# Antioviposition and Reduction ofCallosobruchus chinensis Pic. 1902 (Coleoptera: Bruchidae) Emergence on Phaseolus vulgaris by Dioscorea sansibarensis Powder and Its Chemical Composition

Authors: Godfrey O. Mauti; Peter F. Kasigwa; Justus M. Onguso
Journal: Psyche: A Journal of Entomology
 (2020)

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## Abstract

Callosobruchus chinensis causes damage to the Phaseolus vulgaris seeds. Traditionally, Dioscorea sansibarensis serves as a medicinal plant. Naturally, D. sansibarensis has toxins that protect against herbivores and the surrounding invasive plants in its natural habitat. Phytochemical analysis by thin-layer chromatography (TLC) and laboratory experiments was carried out to determine the activity of D. sansibarensis leaves, bulbils, and yams powders on antioviposition and inhibition of the F1 emergence of C. chinensis. Bioassay data were subjected to nonparametric (Kolmogorov–Smirnov) statistical analysis and a generalized linear model at P≤0.05. Statistically, the powders had an antioviposition activity of 34.3% (R2 = 0.343). A recommendable activity on antioviposition was displayed by the yams powder; treatment by 0.8 g of yams powder had a Wald Chi-Square value of 1.291, P=0.26. Inhibition of F1 emergence was significantly attained by the yams powder; the treatment by 0.6 g of yams powder had a Wald Chi-Square value of 7.72, P=0.01. Statistically, the bulbils powder displayed low antioviposition and inhibition of F1 emergence. Observations on the TLC exposed compounds with similar Rf values; saponin with an Rf value of 0.72 was portrayed in the leaves, bulbils, and yams. A terpenoid and a flavonoid with Rf values of 0.37 and 0.71, respectively, were observed in bulbils and yams but absent in leaves. A terpenoid with an Rf value of 0.49 was visualized in leaves and bulbils but not in the yams powder. The study concluded that the D. sansibarensis yams and leaves powders are viable for application by the farmers in the protection of stored legumes against attack by C. chinensis. However, there may be other diverse interests in other storage insects and other methods of phytochemical analysis that have not been investigated.


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## Body

## 1. Introduction

The common bean,Phaseolus vulgaris (Fabaceae), is one of the principle staple foods and cash crops in Tanzania and serves as an important crop for food security in many countries in sub-Sahara Africa [1]. It is important for many low-income populations and significantly improves their nutrition, aimed at the prevention and treatment of diseases [2]. Several studies have demonstrated Callosobruchus chinensis attacks on P. vulgaris in silos, stacks, and barns throughout the world causing qualitative and quantitative damage by both larvae and the adults [3]. C. chinensis accounts for 60% loss of weight and 66.3% loss of protein content of P. vulgaris [4].Callosobruchus chinensis Pic. 1902, also termed Bruchus chinensis Linnaeus 1758, of the family Bruchidae, has an average lifetime of 22–25 days from egg to the adult stage [5]. The infestation begins in the field during harvest; the bruchid is transported to store with the legumes where it lays eggs on the seed surface at the right atmospheric conditions and its life is completed inside the bean seed as it feeds. Damage by C. chinensis occurs after larvae hatch from the eggs as the first instars penetrate the bean seeds. The bruchids develop within the seeds and emerge as adults leaving damage in the form of the empty feeding chambers [6, 7]. More than one larva can develop in a single grain [8].Studies are being conducted on strategies to reduce common bean damage and losses caused byC. chinensis [4]. Farmers use banned and highly toxic synthetic insecticides in their quest to protect agricultural products, including stored bean seeds. Other setbacks associated with using synthetic pesticides include high costs, health hazards, residue problems, and the development of resistance by treated insects [9].Farmers are opting for exploiting the readily available natural resources such as plants of medicinal importance and have contributed to the suppression of pests. Pretreatment ofVigna radiata seeds with inert clay by farmers led to adult mortality and reduced the oviposition of Callosobruchus chinensis [10]. In recent years, researchers have shifted attention to plant powders for control of insect pests in stored bean seeds, due to their biodegradability and being ecofriendly to the environment, thus becoming an alternative to synthetic pesticides in improving food security [11]. Leaves powders of Ocimum basilica (basil), Azadirachta indica (neem), Dennittia tripetala (pepper fruit), Curcuma longa (black pepper), Anacardium occidentale (nutshell), Alpinia galangal (Siamese ginger), Clusia palmicida (Clusia), and Curcuma zedoaria (zedoary) have been reported to induce antioviposition and reduce the number of emerging F1 progeny of C. chinensis on chickpea, cowpea, and green gram seeds [11–13].Plants store the active compounds in the form of phenols, alkaloids, flavonoids, tannins, and saponins in different parts of their organs for protection against herbivores and pathogens [8]. The genus Dioscorea is the largest genus of the monocotyledonous family Dioscoreaceae. The wild Dioscorea sansibarensis, also called (m)chochoni in Swahili, is a sun-loving ornamental climber that forms a canopy occurring in riverine forests of tropical sub-Sahara Africa, specifically in Madagascar, Zanzibar, and Tanganyika. D. sansibarensis documented history in traditional medicine goes to at least 2000 BC [14, 15]. D. sansibarensis bulbils, yams, stem, and leaves are found to be toxic to pathogens, herbivores, and monkeys; hunters use its extract on arrows to hunt wild pigs [14, 16]. This study investigated the antioviposition and ability to reduce the F1 emergence of the C. chinensis from stored P. vulgaris using D. sansibarensis leaves, bulbils, and yams powders, in addition to determining the chemicals present in the plant parts.


## 2. Materials and Methods

### 2.1. Materials

UndamagedP. vulgaris seeds were brought directly from Kisarawe farm (7° 10′0′ S, 38° 50′0″ E and altitude of 252 m above sea level) after harvest. The seeds were sun-dried and stored with 10 ± 2% moisture content. The commercial synthetic pesticide Actellic 50 EC (100 g/90 kg Syngenta, Switzerland) was purchased from agricultural input suppliers in Dar es Salaam, Tanzania, for use as the check (positive control). Thin-layer chromatography (TLC) plates and analytical grade solvents were purchased from Chem Equip Laboratory supplier in Dar es Salaam, Tanzania.Fresh leaves, bulbils, and yams ofD. sansibarensis were identified and harvested from the Zoology-Botany forest thickets at the University of Dar es Salaam (6° 45′0″ S by 39° 15′0″ E, altitude 80 m above sea level). Morphological identification was done at the Herbarium in the Department of Botany, University of Dar es Salaam, where voucher specimens bearing reference numbers FMM 3910a, 3910b, and 3910c for leaf, bulbil, and yam, respectively, are deposited. The plant materials were brought to the Entomology Laboratory of the Department of Zoology and Wildlife Conservation at the University of Dar es Salaam where they were dried under shade in a well-ventilated area for 5 days. After drying, they were pulverized and sieved using a 60 μm mesh sieve to obtain fine powders.


### 2.2. Qualitative Analysis by Thin-Layer Chromatography

#### 2.2.1. Determination of Alkaloids

Weigh 2.5 g of plant powder into a 100 mL beaker to which 80 mL of 1% acetic acid (1 : 10 w/v) in ethanol at a ratio of 1 : 1 was added and allowed to stand for 5 hours, followed by filtration. The resulting filtrate concentrated on a water bath to one-third of the original volume. Concentrated ammonium hydroxide was added dropwise to precipitation. The precipitate was collected, washed with dilute ammonium hydroxide, and filtered. The dry residue was weighed and calculated as a percentage alkaloid. Thin-layer chromatography (TLC) of isolated alkaloids was performed by wetting the powdered test samples with a solvent mixture of chloroform and methanol at a ratio of 15 : 1 which was also used as the mobile phase for running thin-layer chromatography at room temperature. The TLC plate was visualized under UV light and further detection of alkaloid was aided by spraying with Dragendorff’s reagent [17].


#### 2.2.2. Determination of Flavonoids

Plant powder (2.5 g) was soaked repeatedly with 50 mL of 75% aqueous methanol and filtered through Whatman filter paper at room temperature. The filtrate was dried in vacuo and weighed to calculate the percentage flavanoid. The filtrate was mixed with methanol in the ratio of 1 : 8 (g/mL) and heated on a water bath at 60°C for 5 minutes. Chloroform and methanol at a ratio of 18 : 2 were used as the mobile phase [18].


#### 2.2.3. Determination of Saponins

Weigh 2.5 g of plant powder into a conical flask and 50 mL of 20% aqueous ethanol added. The mixture was subjected to heating over a water bath at a temperature of 57°C for 5 minutes. On cooling, the mixture was filtered and the residue reextracted by 50 mL of 20% ethanol. The alcohol extract was reduced to half the original volume by heating on a water bath at 60°C. On cooling, 25 mL of diethyl ether was added and shaken for 5 minutes. The aqueous layer was recovered and washed with 15 mL of 5% aqueous sodium chloride. The aqueous layer was dried, weighed, and calculated for percentage saponin. TLC analysis was performed by eluting with chloroform, glacial acetic acid, methanol, and water solvent system in a ratio of 48 : 26 : 9 : 6 and spots were visualized under the UV light. Saponins were detected by placing the TLC plate in contact with the iodine vapour [19].


#### 2.2.4. Determination of Phenolics

Weigh 2.5 g of plant powder and transfer it into a conical flask to which 40 mL of 80% acetone (acetone to water at a ratio of 8 : 2) was added. The mixture was heated at 65°C in a water bath for 20 min. The supernatant was decanted and mixed with 80% acetone (acetone to water at a ratio of 8 : 2). The aqueous residue was dried, weighed, and calculated for percentage phenolic compounds. TLC analysis was done by eluting with butan-2-ol/acetic acid/water solvent system in a ratio of 14 : 1 : 5. Then FeCl3-K3Fe(CN)6 was sprayed on the TLC plate for the detection of the phenolic constituents [20].


#### 2.2.5. Determination of Terpenoids

Soak 2.5 g of plant powder in 50 mL alcohol for 24 hours and then filter it. The filtrate was placed in a separating funnel with extraction solvents (hexane : ethyl acetate) at a ratio of 85 : 15 (v/v). The separating funnel was vigorously shaken for 5 min and then left to settle overnight. The hexane layer was decanted to a beaker for air drying. The hexane extract was treated as total terpenoids. TLC analysis was done usingn-hexane : ethyl acetate eluting solvent system in a ratio of 6.2 : 2.4. The TLC plate was visualized under the UV and light then heated to 105°C on a hot plate after spraying with freshly prepared p-anisaldehyde reagent [21].


#### 2.2.6. Calculation of Retention Factor Values

The retention factor (Rf) values of different spots were calculated as follows:(1)Rfvalue=distance traveled by solutedistance traveled by the solvent.


### 2.3. Preparation of Working Concentrations of the Powders

Fine powders of the leaves, bulbils, and yams at 0.2, 0.4, 0.6, and 0.8 g/50 g of bean seed were measured for bioassays. The standard 0.05 g/50 g of the commercial pesticide, Actellic 50, EC was used in the bioassay as the positive control. Clean, undamaged, and sun-driedP. vulgaris seeds were used as the negative control (untreated seeds).


### 2.4. Stock Culture ofCallosobruchus chinensis

In a glass jar (10.3 cm wide and 15 cm deep) containing 350 g of dry and undamagedP. vulgaris beans, approximately 200 females of C. chinensis were placed and the mouth was covered with a muslin cloth. The jar was incubated for 1 week in darkness at 28 ± 2°C and relative humidity of 65 ± 5% to facilitate egg-laying by the C. chinensis. The bruchids were removed and the beans were incubated for mass culturing of the F1 progeny of C. chinensis. The pure culture of the F1 breed was used in experimentation.


### 2.5. Antioviposition onP. vulgaris

Twenty females of 3-day-oldC. chinensis were selected from the culturing jar and added into different jars measuring 6.5 cm wide and 10 cm deep, with 50 g of P. vulgaris seeds treated with the different doses of the leaves, bulbils, and yams powders. Actellic 50 EC was used as the positive control and the untreated seeds were used as a negative control. The experiment was replicated in quadruples. The jars were incubated for 5 days in the dark at 28 ± 2°C with a relative humidity of 65 ± 5% for egg-laying. On the 5th day of incubation, all bruchids were carefully isolated and the eggs on the P. vulgaris seed coat were counted with the aid of a dissecting microscope.


### 2.6. Prevention of the Emergence ofF1 Progeny

Twenty females of 3-day-oldC. chinensis were selected from the culturing jar and added into jars of 6.5 cm width and 10 cm depth with 50 g of undamaged and dry P. vulgaris seeds. Under incubation at 28 ± 2°C with a relative humidity of 65 ± 5% in darkness, the weevils were left to lay eggs for 5 days and the weevils were carefully removed from the jars. Different doses of the leaves, bulbils, yams powders, and the positive control were added to their respective jars and then incubated at 28 ± 2°C with a relative humidity of 65 ± 5%. The experiment was monitored daily until the first adults emerged from the negative control. Emerging C. chinensis were isolated and counted daily for 5 weeks to avoid overlap with their F1 generation.


### 2.7. Statistical Analyses

Antioviposition and reduction ofF1 emergence analyses were performed using the SPSS software (version 20). The analysis involved determination of Poisson distribution by nonparametric (Kolmogorov–Smirnov) test and descriptive statistics of the mean variance. The Poisson distributed data from the antioviposition and F1 emergence were subjected separately to generalized linear model analysis at P≤0.05. In the model of analyzing the antioviposition, the concentrations of the doses were the independent variables as data of antioviposition was the dependent variable. In the analysis of F1 emergence, the concentration was the independent variable while the data obtained from the emerged F1 progeny was utilized as the dependent variable.


## 2.1. Materials

UndamagedP. vulgaris seeds were brought directly from Kisarawe farm (7° 10′0′ S, 38° 50′0″ E and altitude of 252 m above sea level) after harvest. The seeds were sun-dried and stored with 10 ± 2% moisture content. The commercial synthetic pesticide Actellic 50 EC (100 g/90 kg Syngenta, Switzerland) was purchased from agricultural input suppliers in Dar es Salaam, Tanzania, for use as the check (positive control). Thin-layer chromatography (TLC) plates and analytical grade solvents were purchased from Chem Equip Laboratory supplier in Dar es Salaam, Tanzania.Fresh leaves, bulbils, and yams ofD. sansibarensis were identified and harvested from the Zoology-Botany forest thickets at the University of Dar es Salaam (6° 45′0″ S by 39° 15′0″ E, altitude 80 m above sea level). Morphological identification was done at the Herbarium in the Department of Botany, University of Dar es Salaam, where voucher specimens bearing reference numbers FMM 3910a, 3910b, and 3910c for leaf, bulbil, and yam, respectively, are deposited. The plant materials were brought to the Entomology Laboratory of the Department of Zoology and Wildlife Conservation at the University of Dar es Salaam where they were dried under shade in a well-ventilated area for 5 days. After drying, they were pulverized and sieved using a 60 μm mesh sieve to obtain fine powders.


## 2.2. Qualitative Analysis by Thin-Layer Chromatography

### 2.2.1. Determination of Alkaloids

Weigh 2.5 g of plant powder into a 100 mL beaker to which 80 mL of 1% acetic acid (1 : 10 w/v) in ethanol at a ratio of 1 : 1 was added and allowed to stand for 5 hours, followed by filtration. The resulting filtrate concentrated on a water bath to one-third of the original volume. Concentrated ammonium hydroxide was added dropwise to precipitation. The precipitate was collected, washed with dilute ammonium hydroxide, and filtered. The dry residue was weighed and calculated as a percentage alkaloid. Thin-layer chromatography (TLC) of isolated alkaloids was performed by wetting the powdered test samples with a solvent mixture of chloroform and methanol at a ratio of 15 : 1 which was also used as the mobile phase for running thin-layer chromatography at room temperature. The TLC plate was visualized under UV light and further detection of alkaloid was aided by spraying with Dragendorff’s reagent [17].


### 2.2.2. Determination of Flavonoids

Plant powder (2.5 g) was soaked repeatedly with 50 mL of 75% aqueous methanol and filtered through Whatman filter paper at room temperature. The filtrate was dried in vacuo and weighed to calculate the percentage flavanoid. The filtrate was mixed with methanol in the ratio of 1 : 8 (g/mL) and heated on a water bath at 60°C for 5 minutes. Chloroform and methanol at a ratio of 18 : 2 were used as the mobile phase [18].


### 2.2.3. Determination of Saponins

Weigh 2.5 g of plant powder into a conical flask and 50 mL of 20% aqueous ethanol added. The mixture was subjected to heating over a water bath at a temperature of 57°C for 5 minutes. On cooling, the mixture was filtered and the residue reextracted by 50 mL of 20% ethanol. The alcohol extract was reduced to half the original volume by heating on a water bath at 60°C. On cooling, 25 mL of diethyl ether was added and shaken for 5 minutes. The aqueous layer was recovered and washed with 15 mL of 5% aqueous sodium chloride. The aqueous layer was dried, weighed, and calculated for percentage saponin. TLC analysis was performed by eluting with chloroform, glacial acetic acid, methanol, and water solvent system in a ratio of 48 : 26 : 9 : 6 and spots were visualized under the UV light. Saponins were detected by placing the TLC plate in contact with the iodine vapour [19].


### 2.2.4. Determination of Phenolics

Weigh 2.5 g of plant powder and transfer it into a conical flask to which 40 mL of 80% acetone (acetone to water at a ratio of 8 : 2) was added. The mixture was heated at 65°C in a water bath for 20 min. The supernatant was decanted and mixed with 80% acetone (acetone to water at a ratio of 8 : 2). The aqueous residue was dried, weighed, and calculated for percentage phenolic compounds. TLC analysis was done by eluting with butan-2-ol/acetic acid/water solvent system in a ratio of 14 : 1 : 5. Then FeCl3-K3Fe(CN)6 was sprayed on the TLC plate for the detection of the phenolic constituents [20].


### 2.2.5. Determination of Terpenoids

Soak 2.5 g of plant powder in 50 mL alcohol for 24 hours and then filter it. The filtrate was placed in a separating funnel with extraction solvents (hexane : ethyl acetate) at a ratio of 85 : 15 (v/v). The separating funnel was vigorously shaken for 5 min and then left to settle overnight. The hexane layer was decanted to a beaker for air drying. The hexane extract was treated as total terpenoids. TLC analysis was done usingn-hexane : ethyl acetate eluting solvent system in a ratio of 6.2 : 2.4. The TLC plate was visualized under the UV and light then heated to 105°C on a hot plate after spraying with freshly prepared p-anisaldehyde reagent [21].


### 2.2.6. Calculation of Retention Factor Values

The retention factor (Rf) values of different spots were calculated as follows:(1)Rfvalue=distance traveled by solutedistance traveled by the solvent.


## 2.2.1. Determination of Alkaloids

Weigh 2.5 g of plant powder into a 100 mL beaker to which 80 mL of 1% acetic acid (1 : 10 w/v) in ethanol at a ratio of 1 : 1 was added and allowed to stand for 5 hours, followed by filtration. The resulting filtrate concentrated on a water bath to one-third of the original volume. Concentrated ammonium hydroxide was added dropwise to precipitation. The precipitate was collected, washed with dilute ammonium hydroxide, and filtered. The dry residue was weighed and calculated as a percentage alkaloid. Thin-layer chromatography (TLC) of isolated alkaloids was performed by wetting the powdered test samples with a solvent mixture of chloroform and methanol at a ratio of 15 : 1 which was also used as the mobile phase for running thin-layer chromatography at room temperature. The TLC plate was visualized under UV light and further detection of alkaloid was aided by spraying with Dragendorff’s reagent [17].


## 2.2.2. Determination of Flavonoids

Plant powder (2.5 g) was soaked repeatedly with 50 mL of 75% aqueous methanol and filtered through Whatman filter paper at room temperature. The filtrate was dried in vacuo and weighed to calculate the percentage flavanoid. The filtrate was mixed with methanol in the ratio of 1 : 8 (g/mL) and heated on a water bath at 60°C for 5 minutes. Chloroform and methanol at a ratio of 18 : 2 were used as the mobile phase [18].


## 2.2.3. Determination of Saponins

Weigh 2.5 g of plant powder into a conical flask and 50 mL of 20% aqueous ethanol added. The mixture was subjected to heating over a water bath at a temperature of 57°C for 5 minutes. On cooling, the mixture was filtered and the residue reextracted by 50 mL of 20% ethanol. The alcohol extract was reduced to half the original volume by heating on a water bath at 60°C. On cooling, 25 mL of diethyl ether was added and shaken for 5 minutes. The aqueous layer was recovered and washed with 15 mL of 5% aqueous sodium chloride. The aqueous layer was dried, weighed, and calculated for percentage saponin. TLC analysis was performed by eluting with chloroform, glacial acetic acid, methanol, and water solvent system in a ratio of 48 : 26 : 9 : 6 and spots were visualized under the UV light. Saponins were detected by placing the TLC plate in contact with the iodine vapour [19].


## 2.2.4. Determination of Phenolics

Weigh 2.5 g of plant powder and transfer it into a conical flask to which 40 mL of 80% acetone (acetone to water at a ratio of 8 : 2) was added. The mixture was heated at 65°C in a water bath for 20 min. The supernatant was decanted and mixed with 80% acetone (acetone to water at a ratio of 8 : 2). The aqueous residue was dried, weighed, and calculated for percentage phenolic compounds. TLC analysis was done by eluting with butan-2-ol/acetic acid/water solvent system in a ratio of 14 : 1 : 5. Then FeCl3-K3Fe(CN)6 was sprayed on the TLC plate for the detection of the phenolic constituents [20].


## 2.2.5. Determination of Terpenoids

Soak 2.5 g of plant powder in 50 mL alcohol for 24 hours and then filter it. The filtrate was placed in a separating funnel with extraction solvents (hexane : ethyl acetate) at a ratio of 85 : 15 (v/v). The separating funnel was vigorously shaken for 5 min and then left to settle overnight. The hexane layer was decanted to a beaker for air drying. The hexane extract was treated as total terpenoids. TLC analysis was done usingn-hexane : ethyl acetate eluting solvent system in a ratio of 6.2 : 2.4. The TLC plate was visualized under the UV and light then heated to 105°C on a hot plate after spraying with freshly prepared p-anisaldehyde reagent [21].


## 2.2.6. Calculation of Retention Factor Values

The retention factor (Rf) values of different spots were calculated as follows:(1)Rfvalue=distance traveled by solutedistance traveled by the solvent.


## 2.3. Preparation of Working Concentrations of the Powders

Fine powders of the leaves, bulbils, and yams at 0.2, 0.4, 0.6, and 0.8 g/50 g of bean seed were measured for bioassays. The standard 0.05 g/50 g of the commercial pesticide, Actellic 50, EC was used in the bioassay as the positive control. Clean, undamaged, and sun-driedP. vulgaris seeds were used as the negative control (untreated seeds).


## 2.4. Stock Culture ofCallosobruchus chinensis

In a glass jar (10.3 cm wide and 15 cm deep) containing 350 g of dry and undamagedP. vulgaris beans, approximately 200 females of C. chinensis were placed and the mouth was covered with a muslin cloth. The jar was incubated for 1 week in darkness at 28 ± 2°C and relative humidity of 65 ± 5% to facilitate egg-laying by the C. chinensis. The bruchids were removed and the beans were incubated for mass culturing of the F1 progeny of C. chinensis. The pure culture of the F1 breed was used in experimentation.


## 2.5. Antioviposition onP. vulgaris

Twenty females of 3-day-oldC. chinensis were selected from the culturing jar and added into different jars measuring 6.5 cm wide and 10 cm deep, with 50 g of P. vulgaris seeds treated with the different doses of the leaves, bulbils, and yams powders. Actellic 50 EC was used as the positive control and the untreated seeds were used as a negative control. The experiment was replicated in quadruples. The jars were incubated for 5 days in the dark at 28 ± 2°C with a relative humidity of 65 ± 5% for egg-laying. On the 5th day of incubation, all bruchids were carefully isolated and the eggs on the P. vulgaris seed coat were counted with the aid of a dissecting microscope.


## 2.6. Prevention of the Emergence ofF1 Progeny

Twenty females of 3-day-oldC. chinensis were selected from the culturing jar and added into jars of 6.5 cm width and 10 cm depth with 50 g of undamaged and dry P. vulgaris seeds. Under incubation at 28 ± 2°C with a relative humidity of 65 ± 5% in darkness, the weevils were left to lay eggs for 5 days and the weevils were carefully removed from the jars. Different doses of the leaves, bulbils, yams powders, and the positive control were added to their respective jars and then incubated at 28 ± 2°C with a relative humidity of 65 ± 5%. The experiment was monitored daily until the first adults emerged from the negative control. Emerging C. chinensis were isolated and counted daily for 5 weeks to avoid overlap with their F1 generation.


## 2.7. Statistical Analyses

Antioviposition and reduction ofF1 emergence analyses were performed using the SPSS software (version 20). The analysis involved determination of Poisson distribution by nonparametric (Kolmogorov–Smirnov) test and descriptive statistics of the mean variance. The Poisson distributed data from the antioviposition and F1 emergence were subjected separately to generalized linear model analysis at P≤0.05. In the model of analyzing the antioviposition, the concentrations of the doses were the independent variables as data of antioviposition was the dependent variable. In the analysis of F1 emergence, the concentration was the independent variable while the data obtained from the emerged F1 progeny was utilized as the dependent variable.


## 3. Results

### 3.1. Phytochemical Profiling

The leaves, bulbils, and yams contained different compounds with different Rf values (Table 1). Alkaloids were absent on the plant parts. Saponins and terpenoids were spotted in all the plant parts. Two spots of phenolic compounds and three spots of saponins were observed in bulbils. Terpenoids had high intensity in the leaves with an observation of six spots. Five spots of flavonoids were visualized in bulbils (Table 1). The results also showed that the powders had compounds with similar Rf values; saponin with an Rf value of 0.72 was depicted in the leaves, bulbils, and yams. Powders of bulbils and yams had terpenoids with an Rf value of 0.37 and flavonoid of Rf value of 0.71, but were absent in leaves. The bulbils and leaves powders showed terpenoid with an Rf value of 0.49 which was absent in yams powder (Table 1).Table 1
Rf values of chemical compounds found in leaves, bulbils, and yams of Dioscorea sansibarensis.
Rf valuesAlkaloidsFlavonoidsSaponinsPhenolic compoundsTerpenoidsLeaves—0.730.72, 0.84—0.18, 0.29, 0.49, 0.63, 0.80, 0.84Bulbils—0.51, 0.60, 0.69, 0.71, 0.810.61, 0.72, 0.820.37, 0.630.37, 0.49, 0.69, 0.78Yams—0.56, 0.62, 0.710.61, 0.720.520.37, 0.75, 0.80


### 3.2. Antioviposition

Tabulated results showed counts of 423, 459, and 479 eggs on the seeds treated by 0.2 g of leaves powder, 0.2 g of yams powder, and 0.4 g of bulbils powders, respectively (Table2). When the eggs laid on treated seeds are counted against the eggs in the negative control (untreated seeds), 0.2 g of leaves powder, 0.2 g of yams powder, and 0.4 g of bulbils powder exhibited an antioviposition of 36.13%, 30.69%, and 27.61%, respectively (Table 2). Data subjected to statistical analysis (N = 56) showed a Poisson distribution; the Kolmogorov–Smirnov test recorded 3.18 and the descriptive statistics recorded a mean of 398.11 with a variance of 20197.30. The generalized linear model reported a Wald Chi-Square value of 1.291, P=0.26 on the treatment by 0.8 g of leaves powder. Statistical analysis showed an overall antioviposition of 34.3% (R2 = 0.343) by the leaves, bulbils, and yams powders. Treatment by 0.2 g of leaves powder exhibited better antioviposition activity than 0.4 g of leaves and 0.4 g of bulbils powders; they recorded 422.5, 441, and 478.75, respectively, at P=0.51. At P=0.113, the antioviposition activity by 0.6 g of yams powder (338.75) had equivalent antioviposition activity to 0.8 g of leaves powder (299.75). Antioviposition was highly displayed in the treatment by 0.8 g of the yams powder with a statistical result of 286 as the bulbils powder scored the lowest antioviposition effect amongst the test powders.Table 2
Oviposition byCallosobruchus chinensis cultured on Phaseolus vulgaris seeds treated with powders of Dioscorea sansibarensis leaves, bulbils, and yams.
Oviposition ± SEPowder(Conc. g/50 g)Positive controlNegative control0.20.40.60.8Leaves422.5 ± 56.8c441.0 ± 51.6b360.5 ± 39.8c299.8 ± 31.2c,d144.3 ± 15661.5 ± 67.8Bulbils491.8 ± 59.7a478.8 ± 54.4a399.8 ± 46.9c385.5 ± 37.9cYams458.5 ± 65.6b405.0 ± 36.3c338.8 ± 31.6c,d286.0 ± 40.8dTreatment with Poisson distributed data was subjected to a generalized linear model. Means in the table that do not share a letter are significantly different (N = 56; P<0.05).


### 3.3. Inhibition ofF1 Emergence

The firstC. chinensis adult emerged from the negative control (untreated seeds) on the 28th day of incubation from the untreated bean seeds. Graphical results showed that 362, 368, and 387 weevils had emerged from the treatments by 0.2 g of yams and the leaves and 0.4 g of the bulbils powder, respectively (Figure 1). When emerged weevils in treatments are compared to the negative control, 0.2 g of yams powder, 0.2 g of leaves powder, and 0.4 g of bulbils powder exhibited inhibition of weevil emergence from the seeds by 37.02%, 35.98%, and 32.58%, respectively (Figure 1). Kolmogorov–Smirnov test recorded 2.90 and descriptive statistics recorded a mean of 307.71 with a variance of 13727.66. The Poisson distributed data subjected to a generalized linear model reported a Wald Chi-Square value of 2.37, P=0.12 on the treatment by 0.8 g of leaves powder and 7.72, P=0.01 by 0.6 g of yams powder. Statistically, reduction of the F1 emergence was highly displayed in the treatment by 0 g of yams powder where 188.5 weevils emerged from the seeds. Significant activity was displayed in the treatment by 0.2 g of leaves and 0.4 g of bulbils powders recorded 367.75 and 387.25 at P=0.07. Treatments by 0.6 g of yams and 0.8 of leaves powders showed a significance in reduction of the emerging F1 progeny at P=0.332. Statistically, the seeds treated by the bulbils powder recorded a higher number of the emerged weevils than the leaves and the yams powders.Figure 1
EmergingC. chinensis from P. vulgaris seeds treated with D. sansibarensis leaves, bulbils, and yams powders. Means in the graph that do not share a letter are significantly different (N = 56; P<0.05).


## 3.1. Phytochemical Profiling

The leaves, bulbils, and yams contained different compounds with different Rf values (Table 1). Alkaloids were absent on the plant parts. Saponins and terpenoids were spotted in all the plant parts. Two spots of phenolic compounds and three spots of saponins were observed in bulbils. Terpenoids had high intensity in the leaves with an observation of six spots. Five spots of flavonoids were visualized in bulbils (Table 1). The results also showed that the powders had compounds with similar Rf values; saponin with an Rf value of 0.72 was depicted in the leaves, bulbils, and yams. Powders of bulbils and yams had terpenoids with an Rf value of 0.37 and flavonoid of Rf value of 0.71, but were absent in leaves. The bulbils and leaves powders showed terpenoid with an Rf value of 0.49 which was absent in yams powder (Table 1).Table 1
Rf values of chemical compounds found in leaves, bulbils, and yams of Dioscorea sansibarensis.
Rf valuesAlkaloidsFlavonoidsSaponinsPhenolic compoundsTerpenoidsLeaves—0.730.72, 0.84—0.18, 0.29, 0.49, 0.63, 0.80, 0.84Bulbils—0.51, 0.60, 0.69, 0.71, 0.810.61, 0.72, 0.820.37, 0.630.37, 0.49, 0.69, 0.78Yams—0.56, 0.62, 0.710.61, 0.720.520.37, 0.75, 0.80


## 3.2. Antioviposition

Tabulated results showed counts of 423, 459, and 479 eggs on the seeds treated by 0.2 g of leaves powder, 0.2 g of yams powder, and 0.4 g of bulbils powders, respectively (Table2). When the eggs laid on treated seeds are counted against the eggs in the negative control (untreated seeds), 0.2 g of leaves powder, 0.2 g of yams powder, and 0.4 g of bulbils powder exhibited an antioviposition of 36.13%, 30.69%, and 27.61%, respectively (Table 2). Data subjected to statistical analysis (N = 56) showed a Poisson distribution; the Kolmogorov–Smirnov test recorded 3.18 and the descriptive statistics recorded a mean of 398.11 with a variance of 20197.30. The generalized linear model reported a Wald Chi-Square value of 1.291, P=0.26 on the treatment by 0.8 g of leaves powder. Statistical analysis showed an overall antioviposition of 34.3% (R2 = 0.343) by the leaves, bulbils, and yams powders. Treatment by 0.2 g of leaves powder exhibited better antioviposition activity than 0.4 g of leaves and 0.4 g of bulbils powders; they recorded 422.5, 441, and 478.75, respectively, at P=0.51. At P=0.113, the antioviposition activity by 0.6 g of yams powder (338.75) had equivalent antioviposition activity to 0.8 g of leaves powder (299.75). Antioviposition was highly displayed in the treatment by 0.8 g of the yams powder with a statistical result of 286 as the bulbils powder scored the lowest antioviposition effect amongst the test powders.Table 2
Oviposition byCallosobruchus chinensis cultured on Phaseolus vulgaris seeds treated with powders of Dioscorea sansibarensis leaves, bulbils, and yams.
Oviposition ± SEPowder(Conc. g/50 g)Positive controlNegative control0.20.40.60.8Leaves422.5 ± 56.8c441.0 ± 51.6b360.5 ± 39.8c299.8 ± 31.2c,d144.3 ± 15661.5 ± 67.8Bulbils491.8 ± 59.7a478.8 ± 54.4a399.8 ± 46.9c385.5 ± 37.9cYams458.5 ± 65.6b405.0 ± 36.3c338.8 ± 31.6c,d286.0 ± 40.8dTreatment with Poisson distributed data was subjected to a generalized linear model. Means in the table that do not share a letter are significantly different (N = 56; P<0.05).


## 3.3. Inhibition ofF1 Emergence

The firstC. chinensis adult emerged from the negative control (untreated seeds) on the 28th day of incubation from the untreated bean seeds. Graphical results showed that 362, 368, and 387 weevils had emerged from the treatments by 0.2 g of yams and the leaves and 0.4 g of the bulbils powder, respectively (Figure 1). When emerged weevils in treatments are compared to the negative control, 0.2 g of yams powder, 0.2 g of leaves powder, and 0.4 g of bulbils powder exhibited inhibition of weevil emergence from the seeds by 37.02%, 35.98%, and 32.58%, respectively (Figure 1). Kolmogorov–Smirnov test recorded 2.90 and descriptive statistics recorded a mean of 307.71 with a variance of 13727.66. The Poisson distributed data subjected to a generalized linear model reported a Wald Chi-Square value of 2.37, P=0.12 on the treatment by 0.8 g of leaves powder and 7.72, P=0.01 by 0.6 g of yams powder. Statistically, reduction of the F1 emergence was highly displayed in the treatment by 0 g of yams powder where 188.5 weevils emerged from the seeds. Significant activity was displayed in the treatment by 0.2 g of leaves and 0.4 g of bulbils powders recorded 367.75 and 387.25 at P=0.07. Treatments by 0.6 g of yams and 0.8 of leaves powders showed a significance in reduction of the emerging F1 progeny at P=0.332. Statistically, the seeds treated by the bulbils powder recorded a higher number of the emerged weevils than the leaves and the yams powders.Figure 1
EmergingC. chinensis from P. vulgaris seeds treated with D. sansibarensis leaves, bulbils, and yams powders. Means in the graph that do not share a letter are significantly different (N = 56; P<0.05).


## 4. Discussion

In the family Dioscoreaceae, there is limited literature review concerning studies on wildD. sansibarensis especially on chemical analysis and insecticidal potency. Research on D. sansibarensis has surveyed its therapeutic application [22]. In this study, the D. sansibarensis leaves, yams, and bulbils powders were found to possess terpenoids, saponins, and phenolic compounds; these compounds might have been attributed to their biological activities on mortality, antioviposition, and reduction of the emergence of the C. chinensis from the P. vulgaris seeds. Dhandapani and Sabna reported that alkaloids, tannins, and phenolic compounds that are present in Aegle marmelos, Cynodon dactylon, Eclipta prostrata, Moringa pterygosperma, Pongamia pinnata, Sida acuta, and Tridax procumbens protected the plants against pathogens and herbivores [23]. In this study, terpenoid with Rf value of 0.37 and flavonoid of 0.71 were identified. According to Lazarević et al. [24] and Omotoso et al. [25], terpenoids located at Rf values of 0.37 ± 0.03 are associated with the prevention of the egg-laying process of bruchids. Chiluwal et al. [26] state that terpenoids with Rf values of 0.49 ± 0.05 are involved in the larvicidal activity of bruchids, thus preventing the emergence of F1 progeny. Flavonoids with Rf values of 0.71 and 0.72 are associated with antioviposition of C. chinensis [27].The results in this study showed thatD. sansibarensis powders have significant control of C. chinensis in the reduction of oviposition activity and adult emergence. In this study, antioviposition recorded after 5 days on P. vulgaris beans treated with 0.6 g of D. sansibarensis leaves powder was relatively higher as compared to the treatment by 0.8 g of Jatropha curcas leaves powder on cowpea seeds which resulted in a 42% reduction in oviposition after 5 days of infestation by C. maculatus and 50.67% in reduction of emerging F1 progeny [22].The antioviposition and reduction ofC. chinensis emergence might have been influenced by the presence of flavonoids in the leaves, bulbils, and yams. Flavonoids of plant sources are highly involved in the defence mechanisms by exerting toxic effects on the bruchids [28, 29]. Upasani et al. reported that ﬂavonoids extracted from the aqueous shade-dried leaves of Ricinus communis (Euphorbiaceae) showed excellent ovicidal and oviposition deterrent activity and reduced the number of bean seeds damaged and the C. chinensis emerging from cowpeas [30].A considerable amount of saponins present in theD. sansibarensis might have influenced antioviposition and the reduction of the emerging C. chinensis. Saponin in form of diosgenin identified in Trigonella foenum-graecum prevents Tribolium castaneum and Acanthoscelides obtectus from laying eggs on the Zea mays seed coat and interrupts their larval development that led to the reduction of the emerging F1 progeny from seeds [24]. Saponic compounds estimated to appear at Rf values of 0.71 to 0.80 have been highly associated with mortality, antioviposition, larvicidal activity, and emergence of F1 progeny of the family Bruchidae [31]. Saponins from Nicotiana tabacum, Tephrosia vogelii, and Securidaca longepedunculata were found to be toxic to the larvae of C. chinensis and C. maculatus by preventing larval growth and reduction of adult emergence [32]. Saponins isolated from Vigna unguiculata seeds reduced the C. maculatus F1 emergence from P. vulgaris seeds [33].


## 5. Conclusion

Based on the findings in this study,D. sansibarensis powders possess chemicals and have shown potency on C. chinensis in antioviposition and reduction of F1 emergence. D. sansibarensis yams powder is viable for application by the farmers in the protection of P. vulgaris against attack by C. chinensis.



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*Source: 3298479-2020-11-17.xml*