The effect of Quassia extract on eggs and larvae of the apple sawfly Hoplocampa testudinea

was studied. The efficacy of this extract is mainly due to an oral toxicity to the

neonate sawfly larvae. The main active ingredients, Quassin and Neoquassin, were

tested separately. Wheras Quassin has a considerable efficacy also on older larvae,

Neoquassin is less efficient in this case. While Quassin and Neoquassin are found in

different Quassia sources in varying relations to each other and have different efficacy,

they have to be considered separately in the definition of extract quality by the content of

active ingredients. These findings mean, that the “egg maturity” is not important for application

date. Nevertheless, the application must take place before the larvae hatch. It

was shown that low rates of Quassin (4-6 g/ha) can show very good results in the field,

in other cases the rates necessary for good efficacy are much higher. This corresponds

to farmers experience. Several factors as application technique and the condition of the

blossom must be taken in consideration and will be object of further studies.

The side effects of Quassin, Neoquassin and Quassia extract on Aphelinus mali and

other beneficial arthropods were tested. Quassia is harmless to all organisms tested

C.P.W., Zebitz

H., Bathon

H., Vogt

J., Kienzle

J., Zimmer

K., Klopp

K., Yamada

P., Maxin

P., Ternes

English

Organic Eprints

 1) Wollgrasweg 49, D-70599 Stuttgart 2) DLR-Rheinpfalz, KoGa, Walporzheimerstr. 48, D-53474 Ahrweiler 3) Öko-Obstbaugruppe Norddeutschland (ÖON) e.V., D-21635 Jork 4) University of Hohenheim, Institute for Phytomedicine, D-70593 Stuttgart 5) BBA, Institute for Biological Control, Heinrichstr. 243, D-64287 Darmstadt 6) BBA, Institute for Plant Protection in Fruitgrowing, Schwabenheimerstr. 101, D-69221 Dossenheim  Control of the apple sawfly Hoplocampa testudinea Klug in organic fruit growing and possible side effects of control strategies on Aphelinus mali Haldeman and other beneficial insects  Regulierung der Apfelsägewespe im Ökologischen Obstbau und Nebenwirkungen der Strategien auf die Blutlauszehrwespe Kienzle, J. 1); Zimmer, J. 2); Klopp, K. 3); Maxin, P. 3); Yamada, K. 4); Bathon, H. 5);  Zebitz, C.P.W. 4); Ternes, P. 6); Vogt, H. 6) Abstract The effect of Quassia extract on eggs and larvae of the apple sawfly Hoplocampa testu-dinea was studied. The efficacy of this extract is mainly due to an oral toxicity to the neonate sawfly larvae. The main active ingredients, Quassin and Neoquassin, were tested separately.  Wheras Quassin has a considerable efficacy also on older larvae, Neoquassin is less efficient in this case. While Quassin and Neoquassin are found in different Quassia sources in varying relations to each other and have different efficacy, they have to be considered separately in the definition of extract quality by the content of active ingredients. These findings mean, that the “egg maturity” is not important for ap-plication date. Nevertheless, the application must take place before the larvae hatch. It was shown that low  rates of Quassin (4-6 g/ha) can show very good results in the field, in other cases the  rates  necessary for good efficacy are much higher. This corresponds to farmers experience. Several factors as application technique and the condition of the blossom must be taken in consideration and will be object of further studies.  The side effects of Quassin, Neoquassin and Quassia extract on Aphelinus mali and other beneficial arthropods were tested. Quassia is harmless to all organisms tested.  Keywords: Hoplocampa testudinea, Quassia, Quassin, Neoquassin, Aphelinus mali  Introduction Traditionally, in organic fruit growing the apple sawfly Hoplocampa testudinea Klug is controlled by the use of extracts of Quassia wood. The good efficacy of such prepara-tions was shown in 1986 by BLOKSMA (1994), NOACK (personal communication, 1992 and 2001) and HOEHN et al. (1996). Nevertheless, there was always some uncertainty about the best application date and the number of applications necessary in years with a prolonged hatching period of the larvae and high infestation. Really high infestations, however, were rather uncommon in the organic orchards. This situation changend in 1999 and 2000 when the apple sawfly in most parts of Germany caused striking dam-age. The problems seemed to be due mostly to the quality of the Quassia wood (KIENZLE et al., 2002). The main substances cited in literature as active ingredients of Quassia are Quassin and Neoquassin (Dou et al., 1996; Hager 1977 in Wichtl 1997) which are found in different Quassia wood sources in varying ratios to each other (from 1: 0,7 to 1 : 2,7).  Archived at http://orgprints.org/13876/   To develop quality criteria for Quassia wood based on the active ingredients, it is impor-tant to understand whether the effect of Quassin and Neoquassin on the apple sawfly and the non-target organisms is comparable or not. If yes, they could be summarized together, if not, each substance has to be considered separately.  The recommendations for the application date of Quassia are dissenting: There are rec-comendations to spray meeting the stage “eyes of the larva visible”, others favoured the application time when the eggs become opaque. Thus, for the exact determination of the best application date it should be tested which “egg maturity” stage is most sensitive to Quassia. Furthermore, the side effect of Quassia on beneficial organisms, especially on Aphelinus mali Haldeman, one of the most important beneficial arthropods in organic orchards, should be tested.  Material and Methods For the laboratory trials with eggs and neonate larvae (Trial 1) apple blossoms with eggs of the apple sawfly were collected in untreated orchards. The pure substances Quassin and Neoquas-sin or the raw extract were applied by an air brush until runoff. Not earlier than 12 hours after application of these substances to the blossoms containing the eggs, the eggs were transferred to untreated blossoms. Within this time the eggs could adsorb liquid containing the active ingre-dients. The transferred eggs were put on the receptacle of the new blossom and covered with a little piece of moisted filter paper to prevent desiccation. In each treatment, 40 blossoms were used. The peduncle of each blossom was put in an Eppendorf tube with pierced cap filled with water placed in a base of plaster and kept in a little transparent plastic box. Several parameters were monitored from which the feeding signs on the blossoms resulted to be the most important parameter. Three classes of feeding signs were distinguished: Complete feeding gallery, short “stopped” feeding gallery and stings (small superficial feeding signs). For the calculation of the efficacy, a weighted average was calculated using the equation: 1 x complete galleries + 0.5 x “stopped galleries” + 0.1 x stings. With these averages, for the comparison of Quassin and Neo-quassin an “efficacy” (ABBOTT) of Quassin in confront to Neoquassin was calculated adopting Neoquassin as “control”. For statistical analysis only the complete galleries were considered us-ing contingency tables (α = 0.01). For the trials with older larvae fruits with primary or secondary infestation were collected in the field. The larvae were obtained by careful dissection of the gal-leries and placed on treated fruits. The fruits were picked in untreated orchards, the application and the experimental design were similar as for the neonate larvae.  In trial 2, 3 replications with 20, 11 and 18 larvae per replication were used, in trial 3 for each treatment 30 larvae were assessed. Both, the pure substances and the quassia extract with a defined content of quassin were prepared by Trifolio-M GmbH.  The field trials took place at different locations in different regions of Germany in 2002 and 2003. The trials had a randomised block design with 4 replications per treatment (6-12 trees per repli-cation) in organic orchards. In most cases (except Ahrweiler 2002), the substances were applied with a knapsack sprayer. The primary and the secondary infestation were assessed at two dif-ferent times, controlling 50 fruit clusters in each replication for infested fruits.  For the test of the side effects on A. mali, the test substances were diluted in deionised water for application at a volume rate of 600 L/ha. Adult A. mali regardless of sex, and of the age of 36-48 h, were used in the test. The parasitoids were reared at BBA Dossenheim. The exposure cages for the test of contact toxicity consisted of two treated square glass plates (6 cm) and an un-treated acrylic glass ring in between held together with two rubber bands. The acrylic glass ring had four drilled holes, 0.9 cm in diameter. The exposure cage stood in vertical position. The opening at the top was to provide the fructose solution in an Eppendorf tube (0.5 ml, the tip was cut off) with cotton wool. The two holes (left and right side) for ventilation were covered with ny-lon netting to prevent the wasps from escaping.  Archived at http://orgprints.org/13876/   0102030405060708090100Con Ex Q12 Q24 N12 N24 Con Q 12 Q 24 N 12 N 24 Con Q 12 Q 24 N 12 N 24feeding signs in %gallery "stopped" gallery stingA CB010203040Kon Ex Q12 Q24 N12 N24 Kon Q 12 Q 24 N 12 N 24 Kon Q 12 Q 24 N 12 N 24Undisclosed eggs in %CBAOne of these openings was connected to an aquarium pump system. Each pump (200 l/h) aer-ated six cages. The ventilation system prevented a build-up of pesticide vapour within the expo-sure cages.  The opening at the bottom was used for the introduction of the wasps. It was plugged with a cork stopper during the test. For the test of oral toxicity the test units were the same as described above, modified by feeding the wasps with active ingredient diluted in a 25 % fructose solution.  The test was carried out with six replicates, containing five adult wasps of mixed sex each. Wasps were exposed to the dried residues / treated fructose over a period of 48 hours. The two glass plates from each test unit were treated on one side using a laboratory sprayer (Potter Pre-cision Laboratory Tower, Burkard, U.K.). Before application, spray patterns were checked visually for uniformity. Within approximately one hour of application the test units were assembled and five wasps were placed in each exposure cage. The wasps were transferred carefully with a small air pump. As soon as the deposit had dried, the wasps were introduced. After the wasps were transferred into the exposure cages, the test units were placed in an environmental cham-ber and connected to the ventilation system. After 48 hours the test units were examined for wasp mortality using a dissecting microscope. A study was considered invalid if the mean mortal-ity rate in the control was above 12.5 %. Results                     Figure 1: Effect of Quassin (Q) and Neoquassin (N) and Quassia extract (Ex) in different rates (12 and 24g/ha) on eggs and larvae of the apple sawfly when untreated eggs were transferred on treated blossoms (A) or treated eggs in stadium “hya-line” (B) or “eyes of the larva visible” (C) were transferred on untreated blos-soms: Feeding signs on the blossom and number of undisclosed eggs.  There was only a negligible effect of Quassia ingredients on sawfly eggs. The larvae, however, were very sensitive to both ingredients as neonatae (figure 1). The only signifi-cant difference between the variants in figure 1 could be shown between the control and the treatments in trial A. The older the larvae, the lower is the sensitivity to Neoquassin and the higher is the difference between Quassin and Neoquassin (Table 1).  ** Archived at http://orgprints.org/13876/   The effect of Quassin on the older larvae was confirmed by observations in the field, that Quassia had also an effect on secondary infestation.   Table 1: Efficacy (ABBOTT) of both active ingredients and „efficacy“ of Quassin in con-front to Neoquassin on neonate and older larvae in laboratory trials with treated blossoms or fruits  Trial Larvae g/ha Efficacy (ABBOTT) in % "Efficacy" (ABBOTT) in %   ca. Quassin Neoquassin Quassin in confront to Neoquassin 1 neonatae 1.5 71.9 61.0 17.8     3 75.1 71.1   5.7     6 91.6 63.0 45.3   12 80.7 69.5 16.2   18 76.7 75.5   1.6 2 at migration to 2nd fruit   6 68.6 31.4 54.3   12 84.3 39.9 73.8   24 89.9 50.3 79.8 3 soon after migration to 2nd fruit   6 20.6   0.3 20.3   12 41.6 18.1 28.7  0 0 033.326.76.73.330.016.76.76.73.301020304050607080Control Quassin 6 g/ha Quassin 12 g/ha Quassin 18 g/haapplication ratemortality %Oral Toxicity 600 L/ha, Quassin Oral Toxicity 600 L/ha, Extract Contact Toxicity 600 L/ha, Extract  Figure 2: Side effects of Quassin (active ingredient) and Quassia extract on A. mali  In the laboratory, for the neonate larvae, a dose-effect-curve could not be determined because the efficacy was very good also at the lowest concentration applied (table 1). In the field, the results are varying, too (table 2). This variation, however, is not correlated with the infestation rate of the untreated control.  Archived at http://orgprints.org/13876/   Table 2: Efficacy (ABBOTT) and infestation in control in different field trials   Locality Year Quassin g/ha Efficacy of the treat-ment in % Infested fruits on 100 clus-ters in untreated control    Primary infestation Secondary infestation Primary in-festation Secondary infestation 2002   8 66.3 91.5 43.0 41.0  12 80.2 98.8   Lake  Constance I  18 87.2 91.5   2002   8 81.8 90.7 33.0 37.5  12 87.9 92.0   Lake  Constance II  18 93.9 97.3   2002   8 65.7 82.0 105.0 100.0 Lake  Constance III  12 73.3 82.5   2002   6 84.0 95.7   9.5   8.4  12 92.1 96.8   Ahrweiler  18 97.4 96.2   2002   6 30.4 70.0 34.5 65.0  12 37.7 66.9   Jork  18 65.2 91.5   2003   4 92.9 100.0 70.5 28.5  12 97.2 98.2   Lake  Constance   18 93.6 100.0   2003   4 90.7 * 64.0  Jork    6 81.3 *   * Secondary infestation not assessed because of high fruit drop  At recommended field rate (12 g/ha) Quassin and Quassia extract were harmless to Aphelinus mali, Forficula auricularia, Aphidius rhopalosiphi, Coccinella septempunctata and Chrysoperla carnea. The results obtained for the main test organism A. mali are given in figure 2. Neoquassin did not show any effect.  Discussion According to these results, the question which embryonic stage of the sawfly larva in the egg is the most sensitive to Quassia treatments can be answered very simply concluding that it is not at all important that the eggs have contact with Quassia. On the other hand, it is very important that the neonate larvae ingest Quassia directly after hatching before they start to produce galleries. Thus, only primary damage can be controlled.  Tests conducted of YAMADA (2004) demonstrated that the most important mode of ac-tion of Quassia on the larvae is by ingestion and not by contact.  Archived at http://orgprints.org/13876/   Regarding te application time and frequency, this means, that Quassia must be applied before larval hatch. Some results indicate that Quassia is not washed easily off by pre-cipitations. Thus, it does not seem  very important for a good efficacy to carry out the treatment as close as possible to larval hatch. In contrast, it is of main importance that the active ingredients are placed in such a way, that the larvae can ingest them easily: possibly on the receptacle of the blossom. Thus, actually the interest is focused towards best application technique and the optimal shape and condition of the blossom (open or closed, fresh or fading). Nevertheless, the varying results of the dose-effect-trials in the field, which are confirmed by similarly varying experiences in farmers practice cannot yet be totally explained. In most trials and in most of the commercial orchards with uncertain results with low rates it can be excluded that the results are due to an application date that was too late (larvae already hatched). Further research has to be done to allow a reduction of the application rate and, therefore, of the cost. The results obtained until now allow to hope that this will be possible. Another important result of these experiments is the effect of Quassia on secondary in-festation. If the application date is too late and the primary infestation cannot be stopped, the secondary infestation is reduced considerably. Thus, the addition of Nee-mAzal-T/S to the Quassia treatment, which reduces mainly the secondary infestation, does not seem necessary – provided the Quassin rate is high enough.  Concerning the criteria for the definition of the quality of “Quassia wood” by the active ingredients Quassin and Neoquassin it can be confirmed, that Quassin is the most im-portant active ingredient. For the difference in efficacy of both substances on elder lar-vae, it is not possible to summarize both substances together as “Quassinoids” since an extract with high content of Neoquassin and low content of Quassin may have a lower efficacy in control of secondary infestation than an extract with high content of Quassin. The side effects of both Neoquassin and Quassin and the Quassia extract on the bene-ficials tested (Aphelinus mali, Chrysopa carnea, Coccinella septempunctata, Forficula auricularia) can be neglected.   Acknowledgements We are indebted to the BLE, Bonn (Projekt 02OE084) for financing of the project and the fruitgrowers involved in the trials for the possibility to work in their orchards.  Literature  BLOKSMA, J.; WIJNEN, T.; BROUWER, G.; LARVANT, Q. (1994): Bestrijding van de appelzaagwesp met het plantaardige middel Quassia. Onderzoek Fruitteelt. Louis Bolk Instituut, Driebergen DOU, J.D. MCCHESNEY, R.D. SINDELAR, D. K. GOINS, I. A. KHAN, L. A. WALKER (1996): Int. J. of Pharmacognosy, 34: 349-354. HÖHN, H.; HÖPLI, H. U.; GRAF, B. (1996): Quassia und Neem: exotische Insektizide im Obstbau. Schweiz. Z. Obst-Weinbau 3: 62-63 KIENZLE, J.; KOPP, B. & SCHULZ, C. (2002): Control of the apple sawfly (Hoplocampa testudinea) with Quassia amara: Quality and combination with NeemAzal-T/S. In: Proceedings of the 10th Interna-tional Conference on Cultivation Technique and Phytopathological Problems in Organic Fruit-Growing and Viticulture at Weinsberg: 28-34. WICHTL, M. (1997): Teedrogen. Stuttgart, 667 S. YAMADA, K. (2004): Wirkung von Quassin und Neoquassin auf die Larven der Apfelsägewespe Hoplo-campa testudinea Klug. Bachelor thesis, Hohenheim, 2004.  Archived at http://orgprints.org/13876/

Control of the apple sawfly Hoplocampa testudinea Klug in organic fruit growing and possible side effects of control strategies on Aphelinus mali Haldeman and other beneficial insects

Control of the apple sawfly Hoplocampa testudinea Klug in organic fruit growing and possible side effects of control strategies on Aphelinus mali Haldeman and other beneficial insects

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