© 2010 Science From Israel / LPPLtd., Jerusalem

IsraelJournalofPlantSciences Vol.58 2010 pp.13–17DOI: 10.1560/IJPS.58.1.13

*Author to whom correspondence should be addressed.E-mail: fadel@volcani.agri.gov.il

The potential of pomegranate peel and heartwood extracts as a source of new bioacaricides to control the carmine spider mite Tetranychus cinnabarinus

Fauzi abo-Moch,a ibrahiM Saadi,b doron holland,c and Fadel ManSoura,*aDepartmentofEntomology,bDepartmentofSoilandWaterandEnvironmentalScience,and

cUnitofDeciduousFruitTreeScience,AgriculturalResearchOrganization,NeweYa’arResearchCenter,P.O.Box1021,RamatYishay30095,Israel

(Received25November2008;acceptedinrevisedform6April2009)

AbsTrAcT

Extracts of seven pomegranate (Punicagranatum L.) accessions were evaluated for their potential as sources of bioactive ingredients with significant acaricidal activity, which could lead to the development of new and safe bioacaricides. The crude ex-tracts (96% ethanol) of this plant were tested for their acaricidal activity against the carmine spider mite, Tetranychuscinnabarinus Boisduval, under controlled condi-tions. Mortality, repellency, and the number of eggs laid were determined. Extracts from the peel of five pomegranate accessions (C13, P.G.130-31, 7/34, P.G.132-33, and P.G.119-20) caused more than 63% repellency. The other two accessions, P.G.106-7 and P.G.127-28, caused 40% and 50% repellency, respectively, compared with the untreated control. All extracts significantly reduced the number of eggs laid (99%), compared with the controls. Four extracts of four pomegranate accessions (heartwood mixed with a very small ratio of leaves) caused significant repellency and significant reduction in the number of the eggs laid.

Our results show that pomegranate extracts have a good potential as acaricidal agents and that pomegranate accessions differ in their acaricidal activity.

Keywords: pomegranate, spider mite, bioacaricide, plant extract, analysis of acari-cidal activity

InTroducTIon

While pesticides have done much to improve the yields of high-quality agricultural products, the long-term use of chemical pesticides has harmful effects on human health, beneficial organisms, and the environment. Biologically based technologies, such as biological control, are attractive because they can provide last-ing, highly selective, and effective pest control. In this light, the growing need to develop pesticides that are environmentally friendly has gradually heightened the interest in biopesticides. This awareness has led to a steadily increasing movement towards a more environ-ment-oriented, sustainable agriculture with low or no input of toxic synthetic pesticides and other agricultural chemicals, in an attempt to preserve and protect the environment as well as human health ((Zalom et al.,

1992; Isman, 2000; Tsolakis et al., 2004; Rhoda et al., 2006; Ragusa et al., 2007), Considerable efforts are therefore being made worldwide to find biodegradable, environmentally friendly, safer substitutes for synthetic pesticides (Lee, 2004). Two common arthropod pests of universal importance are the tetranychid mite Tet-raychus urticae Koch and Tetranychus cinnabarinus Boisduval. These mites feed on numerous cultivated crops and cause serious damage, reducing the quality and quantity of yields, leading to reduction or total loss of yields (Tomezyk et al., 1989). The economic importance of these mites is constantly increasing be-cause of the development of resistance and resurgence of their populations, due to the nonselectivity of syn-

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thetic pesticides, which damage and may even destroy natural enemies such as predaceous mites and predatory spiders. The use of selective pesticides is suggested as the first step in developing an integrated mite manage-ment strategy. Employing botanical pesticides that are relatively harmless to natural enemies could therefore increase the effectiveness of their predation. This may in turn reduce pesticide applications thus reducing pro-duction costs and environmental pollution. Therefore, during the last 30 years many plant products have been tested as botanical pesticides to control spider mites (Mansour et al., 1993, 2004; Isman, 2000). For ex-ample, some of the essential oils extracted from 14 spe-cies of the family Lamiaceae caused mortality, induced repellency, and reduced egg-laying in adult females of the carmine spider mite (CSM) (Mansour et al., 1986). Different commercial formulations were obtained from the seeds of the neem tree (Azadirachta indica) that were as effective as other botanical pesticides against the two-spotted spider mite (TSM). In different crops these compounds showed ovipositional deterrence, suppressed the emergence of nymphs, and caused re-duction in leaf damage (Schmutterer, 1995; Singh and Singh, 1999; Kumar, 2002; Makundi and Kashenge, 2002). The pesticidal activity of extracts of two Eu-calyptus species (red and spotted gum) in comparison with some biocides and conventional pesticides was evaluated against TSM and Bemisiatabaci Gennadius infesting cotton fields in Egypt and showed very prom-ising acaricidal activity against both insects (Radwan et al., 2000). Because many botanical pesticides are considered to be safe compounds, their essential oils are attractive chemicals for the control of Varroe mites on honeybees, being perceived as “natural” compounds that will not contaminate hive products (Sammataro et al., 1998).

The pomegranate is a deciduous fruit shrub or a small tree that grows up to a height of 5–8 meters. Believed to be native to central Asia, it is widely cultivated through-out the world (Rehm, 1989; Still, 2006; Holland and Bar-Ya’akov, 2008; Holland et al., 2008).

Traditionally, pomegranate fruit has been used in China, India, Japan, and the Mediterranean basin as a medicine to cure a variety of diseases. Recently, the traditional usage was corroborated by scientific data indicating that pomegranates are a good source of antimicrobial, anticancer, and antidiabetic compounds (Seeram et al., 2006). Moreover, different pomegranate accessions contain different amounts of bioactive com-pounds such as punicalagin, punicalin, galagic acid, and ellagic acid (Tzulker et al., 2007).

In a preliminary study we found that pomegranate extracts showed acaricidal activity (Mansour et al.,

2004). The aim of our current work is to evaluate the po-tential of plant extracts (peel and heartwood) of different Israeli accessions of pomegranate as a source of bioac-tive ingredients with significant acaricidal activity that could lead to the development of new bio-safe products for the benefit of sustainable agriculture in the region.

MATerIAls And MeThods

Plant material

Thepomegranate samples used in this study were taken from several different accessions grown in the Newe Ya’ar Research Center of the Agricultural Research Organization in the Yizre’el Valley. This collection was described previously (Bar-Ya’akov et al., 2003, 2008; Holland and Bar-Ya’akov, 2008; Holland et al., 2008). All plants were taken from the same plot and grown under the same agro-technique.

The pomegranate accessions were C13 (DPUN0082 California), P.G.119-20 (‘Rosh Hapered’), P.G.106-7 (‘Malisi’), P.G.127-28 (Black pomegranate), P.G.130-31 (‘Shani-Yonay’), and P.G.132-33 (‘Bint el Basha’), 7/34. These accessions were chosen because they reflect a large variability with respect to peel and aril color, date of ripening, growth habit, and content of chemical compounds in their fruit tissues (Tzulker et al., 2007; Holland et al., 2008).

Preparation of tissue extracts

The colored sections of mature pomegranate fruit peels were removed with a knife. The removed peel was air-dried for 2–3 weeks at room temperature before it was ground to powder. The pomegranate heartwood and leaves (10–20%, w/w) from young branches (50–70 mm in diameter) were immediately cut into small pieces and powdered using a blender.

soxhlet extraction

Samples of dried ground pomegranate peel, or heartwood mixed with small amounts (10–20%, w/w) of leaves were extracted continuously in the Soxhlet extractor us-ing ethanol as an organic solvent (Mathew et al., 2008). A volume of 350 ml of ethanol was put into the round bottom flask of the Soxhlet apparatus. Subsequently, 5 g of pomegranate peel or 10 g of pomegranate heartwood was placed into a thimble and fitted into the Soxhlet extractor, and the apparatus was assembled. The solvent in the setup was heated to 65 ºC and the vapor produced was subsequently condensed by water flowing in and out of the extraction setup. This process of extraction was continued for 4 h, at the end of which, the thimble was removed. The ethanolic pomegranate extract (about

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300 ml) in the round bottom flask was concentrated by vacuum evaporation at 65 ºC to 50 ml of final extract volume. All extracts were kept in a refrigerator at 5 ºC for 4–6 weeks until used in the bioassay.

Maintenance of the carmine spider mites (T. cinnabarinus)

The local strain of T. cinnabarinus used in this study originated from infested kidney bean (Phaseolus vul-garis) leaves that had not been sprayed with pesticides. The mite stock was grown in a controlled climate room at 25 ± 2 ºC, 60 ± 5% rh, with 16 h light supplied by fluorescent lamps yielding a light intensity of ~2000 lux. Rearing was done on 2–3-week-old kidney beans, as described elsewhere (Tadmor et al., 1999). Bean plants were grown in 25 ́ 32 ́ 8 cm-high pots filled with peat-moss and vermiculite (2:1) and maintained in a growth chamber under controlled conditions (26 ± 2 ºC, 60 ± 5% rh, and 16 h light). Mites were transferred from ag-ing plants to younger ones by placing old leaves infested with mites near 7- to 10-day-old healthy seedlings. In-dividual female mites were collected and transferred for bioassay tests using a fine hairbrush.

bioassays for testing acaricidal activity of plant extracts

To investigate the effect of the active fractions or isolated compounds on the spider mites, we used a bioassay de-scribed previously (Mansour et al., 1993). Briefly, bean leaf discs (20 mm in diameter) taken from ~2-week-old plants (grown as described above) were dipped for 5 s in the extracted solutions, placed bottom side up on filter paper in a tray, and air-dried at room temperature before being tested. Concurrently, comparable control discs were prepared and dipped in the extraction solu-tion only or in distilled water. Each filter paper with leaf discs was placed on a sheet of plastic foam, floating on water in an aluminum dish (16 ´ 10 ´ 2.5 cm). On each leaf disc, 5 adult female mites (~5 days old) were placed and used as one replicate. Twelve discs with 60 mites were used for each treatment. The treated mites were incubated under controlled conditions, as described. The number of live, dead, or repelled mites (those that had left the leaf disks) and fecundity (calculated on the basis of eggs laid per treatment) were determined after 48 h of incubation.

statistical analysis

The experiment had a completely randomized design with 5–6 replicates per treatment. The ranking of means was done utilizing Tukey–Kramer’s test (p < 0.05). JMP version 5.0.1 software (SAS Institute) was used.

resulTs And dIscussIon

The effects of the crude extracts from the peel of seven pomegranate accessions and heartwood of four of them on the mean number of live, dead, and repelled mites and on the number of eggs laid during 48 h are summa-rized in Tables 1 and 2.

Mite mortality

Five peel extracts caused significant reduction in the mean number of live mites compared with the controls (Table 1). However, only two extracts, from accessions 7/34 and P.G.132-33, caused significantly more CSM mortality compared with the controls (Table 1). The most active plant extract, which caused 34% mortality, was from accession 7/34. The extracts of the other ac-cessions caused 7–25% mortality.

When four extracts of heartwood mixed with a small amount (~10%, w/w) of pomegranate leaves (from accessions C13, P.G.119-20, P.G.106.7, P.G.127.28) were assayed, they did not cause a higher mortality rate than the untreated controls (Table 2), but they caused a significant reduction in the mean number of live mites compared with the controls (Table 2).

repellency

Five peel extracts (C13, P.G.130-31, P.G.132-33, 7/34, P.G.119-20) caused significant repellency as com-pared with the controls. The most active extracts were P.G.130-31 and P.G.132-33 (Table 1).

When extracts of heartwood mixed with leaves were used, a significant repellency compared with the control caused at least 52% mite repellency. The best extracts were from accessions C13 and P.G.119 -20 (Table 2).

number of eggs laid

All seven peel extracts of pomegranate caused sig-nificant reductions in the mean number of eggs laid (Table 1). The best extracts for causing reductions in the number of eggs laid, as compared with the controls, were C13, P.G.130-31, 7/34, and P.G.132-33 (Table 1). The four heartwood extracts also caused significant reductions in the mean number of eggs laid (Table 2). The order of effectiveness was P.G.127-28, P.G.119-20, C13, P.G.106-7. The heartwood extract from accession P.G.127-28 was more effective than the peel extract. The observed reduction in oviposition is consistent with the data on live and on repelled mites. The lower the av-erage of 48 h fecundity, the lower the percentage of live mites. For example, accessions with 0.16, 0.88, 0.8, 0.6 eggs/5 mites/48 h (C13, P.G.130-31, 7/34, P.G.132-33) had an average of 13%, 8%, 3%, 3% live mites and 68%, 73%, 63%, 72% repelled mites, respectively, whereas

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accessions P.G.106-7 and P.G.127-28 that had 7.16 and 13.16 eggs/5 mites/48 h had approximately 50% and 43% live mites and 40% and 50% repelled mites, respectively (Table 1).

In a previous study we found that an extract of one pomegranate variety showed acaricidal activity (Man-sour et al., 2004). We have not found any former studies regarding the acaricidal activity of pomegranate. There are several studies on the effect of other plant extracts on different species of mites, but we found no investi-gation regarding the carmine spider mite, Tetranychuscinnabarinus. In this study we investigated the acari-cidal activity of different accessions of pomegranate on T.cinnabarinus and found no correlation between acari-cidal activity and antioxidant capacity, total phenols, and gallic acid reported from our laboratory (Tzulker et al., 2007). Direct applications of purified chemicals, such as ellagic acid and gallic acid, on mites also did not show any acaricidal activity (data not shown).

These findings show that both peel and heartwood extracts of pomegranate have a good potential for acari-cidal activity. Moreover, the findings demonstrate that different pomegranate accessions vary in their acari-cidal potential. The chemical nature of the bioactive acaricidal compound is not yet known and its identifica-tion could lead to the development of an effective, envi-ronmentally friendly treatment against spider mites.

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Table 1Effect of peel extracts of 7 different pomegranate accessions on the mortality, repellency, and fecundity of the carmine spider mite T.cinnabarinus (mean values (12 replicates, 5 mites per

replicate) after 48 h of incubation)Treatment Live mites Dead mites Repelled mites Number of % % % % eggsControl (100% water) 75 a 0 c 25 c 84.66 aControl (ethanol 96%) 68 ab 2 c 30 c 71.41 aC13 13 c 19 abc 68 ab 0.16 bP.G.130-31 8 c 19 abc 73 a 0.08 b7/34 3 c 34 a 63 ab 0.08 bP.G.132-33 3 c 25 ab 72 ab 0.16 bP.G.119-20 17 c 15 abc 68 ab 1.16 bP.G.106-7 50 ab 10 bc 40 bc 7.16 bP.G.127-28 43 b 7 bc 50 abc 13.16 bWithin columns, values followed by a common letter do not differ significantly (p < 0.05 Tukey–Kramer test).

Table 2Effect of heartwood and leaf extracts of 4 different pomegranate accessions on the mortality, repellency, and fecundity of the carmine spider mite T.cinnabarinus (mean values (12 replicates,

5 mites per replicate) after 48 h of incubation)Treatment Live mites Dead mites Repelled mites Number of % % % % eggsControl (ethanol 96%) 84 a 0 a 16 b 58.2 aP.G.127-28 28 b 4 a 68 a 0.2 cP.G.119-20 4 b 4 a 92 a 0.2 cC13 12 b 0 a 88 a 1.4 cP.G.106-7 40 b 8 a 52 ab 14.2 bWithin columns, values followed by a common letter do not differ significantly (p < 0.05 Tukey–Kramer test).

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