Liriomyza trifolii (Burgess) (Diptera: Agromyzidae) Control on ...

(Burgess) (Diptera: Agromyzidae) Control on Chrysanthemum by Neem Seed Extract Applied to So iF

Liriomyza

trifolii

HIRAM G. LAREW,2 J. J. KNODEL-MONTZ,2 RALPH E. WEBB,2 ANDJ. DAVID WARTHEN"

J. Econ.Entomol.78:80-84(1985) Applied as a soil drench to chrysanthemums, 0.4% crude neem extract caused significant mortality of late instars and pupae of Liriomyza tn/olii (Burgess)in both research and commercial greenhouses. Insecticidal effects of drenching lasted for 3 weeks. Drenching did not inhibit female feeding or oviposition. Repeated drenching did not significantly inhibit plant growth. Mortality (89%) of prepupae reared from untreated plants occurred when the prepupae were placed on neem-drenched soil. ABSTRACT

Iceberg. The colony was established in March 1982 with adults collected near Ft. Myers, Fla. All chrysanthemums were grown in IO-cm plastic square pots in a mixture of 50% Luna Rock (Pennylsvania Perlite Corp., Le High Valley, Pa.) and 50% Promix (Premier Brands, New Rochelle, N.Y.) under long-day conditions with a LD 16:8 photoperiod. Plants were left unpinched and were fertilized weekly with Peters 20-20-20 (Peters Fertilizer Products, Fogelsville, Pa.). Except when exposed with leafminers, the plants were grown in a glass greenhouse at 23 to 30"C. For each test, plants that were 2 to 4 weeks old and actively growing were randomly placed in a cage for 24 h with 50 to 75 adult leafminers between 1200 and 1500 hours. The cage was illuminated at 5,600 lux with high pressure sodium lamps and incandescent bulbs, and the temperature was maintained at 24°C. Treatment dilutions were prepared from a liquid concentrate (sample A13-42845 [AN 4.57); Biologically Active Natural Products Lab., USDA-ARS, Beltsville, Md.) of neem seed extract (1 part 95% EtOH extract of seed: 1 part 95% EtOH) that contained 2,300 ppm azadirachtin, one of the active insecticidal principles in neem seed extract (Warthen 1979). Tween-20 (Atlas Chemical Industries, Wilmington, Del.), was used to emulsify the extract in water. Effective Concentration. Treatments were water, 0.2% Tween-20, 0.01% neem in 0.005% Tween20,0.1% neem in 0.05% Tween-20, and 0.4% neem Materials and Methods in 0.2% Tween-20. Five plants were used per treatment. On day 0, 150 ml of solution were apThe experiments that were run in research greenhouses were conducted from November 1983 plied to the soil of each pot. This volume was sufficient to cause slight drainage. Plants were exthrough March 1984. Insects used in the experiposed to leafminers on day 3 for 24 h and were ments were removed from a colony of L. trifolii maintained at Beltsville on chrysanthemum cv. then returned to the greenhouse. Three to four days later, the number of first-instar mines (less Thispaperpresentsthe resultsof researchonly.Mentionof a than 1 cm in length) were counted. Before third proprietaryproductdoesnotconstitutean endorsementor a rec- instars began to exit the mines, all leaves longer ommendationfor its useby USDA. than 2.5 cm (petiole + mid vein) were harvested, •Floristand NurseryCropsLab.,B-470,BeltsvilleAgric.Res. passed through an area meter (Li-Cor, Lincoln, Center,USDA-ARS, Beltsville,MD20705. • BiologicallyActiveNaturalProductsLab.,B-306,Beltsville Neb.), and then placed on high-sided trays and left Agric.Res.Center,USDA-ARS, Beltsville,MD20705. in the greenhouse for 7 to 9 days. Prepupae that

THE NEEM TREE, Azadirachta indica A. Juss, is planted extensively in the Old World tropics-e.g., Africa (Nigeria, Northern Cameroon, Gambia), India, and Pakistan, and also has been introduced into Cuba, Haiti, Barbados, and southern Florida (Jacobson 1981, Lewis and Elvin-Lewis 1983). Neem leaves and seeds have long been used as sources of insect repellents and insecticides (Jacobson 1981). As a foliage spray, neem seed extract acts as either a feeding inhibitor or as an insect growth regulator or both against a wide variety of insects (Warthen 1979, Rembold et al. 1982). Neem seed extract applied to foliage is effective against the serious horticultural leaf mining pests Liriomyza sativae Blanchard and L. trifolii (Burgess) (Webb et al. 1983, Fagoonee and Toory 1984). Systemic activity of neem extract in beans against locusts was reported by Gill and Lewis (1971). Our study was done to test for systemic activity of soil-applied neem seed extract against L. trifolii, in chrysanthemums. Experiments were conducted in research greenhouses to determine the most effective concentration of neem extract, the longevity of the insecticidal effect, neem's effect on female feeding and oviposition, and neem's effect on soil-inhabiting third-instar prepupal larvae. Following these experiments, an experiment was conducted to test neem's efficacy against L. trifolii in a commercial greenhouse.

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Table 1. Response of chrysanthemums and L. trifolii to soil applications of crude neem exlracl Mean Treatment

Leaf area (cm2) per plant

No. firstinstar mines per plant

No. pupae per plant

No. adults per plant 14a

Water

334a

68a

26ab

0.2% Tween-20

316a

77a

25ab

13a

0.01% Neem in 0.005% Tween-20

334a

60a

34a

22a

0.1% Neem in 0.05% Tween-20

369a

75a

17b

0.4% Neem in 0.2% Tween-20

332a

60a

0.4c

Ib Ob

Values equal mean from five plants. Means within columns followed by the same letter do not differ significantly (P < 0.05; Duncan's [1951] multiple range test).

emerged from the leaves were placed in gauzecovered vials and left in the greenhouse. Pupae and adults that developed were counted. Longevity of Insecticidal Effect. On day 0, 32 plants were treated with a soil drench of 150 ml of 0.05% Tween-20 per pot; the same number were treated with 150 ml of 0.1% neem extract in 0.05% Tween-20. On days 3, 7, 14, and 21, eight Tweenonly and eight neem-treated plants were exposed to leafminers for 24 h. Data were collected as described in the previous experiment. Effect of Extract on Female Feeding and Oviposition. Treatments were 0.2% Tween-20, 0.1% neem in 0.05% Tween-20, and 0.4% neem in 0.2% Tween-20. Five plants were used per treatment. On day 0, the soil was drenched with 150 ml of treatment solution per pot. On day 3, plants were exposed to leaf miners for 24 h. After exposure, feeding stipples and eggs were counted using a leaf-staining technique (Parrella and Robb 1982). Effect on Soil-inhabiting Prepupae. Treatments were 0.2% Tween-20, 0.1% neem in 0.05% Tween-20, and 0.4% neem in 0.2% Tween-20. Prepupae were collected within 15 min after they emerged from leaves of untreated plants and were placed on the surface of the soil in 25.4-cm pots that had just been drenched with 200 ml of each treatment (this volume resulted in slight drainage). The pots contained no plants. Pots were placed in a growth chamber at 24°C under long-day photoperiod and were misted every other day until adults emerged. Before emergence, fitted caps were placed on the pots so that emerging adults could be trapped and counted. This experiment was done twice. Commercial Greenhouse Experiment. To test neem's effectiveness further, we conducted experiments in a commercial glass greenhouse in Perry Hall, Md. The greenhouse was planted with chrysanthemums of various ages. Daytime temperatures in the greenhouse ranged from 25 to 35°G Leafminer infestations were monitored weekly using three 29.5-by-15.0-cm Sticky Strips (Olson Products, Medina, Ohio). Strips were hung over and along the length of the greenhouse center bed (over nonexperimental plants). Rooted cuttings of

cv. Hartmann's Dignity were planted 10 per row in a section of ground bed (23 by 1.5 m) along the east wall of the greenhouse on 19 March 1984. The crop was grown in topsoil to which peat moss had been added (1 bale/23 m2 of bed space). Plants were grown as single stem disbuds (long day from planting until 10 April; short day from 11 April until blooming; disbudded 14 May; bloomed 13 June 1984). Voucher specimens of adult L. trifolii collected from plants in this experiment have been deposited in the National Museum of Natural History, Washington, D.C. (Lot 84-07884). Experimental plots were 2.3 m long (15 rows per plot; 150 plants total) and were marked off one next to the other along the bed. Plots were partitioned with plastic from the soil surface to 15 cm below ground, and four rows of plants between plots were left as an unsampled buffer zone. There was one plot per treatment and treatments were randomly assigned. Treatments began 1 week after planting and continued biweekly until 3 weeks before blooming. Sampling began 2 weeks after planting and continued biweekly until 2 weeks before blooming. The crop was both treated and sampled a total of five times. At sampling, four plants from each treatment were removed and the leaf area for each plant was taken. Leafminers were reared from the leaves at 24°C (LD 18:6 photoperiod). Treatments included water, 0.1 and 0.4% neem (same source of neem as in previous experiments) applied as soil drenches. No Tween-20 emulsifier was used. We applied 15.6 liters of each treatment to the assigned plot on every treatment date. Based on the research greenhouse studies, this volume should have penetrated the upper 8 to 10 cm of soil. Another plot was sprayed with 75 Trigard (Ciba-Geigy, Greensboro, N.G) in a dose of 140.7 g (AI)/ha (0.125Ibs [AI]/acre), a chemical known to be effective against leafminers. A last plot was sprayed by the growers when they sprayed the rest of the house. They sprayed irregularly with Mavrik (Zoecon, Palo Alto, Calif.) and Pramex (Renick, Lyndhurst, N.J.), and applied Temik (Union Carbide, Jacksonville, Fla.) twice to the soil. All three chemicals were applied in recommended dosage.

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Table 2. Plant growth and insecticidal longevity alter a single application of crude neem extract against L. trifolii on chrysanthemums Mean Challenge day

Leaf area (cm2) per plant

Treatment

No. firstinstar mines

per plant

No. pupae per plant

No. adults per plant

63.7

36.4

3

0.05% Tween-20 0.1% Neem in 0.05% Tween-20

708.8 788.7

71.9

14.3·

O·

7

0.05% Tween-20 0.1% Neem in 0.05% Tween-20

630.6

155.5

89.0

54.9

625.5

160.4

27.1·

O·

14

0.05% Tween-20 0.1% Neem in 0.05% Tween-20

935.9

38.3

13.9

5.1

21

0.05% Tween-20 0.1% Neem in 0.05% Tween-20

1,023.2

93.4

7.3

O·

1,173.7

151.0

84.4

41.1

1,117.5

78.5

13.3·

65.3"

0.25"

Values represent means from eight plants. Neem values that are significantly different from the comparable Tween-20 controls are followed by an asterisk (P < 0.05; Student's I test).

Statistical Analysis. Treatment means for all experiments were compared statistically at the P < 0.05 level with either the Student's t test or Duncan's multiple range test (Duncan 1951). Results and Discussion Effective Concentration. All measured parameters (leaf area, number of mines, pupae, and adults) showed no significant differences between water and Tween-20 treatments (Table 1). Thus, Tween-20 treatments were used as checks for the remaining research greenhouse experiments. There were no significant differences among treatments with regard to mean leaf area or mean number of first-instar miners. The number of pupae, however, was significantly lower when plants were treated with 0.4% neem compared to the other treatments. Both 0.1 and 0.4% neem caused significantly lower adult emergence compared to the other treatments. When mortality in the water check treatment was taken into account using Abbott's (1925) formula, percentage of control between the pupal and adult stages with 0.1 and

Table 3. Response of adult L. trifolii feeding and oviposition on chrysanthemums to soil application of crude neem extract

0.4% neem was 89 and 100%, respectively. A 0.01% neem drench did not reduce leafminer numbers. These results indicated that the tested concentrations of neem did not affect plant growth (as indicated by leaf area; see commercial greenhouse experiment), or the number of first-instar larvae (and thus, presumably the number of eggs laid; see feeding and oviposition experiment). Neem concentrations of 0.1 and 0.4% did, however, negatively affect survival of later instars and pupae. Based on these results, we used either or both of these concentrations in the remaining experiments. Longevity of Effect. For 3 to 21 days after treatment, 0.1% neem caused significant or complete mortality between the pupal and adult stages (Table 2). Thus, the insecticidal effects of a neem soil drench lasted for up to 3 weeks. The fact that day 21 plants grew on average by 53% leaf area between treatment and harvest, coupled with high insect mortality after this growth, suggests that the neem toxins were translocated to new foliage. Effects of Feeding and Oviposition. Although there was a decrease in the mean density of feed-

Table 4. Effect of neem-drenched larvae of L. trifolii

Mean Treatment

0.2% Tween-20 0.1% Neem in 0.05% Tween-20 0.4% Neem in 0.2% Tween-20

Treatment

Density of feeding stipples

Density of eggs

8.85a 8.43a 6.77a

0.60a 0.59a 0.44a

Number per square centimeter of leaf area, 0 = 5 plants. Means within a column followed by the same letter are not significantly different (P < 0.05; Duncan's [1951] multiple range test).

Trial 1 (0

= 27

= 50

No. adults emerged

% Emergence

per treatment)

0.2% Tween-20 0.1% Neem in 0.05% Tween-20 0.4% Neem in 0.2% Tween-20 Trial 2 (0

soil on prepupal

12 I 2

44 4 7

22 9 1

44 18 2

per treatment)

0.2% Tween-20 0.1% Neem in 0.05% Tween-20 0.4% Neem in 0.2% Tween-20

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LAREWET AL.: NEEM CONTROLOF LEAFMINERSON CHRYSANTHEMUMS

Table 5. Resultsof using neem in a commercial greenhouse

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gard dramatically reduced the number of reared adults. When mortality in the water treatment is considered (Abbott 1925), percentage of control Week10 Week6 through the season with 0.4% neem was 98% beTotal Total Mean TreatMean Mean tween the pupal and adult stages. Although 0.1 % no. no. leaf ment no. no. pupaea adults" neem caused some reduction in the total number area pupae adults (cm2) of adults, we thought that control at this concentration was inadequate. Methods used by the 42b Water 1,026a 32a 332 235 "Grower" 1,166a growers also failed to give adequate control. An 30b 23b 261 186 65a 0.1%Neem 1,165a 14b 388 81 important difference between neem and Trigard 0.4%Neem 1,030a 38b 0.5c 189 2 was that, at the tested concentrations, neem-treatOc Trigard 1,297a Oc 4 1 ed plants were damaged by many complete mines n - 4 plantsper treatment.Meanswithina columnfollowed while Trigard-treated plants had only small mines. by the sameletterare notsignificantly differellt(P < 0.05;Dun- Thus, unlike Trigard, neem did not protect the can's[1951]multiplerangetest). crop's foliage from damage. The number of £lies a Total reared from all plants(20 per treatment)up to and reared from the 0.4% neem-treated crop was, includingweek10. however, greatly reduced. Presumably neem would suppress leafminer populations if movement of £lies from untreated areas were kept to a minimum. ing stipples and eggs with increasing concentration In conclusion, these results from research and of neem, neither decrease was significantly differcommercial greenhouses suggest that the insectient from the Tween-20 treatment (Table 3). Thus, cidal constituent of soil-applied crude neem seed a single drench of neem at as high a concentration extract was taken up by chrysanthemums and was as 0.4% did not significantly inhibit adult females from feeding or ovipositing. active both on the plant and soil against leafmiEffect on Prepupal Larvae. When mortality in ners. Based on these results, we suggest that a comthe Tween-20 treatment is taken into account (Ab- mercial formulation of soil-applied neem be developed and tested for use on chrysanthemums bott 1925), percentage of control of prepupallarvae with a soil drench of 0.4% neem was 84 and against L. trifolii. 95% for Trials 1 and 2, respectively (Table 4). Thus, an average percentage of control of 89% was obtained. Neem was active against prepupallarvae Acknowledgment which were not feeding. From a practical viewpoint, these results indicate that by drenching the Rhonda Borisko,David Schrader, and Maureen Gough soil with 0.4% neem, growers would decrease the provided technical assistance. Plant material was donatsurvival of both the soil- and plant-inhabiting stages ed by Yoder Brothers, Inc. of Barberton, Ohio. Joe Begley of Yoder Brothers provided leafminers with which of the insect. we started our colony. George and Howard Rye let us Commercial Greenhouse Experiment. Weekly use their commercial greenhouses for our tests. Garry yellow board catches averaged 1,805 adults per Schnappinger of Ciba-Geigy Corp. provided Trigard. board (range, 645-3,415). We feel this indicated Partial funding for the research was provided by the a consistently large population of leafminers. Data Fred C. Gloeckner Foundation. from the last harvest (week 10) showed the effects of repeated drenchings on plant growth. At this time there were no significant differences in leaf area between treatments (Table 5) and no other References Cited visible signs of phytotoxicity. Thus, five sequential Abbott, W. S. 1925. A method of computing the eftreatments of neem did not cause significant growth fectiveness of an insecticide. J. Econ. Entomo!. 18: inhibition as measured by leaf area. The growers 265-266. stated that all blooms from all treatments were Duncan, D. B. 1951. A significance test for differsaleable. Mean pupal and adult counts are shown ences between ranked treatments in an analysis of from week 6, when the largest mean number of variance. Va. J. Sci. 2: 171-189. adults was reared from the water-treated plot. At Fagoonee, I., and V. Toory. 1984. Contribution to this time neither concentration of neem nor the the study of the biology and ecology of the leafgrower's methods caused significant decreases in miner Liriomyza trifolii and its control by neem. Insect Sci. App!. 5: 23-30. the number of reared pupae compared to water. Gill, J. 5., and C. T. Lewis. 1971. Systemic action of Only Trigard caused a significant decrease in the an insect feeding deterrent. Nature (London) 232: number of pupae. The mean number of reared 402-403. adults from week 6 was significantly lower in both Jacobson, M. 1981. Neem research in the U.S. DeTrigard and 0.4% neem plots than in any of partment of Agriculture: Chemical, biological and the other plots. An indication of treatment effects cultural aspects, pp. 33-42. In H. Schmutterer, K. R. through the season is given by the total number of S. Ascher, and H. Rembold [eds.], Proceedings of the pupae and adults reared from all 20 plants har1st International Neem Conference. German Agency vested from each plot. Both 0.4% neem and Trifor Technical Cooperation, Eschborn.

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Lewis, W. H., and M. P. F. Elvin-Lewis. 1983. Neem (Azadirachta indica) cultivated in Haiti. Econ. Bot. 37: 69-70. Parrella, M. P., and K. L. Robb. 1982. Technique for staining eggs of Liriomyza trifolii within chrysanthemum, celery, and tomato leaves. J. Econ. Entomol. 75: 383-384. Rembold, H., G. K. Sharma, C. Czoppelt, and H. Schmutterer. 1982. Azadirachtin: A potent insect growth regulator of plant origin. Z. Angew. Entomol. 93: 12-17.

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Warthen, J. D. 1979. Azadirachta indica: a source of insect feeding inhibitors and growth regulators. U.S. Dep. Agric., Agric. Rev. Man., Northeast. Ser. 4. Webb, R. E., M. A. Hinebaugh, R. K. Lindquist, and M. Jacobson. 1983. Evaluation of aqueous solution of neem seed extract against Liriomyza sativae and L. tnfolii (Diptera: Agromyzidae). J. Econ. Entomol. 76: 357-362. Received for publication October 1984.

31 May 1984; accepted 15