Effects of Two Plant Extracts on Larval Leafminer Liriomyza ... - BioOne

ECOLOGY AND BEHAVIOR

Effects of Two Plant Extracts on Larval Leafminer Liriomyza trifolii (Diptera: Agromyzidae) in Tomatoes H. S. CIVELEK1

AND

P. G. WEINTRAUB2

J. Econ. Entomol. 97(5): 1581Ð1586 (2004)

ABSTRACT Aqueous extracts from two plants, Urginea maritima L. (Liliaceae) and Euphorbia myrsinites L. (Euphorbiaceae), were tested for their insecticidal activity against the leafminer Liriomyza trifolii (Burgess) on infested tomato, Lycopersicon esculentum Mill., plants in the laboratory and Þeld. Two grams of plant material was extracted with 100 ml of water and then diluted 1:100, 1:50, and 1:25 with distilled water. Diluted plant extract was either applied to the infested tomato leaves or by soil drench and was compared with foliar application of cyromazine. All dilutions of both plant extracts caused signiÞcant control of the leafminer larvae and maintained populations below those of the nontreated control plants in all trials. Only at the most concentrated dilutions (1:25) were the plant extracts statistically similar to the cyromazine treatment. Furthermore, greenhouse yields from all of the foliar treatments were statistically similar to the cyromazine treatment and signiÞcantly better than the nontreated control. Four species of leafminer parasitoids were found in the greenhouse; however, the percentage of parasitism was signiÞcantly less in all treated replicates than in the nontreated control replicates. Aqueous extracts from these two plant extracts exhibited both translaminar and systemic activity and are potential candidates as new organic insecticides. KEY WORDS leafminer, squill, spurge, plant extract

THERE HAVE BEEN MANY studies on the effects of various plant extracts to control or repel insect pests; however, since the advent of synthetic insecticides, botanical insecticides constitute only about 1% of the global marketplace. The primary reason for their lack of use in commercial agriculture is poor efÞcacy compared with synthetic pesticides. Consumers, concerned about pesticide residues on food crops, and governments have responded by legislating and restricting synthetic pesticides. EfÞcacious botanical derivatives can provide an alternative to synthetic pesticides, and agrochemical companies have started to focus on this area (Addor 1995). A key pest in many ßower and vegetable crops, especially tomatoes, is the leafminer Liriomyza trifolii (Burgess) (Diptera: Agromyzidae) (Spencer 1973). Both larvae and adults cause damage: larvae primarily mine the palisade mesophyll (Parrella et al. 1984), and adult females puncture both upper and lower leaf surfaces to feed and lay eggs. Adult ßies have long been known to be resistant to several insecticides (Broadbent and Pree 1989, Keil and Parella 1990, MacDonald 1991, Saito 1994, Civelek 1999). A limited number of insecticides are efÞcacious against the larvae: abamectin, cyromazine, spinosad, and, most recently, bensultap (Civelek and Weintraub 2003). Of the bo1 Mugla University, Faculty of Technical Education, Wood Entomology Laboratory, 48100 Ko¨ tekli, Mugla, Turkey. 2 Department of Entomology, Gilat Research Center, D.N. Negev, 85280, Israel.

tanical insecticides tested to date, only neem-based insecticides are effective against L. trifolii larvae (Azam 1991, Dimetry et al. 1995) and the related pea leafminer, Liriomyza huidobrensis (Blanchard) (Weintraub and Horowitz 1997, Civelek et al. 2002) but are relatively expensive for use in nonorganic agriculture. Extracts of plants from the Liliaceae and Euphorbiaceae have been shown to be effective against a wide range of insects. Some examples of efÞcacious extracts from Euphorbia are water and ethanol extracts from Euphorbia cyparissias L., effective against the codling moth, Cydia pomonella (L.), and the twospotted spider mite, Tetranychus urticae Koch (Velcheva et al. 2001); several compounds isolated from Euphorbia paralias L. have molluscicidal and antifeedant activity against Spodoptera littoralis (Boisduval) (Abdelgalil et al. 2002); Euphorbia fischeriana Steud. is used in China as an anthelmintic and insecticidal ointment (Lee et al. 1991); and Euphorbia hirta aqueous leaf extracts are effective against the blister beetle Zonabris pustulata L. (Oudhia 2000) and against larval Helicoverpa armigera (Hu¨ bner) (Sundararajan 2002). The most well known insecticide of the Lilaceae is the extract of Schoenocaulon officinale, Asa Gray ex. Benth., which is the active ingredient in the commercial insecticide sabadilla. However, to date few other plant extracts with insecticidal activity from Liliaeceae have been reported; for example, Veratrum album (Schurz 1977) and Urginea maritima L. (Hassid et al. 1976, Pascual-Villalobos and Fernandez 1999).

0022-0493/04/1581Ð1586$04.00/0 䉷 2004 Entomological Society of America

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Herein, we are reporting on the effects of aqueous extracts of the Euphorbaceae, Euphorbia myrsinites L., and the Lilaceae, Urginea maritima, on control of leafminer larval L. trifolii in tomato plants and their effects on parasitoids of this pest. Materials and Methods Source Plants. Plants, in the ßowering stage, were collected from wild areas in western Turkey. U. maritima was collected from October to January (only white bulbs) and brought to the laboratory for extraction. E. myrsinites was collected from January to April, and all parts of the plant were brought to the laboratory for extraction. Voucher specimens of each plant were deposited in the Extraction Laboratory (Faculty of Technical Education, Mugla University, Mugla, Turkey). Extract Preparation. Plants were washed thoroughly to remove any soil or debris. Those not immediately used were stored in paper bags at 8⬚C until needed. For U. maritima, bulbs were initially cut into pieces and then placed in a blender to break them into very small pieces. All parts (ßowers, leaves, stems, and roots) of E. myrsinites were similarly ground. Each plant sample (2 g) was extracted with 100 ml of water by using a soxalet hot water extractor (PILZ, HerausWitmann, Heidelberg, Germany) for 4 h. Extracts were stored in a sealed bottle at 5⬚C until use. Each week of the trials fresh extracts were prepared. Laboratory Bioassays. A colony of L. trifolii has been maintained in the laboratory since 2000 on tomato plants at 22 ⫾ 2⬚C, 70 ⫾ 5% RH, and a photoperiod of 14:10 (L:D) h. For the trials, 100 adult ßies were released into cages of potted tomatoes at a ratio of 50 males:50 females, and new ßies were added weekly. Tomato seeds sown in vials were grown until there were four true leaves. They were then removed and transferred to plastic treatment pots; the soil and pots (20 by 20 cm) had been sterilized using 10% formaldehyde (formaldehyde breaks down to carbon dioxide and water, leaving no residue). Soil was composed of one part each potting soil:sand:dried goat-sheep manure. Plants were allowed to adapt in these pots for 2 d before the bioassays started. Untreated tomato plants were placed in a cage (75 by 65 by 50 cm, glass sided) with 100 L. trifolii for 1 wk before initial treatment. One set of Þve plants served as an untreated control for each trial. Two bioassays were used: soil drench and foliar application. Each concentrated extract was diluted to 1:100; 1:50; 1:25 (vol:vol) in distilled water immediately before use. Each trial was conducted four times with Þve replicates each. Five plants were treated with each concentration of both extracts by applying 100 ml of diluted extract to each pot. Excess extract was allowed to drain, and then the potted tomato plants were replaced in the insect cage. This treatment was repeated once a week for 5 wk. Five plants each were treated with plant extract dilutions by foliar application. The soil of each plant was covered with paper toweling (to absorb any runoff from the plant leaves,

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thus preventing both foliar and soil treatment) then each plant was sprayed to runoff. Once the leaves stopped dripping, paper toweling was removed and plants were returned to the cage with ßies. This treatment was repeated once a week for 5 wk. Five plants were treated with Trigard 75 WP (cyromazine 75%, Novartis, applied at the recommended rate of 20 g/100 liters of water) by foliar application as a positive chemical control for comparison. Plants were sprayed weekly as described above. Each week, Þve leaves were removed from each of the treated and nontreated plants, and the number of live larvae was counted and recorded. Each trial was terminated after 5 wk because the plants reached the top of the cages and the leaves began to yellow. Field Trials. Studies were carried out in a commercial greenhouse located in Ortaca, Mugla, Su¨ kru¨ Kilinc, Turkey. The greenhouse (2,500 m2) was made of an iron framework covered with solid polyethylene. Tomato seedlings (variety Astona F1) were planted on 10 October 2001 and 7 October 2002. Plants were watered and fertilized according to local grower practices. Within the greenhouse, 30-m2 plots (42Ð 44 plants) were randomly designated to be treated with one of the plant extracts, Trigard 75 WP (20 g/100 liters of water) or remain as an untreated control. There was a nontreated buffer zone of 1 m between each plot to prevent spray drift to adjacent plots. Each treatment and control was replicated Þve times, and trials were carried out over two seasons. The experimental design was the same for both seasons. Each concentrated plant extract was diluted to 1:100; 1:50; 1:25 (vol:vol) in distilled water immediately before use. Once a week, 50 ml of the appropriate plant extract dilution was applied to the soil at the base of each plant. Once the plants reached 1 m in height, 100 ml was applied. From a height of ⱖ2 m, 200 ml of extract was applied. All foliar treatments were applied with a low-pressure backpack sprayer. Plant extract was applied as a drench when the plants were small (OctoberÐDecember); subsequently 5 liters per 30-m2 replicate was applied. Each week, 10 leaves, removed randomly from each of the treated and nontreated replicates, were brought to the laboratory and examined under a stereomicroscope. Live larvae were counted and recorded. An additional 10 leaves per treatment were collected and placed in plastic insect cages to allow parasitoids to emerge. All parasitoids were sent to Dr. John LaSalle (Commonwealth ScientiÞc and Industrial Research Organization Entomology, Canberra, Australia) for identiÞcation. Five yellow sticky traps were placed in each replicate and changed weekly to monitor adult ßy populations. All tomatoes were harvested and weighed once every 2 wk starting in the middle of January each year. Each trial was terminated after 32Ð33 wk when the plants had Þnished producing fruit. Statistics. Field data for the number of larvae in leaf samples and the number of adult ßies trapped on yellow sticky cards were Þrst transformed by log x ⫹1; when larval and adults populations were plotted over

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CIVELEK AND WEINTRAUB: EFFECT OF PLANT EXTRACTS ON L. trifolii

Table 1. Effect of aqueous extracts of U. maritima and E. myrsinites applied as a foliar spray or soil drench against L. trifolii larvae in potted tomato plants Treatment

Foliar

Drench

Control Ua 1:100 U 2:100 U 4:100 Eb 1:100 E 2:100 E 4:100 Trigardc

2.52 ⫾ 0.04a 1.24 ⫾ 0.04b 1.10 ⫾ 0.04b 0.79 ⫾ 0.04c 1.23 ⫾ 0.05b 1.08 ⫾ 0.05b 0.88 ⫾ 0.04c 0.77 ⫾ 0.04c

2.52 ⫾ 0.04a 1.39 ⫾ 0.04b 1.24 ⫾ 0.04bc 0.99 ⫾ 0.04d 1.20 ⫾ 0.04c 1.21 ⫾ 0.04c 0.98 ⫾ 0.04d 0.77 ⫾ 0.04e

Data are average number of live ⫾ SE larvae from 1 wk after Þrst application until the end of the trial. Different letters within a column indicate differences of P ⬍ 0.05. a U. maritima. b E. myrsinites. c Trigard was applied as a foliar spray.

the time course of these trials, linear relationships with regression coefÞcients ranging from 0.90 to 0.96 were produced. The slopes produced by these regression analyses were separated by GLM. Yield data were analyzed by analysis of variance (ANOVA). Because the number of adult ßies was maintained at a constant weekly density in laboratory trials, and there was no signiÞcant difference between the number of larvae per leaf over the course of the trials, larval data were analyzed by pooling all replicates and evaluating treatment differences by using one- and two-way ANOVAs. Means were separated using TukeyÕs honestly signiÞcant difference (HSD) quantile function, by using JMP 5.0.1a (SAS Institute 2002). All tests were conducted at ␣ ⫽ 0.05 level. Results Laboratory Trials. Each trial consisted of measuring the number of live larvae on Þve leaves from each of Table 2. greenhouse

Þve plants over a period of 5 wk and was repeated four times. At the time of the Þrst treatment, the number of larvae was similar for all plants. From 1 wk after treatment until the end of the trial the number of live larvae remained similar for each treatment. Therefore, the ANOVA was conducted on the number of live larvae observed on each of Þve leaves per Þve replicates over 4 wk (n ⫽ 200 observations per treatment). Results of the number of live L. trifolii larvae from infested tomato plants after foliar treatment with plant extracts, cyromazine, or nontreated control are shown in Table 1. The number of live larvae in the control plants was signiÞcantly larger (pooled trials: F ⫽ 178.60; df ⫽ 7, 3,991; P ⬍ 0.0001) than the number of larvae found in any treatment, even the most diluted plant extracts. The highest concentration of each plant extract applied as a foliar extract was statistically similar to the cyromazine treatment. Results of the number of live L. trifolii larvae from infested tomato plants after soil treatment with plant extracts, foliar cyromazine, or nontreated control are shown in Table 1. As with the foliar treatment, the number of live larvae in the control plants was signiÞcantly larger (pooled trials: F ⫽ 162.24; df ⫽ 7, 3,991; P ⬍ 0.0001) than the number of larvae found in any treatment. The Trigard foliar treatment had signiÞcantly fewer larvae than the highest drench concentration. The foliar application of the plant extracts was more effective than the drench (pooled trials: t ⫽ 4.66; df ⫽ 5,997; P ⬍ 0.0001). Field Trials. Results of the Þeld trials for the application of the plant extracts by foliar and drench applications for both years are shown in Table 2. Regression analyses for the Þrst year showed that there was a signiÞcant difference between the slopes of foliar and drench treatments for larvae (P ⬍ 0.0001) and adults (P ⬍ 0.002), and both larval and adult populations signiÞcantly increased over time in all treatments (larvae, P ⬍ 0.02; adults, P ⬍ 0.0001). All

Effect of aqueous extracts of U. maritima and E. myrsinites applied as a foliar spray against L. trifolii larvae in a tomato First year

Treatment Control Foliar Ua 1:100 U 2:100 U 4:100 Eb 1:100 E 2:100 E 4:100 Drench U 1:100 U 2:100 U 4:100 E 1:100 E 2:100 E 4:100 Trigardc

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Larvae

Second year Adults

Larvae

Adults

3.80 ⫾ 0.06a

673.00 ⫾ 66.14a

6.28 ⫾ 0.05a

721.10 ⫾ 65.83a

1.45 ⫾ 0.03bcd 1.36 ⫾ 0.03cde 1.01 ⫾ 0.03cde 1.53 ⫾ 0.03bcd 1.40 ⫾ 0.04cde 1.03 ⫾ 0.03de

227.99 ⫾ 20.59b 214.78 ⫾ 19.51bc 134.69 ⫾ 13.49bc 224.52 ⫾ 20.28bc 132.18 ⫾ 11.36bc 103.72 ⫾ 10.82c

2.10 ⫾ 0.06a 1.72 ⫾ 0.05a 1.41 ⫾ 0.04a 2.46 ⫾ 0.06a 2.06 ⫾ 0.05a 1.46 ⫾ 0.04a

212.47 ⫾ 19.05bc 210.66 ⫾ 18.68c 116.39 ⫾ 11.00c 236.57 ⫾ 23.02bc 206.45 ⫾ 20.78bc 125.02 ⫾ 13.08c

1.94 ⫾ 0.04bc 1.54 ⫾ 0.04cde 1.15 ⫾ 0.03cde 1.74 ⫾ 0.04bcd 1.52 ⫾ 0.04ab 1.23 ⫾ 0.03cde 0.69 ⫾ 0.02e

322.61 ⫾ 30.39b 248.44 ⫾ 22.82bc 221.63 ⫾ 22.49b 313.43 ⫾ 32.76bc 191.44 ⫾ 18.31bc 139.83 ⫾ 11.60c 56.04 ⫾ 6.32c

2.15 ⫾ 0.06a 1.87 ⫾ 0.05a 1.68 ⫾ 0.05a 2.48 ⫾ 0.06a 2.31 ⫾ 0.06a 1.78 ⫾ 0.05a 1.04 ⫾ 0.04a

392.59 ⫾ 34.89b 228.59 ⫾ 19.51bc 212.27 ⫾ 18.77bc 286.75 ⫾ 26.27b 229.51 ⫾ 20.71bc 208.87 ⫾ 19.44bc 60.93 ⫾ 5.93c

Data indicate average number of live larvae ⫾ SE and adult ßies ⫾ SE from 1 wk after Þrst application until the end of the trial for 2 yr. Different letters within a column indicate differences of P ⬍ 0.05 of the slope of the regression analysis. a U. maritima. b E. myrsinites. c Trigard was applied as a foliar spray.

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Table 3. Effect of aqueous extracts of U. maritima and E. myrsinites applied to the foliage and soil against L. trifolii larvae in a tomato greenhouse on yield Treatment

Foliar

Soil

Control Ua 1:100 U 2:100 U 4:100 Eb 1:100 E 2:100 E 4:100 Trigardc

20.09 ⫾ 0.68c 26.70 ⫾ 0.92b 27.90 ⫾ 1.04ab 30.94 ⫾ 1.04a 26.86 ⫾ 0.77b 27.87 ⫾ 0.92ab 29.59 ⫾ 1.05ab 29.91 ⫾ 1.08ab

20.09 ⫾ 0.68c 23.80 ⫾ 0.93bc 27.26 ⫾ 0.96ab 28.58 ⫾ 0.98a 21.96 ⫾ 0.76c 27.67 ⫾ 0.81a 27.79 ⫾ 0.88a 29.91 ⫾ 1.08a

Data indicate average weight ⫾ SE of tomatoes for both years. Different letters within a column indicate differences of P ⬍ 0.05. a U. maritima. b E. myrsinites. c Trigard was applied as a foliar spray.

treatments reduced the average number of larvae to two or less per leaf; however, cyromazine had the fewest. Plant extract treatments signiÞcantly reduced the number of adults (three- to six-fold) compared with the nontreated control. Whereas cyromazine had the greatest reduction in the number of adults, the regression analysis showed that the slope of the cyromazine treatment was not signiÞcantly different than those of the plant extract foliar treatments (excluding the most dilute treatment with U. maritima). Most of the drench treatments with plant extracts reduced the number of adults trapped and compared favorably with the cyromazine foliar treatment. Regression analyses for the second year showed that there was a signiÞcant difference between the slopes of foliar and drench treatments for adults (P ⬍ 0.0001), but not larvae, and both larval and adult populations signiÞcantly increased over time in all treatments (larvae, P ⬍ 0.001; adults, P ⬍ 0.0006). All treatments reduced the average number of larvae to two or less per leaf; however, comparison of the slopes or intercepts generated by regression analysis showed not signiÞcant differences. All foliar and drench applications of plant extracts signiÞcantly reduced the number of adults (2- to 4.8-fold) compared with the nontreated control; however, all foliar plant extract applications compared favorably with the cyromazine treatment, but not all drench treatments. Results of the yield are shown in Table 3. There was no difference between the yields for the 2 yr. All treatments had signiÞcantly heavier yields than the control (pooled foliar trials: F ⫽ 12.48; df ⫽ 7, 792; P ⬍ 0.0001; pooled drench trials: F ⫽ 15.34; df ⫽ 7, 792; P ⬍ 0.0001). A comparison of the two application methods of the plant extracts showed that the foliar application had signiÞcantly heavier yield (F ⫽ 15.30; df ⫽ 1, 1,198; P ⬍ 0.001). Four species of parasitoids were found: Diglyphus isaea (Walker) (average 16.44 per week), Neochrysocharis formosa (Westwood) (average 13.90 per week), Diglyphus crassinervis Erdo¨ s (average 4.43 per week), and Chrysocharis pentheus (Walker) (two specimens collected once). There were no signiÞcant differences in the number of parasitoids from the soil applications

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and the nontreated control in either year. There were signiÞcantly more parasitoids from the nontreated control replicates than in any of the foliar treatments for both years (F ⫽ 6.95; df ⫽ 7, 512; P ⬍ 0.0001). There was an average of 3.39 parasitoids per 50 leaves per week in the control plots and between 1.1 and 1.7 parasitoids per 50 leaves per week in the treatments. Discussion The chemistry of U. maritima, commonly known as squill, is complex but well studied, because of its cardiac glycoside (medicinal) and raticide activity; but relatively little is known about its insecticidal activities. The active compounds are L-azetidine-2-carboxylic acid (AZA) (Hegnauer 1970), and the bufadienolides scilliroside, scilla glycoside, and aglycones (Verbiscar et al. 1986). Hassid et al. (1976) were the Þrst to examine the insecticidal properties of extracts of U. maritima. They showed that the foliage of U. maritima was highly toxic to lepidopterous larvae, such as S. littoralis, and methanol extracts of dried leaves (which contained AZA), when incorporated into artiÞcial diet, also caused 100% mortality. Subsequently, Adeyeye and Blum (1989) showed that AZA incorporated into the artiÞcial diet of another lepidopteran species, Helicoverpa zea (Boddie), disrupted growth and development. Pascual-Villalobos and Fernandez (1999) showed that ethanol extracts of red and white U. maritima bulbs of different ploidy differed in their topical and dietary effects on the stored food pest Tribolium castaneum (Herbst). In their studies, the white bulbs of higher ploidy (tetra- and hexaploids) had more activity than white triploids or red bulbs. Whereas this was sufÞcient to show insecticidal properties in general, it could not be used in a practical manner. We used aqueous extracts of pentaploid (n ⫽ 50) white bulbs from U. maritima and applied these extracts as an insecticide would be used against a pest: as a spray and through the irrigation system. There was no signiÞcant difference between the numbers of live larvae found in the foliar application of 1:25 dilution of U. maritima-treated tomato leaves versus cyromazinetreated leaves in all four laboratory trials and overall in the Þeld trials. In fact, even though there were signiÞcant differences between the number of live larvae in the 1:100 and 1:50 dilutions of U. maritima extract versus cyromazine, these differences were not biologically signiÞcant because all treatments reduced the number of larvae to two or fewer per leaf (the level where treatment is recommended), and Kotze and Dennill (1996) showed that low levels of L. trifolii infestations on tomatoes actually increase yield. The soil treatment, although effective, was not as efÞcacious as the foliar treatment. This means that the aqueous plant extract of U. maritima is probably both translaminar and systemic in action. E. myrsinites is native to Eurasia but has invaded other continents, and the milky sap is known to cause mild skin irritation (Spoerke and Temple 1979, Eberle et al. 1999). Recent work on an acetone extract of the

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plant from Turkey has revealed four new diterpene esters in addition to the previously known cycloartane-type triterpenoids and betulin (Oksuz et al. 1995). It is unknown whether these chemicals also are found in the aqueous extracts of E. myrsinites and whether they are responsible for the insecticidal effects. In foliar and soil applications, the most concentrated extract applied compared favorably with the commercial insecticide cyromazine and all dilutions reduced the number of larvae to two or fewer per leaf. As with the aqueous extract of U. maritima, the aqueous extract of E. myrsinites seem to be both translaminar and systemic in action. Four parasitoid species invaded the greenhouse: D. isaea, D. crassinervis, N. formosa, and C. pentheus. The level of parasitization in the control replicates was ⬇1.4%, relatively low because trials were run in a commercial greenhouse. Unfortunately, the two plant extracts also reduced the number of parasitoids, most probably due to host death. This research was initiated when the Þrst author observed that some growers were putting these plants in their irrigation tanks, thus creating a crude extract. Other growers were boiling the plants in large cauldrons then adding the solution to their irrigation tanks. These plants are plentiful in Turkey and could serve as an inexpensive and organic alternative to commercial insecticides. Research is in progress with these two potential insecticides to identify the active ingredients in the aqueous fractions and to compare the insecticidal activity of the aqueous and ethanol extractions of U. maritima.

Acknowledgments We thank John LaSalle for identiÞcation of the parasitoid species, Aysen Melda Colak for technical assistance, and Ruth Marcus for assistance with regression analysis. We also acknowledge The ScientiÞc and Technical Research Council of Turkey (TUBITAK-TOGTAG-2836) and Mugla University, Committee of ScientiÞc Research Projects, for supporting this study.

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Verbiscar, A. J., J. Patel, T. F. Banigan, and R. A. Schatz. 1986. Scrilliroside and other scilla compounds in red squill. J. Agric. Food Chem. 34: 973Ð979. Weintraub, P. G., and A. R. Horowitz. 1997. Systemic effects of a neem insecticide on Liriomyza huidobrensis larvae. Phytoparasitica 25: 283Ð289. Received for publication 19 February 2004; accepted 5 July 2004.