MORTALITY AND REPELLENT ACTIVITY OF CLERODENDRUM VISCOSUM VENT. (VERBENACEAE) AGAINST SITOPHILUS ORYZAE (COLEOPTERA: CURCULIONIDAE)HTML Full Text
MORTALITY AND REPELLENT ACTIVITY OF CLERODENDRUM VISCOSUM VENT. (VERBENACEAE) AGAINST SITOPHILUS ORYZAE (COLEOPTERA: CURCULIONIDAE)
Talukdar Muhammad Waliullah* 1, Akter Mst Yeasmin 1, Islam Md. Wahedul 2 and Hassan Parvez 2
Department of Bioscience 1, Molecular and Cell Biology Laboratory, GSST, Shizuoka University, 836-Oya, Suruga-ku, Shizuoka 422-8529, Japan.
Institute of Biological Sciences 2, University of Rajshahi, Rajshahi - 6205, Bangladesh.
ABSTRACT: In continuation of our study we have screened out the root, leaf, and stem of C. viscosum against rice weevil, S. oryzae extracted into ethanol, ethyl acetate, and chloroform. A correlation was found between the insecticidal and repellent activity observed by residual film assay technique and repellency test. It is interesting to note that these plant extracts showed higher to less mortality activity by using 3.93, 1.97, 0.98, 0.49 and 0.25 mg-cm-2 doses. From the overall assessment suggested that the root extracts showed most potency. Ethanol fraction of root extract exposed 44-84% mortality with 0.39 mg/ml of LC50 value at 72 h of exposure and 95% confidence limit was 0.19 - 0.80. Repellency test carried out ethanol fraction. All the extracts showed 100% repellency (Class V category repellency) in dose no 1 to a dose no 5. The degree of repellency response among the three parts of the test insect was significantly different (P<0.05). Class V category repellency was observed for root and leaf up to dose 0.06 mg-cm-2 (Dose no 7). Whereas, the lowest dose at which the stem exhibited class V category repellency at 0.12 mg-cm-2. The root extract showed strong repellent activity than the leaf and stem. The susceptibility of contact toxicity in the intensity of solvents was found ethyl alcohol > ethyl acetate > chloroform fraction. The dose-mortality order of the C. viscosum extracts is root > stem > leaf.
Insecticidal activity, Residual film assay, Contact toxicity, Medicinal plant, Ethyl alcohol
INTRODUCTION: Today, humans are waging an undeclared war against insects in the competitive struggle for existence, and almost no crop in the World is free from attack by insects, at least to some degree 1. Farmers have been using plant extracts in pest control for centuries. Botanical insecticides are one of the best alternatives for hazardous chemical insecticides.
Phytochemicals can induce different types of abnormalities in insects that could safely be used for insect pest control 2. This method of pest control provides an ideal source of low cost, safe and effective pesticides. Extracts of plant products rely on the solubility of the active compounds. Different solvents are utilized, the most commonly used as acetone, chloroform, ethanol, hexane, petroleum ether, ethyl acetate or methanol. Extracts have been applied in various forms of insect bioassay; residual film assay technique or topical application as mortality test, repellency trials, etc.
The plant bhant tree, Clerodendron viscosum Vent. (Verbenaceae), exhibits a wide spectrum of folk and indigenous medicinal uses 3, 4, 5, 6 but little information about its bio-efficacy against stored product insect pest rice weevil, Sitophilus oryzae L. (Coleoptera: Curculionidae). This scarcity was addressed in the present experiment using three solvents (ethanol, ethyl acetate, and chloroform) carrying on root, leaf and stem extracts. S. oryzae is the most widespread and destructive major insect pest of stored cereals throughout the world. Female rice weevil oviposits directly into the seeds and completes larval development inside the seeds and emerges as adults. S. oryzae has been reported to developed resistance to synthetic insecticides 7. The plant-derived chemicals have been used as potential seed protectant (insecticides and antifeedants) often begins with the screening of plant extracts 8. It is logical to expect biologically active compounds to be produced by plants as a chemical defense measure against their enemies 9.
MATERIALS AND METHODS:
Plant Collection and Identification: The plant specimen was collected from Rajshahi University campus, Rajshahi-6205, Bangladesh. Identification of voucher specimen was confirmed at the taxonomical section, Department of Botany, Rajshahi University, Bangladesh and dried in the shade. The Plant materials root, leaf, and stem were powdered in a grinding machine. Before grinding the plants were well dried in an oven at 37 ± 0.5 °C for overnight.
Insects: Adults of S. oryzae were collected from the wheat infested rearing cultures maintained in the control temperature (CT Room 30 ± 0.5 °C) and 70-80% RH with photoperiod 12h L: 12h D), Integrated Pest Management (IPM) laboratory, Institute of Biological Sciences, the University of Rajshahi for five years. A standard mixture of sterilized (at 60 °C for 24h) whole-wheat flour with powdered dry yeast in a ratio of 19:1 was used as food medium in the experiments 10, 11, 12.
The Output of Extracts: The root, leaf and stem were extracted separately with ethyl alcohol, chloroform, and ethyl acetate. The extracts condensed using rotary evaporator and weighed. The yield of the extracts according to solvents and plant parts are presented in Table 1.
TABLE 1: EXTRACTS FROM DIFFERENT PARTS OF C. VISCOSUM WITH DIFFERENT SOLVENTS
|Plants part||Solvents||Wt. of dust. (g)||Wt. of ext. (g)|
Pilot Experiment: Same ages of 25 adult pests were used for making different doses of this experiment with five replications. The pilot experiments were carried out by indications made by the produces for the users, to obtain doses in which mortality rate was in between 12-84% Table 3. The actual doses were calculated from the amount of extract present in 1 ml of the solution, and then the amount of active ingredient was also worked out. The calculated amount of the active ingredient of the extract was expressed in mg-cm-2.
Bioassay: From the result of the pilot experiment, a dose was selected. That was considered as the stock solution, and then the final doses were made by serial dilution technique with the same solvent at the proportion of 1: 2 in treatments.
Five different condensed extracts viz. 12.5, 10.0, 6.25 and 3.125 gm were dissolved in the requisite amount of respective solvents and transferred uniquely into 3.93, 1.97, 0.98, 0.49 and 0.25 mg-cm-2 concentrations by measuring the dry-weight of extracted materials and divided by the surface area of a petri dish (90 mm) according to the residual film method 13. The extract was dispersed into each petri dish for each dose. After evaporating the solvents from the petri dish 25 same-aged (3-5 day old) adult, S. oryzae was released separately by an aspirator sub sequentially control batch was maintained with respective solvent only. Then the petri dish kept within an incubator at 30 ± 0.5 °C. The mortality was recorded after 24, 48 and 72h of exposure. The dose was calculated by measuring the dry-weight of the crude extracts applied in petri dish divided by surface area of the respective petri dish.
Statistical Analysis: Mortality percentage was corrected using Abbott’s formula 14. Then this percentage of mortality was subjected to statistical analysis according to Finney 15.
Repellency Test: To study the repellent activity of plant extracts on the basis of McDonald 16 with some modification, filter paper strips (Whatman no. 40) 9 cm in diameter cutting in half. To make stock solution 10 mg-dried samples was dissolved into 1 ml ethyl alcohol and considered as the highest dose. The eight doses of 3.93, 1.97, 0.98, 0.49, 0.25, 0.12, 0.06 and 0.03 (mg-cm-2) were made by serial dilution technique at the proportion 1:2 from the stock solution and were used in repellency treatments of the subjected insects. One ml solution of plant extract from the respective dose was applied uniformly on the half of filter papers with a micropipette. Then the treated half circles were air dried to evaporate the solvent completely.
Attaching a treated half to an untreated half circle of the same dimension by transparent, non-toxic adhesive tape full circle then remake one. Ten insects were released on the center of each filter paper circles, and Petri dishes were covered immediately. There were three replications for each treatment. Counts of the insects present on each strip were made at one-hour interval up to five hours observation. The averages of counts were converted into percentage repulsion (PR) using the formula of Talukder and Howse 17, 18;
PR = (Nc– 5) × 20
Where, Nc is the number of insects present in the control half. Positive values (+) indicated repellency and negative values (-) attraction. The average values were then categorized according to the following scale presented in Table 2.
TABLE 2: AVERAGE VALUE OF REPELLENCY RATE OF S. ORYZAE
|Class||Repellency rate (%)||Class||Repellency rate (%)|
|0||>0.01 to <0.1||III||40.1 to 60|
|I||0.1 to 20||IV||60.1 to 80|
|II||20.1 to 40||V||80.1 to 100|
Statistical Analysis: Repellency activity test was conducted according to a completely randomized experimental design with three replications for each treatment. Using Duncan’s Multiple Range Test (DMRT) as described by Steel and Torrie 19 did repellent significance. Data on percentage were subjected to arcsine transformation of the proportion before analysis and were transformed back to the percentage of presentation.
RESULTS AND DISCUSSION: The mortality (%) of S. oryzae adults treated with root, leaf, and stem of C. viscosum extracts within three solvents. Ethyl alcohol root and stem extract of C. viscosum showed highest mortality activity 44-84% at 72h of exposure and 40-80% mortality for root extract; 36-76% mortality for stem extract at 24h of observation period within 0.25 and 3.93 mg-cm-2 doses. LC50 value was 0.39 and 0.51 mg/ml for root extract; 0.43 and 0.88 mg/ml for stem extract at 72h and 24h of exposure. Leaf extract exposed moderate activity 32-72% mortality at 72h of exposure and 28-60% mortality at 24h of exposure with 3.93 and 0.25 mg-cm-2 doses. LC50 value was 0.93 and 1.81 mg/ml at 72h and 24h of exposure Table 3. Regression equations are presented in Fig. 1.
Ethyl acetate fraction of root also exposed significant mortality40-80% and 28-68% with the recorded LC50 value were found 0.66 and 1.30 mg/ml respectively at 72h and 24h of exposure within 0.25 and 3.93 mg-cm-2 doses. Leaf and stem extracts of ethyl acetate fraction were also showed promising activity, 40-80% and 28-56% mortality with 0.60 and 2.40 mg/ml of LC50 value for leaf; 48-76% and 32-60% mortality with 0.41 and 1.25mg/ml of LC50 value for stem respectively at 72h and 24h of exposure period within 0.25 and 3.93 mg-cm-2 doses Table 4. Regression equations are shown in Fig. 2.
TABLE 3: MORTALITY (%) OF S. ORYZAE
|Kill||% Kill||Kill||% Kill||Kill||%Kill|
|Ethyl alcohol extract|
|Ethyl acetate extract|
No of insects = 25
FIG. 1: PROBIT REGRESSION LINES BETWEEN PROBIT MORTALITY OF S. ORYZAE AND LOG DOSE (+1 mg-cm-2) OF ETHYL ALCOHOL EXTRACTS OF C. VISCOSUM AFTER 24, 48 AND 72h EXPOSURE
TABLE 4: 95% CONFIDENCE OF MORTALITY RANGED OF S. ORYZAE
|24h of Exposure|
|72h of Exposure|
|Ethyl alcohol||0.26 - 1.00||0.81 - 4.00||0.48 - 1.61|
|0.19 - 0.80||0.53 - 1.64||0.21 - 0.87|
|Ethyl acetate||0.67 - 2.42||0.73 - 7.81||0.53 - 2.95|
|0.35 - 1.21||0.30 - 1.22||0.15 - 1.17|
|Chloroform||1.31 - 25.24||65.53 - 3058.8||1.15 - 65.31|
|0.90 - 6.44||0.96 - 63.40||1.07 - 9.45|
FIG. 2: PROBIT REGRESSION LINES BETWEEN PROBIT MORTALITY OF S. ORYZAE AND LOG DOSE (+1 mg-cm-2) OF ETHYL ACETATE EXTRACTS OF C. VISCOSUM AFTER 24, 48 AND 72h EXPOSURE
Contact toxicity of chloroform fraction showed lesser to moderate activity. 24-56% mortality rate with 2.41 mg/ml of LC50 value at 72h of observation; 16-48% mortality with 5.76 mg/ml of LC50 value at 24h of observation for root extract. The stem extract of chloroform fraction showed 20-52% mortality rate with 3.17 mg/ml of LC50 value at 72h of observation; 16-40% mortality with 8.65 mg/ml of LC50 value at 24h of observation and the toxicity result of leaf extract was 20-44% mortality with 7.79 mg/ml of LC50 value at 72h of exposure; 12-28% mortality with 44.77 mg/ml of LC50 value at 24h of experiment period respectively within 0.25 and 3.93 mg-cm-2 doses Table 4. Regression equations are shown in Fig. 3. The 95% Confidence of the mortality of common stored product coleopteran insect pest S. oryzae is presented in Table 5.
FIG. 3: PROBIT REGRESSION LINES BETWEEN PROBIT MORTALITY OF S. ORYZAE AND LOG DOSE (+1 mg-cm-2) OF CHLOROFORM EXTRACTS OF C. VISCOSUM AFTER 24, 48 AND 72H EXPOSURE
TABLE 5: REPELLENCY EFFECT OF ETHYL ALCOHOL EXTRACT ON S. ORYZAE
|Mean repellency values||Mean repellency %|
|S. Nos.||Dose (mg-cm-2)||Root||Leaf||Stem||Root||Leaf||Stem|
Means within a column having the same letter are not statistically significant (P < 0.05) according to Duncan’s Multiple Range Test.
From the result of the current investigation, the overall assessment suggested that the root extracts showed most potency using three kinds of fraction. Though, ethyl alcohol root and stem extracts exposed the equal toxicity 44-84% mortality at 72h of exposure but the LC50 value (0.39 mg/ml) of root extract is more effective than the LC50 value (0.43 mg/ml) of stem extract along with 95% confidence limit 0.19-0.80 at 72h of observation. The susceptibility of contact toxicity in the intensity of solvents was found ethyl alcohol > ethyl acetate > chloroform fraction. The dose-mortality order of the C. viscosum extracts is root > stem > leaf against S. oryzae.
FIG. 4: REPELLENCY EFFECT OF ETHYL ALCOHOL EXTRACT OF ROOT ON S. ORYZAE
By the susceptibility of contact toxicity in the intensity of solvent was ethyl alcohol > ethyl acetate > chloroform fraction. So, the repellent activity was carried out using ethyl alcohol fraction of root, leaf, and stems of C. viscosum. All the extracts showed 100% repellency (Class V category repellency) in dose no 1 to a dose no 5. The degree of repellency response among the three parts of the test insect was significantly different (P < 0.05). Class V category repellency was observed for root and leaf up to dose 0.06 mg-cm-2 (Dose no 7). Whereas, the lowest dose at which the stem exhibited class V category repellency at 0.12 mg-cm-2 (Dose no 6) presented in Table 5. The root extract showed strong repellent activity than the leaf and stem Fig. 4.
The above findings of the present study are in general agreement with the findings of Yankanchi and Gadache 20. Our study supports the observation of Husain and Rahman 21; Husain and Hasan 22 who conducted repellent and toxicity experiment on the larvae and adults of T. confusum. In continuation of our search for substances of plant origin with insecticidal effects, we have screened out root, leaf, and stem of C. viscosum, extracted into ethyl alcohol, ethyl acetate and chloroform. A correlation was found between the insecticidal and repellent activity observed by residual film assay technique and repellency test. It is interesting to note that these plant extracts showed higher to less mortality activity. Our previous study indicates promising antibacterial and antifungal properties of C. infortunatum against life-threatening pathogens 23.
CONCLUSION: In conclusion, the present research revealed that the C. viscosum extracts have strong insecticidal and repellent effect against S. oryzae.
CONFLICT OF INTEREST: Nil
- Berenbaum MR: Bugs in the system: Insects and their impact on human affairs. Perseus Publishing 1995; 377.
- Sreelatha C and Geetha PR: Pesticidal effects of Clerodendron infortunatumon the fat body of Oryctes rhinoceros (Linn.) male. Journal of Biopesticides 2011; 4(1): 13-17.
- Moldenke HN: Notes on the genus Clerodendrum (Verbenaceae). Phytologia 1985; 57: 334-365.
- Rueda RM: The genus Clerodendrum (Verbenaceae) in Mesoamerica. Annals of the Missouri Botanical Garden 1993; 80: 870-890.
- Hsiao JY and Lin ML: A Chemotaxonomic study of essential oils from the leaves of genus Clerodendrum (Verbenaceae) native to Taiwan. Botanical Bulletin of Academia Sinica 1995; 36: 247-251.
- Steane DA, Scotland RW, Mabberley DJ and Olmstead RG: Molecular systematics of Clerodendrum (Lamiaceae): its sequences and total evidence. American Journal of Botany 1999; 86 98-107.
- Benhalima H, Chaudhry MQ, Mills KA and Price NR: Phosphine resistance in stored-product insects collected from various grain storage facilities in Morocco. Journal of Stored Products Research 2004; 40: 241-249.
- Pavela R: Possibilities of botanical insecticide exploitation in grain protection. Journal of Pest Technology 2007; 1: 47-52.
- Oly WT, Islam W, Hasan P and Parween S: Antimicrobial activity of Clerodendrum viscosum (Verbenaceae). International J of Agriculture & Biology 2011; 13: 222-26.
- Park T: Beetles, competition and population. Science 1962; 138: 1369-1375.
- Zyromska-Rudzka H: Abundance and emigration of Triboliumin a laboratory model. Ekologia Polska- Seria A 1966; 14: 491-518.
- Khalequzzaman M, Khanam LAM and Talukdar D: Growth of Tribolium confusum Duval on wheat flour with various yeast levels. International Pest Control Magazine 1994; 36: 128-130.
- Busvine JR: A critical review of the techniques for testing insecticides: Commonwealth Agricultural Bureaux London 1971; 269-288.
- Abbott WS: A method of computing the effectiveness of an insecticide. J of Economic Entomol 1925; 18: 265-67.
- Finney DJ: Probit Analysis: A statistical treatment of sigmoid response curve. Cambridge University Press London 1947; 333.
- Mcdonald LL, Guy RH and Speirs RD: Preliminary evaluation of new candidate materials as toxicants, repellents and attractants against stored product insects Agricultural Research Service, U.S. Department of Agriculture, Washington DC, Marketing Report No. 882, 1970.
- Talukder FA and Howse PE: Deterrent and insecticidal effects of extracts of pithraj, Aphanamixis polystachya (Meliaceae), against Triboloum castaneum in storage. Journal of Chemical Ecology 1993; 19: 2463-2471.
- Talukder FA and Howse PE: Evaluation of Aphanamixis polystachya as a source of repellents, antifeedants, toxicants and protectants in storage against Tribolum castaneum (Herbst). Journal of Stored Product Research 1995; 31: 55-61.
- Steel RG and Torrie JH: Principle and procedures of statistics. In. Membrane Transport Neuberger A, van Deenen LLM (eds.), Elsevier, Amsterdam 1960; 1-28.
- Yankanchi SR mand Gadache AH: Grain protectant efficacy of certain plant extracts against rice weevil, Sitophilus oryzae (Coleoptera: Curculionidae). Journal of Biopesticides 2010; 3(2): 511-513.
- Husain MM and Rahman M: Repellent effect of indigenous plant Bhant (Clerodendron viscosum) Leaf on Tribolium castaneum Herbst. Bangladesh Journal of Scientific and Industrial Research 2006; 41(1-2): 67-72.
- Husain MM and Hasan MR: Repellency of Indigenous Plant, Bhant (Clerodendron viscosum) Leaf on Tribolium confusum Duval. Bangladesh Journal of Scientific and Industrial Research 2008; 43(2): 267-272.
- Waliullah TM, Yeasmin AM, Ashraful A, Wahedul IM and Parvez H: Antimicrobial potency screening of Clerodendrum infortunatum International Research Journal of Pharmacy 2014; 5(2): 57-61.
How to cite this article:
Waliullah TM, Yeasmin AM, Wahedul IM and Parvez H: Mortality and repellent activity of Clerodendrum viscosum Vent. (Verbenaceae) against Sitophilus oryzae (Coleoptera: Curculionidae). Int J Pharmacognosy 2014; 1(4): 250-57. doi link: http://dx.doi.org/10.13040/ IJPSR.0975-8232.IJP.1(4).250-57.
This Journal licensed under a Creative Commons Attribution-Non-commercial-Share Alike 3.0 Unported License.
T. M. Waliullah*, A. M. Yeasmin, I. M. Wahedul and H. Parvez
Molecular and Cell Biology Laboratory, Bioscience Department1, GSST, Shizuoka University, 836-Oya, Suruga-ku, Shizuoka 422-8529, Japan.
15 January 2014
23 February 2014
26 March 2014
01 April 2014