Egypt. J. Plant Prot. Res. Inst. (2020), 3 (1): 493 - 501

Egyptian Journal of Plant Protection Research Institute

www.ejppri.eg.net

Evaluating the insecticidal efficiency of some legumes extracts against zonata (Diptera: )

Mahenaz, A.A. Gab Alla and Basant, S. M. El-Banna Plant Protection Research Institute, Agricultural Research Center, Sabahia, Alexandria, Egypt. ARTICLE INFO Abstract: Article History Insecticidal and biological activity of three legumes; Lupinus Received: 17 / 2 / 2020 luteus L., Glycine max L. and Cicer arietinum; seeds ethanolic extracts Accepted: 31/3/2020 against adult and egg stages of peach Bactrocera zonata Keywords (Saunders) (Diptera: Tephritidae) was investigated under laboratory Control, Bactrocera conditions. The phytochemical screening of ethanolic extract of the zonata, Lupinus luteus tested three legumes showed the presence of alkaloids, steroids, , Glycine max and flavonoids, terpenoids, tannins and saponins in G. max extract and Cicer arietinum. absence of saponins in C. arietinum . Also, terpenoids, flavonoids and tannins are absent in L. luteus ethanolic extract. Ethanolic extracts of the tested legume seeds achieved variable toxicity against adult and egg stages of B. zonata. Ethanolic extracts of G. max and C. arietinum -1 seeds (LC50 = 366.2 and 437.3 mg L , respectively) were more toxic against adults than L. luteus seeds ethanolic extract (LC50 = 627.5 mg L-1) after 8 days of exposure. Ethanolic extract of C. arietinum achieved the higher ovicidal activity (Egg hatchability reached to 0% at 1000 mg / L) compared to the other extracts. G. max and C. arietinum seeds ethanolic extracts were the most potent on the adult fecundity with 2.0 and 0.0 egg laid / female at 1000 mg / L, respectively compared to 49.0 egg / female in control. The tested ethanolic extracts of the three tested plant seeds reduced the adult longevity of B. zonata and the effect was concentration dependent. Results of the present study suggest that, plant extracts can be an effective tool in integrated pest management programs for the control of fruit . Introduction The peach fruit fly Bactrocera South and South- East Asia (Kapoor, zonata (Saunders) (Diptera: Tephritidae) 1993 and Duyck et al., 2004). The is a serious pest of in Egypt and presence of this insect reduces the quality many world regions. It attacks a wide of fruits and subsequently negatively range of fruit and vegetable hosts (White affects their exportation (Shehata et al., and Elson-Harris, 1994). Saafan et al. 2008). Damage is caused mainly by the (1993) recorded B. zonata in many larvae, which feed during the growth and orchards in several Egyptian development of the fruit (Stonehouse et Governorates. Peach fruit fly originates in al., 2005). Therefore, the control of

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Gab Alla and El-Banna, 2020 larvae is difficult using a single control tested against Bactrocera zonata adult. measure (Dhillon et al., 2005). Effects on the fecundity and fertility of Control methods of this insect are ranged treated adults were also evaluated. from foliage and spraying by the Materials and methods specific , bait-application, 1.Tested insect: B. zonata pupae were male annihilation techniques, releases of obtained from the Department of Pests sterilized flies and , and Horticultural Crops, Plant Protection cultural controls (Khan et al., 2017). The Institute Research, Agricultural Research unwise use of synthetic Center. Emerged adults were introduced resulted in many environmental to cages of 30 cm- 30 cm - 30 cm size. problems, health problems, development Cages were enclosed with mesh screens of insecticide resistance and natural and have cloth sleeves at front and back enemies toxicity (Victor, 2009). sides for the introduction of the food Therefore, there is a need to search about (hydrolyzed yeast and sugar of ratio 1:3) more effective and safe alternative (Khan et al., 2016) and oviposition trays methods to these synthetic insecticides. (El-Minshawy et al., 1999). The insect Between these alternatives are the plant larvae were reared under the laboratory natural products. conditions (25 °C ± 2; 65% ± 5 RH.) on a Plants synthesize many aromatic semi-artificial diet (Wheat bran 500 gm, secondary metabolites, like phenols, molasses 250 gm, dried yeast 150 gm, phenolic acids, quinones, flavones, citric acid 4 gm and sodium benzoates 6 flavonoids, flavonols, tannins and gm in 1liter water) according to Awad coumarins which can negatively affect (1993). (Cowan, 1999). These compounds 2.Tested plants: can be used as repellents, feeding Seeds of three legumes; L. luteus, G. deterrents, toxins, defense chemicals and max and C. arietinum, ethanolic extracts growth regulators. Many are also were used to study its potential biocidal effective against biting Diptera, activity against the tested insect B.zonata. especially those volatile components Dried legume seeds were purchased from (Gurjar et al., 2012). It is documented local markets, Alexandria Governorate. that, the leaf extracts of eucalyptus The plants were identified (Eucalyptus globulus) shows efficacy by=Department of Horticultural against Bactrocera cucurbitae (Ali et al., Crops,Agricultural Research Center, 2011). In addition, Mahmoud and Shoeib Alexandria. (2008) showed that low concentrations of 3.Plant extraction: neem can be applied effectively as The legumes seeds=were sterilant and oviposition deterrent for the pulverized into fine=powder using a peach fruit fly populations. Neem blocks grinding mill. The extraction of the the ovarian development and can be used investigated plants was carried out as safe alternative of insecticides for the according to the method of (Mbatchou et control of a species (Mahfuza et al., al., 2011). Powder of each of L. luteus , 2007). In the present study, the toxic G.. max and C . arietinum seeds (200g) effects of ethanolic extract of three were soaked in (700 ml) of ethanol 98% legume plant seeds; Lupinus luteus L. (L. for two weeks with intermittent shaking. luteus), Glycine max L (‎G. max) and The extracts were separately evaporated Cicer arietinum L (C. arietinum), were to dryness at room temperature to obtain

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the crude extracts (ethanol extracts). This added to 2 ml of distilled water and procedure was repeated 10 times. The boiled for 3:5 minutes. The resultant resulting crude extracts were stored in mixture was filtered, allowed to cool with glass vials pack closed at (2-4°C) until the filtrate shaken=vigorously. Honey used for phytochemical and bioassay comb froth higher than the aqueous layer assessment experiments. was taken as strongly positive for 4. Qualitative phytochemical screenings: saponins. The extracts obtained from 5.Insecticidal activity of legumes ethanol extract, were subjected to extract: preliminary phytochemical analysis tests The=bioassay=experiments=were= to identify the main chemical groups such carried=out=using=the=extract=of the as alkaloids, steroids, flavonoids, investigated plants to test its potential saponins and tannins according to biocidal activity against B. zonata. Series Mbatchou et al. (2011). of concentrations of each botanical 4.1.Alkaloids test (Wagner’s test): extract from the three legumes were One ml of each extract was treated prepared in ethanol and tested against B. with drops of Wagner’s reagent (Dissolve zonata adults. Tween-twenty was used as 2 g of iodine and 6 g of potassium iodide an emulsifier. Twenty adults of B. zonata in 100 ml distilled water) and observed (Males and Females) 10 days old gravid for the formation of reddish brown flies (Rehman et al., 2009a) put in plastic precipitate. container containing food (hydrolyzed 4.2. Flavonoids test (Willistatter test): yeast and sugar of ratio 1:3) and water. To an ethanol solution of each (Sultana et al., 2013). The tested extract extract, a piece of magnesium ribbon was was put as a layer on the banana peel in added, followed by dropwise addition of the plastic container. Emulsifier and concentrated HCl. Colors ranging from ethanol solution without extract was orange to red indicated flavonoids. added to a piece of banana peel and kept 4.3. Terpenoids and steroids test as untreated control. Each treatment in (Liebermann Buchart test): addition to control was replicated three A small quantity of each extract was time. The plastic container covered with dissolved in trichloromethane, and a muslin cloth and tight with rubber bands minimum volume of concentrated and kept under laboratory conditions. The sulphuric acid added to its content. A adult mortality and number of laid eggs blue or green color or a mixture of these /female was recorded after 2, 4, 6 and 8 two shades was taken as positive test for days of treatment. The eggs removed and terpenoids compounds and the formation counted from banana peel by pin and of dark pink or red color taken as positive placed in a petri dish having wet black test for Steroids compounds. cloth to calculate egg hatchability%. 4.4. Tannins test: Mortality percentages were calculated Each plant extract (0.2 ml) was re- and subjected to probit analysis according extracted with ethanol. The solution to Finney (1971). The concentration obtained was later treated with 5% ferric which cause 50% mortality (LC50) and chloride. A blue-black or blue-green the time required for 50% mortality appearance was taken as positive test for (LT50), confidence limits and slope ± SE tannins. were calculated. 4.5. Saponins test: 6. Statistical analysis: A small portion of each extract was

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Data were subjected to one-way present in the G. max L ethanolic extract. ANOVA test (Duncan’s Multiple Alkaloids, steroids and saponins were Comparison Range Tests at 0.05% level). identified in L. luteus extracts. C. The means were separated through arietinum ethanolic extract contains Tukey’s HSD (Honest Significant alkaloids, steroids, flavonoids, terpenoids Difference) test at a significance level of and tannins. The variability in active 0.05 probability. Percentage repellency compounds in each extract is expected to (PR) data was calculated by applying this affect their biological activities. Results Schneider-Orelli's formula (Püntener, of the present study is in accordance with 1981). many previous studies. Phytochemical analysis indicated the presence of flavonoids, alkaloids, steroids, and tannins in L. luteus, C. arietinum and G. Results and discussion max ethanolic extracts (Maknickiene et 1. Phytochemical screening: al., 2013; Mamta et al., 2013 and The phytochemical screening of Kumaran and Citarasu 2015). The ethanolic extracts showed the presence of variability in active compounds and their different types of active constituents concentrations in the extracts are (Table, 1). Alkaloids, steroids, flavonoids, expected to affect in the biological terpenoids, tannins and saponins were activities against pest (Elena et al., 2016). Table (1): Phytochemical screening of various extracts of tested plants.

Phytochemical Constituents Experimental Plants Alkaloids Steroids Terpenoids Flavonoids Tannins Saponins Lupinus luteus + + - - - + Glycine max + + + + + + Cicer arietinum + + + + + - 2. Toxicity of tested plant extracts arietinum act more fast against B. zonata against Bactrocera zonata adults: adults with LT50 values 4.5 and 5.2 days Toxicity of ethanolic extracts of compared to L. luteus extract with LT50 three legume plants L. luteus, G. max and value 15.5 days at 500 mg / L (Table, 3). C. arietinum seeds against the B. zonata Botanical insecticides are good adults after 6 and 8 days of treatment are alternatives to chemical insecticides and presented in Table (2). Ethanolic extracts approved to be effective in insect control of G. max and C. arietinum seeds (LC50 = (Rehman et al., 2009b). 366.2 and 433.9 mg L-1, respectively) The over use of chemical were more toxic than L. luteus seeds pesticides causes many environmental -1 ethanolic extract (LC50 = 627.5 mg L ) and health problems. Therefore, It is after 8 days of exposure. According to recorded that, Lupinus species are a confidence limits, the toxicity of source of alkaloids, which provide the ethanolic extracts of both G. max and C. plants with protection from adverse arietinum seeds against B. zonata adults weather conditions, microorganisms, after 8 days of exposure was comparable. fungi, insects, and herbivores (Rybiński Toxicity of the three tested plant extracts et al., 2018). The ethanolic extract of against B. zonata adults is time dependent marine algae Callyspongia crassa and (Table, 2). According to the LT50 values Grayella cyathophora achieved of the tested extracts, G. max and C. considerable toxicity against B. zonata

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under laboratory conditions (Elnagar et egg hatching of Bactrocera cucurbitae al., 2018). Acetone and water extracts of (Coquillett) in a dose dependent manner A. Cunn. bark (Kaur et al., 2010). Siddiqi et al. (2011) significantly prolonged the larval period reported that the acetone extract of and total developmental period, Curcuma longa achieved high mortality decreased percentage pupation, percentages to B. zonata and caused high percentage emergence, oviposition and inhibition of pupa formation. Table (2): Insecticidal effect of different concentrations of extracts on adult of Bactrocera zonata. Extracts Exposure period 2 LC50 Confidence limits Slope±SD Chi Tabulated Lupinus luteus 6days 712.5 670.8-755.3 7.3±0.7 3.8 8days 627.5 592.9– 659.3 7.5±0.6 3.8 6days 483.8 410.4-536.4 4.4±0.7 3.8 Cicer arietinum 8days 433.9 373.1-473.6 7.6±1.3 3.8 6days 442.6 328.3-514.9 3.4±0.7 3.8 Glycine max 8days 366.2 254.4-436.2 4.3±0.9 3.8

Table (3): LT50 values for adult of Bactrocera zonata exposed to plants extract at concentration of 500 mg / L. 2 Extracts LT50 Confidence limits Slope±SD Chi Tabulated C Glycine max 4.5 4 - 5 2.7±0.2 9.5 0.8 Cicer arietinum 5.2 3.8-6.6 3.2±0.3 9.5 0.8 Lupinus luteus 15.5 11.4-20.4 3.5±0.3 9.5 0.8 2. Ovicidal activity and effects on pronounced reduction in egg production some biological aspects of tested and egg viability when Phthorimaea plant extracts against Bactrocera operculella were exposed to the vapours zonata: arising from paper treated with 220 μl of Effects of the three legume plants Citrus sinensis. L. luteus , G. max and C. arietinum seeds The number of laid eggs / female ethanolic extracts on the B. zonata egg was highly affected when adults of B. hatchability are presented in Table (4). It zonata where exposed to the ethanolic is clear that, C. arietinum seeds ethanolic extracts of the three tested plant seeds extract had the higher activity where the (Table, 4). Glycine max L and C. egg hatchability reached to 0% at arietinum seeds ethanolic extracts were concentration of 1000 mg / L compared the most potent with 2.0 and 0.0 egg laid to 88.5% in control. The egg hatchability / female at 1000 mg / L, respectively reached to 69.8 and 58.4% when the B. compared to 49.0 egg / female in control. zonata eggs where treated by L. luteus L. The tested ethanolic extracts of the three or G. max L. at 1000 mg / L compared to tested plant seeds reduced the adult 88.5%in control. The ovicidal activity of longevity of B. zonata and the effect was the tested three plant ethanolic extracts is concentration dependent (Table, 4). The concentration dependent. Similar type of adult longevity reached to 6.7days when response in insects caused by plant adults was exposed to C. arietinum seeds extracts has also been reported from other ethanolic extract at 1000 mg / L laboratories. Sharaby (1988) reported compared to 59.3 days in control.

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Table (4): Effects of the tested plants extracts on egg hatchability, number of egg/female and longevity (days) for Bactrocera zonata. Concentration Egg hatchability(%) Number of egg/female Time of Treatments (mg/L) ±SD ±SD exposure(days) ±SD Control 0.0 88.5±9.1a 49.0±8.1a 59.3±1.2a 500 76.5±8.2b 12.3±0.4 b 32.0±3.5b Lupinus luteus 750 71.2±5.7b 11.25±1.3b 12.7±1.2c 1000 69.8±1.6bc 5.0±3.0cd 9.3±2.3de 500 75.0±8.3b 5.5±3.5c 11.3±1.2cd Glycine max 750 65.5±1.7cd 2.0±0cd 8.0±0ef 1000 58.4±8.3d 2.0±1.0cd 7.3±1.2ef 500 33.3±0e 1.1±0.2cd 11.3±1.2cd Cicer arietinum 750 33.3±0e 1.0±0cd 7.3±1.2ef 1000 0.0f 0.0d 6.7±2.3f Means with the same letters in the same column are not significantly different according to L.S.D. test at 0.05 level of probability. Insecticidal properties of any plant control of fruit flies, unless targeting the extracts depend on the active constitutes. fruit fly adults in abandoned areas and Saponins have strong detrimental effects vegetation. This behavior of flies on insects, causing mortality, growth suggests that, such control strategies may retardation and decreased fecundity (De be useful as an integrated pest Geyter et al., 2007). In addition, lower management (IPM) approach. Therefore, food intake of insects fed on saponin- plant extract formulations affecting the containing plant extracts was recorded oviposition have an added advantage over (Taylor et al., 2004 and Golawska et al., synthetic insecticides and can be a tool in 2006). According to Ishaaya (1986) integrated pest management programs for saponins slow down the passage of food the control of fruit flies (Khattak et al., through the insect gut. Perhaps they 2006). reduce the digestibility of the food by References inhibiting the secretion of digestive Ali, H.; Ahmad, S. ; Hassan, G. ; Amin, enzymes (Proteases and amylases) A.; Hussain, Z. and Naeem, H. (Golawska et al., 2006). Shimada (2006) (2011): Bio efficacy of different reported tannins to form irreversible plant extracts against fruit complexes with proline rich protein fly in bitter gourd. Pakistan J. Weed resulting in the inhibition of cell protein Sci. Res., 17(2):143-149. synthesis. Steroidal compounds are of Awad, A. I. (1993) : Biological and importance and interest due to their genetical studies on the relationship with various anabolic Mediterranean fruit fly Ceratitis hormones including sex hormones capitata (Wied.). M. Sc. Thesis, (Okwu, 2001). Hence, the presence of Fac. Agric. Alexandria University. these compounds in the tested plant Cowan, M.M. (1999) : Plant products as extracts corroborates the insecticidal antimicrobial agents. Clinical activities observed. Microbiology Reviews, 12 : 564- The systemic insecticides are not 582. the preferred choice for fruit flies control De Geyter, E.; Lambert, E.; Geelen, D. in fruit crops. Furthermore, the and Smagghe, G. (2007): Novel insecticides having contact action advances with plant saponins as remained insufficient to give successful natural insecticides to control pest

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