INTRODUCTION
Introduction
Cruciferous vegetables such as broccoli, cabbage, cauliflower, kale, and collards are highly valued in Vietnam for their rich nutrients and economic significance These vegetables are extensively cultivated in regions like Sapa, Son La, and Đà Lạt Due to their short growth cycle, they are planted continuously and harvested in staggered batches Their soft stems and nutrient-rich leaves thrive in Vietnam's warm and humid climate; however, they are susceptible to various pests, including silkworms, Pieris rapae, and click beetles, which significantly impact their productivity and quality Among these pests, Pieris rapae poses the greatest threat, appearing 14 to 15 times a year with high population densities.
Pieris rapae, a small to medium-sized butterfly from the Pieridae family, is thought to have originated in the Eastern Mediterranean and is now widespread across Europe and Asia This butterfly is easily identifiable by its white wings adorned with small black dots The young caterpillars feed on the green parts of vegetable leaves, and as they mature, they consume the leaf blades, leaving only the veins intact Their waste can damage the leaves, and if their population density is high, they can severely defoliate vegetable fields.
Farmers primarily rely on chemical methods to manage Pieris rapae, utilizing pesticides extensively in vegetable-growing regions While chemical control is effective, it poses significant risks to organisms, ecosystems, and human health These concerns have led to an increased interest in biological pesticides, which offer a more environmentally friendly alternative for pest management.
Plant extracts are widely used as insecticides, but this method has limitations, including susceptibility to environmental conditions, slow action, and unpredictability Therefore, a study was conducted to identify plant extracts that demonstrate high effectiveness as insecticides.
Purpose and requirement
Testing the effect of Kaempferia galanga extract to control Pieris rapae
Testing the toxicity of Kaempferia galanga extracts on young caterpillars
Testing the antifeeding effect of Kaempferia galanga extracts on young caterpillars.
Research location
The study was performed at Department of Plant Biotechnology ,Faculty of Biotechnology and Institute of Agro- Biology in Vietnam Nation University of Agriculture
LITERATURE REVIEW
Pieris rapae
Figure 2.1 Butterfly and larvae of Pieris rapae
Pieris rapae, a butterfly species belonging to the family Pieridae, is characterized by its white wings adorned with small black dots This species primarily feeds on cruciferous crops, including cabbage, kale, bok choy, and broccoli Originally found in temperate regions of Europe, Africa, Asia, and North America, Pieris rapae has now spread to Australia, New Zealand, southern Mexico, and Hawaii The first recorded sighting of this imported butterfly in North America dates back to 1860.
The life cycle of Pieris rapae consists of four stages: egg, larvae, pupae, and butterfly, taking three to six weeks to complete, influenced by weather and temperature In Vietnam, the imported cabbageworm is present year-round, with the highest population occurring from autumn to winter when food sources are abundant.
Figure 2.2 Eggs of Pieris rapae
The egg, characterized by its yellow color and distinctive surface ridges, is typically laid on its end on the undersides of leaves These eggs are found individually rather than in clusters and generally hatch within a period of 3 to 7 days (Ronald, 2007).
The green caterpillar features a slender yellow stripe along its back and intermittent yellow stripes on each side It undergoes five instar stages, with body lengths measuring approximately 0.4, 0.6, 0.97, 1.5, and 2.2 cm At maturity, the average body length of each instar ranges from 2 to 3 cm The larvae require about 15 days to complete their development, with a range of 11 to 33 days, thriving best in conditions of 20-24°C and 75% - 85% humidity (Muggeridge, 1942).
Figure 2.3 Five stages of Pieris rapae larvae
The 1st instar larvae of Pieris rapae, upon hatching, exhibit an ivory-white or milky-white body, occasionally appearing pale yellow, with an average length of 2.76 ± 0.043 mm and a body width of 0.40 ± 0.0047 mm In the 2nd instar, the larvae transition to a green color resembling that of leaves, with increased body hair, averaging 6.75 ± 0.11 mm in length and a body width between 0.52 and 0.67 mm By the 3rd instar, the larvae are predominantly green with a light brown head, measuring an average body length of 13.45 ± 0.10 mm (ranging from 12.40 to 14.23 mm) and a body width of 0.96 ± 0.01 mm (ranging from 0.83 to 1.12 mm).
Pieris rapae exhibits the highest fluff density among its species, with an average body length of 19.11 ± 0.15 mm, ranging from 17.36 to 20.45 mm, and an average body width of 0.96 ± 0.013 mm, with measurements between 1.34 and 1.58 mm The fifth-stage larvae are particularly voracious, reaching maximum body sizes while maintaining a green coloration and a fluffy exterior, with body lengths varying from 25.89 to 30.12 mm and widths from 1.74 to 2.21 mm (Huong, 2011).
Pupae typically exhibit a green coloration, although gray or tan variants are also common They feature sharp angular projections on both the back and front These pupae are secured to the undersides of leaves using silk at the tail and a silk rope around the midsection Measuring approximately 3/4 inch in length, they can be located on the host plant or in sheltered areas up to 15 yards away The pupal development process takes about 1 to 2 weeks to complete (Ronald, 2007).
Figure 2.4 Pupae of Pieris rapae
The butterfly has a wingspan of approximately 1.5 inches, featuring black-tipped forewings Females display two black spots on each forewing, while males have only one Viewed from below, the wings appear yellowish with faint black spots visible The butterfly's body is densely covered in hair, which is white in females and darker in males Adults typically live for about three weeks, with females laying between 300 to 400 eggs They are most active during the day, frequently moving between crops and flowering weeds to feed.
Figure 2.5 Adult of Pieris rapae
The Pieris rapae larvae are highly voracious feeders, initially consuming their own eggshell before targeting the leaves of their host plant They burrow into cabbage, feeding on new sprouts, while the mustard oil present in the host plant renders them unappealing to birds To optimize nitrogen intake, these larvae adjust their feeding rates, consuming food more rapidly in low-nitrogen environments, although this may compromise their efficiency in absorbing other nutrients Despite these adaptations, no significant growth rate differences have been noted between larvae in varying nitrogen conditions (Scott, 1986) As a serious pest, the caterpillar is responsible for substantial annual crop damage, amounting to hundreds of thousands of dollars (Holland, 1931).
Larvae exhibit a behavior of dispersing their damage across plants, primarily feeding during the day while frequently changing their position to new leaves or different parts of the same leaf (Maurico, 1990) This adaptive strategy helps them avoid detection by visually-oriented predators Despite being cryptic, Pieris rapae larvae tend to remain exposed in sunlight rather than hiding beneath leaves Additionally, the growth of the larvae is significantly influenced by the condition of the host plant, particularly from the second stage onward.
8 gnaw leaf blade and remain only the veins Their waste can spoil the leaves
So if the high density is left, the field of vegetables will be bare and frayed.
Some researchs on Pieris rapae
For the past three decades, researchers have focused on plant extracts and phytochemicals to create insecticides that minimize risks to human health and the environment Insects can cause annual damage ranging from 10% to 20% (Ferry et al 2004) To combat the impact of Pieris rapae on cruciferous vegetables, numerous studies worldwide have explored various plant extracts, yielding promising results.
Seyedeh et al ( 2011) performed a study on Pieris rapae with two medicinal plants ( Artemisia annua L and Achillea millefolium L.) The
Extracts of Artemisia annua and Achillea millefolium were obtained using methanol as a solvent, following the procedure by Warthen et al (1984) Toxicity tests conducted on third-stage larvae of Pieris rapae at concentrations of 0.625%, 1.25%, 2.5%, 5%, and 10% revealed that after 48 hours, the LC50 and LC25 values for Artemisia annua were 9.387% and 4.19%, respectively, while Achillea millefolium exhibited lower LC50 and LC25 values of 3.645% and 1.69%, indicating higher toxicity Deterrency tests showed that after 24 hours, Achillea millefolium extract demonstrated a deterrency rate of 44.185%, significantly higher than Artemisia annua's 29.826% Overall, both extracts exhibited toxic and antifeeding effects on Pieris rapae larvae.
Also performed experiments on Pieris rapae, Manal (2015) et al used four plant extracts of vinca, ak, neem and chinaberry The hexane, acetone
In an experiment using a 9 and ethanol (1:1:1) solvent for extraction, cabbage leaves were treated with varying concentrations from 500 to 15,000 ppm and fed to third-stage larvae Mortality was monitored daily over a period of 7 days, revealing that all four types of plant extracts were lethal to the larvae at all concentrations Higher extract concentrations correlated with increased mortality rates, with the ak tree extract exhibiting the highest mortality at 93.33% at 10,000 ppm after 7 days Neem and vinca extracts followed closely, each causing 86.67% mortality at their highest concentrations Notably, all plant extracts resulted in over 60% mortality at concentrations of 5,000 ppm and above In conclusion, the ak extract demonstrated the most toxic effect on young caterpillars of Pieris rapae, followed by neem and vinca extracts, while chinaberry extracts resulted in 80% mortality at the highest concentration.
Esmat et al (2019) investigated the oviposition and antifeeding effects of various plant oils, including garlic, mint, thyme, camphor, colocynth, cumin, fenugreek, and orange, on Pieris rapae They conducted tests using concentrations of 250, 500, and 1000 µg/L for all the oils The study focused on the impact of these oils on the larvae at different developmental stages.
4 were used The researchers found out that, the higher the oil concentration leads to anti-feeding effects more efficient At the highest concentrations
At a concentration of 1000 àg/L, Garlic, Mint, and Fenugreek exhibited significant effects with efficacy rates of 87.21%, 86.93%, and 79.13%, respectively At 500 àg/L, Garlic, Mint, and Camphor showed effects of 76.64%, 71.90%, and 64.09%, while Thyme and Fenugreek also demonstrated effects exceeding 50% Notably, Cumin and Orange oils did not exhibit any antifeeding effects across all concentrations Oviposition testing revealed that the impact of plant oils on oviposition was concentration-dependent, with minimal effects at lower concentrations At 1000 àg/L, Mint, Thyme, and Garlic oils significantly reduced oviposition by 91.97%, 84.26%, and 76.71%, respectively.
10 were not effective, Orange with 29.60%, Fenugreek with 28.70% and Cumin with 39.76%
Research by Seyedeh et al (2011) and Manal (2015) demonstrated that extracts from dried leaves of medicinal plants exhibit significant toxic and antifeeding effects on Pieris rapae, with efficacy increasing alongside extract concentration Manal's experiment revealed that surviving larvae displayed defects Additionally, findings from Esmat et al indicate that garlic and mint oils are highly effective in reducing both the larvae of Pieris rapae and the oviposition of the white butterfly.
Kaempferia galanga L
Kaempferia galanga L., a native plant of Vietnam, is known for its insect-resistant active ingredients that have been extensively studied This perennial grass features stems that can live for many years, with roots that occasionally develop into small, egg-shaped bulbs.
Kaempferia galanga, known for its fragrant and spicy rhizomes, produces adjacent tubers with a golden brown outer shell and a nearly round light yellow to white fibrous cross-section measuring 2-3 cm This plant typically bears fruit from May to July and thrives in light, moisture, and drought conditions Young leaves emerge from April to May, growing rapidly during the summer before flowering The flowers bloom each morning and wilt by 10 o'clock, while the above-ground parts of the plant wither in winter Kaempferia galanga is distributed across India, Laos, Cambodia, China, Malaysia, Indonesia, and Vietnam, where it grows wild in various locations, often under forest canopies, and is cultivated for its ornamental and medicinal tubers.
Dried Kaempferia galanga rhizomes are rich in essential oils, comprising approximately 2.4–3.9% Key components include p-methoxycinamic acid, ethyl cinamate, and p-methoxy ethylcamide, along with other compounds such as n-pentadecan, A3-carene, camphen, O-methoxy ethylcamide, borneol, cinamic aldehydes, cineol, kaempferol, and kaempferid (Singh, 2013).
Kaempferia galanga rhizomes have a long history of use in traditional medicine, particularly in the form of alcoholic maceration applied as a liniment for rheumatism (Kanjanapothi, 2004) Additionally, the decoctions and sap from its leaves may possess hallucinogenic properties, potentially linked to unidentified chemical components in the plant's essential oil (Thomas, 2002).
Singh et al (2013) identified key compounds in the methanolic extract of K galanga, including ethyl cinnamate, ethyl pmethoxycinnamate, and p-methoxycinnamic acid, which exhibit insecticidal properties Notably, the ethyl esters of cinnamate and pmethoxycinnamate demonstrate antagonistic effects against the polyphagous larvae of Spodoptera littoralis, with an LD50 range of 0.18-0.74 µmol vial⁻¹ (Pandji, 1993) at doses of 1250 and 2500 ppm.
12 respectively of extract against subsistence and growth of neonate larvae
S.littoralis in relative to controls
Ranjan Dash et al (2017) demonstrated the effectiveness of Kaempferia galanga L rhizome extracts against S oryzae using a cold extraction method The extracts were diluted with acetone (ACR), petroether (PEF), chloroform (CHF), and methanol (MEF) at various concentrations (2.5, 5, 10, 20, 40, 50, 60, 70, and 80 mg/ml) and tested over 12 and 24 hours After 12 hours, the methanolic extract (MEF) at 80 mg/ml exhibited the highest mortality rate of 90%, while ACR and CHF showed a mortality rate of 70%, and PEF had the lowest at 50% Notably, all extracts achieved 100% mortality at the concentration of 80 mg/ml after 24 hours.
Kaempferia galanga was effecticallarvicidal activity on the second stage of dog roundworm larva, Toxocaracanis (KIuchi,1988) It was showed larvicidal activity against Culexquinquefasciatus with LC50 values of 50.54ppm
(Pitassawat,1998) The hexane fraction, dichloromethane fraction and methanolic fraction of Kaempferia galanga extract effected larvicidal and repellent activities The hexane fraction possesses larvicidal potency against
Kaempferia galanga L extract exhibits effects against various parasites and insects However, there is a lack of research evaluating the toxicity and antifeeding effects of this extract on Pieris rapae, both in Vietnam and globally.
MATERIALS AND METHODS
Materials
Larvae of Pieris rapae L (Lepidoptera: Pieridae)
The flask, petri, pipet, eppendorf tubes 1.5 ml, filter paper,
The container to feed larvae
Methods
Cabbage seeds, specifically the hybrid x2 of Nong Nghiep 1, were soaked in water for 8 hours and then incubated at 35°C for 24 hours before being planted After 12 days, the seedlings were transferred to pots, with 40 seedlings planted in the field and 300 in a greenhouse The cabbages received daily watering and NPK Dau Trau fertilizer every 20 days, without the use of pesticides The cabbage leaves were utilized for feeding young caterpillars during experiments.
Figure 3.2 The process of growing cabbage
The larvae at all stage of Pieris rapae were collected in the field in
The Institute of Agro-Biology at the Vietnam National University of Agriculture conducted a study where larvae were placed in boxes measuring 65×25×20 cm and fed cabbage leaves To maintain a healthy environment, these boxes were cleaned daily, and the larvae were kept at a temperature range of 20°C to 25°C for optimal growth.
To maintain optimal conditions for the white butterfly, the air humidity was kept between 65% and 75% After the larvae transformed into pupae, they were transferred to a net cage measuring 1.5×1.2×1m, which was placed in a greenhouse at temperatures of 20°C to 25°C and humidity levels of 60% to 70% Seven cabbages were introduced into the cage to serve as a food source for the butterflies, which would lay their eggs on these plants, allowing the larvae to feed and be utilized for experiments Additionally, three petri dishes containing cotton soaked in a honey solution (with a ratio of 8 parts water, 1 part sugar, and 1 part honey) were placed in the cages to provide essential nutrition for the butterflies.
The extract of Kaempferia galanga L was obtained using a methanol solvent following the method of Warthen et al Dried rhizomes were ground into flour, and 15g of this flour was mixed with 150ml of 85% methanol in a jar, which was then kept at room temperature for 1 hour The mixture was refrigerated at 4°C for 48 hours, followed by stirring for 1 hour with a magnetic stirrer After filtering the mixture through filter paper, a clear solution was obtained The solvent was then evaporated using a rotary vacuum evaporator at 38°C, and the crude extract was dissolved in 5ml of methanol.
16 used as origin solution This origin solution was diluted with distilled water at different concentrations to use for experiments
Figure 3.4 Preperation steps of the Kaempferia galanga L extract
The extract solution was diluted to five concentrations: 0.5%, 1%, 2%, 4%, and 8%, with an 8% methanol control Each formulation was tested on 30 stage 3 larvae, with three repetitions Prior to the experiment, the larvae were starved for 4 hours and then fed cabbage leaves treated with the extract solution The LC50 value was determined using SPSS after 24 and 48 hours.
Formula Extract concentration Stock solution (àl) Distilled water (àl)
Figure 3.5 Steps of toxicity tests
An anti-feeding test was conducted using two concentrations of 0.5% and 1%, with control samples consisting of 0.5% and 1% methanol Cabbage leaves were treated by dipping them into the solution and then dried for 30 seconds at room temperature Following a 4-hour starvation period, larvae were fed the treated leaves.
10 larvae and repeated three times Consumption was determined by using ImageJ software The index of antifeeding was calculated as:
C : The consumption of control leaf
Figure 3.6 Steps of antifeeding test
Experiments
Experiments 1 Testing toxicity of the Kaempferia galanga extract on Pieris rapae larvae
In this research content, Kaempferia galanga L would be extracted on methanol solvents
The stock extract was diluted to various concentrations (0.5%, 1%, 2%, 4%, and 8%) and tested on Pieris rapae larvae To assess the toxic effects of the extract, the number of dead larvae was recorded and evaluated after 24 and 48 hours.
Experiment 2 Testing the effect of Kaempferia galanga extracts on the antifeeding of Pieris rapae larvae
The stock extract was diluted at 0.5%, 1% concentration and tested to the antifeeding and calculare area of leaf infestation in the control and experimental treatments
RESULTS AND DISCUSSIONS
Experiment 1 Testing toxicity of the Kaempferia galanga extract on
Testing toxicity of the Kaempferia galanga extract were performed on 30 Pieris rapae larvae at stage 3 After 24 hours and 48 hours, the obtained results are presented in table 4.1 and Figure 4.1
Table 4.1 Mortality rate of larvae during Kaempferia galanga L treatment
Mortality rate (%) After 3 h After 24 h After 48 h
Figure 4.1 Mortality rate of larvae after 24 hours of treatment y = ‐0.3541x 2 + 15.348x + 0.459
Treatment with Kaempferia galanga extract at concentrations of 0.5%, 1%, 2%, and 4% resulted in larvae crawling out of the leaves, while at 8% concentration, the surviving larvae showed no movement In the initial 3 hours, larvae in the control group and those exposed to 0.5% and 1% concentrations began feeding on the leaves However, larvae treated with 2% to 4% of the extract did not consume any leaves, and at 8% concentration, half of the larvae were found dead.
The study revealed that mortality rates increased with higher concentrations of the extract after 24 hours The LC50 value for Kaempferia galanga was determined to be 2.474% with a 95% confidence interval, indicating a high level of toxicity to Pieris rapae larvae due to the low LC50 values.
The Kaempferia galanga extract was prepared following the method of Warthen et al (1984), using 30g of dried plant material in 300 ml of 85% methanol According to Seyedeh et al (2011), the LC50 values for the A annua extract were reported.
A millefolium extract on Pieris rapae larvae were 9.387% and 3.645%
Compare with the analytical results in Table 4.2, the LC50 value for
Kaempferia galanga extract was lower, so Kaempferia galanga extract was more toxic than A annua and A millefolium extracts
Extract of Kaempferia galanga contain ethyl esters of cinnamate and pmethoxycinnamate that are antagonistic toward the polyphagous of
Spodoptera littoralis larvae (Pandji, 1993) The hexane fraction, dichloromethane fraction and methanolic fraction of Kaempferia galanga extract effected larvicidal against C quinquefasciatus (Pitassawat,1998)
Rhizome extracts of Kaempferia galanga demonstrated a significant effect against S oryzae, achieving 100% mortality at a concentration of 80 mg/ml after 24 hours (Dash, 2017) This study highlights the potential of Kaempferia galanga rhizome extract as an effective treatment.
23 showed potent activity against Pieris rapae Thus, the Kaempferia galanga extracts has high potential in control pests
Table 4.2 Dose–response parameters of Pieris rapae after 24 hours
Plant extract N LC50 (95%CI) dfᵇ SE
Figure 4.2 Testing toxicity of (4%) Kaempferia galanga extract on Pieris rapae larvae
Experiment 2 Testing the effect of Kaempferia galanga extract on the anti-feeding of Pieris rapae larvae
Testing the effect of Kaempferia galanga extracts on the antifeeding of
The study involved 30 third-stage Pieris rapae larvae, with three control samples and three treatment groups Leaf consumption was assessed by photographing the leaves and measuring their area at 3, 6, and 24 hours The results of the leaf area consumption by Pieris rapae larvae are detailed in Table 4.3, Figure 4.3, and Figure 4.4.
Table 4.3 Deterrence percentage acreage leaves of Kaempferia galanga extract on Pieris rapae larvae
Fomula Concentration Mean Difference between means (B - A) ± SEM
Figure 4.3 The leaf consumption of larvae in the treatment with 0.5%
Figure 4.4 The leaf consumption of larvae in the treatment with 1%
The study found no significant difference in leaf consumption between the control and treatment samples at both 0.5% and 1% concentrations Specifically, the control samples exhibited leaf consumption areas of 32.28 and 39.24, while the treatment samples showed areas of 25.93 and 41.40 for the same concentrations The P-values for the 0.5% and 1% extract treatments were 0.3646 and 0.7671, indicating that neither concentration was effective in reducing feeding behavior.
As announced by Seyedeh et al in 2011, Achillea millefolium L extract (with deterrence was 44.185%) and Artemisia annua L extract (29.826%)
Similarly, Kaempferia galanga extract was low effective Kaempferia galanga extract was not high effectively antifeeding as well as Garlic, Mint,
Fenugreek oils with 87.21%, 86.93%, 79.13% as reported by Esmat et al in
Figure 4.5 The effect of Kaempferia galanga extract on the antifeeding of
CONCLUSIONS AND SUGGESTIONS
Conclusions
- Kaempferia galanga extract was toxic to Pieris rapae larvae, at 8% concentration the mortality was 100% after 24h The LC50 values for
Kaempferia galanga were 2.474% at 95% CI
- At 0.5% and 1% concentration, Kaempferia galanga extract did not have antifeeding effect on Pieris rapae larvae.
Suggestions
Due to the limited time and research conditions and not have expanded the topic, so we would like to give some suggestions as follows:
- Testing toxicity of the Kaempferia galanga extract on Pieris rapae larvae at stage 4 and stage 5
- Testing the effect of Kaempferia galanga extract on oviposition of
- Testing the self-defense respone to Kaempferia galanga extract in cabbage plants
- Combine Kaempferia galanga extract with some binders to make the extract exist longer and stable in the environment
The study by Hashemnia S Seyedeh et al (2011) investigates the impact of crude leaf extracts from Artemisia annua L and Achillea millefolium L on the toxicity, development, feeding efficiency, and chemical activities of the small cabbage butterfly, Pieris rapae L The findings, published in Pesticide Biochemistry and Physiology, highlight the potential of these plant extracts as natural pest control agents, offering insights into their effectiveness against this specific lepidopteran pest.
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LOG Transform Cannot be Done 0
Number of Responses > Number of Subjects 0
Number of Iterations Optimal Solution Found
Parameter Estimate Std Error Z Sig 95% Confidence Interval
Intercept -1.025 171 -6.000 000 -1.196 -.854 a PROBIT model: PROBIT(p) = Intercept + BX (Covariates X are transformed using the base 10.000 logarithm.)
The PROBIT Pearson Goodness-of-Fit Test yielded a statistic of 10.901 with 3 degrees of freedom and a significance level of 012 Since this significance level is below 050, a heterogeneity factor is applied in calculating the confidence limits Additionally, it is important to note that statistics derived from individual cases may differ from those based on aggregated cases.
Number concentration Number of Subjects Observed
Responses Expected Responses Residual Probability
Probability 95% Confidence Limits for concentration
95% Confidence Limits for log(concentration)b
Estimate Lower Bound Upper Bound Estimate Lower Bound Upper Bound
.990 22.632 6.933 3959029.003 1.355 841 6.598 a A heterogeneity factor is used b Logarithm base = 10.