantea were extracted and then tested to determine the class of compounds which included total alkaloids, tannins, phenols, flavonoids, saponins and quantitative tests of terpenoids. The total alkaloids, tannins, phenols, flavonoids, and saponins were performed using spectrophotometry, while terpenoids were performed using thin layer chromatography [16].

2.6 Data analysis

Probit analysis [17] was done using SAS JMP Pro.v13.2.1 to obtain LC50 and LC95 on test insects. Antifeedant was calculated using formula [16]: antifeedant no choice test = (C-T) / C x 100%, antifeedant choice test = (C - T)/(C + T) x 100. C = leaf area consumed leaf in control, T = leaf area consumed from treatment. ANOVA was done using SPSS 16.0 at α = 5% and if significantly different, a post-hoc test using DMRT was done.

3. Result and Discussion

3.1 Toxicity test

Toxicity against P. xylostella from the leaf dipping bioassay (Table 1) demonstrated probit analysis with LC50 estimation of 2.958 µgl-1 with 95% CI between 2.415 µgl-1 to 3.565 µgl-1. Toxicity of C. gigantea extract showed P. xylostella larvae was higher as concentration increased even though the less leaf consumed. Research [18] showed high concentration of Argemone ochroleuca extract resulted in high mortality of Spodoptera frugiperda larvae even though only a small portion of leaves are consumed. A decrease in feeding rates is a result of feeding deterrent or sublethal toxicity [19]. This implies that insect death is a cause of a combination between starvation and compounds contained in C. gigantea extract. According to the study [20], all parts of the C. gigantea plant contain toxins in the leaves, flowers, sap, stems and roots. Bioactive compounds cause poisoning, knockdown, locomotive instability, paralysis and slow death, and inhibition of eating behavior [21]. Symptoms of P. xylostella larvae after consuming diets were slower to no movement of larvae from diets and larvae color turn pale green to light brown to blackish brown and finally die. Toxicity of P. xylostella larvae using spraying application (Table 1) had a lethal concentration (LC50) estimation of 3.944 µgl-1 with 95% CI between 3.219 µgl-1 to 4.794 µgl-1. After application, larvae moved in a random direction but became calmer when moved to untreated diets. However, after 24 hours, several larvae died and mortality increased until 48 hours with symptoms of exposed insects to experience corrosion, browning, and died.

Table 1. Toxicity of Calotropis gigantea leaf extract using dipping and spraying method on 3rd instar Plutella xylostella

Method

Volume

n

Slope (± SE)

LC50

df

Dip

40

1200

0.480 (± 0.058)

2.958 (2.4146 - 3.565)

3

Spray

40

1200

0.430 (± 0.042)

 3.944 (3.2198 - 4.7947)

3

Dip: Bok choy leaf discs were dipped into Calotropis gigantea leaf extract, larvae were placed into containers with diets and observed 72 hours before mortality count. Spraying: Larvae were placed into a petri dish and sprayed with Calotropis gigantea leaf extract

Table 2. The antifeedant activity of Calotropis gigantea leaf extract using choice and no-choice test of third instar Plutella xylostella larvae (n = 1000) at 3, 6 and 9 hours

Concentration (µgl-1)

Antifeedant index + SE

No-Choice test

Choice test

3 hours

6 hours

9 hours

3 hours

6 hours

9 hours

15

83.30 ± 5.43b

87.72 ± 2.97d

89.83 ± 1.63d

91.79 ± 5.43a

87.95 ± 4.85a

80.32 ± 6.27a

7.5

79.19 ± 3.93b

73.47 ± 7.01cd

81.61 ± 4.33d

72.46 ± 7.01ab

64.84 ± 6.96a

66.74 ± 5.53a

3.75

74.83 ± 4.55b

48.98 ± 9.24bc

63.15 ± 7.41c

63.63 ± 7.58ab

61.98 ± 6.72ab

58.20 ± 5.49ab

1.875

49.05 ± 7.26a

34.05 ± 9.27ab

29.21 ± 9.71b

63.62 ± 7.90bc

57.68 ± 6.72b

48.21 ± 5.56b

0.937

38.23 ± 6.41a

11.24± 13.90a

3.82 ± 3.39a

48.82 ± 8.10c

46.08 ± 5.81c

48.09 ± 5.26c

Note: SE (Standard error). Mean antifeedant index ± SE followed by di same letter in the same column at each observation time were not significantly different based on Duncan (P ˂ 0.05)

Toxicity of C. gigantea leaf extract when applied using spraying method caused higher mortality as concentration increased. Mortality level is affected by concentration, the content of bioactive compounds and physiological characteristics of P. xylostela. In general, toxicity is affected by dosage, application time, and individual characteristics, such as the sensitivity of action cites, and development stage [22]. P. xylostella larvae have soft and fatty cuticles causing compounds in the extract can easily penetrate the cuticle. Cuticles have physical characteristics consisting of chitin, protein, water, mineral, and tanner substance and relevance that affect the mechanical or elasticity aspect of the cuticle [23]. The structure and thickness of the cuticle affect insecticide penetration levels [24].

3.2 Antifeedant activity

The antifeedant activity of the non-choice test (Table 2) at 3 hours after application of C. gigantea extract at each concentration showed a different index of antifeedant. Concentration 15 to 0.937 µgl-1 significantly (p < 0.05) reduce leaf consumption of P. xylostella. Therefore, 15, 7.5, 3.750 µg l-1, C. gigantea leaf extract decreased leaf consumption of P. xylostella by 83.30%, 79.19% and 74.83% compared to concentration of 1.875 and 0.937 µgl-1 decreased consumption of 49.05 and 38.23%. Antifeedant activity at 6 hours, C. gigantea leaf extract significantly decrease leaf consumption of P. xylostella by 87.72; 73.47; 48.98; 34.05 and 11.24%. The concentration of 15 and 7.5 µgl-1 demonstrated the highest antifeedant activity compared to other concentrations. Antifeedant active at 9 hours, each concentration of C. gigantea leaf extract decreased leaf consumption of P. xylostella by 89.83; 81.61; 63.15; 29.21 and 38.23%. At all observation times (3.6 and 9 hours) the antifeedant activity showed a concentration of 15 µgl-1 giving the highest antifeedant activity and significantly different with 1.87 and 0.937 at 3 hours and 3.75, 1.875 and 0.937 µgl-1 at 6 and 9 hours but not significantly different from the others. The concentration of 15 and 7.5 µgl-1 at 9 hours of C. gigantea leaf extract had higher inhibition compared to 3 and 6 hours.

Choice antifeedant activity (Table 2) at 3 hours showed that leaf consumption was significantly affected by C. gigantea leaf extract by 91.79; 72.46; 63.6; 63.62 and 48.82%. Choice test at 6 hours demonstrated antifeedant activity by 87.95; 64.84; 61.98; 46.08 and 45.85%. At 9 hours, antifeedant activity significantly decreases leaf consumption of P. xylostella larvae by 80.32; 65.20; 58.20; 48,12 and 48.09%. At a concentration of 15 µgl-1 in all observations (3, 6, and 9 hours) showed the highest antifeedant activity but not significantly different from 7.5 µgl-1 and 3.75 µgl-1 but significantly different from the others. The antifeedant activity of C. gigantea leaf extract at 6 and 9 hours showed a decrease of inhibition at untreated control. At the beginning, all larvae moved to diets. However, after 2-3 hours, larvae started to consume leaf disc, except for concentration at 15 µgl-1.

Antifeedant activity from the no-choice test with C. gigantea leaf extract caused leaf consumption of P. xylostella to decrease based on leaf extract concentrations. Feeding levels decrease respectively to leaf extract concentrations and are consistent with similar research on H. armigera [12]. A higher antifeedant index shows a decrease in feeding [25]. At the beginning of this bioassay, larvae moved randomly and did not directly consume disc leaves. Larvae would lightly consume leaves and continue to move again. Results from other research showed that azadirachtin affected S. litura selectivity of azadirachtin odor over time [26]. According to [1] chemical compounds in plants have to potency to affect insect behavior by deterrence and repellency.

Antifeedant activity on P. xylostella larvae at concentrations of 15 and 7.5 µgl-1 at 3, 6 and 9 hours showed an increase of inhibition compared to other concentrations. An increase of antifeedant activity at that concentration demonstrated that leaf extract still possessed antifeedant activity against P. xylostella. Antifeedant activities at 3, 6 and 9 hours showed high antifeedant activity. At concentrations of 15 µgl-1 at 3 hours showed feeding inhibition by 91.79%; however, at 6 and 9 hours, inhibition decrease to 87.95 and 80.32%.

The antifeedant test was carried out with different methods, namely choice and no-choice. In the no-choice antifeedant test, feed at each concentration was placed in one petri dish and untreated feed was put in another petri dish, then twenty larvae were added in each treatment given the opportunity to consume the provided feed. The untreated feed was intended as a control to be used as a comparison in determining antifeedant activity. While the choice of antifeedant feed was given to each treatment and without treatment was placed in the same petri then 20 larvae were added to choose the feed and then the antifeedant activity was measured. Research of [27] showed that essential oil of clove leaves inhibited the feeding of S. litura. According to previous researches, secondary metabolites modulate insect behavior [28, 29]. Antifeedant activity stimulates specific feeding resistant nerves chemoreceptors on insect mouth parts that work synergistically with other receptors [25].

According to [30], the appearance, taste and odor of the extract can change the behavior of insects. Biting speed is considered to be more effective as an antifeedant than a repellent Apart from odor, the behavior of P. xylostella larvae was also greatly influenced by the concentration and chemical compounds found in the extract. The chemical stimulants that act on certain concentrations against insect receptors might affect host acceptance [31]. The behavior of P. xylostella larvae towards the chosen feeds indicated that C. gigantea leaf extract is considered a deterrent. Repellent and deterrent compounds in the feeds cause insects to stop feeding [32].

3.3 Compound group identification in C. gigantea leaf extract

The compound content of C. gigantea leaf extract consisted of alkaloids (0.90%), tannin (5.94%), phenol (9.88%), flavonoids (2.20%), saponin (4.95%), and terpenoid (positive) (Table 3). These results showed that the phytochemicals contained in C. gigantea leaves were the secondary metabolites. Research [11], the composition of compounds in C. gigantea leaf extracts were alkaloid, carbohydrates, glycoside, phenolic/tannin, protein or amino acid, flavonoid, saponin, sterol, and resin. Alkaloid, saponin, glycoside, tannin, flavonoid, terpenoid are used for plant defense against herbivores and have been reported to possess insecticidal properties [33].

The compounds considered as toxic against P. xylostella were saponins, alkaloids, tannins, and terpenoids. Saponins are toxic and important for plant defense [34]. Saponins are toxic and antifeedant [35]. Some saponins form complexes with proteins [36] that inhibit proteinase and affect insect digestion [27]. Saponins indirectly affect the insect gut microbiome, several digesting enzymes, and mucous membranes. Saponins strongly affected lepidoptera cells, especially midgut primer cells [37]. Alkaloids act to defend plants from herbivores [27], is toxic [38], inhibit amylase activity, breaks plasm membranes and causes death [39]. Tannins are also toxic against several insects by affecting the growth and development of insect by binding with protein, decreasing nutrition absorption, and causing lysis in the midgut [40].

The compound composition of C. gigantea leaf extract that act as antifeedant is terpenoid, alkaloid, saponins, tannin, phenol and flavonoid.

Table 3. Chemical composition of Calotropis gigantea leaf extract

Group of compounds

A total compound in material (% w/w)

Alkaloid

0.90

Tannin

5.94

Phenol

9.88

Flavonoid

2.20

Saponin

4.95

Terpenoid

Positive

Note: positive (extract contains Terpenoids)

In general, alkaloid is a feeding deterrent for insects [29] [26]. Saponins act as antifeedant [35]. Tannins act as feeding deterrents [40]. Phenol acts as defend compounds against herbivores [41] and can be a potential compound for insect management [38].

4. Conclusion

C. gigantea leaf extract had contact toxicity and antifeedant activity against P. xylostella larvae. Mortality and antifeedant increase as the concentration of C. gigantea increased on the no-choice test and choice test. The compound composition of saponin, alkaloid, flavonoid, tannin, phenol, terpenoid within C. gigantea leaf extract determined toxicity and antifeedant activity.

Acknowledgement

The author would like to thank the Directorate General of Education and Culture of the Republic of Indonesia for funding the first author doctoral degree through the Beasiswa Unggulan (BUDI-DN). The author would also like to thank Sriyanto Harjanto for assisting throughout the research.

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