SITUATION ABOUT REGISTRATION AND APPLICATION OF NEEMAZAL-T/S IN SWITZERLAND 1998 3 Markus Zuber 3

EXPERIMENTS WITH AZADIRACHTIN TO REDUCE THE COMMON COCKCHAFER (MELOLONTHA MELOLONTHA L.) AND SOME LEAF-EATING INSECTS FROM THE ORDER LEPIDOPTERA 6 Henryk Malinowski x), Danuta Woreta x), Jacek Stocki xx) 6

FIELD INVESTIGATIONS ON THE EFFECT OF NEEMAZAL-T/S (3 L/HA) ON THE GRAPE LEAFHOPPER EMPOASCA VITIS (GOETHE) IN VITICULTURE 12 Karl-Josef Schirra, Friedrich Louis 12

WEITERE ERGEBNISSE BEIM EINSATZ VON NEEMAZAL-T/S GEGEN EICHENPROZESSIONSSPINNER (THAUMETOPOEA PROCESSIONEA LINNE) IM LAND BRANDENBURG 17 M. Lehmann, A. Fieguth; Pflanzenschutzdienst beim Landesamt für Ernährung, Landwirtschaft und Flurneuordnung, Frankfurt (Oder) 17

LABORATORY TRIALS WITH NEEMAZAL-T/S ON THE ALLERGENIC FOREST PEST THAUMETOPOEA PROCESSIONEA (L.) 21 M. Breuer & A. De Loof 21

NEEMAZAL-T/S ANWENDUNG ZUR BEKÄMPFUNG DER APFELBAUMGESPINSTMOTTE (YPONOMEUTA MALINELLUS ZELL.) 29 M. Lehmann, E. Bartelt; Pflanzenschutzdienst beim Landesamt für Ernährung, Landwirtschaft und Flurneuordnung, Frankfurt (Oder) 29

DIE WIRKUNG VON NEEMAZAL-T/S AUF DEN FORTPFLANZUNGSFRAß DES GROßEN BRAUNEN RÜSSELKÄFERS (HYLOBIUS ABIETIS L.) 33 Anne Luik 33

THE EFFECT OF NEEMAZAL-T/S ON THE MORTALITY OF MITE TETRANYCHUS URTICAE KOCH AND SOME INSECTS - APHIS GOSSYPII GLOV. AND THRIPS TABACI LIND. 38

DIE WIRKUNG VON NEEMAZAL-T/S AUF DIE STERBLICHKEIT DER MILBE TETRANYCHUS URTICAE KOCH UND EINIGER INSEKTEN - APHIS GOSSYPII GLOV. UND THRIPS TABACI LIND. 38 Külli Hiiesaar, Anne Luik, Aare Kuusik and Luule Metspalu 38

STANDARDISATION OF ANALYTICAL METHODS FOR NEEM BASED PRODUCTS - LATEST DEVELOPMENTS 43 Peter Förster 43

1 RISK ESTIMATION OF MOULD GROWTH IN NEEMAZAL AND ITS FORMULATION 50 Christine Kliche-Spory, Hubertus Kleeberg 50

ENVIRONMENTAL BEHAVIOUR AND AQUATIC ECOTOXICITY OF AZADIRACHTINA 58 Luc Pussemier 58

AUSWIRKUNGEN VON NEEMAZAL-T/S AUF DIE BRUT VON HONIGBIENENVÖLKERN (APIS MELLIFERA CARNICA L., HYMENOPTERA, APIDAE). ERGEBNISSE EINES HALBFREILANDVERSUCHES. 64 Boris Leymann (1), Werner Mühlen (2), Alois Edelmann (1) 64

NEEM ACTIVITY AGAINST MICROORGANISMS: AZADIRACHTIN A IN BACTERIAL AND FUNGAL AGAR DIFFUSION TESTS 71 Detlef F. Hein & Hans E. Hummel 71 Martin Weidenbörner 71

PHYTOTOXICITY OF NEEM TERPENOIDS TOWARDS ARABDOPSIS THALIANA. 76 Hilde Bettum, Peter J. Dominy and Robin H. C. Strang 76

USE OF PMR SPECTRAL DATA IN QUALITY CONTROL OF NEEM PESTICIDES 81 Koteppa Pari, C. Devakumar 81

EFFECTIVENESS OF SOME EXTRACTS OF NATURAL PRODUCTS IN CONTROLLING PATHOGENIC SOIL-BORNE FUNGI 99 Dr. P. Chintea1, A. Buliga, Mariana Mihăilă2, Dr. Maria Oprea3 99

EINSATZ VON PHEROMON-FALLEN FÜR DIE ERBSENWICKLERÜBERWACHUNG 1996 - 1997 119 Klose E. 119

PHYLLONORYCTER-PHEROMONES IN PYRACANTHA-COCCINEA-CULTURE - TESTING MONITORING, INDIRECT DETERMINATION AND CONTROL OF LEAFMINERS ON PYRACANTH 122 Manfred Lehmann 122

THE USE OF PHEROMONES AND PHEROMONE TRAPS IN FOREST PROTECTION IN POLAND IN THE YEARS 1980-1997 128 Jacek Stanislav STOCKI 128

POSSIBLE USE OF PHEROMONE TRAPS FOR POPULATION DENSITY MONITORING OF OAK ROLLERS (TORTRIX VIRIDANA L. AND ALEIMMA LOEFLINGIANA L.). 134 Marek Turcani 134 2 SITUATION ABOUT REGISTRATION AND APPLICATION OF NEEMAZAL-T/S IN SWITZERLAND 1998

MARKUS ZUBER

Andermatt BIOCONTROL AG, CH-6146 Grossdietwil Abstract

NeemAzal-T has been registered provisionally in Switzerland in 1995 for the use against the rosy apple aphid Dysaphis plantaginea.

One year later the registration was changed to NeemAzal-T/S. The very good efficacy against the rosy apple aphid was a big relief for organic apple growers.

Based on the good results against aphids in greenhouses NeemAzal-T/S could also be registered on ornamental plants in 1998. Open questions about possible toxic effects now threaten the continuation of the registration after 1998. The circumstances as well as some field experiences are discussed. Registration

Based on the good results obtained with NeemAzal-T + rape seed oil against the rosy apple aphid Dysaphis plantaginea in Switzerland and in Germany (Schulz et al., 1994) NeemAzal-T was registered for large scale field trials in 1995. Parallel to the successful introduction of NeemAzal-T into organic fruit growing NeemAzal-T/S was tested as well. As a consequence of the good results with Neem- Azal-T and NeemAzal-T/S the latter was registered provisionally in 1996. In 1997 Andermatt BIOCONTROL AG also applied for a registration against aphids, sciarids, leafminers and whiteflies in ornamentals and vegetable crops. Thanks to the good results against some aphid species NeemAzal-T/S was also registered against aphids on ornamentals in early 98. In the same time the federal office for public health (BAG) claimed that NeemAzal-T/S or Azadirachtin could possibly cause cancer. This risk estimation was mainly based on the study by Rosenkrantz & Klopman (1995) which assumed Azadirachtin to be carcinogenic according to its molecular structure. Andermatt BIOCONTROL AG is optimistic that together with the new toxicological studies carried out by E.I.D. Parry (the producing company of the raw extract) the theory about carcinogenic effects of Azadirachtin can be denied. Experiences form practice

In Switzerland there is a total surface of apple orchards of about 7500 ha. Organic apple orchards covered a surface of about 100 ha in 1997. In 1995 about 30 ha of organic orchards were treated with NeemAzal-T + rape seed oil - with very good success. In 1996 already about 100 ha were treated with NeemAzal-T/S. With the amount of 3-4 l/ha the effect was very good as well. In 1997 we can expect that more than 2/3 of 300 ha treated with NeemAzal-T/S were IPM orchards. In most of the Practice Oriented Results on Use and Production of Neem Ingredients and Pheromones VIII 3 H. Kleeberg & C. P. W. Zebitz (eds.) Copyright 2000 by Druck & Graphic, Giessen cases the results were convincing. There were however some problems in the south- western part of Switzerland, in the Valais. The climatic conditions were very favourable to the rosy apple aphid quite early in spring. The treatments were carried out very early as well. This lead to the fact that during the application there was not enough foliage for taking up the active ingredient. Moreover the efficacy lasted not long enough to prevent a mass outbreak in the end of May. As a result it will be important to test the splitting model which has been tested by Kienzle et al. in 1997 (personal communication) with one treatment in the red bud stage and a later treatment after blossoming. Several field trials were carried out in 1997. The following table gives a short overview without going into details. Many of the results will be checked again within the next few years. target insect crop effect Psylla sp. pear ++ Anthonomus pomorum apples - Leptinotarsa decemlineata potatoes +/- Sciarids ornamentals + Leafhoppers medical plants ++

4 Open questions

As already reported in earlier workshops NeemAzal-T/S causes strong phytotoxic effect on many varieties of pears. Moreover strong phytotoxic effects occurred when NeemAzal-T/S was applied on the apple variety Gala in Mid-June. Growth depression was found when applying NeemAzal-T/S to begonia cuttings. Phytotoxic or at least early ageing symptoms could be detected on grape leaves after several applications with Fu-3, a by-product form the extraction of NeemAzal. In respect to the registration for NeemAzal-T/S on ornamental plants, more should be known about susceptibility or tolerance of plants against NeemAzal-compounds. For IPM farming more must be known about miscibility problems of NeemAzal-T/S with conventional fungicides. Conclusion

NeemAzal-T/S is a very important tool for biological control of different pest insects. Although its efficacy is comparable to the one of conventional insecticides the relatively high price will have a big influence on the acceptance in the IPM market. Studies on miscibility, residues and possible negative side effects (toxicology, phytotoxicity) will be necessary for a successful use in the future in integrated pest management. Literature

Rosenkrantz, H.S. & Klopman, G., 1995: An examination of the potential ”genotoxic” carcinogenity of a biopesticide derived from the Neem tree. Environmental and Molecular Mutagenesis 26: 255-260. Schulz, Ch. & Kienzle, J., 1996: Effects of different NeemAzal formulations on Dysaphis plantaginea. Proceedings of the 4th workshop ”Neem ingredients and pheromones”. 45-49.

5 EXPERIMENTS WITH AZADIRACHTIN TO REDUCE THE COMMON COCKCHAFER (MELOLONTHA MELOLONTHA L.) AND SOME LEAF-EATING INSECTS FROM THE ORDER LEPIDOPTERA

X) X) XX) HENRYK MALINOWSKI , DANUTA WORETA , JACEK STOCKI x) Forest Research Institute, Bitwy Warszawskiej 1920r. nr. 3, 00 - 973 Warsaw, Poland xx) General Directorate of State Forests, Wawelska 52/54, 00 - 922 Warsaw, Poland Abstract

The effect of Azadirachtin (as 10 g/l EC NeemAzal-T/S, Trifolio M-GMBH) on the production of eggs by common cockchafer (Melolontha melolontha L.) females and on the mortality of the nun moth (Lymantria monacha L.), pine (spinner) moth (Dendrolimus pini L.) and pine looper (Bupalus piniarius L.) larvae under laboratory conditions was studied. 80 - 90% of the common cockchafer females fed during 14 days with oak (Quercus robur L.) leaves treated with Azadirachtin at a concentration of 0.03% were not able to produce eggs. Feeding inhibition and decreased vitality were also observed. The preliminary studies showed that the concentration of Azadirachtin giving minimum 90% mortality of above mentioned species of Lepidoptera larvae can be established on the level of 0.025%. The experiments will be continued in order to obtain more detail information on the effect of Azadirachtin on Lepidoptera larvae. 1. Introduction

Introduction of insecticides characterised by novel modes of action and by good environmental properties to forest protection is desirable. The products from the neem tree (Azadirachta indica A. Juss) have a number of properties useful for insect control (repellency, feeding and oviposition deterrence, insect growth regulator) and are considered as safe for the environment (low mammalian toxicity, low persistence) (Koul et al. 1990; Schmutterer 1990). The products from the neem tree are also characterised by low toxicity against nonphytophagous insect species, including natural enemies of pest insects (Hoelmer et al. 1990; Stark 1992, McCloskey et al. 1993). The laboratory and field trials conducted so far on the use of insecticides based on Azadirachtin in forest protection were very promising (Schnetter et al. 1996; Rohde 1996). Although the high costs of neem products actually limits their use in a large scale, it seems that further experiments are required to determine for example the lowest effective dose for the particular species controlled. The aim of this study was to evaluate the effect of Azadirachtin on egg production by common cockchafer females and on the mortality of the nun moth (Lymantria monacha L.), the pine moth (Dendrolimus pini L.) and the pine looper (Bupalus piniarius L.) larvae.

Practice Oriented Results on Use and Production of Neem Ingredients and Pheromones VIII 6 H. Kleeberg & C. P. W. Zebitz (eds.) Copyright 2000 by Druck & Graphic, Giessen 2. Materials

1. Botanical insecticide NeemAzal-T/S as EC formulation containing 1% of Azadirachtin A, kindly obtained from Trifolio-M GmbH (Lahnau, Germany) was applied in laboratory tests. 2. Insects The following insect species were used:

− common cockchafer (Melolontha melolontha L.) adult females and males collected in a short time after emergence from the soil – the insects originated from two forest districts: Olsztynek and Czarne Człuchowskie − second/third instar larvae of the pine (spinner) moth (Dendrolimus pini L.), − second instar larvae of the nun moth (Lymantria monacha L.), − second instar larvae of the pine looper (Bupalus piniarius L.). The larvae of the above Lepidoptera species were collected from pine forest stands of different forest districts or were reread in laboratory. 3. Methods

1. Experiments with common cockchafer adults

The common cockchafer adults, males and females, were fed with oak (Quercus robur L.) leaves, which were dipped for five seconds in NeemAzal-T/S water suspension at the Azadirachtin concentration of 0.03%. In the experiments we only used those females in which the production of eggs were not observed (i.e. their ovaries were empty). Twenty oak bunches (treated with Azadirachtin) with 5 males and 5 females on each placed in water were used in each of the two experiments. The same procedure was used for control treated with water. After 10 - 14 days, females were killed and the production of eggs in their ovaries were checked. 2. Experiments with Lepidoptera larvae

Aqueous suspensions at five different Azadirachtin concentrations (0.1; 0.05; 0.025; 0.01 and 0.005%) were prepared for tests of the insecticide. Bunches of Scots pine twigs were dipped for five seconds in each concentration and left to dry. Lepidoptera larvae were put on the treated bunches. The bunches with larvae were then placed in water. Three replicates of 15 larvae each were used for each concentration. The control bunches were treated with water. Percent mortalities were calculated 10 to 12 days after treatment. Immobile larvae which showed no response to a repeated tactile stimulus were evaluated as dead. At the same time, other observations concerning the abnormalities in development were noted.

7 4. Results and discussion

The effect of NeemAzal-T/S used at Azadirachtin concentration of 0.03% on the production of eggs by common cockchafer females fed during 14 days with treated oak leaves in comparison to controls is given in Fig. 1. The data showed that in first experiment with NeemAzal-T/S about 80% and in second one about 90% of females were not able to produce the eggs. In contrast, we observed in controls only about 10 and 5% of females which did not produce the eggs. It means that under laboratory conditions the period of time was to short for some females to finish oogenesis. However in experiments 1 and 2, 20 and 10% of females, respectively, fed with NeemAzal-T/S treated leaves were able to produce small number of eggs.

% females without eggs

100 90 Experiment 1 Experiment 2 80 70 60 50 40 30 20 10 0 CACA

Fig. 1. Effect of Azadirachtin (A) on the production of eggs by common cockchafer females fed with oak leaves treated with Neem Azal T/S water solution at Azadirachtin concentration of 0.03% in comparison to controls (C). Experiment 1 - females from Olsztynek forest district. Experiment 2 - females from Czarne Człuchowskie forest district.

The mean number of eggs produced by one common cockchafer female in controls and in experiments taking into consideration only these females which produced the eggs is presented in Fig. 2. The mean numbers of eggs per one female in controls were 25 and 28, whereas the mean numbers of eggs per one female found in ovaries of insects fed with treated leaves were a half of those found in controls. The causes why some females fed with Azadirachtin treated leaves had eggs in their ovaries is unknown. It seems highly likely that those insects fed on untreated leaves of forest trees before they were collected. On the other hand it is known from literature (Dorn 1995, Crisofaro et al. 1996) that some number of eggs can be produced by females after consumption of Azadirachtin treated leaves.

8 Number of eggs per female

30 Experiment 1 Experiment 2 25

20

15

10

5

0 CACA

Fig. 2. Mean number of eggs from one common cockchafer female in controls (C) and in combinations with Azadirachtin (A) taking into account only those females which were able to produce the eggs. Experiment 1 - females from Olsztynek forest district. Experiment 2 - females from Czarne Człuchowskie forest district. It has been additionally observed that common cockchafer beetles, fed on oak leaves treated with Azadirachtin, reduced significantly the consumption of food (in comparison to control) and diminished their vitality. Results presented here confirm those obtained in Germany (Rohde 1996, Schnetter et al. 1996).

9 Table 1: The toxicity of NeemAzal-T/S against three species of Lepidoptera larvae Instar and species of Lepidoptera larvae Concentration of Azadirachtin giving minimum 90 % mortality after 10 days second third instar larvae of the pine 0.025% (spinner) moth (Dendrolimus pini L.) second instar larvae of the nun moth 0.025% (Lymantria monarcha L.) second instar larvae of the pine looper 0.025% (Bupalus piniarius L.)

In this preliminary experiment conducted under laboratory conditions, the concentrations of Azadirachtin giving minimum 90% mortality of larvae fed on treated foliage during 10 days was established (Table 1). All tested species of Lepidoptera larvae were susceptible to Azadirachtin. A minimum of 90% mortality of tested larvae was obtained at the concentration of Azadirachtin on the level of 0.025%. The larvae feeding on treated pine bunches reduced food intake and, at higher concentrations of Azadirachtin, died in a few days. It has been observed that larvae had difficulties with moulting and the successful moult was not seen. The laboratory tests with Lepidoptera larvae will be repeated to obtain more detailed information on the activity of Azadirachtin based compounds. The experiments conducted in laboratory are very promising and can serve as a base for further investigation in the field in order to establish the dose for practical application of products containing Azadirachtin as active ingredient.

10 Literature

Crisofaro M., Dillio V., Marchini D., Nobili P., Dallai K., Cirio V. 1996. Effect of an Azadirachtin based compound on the fecundity of mediterranean fruit fly Ceratitis capitata (Wied.) (Diptera: Tephritidae): structural and ultrastuctural analysis. XX Intern. Congress of Entomology, Aug. 25-31, 1996, Firenze, Italy, Proceedings, 15 F (15-103), 474. Dorn A. 1995. Heteroptera: true bugs. In: Schmutterer H. (ed.) „The neem tree, Azadirachta indica A. Juss and other Meliacaeous plants. VCH, Weinheim, New York, Basel, Cambridge, Tokyo, 255-267. Hoelmer K.A., Osborne L.S., Yokomi R.K. 1990. Effects of neem extracts on beneficial insects in greenhouse culture, pp. 100-105. In: J.C. Locke and R.H. Lawson (eds), Neem’s potential in pest management programs. U.S. Dep. Res. Serv. 86. Koul O., Isman M.B., Ketkar C.M. 1990. Properties and uses of neem, Azadirachta indica. Can. J. Bot. 68, 1-11. McCloskey C.J., Arnason J.T., Donskov N., Chenier R., Kaminski J., Philogène B.J.R. 1993. Third trophic level effects of Azadirachtin. Can. Entomol. 125, 163-165. Rohde M. 1996. Experiments to reduce Melolontha hippocastani F. damages in the Hessian Rhein - Main - Plain. Proc. of the meeting „Integrated control of soil pests”, Freiburg, Germany, 23-25 October, 1995. IOBC/WPRS Bulletin vol. 19(2), 89-94 Schmutterer H. 1990. Properties and potential of natural pesticides from the neem tree, Azadirachta indica. Annu. Rev. Entomol. 35, 271-297. Schnetter W., Mittermüller R., Fröschle M. 1996. Control of the cockchafer Melolontha melolontha in the Kraichgau with NeemAzal-T/S. Proc. of the meeting „Integrated control of soil pests”, Freiburg, Germany, 23-25 October, 1995. IOBC/WPRS Bulletin vol. 19(2), 95-99. Stark J.D. 1992. Comparison of the impact of a neem seed-kernel extract formulation, Margosan O and Chlorpyrifos on non-target invertebrates inhabiting turf grass. Pest. Sci. 36, 293-300. Woreta D. 1997. Insecticidal activity of preparations used against Melolonthinae (In Polish). Doctoral thesis pp. 126, Forest Research Institute.

11 FIELD INVESTIGATIONS ON THE EFFECT OF NEEMAZAL-T/S (3 L/HA) ON THE GRAPE LEAFHOPPER EMPOASCA VITIS (GOETHE) IN VITICULTURE

KARL-JOSEF SCHIRRA, FRIEDRICH LOUIS

Staatliche Lehr- und Forschungsanstalt für Landwirtschaft, Weinbau und Gartenbau (SLFA)

Fachbereich Phytomedizin, Sachgebiet Entomologie

Breitenweg 71

67435 Neustadt an der Weinstraße Abstract

In 1997 a field trial was conducted at Deidesheim/Pfalz to investigate the effect of NeemAzal-T/S on the second generation of the grape leafhopper Empoasca vitis (Goethe) in viticulture. In August two applications were carried out with an interval of one week. The efficacy of NeemAzal-T/S (0,3%) against larvae and nymphs of Empoasca vitis reached between 44% and 62%, which is in general not sufficient enough to control this leafhopper in vineyards. Introduction

Empoasca vitis (Goethe) is known to be a native inhabitant of German vineyards. Since the beginning of the 90´s the grape leafhopper has become an increasing problem building up high population densities and spreading in most viticultural regions, with a striking emphasis on warmer areas like the „Pfalz“ or „Baden-Württemberg“. The reasons for this phenomenon are unknown. One could possibly be the long periods of hot and dry weather during summer/fall of the years 1990 until now, offering superb living conditions for this thermophilic species. Especially the second larval generation, which normally appears from July to the end of August, can cause severe damage on vine leaves having negative influence on the ripening of the grapes. At present no chemical agents are registered in German viticulture to combat the grape leafhopper. In 1997 at the SLFA Neustadt investigations were started to get to know more details on the biology as well as the biological and chemical control of Empoasca vitis. A screening was started to check different chemical and biological compounds against this pest. One of the products tested was the biological insecticide NeemAzal-T/S (3 L/ha). The insecticidal ingredients (mainly Azadirachtins) can be found in seeds of the neem tree Azadirachta indica A. Juss. In many trials these agents have shown to be highly efficient in controlling a large number of pest specimens (Schmutterer 1995). During a field trial in 1997 the potential effect of NeemAzal-T/S (3 L/ha) was examined on the second generation of Emposaca vitis. Furthermore the correlation between leafhopper-infestation and quality of harvest was investigated.

Practice Oriented Results on Use and Production of Neem Ingredients and Pheromones VIII 12 H. Kleeberg & C. P. W. Zebitz (eds.) Copyright 2000 by Druck & Graphic, Giessen Material and Methods

Trial site The field experiment was carried out in a vineyard at Deidesheim, which belongs to the viticultural region of the „Pfalz“. Details concerning the test site are summarised in Table 1. The vineyard was situated near the slopes of a forest („Pfälzer Wald“). It was managed according to the guidelines of integrated pest management.

Table 1: Trial site

location: Deidesheim/Pfalz area (hectare): 0,3 length (m): 120 width (m): 25 vine-variety: Spätburgunder Regent planting date: 1989

GRASS PATH

N1 C1 N2 1x 25 2x 25 1x 25 C2 U N D E P G R N3 R A G 2x 25 A T R N4 S H O S W T H N5 C3 N6 1x 25 2x 25 1x 25 C4

PATH row: 1 2 3 4 5 6 7 8 9 10 11 12 13 N S S S R R S S R R S S R R

: untreated check plot (4 rows x 12 vines) : treated vineyard

: sampled vines (N1 - N6: treated ; C1 - C4: control)

: vine row 1 x 25: one sample of 25 leaves per sampling date

S: variety Spätburgunder R: variety Regent

Figure 1: Design of the trial site and the sampling places, Deidesheim/Pfalz, 1997

13 Application

NeemAzal-T/S was applied two times: the first application (T1) was conducted on

05.08.97 (vines: BBCH-code stage about 75-77), the second application (T2) one week later on 13.08.97 (vines: BBCH-code stage 75 - 81). Both applications were carried out with commercial equipment using 1000 l/ha of water. The two control plots were left untreated (see Figure 1). Sampling Each sample consisted of 25 mature leaves always collected from the same insertion levels (lower, medium and upper parts of the vines). Immediately after collection the leaves were put into containers partially filled with water and a little detergent. The evaluation was carried out by using a special washing method. Before the two NeemAzal-T/S-applications between 1st of July and 4th of August 8 samplings each on 6 different places of the trial site were taken to get a survey of the mean individual density of leafhoppers per leaf .

Samples were taken one week after the first NeemAzal-T/S-application (T1 + 7), 8 days after the second application (T2 + 8) and 17 days after the second application (T2 + 17). Sampling places are indicated in Figure 1. Only larval- and nymphal stages were examined, for these are generally the development stages to be controlled. Results

Assessment of infestation The first assessment was conducted on 01.07.97. Looking at Table 2 there was a permanent decrease in mean larval density up to the 21st of July. Compared to the larval decrease, the appearance of adult Empoasca vitis increased in a striking way (visual observation). At the end of July the larval density began to increase again due to the hatching and development of the second leafhopper generation. The first NeemAzal-T/S- application was carried out at the beginning of August during a period of a mean density of about 1,37 larvae/leaf, which is still below the recommended threshold of 3 - 5 larvae per leaf. The reason for applying at that time was the relatively heavy leaf-damage on the vines and the already late time of season.

14 Table 2: Mean individual density of leafhoppers per leaf before treatment with NeemAzal-T/S, Deidesheim/Pfalz,1997

sampling date mean no. of leafhopper- larvae per leaf (n=150) 01.07.97 1,94 07.07.97 1,27 10.07.97 0,74 14.07.97 0,61 21.07.97 0,49 28.07.97 0,65 31.07.97 1,19 04.08.97 1,37

Effect of NeemAzal-T/S on larval/nymphal stages of Empoasca vitis

The results are presented in Figure 2. The degree of effectiveness was calculated according to ABBOTT (1925). In all samples the number of larval stages went down over the samplings due to the development of the second generation of adults. One week after the first NeemAzal-T/S-treatment the mean larval density in the control-samples reached about 2,4 individuals per leaf. At the same time the Neem-samples showed a mean abundance of 1,4 leafhoppers/leaf which was approximately 45% below the density of the untreated leaves. 8 days after the second Neem-application the efficacy was quite noticeable and resulted in a larval decrease of about 44% in the Neem- samples. Another 9 days later (T2 + 17) the differences between the average number of larvae per leaf in untreated and treated samples rose up: compared to the untreated control-samples there was a decrease of 62% in the treated samples.

larvae/nymphs per leaf (x) 2,5 control samples (n = 4) treated samples (n = 6)

2 Treatment 1 (T1): 05.08.97 Treatment 2 (T2): 13.08.97 44,70% 1,5

1 43,50%

62,40% 0,5

0 T1 + 7 T2 + 8 T2 + 17 time of sampling

Figure 2: Efficacy (%) of NeemAzal-T/S on Empoasca vitis, Deidesheim/Pfalz 1997

15 Discussion

The first application of NeemAzal-T/S resulted in a leafhopper reduction of about 44%. One week after the second application the efficacy reached 45 %. 17 days after the second treatment the samples showed a decrease of leafhopper larvae of 62% in the samples taken off the treated site. Looking at the results in general the impact of NeemAzal-T/S on Empoasca vitis was not sufficient. One reason for that could be that the larval density at the time of application was too high already for a more effective use of NeemAzal-T/S. In viticulture an application is recommended when the target pest has reached the damage threshold, which provisionally is 3 - 5 larvae per leaf for Empoasca vitis. In the trial NeemAzal-T/S was applied when the second larval generation reached a level of about 1,5 on average. This was possibly too high already to conduct a successful treatment. Another aspect is that Neem-Products may have a high impact on the reproduction, the development and feeding behaviour of pest insects (Schmutterer 1995). In the case of Empoasca vitis therefore further field experiments are necessary to acquire additional results. Especially an early application of NeemAzal-T/S at the beginning of the larval development of the first Empoasca vitis-generation needs to be tested. References

Abbott, W.S. (1925): A method of computing the effectiveness of an insecticide. J. Econ. Entomology 18, 265 - 267 Louis, F. u. K.-J. Schirra (1997): Grüne Rebzikade - ein Problem? Das Deutsche Weinmagazin 14, 12. Juli 1997, 28 - 30 Remund, U. u. E. Boller (1995): Untersuchungen zur Grünen Rebzikade. Schweiz. Zeitschrift für Obst- und Weinbau, 200 - 203 Schmutterer, H. (1995): The Neem Tree: source of unique natural products for integrated pest management, medicine, industry and other purposes. VCH Weinheim, New York, Basel, Cambridge, Tokyo 1995, 696 pp.

16 WEITERE ERGEBNISSE BEIM EINSATZ VON NEEMAZAL-T/S GEGEN EICHENPROZESSIONSSPINNER (THAUMETOPOEA PROCESSIONEA LINNE) IM LAND BRANDENBURG

(FURTHER RESULTS IN USING NEEMAZAL-T/S AGAINST OAK PROCESSION MOTH (THAUMATOPOEA PROCESSIONEA LINNE)

M. LEHMANN, A. FIEGUTH; PFLANZENSCHUTZDIENST BEIM LANDESAMT FÜR ERNÄHRUNG, LANDWIRTSCHAFT UND FLURNEUORDNUNG, FRANKFURT (ODER) Summary

Last year tests on NeemAzal-T/S against oak procession moth were continued in 1997. The infested avenues showed egg and larval density lower than 1996. The application of Neem by spraying (1 % concentrated) and by stem brushing (10 % concentration) were compared to Foray 48 B (Bacillus thuringiensis) and untreated. The results were 1% Neem efficacy as well as Foray and a detectable but less effect of Neem brush application. The population of oak procession moth collapsed in July at the whole avenue treated and untreated. Einleitung

Der seit mehreren Jahren in den nordwestlichen und westlichen Kreisen des Landes Brandenburg an Eichen im Straßenbegleitgrün und im Kommunalgrün als Blattschädling auftretende Eichenprozessionsspinner war 1997 an den bekannten Befallsstandorten erneut nachweisbar. Die vor dem Aktivitätsbeginn ermittelten Eigelegedichten und der lebensfähige Inhalt der Gelege erreichten an den "alten" Standorten jedoch nicht die Werte des Vorjahres. Nur im Zuge der Expansion des Schädlings in neu besetzte Standorte zeigten hohe Eidichten und hohe Werte schlupfbereiter Raupen. Mit einer Fortsetzung der Kahlfraßschäden und mit Beschwerden aus der Bevölkerung über gesundheitliche Beeinträchtigungen im Befallsgebiet mußte 1997 gerechnet werden. Deshalb wurden die im Vorjahr in Angriff genommenen Versuche mit NeemAzal-T/S zur Bekämpfung der Prozessionsspinner-Raupen 1997 fortgesetzt, um noch offene Fragen zu klären. Vorbereitung und Durchführung der Behandlung

Das Versuchsobjekt stellte eine Teilmenge eines 275 Bäume umfassenden Eichenbestandes dar, der bereits im Jahre 1996 befallen und behandelt worden war. Die Stieleichen (Quercus robur) hatten eine Kronenhöhe von ca. 20 bis 25 Metern. Eigentümer und Bewirtschafter der Allee wurden durch das Brandenburgische Straßenbauamt Kyritz repräsentiert, das dankenswerterweise sein Einverständnis zur Versuchsanlage und die materielle Unterstützung dafür gab. Die im Winter 1996/97 festgestellte Eibesatzdichte des Eichenprozessionsspinners lag beim Untersuchungsobjekt mit 1 Gelege auf 10 Meter untersuchter Triebspitzenlänge im Mittel der Untersuchungen des Landes. Die Anzahl schlupfbereiter Raupen war im

Practice Oriented Results on Use and Production of Neem Ingredients and Pheromones VIII 17 H. Kleeberg & C. P. W. Zebitz (eds.) Copyright 2000 by Druck & Graphic, Giessen Vergleich zu den anderen Standorten mit 71 % überdurchschnittlich hoch, blieb jedoch 20 % unter den Durchschnittswerten des Vorjahres. Der Schlupf der Raupen setzte ab 25. April, also relativ spät, ein. Anhaltende kühl-feuchte Witterung, zögernder Austrieb der Eichen, allgemeine phänologische Verspätung und Unsicherheiten in der Terminwahl der Behandlung verzögerten die Behandlung bis zum 17. Mai. Die vorgesehene Behandlung mit NeemAzal-T/S in Spritz- und Streichapplikation und Foray 48 B (Bacillus thuringiensis - in Spritzapplikation) wurde an jeweils acht aufeinanderfolgend stehenden Bäumen je Variante ohne Wiederholung durchgeführt. Die Präparate wurden in der entsprechenden Konzentration in ca. 10 l Wasser je Baum angewandt. Die Streichapplikation in der Konzentration von 10 % erfolgte mittels Pinsel ungefähr in Augenhöhe am Stamm. Anwender war ein im Land registriertes Dienstleistungsunternehmen, dem an dieser Stelle für die Bereitschaft zur Mitarbeit gedankt sei. Die Streichapplikation erwies sich als schlecht realisierbar, weil die Emulsion an der Rinde der Bäume nicht sofort und vollständig haftet.

Tabelle 1: Daten zur Behandlung

Variante Anzahl Präparat Applikationsform Konzentration 1 8 Bäume Foray 48 B Krone Spritzen 4 l/ha 2 8 Bäume NeemAzal-T/S Krone Spritzen 1,0 % 3 8 Bäume NeemAzal-T/S Stamm Streichen 10,0 % 4 8 Bäume unbehandelt - -

Während der Applikation war die Witterung sonnig, trocken; die Lufttemperatur lag bei 23°C; die Windstärke 1-2; die Luftfeuchte betrug ungefähr 65 %. Die Behandlung wurde in der Zeit von 14-15 Uhr vorgenommen. Zum Zeitpunkt der Applikation waren noch keine Fraßschäden zu erkennen. Ergebnis

Zur Feststellung der Fraßaktivität der Raupen wurden im Kronentraufbereich jeweils für 12 Stunden ca. zwei Meter vom Stamm entfernt Plastikschalen für eine Kotfallmessung deponiert. Die Schalen hatten eine Kantenlänge von 26 x 33,5 cm = 871 cm².

18 Tabelle 2: Ergebnis der Kotfallzählung Anzahl Kotkrümel in 4 Schalen innerhalb 12 Stunden

Datum Variante 1 Variante 2 Variante 3 Variante 4 der Kontrolle in % in % in % in %

26.05. 6 1,3 0 0 150 33,2 452 100

03.06. 8 4,5 3 1,7 61 35,1 174 100

19.06. 75 55,6 3 2,2 19 14,5 135 100

Summe 89 11,7 6 0,8 230 30,2 761 100

Tabelle 3: Einschätzung der Blattmasseverluste und Prozessionsaktivität an jeweils acht Bäumen, Zeitpunkt 23.06.

Variante Blattmasseverlust in % Anzahl Bäume mit Anzahl zu ungeschädigt Prozessionen Prozessionen, Größe der Prozessionen 1 Foray 48 B 0 1 2 200 Raupen 2 Neem 1% 5 0 0 0 3 Neem 10 % 15 ... 20 1 1 150 Raupen 4 Unbehand. 30 8 12 bis 500 Raupen

Ab Anfang Juli kam es im Unterschied zum Vorjahr zu einem deutlichen Rückgang der Raupenaktivität und zum eindeutigen Zusammenbruch der Population. Damit fiel die langanhaltende Fraßleistung der Raupen aus und die erwarteten Unterschiede in der Reaktion der Bäume in Form eines differenzierten Neuaustriebes waren nicht nachweisbar. Die Kotfallzählmethode erwies sich bei der geringen Anzahl behandelter Objekte als nur eingeschränkt verwendbar. Hier wurde anscheinend die untere Grenze der Auswertbarkeit unterschritten. Eine gegenseitige Beeinflussung der Varianten und vor allem eine Migration der Raupen zu den zeitweilig befallsfrei gestalteten Varianten muß angenommen werden.

19 Diskussion

Ein deutlicher Behandlungseffekt war bei allen drei Applikationsvarianten gegenüber "Unbehandelt" erkennbar. Trotz der beschriebenen teilweisen Unsicherheiten in der Aussagefähigkeit der Ergebnisse ist in drei Wertungsgängen - Kotfallzählung, Ermittlung des Blattmasseverlustes und Zählung der Raupenprozessionsaktivität - eine sehr gute Wirkung der einprozentigen Spritzapplikation mit NeemAzal-T/S festzustellen. Sie liegt nur unwesentlich unter der bekannten sehr guten Wirkung von Foray 48 B. Die Wirksamkeit der zehnprozentigen NeemAzal-T/S-Streichapplikation ist nachweisbar, reicht jedoch in der Dauer und im angestrebten Zweck "Verminderung des Kahlfraßes " nicht aus. Bei einem stärkeren Befallsdruck und ungehinderter Entwicklung der Raupen bis zum Ende wären die Unterschiede voraussichtlich deutlicher ausgefallen. Über Streichformulierungen - pastöse, gut haftende Präparate mit einem die Aufnahme des Wirkstoffes in die Rinde fördernden "tractor" - sollte nachgedacht werden. Eine gezielte Anwendung von NeemAzal-T/S gegen die Raupen des Eichenprozessionsspinners kann empfohlen werden, wenn die Aufwandmenge von 100 ml / Baum bzw. 1 % gewählt wird, das Maximum der Raupen sich im ersten Entwicklungsstadium befindet, während der Applikation niederschlagsarmes und warmes Wetter herrscht und die Raupen Fraßaktivität zeigen.

20 LABORATORY TRIALS WITH NEEMAZAL-T/S ON THE ALLERGENIC FOREST PEST THAUMETOPOEA PROCESSIONEA (L.)

M. BREUER & A. DE LOOF

Zoological Institute, Laboratory for Developmental Physiology and Molecular Biology, Katholieke Universiteit Leuven, Naamsestraat 59, B-3000 Leuven (Belgium) Abstract

The efficacy of NeemAzal-T/S, a formulation of the active ingredients from the Neem tree Azadirachta indica, on the 2nd and 4th larval instar of Thaumetopoea processionea was studied under laboratory conditions. The caterpillars were kept on oak twigs, that were sprayed with different concentrations of NeemAzal-T/S (0.3, 0.5 and 1%).

A clear antifeedant effect could be seen and the growth of the larvae was retarded. After 4 days, the caterpillars were lethargic and 100% mortality was attained within a period of 8 - 12 days, depending on the age of the larvae. Most were not able to shed the old exuvia. NeemAzal-T/S proved to be an efficient insecticide against the oak processionary caterpillars. 1. Introduction

Year after year, mass appearance of oak processionary caterpillars, Thaumetopoea processionea (L.), causes considerable problems in certain regions of western, southern and central Europe. The larvae can completely defoliate oak stands. The affected trees resprout, but the wood growth is slowed down and the vitality of the trees is weakened (JUPE 1956, MAKSYMOV 1978). Even more serious is the health hazard caused by these insects. From the third instar onwards, the caterpillars carry urticating hairs, which can cause strong allergic reactions of the skin, the eyes and respiratory mucous membranes. In some cases anaphylactic shock situations were reported (LAMY & NOVAK 1987, LAMY 1990). Chemical treatment of the oak trees is often not acceptable for many reasons. The application of insecticides in urban areas (oak trees in avenues, parks etc.) holds a risk for humans and cattle. Moreover, the majority of the chemicals are not very selective and disturb the ecological balance even more. It has therefore become necessary to search for alternative, safe and environmentally friendly pesticides. For many years, plant extracts with insecticidal properties have become more and more important. A lot of research has been done on the neem tree, Azadirachta indica A. Juss. The active insecticidal ingredients of this species seem to be readily degraded in the environment and mostly affect plant feeding and sucking insects. The natural antagonists are spared in most cases (SCHMUTTERER 1995a, RUCH et al. 1997). The present investigation reports attempts, under laboratory conditions, to estimate the efficacy of NeemAzal-T/S, a formulation of the active ingredients of the neem tree. 2. Materials and Methods

NeemAzal-T/S was sprayed on small twigs of Quercus robur L. and Q. rubra L. in concentrations of 0.3%, 0.5% and 1% (v/v). As control, treatment was done with water. After drying, 10 larvae of T. processionea, obtained from a culture (egg masses collected

Practice Oriented Results on Use and Production of Neem Ingredients and Pheromones VIII 21 H. Kleeberg & C. P. W. Zebitz (eds.) Copyright 2000 by Druck & Graphic, Giessen near Ravels, North Belgium), were put on each branch. The twigs were placed in glass tubes containing water. Glycerol was smeared around the tubes to hinder wandering. Experiments were carried out with 2nd (5 twigs/concentration) and 4th instar larvae (3 twigs/concentration). These experiments were carried out under a fume hood, due to the allergenic property of the older larvae. Periodically the weight of the larvae and the dry faeces as well as the number of living, lethargic and dead caterpillars were registered. To ensure statistical validity of the gained data, a simple variance analysis and a t-test was carried out (KÖHLER et al. 1984). Significance to the corresponding control is designated as follows: '*' P<0.05, '**' P<0.01, '***'P<0.001. 3. Results

The investigations with the 2nd larval instar of T. processionea showed quite large differences between the experimental variants (Fig. 1). The animals in the control grew steadily and gained about 150 mg in 20 days, whereas those treated with NeemAzal-T/S approximately maintained their mass and size from the beginning of the experiment. The faecal production was accordingly high in the control and increased with the age of the larvae (Fig. 1). In contrast, caterpillars feeding on oak twigs sprayed with different concentrations of NeemAzal-T/S produced significantly lower quantities of faeces in the first four days and almost none after this period. This shows that the plant extract preparation has a quite strong antifeedant effect on the processionary caterpillars. Mortality did not occur within the first days, but larvae on treated twigs were quickly inactive, mostly sitting or hanging in one place (Fig. 2). These animals, which moved only after touching, were counted as 'lethargic' larvae. Already after 4 days, most of the larvae were in this lethargic stage. 100% mortality could be observed after 8 days on all plants, sprayed with NeemAzal-T/S, even with the lowest concentration of 0.3 % (Fig. 1).

22 % of dead (lethargic) larvae 180 8 8 8 + control 160 2 (98) 100 100 × 0.3% ] 140 2 (98) 100 100 ' 0.5% 2 (98) 100 100 ( 1% 120

ht [mg 100 80 60 rval weig 40 La 20 0 0 2 4 6 8 10 12 14 16 18 20 300 Control 250

200

150

100

50 g] m 0 0 2 4 6 8 10 12 14 16 18 20 100 ction [ 0.3% 50 odu r

p ** 0 *** 0 2 4 6 8 10 12 14 16 18 20

ecal 100

Fa 0.5% 50 ** 0 *** 0 2 4 6 8 10 12 14 16 18 20 100 1% 50 ** 0 *** 0 2 4 6 8 101214161820 Days after start of experiment

Fig. 1. Faecal production, growth and number of dead and lethargic larvae (cumulative) of Thaumetopoea processionea on oak twigs treated with different concentrations of NeemAzal-T/S. 2nd instar larvae were used for the experiment.

23 a b

Fig. 2. Larvae of Thaumetopoea processionea after treatment with NeemAzal-T/S. (a) Inactive, 'lethargic' larvae hanging in a twig. (b) Lavae, which died during moulting. The larval body was partly covered by their old cuticle. Rather similar results were obtained after treatment of the 4th instar (Fig. 3). After 4 days the larvae on twigs treated with 0.3 and 0.5% NeemAzal-T/S stopped feeding and faecal production. Consequently, larval weight stagnated or even decreased. In the control a high amount of faeces was observed and the larvae grew normally. It was very obvious that NeemAzal-T/S treated larvae quickly separated from the group whereas control animals always aggregated at one place, if they were not eating. The mortality appeared mainly in connection with the moulting. The animals were unable to remove the old skin and were often 'caged' by their own exuvia (Fig. 2). 4. Discussion

NeemAzal-T/S was tested as an alternative insecticide against T. processionea under laboratory conditions. The preparation proved to be quite efficient against the 2nd and 4th larval instars. The efficacy was very similar between the treatments, done with 0.3, 0.5 and 1% solution. The concentrations used already seemed to be too high under laboratory conditions but they were chosen in view of field trials. The retardation of growth was the most obvious effect during the experiment. This can be a direct consequence of the reduced food ingestion. However, several investigations pointed out that reduction of growth appeared not to be primarily caused by the starvation, but by an intake of toxic compounds from the Neem tree or of other Meliaceae species (e.g. FAGOONEE 1984, SCHLÜTER & SCHULZ 1984, WILPS 1987, BREUER & SCHMIDT 1995, 1996, BREUER & DE LOOF 1998). In most of the treated T. processionea larvae, disruption of moulting could be observed. In lepidopterans, disturbance of metamorphosis is frequently reported after treatment with neem extracts or the ingredient Azadirachtin (e.g. SCHMUTTERER et al. 1983, TANZUBIL & MCCAFFERY 1990, KOUL & ISMAN 1991).

24 % of dead (lethargic) larvae control 10 13 0.3% 0 (100) 100 0.5% 0 (100) 100 200 180 160

g] 140 120 100 80 l weight [m a

v 60

Lar 40 20 0 024681012

700 Control 600 500 g] 400 [m 300 200 tion 100 0 oduc 0 2 4 6 8 10 12 al pr 200 * 0.3% 100 Faec 0 024681012 200 * 0.5% 100 0 0 2 4 6 8 10 12 Days after start of experiment

Fig. 3. Faecal production, growth and number of dead and lethargic larvae (cumulative) of Thaumetopoea processionea on oak twigs treated with different concentrations of NeemAzal-T/S. 4th instar larvae were used for the experiment.

25 These effects are probably due to an influence of the plant compounds on the hormonal control of the moulting. It is, of course, also conceivable that this can be seen in connection to reduced ingestion. For several insects, starvation caused changes of the neuroendocrine system and this indirectly influences the moulting process (SLAMA 1978, CYMBOROWSKI et al. 1982, REMBOLD 1985). During the experiment the larvae stopped feeding very soon, therefore no extensive feeding damages were found on the oak twigs. NeemAzal-T/S seems to be a very potent antifeedant against T. processionea. This can not be seen as a general effect of neem preparations because the antifeedant potency varies between insect species (for review see SCHMUTTERER 1995b). In larvae of different lepidopteran species, Azadirachtin has been shown to stimulate at least one deterrent neurone in the maxillary stylonica (SCHOONHOVEN 1982, SIMMONDS & BLANEY 1985). An interesting observation was that the larvae, kept on treated oak leaves, separated from each other, while untreated caterpillars always aggregated. This could also be registered after treatment of pine processionary caterpillars, T. pityocampa with a Melia azedarach extract: the individuals neither clustered in groups nor built nests (BREUER and DEVKOTA 1990). From this species, it is known that single caterpillars are not able to survive and even small groups have a lower probability for a successful development (BREUER 1997). Since the loss of this social behaviour could be observed even after spraying of very low concentrations, it may be an important aspect for practical application, especially on huge trees, where treatment can not often be done completely. Acknowledgements: The authors are grateful to Dr. H. Kleeberg from Trifolio-M GmbH, Lahnau, Germany, for providing NeemAzal-T/S.

26 References

BREUER, M. (1997): Einfluß der Sonnenenergie auf die Entwicklung von Thaumetopoea pityocampa (Den. & Schiff.).- Mitt. dtsch. Ges. allg. angew. Ent. 11, 705-708. BREUER, M. & DE LOOF, A. (1998): Efficacy of an enriched Melia azedarach L. fruit extract for insect control.- Practice Oriented Results on Use and Production of Neem Ingredients and Pheromones; 6. Workshop, Trifolio-M, Lahnau, 1997 (in press). BREUER, M. & DEVKOTA, B. (1990): Control of Thaumetopoea pityocampa (Den. & Schiff.) by extracts of Melia azedarach L. (Meliaceae).- J. Appl. Ent. 110, 128-135. BREUER, M. & SCHMIDT, G.H. (1995): Influence of a short period treatment with Melia azedarach extract on food intake and growth of the larvae of Spodoptera frugiperda (J.E. Smith) (Lep., Noctuidae).- Z. PflKrankh. PflSchutz 102, 633-654. BREUER, M. & SCHMIDT, G.H. (1996): Effect of Melia azedarach extract incorporated into an artificial diet on growth, development and fecundity of Spodoptera frugiperda (J.E. Smith) (Lep., Noctuidae).- Z. PflKrankh. PflSchutz 103, 171-194. CYMBOROVSKI, B., BOGUS, M., BECKAGE, N.E., WILLIAMS, C.M. & RIDDIFORD, L.M. (1982): Juvenile hormone titers and metabolism during starvation induced super-numerary larval molting of the tobacco hornworm, Manduca sexta.- J. Insect Physiol. 28, 129-136. FAGOONEE, I. (1984): Effect of Azadirachtin and of a neem extract on food utilization by Crocidolomia binotalis. Proc. 2nd Int. Neem Conf. (Rauischholzhausen, 1983), 211- 224. JUPE, H. (1956): Der Eichenprozessionsspinner - Massenauftreten in der Altmark. Ent. Z. 66 (17), 193-199. KÖHLER, W., SCHACHTEL, G. & VOLESKE, P. (1984): Biometrie. Einführung in die Statistik für Biologen und Agrarwissenschaftler.- Springer-Verlag, Berlin, Heidelberg, 255 pp. KOUL, O. & ISMAN, M.B. (1991): Effects of Azadirachtin on the dietary utilization and development of the variegated cutworm Peridroma saucia. J. Insect Physiol. 37, 591-598. LAMY, M. (1990): Contact dermatitis (erucism) produced by processionary caterpillars (Genus Thaumetopoea). J. Appl. Ent. 110, 425-437. LAMY, M. & NOVAK, F. (1987): The oak processionary caterpillar (Thaumetopoea processionea L.) an urticating caterpillar related to the pine processionary caterpillar (Thaumetopoea pityocampa Schiff.) (Lepidoptera, Thaumetopoeidae). Experientia 43, 456-458. MAKSYMOV, J.K. (1978): Thaumetopoeidae, Prozessionsspinner. In: SCHWENKE, W.: Die Forstschädlinge Europas, 3. Bd., p. 391-404. REMBOLD, H. (1985): Wozu Grundlagenforschung über Biochemie der Insekten.- In: BÖGER, P. [Ed.]: Physiologische Schlüsselprozesse in Pflanze und Insekt.- Universitätsverlag Konstanz GmbH, Konstanz, p. 191-212. RUCH, B., KLICHE-SPORY, C., SCHLICHT, A., SCHÄFER, I., KLEEBERG, J., TROß, R. & KLEEBERG, H. (1997): Practice Oriented Results on Use and Production of Neem Ingredients and Pheromones; 5. Workshop, Trifolio-M, Lahnau, 1996, 15-20. SCHLÜTER, U. & SCHULZ, W.D. (1984): Structural damages caused by neem in Epilachna varivestis: A summery of histological and ultrastructural data. I. Tissues affected in larvae. Proc. 2nd Int. Neem Conf. (Rauischholzhausen, 1983), 227-236.

27 SCHMUTTERER, H. (1995a): Side effects on beneficials and other ecological important non-target organisms.- In: SCHMUTTERER, H.: The Neem Tree Azadirachta indica A. Juss. and Other Meliaceous Plants. Sources of Unique Natural Products for Integrated Pest Management, Medicine, Industry and other Purposes.- VCH Verlagsgesellschaft, Weinheim, FRG, p. 375-384. SCHMUTTERER, H. [ED.] (1995b): The Neem Tree Azadirachta indica A. Juss. and Other Meliaceous Plants. Sources of Unique Natural Products for Integrated Pest Management, Medicine, Industry and other Purposes.- VCH Verlagsgesellschaft, Weinheim, FRG, 696 pp. SCHMUTTERER, H., SAXENA, R.C. & HEYDE, V.D.J. (1983): Morphogenetic effects of some partially-purified fractions and methanolic extracts of neem seeds on Mythimna separata (Walker) and Cnaphalocrocis medinalis (Guenée). J. Appl. Ent. 95, 230- 237. SCHOONHOVEN, L.M. (1982): Biological aspects of antifeedants. Ent. Exp. Appl. 31, 57-69. SIMMONDS, M.S.J. & BLANEY, W.M. (1985): Some neurophysiological effects of Azadirachtin on lepidopterous larvae and their feeding response.- Proc. 2nd Int. Neem Conf. (Rauischholzhausen, 1983), 163-179. SLAMA, K. (1978): The principles of antihormone action in insects. Acta ent. bohemoslov. 75, 65-82. TANZUBIL, P.B. & MCCAFFERY, A.R. (1990): Effects of Azadirachtin and aqueous neem seed extracts on survival, growth and development of the African armyworm, Spodoptera exempta.- Crop Protection 9, 383-386. WILPS, H. (1987): Growth and adult molting of larvae and pupae of the blowfly Phormia terrae-novae in relationship to Azadirachtin concentrations. Proc. 3rd Int. Neem Conf. (Nairobi, 1986), 299-314.

28 NEEMAZAL-T/S ANWENDUNG ZUR BEKÄMPFUNG DER APFELBAUMGESPINSTMOTTE (YPONOMEUTA MALINELLUS ZELL.)

(NEEMAZAL-T/S AGAINST YPONOMEUTA MALINELLUS ZELL.)

M. LEHMANN, E. BARTELT; PFLANZENSCHUTZDIENST BEIM LANDESAMT FÜR ERNÄHRUNG, LANDWIRTSCHAFT UND FLURNEUORDNUNG, FRANKFURT (ODER) Summary

Apple trees as a part of official greens in a village of the land Brandenburg in 1996 were infested by the larvae of Yponomeuta malinellus. Expecting open air celebrations in the summer of 1997 it was necessary to prevent damage on the trees and irritations of the citizens. NeemAzal-T/S was applied by a ground based technique in an amount of 50 ml/tree in 10 l water. The results: Good effects against Y. malinellus and larvae of tortricids, less effect on Aphis pomi and no effect on Psylla mali. A "greening effect" by Neem was detected from August up to October. NeemAzal-T/S can be recommended for the purpose to control Yponomeuta malinellus and tortricids. Einleitung

In der Brandenburgischen Gemeinde Neutrebbin, Kreis Märkisch Oderland, wurde im Jahre 1996 der gesamte Bestand der Apfelbäume in einer extensiv bewirtschafteten Angerbepflanzung von der Apfelbaumgespinstmotte (Yponomeuta malinellus Zell.) kahlgefressen. Es kamen massive Beschwerden der Bevölkerung, die sich von den abbaumenden Raupen belästigt fühlte. Zur Vorbereitung eines Jubiläums- und Dorffestes im Sommer 1997 auf dem Dorfanger sollten Maßnahmen getroffen werden, die eine Wiederholung des Starkbefalls unterbinden. Die ursprügliche Empfehlung, Bacillus thuringiensis-Präparat gegen die Gespinstmottenraupen einzusetzen, wurde nach Kenntnis des Befallsdruckes anderer Schaderreger des Apfels revidiert. Wegen des z.T. starken Befalls mit weiteren Obstbaumschädlingen (Tabelle 1) wurde auf den versuchsweisen Einsatz von NeemAzal-T/S orientiert. Diese Entscheidung stellte die umwelttoxikologisch und auch "kommunalpolitisch" günstigste Lösung dar. Vorbereitung und Durchführung der Behandlung

Das Ergebnis der Kontrolle unmittelbar vor der Behandlung - Besatzdichten mit Schädlingen nach dem Schlupf aus den Überwinterungsstadien und nach dem Austrieb - bestätigte die Berechtigung der Bekämpfungsentscheidung. Hauptschaderreger waren Grüne Apfelblattlaus (Aphis pomi de Geer), Apfelblattsauger (Psylla mali Schmidtb.), Knospenwicklerraupen (Spilonota ocellana F., Olethreutes variegana Hb.) und Apfelbaumgespinstmotte (Yponomeuta malinellus Zell.) (Tabelle 2).

Practice Oriented Results on Use and Production of Neem Ingredients and Pheromones VIII 29 H. Kleeberg & C. P. W. Zebitz (eds.) Copyright 2000 by Druck & Graphic, Giessen Die Behandlung wurde am 2. Mai 1997 nach der Terminvorgabe des Pflanzenschutzdienstes durch ein im Land registriertes Dienstleistungsunternehmen mit bodengebundener Technik durchgeführt. Die Raupen der Gespinstmotte waren zu diesem Zeitpunkt im Begriff, die ersten Gespinste an den Blütenbüscheln herzustellen und konnten zum Teil noch frei fressend angetroffen werden. Daten zur Behandlung - Aufwandmenge: 50 ml NeemAzal-T/S je Baum in 10 l Wasser je Baum - Uhrzeit der Applikation: von 18 Uhr bis 21 Uhr - Wetter: sonnig, 18°C, Windstärke <3m/sek., Luftfeuchte 40 % Ergebnis der Behandlung Die Wirksamkeit der Behandlung wurde in drei aufeinander folgenden Kontrollen überprüft. In der ersten Kontrolle 12 Tage nach der Behandlung konnte ein guter Abtötungserfolg gegen Gespinstmottenraupen festgestellt werden.. Dieses Ergebnis bestätigten die darauffolgenden Kontrollen (Tabellen 3, 4 und 5). Die in der ersten Erfolgsbonitur festgestellte Wirkung gegen Mehlige Apfelblattläuse war unbefriedigend, der Befall brach jedoch im gesamten Bestand, also auch in der unbehandelten Kontrolle (UK), kurze Zeit nach der 1. Bonitur Anfang Juni durch normale Abwanderung ab. Eine Wirkung auf den Apfelblattsauger konnte nicht nachgewiesen werden. Die Wicklerraupen - Grauer Knospenwickler und Roter Knospenwickler - reagierten ebenfalls deutlich auf die Anwendung von Neem. Ende August waren optisch eindeutig nachweisbare "Greening"-Effekte durch die Neem- Behandlung festzustellen. Diskussion

Die Anwendung von NeemAzal-T/S gegen Apfelbaumgespinstmotte und Knospenwickler kann als günstige Lösung für eine notwendige Bekämpfung dieser Schaderreger sowohl im biologischen Obstbau und Extensivobstbau als auch, die Beibehaltung der bisher geringen umwelt- und humantoxikologischen Auflagen vorausgesetzt, im öffentlichen Grün innerhalb urbaner Bereiche empfohlen werden. Das Mittels erwies sich in der angewandten Aufwandmenge als gut wirksam gegenüber den Zielarten. Es kam trotz kritischer Fragen der Anlieger während der Behandlung zu keinen Beschwerden über unbeabsichtigte Nebenwirkungen.

30 Tabelle 1: Ergebnis der Fruchtholzprobenuntersuchung, Anzahl Schaderregerstadien je 1 m Fruchtholz, Termin 23. Januar 1997, Standort Spinnmilben Blattläuse Apfelblatt- Schildläuse Blutläuse Wickler sauger Gespinst- motte Kiebitz- 28 Eier 51 Eier* 33 Eier 2 Schilde - - winkel - K.-Marx- - 184 Eier* - - 3Kol. 0,5 Straße 8 Gelege*

Anmerkung: * Befall über dem kritischen Wert (Warnschwelle nach GOTTWALD)

Tabelle 2: Ergebnis der Bonitur vor der Behandlung, jeweils 100 Blütenbüschel bzw. Blattbüschel, Termin 2. Mai 1997, Entwicklungsstadium Grüne Knospe Standort Knospen- Blattläuse Blattsauger Gespinstmotte wickler bef.Büschel/ bef.Büschel bef.Büschel/ Anzahl /Anzahl Raupen

Kiebitzwinkel 6 Raupen* 37* 356 62 606 16* 38

K.-Marx-Str. 8 Raupen* 50* 470 14 75 5* 13

Anmerkung: * Befall über dem kritischen Wert (Schadensschwelle nach GOTTWALD)

Tabelle 3: Ergebnis der 1. Bonitur nach der Behandlung, jeweils 25 Blütenbüschel bzw. Blattbüschel, Termin 14. Mai 1997, Entwicklungsstadium Vollblüte

Variante Standort Anzahl Anzahl Gespinstmotte Wickler (u.a.) Blattläuse Blattsauger Anzahl der Anzahl Raupen Nester * / Puppen UK , je 2 Kiebitzwinkel 65 42 2 2 Bäume Karl-Marx- 42 10 5 (+ 2 leer) 3 Straße Summe 107 51 7 5 Neem, je 10 Kiebitzwinkel 21 89 0,5 (+ 1 leer) (1 Phyllonor.) Bäume Karl-Marx- 65 13 0,5 (+ 2 leer) 1 Straße (+ 8 Coleoph.) Summe 86 102 1 1 Wirkungsgrad 19,7 % - 85,8 % 80,0 %

Anmerkung: * Als "Nest" wurde ein Gespinst mit Raupeninhalt von mehr als 10 lebenden Tieren gewertet.

31 Tabelle 4: Ergebnis der 2. Bonitur nach der Behandlung, Termin 4. Juni 1997, Entwicklungsstadium Beginn der Fruchtbildung

Variante Standort Anzahl Gespinstmottennester an 1m² Kronenoberfläche Bemerkungen UK, 2 Bäume Kiebitzwinkel 6 ... 10 K.-Marx-Str. < 1 kleine Nester Summe 8 Neem, 10 Bäume Kiebitzwinkel 2,2 K.-Marx-Str. < 1 Summe 2,2 Wirkungsgrad 72,5 %

Tabelle 5: Ergebnis der 3. Bonitur nach der Behandlung, Termin 3. Juli 1997, Entwicklungsstadium Fruchtbildung Variante Standort Anzahl Gespinstmottennester an 1 m² Kronenoberfläche Bemerkungen UK, 2 Bäume Kiebitzwinkel 8-10 beginn. Verpuppung K.-Marx-Str. < 1

Summe 9

Neem, 10 Bäume Kiebitzwinkel 0,6 kleine Nester, Raupen geschädigt K.-Marx-Str. < 1

Summe 0,6

Wirkungsgrad 93,7 %

32 DIE WIRKUNG VON NEEMAZAL-T/S AUF DEN FORTPFLANZUNGSFRAß DES GROßEN BRAUNEN RÜSSELKÄFERS (HYLOBIUS ABIETIS L.)

INFLUENCE OF NEEMAZAL-T/S ON THE MATURATION FEEDING OF THE LARGE PINE WEEVIL

ANNE LUIK

Institut für Pflanzenschutz, Estnische Agraruniversität, Tartu, Estland. Institute of Plant Protection, Estonian Agricultural University, Tartu, Estonia Abstract

Frischgeschlüpfte Larven des großen braunen Rüsselkäfers befressen die Rinde und das Kambium der jungen Nadelhölzer dicht oberhalb des Wurzelknotens um fortpflanzungsfähig zu werden. Befressene Pflanzen verkümmern und sterben bei starkem Befall ab. Um festzustellen wie NeemAzal-T/S (1 % Azadirachtin) auf den Fraß der Rüsselkäfer wirkt, wurde eine Versuchsreihe im Labor durchgeführt, wo die Käfer zwischen den unbehandelten Kontrollkiefersprossen und dem mit NeemAzal bearbeiteten Kieferspross wählen konnten. Es wurde festgestellt, dass die Behandlung der Kiefertriebe mit NeemAzal den Fraß, abhängig von der Konzentration der Wasseremulsion und dem Geschlecht des Rüsselkäfers gehemmt hat. Schon gebrauchte niedrige Konzentration (5%) hat signifikant die Frassaktivität bei den Männchen während 24-72 Stunden gehemmt, die Weibchen hat das ganz gering beeinflußt (Tabelle 1). Die höhere Konzentration (10%) hat auch die Weibchenfrassaktivität signifikant gehemmt aber nur während 24-48 Stunden. Es wäre wichtig in der Natur zu kontrollieren wie NeemAzal die jungen Nadelhölzer beeinflusst und könnt die Stammbearbeitung mit höheren NeemAzalkonzentrationen die Rüsselkäferschädigung vorbeugen. 1 Introduction

The large pine weevil, Hylobius abietis L. (Coleoptera, Curculionidae), is one of the most destructive insects in conifer reforestation areas in the Palearctic region. Adults hibernate in forest litter. Active spring movement of weevils starts generally in the second half of May when temperatures are around 1°C (Christiansen & Bakke, 1968; Längström, 1982). Usually in Northern European conditions mass flight occurs from the middle of May till the second half of June (Eidmann, 1974; Solbreck & Gyldberg, 1979; Lekander et al., 1985). At this time weevils concentrate by olfaction to fresh clear cut areas where they are looking for maturation feeding on the cambial layers of young conifer tree stems and shoots and for oviposition on the roots of the stumps in recent felled conifer trees or in the logging slash mixed with soil and ground litter (Nordlander et al., 1987; Luik & Voolma, 1989). Heavy conifer seedling mortality is common, particularly where seedlings have been planted on recently clear-cut areas (Bejer-Petersen et al., 1962; Eidmann, 1974; Heritage, et al., 1989). To protect young trees, it would be important to detect natural compounds which would have a high deterring or repellent activity for the weevils. of H. abietis. Recently new interest has arisen to natural botanical insecticides. They are environmentally more harmless than synthetical pesticides and acting in many insects in different ways (Schmutterer, 1990,1992; Bergen,1994 ). Among natural pesticides the compounds from neem (Azadirachta indica A. Juss) have a number of properties useful for insect pest management. These include repellency, feeding and oviposition

Practice Oriented Results on Use and Production of 33 Neem Ingredients and Pheromones VIII H. Kleeberg & C. P. W. Zebitz (eds.) Copyright 2000 by Druck & Graphic, Giessen deterrence, insect growth regulator activity, low mammalian toxicity and low persistence in the environment (Schmutterer, 1990; Koul, 1992). Neem is also less toxic to nonphytophagous insect species than many conventional insecticides, including pest natural enemies and insects-pollinators (Hoelmer et al., 1990; McCloskey et al. 1993; Nauman et al., 1994b). Some experiments are made with neem and other plant compounds also for forest pest management. The neem seed extracts had systemic influence on bark beetle Dendroctonus ponderosae. Lodgepole pines treated with neem were less attacked by D. ponderosae and mortality of larvae was increased on applied trees (Naumann et al., 1994a). In weevils Hylobius pales the application of pine logs with neem extract significantly inhibited feeding during 24 h, some other natural compounds as borneol, bornyl acetate, cucurbitacin, limonin, myrcene S(+) and (R(-) carvone and verbenone also acted as feeding deterrents during that time (Salom et al., 1994). In choice feeding tests where weevils of H. abietis chose between Scots pine twigs treated with plant extracts and untreated ones it was explained that 20% water extracts of Asarum europaeum and Narcissus poeticus influenced H. abietis as feeding stimulants. The extracts of Allium sativum, Taxus baccata, Primula vera and Heracleum sosnowsky acted as feeding deterrents for a short term (24-48 h) in laboratory conditions (Luik, 1997). The aim of the present study was to establish the influence of neem formulation NeemAzal -T/S (l% Azadirachtin) on the maturation feeding of H. abietis. 2 Material and methods

The spring-summer period in 1997 was cold for a long time in Estonia and therefore the active movement of weevils occurred later than usual - in the second half of June and July. The weevils of H. abietis were collected from trapping holes from a fresh clear cutting area in July in the forest district of Räpina in South -Estonia The 10 cm long Scots pine twigs with 5 mm diameter were halved, one half was treated With NeemAzal-T/S, the other was an untreated control twig. NeemAzal-T/S formulation is produced by Trifolio-M GmbH in Germany. It was used for making of 5 % and 10% water emulsions for pine twigs dipping for 5 seconds. After dipping to Neem/Azal T/S emulsion the pine twigs were dried for 5 seconds on filter paper and put together with the control half to petri dish. A treated and an untreated twig, were placed in individual moistened paper sleeves (to prevent contact between twigs) within petri dish. One weevil, starved for 12h was placed in each dish (100x15 mm). The petri dishes were exposed at natural light conditions in temperature + 20±1°C. In every variant of choice test 35-40, weevils were used and every specimen was tested only one time. An area estimate of weevil feeding on cambial tissue was made by the help transparent mm paper after 24 h, 48 h and 72 h for each twig. Treatment tests were replicated two times. The sex of beetles was established for estimation of feeding differences between sexes. The mean feeding area of weevil per different periods and their standard deviation was calculated. The significance of differences was controlled by Student-t test. 3 Results

The effect of NeemAzal-T/S on feeding of H. abietis weevils depended on concentration, feeding time and weevils' sex. After 24h weevils on average fed significantly less on twigs treated with 10% emulsion of NeemAzal-T/S , whereas the feeding of males was more inhibited than of females (Table 1) . In females this concentration depressed feeding also during 48 h but not significantly during 72h, at the same time males were very strongly influenced by this concentration. In males also the lower concentration (10%) of NeemAzal-T/S inhibited feeding on treated twigs significantly during all the testing time (24-72 h) whereas in females it influenced only slightly.

34 Table 1. Mean feeding area (mm² ± standard deviation) of pine Weevil females (F) and males (M) depending on treatment of pine twigs and feeding time.

Treatment, sex 24 hours 48 hours 72 hours NeemAzal 5% F 7.8±3.6 15.8±4.3 16.8±4.7 Cont.F 10.4 ±3.0 16.1±4.1 19.7 ±4.5 NeemAzal 5% M 3.3 ±1.0* 4.4 ±1.2* 8.2 ±2.4* Cont. M 8.5 ± 3.5“ 16.4 ±5.4“ 19.0 ±6.1“,

NeemAzal 10% F 0.8 ± 0.3 3.8 ±1.2 14.1 ±2.2 Cont. F 9.0 ± 2.5“ 13.8 ±3.3“ 18.7 ±5.7“ NeemAzal 10% M 0.3 + 0.1* 1.5 ±0.6* 2.0 ±0.9* Cont.M 8.1 ±1.6“ 13.2 ±2.7“ 17.7 ±4.6“

*Significant difference between males and females by Student t-test, p < 0.05 , “significant difference between treated and untreated control twigs by Student t-test, p-< 0.05

4 Discussion

A phytophagous insect finds a host-plant by certain chemical stimuli, whereas secondary metabolites give the species specific chemistry which has profound importance in the selection of hosts by phytophagous insect (Bernays, Chapman,1994). On the other hand, secondary compounds of plants are a part of the plants' defence against plant-feeding insects and other herbivores (Rosenthal and Janzen, 1979) and therefore they can actively influence insects. With the treatment of a host-plant with non-host compounds its smell and taste can become nonacceptable for a phytophagous insect. From the ecological point of view of the pest control the most promising are plant-produced compounds which are acting as repellents, inhibitors, or antifeedants as they only disorientate insects but do not directly kill them. What kind of compounds act as repellents or deterrents depends also on insect species (Bernays, Chapman, 1994). NeemAzal-T/S contains Azadirachtin - a secondary metabolite of the neem tree. In tests with weevils of H. pales Azadirachtin had antifeedant activity and it was stronger with higher concentration (Salom et al., 1994). In the case of H. abietis the antifeedant influence of NeemAzal also depended on concentration. The data suggested that males were more sensitive when they reacted to lower concentrations of Azadirachtin than females (Table 1). The further study is needed for the estimation of phytotoxity of NeemAzal on coniferous seedlings. It is necessary to evaluate whether antifeedant activity of NeemAzal for pine weevils in a higher concentration can be maintained in forest conditions on live seedlings and for how long. 5 Summary

Laboratory experiments established that NeemAzal-T/S (contents l% Azadirachtin) has antifeedant activity for weevils of H. abietis and it was stronger in higher (10%) concentration than in lower (5%) concentration whereas males reacted significantly already to lower concentration.

35 Acknowledgements belong to Valli Viidalepp for laboratory assistance. 6 References

Bejer-Petersen, B., Juutinen, P.-, Kangas, E.; Bakke, A. Butovitsch, V.; Eidmann, H.; Hedqvist, K, J.; Lekander, B. 1962: Studies on Hylobius abietis L. development and life cycle in the Nordic countries. Acta Ent. Fenn. 18, 1-107. Berger, A. 1994: Using natural. pesticides: current and future perspectives. Alnarp, Swedish University of Agricultural Sciences. Bernays,E.A., Chapman,R.F. 1994: Host-plant selection by phytophagous insects. New York., Chapman&Hall. Christiansen, E.; Bakke, A. 1968: Temperature preference in adults of Hylobius abietis L. (Coleoptera:Curculionidae) during feeding and oviposition. Z. ang. Ent. 62, 83-89. Eidmann, H. H. 1974: Hylobius Schönh. In: Die Forstschädlinge Europas. Bd. 2., Schwenke, W. (Hrsg.) Hamburg u. Berlin, 272-303. Heritage, S. G.; Collins, S.; Evans, H. F. 1989: A survey of damage by HyIobius abietis and Hylastes spp. in Britain. In: Insects Affecting Reforestation: Biology and Damage. Ed. by Alfaro, R. I., Glover, S. G. Victoria, Canada, Forestry Canada, 28- 33. Hoelmer K.A.; Osbome L.S.; Yokomi, R.K. 1990: Effects of neem extracts on beneficial insects in greenhouse culture. In: Lovke, J.C.; Lawson, R.H.(Eds.) Neem´s Potential in pest management programs. U.S. Dep. Agric. Res. Serv.86, 100-105. Koul, O.1992: Neem allelochemicals and insect control. In: Allelopathy; basic and applied aspects. Ed. By Rizvi, R.S.J.H.,Rizvi, V.J. London, Chapman and Hall Ltd., 389- 412. Längström, B. 1982: Abundance and seasonal activity of adult Hylobius weevils in reforestation areas during first years following felling. Comm. Inst. For. Fenn. 106: 1-3. Lekander, B.; Eidmann, H. H; Bejer, B.; Kangas, E. 1985: Time of oviposition and its influence on the development of Hylobius abietis (L.) (Col. Curculionidae), Z. ang. Ent. 100: 417-421. Luik,A. 1997: Influence of plants on insects.(In Estonian with English summary). Tartu, AS Tartumaa. Luik, A.; Voolma, K. 1999: Some aspects of the occurrence, biology and coldhardiness of Hylobius Weevils. In: Proc. IUFRO working group of Insects Affecting Reforestation. Ed. by Alfaro, R. I., Glover, S. G. Victoria, Canada, Forestry Canada, 28-33. McCloskey, C.; Arnason, J.; Donskov, N.; Chenier, R.; Kaminski, J.;Philogene, B. J. R. 1993 :Third trophic level effects of Azadirachtin. Can. Entomol. 125,163-165. Naumann, K.; Rankin, J. L.; Isman, B. M. 1994a: Systemic action of neem seed extract on mountain pine beetle. For. Ent. 87, 1580-1585. Naumann, K., Currie, R.W.; Isman, M.B. 1994b: Evaluation of the repellent effect of a neem insecticide on foraging honey bees and other pollinators. Can. Entomol. 126, 225- 230. Nordlander, G. 1987: A method for trapping Hylobius abietis (L.) with a standardized bait and its potential for forecasting seedling damage. Scand. J. For. Res. 2, 199-202.

36 Rosenthal, G.A.; Janzen, D.H. (Eds.) 1979: Herbivores their interaction with secondary plant metabolites. New York., Academic Press. Salom, S.M., Carlson, J.A., Ang, B.N., Grosman, D.M., Day, E.R. 1994: Laboratory evaluation of biologically based compounds as antifeedants for the pales weevil, Hylobius pales (Herbst) (Coleoptera: Curculionidae). J. Entomol. Sci. 29, 407- 417. Schmutterer, H. 1990: Properties and potential of natural pesticides from neem tree, Azadirachta indica. Ann. Rev. Entomol. 35, 271-297. Schmutterer, H. 1992: Higher plants as sources of novel pesticides. In: Insecticides: Mechanism of Action and Resistance, Andover, Intercept Ltd., 3-15. Solbreck, C.; Gyldberg, B. 1979: Temporal flight pattern of the large pine weevil, Hylobius abietis L. (Coleoptera, Curculionidae), with special reference to the influence of weather; Z. ang. Ent. 88, 532-536.

37 THE EFFECT OF NEEMAZAL-T/S ON THE MORTALITY OF MITE TETRANYCHUS URTICAE KOCH AND SOME INSECTS - APHIS GOSSYPII GLOV. AND THRIPS TABACI LIND.

DIE WIRKUNG VON NEEMAZAL-T/S AUF DIE STERBLICHKEIT DER MILBE TETRANYCHUS URTICAE KOCH UND EINIGER INSEKTEN - APHIS GOSSYPII GLOV. UND THRIPS TABACI LIND.

KÜLLI HIIESAAR, ANNE LUIK, AARE KUUSIK AND LUULE METSPALU

Institute of Plant Protection, Estonian Agricultural University, Tartu, Estonia

Institut für Pflanzenschutz, Estnische Agraruniversität, Tartu, Estland Abstract

Die Wirkung des Pflanzenschutzmittels NeemAzal-T/S (1% Azadirachtin) in den Konzentrationen 0,1% und 0,5% wurde im Labor auf Eiern, Erstlarven und diapausierenden Adulte der Milbe T. urticae festgestellt. Alle Milbenstadien wurden topikal mit 1 µl des Mittels behandelt. Bei der Blattlaus A. gossypii wurden die jungen Larven mit NeemAzal-T/S übergossen. Die Thripslarven und Adulten wurden topikal mit 1 µl des Mittels behandelt. Die Entwicklungsstörungen und Sterblichkeit aller Versuchsobjekte wurden während eine Woche ermittelt.

Die Bearbeitung mit der höheren Konzentration (0,5%) des Mittels hat bei der Milbe hohe Sterblichkeit hervorgerufen. So verdarben 85,9% der Eier. In einigen Eiern hatte sich die Embryonalentwicklung fortgesetzt, die Larven konnten jedoch nicht ausschlüpfen. Im Vergleich zu den unbehandelten Eiern der Kontrolle schlüpften die Larven der am Leben gebliebenen Eier 1-2 Tage später. Durch die Behandlung mit dem 0,1%igen Mittel konnten sich 24,6% der Eier nicht weiterentwickeln; 15% der Larven starben ab. Mit dem 0,5% Mittel wurden 70,4% der Larven getötet. Von diapausierenden Adulten starben 19,4% respektive 33%.

Bei den Blattläusen wurde die Sterblichkeit am dritten Tag nach der Behandlung ausgewertet. Am siebenten Tag waren die geschädigten und toten Larven gelb gefärbt. Die Larvensterblichkeit 31,4% bzw. 74,1% korrelierten entsprechend zu den verwendeten Konzentrationen (0,1 und 0,5%).

Bei den Thripsen hat die Behandlung mit dem 0,1%igen Mittel 40,2% und mit dem 0,5%igen Mittel 89,9% der Individuen abgetötet. Die Sterblichkeit war schon am zweiten Tag nach der Behandlung festzustellen.

Bei allen Versuchstieren hat die Anwendung des höher konzentrierten Mittels schnellere und höhere Sterblichkeit verursacht. 1 Introduction

The neem tree, Azadirachta indica, is widely spread in tropical regions, has proved to be one of the most universal sources of natural pesticides for a great number of insect and mite pests. So far, the seed kernel extract of neem, known as Azadirachtin, has been most thoroughly tested, and it has been extracted in bigger quantities than other components of neem ( Schmutterer et al. 1981; Schmutterer & Zebitz 1984; Ascher

Practice Oriented Results on Use and Production of 38 Neem Ingredients and Pheromones VIII H. Kleeberg & C. P. W. Zebitz (eds.) Copyright 2000 by Druck & Graphic, Giessen 1995). Azadirachtin has been tested for the control of mites, nematodes and the insects of different orders in different regions of the world (Jacobson 1987, Parmnal 1987). The wide spectrum of action of the preparation has been established. In numerous studies the repellent, antifeedant and deterrent properties of Azadirachtin, but also its effects on female fecundity and neuroendocrine system, are shown (Ruscoe 1972; Schmutterer et al. 1981; Saxena 1987; Schmutterer 1987; Addor 1995) Since Azadirachtin has often caused morphogenetic effects, it has been first of all characterised as the insect growth regulator (IGR). In literature low toxicity of Azadirachtin, especially in the case of moderate doses, is emphasised, but at the same time the fact that insects perished on the following day after the treatment is evident (Saxena et al. 1984; 1984a). From the point of view of plant protection, it is not very important through which mechanisms the action of the neem preparation is revealed if it leads to a visible decrease in the number of pests. From the practical aspect the direct quick killing effect is often essential. The aim of the present paper was to examine the toxic effect of a NeemAzal-T/S (1% Azadirachtin, produced by Trifolio-M GmbH) on three of the most widely spread pests in our greenhouses Tetranychus urticae (Acari, Tetranychidae), Aphis gossypii (Homoptera, Aphididae) and Thrips tabaci ( Thysanoptera,Thripidae). 2 Materials and methods

The toxicity of 0.5% and 0. 1% water emulsions of NeemAzal-T/S was studied at different developmental stages of two spotted spider mite (T. urticae), in the larvae of aphids A. gossypii and in the second instar larvae and the adults of tobacco thrips T. tabaci. The pests were gained from highly infested cucumber plants grown in a greenhouse. The experiments were carried out at the temperature of 24±1°C and in natural light conditions. In the tests the methods of toxic studies described by Helle & Overmeer (1985) were used, according to which the efficiency of preparations is estimated by the direct mortality of the individuals. For test objects cucumber leaf sectors were used as nutrient substrates. Sectors were cut out of the leaves so that veins would remain on the edges of the cuttings to avoid the flowing of water on the leaf and its wetting. The leaf sectors were placed to float on water in petri dishes. The dishes were left uncovered to avoid too high air humidity. Floating on water, the leaf stays fresh and the pests have no possibility to leave it. Water was changed and the dishes were disinfected every day, in that way the leaves remained green until the end of the experiment. The method of topical application was mainly used for treatment of insects. A micro-syringe was exploited for dosing of NeemAzal-T/S water emulsion. All these operations were made under stereo-microscope at a magnification of 16x. All the experiments were made in five replications. The number of treated individuals (N) is marked in figures 1 and 2. For the statistic evaluation of differences in the results between tests variants Chi² test was used. 3 Results

Tetranychus urticae. To obtain eggs of a certain age, five adult females were placed on each leaf disc and left there for 24 hours. The deposited eggs were counted and each of them was treated with 1 µl of the emulsion. From the moment of their treatment, the eggs were observed under a microscope in daily intervals during one week. From untreated control eggs larvae hatched on 4-5th day after oviposition and 14.5% of eggs perished. Thrips tabaci. Tobacco thrips lays its eggs into plant tissue or between the petals and sepals of flowers which makes it difficult to study them. That is why only the postembryonic stages of development, second instar larvae and the adults of unknown ages were used in the experiments. To replace the larvae and the adults of thrips on new leaves, they had to be administered a mild narcosis. The cucumber leaves with thrips

39 were submerged into water for a minute and then the wet individuals were placed on a clean and dry piece of leaf. The treatment was not started until the thrips had completely recovered and become active again. Each individual was treated dorsally with 1-2 µl of emulsion. Mortality was determined on the 5th day of the experiment. The death rate was 89.9% when 0.5% emulsion was used (Fig.2) . There was possible to identify death cases already on the 2nd or 3rd day because they did not move any more. In the treatment with 0.1% emulsion, 40.2% of the individuals died, but in the control the percentage of mortality was only 3.7%. In the 5th day of the observation the dead individuals had turned brown, haemolymph had spread out of their bodies and they had stuck to the leaf. The death rate comparison between the different treatments and their comparison with the control variant showed significant differences (p<0.01). 4 Discussion

In the favourable conditions of greenhouse the generation of mites, aphids and thrips lasts less than two weeks. To save the infested plants, the pest-control is almost always necessary. Under the pressure of permanent pesticides' treatments the resistance to synthetical pesticides can develop in pests quickly. This is one, but not the only reason why the alternative means of plant protection should be searched for. The direct and indirect effect of Azadirachtin on mites has been studied by Mansour et al. (1987) and they have discovered that low concentrations are not dangerous to predators, but at the same time they reduce the fecundity of harmful mites. Consequently, Azadirachtin could be joined into the system of integrated pest control. In our tests with different developmental stages of two spotted mite, the most susceptible ones to Azadirachtin turned out to be eggs, while some of the eggs had completed their embryonic development, but their larvae died while hatching. Schauer & Schmutterer (1981) have also found a strong toxic action on the eggs of red spider mite treated with the extract of neem. In some cases no larvae hatched at all and in other cases larvae hatched but had no vitality, they did not feed and died. The authors consider starving to be one of the possible causes of their dying. The concentration of 0.5% proved to be toxic enough for larvae as well, but the larvae who survived, moulted two days later than in the control. The prolonged time between moulting has been explained with the reduction of ecdyson in haemolymph (Rembold et al 1987). Schmutterer (1984) does not exclude the possibility that there may be an indirect effect on moulting and metamorphosis were due to the reduced consumption of food, larvae do not reach a weight necessary for inactivating corpora allata. Schauer (1984) has thoroughly studied the action of neem extracts on aphids but using topically applied moderate doses, she did not manage to attain toxic effects. When the larvae of aphid fed on the leaves treated with Azadirachtin, they died during moulting. They could not free themselves from their old exuviae, although morphological changes were not perceived. In the tests with 0.5% NeemAzal-T/S emulsion 85.5% of the eggs were killed. By the last day of the observation the colour of perished eggs had turned light brown and their shells were wrinkled. A part of the eggs had passed their embryonic development, but the larvae died, being unable to emerge from their shells. In the treatment of eggs with 0.1% NeemAzal-T/S emulsion their death rate remained low, reaching only 24.6% and showing insignificant difference with the control (p=0.9). Nevertheless, there was a significant difference between compared concentrations (p<0.01).

40 The first instar larvae of the spider mite were used in the tests. They were placed on the parts of cucumber leaves with the help of a needle and treated topically with 1 µl of emulsion. Mortality was determined on the 5th day after the treatment. Larvae were considered to be dead when they did not respond to touch and their colour faded. The toxic effect of NeemAzal-T/S on mite larvae depended on the concentration. The treatment with 0.5% and 0.1% emulsions killed 70.4% and 15,1% of larvae respectively. Statistically significant mortality was caused only by 0.5 % water emulsion of NeemAzal- T/S (p<0,01). The red coloured pre-diapausing females as adults were used in the experiment. Each specimen was treated with 1-2 µl of the emulsion. Mortality was determined on the 7th day. Treatment with 0.5% NeemAzal-T/S emulsion killed 33% of adults. The mortality of control adults and of sample treated with 0.1% emulsion was 5.9% and 19.4% respectively. There were no significant differences in the mortality rate between the control variant and the variants of different concentrations of NeemAzal-T/S . The low sensitivity of the pre-diapausing adults could be explained by the physiological changes which take place in the organism. In diapausing organisms the metabolism rate is low and therefore it could be less sensitive to unfavourable environmental factors. Aphis gossypii. Two female individuals were placed on each leaf cutting and left there for 48 hours. Then the adults were removed and larvae were counted and treated with NeemAzal-T/S water emulsions. As the larvae of aphids are covered with a hard wax layer they were overflown with the emulsion for 2 minutes. In the control, distilled water was used for submerging the larvae. When two minutes had passed, the rest of the liquid was removed using filter paper and a syringe, but a certain amount of the emulsion remained on the surface of the leaf. The estimation of mortality was made in daily intervals during one week. The ultimate death rate was estimated on the 7th day. During the first two days all the larvae were active when both 0.1% and 0.5% emulsions were used and there were no differences noticed as compared with the control. The leaves did not show any signs of phytotoxicity either but the concentrations of Azadirachtin that are higher than 2% may have phytotoxic effects on leaves. On the 3rd and 4th day it was difficult to establish whether the larvae were alive or dead, there were no external changes to be seen, but their reaction to the touch was only slightly perceivable. On the 5th day the death among the larvae was identified as they did not respond to the touch anymore. The experiment was finished on the 7th day. At this time cuticle of larvae had already become wrinkled and turned brown. The control larvae had already moulted in the 7th day. The mortality of the larvae was in correlation with the concentration of the used emulsion. 0.50% and 0.1% emulsions resulted in 74.1% and 31.4% mortality respectively (Fig.2). In the control variant, only 7.8% of larvae perished, showing a significant difference from the treated samples. Toxicosis and rapid killing is only one effect of Azadirachtin on the aphid A. gossypii. 5 References

Chiracanthium mildei. In : Natural Pesticides from Neem Tree and other Tropical Plants. Proc. of the 3rd Int. Neem Conf. Nairobi, Kenia, 1986, 577-587. Parmal, B.S. 1987: An overview of neem research and use in India during the years 1983-1986. In : Natural Pesticides from Neem Tree and other Tropical Plants. Proc. of the 3rd Int. Neem Conf. Nairobi, Kenya, 1986, 55-80. Rembold, H., Uhl, M. , Müller, Th. 1987: Effect of Azadirachtin A on hormone titers during the gonadotropic cycle of Locusta migratoria. In : Natural Pesticides from Neem

41 Tree and other Tropical Plants. Proc. of 3rd Int. Neem Conf. (Nairobi, Kenya, 1986), 289-298. Ruscoe, C.N.E. 1972: Growth disruption effects of on insect antifeedant. Nature, New Biol. 236, 159-160. Saxena, R.C., Justo, H.D.; Epino, P.B. 1984: Evaluation and utilisation of neem cake against the brown planthopper, Nilaparvata lugeiis. In: Natural Pesticides from the Neem Tree and other Tropical Plants. Proc. 2nd Int. Neem Conf. Rauischholzhausen, 1983, 391-402. Saxena, R.S., Epino, P.B., Tu Cheng-Wen; Puma, B.C. 1984a: In: Natural Pesticides from the Neem Tree and other Tropical Plants. Proc, 2nd int. Neem Conf Rauischholzhausen, 1983, 403-412. Schauer, M.; Schmutterer, H. 1981: Effects of neem kernel extracts on the two spotted spider mite, Tetranychus urticae. In: Natural Pesticides from the Neem Tree and other Tropical Plants. Proc. 1st Int. Neem Conf. Rottach-Egern 1980, 259-266. Schauer, M. 1984: Effects of variously formulated neem seed extracts on Acyrthosiphon pisum and Aphis fabae. In: Natural Pesticides from the Neem Tree and other Tropical Plants. Proc. 2nd Int. Neem Conf. (Rauischholzhausen, 1983), 141-150. Schlüter, U.; Schulz, W.-D. 1984: Structural damages caused by neem in Epilachna varivestis: A summary of histological and ultrastructural data. 1.Tissues affected in larvae. In: Natural Pesticides from the Neem Tree and other Tropical Plants. Proc. 2nd Int. Neem Conf. (Rauischholzhausen, 1983), 227-236. Schmutterer, H., Ascher, K.R.S.; Rembold, H. 1981: Natural pesticides from the neem tree (Azadirachta indica A. Juss). In: Natural Pesticides from the Neem Tree and other Tropical Plants. Proc. 1st Int. Neem Conf. (Rottach-Egern, 1980), 297 pp. Schmutterer, H. 1984: Neem research in the Federal Republic of Germany since the First International neem conference. In: Natural Pesticides from the Neem Tree and other tropical Plants. Proc. of the 2nd Int. Neem Conf. (Rauischholzhausen,1983), 21-31. Schmutterer, H. 1987: Fecundity reducing and sterilising effects of neem seed kernel extracts in the Colorado potato beetle, Leptinotarsa decemlineata. In: Natural Pesticides from the Neem Tree and other tropical Plants. Proc. of the 3rd Int. Neem Conf (Nairobi, Kenya, 1986), 351-260. Schmutterer, H.; Zebitz, C.P.W. 1984: Effect of in ethanolic extracts from seeds of single neem trees of African and Asian origin, on Epilachna variivestis and Aedes aegypti. In: n: Natural Pesticides from the Neem Tree and other tropical Plants. Proc. of the 2nd Int. Neem Conf. (Rauischholzhausen,1983), 83-90.

42 STANDARDISATION OF ANALYTICAL METHODS FOR NEEM BASED PRODUCTS - LATEST DEVELOPMENTS

PETER FÖRSTER

PESTICIDE SERVICE PROJECT, GTZ Background: The Experiences of GTZ neem projects so far

Since more than twenty years GTZ on behalf of the Ministry for Economic Co-operation of the Federal Republic of Germany is promoting research, dissemination and use of plant-derived pesticides. In the beginning the focus was placed on research to screen and investigate the properties of plant-derived pesticides and assess their potential for pest control. The results of all this activities were that extracts of the neem tree were most suitable for IPM concepts. When it became obvious that neem extracts revealed good pest control properties along with their non toxic properties for mammals and non-target organisms, GTZ started to promote their use on small-farmers level, presuming that crude water extracts of neem seeds or kernels would have the largest potential of being used by the target group: 'small farmers in developing countries'. It was presumed that neem would be per se accepted due to the availability of the raw material more or less for free and that the small farmers would produce their own pesticide which would even have low impact on the environment. What were the experiences drawn from all activities? 1.) There is a potential for simple neem extracts being used on small farmers level 2.) Neem was not applied to such an extent as possible. What were the reasons? Economical analysis of the project activities indicated that the potential of home-made pesticides based on neem is limited by its high production costs and the limited availability of the raw material (neem seeds) concerning the time period (HELLPAP & LEUPHOLZ 1996: in GATE, GTZ 4/96). The costs are determined by the opportunity costs, say: alternative labour. It appeared that at many sites neem kernels are ready for harvesting when the small farmers have labour peaks anyway due to the fact that land has to be prepared, crops to be harvest etc. The availability of the kernels is limited to a certain time period if not processed for storage which would cause additional costs. Perspectives for the use of neem derived pesticides

Still following the aim to promote the use neem the consequence for development concepts would be to decrease the cost for plant protection products derived from the neem tree.

Practice Oriented Results on Use and Production of Neem Ingredients and Pheromones VIII 43 H. Kleeberg & C. P. W. Zebitz (eds.) Copyright 2000 by Druck & Graphic, Giessen This can be achieved by two ways: - either by using by-products of neem i.e. oil for preparing soap, firewood, fodder etc. or by - decreasing the production costs for pesticides which is usually achieved in the economic world by mass production and specialisation of the manufacturer. Of course both strategies should be pursued. While in the beginning the use of neem for plant protection was restricted to small farmers level, in future the great potential of commercial products should be used. The development during recent years already indicates that the significance of commercial neem formulation for plant protection is increasing. The PESTICIDE SERVICE PROJECT of GTZ

As one well known GTZ activity in the field of neem 'The Natural Pesticide Project' was backing all this activities from 1987 to 1995 as a supra regional project. In 1995 the 'Pesticide Service Project' of took over in 1994 the task to promote the use of non-synthetic pesticides as one working area among others. The following services were offered:

Table 1 Services offered by the working field 'NON-SYNTHETIC PESTICIDES' of the GTZ- PESTICIDE SERVICE PROJECT:

- advisory service on production, application and dissemination of neem based pesticides

- conducting fact finding studies, seminars, workshops

- financing studies on specific aspects related to non-synthetic pesticides

- question & answer service on alternatives to synthetic pesticides

- providing access to technical leaflets, reports, surveys, documents

- documentary on alternatives to synthetic pesticides

- conducting surveys on the use of neem and neem related activities world wide and country specific

- analytical quality control and product standardisation

- legal regulations i.e. for registration of non-synthetic pesticides

- creating awareness for non-synthetic pesticides among political decision makers on international conferences and seminars

Target groups are Non-Governmental Organisations, Governmental Organisations, projects, small-business operators in partner countries, decision makers, self-help initiatives

44 The need for uniform analytical standards

Neem extracts are in the process of registration or already registered for certain applications in several countries. However, the extensive application of commercial neem-based pesticides is still restricted by two bottle necks: 1.) lack of high quality standards for neem products and 2.) legal regulations for registration For both uniform analytical standards are required which are viable all over the world. This was the situation like when the Pesticide Service Project came in. In October (16 - 18), 1996 the 'International Working group Neem Analysis' met near Stuttgart invited by the Pesticide Service Project of GTZ. In co-operation with Dr. Wieland, Schopfheim, a symposium on “Analysis and Standardisation of Neem Extracts” has been conducted. Invitations were sent out to scientists, to companies dealing with preparation of neem extracts, and to members of governmental organisations in India, Pakistan, Thailand, Sweden, Australia, Canada, Germany, England and USA. 13 Lectures were presented, mostly dealing with extraction and separation techniques, and with the isolation of pure Azadirachtin. Abstracts and manuscripts of the lectures are planned to be published in 1998 by GTZ. Aim of these activities co-ordinated by the Pesticide Service Project was to work out a world wide excepted and CIPAC registered analytical method for neem based pesticides.

Table 2 CIPAC - (Collaborative International Pesticides Analytical Council) is a organisation based in UK, was found 1957 to promote international agreement on

- methods for the analysis of pesticide products and of such other substances as the Council may .... determine.

- methods for physico-chemical evaluation of technical pesticide materials and formulations

- methods for correlating biological efficacy with physical and chemical properties of pesticide products

- foster inter-laboratory collaborative analysis among interested laboratories

- to publish agreed methods ...... etc. in close collaboration with FAO and WHO, when accepted. Analytical methods are published in 'FAO Plant Protection Bulletin' as FAO Specifications for Plant Protection Products

45 So once an analytical method is registered at CIPAC a world wide analytical procedure would be available. It is hoped that this would make the registration of standardised commercial products based on neem easier and at the same time would allow to control the international trading with neem-based pesticides. The symposium was organised to work out suggestions how to prepare and to standardise neem extracts for application in developing countries. Two main subjects were chosen: - 1. Which ingredients should be used as leading compounds for a standardised analytical method? - 2. Which chemical standard procedures should be used for analysis?

Analytical Methods

1. Should the discussion be restricted to Azadirachta indica or should other Azadirachta species such as A. excelsa or A. siamensis be included? Seeds of A. excelsa and A. siamensis are interesting starting materials for pesticide production as they contain, together with Azadirachtin and 3- tigloylazadirachtol, additional bioactive compounds) which are in some cases more active than Azadirachtin itself. However the participants agreed that these species should not be considered further as they are available in limited areas only, and because the recommendations worked out for A. indica are valid also for A. excelsa and A. siamensis. 2. Standard procedure for analysis a) Which standard method should be recommended for work up of the seeds? A long discussion was devoted to considerations on advantages and disadvantages of various solvents for extraction, such as water, methanol, ethanol, and chlorinated hydrocarbons. At the end the participants agreed that methanol should be chosen as the most suitable solvent. To report all details of the analytics in this paper will lead to far. c) Which analytical method should be used? At present HPLC is the only reliable method to analyse neem extracts. Other proposed methods need further research. The water content of the seeds should be determined using the method published by International Seed Testing Association (ISTA) in: International Rules for Seed Testing, Rules 1996: Seed Sci. & Technol., 24, Supplement, ISTA Zürich 1996, ISSN 0251-0952, ISBN 3-906 549-27-5. d) Which reference samples should be used for analysis and standardisation? Many investigations have shown that the biological activity of neem extracts is not only due to the Azadirachtin content. Nevertheless for the present solely Azadirachtin should serve as the reference compound for analysis and standardisation because it is the only compound belonging to that group of active

46 ingredients which can be isolated sufficiently pure in sufficient quantities. Standard samples (0.5 - 1 mg) should be available as stock solutions of known concentration in methanol, not in solid form, and information about purity, durability, and storage conditions should be provided with the samples. RING ANALYSIS

The participants agreed that recommendations cannot be given before ring analysis have been carried out. Ring analyses were carried out by 10 laboratories (6 from developed countries and 4 from developing countries) Pakistan, India, Thailand, Kenya. England, USA, 4 Germany. Azadirachtin Standard and neem seeds were distributed along with a detailed description of the analysis method, based on the method worked out by ERMEL & CHIRATHMAJAREE, Bangkok, and modified by Wieland and by Troß, Germany. Results discussed by the international working group NEEM ANALYSIS

The results were collected by GTZ Pesticide Service Project and discussed among the members of the working group, selected from GTZ and from laboratories involved in the ring analysis during a second symposium in September 1997 at GTZ Headquarter. Not all of the laboratories got finally involved in the ring analysis as scheduled. Results were received from Thailand, Kenya, UK, Pakistan, 4 Germany, there was no reply from, USA and India. 1. Results and suggestions for modifications The results collected and summarised were discussed with regard to 1) water content of the seeds, 2) method of extraction, preparation of seed material, 3) analysis for Azadirachtin content. 1. Water content

Table 3: Results of the ring analysis 'Azadirachtin'; average water content Laboratory Temperature time content % A 103 ° 17 h 7,28 B 103 ° 18 h 8,0 C 103 ° 17 h 5,85 D 130 ° 1 h 5,5 E 105 ° 24 h 6,91 F 130-133 ° 2 h 8,5 G 103 ° 72 h 7,42 H 103 ° 18 h 6,64 Average 7,01

47 The values as determined by the eight laboratories do not differ very much from the mean. They are acceptable, however, they seem to be somewhat dependent on the method of the seed preparation used prior to drying (cutting, grinding, pre-drying, a.s.o.). Agreement was achieved that in future analyses the seeds should be ground, and the drying procedure should be carried out according the instructions published by ISTA. Drying will reduce not only the water content but will cause also loss of volatile compounds. Is was therefore suggested to replace the term “water content” by “weight losses by drying”. 2. Method of extraction, preparation of seed material

Table 4: Results of the ring analysis 'Azadirachtin' (content of AzadirachtinA in mg/kg) Laboratory sample 1 sample 2 sample 3 average A 3,61/3,63 3,79/3,91 3,67/3,60 3,74 B 2,83//2,83 2,90/2,88 3,07/3,10 2,94 C 2,34/2,39 2,37/2,37 2,37/2,48 2,39 D 1,56/1,50 1,58/1,54 1,57/1,56 1,55 E 6,36/7,89 7,61//7,32 6,80/6,63 7,01 F 1,83/1,82 1,85/1,86 1,85/1,86 1,83 G 2,78/2,68 2,84/2,83 2,51/2,53 2,69 H 2,93/2,99 2,75/2,83 2,93/2,96 2,89

Two laboratories used Ultra-Turrax (A+B), the others (C-H) used mortars for grinding the seed material. The two different methods may lead to different grades of homogenisation and hence, to virtually different Azadirachtin contents. It is very difficult to get good homogenisation in a mortar if the oil content of the seeds is high. The size of the particles is also dependent on the mode of grinding in a mortar. From the results it was therefore concluded that an Ultra-Turrax should be preferred over mortar grinding. The material should be seed kernels instead of seeds, and grinding time 3 minutes. 3. Analysis for Azadirachtin content

The Azadirachtin contents found by HPLC in the different laboratories varied in a range from 1.55 to 7.01 mg/g, the major part being around 2.7 mg/g. These big differences in the results were, in part, certainly due to the different quality of the chromatograms, and raised a number of questions, statements, and suggestions for modifications of the method: a) It turned out that some laboratories used peak heights, and others peak areas. The discussion resulted in a statement that in future analyses only peak areas shall be used. In order to work out an analytical method to give reproducible results to be published it is necessary to do calibration of the columns, and to do statistics including calculation of standard deviations. b) A column oven is not necessary. c) Columns should be washed after each third run. d) Weighing of small samples with a precision of 0.1 mg to prepare standard solutions is problematic. Therefore it was suggested to adapt a method

48 used by Sigma Company. Trifolio will practise this procedure to prepare standards for future analyses. e) Azadirachtin solutions are not stable if kept in refrigerators. If stored at -15 °C in acetonitrile/water mixtures or methanol they are stable up to 1 year. Lipophilic Leads

As a result of the discussion lipophilic leading compounds were stated not to be very recommendable. There is no doubt that synergistic effects are operating if neem oil is applied, but the active principles involved in those effects are not known. The main activity is antifeedant, not insecticidal. Marker compounds could be salannin and its derivatives. However, it might be better to establish a method to determine Azadirachtin in neem oil, e.g. by solid phase extraction followed by HPLC, and to work out methods for the analysis of formulations like NeemAzal-T/S, which can be done by loading the formulation directly on top of the column and eluting it with acetonitrile/water 60/40 or methanol. Treatment of neem oil with hexane to remove the fat before dissolving the residue in methanol is not recommendable because Azadirachtin is somewhat soluble in hexane which then is acting as a carrier lowering the Azadirachtin content. Neem oil and formulations (NeemAzal-T/S) were included into the second ring analysis. A second round of ring analysis have been started end of 1997 including 10 laboratories from 7 northern countries and 3 developing countries. Samples of seeds, oil, and formulations, together with modified instructions for analysis has been distributed end of 1997. The participants were advised to store the seeds in cool and dry environment, and to discard seeds contaminated by fungi. Deadline for submission of the results will be end of February 1998. Future activities

It is expected that the results will be available in May 1998 and be published end of this year by GTZ.

49 RISK ESTIMATION OF MOULD GROWTH IN NEEMAZAL AND ITS FORMULATION

NEEMAZAL-T/S

CHRISTINE KLICHE-SPORY, HUBERTUS KLEEBERG

Trifolio-M GmbH, Sonnenstr. 22, D-35633 Lahnau

Abstract

NeemAzal (NA) and NeemAzal-T/S (NA-T/S) were subject to an evaluation of the risk of an infection by aflatoxigenic fungi. General factors causing microbial, especially fungal, infection and growth were compared to physical and chemical attributes found in NA and NA-T/S. The results indicate that mould growth in NA and NA-T/S is not to be expected. 1. Introduction: General Factors for Microbial Infection and Growth

Many natural products are subject to microbial destruction by bacteria, yeasts or by fungi. The attack of spores, conidia or mycelium from mould may happen in or on raw materials or later on during the manufacturing process. This microbial infection of a natural product is due to growing conditions, hygienic and physical aspects of harvest, storage and product refinement. Another aspect for the microbial growth is the chemical composition of the substrate. The knowledge of these factors is important, because especially moulds or their secondary metabolites may induce acute or chronic effects of poisoning or allergenic reactions to mammals. This may happen by oral or inhalative intake of contaminated products and also by contact with the skin 1. 2. Mycotoxins

Toxic metabolites (mycotoxins) can be developed by many different species of fungi. The most important species belong to the genii of Alternaria, Chaetonium, Cladosporium, Fusarium, Penicillium and especially to Aspergillus. Almost 50 species of Aspergillus have been listed as capable of producing mycotoxins. Among them the most important group of toxigenic aspergilli are the aflatoxigenic moulds Aspergillus flavus, Aspergillus parasiticus and the much less common species Aspergillus nomius. A. flavus is widely distributed in nature, but A. parasiticus is probably less widespread, it occurs mainly in tropical and in subtropical regions 2.

Figure 1: Aflatoxigenic fungi. (A) A. flavus head (x 216); (B) A. flavus conidia (x 1350); (C) young A. parasiticus heads (x 216); (D) A. parasiticus conidia (x 1350)²

Practice Oriented Results on Use and Production of Neem Ingredients and Pheromones VIII 50 H. Kleeberg & C. P. W. Zebitz (eds.) Copyright 2000 by Druck & Graphic, Giessen Table 1: Significant mycotoxins produced by Aspergillus species and their toxic effects ² Mycotoxin(s) Toxicity Species producing Aflatoxins B1 and B2 Acute liver damage, A. flavus, A. parasiticus, A. cirrhosis, carcinogenic (liver), nomius teratogenic, immunosuppressive Aflatoxins G1 and G2 Effects similar to those of B A. parasiticus, A. nomius Aflatoxins: G1 toxicity less than that of B1. Cyclopiazonic acid Degeneration and necrosis A. flavus of various organs, tremorganic, low oral toxicity Ochratoxin A Kidney necrosis (especially A. ochraceus and related pigs), teratogenic, species immunosuppressive, carcinogenic Sterigmatocystin Acute liver and kidney A. versicolor, Emericella spp. damage, carcinogenic (liver) Fumitremorgens Tremorganic (rats and mice) A. fumigatus Territrems Tremorganic (rats and mice) A. terreus Tryptoquivalines Tremorganic A. clavatus Cytochalasins Cytotoxic A. clavatus Echinulins Feed refusal (pigs) Eurotium chevalieri, E. amstelodami

In table 1 significant mycotoxins, that are produced by Aspergillus species are listed. Aflatoxins are both acute and chronical toxins for animals and humans. The acute lethal dose for adult humans is estimated to be of the order of 1-10 mg/kg bodyweight 3. Figure 2 shows the chemical formula of the most important toxic metabolites from A. flavus and A. parasiticus, Aflatoxin B1, B2, G1 and G2. The letters B and G refer to the fluorescent colours (blue and green) observed under long-wave UV-light. The chemical structure of Cumarin is common in all Aflatoxins. In a worldwide study it turned out, that only about 35% of all isolated strains of A. flavus 4 produce the mycotoxins B1 and B2 .

51 Figure 2: Most important toxic metabolites from A. flavus and A. parasiticus, Aflatoxin B1, B2, G1 and G2 ³.

Aflatoxin B1 Aflatoxin B2 CAS Nr.: [1162-65-8] CAS Nr.: [7220-81-7] O O O O

O O H H

O O

H O OMe H O OMe

Aflatoxin G1 Aflatoxin G2 CAS Nr.: [1165-39-5] CAS Nr.: [7241-98-7] O O O O

O O O O H H

O O

O OMe O OMe H H

3. NeemAzal, NeemAzal-T/S and Mycotoxins

Aspergillus flavus and Aspergillus parasiticus have a strong affinity for nuts and oilseeds. In some cases infestation occurs before harvesting. Peanuts, for example are invaded while still in the ground. In other products, insect damage allows the entry of aflatoxigenic moulds. Infection also may take place during poor drying and/or unprofessional storage conditions. NeemAzal is a product from oilfruit, from neem seed kernels (NSK). EID Parry, the producer of NA, buys neem seed kernels from different farmers in India. They consult the farmers in the proper handling of NSK, to avoid infestation. After arrival in the factory the kernels are controlled, than washed and dried under the best possible conditions. Nevertheless the infestation with mould can’t be excluded completely and will vary. Steps during the production of NA are treatments/extractions with organic solvents. Living cells do not survive this procedure. Spores or other living parts from fungi in NSK can’t get into the product. NeemAzal is dried subsequent to the extraction. The drying temperature lies between 30 - 50 °C. Residues of the organic solvents are removed by this procedure. After drying the product is directly packed into plastic bags to minimise the risk of reinfection with mould and to keep it dry. The product is stored cool and in the dark. At Trifolio-M GmbH, NeemAzal is also handled with greatest care. It is stored dry and cool, with a maximum temperature of 16 °C and also kept in the dark. Containers are kept close, so that the danger of reinfection is quite low.

52 Aflatoxins may accompany Azadirachtin during the extraction process and hence may be present in NeemAzal and consequently also in NeemAzal-T/S. Therefore, the content of Aflatoxins in each batch of NeemAzal is controlled. Batches that contain more than 100 mg/kg Aflatoxins are not used for further processing to NeemAzal-T/S. In this context, some results are interesting: We determined the content of Azadirachtin A (AzA) and of Aflatoxin B1 in NSKs and NeemAzal produced from these kernels. A batch of NSK with a high content of Aflatoxin B1 was chosen in order to increase the accuracy of the analytical determination. The analytical results (see table 2) show that AzA is enriched to a higher degree in NeemAzal than Aflatoxin B1 is. But the results also show that the content of Aflatoxins can not be reduced sufficiently by the production process. Thus care has to be taken for a low Aflatoxin content in the starting material (see above).

Table 2: Enrichment of Aflatoxins and Azadirachtin A in NeemAzal

Substance Neem seed kernels NeemAzal enrichment (NA/NSK) (NSK) (NA)

Azadirachtin A 5 g/kg 340 g/kg 68

Aflatoxin B1 9,2 mg/kg 202 mg/kg 22

The development of the NeemAzal-technology during the last years shows that the Aflatoxin content is continuously decreasing from 50 to 500 mg/kg (batches with above 100 mg/kg were rejected) to now between 10 to 50 mg/kg. This shows that the steps taken for control and in the production contribute to a significant improvement of the quality of NeemAzal. Of course, it is the aim to decrease the Aflatoxin content of NeemAzal below the limit permitted for food.

The maximum level of Aflatoxins allowed in food in Germany is 4 mg/kg (2 mg/kg B1, 4 mg/kg Aflatoxins in total) 5. With a maximum content of 100 mg/kg Aflatoxins in NeemAzal, the formulation NeemAzal-T/S contains less than the limit of Aflatoxin content for food in Germany, which has been agreed upon during the registration process. 4. Physical and Chemical Conditions for Growth of Fungi/Formation of Mycotoxins

In general: Aspergilli are widespread all over the world. Therefore the reinfection of NeemAzal or its formulation NeemAzal-T/S with mould can’t be fully excluded, although the products are produced and handled with great care. The growth of fungi and the formation of secondary metabolites depend on many factors (see table 3 for summary).

53 Table 3: Main Factors for growth of fungi and formation of secondary metabolites 1 Temperature Activity of water pH-value of the substrate Content of Oxygen Chemical composition of the substrate

Temperature and water activity Many fungi have similar growth patterns. Aspergillus flavus and Aspergillus parasiticus grow at temperatures ranging from 10 - 12 °C to 42 - 43 °C, with an optimum near 32 - 33 °C, with Aflatoxins being produced at 12 - 40 °C 2. Figure 3 shows the correlation between temperature and water activity. The content of free water - water that is not bound as hydrate in a substrate- is of fundamental importance for the growth of fungi and for the development of secondary metabolites.

4 The definition of water activity aw is as follows :

aw = PD/PS

PD = vapour pressure of water in the substrate PS = vapour pressure of pure water at the same temperature

The correlation between relative humidity R.H. and water activity aw is:

. R. H. (%) = 100 aw

The optimum water activity aw for growth of A. flavus and A. parasiticus is near 0.99. At this point the temperature must reach its optimum, otherwise growth will not be possible. Aflatoxins are generally produced in greater quantity at higher aw -values (0.98 - 0.99). 2 The toxin production ceases at or near aw = 0.85 . In general, mycotoxin production is hardly possible below a water activity of 0.83. This value of 0.83 frequently corresponds to a water content (free water) of 17 % in many substrates. Most species of mould need a water activity of 0,8 to 0,85 (minimum) for their growth. Some species that belong to the Aspergillus glaucus group, however, are able to grow at lower aw-values (for example: Aspergillus echinulatus: aw (min.) = 0,62 . Some mathematical calculations showed, that fungal growth in a substrate with a water content lower than 0.61 is highly unlikely to happen 4.

54 Figure 3: Influence of water activity and of temperature:

______growth of Aspergillus flavus and Aspergillus parasiticus; ------formation of Aflatoxin B1

pH-Value of the substrate: Growing of A. flavus and A. parasiticus can take place in the pH-range of 2.0 up to 10.5 (A. parasiticus) or 11.2 (A. flavus). Aflatoxin production has been reported for A. parasiticus only between pH 3.0 and 8.0, with an optimum near pH 6.0 2. Content of oxygen: Both moulds can reduce the formation of Aflatoxins when the content of oxygen in the atmosphere is reduced. The growth of mould is very often reduced by the increase of carbon dioxide up to 5 %. Chemical composition of the substrate: Fungi are heterotroph, that means, they need organic substrates that they cannot produce themselves, for their growth. Favoured substances are different types of sugars and other substrates of low molecular mass that are soluble in water. Proteins and lipoids + - can also be metabolised in many cases. Nitrogen can be used as NH4 or NO3 or as organic nitrogen. In Table 4 conditions that are necessary for the development of aflatoxins and properties and storage conditions of NA and NA-T/S are compared. The water activity of NeemAzal and NeemAzal-T/S is restricted to hydrate water. The absolute content of water (free and bound) in different batches of NeemAzal was determined by Karl-Fischer titration. The value was determined to 1,3 % (max. content: 2 %) in an average. The minimum water activity for formation of mycotoxins in general is 0,83. Aspergilli need a minimum water activity of 0,85. This leads to the result, that the formation of Aflatoxins in NeemAzal is

55 unlikely, since the water is hydrate-water. The same can be assumed for NeemAzal-T/S with about 5% as the average content of total water.

Table 4: Formation of Aflatoxins/Properties and storage conditions in NeemAzal and NeemAzal-T/S

Factor Formation of Aflatoxins NeemAzal/NeemAzal-T/S

Temp. optimum: 32-33 °C max: 20 °C

aw min.: 0,85 aw (NA, NA-T/S) = unknown (ca. 18% (free) water) NA: 1-2 % water (total) mean value: 1,3 %** NA-T/S: max = 4 % total water pH 2,0 - 11,2 @ neutral optimum: @ 6,0

O2 low influence, product stored in closed containers reduction of O2 can lead to reduction of Aflatoxins light low influence kept in the dark chemical ingredients sugar, low molecular NA: substances (soluble in limonoids: ca. 60 % water), proteins, lipoids. fatty acids and lipids: ca. 4 % partially characterised material*: ca. 34 %

NA-T/S additionally: Plant oil and surfactants

*: like: water-soluble saccharides or polysaccharides, water-soluble low-molecular weight lignins. **: internal results Trifolio-M GmbH.

56 Conclusion:

The evaluation concerning NeemAzal, its formulation NeemAzal-T/S and their content of Aflatoxins led us to the following results: Both products may contain Aflatoxins within given limits. These Aflatoxins originate from the Neem Seed Kernels already and are only partially removed by the manufacturing process efficiently. During production, transportation and storage both NA and NA-T/S are handled with greatest care. Improvements of the production process reduced the Aflatoxin content during the last years drastically. Nevertheless we can’t fully exclude the reinfection with mould. The growth of mould, however, and the formation of Aflatoxins are unlikely, because the water content of both products is very low. The absence of "free water" does not permit fungi to develop mycotoxins. Furthermore NeemAzal and NeemAzal-T/S are stored at low temperature, which prevents mould formation as well. Experimental studies for the determination of changes in the Aflatoxin content of NeemAzal and NeemAzal-T/S during storage are underway. Literature

E. Lück; M. Jager; Chemische Lebensmittelkonservierung, 3. Auflage 1995, Springer- Verlag; Berlin Heidelberg; ISBN 3-540-57607-X. M. P. Doyle; T. J. Montville; L. R. Beuchat; Food Microbiology; Fundamentals and Frontiers; S. 394-398; 1997, ASM Press Washington D.C. Giftpflanzen, Pflanzengifte; Roth, Daunderer, Kormann; 3. Auflage 1988; ecomed Verlagsgesellschaft mbH, Landsberg München; ISBN 3-609-61810-4. J. Reiß: Schimmelpilze. Lebensweise, Nutzen, Schaden, Bekämpfung. S. 47-187, 2. Auflage 1998, Springer-Verlag, Berlin Heidelberg. Deutsche Aflatoxin-Höchstmengenverordnung 1990.

57 ENVIRONMENTAL BEHAVIOUR AND AQUATIC ECOTOXICITY OF AZADIRACHTINA

LUC PUSSEMIER

Veterinary and Agrochemical Research (CODA-CERVA) Leuvensesteenweg 17, B-3080 Tervuren (Belgium) Abstract

A preliminary assessment has been made in order to evaluate the environmental risks linked to the use of the botanical insecticide AzadirachtinA, the main insecticidal component of NeemAzal- T/S. After a survey of the main physico-chemical properties, an analysis of data dealing with the persistence and the mobility of AzadirachtinA in soil has been carried out. Using these data, Predicted Environmental Concentrations (PECs) were calculated for surface waters and groundwater, and compared with toxicity data for 3 representative aquatic organisms. The Toxicity/Exposure Ratios (TERs) obtained were compared with the requirements of European Directive 91/414 and the conclusion was drawn that the environmental behaviour and toxicity properties of AzadirachtinA were in accordance with the requirements of the European legislation for plant protection products. Introduction

In the European Union, any plant protection product must be granted an authorisation in order to be placed on the market according to directive 91/414. This means that a new active ingredient and at least one preparation containing this active ingredient must be evaluated according to very precise instructions and according to an official decisional procedure (the so-called Uniform Principles). This procedure is also valid for the biopesticides (bacterial, fungal and viral preparations that can be used for plant protection) as well as for botanical pesticides. Therefore, taking into consideration all the information already available for AzadirachtinA and the commercial preparation NeemAzal, it was decided to prepare a preliminary assessment and to evaluate the possible risks of soil and water pollution linked to the use of the botanical insecticidal active ingredient AzadirachtinA. Material and Methods

Pertinent physico-chemical and biological data were taken from the literature (Ruch et al., 1996) as well as from confidential information provided by Trifolio-M GmbH, Germany (Kleeberg, personal communication, 1997). The assessment has been carried out according to the instructions given in directive 91/414 (Annex II, Annex III and Annex VI)

Practice Oriented Results on Use and Production of Neem Ingredients and Pheromones VIII 58 H. Kleeberg & C. P. W. Zebitz (eds.) Copyright 2000 by Druck & Graphic, Giessen Results

1) Summary of the main physico-chemical properties of Azadirachtin A

All properties were determined from NeemAzal solutions containing typically 34 +/- 9 % of Azadirachtin A. No pKa value is given for the compound: it is thus assumed that the chemical is non ionisable under environmental relevant pH. Water solubility: 2.9 g/L This high water solubility means that this unionizable compound will show a high mobility in the soil environment. Stability in water solutions: Typical half-lives (DT50) are given for AzadirachtinA in different aqueous buffers at 30°C (Table 1).

Table 1: Half-life (DT50) of AzadirachtinA at 30 °C in different buffer solutions. pH DT50 (hour (days) s)

4 236 9.8 7 105 4.4 8 21 0.9

From these data it can be said that the product is unstable in alkaline water solutions. The nature of the hydrolysis products is unknown. 2) Behaviour in the soil environment

Leaching behaviour in German Standard soils. The study has been carried out according the Guidelines of BBA (Richtlinie IV/4-2, 1986). The results (percent of applied product that can be found in the leachate after applying a volume of water equivalent to 200 mm rainfall) are shown in Table 2.

59 Table 2: Percent of applied product found in leachate (soil column leaching study) Soil number Soil type % OC % in leachate

2.1 sandy soil 0.62 90.4 2.2 humic sand 2.32 55.1 2.3 loamy sand 1.22 42.1

The compound is thus very mobile in the soil environment, which is in accordance with its high water solubility. Organic matter seems to be one parameter influencing the leaching (less product found in the leachate of the soil columns with a higher organic matter content). Adsorption/desorption The study has been carried out according to OECD guideline Nr 106. The soils are those selected for the soil column leaching study (Standard German soils). The results are expressed under the form of a mean soil/water partition coefficient (Kmean):

Kmean = 0.000419 l/g It is assumed that these adsorption measurements have been performed in the 3 standard soils used for the leaching experiment. Therefore a Koc value is proposed using the OC contents as mentioned in the leaching study (average Organic Carbon content = 1.39%). Assuming that soil organic matter is the main adsorbing material in soil, a mean Koc value can calculated as follows: Koc = (0.000419 * 100)/1.39 = 0.03 l/g or 30 l/kg The compound can thus be considered as very mobile in the soil environment according to the scale proposed by McCall (Annual book of ASTM standards, 1988) Degradation in soil This study has been performed in only one soil. Normally, the degradation rate has to be provided in 3 or 4 soils. There is no information about metabolization pathway, rate of mineralization, importance of bound residues. There is also a lack of information about the soil physico-properties (at least in the summary of the study). The results can be summarised as follows: DT50 = 1.9 d and DT90 = 9 d The parent product is thus quickly dissipated in the soil under investigation. This is in accordance with the low chemical stability observed in aqueous medium. It has to be pointed out, however, that there is still a lack of information about possible relevant metabolites and about dissipation rate in soils covering a large pH range: it is feared, indeed, that the product could be more stable under acidic conditions.

60 3) Assessment of the risks of water pollution

Determination of the GUS index Using a DT50 of 1.9 d and a Koc of 30 ml/g, the calculated GUS index is 0.70 which means that leaching to deeper soil layers is improbable (GUS < 1.8). This is mainly due to the rapid degradation of the parent chemical in the soil. Leaching under Standard Dutch scenario (PESTLA model) Using the PESTLA model (version 1.1 from Boesten and van der Linden, 1991) and using the obtained values for pesticide persistence and adsorbability (DT50 = 1.9d; Koc = 30 ml/g), the predicted concentration in groundwater between 1 and 2 m depth will be less than 0.001 µg/l after application of 1 kg of active ingredient/ha according to the standard Dutch scenario (sandy soil with 4.7 % of OM cropped with maize; total rainfall of 860 mm, application of pesticide in spring). As a matter of fact, the risks will be even more reduced for application rates lower than 1 kg/ha (100 g/ha seems to be a reasonable worst case). It must be stressed, however, that a slight difference in DT50 or Koc values could change dramatically the concentration predicted with the models. Since the DT50 is given for only one soil (with unknown information about physico-chemical properties), the results of mathematical modelling must be taken with great caution. Predicted environmental concentrations in surface water (Drift tables from Ganzelmeier et al., 1993) Using the drift Tables proposed by Ganzelmeier and officially used by the German authorities involved in pesticide registration, one can propose (Table 3) different predicted concentrations (PECsurface waters) for different distances between the treated field and the surface water body (0 m distance means direct overspray). This is done assuming an application rate of 100g active ingredient per ha and a water body of 0.3 m depth.

61 Table 3: PECsurface waters according to the drift Tables of Ganzelmeier (application rate = 100 g a.i./ha)

Distance from treated field 0 m 1 m 3 m 5 m 10 m

Drift (in % of applied) 100 4.0 1.0 0.6 0.4

Concentration in water(1) (in µg/l) 33 1.32 0.33 0.2 0.13

(1) assuming a water body of 0.3 m depth

4) Risk evaluation for aquatic organisms

Acute toxicity studies Fish species (OECD guideline 203): - rainbow trout (Oncorhynchus mykiss) LC50 (96h) : 160 mg/l NeemAzal-T/S (1.60 mg/L AzadirachtinA) - common carp (Cyprinus carpio) LC50 (96h) : > 100 mg/l NeemAzal-T/S (> 1.00 mg/L AzadirachtinA)

Crustacean species (immobilisation test: OECD guideline 202) - Daphnia (Daphnia magna) EC50 (48h): 1000 mg/l NeemAzal-T/S (6.57 mg/l AzadirachtinA)

Algal species (OECD guideline 201) - Scenedesmus subspicatus EC50 (72h): > 22 mg/l NeemAzal-T/S (> 0.22 mg/l AzadirachtinA)

Bioaccumulation The proposed Pow value for the different Azadirachtins is of the order 10 (log Pow about 1). No bioaccumulation studies are required: the product is considered as non bioaccumulable. Calculation of TER (Toxicity/Exposure Ratio for surface waters) PEC (1m) = 1.32 µg/l PEC(overspray)= 33µg/l

TERfish = (1600/1.32) = 1210 = (1600/33) = 48 TERdaphnia= (6570/1.32) = 4988 = (6570/33) = 199 TERalgae = (>220/1.32) = >170 = (>220/33) = >6.7

62 Ecotoxicological assessment According to Uniform Principles (Annex VI of European Directive 91/414), no authorisation should be given if:

TERfish < 100 TERdaphnia <100 (When short term exposure is considered) or TERalgae < 10 or BCF > 1000 (for readily degraded active ingredients) BCF > 100 (for nor readily degraded active ingredients)

In case of AzadirachtinA, all these conditions are met when considering treatment of fields adjacent to water bodies. In case of direct spraying of water bodies (e.g. for mosquito control) a case by case assessment will be needed using long term toxicity data (especially for fish). Conclusion

Despite a high mobility in soil, Azadirachtin does not seem to present an important risk of groundwater pollution (thanks to rapid degradation). After this preliminary evaluation it seems reasonable to propose this insecticide in the framework of an agriculture more compatible with the environment. It seems important, however, to complete this assessment by gaining more information about the degradation in soil (degradation rate in different soils with contrasted physico- chemical properties such as pH, and additional information about the transformation products). This information is needed in order to evaluate the risks under a large range of pedo-climatic conditions. In case of treatment with AzadirachtinA at the recommended dosage, all conditions are fulfilled for a safe use of the product in fields adjacent to water bodies. Under such conditions, the product can be considered as non toxic for the aquatic organisms. References

Boesten and van der Linden. Modelling the influence of sorption and transformation on pesticide leaching and persistence. J. Environ. Qual. 20, 1991, p.425-435 Ganzelmeier M., Köpp H., Spangenberg R. and Streloke M. Wann Pflanzenschutzmittel Abstandsauflagen erhalten. Pflanzenschutz-Praxis, 3, 1993, p 14-15 Ruch B., Kliche-Spory Ch., Schlicht A., Schäfer I., Kleeberg J.,Tross R. and Kleeberg H. Summary of some environmental aspects of the Neem ingredient NeemAzal and NeemAzal-T/S. In “Practice Oriented Results on Use and Production of Neem- ingredients and Pheromones V”; H. KLEEBERG &C.P.W. ZEBITZ (Eds) 1997, 15- 20

63 AUSWIRKUNGEN VON NEEMAZAL-T/S AUF DIE BRUT VON HONIGBIENENVÖLKERN (APIS MELLIFERA CARNICA L., HYMENOPTERA, APIDAE). ERGEBNISSE EINES HALBFREILANDVERSUCHES.

BORIS LEYMANN (1), WERNER MÜHLEN (2), ALOIS EDELMANN (1)

(1) Universität Bielefeld, Fakultät Biologie, Abteilung Morphologie und Systematik der Tiere, Morgenbrede 45, D-33501 Bielefeld, Germany (2) Landwirtschaftskammer Westfalen-Lippe, Institut für Pflanzenschutz, Saatgutuntersuchung und Bienenkunde (IPSAB), Nevinghoff 40, D-48 147 Münster Zusammenfassung

NeemAzal-T/S wurde bei der Honigbiene (Apis mellifera L.) im Halbfreilandtest auf brutschädigende Wirkungen geprüft. In den drei Versuchsgliedern wurde neben NeemAzal-T/S als Referenzmittel Alsystin WP 25 ausgebracht. Die Kontrolle wurde nur mit Wasser behandelt. Es wurden zwei Wiederholungen durchgeführt. Individuelle Brutzellen wurden in ihrem Entwicklungsverlauf beobachtet. Eine prozentuale Unterteilung in geschädigte und normalentwickelte Zellen machte eine Einstufung der Brutschädigung der Pflanzenschutzmittel möglich. Im Zeltversuch konnte für NeemAzal-T/S weder eine brutschädigende Wirkung noch eine akute Toxizität nachgewiesen werden. Einleitung

Da NeemAzal-T/S auf Entwicklungsstadien von Insekten wirkt, liegt die Vermutung nahe, daß dieses Produkt auch auf die Entwicklungsstadien von Honigbienen wirken könnte. Laborversuche zur Toxizität von Pflanzenschutzmitteln (CZOPPELT 1991, WITTMANN 1982) sowie qualitative Fütterungsversuche (OOMEN und DE RUIJTER 1992) und Halbfreiland- bzw. Freilandversuche (MÜHLEN 1996) lassen keine Einschätzung der tatsächlichen Gefährdung der Brut in Bienenvölkern zu. Aufbauend auf der bei MÜHLEN (1996) beschriebenen Methode sowie auf der Grundlage der EPPO-Guideline 170 (OEPP/EPPO 1992) und dem Decision making Scheme (OEPPO/EPPO 1993) wurde eine Testmethode entwickelt, die es erlaubt, den Einfluß von Insektenwachstumsregulatoren (IGR) auf die Entwicklung von Honigbienenvölkern quantitativ zu beschreiben. Material und Methode Für die Versuche wurden folgende Substanzen und Aufwandmengen ausgewählt:

• Prüfmittel: NeemAzal-T/S: NeemAzal-T/S (Trifolio - M) mit dem Wirkstoff Azadirachtin A ist für adulte Honigbienen nicht toxisch. Es wird allerdings vermutet, daß NeemAzal-T/S auf Entwicklungsstadien der Bienen einen hemmenden Effekt ausübt. NeemAzal-T/S wurde mit der verdoppelten, höchsten zuzulassenden Aufwandmenge von 6,0 l auf 400 l Wasser/ha getestet. • Referenzmittel: Alsystin WP 25: Alsystin WP 25 (Bayer, Leverkusen) mit dem Wirkstoff Triflumuron besitzt eine bekannte brutschädigende Wirkung und wurde in der vorliegenden Studie in erhöhter Dosis mit 800 g in 400 l Wasser/ha ausgebracht (Faktor 10 nach Schmidt, Bayer,

Practice Oriented Results on Use and Production of Neem Ingredients and Pheromones VIII 64 H. Kleeberg & C. P. W. Zebitz (eds.) Copyright 2000 by Druck & Graphic, Giessen Ringversuch AG Bienenschutz 1994) (siehe auch MÜHLEN 1996). • Kontrolle: Wasser: Die Kontrolle wurde mit 400 l Wasser behandelt, da die Formulierungen in wäßriger Lösung ausgebracht werden. Für die Versuche wurden Großzelte, sogenannte Tunnelzelte, angefertigt. Die Größe der Tunnelzelte betrug 12 m x 4 m x 2 m (L x B x H). Dies entspricht einer Grundfläche von 48 m² und einem Raumvolumen von 96 m³. Der Boden des Innenrandes der Tunnelzelte war mit Schattierungsleinen von 30 cm Breite ausgelegt. Dieser Randbereich diente zur Registrierung der Mortalität im Zelt. Vor den Bienenvölkern wurden Gary-Fallen eingesetzt. Gary-Fallen sind Rückhalte-Systeme für die im Stock gestorbenen Bienen. Diese Fallen bestanden aus einem seitlich bienendicht geschlossenen Drahtkäfig mit einer Maschenweite von 3 mm und einem bienendurchlässigen Deckel mit 8 mm Maschenweite. Abfliegende Bienen konnten durch diese weiten Maschen im Deckel der Falle den Bienenstock verlassen. Im Stock gestorbene Bienen sammelten sich auf dem Boden der Fallen und wurden hier zu den Boniturzeiten abgesammelt und registriert.

Ein Flugaktivitätsmessgerät (BeeSCAN) wurde in den Zelten eingesetzt (STRUYE et. al. 1994). Das BeeSCAN-Gerät ist eine microprozessorgesteuerte Einheit, die über Lichtschranken den Ein- und Ausflug von Honigbienen am Stock registriert. Es ist möglich, das individuelle Flugaktivitätsmuster der Prüfvölker im Tageslauf zu dokumentieren. Die Differenz der Ein- und Ausflüge gibt die tatsächlichen, absoluten Bienenverluste pro Tag wieder. Es kann sichergestellt werden, daß tatsächlich eine Spritzung in den vollen Bienenflug gegeben ist. Ferner können Repellenteffekte mit diesem Gerät dokumentiert werden. Anhand der Flugdaten kann im Vergleich der Versuchsglieder das Verhalten und die Mortalität zur Beurteilung des Pflanzenschutzmittels herangezogen werden. Als Versuchsvölker wurden in jedes Großzelt weiselrichtige Kleinstvölker in Schaukästen mit jungen Brutstadien eingestellt. Die Größe der Versuchsvölker betrug etwa 4 000 Bienen. Die Schaukästen erlaubten es, die Brutnester der Völker ohne Öffnen der Beuten beobachten zu können. Jeweils zwei Waben Deutsch-Normal-Maß (DNM: 35,4 cm x 20,7 cm; ca. 7 dm2 je Wabenseite, ges. 14,6 dm2) waren übereinander angeordnet und von beiden Seiten durch eine Glasscheibe geschützt. Durch die Glasscheiben war es möglich, direkt auf die Waben bzw. in die Brutzellen zu blicken, ohne die Völker öffnen oder Waben entnehmen zu müssen. Dies verhinderte ein zu starkes Auskühlen des Brutnestes während der Bonituren und beunruhigte die Bienen nicht wesentlich. Zwei mit Isoliermaterial ausgekleidete Holzklappen deckten die Glasscheiben ab. Das Brutnest wurde zur besseren Übersicht auf eine Wabe beschränkt. Als obere Wabe wurde eine frisch ausgebaute, helle Wabe ausgewählt, um die Eier und junge Maden am Grunde der Zellen besser erkennen zu können. Diese Wabe wurde mindestens fünf Tage vor Behandlung eingehängt, damit Eier und junge Maden als empfindlichste Brutstadien zum Zeitpunkt der Applikation vorhanden waren (worst case conditions). Als untere Wabe wurde eine volle Honigwabe eingesetzt. Diese Honigwabe war größtenteils verdeckelt. Die Verdecklung hält die Bienen davon ab, sofort auf diesen Honig zuzugreifen. Bei Futterengpässen stellt sie allerdings eine Reserve dar. War keine Futterwabe vorhanden, wurde statt dessen eine Futtertasche mit Futterteig eingesetzt. Eine natürliche Tracht wird dem Futterteig jederzeit vorgezogen.

65 Als Versuchspflanzen wurden Phacelia (Phacelia tanacetifolia Benth.) sowie Raps (Brassica napus) gewählt. Beide Testpflanzen besitzen sehr lange Blühperioden. Die Antheren in den Blüten stehen über den Kelchen. Dies stellt ideale Prüfbedingungen (worst case conditions) dar, da bei Applikation auch der für die Ernährung der Brut wichtige Pollen von der Spritzbrühe getroffen wird. Auf Overhead-Folien wurde festgehalten, in welchen Entwicklungsstadien sich die verschiedenen Zellen auf der Brutwabe befanden. Die Folie wurde auf der Glasscheibe des Beobachtungsstocks über das Brutnest gebracht und hier fixiert. Kurz vor Applikation wurden auf der Brutwabe jeweils ca. 100 Eizellen und jüngste Maden (Alter 1-3 Tage) individuell auf der Folie markiert. Durch die Folie und die Glasscheibe wurden die Zeichen für die verschiedenen Brutstadien mit einem roten Folienschreiber auf die Folie gezeichnet. Die genaue Position der Zellen wurde später auf eine zweite Folie übertragen. Diese Folie stellte nun die Schablone für die nächsten Zählungen dar. Sie wurde 21 mal auf Folie kopiert. Eine dieser Folien diente bei der Auswertung als Zählschablone. Hier wurden Zahlen mit grünem Folienschreiber eingetragen, durch die die Zellen für die Auswertung individuell numeriert wurden. Die Entwicklung individuell markierter Zellen ließ sich so verfolgen und bewerten. Ergebnisse

Mortalität adulter Bienen: Der tägliche Totenfall wurde mindestens fünf Tage vor Applikation aufgenommen. Die erhöhten Werte der ersten zwei Tage in Graphik I. und II. sind dem erhöhten Decken- und Eckenflug älterer Flugbienen zuzuschreiben. Diese alten Flugbienen konnten sich nicht an die neue Situation im Zelt gewöhnen. Nach Applikation der Mittel ergaben sich keine signifikanten Änderungen im täglichen Totenfall. Er schwankt um 40 bis 50 Bienen pro Tag. Eine akute Toxizität kann also ausgeschlossen werden. Aufgrund der sehr unterschiedlichen Flugaktivität, unterliegt auch die täglich registrierte Mortalität normalen, witterungsbedingten Schwankungen.

66 Anzahl Bienen

200

Prüfmittel:NeemAzal T/S 180 Referenzmittel:Alsystin WP 25 160 unbehandelte Kontrolle 140 Applikation 120

100

80

60

40

20

0 Tage -5-4-3-2-10123456789101112131415161718192021

Graphik I: Mortalitätsraten adulter Bienen in der 1. Wiederholung

Anzahl Bienen

200

Prüfmittel: NeemAzal T/S 180 Referenzmittel: Alsystin WP 25 160 unbehandelte Kontrolle: Wasser 140

120

100 Applikation

80

60

40

20

0 Tage -5 -4 -3 -2 -1 0 1 2 3 4 5 6 7 8 9 101112131415161718192021

Graphik II: Mortalitätsraten adulter Bienen in der 2. Wiederholung

67 Tabelle I: Brutkontrolle Halbfreilandprüfung

1. Wiederholung Kontrollierte Zellen Prüfmittel: Vergleichsmittel: unbehandelte NeemAzal-T/S Alsystin WP 25 Kontrolle Gesamtzahl Zellen 153 187 189 Normale Entwicklung 133 18 162 Abgestorbene Zellen 20 169 27 Normale Entwicklung (%) 86,9 9,6 85,7 Abgestorbene Zellen (%) 13,1 90,4 14,3 2. Wiederholung Kontrollierte Zellen Prüfmittel: Vergleichsmittel: unbehandelte NeemAzal-T/S Alsystin WP 25 Kontrolle Gesamtzahl Zellen 359 359 128 Normale Entwicklung 282 2 109 Abgestorbene Zellen 77 358 19 Normale Entwicklung (%) 78,6 0,6 85,2 Abgestorbene Zellen (%) 21,4 99,4 14,8 Mittelwerte aus 1. und 2. Wiederholung Mittelwert norm. Z. (%) 82,8 5,1 85,5 Mittelwert abgest. Z. (%) 17,2 94,9 14,5

68 Ergebnisse der Brutbonituren 100 90 80 70 60 % 50 40 30 20 10 0 Kontrolle NeemAzal Alsystin Kontrolle NeemAzal Alsystin (Wdh. I) (Wdh. I) (Wdh. I) (Wdh. II) (Wdh. II) (Wdh. II)

Normalentwicklung Geschädigte Zellen

Graphik III: Ergebnisse der Brutbonituren aus 1. und 2. Wiederholung

Mortalität in der Bienenbrut: Tabelle I zeigt die Zusammenfassung der Ergebnisse der Brutbonituren. Ausgezählt wurden einzelne Zellen, deren Entwicklung bewertet wurde. In der Kontrolle entwickelten sich in der ersten Wiederholung 85,7% der Brutzellen normal. In der zweiten Wiederholung waren es 85,2% der beobachteten Zellen. 14% der Brutzellen starben in der Kontrolle ab (1. Wdh.: 14,7%; 2. Wdh.: 14,8%). Bei Behandlung mit NeemAzal-T/S entwickelten sich 86,9% der Bienen in der ersten Wiederholung normal, in der zweiten Wiederholung waren es 78,6%. Die Behandlung mit Alsystin WP 25 führte zu einem sehr hohen Brutverlust von 90,4% in der ersten und 99,4% in der zweiten Wiederholung. Geschädigte Zellen wurden zu einem großen Teil erst sehr spät, oft Tage nachdem der Zelldeckel eingefallen war, ausgeräumt. Die toten Larven und Puppen befanden sich in sehr unterschiedlichen Entwicklungsstadien. Graphik I zeigt die relativen Anteile der geschädigten und normalentwickelten Zellen. Die Werte der Kontrolle stimmen mit Literaturwerten von 15 bis 20% Brutverlust bei unbehandelten Völkern gut überein. Die Werte des Prüfmittels entsprechen den Werten der Kontrolle. Die Referenzsubstanz führt fast zu einem vollständigen Brutverlust. Die Versuchsvölker beflogen ruhig und gleichmäßig den Pflanzenbestand. Ein Repellenteffekt in Form von Meidung der Versuchsfläche konnte nicht beobachtet werden. Eine akute Toxizität von NeemAzal-T/S wurde nicht beobachtet. NeemAzal-T/S kann auf der Grundlage dieses Tests daher als „bienenungefährlich“ eingestuft werden. 69 Literatur

CZOPPELT, CH. 1991: Toxizitätsmessungen mit dem Juvenoid Fenoxicarb an Bienenlarven im in vitro Aufzuchttest. Apidologie 22, 457-459. GARY, N. E. 1960: A Trap to quantitatively recover dead and abnormal honeybees from the hive. Journal of Economic Entomology Bd.53, H.5, S. 782-785. MÜHLEN, W., 1996: Implications of the IGR Alsystin WP 25 on the Development of Honeybee Colonies under Field and Semi-Field Conditions. Proc. of the 6th International Symposium on Hazards of Pesticides to Bees. Braunschweig. OEPP/EPPO 1992: Guideline on Test Methods for Evaluating the Side-Effects of Plant Protection Products on Honeybees. Bulletin OEPP-EPPO Bulletin 22, 203-216. OEPP/EPPO 1993: Decision-Making Scheme for the Environmental Risk Assessment of Plant Protection Products. Bulletin OEPP-EPPO Bulletin 151-165. OOMEN, P.A., DE RUIJTER, A., VAN DER STEEN, J. 1992: Method for Honeybee Brood Feeding Tests with Insect Growth-Regulating Insecticides, Bulletin OEPP-EPPO Bulletin, 22, 613-616. STRUYE, M.H., MORTIER, H.J., ARNOLD, G., MINIGGIO, C. UND BORNECK, R. 1994: Microprocessor-controlled Monitoring of Honeybee Flight Activity at the Hive Entrance. Apidologie 25, 384-395. WITTMANN, D. 1982: Entwicklung von Testverfahren und Experimente zur Beurteilung von Insektizid - Wirkungen auf Bienenlarven. Dissertation, Universität Tübingen

70 NEEM ACTIVITY AGAINST MICROORGANISMS: AZADIRACHTIN A IN BACTERIAL AND FUNGAL AGAR DIFFUSION TESTS

DETLEF F. HEIN & HANS E. HUMMEL

Justus-Liebig-University, Biological and Biotechnical Plant Protection, Institute of Phytopathology and, Applied Zoology, Ludwigstr. 21B, 35390 Giessen, Germany

MARTIN WEIDENBÖRNER

Justus-Liebig-University, Institute of Applied Microbiology, Senckenbergstr. 3, 35390 Giessen, Germany Abstract

The ingredients of the neem tree Azadirachta indica A.Juss offer a natural source useful for pest management that is both sustainable and environmentally compatible. So far, Azadirachtin A is well known as a 'phagorepellent' and as a 'modifier' of insect development. We investigated the effect of Azadirachtin A on fungal and bacterial growth with the agar diffusion method. The mould Penicillium chrysogenum and the yeast Saccharomyces cerevisiae were treated in vitro with an Azadirachtin A solution (50 µg/ml). The bacterium Escherichia coli was treated with acetone solutions containing 25, 50, and 75 µg/ml Azadirachtin A. P. chrysogenum and S. cerevisiae showed no retardation of growth compared to solvent and untreated control. No formation of inhibition zones can be observed in the E. coli in the Azadirachtin A, solvent, and untreated variant. The control variant with a standardized grapefruit seed extract (Citricidal™) gave clear retardation of growth. Substances other than Azadirachtin A seem to be responsible for the antifungal and antibacterial effects of neem extracts observed in investigations published by other authors. Introduction

Nature offers a broad range of highly specific and effective natural products. Neem extracts are well known to be used in sustainable and environmentally sound pest management. So far, however, most observations are published with neem as a weapon against insect pests [4, 5, 6, 7]. Because of the worldwide damages caused by fungal diseases it is important to use fungicides which can be claimed as ecologically compatible. During their search for active ingredients chemical companies orientate more and more on lead structures derived from natural molecules (e.g. the molecular basic structure of strobilurin led to the synthetic fungicide 'kresoxim-methyl' [1]). We are looking for neem ingredients which can be used as fungicides. Some hints are given that neem seed kernels contain active ingredients which can retard bacterial and fungal growth [2]. Which substance(s) is/are responsible for the observed effects is still unknown. The effectiveness of Azadirachtin A on growth of Escherichia coli, Saccharomyces cerevisiae, and Penicillium chrysogenum was investigated.

Practice Oriented Results on Use and Production of Neem Ingredients and Pheromones VIII 71 H. Kleeberg & C. P. W. Zebitz (eds.) Copyright 2000 by Druck & Graphic, Giessen Material and Methods

Different concentrations of Azadirachtin A solutions were tested in vitro against E. coli, S. cerevisiae, and P. chrysogenum. The pure Azadirachtin A was dissolved in acetone. Bacteria The filter paper diffusion test was used to investigate the effect of pure Azadirachtin A on E. coli. The technique is able to show antibacterial activity of test substances by the formation of inhibition zones vs. control. Nutrition agar was prepared in 9-cm Petri dishes. A volume of 0.1 ml bacterial suspension was placed in the center of the agar in the Petri dishes. A Drigalski spatula was used to spread the suspension on the surface of the solid agar. Sterile filter paper disks (6 mm diameter) were dipped until saturation was achieved into the test solutions, containing 25, 50, and 75 µg/ml Azadirachtin A. After solvent evaporation, four of the disks, each prepared with the solvent (acetone) or the Azadirachtin A solutions in different concentrations were placed on the prepared agar surface (see fig. 1, on the right). The Petri dishes were kept cool for 2 days to allow the substance to diffuse into the agar and were then put into the incubator for 24h. Each variant was measured in at least 11 replicates. Totally untreated Petri dishes with E. coli suspension served as growth control.

Fig. 1: Experimental setup of fungal and bacterial agar diffusion tests

Fungi The effect of Azadirachtin A on S. cerevisiae and P. chrysogenum was investigated with the agar diffusion method. MEA agar was prepared in 9-cm Petri dishes. The test solution contained 50 µg/ml Azadirachtin A dissolved in acetone. 0.1 ml of this solution was spread around by a Drigalski spatula on the agar surface. In one case, the agar was inoculated with a small piece of P. chrysogenum from a colony and was put with a needle on the agar surface as a dot (see fig. 1, in the middle), in the other case the inoculation with S. cerevisiae happened with a loop in form of a line (see fig. 1, on the left). Preparations with acetone or without any solvent served as controls. The Petri dishes were kept cool for 2 days and were then put into the incubator. The growth of the fungi was measured and compared to control.

72 Results

Bacteria The treatment of E. coli in the agar diffusion test showed no growth inhibition zones in all tested concentrations and in the solvent control. The treatment with a commercial standardized grapefruit seed extract (Citricidal™) gave clear retardation of growth. Fungi Both, the 50 µg/ml Azadirachtin A treatment of S. cerevisiae and P. chrysogenum showed no effect of growth inhibition in comparison to controls. The results are shown in fig. 2 and fig. 3.

80

70

60 m] [m 50 th

ow solvent control r 40 aza 50 ppm G untreated

30

5101520 Days post application

Fig. 2: Growth curve of Penicillium chrysogenum after Azadirachtin A (aza) treatment with a 50 µg/ml acetone solution

73 13

12

11

10

9

th [mm] 8

ow solvent control r 7 aza 50 ppm G untreated 6

5

5 10152025 Days post application

Fig. 3: Growth curve of Saccharomyces cerevisiae after Azadirachtin A (aza) treatment with a 50 µg/ml acetone solution

Other authors could show an influence of neem extracts or commercial neem formulations on microorganisms more or less clearly. Eppler [3] found, if at all, a very low effect of different extracts (e.g. leaves, bark, kernels, oil) and Azadirachtin A on bacteria. But sometimes the observed effects were not dose dependent (lower concentrations gave larger inhibition areolas than higher ones). E. Coventry and E.J. Allan [2] showed some effects with commercial neem formulations tested on bacteria and fungi. In this case, the bacteria Bacillus cereus, B. mycoides, B. thuringiensis, B. subtilis, Erwinia carotovora, Pseudomonas syringae, Agrobacterium tumefaciens, and the fungus Gaeumannomyces graminis were treated with NeemAzal-F, NeemAzal-T/S, the respective formulation additives Azasolv and Blank and NeemAzal powder. The 1 % NeemAzal powder solution inhibited all tested bacteria with the exception of A. tumefaciens. A 10 % NeemAzal-F solution inhibited the bacteria as well but a significant difference to the formulation additive Azasolv could not be observed. NeemAzal powder could retard the growth of G. graminis but only in very high concentrations (500 ppm Azadirachtin A equivalent). The treatment with NeemAzal-T/S and NeemAzal-F showed no significant growth reduction compared to controls. NeemAzal-T/S showed no effect neither in the bacteria nor in the fungi test. The observed effects show that NeemAzal powder has antibacterial and antifungal activity. But it is unknown which substance(s) is/are responsible for the inhibition of growth. In our case, Azadirachtin A does not inhibit the tested organisms P. chrysogenum, S. cerevisiae, and E. coli. These microorganisms are insensitive to Azadirachtin A. It might be that the tested concentrations are too low. But according to future aspects of input in plant protection there is a financial border line where too much input of highly active ingredients is not economic under the actual social circumstances. Another reason could

74 be a negligible diffusion of Azadirachtin A in the agar, although it is slightly soluble in water. All in all it seems not to be implausible that Azadirachtin A is ineffective against the tested microorganisms. In insects there is a different mode of action than could be expected in bacteria and fungi. In view of the positive results with neem kernel extracts it is promising to search for the substances actually responsible for the antibacterial and antifungal effects of neem in further investigations. Acknowledgements

The authors appreciate the kind encouragement of Prof. Dr. J.C.G. Ottow, Mrs. Linda Thiele-Eichenberg and Mrs. Rita Geißler-Plaum provided technical help with the bacterial and fungal cultures. Financial support for the extraction and purification of Azadirachtin A came from the ‘AZTEC’ project funded by the EU commission under AIR2-CT94-1343. References

[1] BASF Aktiengesellschaft, 1998: Internet page of BASF AG: http://www.basf.de. [2] Coventry, E.; E.J. Allan, 1997. The effect of neem-based products on bacterial and fungal growth. In: H. Kleeberg & C.P.W. Zebitz (eds.), Practice Orientated Results on Use and Production of Neem-Ingredients and Pheromones V. Druck & Graphic, Giessen, pp. 237-242. [3] Eppler, A., 1993. Untersuchungen zum hemmenden Einfluss von Niem auf Bakterien. Med. Fac. Landbouw. Univ. Gent 58/3a, 1145-1153. [4] Schmutterer, H.; K.R.S. Ascher; H. Rembold (eds.), 1981. Natural pesticides from the neem tree Azadirachta indica A. Juss. Proceedings of the 1st Internat. Neem Conference, Rauischholzhausen, FRG, 25-28 May, 1983. Rossdorf: typo-druck- rossdorf GmbH. [5] Schmutterer, H.; K.R.S. Ascher (eds.), 1985. Natural pesticides from the neem tree Azadirachta indica A. Juss and other tropical plants. Proceedings of the 2nd Internat. Neem Conference, Rauischholzhausen, FRG, 25-28 May, 1983. Schriftenreihe der GTZ; No. 161. Rossdorf: TZ-Verlagsgesellschaft, ISBN 3-88085- 156-5. [6] Schmutterer, H.; K.R.S. Ascher (eds.), 1987. Natural pesticides from the neem tree Azadirachta indica A. Juss and other tropical plants. Proceedings of the 3rd Internat. Neem Conference, Nairobi, Kenya, 10-15 July, 1986. Schriftenreihe der GTZ; No. 206. Rossdorf: TZ-Verlagsgesellschaft, ISBN 3-88085-372-X. [7] Schmutterer, H. (ed.), 1995. The Neem Tree Azadirachta indica A.Juss and other meliaceous plants. VCH Weinheim, pp. 696, ISBN 3-527-30054-6.

75 PHYTOTOXICITY OF NEEM TERPENOIDS TOWARDS ARABDOPSIS THALIANA.

HILDE BETTUM, PETER J. DOMINY AND ROBIN H. C. STRANG

Division of Biochemistry and Molecular Biology, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow, G12 8QQ Introduction

The literature on neem extracts, as summarised by Schmutterer (1995), contains many references to the phytotoxicity of crude neem extracts, when applied at high concentration to various plants. Many of these effects are attributed to "neem oil", which is notoriously variable in its content of the terpenoids. Occasionally, the adverse effects have been due to the use of aqueous kernel extracts, which suggests that the active compound is mainly azadirachtin A, the only major terpenoid with moderate water- solubility, (Freisewinkel (1989), Srivastava and Parmar (1985). Few reports deal with fully characterised neem extracts, but Nisbet (1992), in the course of a study of the systemic effects of pure azadirachtin A, observed that exposure of the roots of young tobacco plants (N. clevelandia ) to a solution of 500 ppm of the pure terpenoid, caused a long- term retardation of growth. A study using NeemAzal T/S as a spray on fruit trees against aphids (Schulz et al. 1997, M. Zuber, (personal communication, 1997) suggest that this commercial product, in which azadirachtin is the active compound, may have phytotoxic effects on some of the species examined. The aim of the study reported here was to use Arabdopsis thaliana, a crucifer of short life cycle, to test the phytotoxicity of a mixture of neem terpenoids similar to some used commercially. An added advantage in using A. thaliana is that its cells can be readily grown in liquid culture, thus allowing a defined concentration of the neem to be applied directly to the growing vegetatious plant cells. Methods and materials

Arabdopsis thaliana a) The cells were cultured under sterile conditions, at room temperature, in an appropriate liquid nutrient medium under low intensity light, with continuous shaking, for periods of up to 7 days.

Practice Oriented Results on Use and Production of Neem Ingredients and Pheromones VIII 76 H. Kleeberg & C. P. W. Zebitz (eds.) Copyright 2000 by Druck & Graphic, Giessen Duplicate samples were taken at regular intervals and centrifuged under standard conditions, in order to obtain a measure of cell growth, as "packed cell volume". From this, rates of growth were calculated. b) Germination and growth of seedlings Seeds which had been washed, sterilised, and dried, were placed on to nutrient agar plates, and after a 72h exposure to cold conditions (4°C), were allowed to germinate and grow for 18 days at 23°C under moderate light. Plant development was estimated by measuring the extent of root growth. Neem terpenoid mixture The extract used (Fig. 1) was one obtained from deoiled neem kernels by methanolic extraction. It contained neem terpenoids ranging in polarity from the azadirachtins to those such as nimbin and salannin.

Figure 1. Resolution by HPLC of the terpenoids in the neem extract used in this work. The column used was a C6 silica-based reverse phase analytical column. The terpenoids were eluted by means of a linear gradient of methanol in water rising from a starting mixture of 50/50 methanol/water to 100% methanol, over a period of 10 min. The rate of pumping was 1 ml/min. Detection was by UV light absorbance at 220nm. Some of the major compounds are: a: azadirachtin A; b: azadirachtin B, c: nimbin; d: salannin. In this particular extract, azadirachtin A represents 15% of the terpenoids by weight.

77 Inclusion of the neem mixture in the growth media. Of the neem terpenoids only azadirachtins (mainly A and B) are water-soluble to any extent. For this reason the terpenoids were dissolved in dimethylsulfoxide (DMSO) before being introduced into the aqueous media. Preliminary experiments showed that at a concentration of 1% (v/v) the solvent had no effect on the growth of cultured cells, although it did reduce the growth of the seedlings. Under these conditions, it was possible to include the terpenoid mixture at a maximum concentration of 700 ppm, although this represented the limit of solubility. The solution in DMSO was filter-sterilised before addition to the growth media. Results and Discussion.

In terms of phytotoxicity, the results (Fig. 2) were almost exactly the same for cells in liquid culture and developing seedlings. At concentrations of the terpenoid mixture of 70 ppm and lower, there was no negative effect on the growth and development of Arabdopsis thaliana. At a concentration of 700 ppm, growth of the seedlings was severely restricted and the cells in culture were killed, losing all colour in the process. Thus the EC50 lies between 70 and 700 ppm and can be rather arbitrarily assigned a figure of 250 ppm. The developing seedlings presented some interesting features. Even the highest concentration of terpenoids used had no effect on the number of seeds germinating. There was evidence that the lowest concentration of the terpenoids (0.7 ppm) produced a 16%, statistically-significant stimulation of root growth, compared to the controls grown in the presence of DMSO. This effect was not evident at the higher concentrations. The results offer no indication as to the mode of action of the terpenoids on plant cells, but the fact that the seeds all germinated suggests that the effect of the terpenoids reported here is on some aspect of cellular division. Although there was little root growth, the seed leaves of the plants at the highest concentration of the terpenoids showed no sign of withering. It would be a mistake to generalise too widely from these results on the phytotoxicity of the mixture of the terpenoids. Just as there are considerable differences in sensitivity to the compounds between insect species, the same is likely to apply to plants, In fact, the results of Schulz et al. (1997), showed that different commercial cultivars of pear trees showed widely differing susceptibilities to NeemAzal applied to leaves as spray containing a concentration of 30 ppm azadirachtin. There are, however, a few previous reports on phytotoxicity of pure terpenoids which are generally consistent, in quantitative terms, with the results reported here. The results obtained by Nisbet (1992) with 500 ppm pure azadirachtin A applied to the roots of tobacco have already been mentioned, and Verma (1974) found that mixtures of nimbin and nimbidin at a concentration greater than 250 ppm were toxic to C. amaranticolor when applied in the same fashion. As a rule of thumb, therefore, it is reasonable to assume that several (or possibly most) of the neem terpenoids, when presented to some types of vigorously-dividing plant cells in concentrations in excess of 250 ppm will retard growth.

78

Figure 2. The effect of different concentrations of neem terpenoids on the growth of a) cultured cells, b) young plants of Arabdopsis thaliana. a) the results represent the growth of cells in liquid culture as packed cell volumes (PCV) . Each point represents the rate of growth of duplicate cultures over a period of 7 days. b) the results are taken from photographs, Plant growth was estimated by the length of the roots after 18 days. Each point is the average of at least 15 separate measurements" and the lines are the standard

79 References

Freisewinkel, D. (1989), Diploma Thesis, University of Giessen, BRD. Nisbet, A. (1992) Ph. D. Thesis, University of Glasgow, UK. Schmutterer, H. (1995) The Neem Tree, VCH Verlagsgesellschaft mbH. Weinheim, BRD. Schulz, C., Kienzle, J., Hermann, P., and Zebitz, C. P. W. (1997) Gesunde Pflanzen 49, 95. Srivastava, K.P. and Parmar, B.S. (1985), Neem Newsletter 2, 7. Varma, V.S. (1974), Acta Microbiol. Polon 6, 9.

80 USE OF PMR SPECTRAL DATA IN QUALITY CONTROL OF NEEM PESTICIDES

KOTEPPA PARI, C. DEVAKUMAR

Division of Agricultural Chemicals, Indian Agricultural Research Institute New-Delhi-1 10 0129 INDIA Neem elaborates a vast array of Tetranortriterpenoids and Neem products find applications as Neem-pesticides and Neem-drugs. The bio-activity of Neem is not due to a particular constituent though, Azadirachtin A is important. There is a dire need for a simple and cheap method for the quality control of Neem pesticides and their bio-active products. Use of HPLC though very reliable and quantitative, is sophisticated, costly and requires standards. This is a major constraint in developing countries. More orten, a semi- quantitative profile is adequate. We propose that proton nmr spectra of Neem-products can serve as an indispensable tool in understanding the chemical profile of a product. We have catalogued the PMR spectral data of all known limonoids, identified a set of diagnostic chemical shifts for each and every key molecule and carried out a correlation analysis with products whose chemical profiles are known. The technique, we call PCCOR has been tested with commercial Neem pesticide samples of some reputed companies. It will be immensely useful in routine quality control and shelf life evaluations.

Practice Oriented Results on Use and Production of Neem Ingredients and Pheromones VIII 81 H. Kleeberg & C. P. W. Zebitz (eds.) Copyright 2000 by Druck & Graphic, Giessen 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 EFFECTIVENESS OF SOME EXTRACTS OF NATURAL PRODUCTS IN CONTROLLING PATHOGENIC SOIL-BORNE FUNGI

1 2 3 DR. P. CHINTEA , A. BULIGA, MARIANA MIHĂILĂ , DR. MARIA OPREA

1Institute of Genetics, Kishinev, Moldavia

2BIOTEHNOS, Bucharest, Romania

3Research Institute for Plant, Protection, Bucharest, Romania ABSTRACT

Use of natural extraction products aiming to diminishing antifungal synthetic consumption is an ecological problem of agriculture.

The natural products Moldstim and Ecostim, applied to tomato seed as an imbibition solution induced more rapid development of seedlings, thus reducing the critical period of susceptibility to attack by fungal soil – borne pathogens.

In presence of inoculum plant emergence was significantly influenced in case of pathogenic action of the fungi: Pythium debaryanum, Fusarium oxysporum f. sp. lycopersici, Sclerotinia sclerotiorum, and Verticillium dahliae.

Increased growth and development of plantlets have been recorded in the variants treated, marked by distinctly significant gains in biomass accumulation.

Cucumber seed treated with Moldstim, Ecostim and Pavstim sown in presence of soil – borne pathogenic fungi resulted in healthy plantlets with biomass accumulation and development rhythm of vegetative organs superior to untreated check, while as biochemical effect increased concentrations of glucids have been detected. INTRODUCTION

Steady use of pesticides and ignorance of their side – effects had negative outcomes on environment ( Vendrig, 1964 ). Having in view progress of scientific research and evermore severe requirements for people health, at present selective methods from ecological standpoints are developing, in order to limit contacts between the basic components of agrocenoses and the pest control materials ( Balasova, 1984, 1990; Volanet,1992 ). The use of natural extraction products to limit excessive applications of antifungal synthetic compounds constitutes an optimal solution for the aforementioned issues, being a research subject of Institute of Genetics – Kishinev and Institute of Biotechnology ( BIOTEHNOS ).

Practice Oriented Results on Use and Production of Neem Ingredients and Pheromones VIII 99 H. Kleeberg & C. P. W. Zebitz (eds.) Copyright 2000 by Druck & Graphic, Giessen MATERIALS AND METHODS

The growth bioregulators Moldstim, Ecostim and Pavstim, extracted at pilot – laboratory of Institute of Genetics – Kishinev, scale from Capsicum annuum seeds devoid of biological value, Lycopersicum esculentum leaves and Solanum melongena roots have been trialled in greenhouse. These products were characterized by their dry matter and saponin contents, being framed in the class of steroid saponations. The test fungi used in experiments with tomato and cucumber behavior were telluric pathogen species: Pythium debaryanum, Fusarium oxysporum f. sp. lycopersici, Sclerotinia sclerotiorum, and Verticillium dahliae. Tomato and cucumber seed has been treated with one of the growth bioregulators: Moldstim, Ecostim and Pavstim at concentrations: 0.01%, 0.05%, 0.08%, by 2 hrs immersion before sowing. Seeds were sown in pots containing sterile soil in which the inoculum has been concomitantly applied. A check was used, where seeds had been treated with biostimulators but fungal infection was present in each variant, and another check with seed not treated with stimulators and without fungi in soil. Observations started at emergence of the first seedlings, recording the emergence percentage and evolution of plantlets and of mature. The results have been statistically assessed. RESULTS

The bioregulators Moldstim and Ecostim have been tested in a glasshouse during 1994 – 1997, having to study the behavior of tomato seedlings to pathogenic action of soil borne fungi: Pythium debaryanum, Fusarium oxysporum f.sp. lycopersici, Sclerotinia sclerotiorum and Verticillium dahliae. Tomato seeds have been kept for 24 hrs in bioregulator solutions 0.01%, 0.05%, 0.08%. Significant results have been obtained with 0,08% solution, which will be discussed here. From table 1 it results that Moldstim at 0.08% applied to tomato seeds prior to sowing resulted in an emergence percentage higher than the untreated but infected check, and also than the untreated and not infected check. Thus, when Pythium debaryanum was present, emergence reached 90%, compared to untreated check with 54%; in presence of Sclerotinia sclerotiorum the emergence percentage was 95.8%, by 10.8% higher than in the infected and untreated check; for Fusarium oxysporum f. sp. lycopersici emergence was clearly superior in treated seeds over the infected but not treated check, by 24.6%; the same effect was noticed with Verticillium dahliae seed infection, where emergence percentage was by 24.4% higher in the treated variant. Thus, the treatment with Moldstim 0.08% positively influenced growth of plantlets, these being definitely protected from diseases induced by soil borne pathogenic fungi. When two true leaves were formed biometrical observations showed that the height of plants from treated seed exceeded by 7.9 – 9.95 cm that of the check. In the same phenophase average plant weights in treated seeds were by 19.2 – 33.8 g higher than those of plants derived from untreated seed and developed in infected soil. In variants infected by Sclerotinia sclerotiorum and Verticillium dahliae, fungi able to also infect mature plants, no disease symptoms were noticed in surviving plants from seed treated with Moldstim 0.08%. 100 Tomato seed treated with Ecostim 0.08% before sowing gave satisfactory results by obtaining healthy transplants (tables 1 and 2). Higher emergence was obtained with treated seed, in presence of a soil borne pathogen. Thus, the percentage of emerged plants from seed treated was by 28.4% higher in a soil infected by Pythium debaryanum than in the untreated and contaminated check. In variants infected by Fusarium oxysporum f.sp. lycopersici, Sclerotinia sclerotiorum and Verticillium dahliae plant emergence percentages were also higher in the treated variants. In the two true leaves phase plant heights in treated variants were by 4.11 – 23.46 cm higher than in the check, while their weight overpassed it by 13.48 – 23.6 g (table 3 ).

101 Table 1

INFLUENCE OF SOME BIOREGULATORS ON TOMATO EMERGENCE

UNDER THE ACTION OF SOME SOIL – BORNE PATHOGENIC FUNGI

Pythium debaryanum Fusarium oxysporum Sclerotinia sclerotiorum Verticillium dahliae Varia Conc Emer- Mt1 Mt2 Emer- Mt1 Mt2 Emer- Mt1 Mt2 Emer- Mt1 Mt2 (%) gence gence gence gence nt D S D S D S D S D S D S ( % ) D S D S ( % ) ( % ) ( % )

90.0 36 xxx 14 x 84.4 24.6 xxx 20.4 xxx 95.8 10.8 xxx 27.8 xxx 95.6 22.4 - 9.59 - Moldstim 0.08 Ecostim 0.08 82.4 28.4 xxx 6.4 - 70.0 10.2 xxx 6 xxx 87.0 2.0 - 19.0 xxx 87.0 13.8 - 1.0 -

Mt1 54.0 0 - -22.0 oo 59.8 0 - -4.2 ooo 85.0 0 - 17.0 xxx 73.2 0 - -12.8 -

Mt2 76.0 22 xx 0 - 64.0 4.2 xxx 0 - 68.0 -17 ooo 0 - 86.0 12.8 - 0 -

CL 0.1% = 25.63525 CL 0.1 = 4.019453 CL 0.1% = 9.476412 CL 0.1% = 94.7577

CL 1% = 18.13703 CL 1% = 2.84377 CL 1% = 6.704595 CL 1% = 67.04141 CL 5% = 12.93636 CL 5% = 2.028344 CL 5% = 4.782099 CL 5% = 47.81775

Mt1 - check without bioregulator and infected by fungus D – difference

Mt2 - check without bioregulator and not infected by fungus S – signification

Table 2

INFLUENCE OF SOME BIOREGULATORS ON TOMATO PLANTS

GROWTH UNDER THE ACTION OF SOME SOIL – BORNE PATHOGENIC FUNGI

Pythium debaryanum Fusarium oxysporum Sclerotinia sclerotiorum Verticillium dahliae Conc Variant Mt Mt Mt Mt Mt Mt Mt Mt ( % ) Height 1 2 Height 1 2 Height 1 2 Height 1 2 ( cm ) D S D S ( cm ) D S D S ( cm ) D S D S ( cm ) D S D S Moldstim 0.08 17.8 9.35 xxx 5.54 xxx 20.54 7.5 xxx 7.56 xxx 17.8 7.28 xxx 4.47 xxx 16.28 7.81 xxx 3.03 xxx Ecostim 0.08 27.18 18.7 xxx 14.8 xxx 23.46 10.4 xxx 10.5 xxx 21.32 10.8 xxx 7.98 xxx 12.58 4.11 xxx -0.65 o

Mt1 - 8.48 0 - -3.82 oo* 13.04 0 - 6.00 - 10.54 0 - -2.8 ooo 8.46 0 - -4.77 ooo Mt2 - 12.3 3.82 xx 0 - 12.98 -6.0 - 0 - 13.34 2.80 xxx 0 - 13.24 4.77 xxx 0 -

CL 0.1% = 4.245936 CL 0.1% = 0.99314 CL 0.1% = 1.886625 CL 0.1% = 0.9420764 CL 1% = 3.004015 CL 1% = 0.7065 CL 1% = 1.334794 CL 1% = 0.6665223 CL 5% = 2.142634 CL 5% = 0.50117 CL 5% = 0.9520508 CL 5% = 0.4756017

Mt1 - check without bioregulator and infected by fungus D – difference Mt2 - check without bioregulator and not infected by fungus S – signification

Table3

INFLUENCE OF SOME BIOREGULATORS ON TOMATO VEGETATIVE MASS

UNDER THE ACTION OF SOME SOIL – BORNE PATHOGENIC FUNGI

Pythium debaryanum Fusarium oxysporum Sclerotinia sclerotiorum Verticillium dahliae Conc Variant Average Mt1 Mt2 Average Mt1 Mt2 Average Mt1 Mt2 Average Mt1 Mt2 (%) weight weight weight weight ( g ) D S D S ( g ) D S D S ( g ) D S D S ( g ) D S D S

Moldsti 0.08 91.9 23.3 xxx 17.5 xxx 84.0 19.2 - 9 - 95.6 32 xxx 28.6 xxx 94.8 33.8 xxx 19.8 xxx m Ecostim 0.08 84.08 13.4 xxx 8.69 xxx 84.4 19.6 - 9.4 - 86 22.4 xxx 19.0 xxx 84.6 23.6 xxx 9.6 xxx

Mt1 - 70.6 O - -3.80 - 64.8 0 - -10.2 - 63.3 0 - -3.42 - 61.0 0 - -14 ooo Mt2 - 74.4 3.80 - 0 - 75.0 10.2 - 0 - 67.0 3.4 - 0 - 75 1 xxx 0 -

CL 0.1% = 7.564515 CL 0.1% = 76.74832 CL 0.1% = 9.273452 CL 0.1% = 4.424844 CL 1% = 5.351921 CL 1% = 54.30077 CL 1% = 6.561 CL 1% = 3.132917 CL 5% = 3.817295 CL 5% = 38.7304 CL 5% = 4.679679 CL 5% = 2.232917

Mt1 - check without bioregulator and infected by fungus D –-difference

Mt2 - check without bioregulator and not infected by fungus S – signification

Seedling showed resistance during their development and after this phase. Ungerminated seeds extracted from soil were in the phase of germinated – dry rootlet, due to occurrence of soil – borne pathogenic fungi in each variant. Table 4 exhibits the percentages of germinated and dead seeds in each variant. The products Pavstim at 0.01% was applied to cucumber seeds 24 hrs before sowing. In greenhouse, seeds were sown in pots with soil infected by one of the pathogenic fungi: Pythium debaryanum, Fusarium oxysporum f. sp. lycopersici, Sclerotinia sclerotiorum and Verticillium dahliae. Under these conditions seed treated with the bioproduct better crossed the seedling phenophase, positively influencing emergence. Plants developed well, and biometrical measurements demonstrated their vigor. Differences between the untreated and infected check and the variants treated with Pavstim 0.01%, regarding seed germination and emergence were significant ( table 5 ), differences ranging from 4 to 9%. But the evolution of treated variants was superior to that of untreated check. At the stage of 2 true leaves, these plants had the root – neck diameter by 0.063 – 0.103 cm higher than the untreated check and foliar surfaces were larger by 5.2 – 7.7 cm2. Root masses exceeded by 16 – 32 mg the mass of the untreated check. During the development of plants in the same infected soil possibility existed for telluric fungi to induce infections. The fungi Fusarium oxysporum f. sp. lycopersici and Verticillium dahliae induce tracheomycoses also in mature plants; however this did not happen, the plants remained healthy. Likewise, Sclerotinia sclerotiorum is a potential pathogen during plant development, nevertheless no disease symptom was noticed. Ungerminated seeds, or germinated and dead before emergence have been examined in the laboratory. Germinated seeds in the phase of rootlet were populated by one of the pathogenic fungi occurring in soil, and the rootlets became brown as a result of the attack. The fungus Pythium debaryanum induced severe attack, the percentage of emerged plants in the untreated check and not infected was rather close to variant with seeds treated by Pavstim 0.01%. In the presence of the fungus Fusarium oxysporum f. sp. lycopersici, the variants protected with Pavstim 0.01% exhibited emergence percentages slightly higher than the check variant ( untreated and infected ). The same effect was also remarked with infections by Sclerotinia sclerotiorum and Verticillium dahliae, where the attack on seedlings and germinated seeds was obvious, as reflected by the low emergence percentages.

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Table 4

PERCENTAGE OF SEEDS ATTACKED BY SOIL – BORNE PATHOGENIC FUNGI

Soil – borne fungi Products Pythium Fusarium Sclerotinia Verticillium debaryanum oxysporum sclerotiorum dahliae Moldstim 0,08% 10.0 15.6 4.2 4.8

Ecostim 0,08% 12.6 30.0 13.0 13.0

Mt1 46.0 40.2 15.0 24.8

Mt2 24.0 36.0 32.0 14.0

Mt1 - Untreated infected check Mt2 - Untreated not infected check

Table 5

INFLUENCE OF PAVSTIM 0.01% APPLIED TO CUCUMBER SEED

ON PLANT DEVELOPMENT UNDER THE PATHOGENIC ACTION OF SOME SOIL – BORNE FUNGI

Root – neck Emergence Stem length Foliar surface Radicular mass Foliar mass Variant diameter ( % ) ( cm ) ( cm2 ) ( mg ) ( mg ) ( cm ) Pythium 44 0.356 5.6 12.6 121 34 debaryanum Fusarium 48 0330 6.8 12.8 75 34 oxysporum Sclerotinia 37 0.366 7.2 13.1 53 21 sclerotiorum Verticillium 70 0.303 6.1 5.0 46 8 dahliae Mt 1 36 0.266 5.0 5.2 45 22 +P. debaryanum Mt 1 44 0.233 5.8 5.2 10 27 +F. oxysporum Mt 1 28 0.250 6.3 4.8 26 8 +S. sclerotiorum Mt 1 68 0.253 5.2 2.7 20 4 +V. dahliae Mt 2 72 0.233 5.0 7.7 15 3

Mt1 - check untreated and infected Mt2 - check untreated and not infected

CONCLUSIONS

1. The biostimulators Moldstim and Ecostim applied to tomato seeds at conc. of 0.08% provided resistance to seedlings, enabling these to cross more rapidly the phenophase which is susceptible to attack by soil borne pathogenic fungi: Pythium debaryanum, Fusarium oxysporum f. sp. lycopersici, Sclerotinia sclerotiorum and Verticillium dahliae. 2. The bioregulators Moldstim and Ecostim at 0.08% applied to tomato seeds determined plant development, ensuring superior vegetative mass over that resulted from untreated seeds, thus further providing plant resistance to possible diseases equally caused by soil pathogens ( Sclerotinia sclerotiorum Verticillium dahliae and Fusarium oxysporum f. sp. lycopersici ). 3. The bioregulator Pavstim applied to cucumber could provide protection to plantlets from the pathogenic action of soil – borne fungi: Pythium debaryanum, Fusarium oxysporum f. sp. lycopersici, Sclerotinia sclerotiorum and Verticillium dahliae, securing vigorous plant development and resistance to potential soil – borne pathogens. REFERENCES

Balasova I. T., Chintea P. C., Lazurevschii G. V., Timina O. O., Rasciuc V. S., Mascenco N. E., Bobeica V. A., Demidov E. S., 1984. Vtoricnae metabolica rastenii ( steroidnae glicozida ) cac factora fitoimuniteta. V. Chinghe “Ghenetica i selectia s-h rastenii na usticivosti v Moldavii”, Kishinev, Stiinta, 34 - 53 Balasova I. T.,1990. Steroidal glycosides as plant resistance inductors. Acta Agronomica Hungarica, 39 ( 1 – 2 ): 183 –191. Vendrig I. C., 1964. Growth–regulating activity of some saponins nature.N4951:1301- 1302 Volanet A. P., Karosa S. E., Chintea P. C., Lupescu G. A., 1992. Activitatea auxinica a glicosidelor steroidale. Ref. acad. St. Bielorusia, vol. 36, 3 – 4.

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EINSATZ VON PHEROMON-FALLEN FÜR DIE ERBSENWICKLERÜBERWACHUNG 1996 - 1997

KLOSE E.

I.G. Saatzucht GmbH, Biendorf 1. Einleitung

Durch den schon seit vielen Jahren relativ hohen Anteil an Erbsenflächen in Sachsen- Anhalt hat sich der Befall der Bestände mit Erbsenwickler als Problem bei der Erzeugung von hohen Stufen in der Saatgutwirtschaft erwiesen. Die Wickler (Cydia nigricana, Lep., Tortricidae) fressen an den Samen, und das auf Kosten der Keimfähigkeit und des Ertrages. Um den Flug der Falter zu überwachen und rechtzeitig Bekämpfungsmaßnahmen einleiten zu können, hat sich der Einsatz von Pheromon-Fallen als gutes Hilfsmittel erwiesen. 2. Material und Methoden

In den Jahren 1996 und 1997 haben wir unsere gesamten Erbsenbestände mit Tripheron®-Pheromonfallen der Firma Trifolio-M überwacht. Im Jahre 1996 setzten wir 10 Fallen und im Jahre 1997 14 Fallen zur Überwachung ein. Die Fallen werden kurz vor Blühbeginn auf gut erreichbaren Stellen im Bestand angebracht. Die Kontrolle der Fallen erfolgte in einem Abstand von 2 - 8 Tagen, entsprechend der Flugintensität. 3. Ergebnisse

Die Überwachung des Erbsenwicklerfluges der Jahre 1996 und 1997 haben gezeigt, daß sich der Falterflug über mehrere Wochen im Jahr erstreckt. Die Ergebnisse sind in dem Diagramm dargestellt. Dabei ist die Zahl der gefangenen Falter sehr differenziert. Um die günstigsten Zeitpunkte für den Einsatz der Insektizide zu bestimmen, kann die Zahl der Falter in einer bestimmten Zeitspanne herangezogen werden. Mit Hilfe der Pheromonfallen kann so der Zeitpunkt der Behandlung gut festgelegt werden. Dabei sind auch die Witterung und somit die Entwicklung der Larven zu berücksichtigen. Bei warmem Wetter entwickeln sich die Larven schneller und es muß nach ca. 7 Tagen ab Flughöhepunkt gespritzt werden. Bei kühlem Wetter beträgt die Wartezeit etwa 10 Tage. Eine mehrmalige Spritzung ist bei starkem Flug unbedingt nötig. 4. Auswertung

Die Fangergebnisse wurden jeweils in einer Tabelle festgehalten. Dabei war zu beobachten, daß bei warmer bis heißer Witterung der Flug der Falter sehr intensiv war, während bei kühler Witterung nur geringe Fangergebnisse zu verzeichnen waren. Entsprechend der Flughöhpunkte wurden die nächstmöglichen Spritztermine festgelegt. Gespritzt wurde in den frühen Morgen-, bzw. späten Abendstunden. Dadurch wurden sowohl die bereits geschlüpften Larven, als auch die Falter bekämpft, welche zu diesen Zeiten ihre Flughöhepunkte hatten.

Practice Oriented Results on Use and Production of Neem Ingredients and Pheromones VIII 119 H. Kleeberg & C. P. W. Zebitz (eds.) Copyright 2000 by Druck & Graphic, Giessen

5. Zusammenfassung

Mit Hilfe der Fangergebnisse von Pheromon-Fallen ist es möglich, günstige Spritztermine zur Bekämpfung des Erbsenwicklers (Cydia nigricana) festzustellen. Dabei ist jedoch auch die Entwicklung der Larven zu beachten. Je nach Witterung wird 7 - 10 Tage nach dem Flughöhepunkt z.B. mit Pyrethroiden gespritzt.

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Phyllonorycter-Pheromone (Lep., Gracillariidae) in Pyracantha coccinea-Kultur - ein Versuch zum Aktivitätsnachweis, zur indirekten Artbestimmung und zur Bekämpfung von Miniermotten an Feuerdorn PHYLLONORYCTER-PHEROMONES IN PYRACANTHA- COCCINEA-CULTURE - TESTING MONITORING, INDIRECT DETERMINATION AND CONTROL OF LEAFMINERS ON PYRACANTH

MANFRED LEHMANN

Landesamt für Ernährung, Landwirtschaft und Flurneuordnung – Pflanzenschutzdienst, Ringstraße 1010, 15236 Frankfurt (Oder) Einleitung

In der Baumschule eines Gartenbaubetriebes in Südbrandenburg trat im Winter 1996/97 "plötzlich und unerwartet" ein Blattminierschaden an getopftem Feuerdorn (Pyracantha coccinea) im ungeheizten Gewächshaus auf. Als Verursacher wurde eine Miniermotte der Gattung Phyllonorycter festgestellt, ohne jedoch die Art bestimmen zu können. Der Befall an dieser Wirtspflanze war bis 1995 im Betrieb unbekannt, wurde anscheinend im Herbst 1995 durch Pflanzenzukäufe aus Norddeutschland eingetragen und vermehrte sich seitdem im Gewächshaus stark und im Freiland auf Beeten sowie an einer Pyracantha-Hecke im Betriebsgelände nachweisbar. Weitere vereinzelte Nachweise gelangen im Spätsommer 1997 in Pyracantha-Beständen in Grünanlagen eines anderen Betriebes in ungefähr 500m Entfernung vom erwähnten Baumschulbetrieb. Im ersten Halbjahr 1996 kam es an getopfter Ware erstmalig zum auffälligen Minierschaden, der durch eine Insektizidbehandlung eingeschränkt wurde. Erst die Folgegeneration erreichte im Herbst 1996 eine Befallsstärke, die an allen ca. 5 800 Pflanzen 100 %igen Befall des Laubes (je Blatt eine oberseits erkennbare Faltenmine oder das Blatt bereits über der Mittelrippe zusammengefaltet) und an weiteren 5 000 Jungpflanzen 5...10 %igen Befall der Blätter umfaßte. Dieser starke Befall wurde zu spät erkannt und im Laufe der Wintermonate mit mehreren teilwirksamen Insektizidbehandlungen zurückgedrängt. Eine Parasitierung des eingetragenen Larven- und Puppenmaterials war nicht feststellbar, obwohl MEY (1991) und BATHON (1984) zumindest im Freiland eine sehr starke generelle Parasitierung bei allen Phyllonorycter-Arten beschreiben. Die Artzugehörigkeit der Miniermotten und ihr weiterer Entwicklungsverlauf sowie Termine für Folgemaßnahmen zur Bekämpfung waren unklar. Alle drei Fragen sollten mit dem Einsatz von Pheromonen von Ph. blancardella Fabricius und Ph. corylifoliella Hübner aus der Produktpalette von Trifolio-M GmbH geklärt werden, in der Hoffnung, damit auch Hinweise zur Artzugehörigkeit erhalten zu können. Aktivitätsüberwachung

Am 2. Mai wurde im Bestand im Gewächshaus erster starker Flug der Falter festgestellt. Es wurden sofort die Fallen (je Art eine Delta-Falle mit Leimeinlage und Practice Oriented Results on Use and Production of Neem Ingredients and Pheromones VIII 122 H. Kleeberg & C. P. W. Zebitz (eds.) Copyright 2000 by Druck & Graphic, Giessen

Gummistopfen-Dispenser) in Höhe der Pflanzenspitzen plaziert. Beide Pheromone waren schon am ersten Einsatztage fängig und repräsentierten sehr gut den Anflug von im Bestand vorhandenen Faltenminiermottenarten. Die Fallen wurden im Mai durch den Pflanzenschutzdienst wöchentlich kontrolliert. Danach setzte der betroffene Betrieb die Kontrollen selbständig fort und die Ergebnisse wurden nur noch fallweise registriert. Der plötzliche Abbruch des starken Fluges (Tabelle) wurde durch eine gezielte Behandlung mit Tamaron am 5. Mai erreicht. Die Wirkung dieser Applikation war so gründlich, daß bis zum 27. Mai im Bestand des Gewächshauses keine lebenden Larven, Puppen und Falter mehr auftraten, alle Mineninhalte (Larven, Puppen, bereits geschlüpfte Falter) abgetötet waren und die Pheromonfänge am Pheromon der Ph. corylifoliella auf nahezu 0 zurückgingen. Eine für den 9. Mai vorgesehene Wiederholungsbehandlung wurde aufgrund des Fangergebnisses abgesetzt. Es lag die Vermutung nahe, daß die Pheromone von Ph. corylifoliella und Ph. blancardella auch diese Arten fingen und damit die Artzugehörigkeit geklärt wäre. Eine Überprüfung des Materials (gefangene Falter in der Falle Ph. corylifoliella, Handfänge vereinzelt im Bestand fliegender Falter) durch Manfred Gerstberger, Berlin, brachte jedoch am Pheromon der Ph. corylifoliella eine dritte Art: Phyllonorycter leucographella Zeller. Die Fänge der Falle Ph. blancardella und vereinzelte Handfänge der Falter dieser Art konnten als vermutlicher Zuflug aus dem Freiland eingeordnet werden. Phyllonorycter corylifoliella wurde nicht gefangen. Die ursprünglich mediterrane Art Phyllonorycter leucographella, die Feuerdornminiermotte, ist als Besiedler und Schädling an Pyracantha coccinea in urbanen Grünanlagen bekannt, dehnt seit Anfang der 80er Jahre ihr Areal in Mitteleuropa aus (MEY, 1991; BATHON, 1984) und hatte Mitte der 80er den Harz und den Raum Berlin erreicht. Die Art wurde im vorliegenden Fall auch, wie bisher allgemein beobachtet, anscheinend mit Pflanzenmaterial eingetragen (BATHON, 1984). Das Fraßverhalten der Raupen - Fortsetzung des Fraßes am festhaftenden Laub während des gesamten Winters ohne Diapause - bestätigte den ausschließlichen Befall mit der Art Ph. leucographella. Die Fänge und Begleitfänge von Ph. blancardella repräsentieren eine aus dem Freiland von Malus-Kulturen zufliegende Begleitart. Diese Art ist in der Pyracantha-Kultur als Schaderreger nicht nachweisbar, obwohl der Flug mit Hilfe der entsprechenden Pheromonfalle zu den bekannten Aktivitätszeiten festgestellt wurde. Der starke Anflug an das Pheromon von Ph. corylifoliella läßt sich aus der sehr engen Verwandschaft beider Arten erklären, die an nahe verwandten Wirtspflanzenarten minieren und von denselben Parasitoiden besiedelt werden (GERSTBERGER, 1997 unveröffentlicht; MEY, 1991). Derartige artübergreifende Pheromonfänge sind nach eigenen Erfahrungen besonders bei Mottenarten häufig zu beobachten.

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Tabelle: Pheromonfangergebnisse und Bekämpfungsmaßnahmen Datum Ereignis Minen je Pflanze Phyll. blancardella Phyll. corylifoliella 23.3. 97 Blattminierung 100 %

2.5. 97 Erstauftreten der Falter, x ? Aufhängen der Fallen x ? 5.5. 97 Tamaron-Einsatz

6.5. 97 Falterfang 12 236 * 9.5. 97 Falterfang 2 8 * 16.5. 97 Falterfang 4 4 * 23.5. 97 Falterfang 2 1 * 17.6. 97 Falterfang (zahlreich)** 19.6. 97 Unden flüssig-Einsatz

2.7. 97 Unden flüssig-Einsatz

5.7. 97 Blattminierung / Falterfang/Klebeflächen- und 4,1 17 Pheromondispenserwechsel 0 * 17.7. 97 Falterfang 0 0 * 29.7. 97 Falterfang 104 3 * 8.9. 97 Blattminierung / Falterfang 0 ca. 300 0 *

Anmerkung: * wurde determiniert als Phyllonorycter leucographella ** nach Angaben des Betriebes, Fanghöhe wurde nicht aufgezeichnet

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Bekämpfung

Während in den Wintermonaten bei relativ frostfreiem Wetter durchgeführte mehrmalige Behandlungen mit Phosphorsäureester-Präparaten nur unbefriedigende Wirksamkeit zeigten, brachte jeweils eine gezielte Behandlung mit Tamaron bzw. Unden flüssig zum Zeitpunkt des Massenfluges der Falter, beginnender Eiablage und erster vereinzelter Gangminen mit lebendem Inhalt eine praktisch 100-prozentige Wirksamkeit. Die zur Junibehandlung im Abstand von einer Woche vorgenommene Wiederholung der Unden-Applikation war überflüssig. Eine am 5.7. durchgeführte Bonitur des frischen Minenbesatzes ergab an einjährigen Jungpflanzen nach allgemein gutem Neuaustrieb eine durchschnittliche Anzahl von 4,1 Minen je Pflanze (Streuung von 1 bis 8). Zu 90 % waren es Platz- bzw. Gangminen und zu 10 % bereits Faltenminen. Die Inhalte aller Minen war jedoch tot. Zum selben Zeitpunkt konnten an einer nicht behandelten Pyracantha-Hecke im Freiland vereinzelt Gangminen (< 1 % der Blätter) mit lebendem Inhalt und auch schwärmende Falter ermittelt werden. Auch alle anderen Gehölze im Gewächshaus (vorrangig Prunus-Arten, Cotoneaster- Arten, Evonymus spec., Berberis thunbergii, Spiraea spec., Cytisus spec.) waren frei von Miniermottenbefall. Dieser Zustand hielt, nun einschließlich der Pyracantha- Pflanzen, an bis Ende September des Untersuchungsjahres. Ein Ausweichen der am Feuerdorn minierenden Mottenart auf einen anderen Wirt oder eine Beteiligung einer anderen schädlich wirkenden Miniermottenart am Kulturpflanzenbefall in diesem Kulturraum ist damit für das Untersuchungsjahr nicht nachweisbar. Die im Freiland lebenden Populationsteile der Ph. leucographella können als Bedrohung der Pyracantha-Kultur im Gewächshaus weitgehend vernachlässigt werden, jedoch jeder Zuflug in den geschützten Anbau und jeder Eintrag von Entwicklungsstadien mit dem Pflanzenmaterial führen zum Wiederaufbau eines Schadauftretens und müssen erfaßt werden. Konsequenzen

- Die seit fast 20 Jahren in Mitteleuropa in Ausbreitung befindliche Faltenminiermottenart Phyllonorycter leucographella hat spätestens 1995/96 nachweislich Südbrandenburg erreicht. - Sie kann, solange ihr Pheromon nicht bekannt und für den Einsatz verfügbar ist, in Baumschulen mit dem Pheromon der Apfelblattmotte Phyllonorycter corylifoliella überwacht werden. Eine Aktivitätserfassung ist erforderlich, sobald erste Minierschäden das Vorhandensein der Miniermotte anzeigen. Sie sollten von Ende März bis Mitte/Ende September durchgeführt werden. - Die Reaktion im Pheromonfang in der ersten Generation und in der anscheinend aus dem Freiland zugeflogenen Generation 1 B ist so eindeutig, daß daraus ein exakter Bekämpfungstermin eines zu erwartenden Schadauftretens abgeleitet werden kann: innerhalb von zwei bis drei Tagen nach deutlicher, starker Flugaktivität Einsatz eines Präparates mit Kontakt- und/oder System- bzw. Translaminarwirkung. Der kritische Aktivitätswert könnte mit 10 Tieren pro Falle und Tag vorläufig angenommen werden. - Es muß mit bis zu drei Flughöhepunkten, also mit bis zu drei gezielten Behandlungen gerechnet werden. Die Faltergeneration 1 A stammt von den im Kulturraum überwinterten Individuen und fliegt im April bis in den Mai hinein. Die Faltergeneration 1 B ist freilandbürtig, dringt in den Kulturraum ein und fliegt bis in den Juni hinein. Eine

125 zweite Freilandgeneration ist im August bis September zu erwarten (BATHON), die ebenfalls in den geschützten Anbau eindringt, aber auch mit "Glashaus-Tieren" untermischt sein kann. - Das Fehlen von Parasitoiden im vorliegenden Fall im Gewächshaus zeigt die vollständige Eliminierung von Nützlingen durch nachweislich mehrmalige Applikation breitwirksamer, nicht Nützlinge schonender Insektizide an (z.B. Tamaron, Unden flüssig). Zur Unterstützung und Reaktivierung der Parasitoidenfauna sollte nach einer "Radikalkur" zu selektiv wirkenden Produkten übergegangen werden. Nebeneffekt wäre eine weitgehende Schonung der Arbeitskräfte, die in Baumschulbetrieben während und nach der Applikation durch die Bestände gehen müssen und bei Pflegearbeiten Hautkontakt mit kontaminierten Pflanzen nicht vermeiden können. Wenn nicht zwingende Gründe andere Präparate erforderlich machen, gebührte den "Häutungshemmern" Dimilin, Insegar, Alsystin oder NeemAzal der Vorrang. - Eine intensive Aktivitätsüberwachung und Bekämpfung in Freilandbeständen der Wirtspflanze Feuerdorn wird nicht für erforderlich gehalten. Befallenes Material kann in Baumschulen befallsfrei gemacht werden. Symptombehaftete Baumschulware ohne lebenden Inhalt kann nach Aussagen von Kunden des betroffenen Betriebes ohne Bedenken im Freiland gepflanzt werden. - Ein Eintrag bzw. Wiedereintrag in Bestände im geschützten Anbau ist jederzeit möglich und sollte zu den obengenannten Konsequenzen führen. Zusammenfassung

Die in Deutschland in Expansion befindliche Feuerdornminiermotte hat nachweislich Baumschulbestände in Südbrandenburg befallen und sich auch im Freiland verbreitet. Zur Erfassung des Aktivitätsverlaufs und versuchsweisen Artbestimmung wurden verfügbare Miniermottenpheromone verwandter Arten eingesetzt. Eine gezielte chemische Bekämpfung gelang nach Ermittlung des Aktivitätszeitpunktes unter Verwendung eines Pheromons von Phyllonorycter corylifoliella. Hinweise zur weiteren Überwachung und Bekämpfung der Art werden gegeben. Summary

The pyracanth-leafminer, expanding in Germany, has detectably occupied tree nurseries in the south of the Land Brandenburg and has dispersed in outdoor plant populations. Availalable pheromones of leafminers have been used for monitoring and experiments on determination. A specific control succeeded after finding out the time of activity with the help of the pheromone of Phyllonorycter corylifoliella. Informations on further monitoring and control are given. Danksagung: Herrn Manfred Gerstberger, Berlin, danke ich für die Durchsicht des Faltermaterials und die Überlassung von Literaturhinweisen und zusammenstellten Informationen.

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Literatur:

BATHON, H. (1984): Die Feuerdornminiermotte, Phyllonorycter leucographella Zeller, ein für Deutschland neuer Kleinschmetterling (Lep.: Gracilariidae). Entomologische Zeitschrift 94, 151-157. ECKSTEIN, K. (1933): Die Schmetterlinge Deutschlands, 5. Band: Die Kleinschmetterlinge Deutschlands. Stuttgart. MEY, W. (1991): Über die Bedeutung autochthoner Parasitoidenkomplexe bei der rezenten Arealexpansion von vier Phyllonorycter-Arten in Europa (Insecta, Lepidoptera, Hymenoptera).- Mitt. Zool. Mus. Berlin 67, 12ff.

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THE USE OF PHEROMONES AND PHEROMONE TRAPS IN FOREST PROTECTION IN POLAND IN THE YEARS 1980-1997

JACEK STANISLAV STOCKI

Dyrekeja Generalna, Lasow Panstwowyc, Wawelska 52/54, Warszawa 00-922, Poland There is no other country in Europe, where the negative effect of insects outbreaks are so intense and diverse as in Poland. This situation is the effect of the high susceptibility of Polish forests to insect pests and diseases, as well of the influence of numerous biotic and abiotic factors, including air pollution. The localisation of the country on the map of Europe is an additional and influential factor. Poland is situated in the contact zone between continental - east and oceanic - west climates; in effect, the dominating insects in Polish entomofauna are species characteristic for both types. More than 50 species of this fauna may cause periodical, or permanent threats to forests. Forests cover 27,8% of the territory of Poland or about 8,7 million hectares. The dominating species are conifers - Scots pine is occupying 70%, Norway spruce - 6,5%, Silver fir - 2,7%, and the others as Larch and Douglas fir - about 0,3% of the area of forests. Among the broad leaved tree species ( 20% in total) the main ones are oaks, beech, birch and alder. It is evident, that the threat of stand infestation caused by insect pests is closely related with the species composition and age structure of stands. The general problems with forest protection are therefore associated with the Scots pine stands growing on most of the territory of Poland, and with the Norway spruce which is dominating species in mountain regions. In both cases, the main predisposing factor is a single-species composition of stands, usually artificially planted on poor forest sites, with an incorrect age and spatial structure. This factor, accompanied with unfavourable weather conditions, reduces the primary resistance of stands and, thus, it affects its susceptibility to insect outbreaks. The overall area of forest in which control treatments against insect pests were made in the period of 1946-1997, was about 10 million hectares. In the last 50 years the total area of the stands infested by the defoliating insects has been continuously increasing (table 1). In 1946-1950 three following insect pests species: Lymantria monacha L., Dendrolimus pini L., and Acantholyda posticalis Mats. = A. nemoralis Thoms. were controlled on the area of 82 thousand hectares. In 1951-1960 six defoliating species (three mentioned above and: Panolis flammea Schiff., Bupalus piniarius L. and a few species of Diprionidae) were treated on 269 th.ha. In the 60´s, the area treated against these insects was about 600 th. ha, and in the 70´s it increased to 800 thousand ha. The greatest infestation - was recorded in 1980´s, when seven major insect pests (six mentioned above, and Neodiprion sertifer Geoffr.) were treated in Scots pine stands on the total area of 6.1 million ha.

Practice Oriented Results on Use and Production of Neem Ingredients and Pheromones VIII 128 H. Kleeberg & C. P. W. Zebitz (eds.) Copyright 2000 by Druck & Graphic, Giessen

Table 1. Chemical and biological control of Scots pine defoliators in 1946 – 1995 (hectares)

Year Dendrolimus Lymantria Panolis Bupalus Diprion pini monacha flammea Piniarius pini 1946 100 1947 500 1948 100 2000 1949 2536 1000 1950 3131 2345 1951 6195 7822 1952 115 7841 36 1953 220 1954 15 1955 60 1956 478 2144 2593 1320 230 1957 1684 2371 14641 1262 10 1958 1613 1959 6114 5189 1960 179 1961 8 408 153 1962 2656 46424 565 1963 742 14268 1964 13400 170 15 1270 1965 659 784 484 1760 1966 8021 19109 7350 190 1967 9503 24892 1900 1968 2759 676 85 1969 8488 866 1970 9790 1081 1971 9667 1827 2750 100 1220 1972 17842 1049 2065 2090 1973 1644 5085 1232 592 1974 1630 91112 100 1975 1844 420 1170 1976 1977 1978 190 20378 18617 3050 1979 179124 684 1980 509143 1981 1736362 1982 1670 2303189 1983 6500 1423791 1984 2707 138688 1985 1174 633 87530 1986 1559 3800 1987 301 12397 290 1988 3533 178637 45880 1186 1989 1261 7625 237 421 1990 90 1361 1991 350 2531 155096 1992 4198 62631 500 11856 113210 1993 46260 109321 3581 144381 1994 55000 736900 21800 37100 1995 27226 1731 515 172394 Total 232877 7420923 332399 75652 732827 129

After 1991 stand infestation by these pests continued to increase again. They were mainly Scots pine stands, to which chemical control against Lymantria monacha L. was applied 41 times on a total area of near 7,5 million ha between 1945 - 1997. In 1996 more then 25 insect species and multispecies groups of insect pests were treated on 185 th. ha, in 1997 - more than 30 on 90 th. ha. Studies on the use of semiochemicals (chemical informers, mainly pheromones) in the forest protection have been carried out in Poland in the Forest Protection Section of the Forest Research Institute since 1975. Several dozen of biologically active compounds - potential pheromones, kairomones and allomones of insects were tested in co-operation with "CHEMIPAN" - The Experiment Section of The Institute of Physical Chemistry of The Polish Academy of Sciences in order to check their attractiveness or repelling activity towards selected species of butterflies and beetles. The domestic production of the following pheromones and attractants started by CHEMIPAN on a commercial scale was the effect of those studies:

LYMODOR - for Lymantria monacha (Lymantriidae, Lep.) PANODOR - for Panolis flammea (Noctuidae, Lep.) RHYODOR - for Rhyacionia buoliana (Tortricidae, Lep.) COLODOR - for Coleophora laricella (Coleophoriidae, Lep.) TORTRODOR- for Tortrix viridana (Tortricidae, Lep.) IPSODOR - for Ips typographus (Ipidae, Col.) TRYPODOR - for Trypodendron lineatum (Ipidae, Col.) TOMODOR - for Tomicus piniperda (Ipidae, Gol.) HYLODOR - for Hylobius abietis ( Curculionidae, Col.)

The dispenser mentioned above are used in various traps for detection, forecasting and trapping of insects. Mostly, the following traps are used:

- IBL-I, funnel trap for capturing bigger butterflies in particular Lymantria monacha, Panolis flammea and others,

- IBL-2, triangle shaped trap for capturing flying beetles, particularly efficient for Ips typographus, Trypodendron lineatum, Pityogenes chalcographus and others,

- IBL-3, vertical multicone funnel trap for capturing flying beetles, in particular Tomicus piniperda, T.minor, Ips & Trypodendron,

- IBL-4, ground trap for capturing Curculionidae, especially Hylobius abietis and H. inastri

- Adhesive traps PL-1 "wing" and PL-2 "delta” types for capturing small butterflies as Rhyacionia buoliana, Zeiraphera griseana, Coleophora laricella, Tortrix viridana and other tortricids. These traps are made of polypropylene with three or one changeable sticky floors. Between 1984-1994 State Forest Districts and National Parks purchased 1.340.779 pieces of pheromones and 572.893 different types of traps. Numbers and kinds of traps used in State Forest Holding and National Parks are presented at Fig. 1.

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The most often used traps are: IBL-1 (130.049 pieces), IBL-3 (121.915) and PL-2 (11.088). The number of IBL-4 type traps have been increasing from year to year. For the years 1993 - 1995 State Administration Forest District were used about 43.881 pieces of traps, in 1996 – 335.598 pieces and in 1997, 396.848 pieces. Kinds and numbers of pheromones and attractants being used in Polish Forests are shown on Fig. 2. The following pheromones were mostly used together with traps: LYMODOR (520.395 pieces), IPSODOR (243.125), RHYODOR (188.330), TRYPODOR (176.010). Apart from pheromones and traps made in Poland, the foreign ones German, American, Romanian, Czech, Swedish, Russian and some others were tested with various effects. Already in the initial stage of their use, pheromones are of great concern not only to researchers, but also to forest administrative personnel as well.

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Between 1975-1977 the Forest Protection Department of the Forest Research Institute in Warsaw worked out an introductory method of the monitoring Rhyacionia buoliana by using sexual pheromone octan-trans-9-dodecenyl and glue traps of Zoecon Company. After installation of 50 traps on the area of 2,2 hectares bud and needles damage lowered from 60% in 1975 to 10% in 1977 year. The use of pheromones for forecasting and controlling of Lymantria monacha has also been studied since 1975. The traps of IBL-1 type containing the Lymodor pheromone has been used since 1984. In Poland and other countries a method evaluating the degree of stand threat by Lymantria monacha on the basis of the number of male individuals captured into pheromone traps has been put in practice. At forest service request the approximate numbers of captured butterflies indicating the degree of threat have been determined too. Since 1989-1993 high numbers of captured butterfly-males of Coleophora laricella were attained using glue pheromone traps of PL-I and PL-2 types hung up at the height of about 2 meters with the COLODOR pheromone. The results of the investigation show, that just of two pheromone traps should be used for predicting the occurrence of the C. laricella pest; they should be located at the distance of about 25 m. from each -other. Since 1977 a multi-objective research has been carried out on the use of pheromones and attractants for the control of bark beetles - especially Ips typographus and the curculionid Hylobius abietis. The greatest number of I. typographus beetles in a single trap amounted to 7000 individuals in 1980. Similar experiments were carried out with

132 pheromones of Trypodendron lineatum (TRYPODOR) and Pityogenes chalcographus. In the recent years much attention has been paid to the research on primary attraction in H. abietis and Tomicus piniperda. Out of five tested types of traps the most effective were traps of IBL-3. One artificial trap with TOMODOR makes it possible to catch about 300 to 1500 beetles within the swarming - flight period. Catches of more than 2000 beetles per single trap have already been recorded. Normally about 250-400 individuals of this species are captured in an average natural log trap of Scots pine trunk. Between 1991 and 1992, a new original trap for capturing Hylobius abietis - one of the most dangerous culture pests - was worked out from the original attractant called HYLODOR. This led to the introduction of a new, more effective and less time consuming method of large pine weevil control into practice. The proper location of the IBL-4 ground-trap with HYLODOR in the plantation results in the captures of several hundred beetles of this curculionid within one vegetation season. In all investigations, IBL-4 traps were capturing pests definitely better, than pine trap logs especially in deposits in one-year-old cultures with coniferous plants. TORTRODOR were tested in the years of 1991-1994 in broad leafed stands with oaks very heavy infested by tortricids. Both, PL-I and PL-2 traps with this pheromone are very effective in controlling butterflies-males of Tortrix viridana, Cacoecia xylosteana, Archips crataegana and Archips podana. Summarising, this is necessary to say, that efficient pheromone monitoring systems of tested species of forest insect pests must consider the following principles:

- the permanent observation of plots of biological monitoring network must - first of all - include the anticipated pest outbreak centres,

- in the monitoring period, the type of trap and chemical composition of synthetic pheromone should not be changed,

- every year, the pheromone traps should be mounted in the same location and position,

- in all observation plots the pheromone traps should be located prior to the mating season of the insects,

- in all observation plots the traps should be checked and at the same time the individual pest species identified,

- the data of the catches in individual pheromone traps should be supplemented with the information about weather conditions and standard characteristics. In practice, it means that pheromone traps used are not supplied with instruction containing reliable information about the range of their use and methods of interpretation of results obtained with their aid. It still requires many additional studies of varying behaviour of insects, toward pheromones in traps, depending on their age, density, sex ratio and other biotic factors.

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POSSIBLE USE OF PHEROMONE TRAPS FOR POPULATION DENSITY MONITORING OF OAK ROLLERS (TORTRIX VIRIDANA L. AND ALEIMMA LOEFLINGIANA L.).

MAREK TURCANI

Forest Research Institute, Research Station Banská Stiavnica, SLOVAK REPUBLIC. Abstract

Development of pheromone monitoring system for T. viridana and A. loeflingiana is decribed. Between population density T. viridana caterpillars and the number of captured males by using pheromone traps was found close correlation relationship during some years (coefficient of determination: 1990 - 0.01, 1991 - 0.88, 1992 - 0.76, 1993 - 0.32). Between population density A. loeflingiana caterpillars and number of captured males by using pheromone traps was found close correlation relationship during the whole study period (coefficient of determination: 1991 - 0.92, 1992 - 0.83, 1993 - 0.96). On the basis of the observed correlation, it is possible to state, that there is the possibility to estimate population densities approximately by using pheromone traps. 1. Introduction

Leafeating insect have defoliated over 10 thousands hectares of forest stands in Slovakia in average every year during the period 1972 - 1996. Besides the gypsy moth (Lymantria dispar L.), the most serious defoliators of broadleaved stands (oak stands mainly### are Geometridae and Tortricidae (Fig. 1). Tortricidae are presented by following species mainly: Tortrix viridana, Aleimma loeflingiana and Archips sp. Tortricidae are typical pests of floodplain forests and forest stands on warm south facing slopes in the lowland (below 400 m a.s.l.). They infest Quercus robur (flood - plain forests) and Quercus pubescens (dry places) mainly. Majority of these stands are characterized by bad health as a result of longterm oak decline. Defoliation of these stands can cause an increase in tree mortality in subsequent years. During the period from 1981 - 95 more than 5.5 mil m 3 trees were killed by oak decline (Fig.2).

Practice Oriented Results on Use and Production of Neem Ingredients and Pheromones VIII 134 H. Kleeberg & C. P. W. Zebitz (eds.) Copyright 2000 by Druck & Graphic, Giessen

25000

20000 Tortricidae Geometridae 15000

hectares 10000

5000

0 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 year

Fig. 1: The trend of oak stands defoliation (in hectares) caused by Geometridae (Erannis defoliaria and Operopthera brumata mainly) and leafroller (Tortrix viridana, Aleimma loeflingiana and Archips sp. mainly) moths during the period from 1972-1996

2500

2000

1500

1000

500

0 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 year

Fig. 2: The trend of oak decline (in thousands of m 3 of killed trees ) in Slovakia during the period from 1981 - 1995.

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Therefore attacked stands are treated by biopesticide (B.t. preparations mainly###. For evaluation of oak rollers population density in Slovakia egg and caterpillar counting is especially used (by using sample branches). Both methods may be regarded as very time-consuming processes. Outbreaks of oak rollers in Slovakia occur suddenly and forest staff usually observe it after the beginning of an outbreak, when heavy defoliation is present in large areas. In order to recognize population densities at the beginning of outbreaks we started to prepare a monitoring system by using pheromones. 2. Material and Methods

Since 1986, we have tested different types of traps (Fig.3) and dispensers in Slovakia. Likewise we have optimalized different doses of pheromones. Optimization was finished in 1990 and a trap set (trap, pheromone, dispenser), which has been used during the next 3 - 4 years is as follows:

Fig.3: Types of traps, used in testing experiments A - Zinc coated sheet (sticky) B -Polish lantern (dry) C - Delta Kishinyev + insecticide (dry) D - Czech barrier lantern (dry) E - Delta Kishinyev open (sticky) F- Wing trap (sticky) G - Mushroom + insecticide (dry) H - Disk + insecticide (dry)

Pheromones: For Aleimma loeflingiana 100 ###g Z11 - 14Ac + E11 - 14 Ac (1:1) For Tortrix viridana 50 ###g Z11 -14 Ac + Z11 - 14OH (9:1). Dispenser: Conical rubber cap. Traps: For both species as an optimal trap were established: modified Wing trap (now called BETA) and Mushroom + insecticide as well.

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Fig. 4 Network of the study plots for Tortrix viridana (Ax) and Aleimma loeflingiana (Bx) population density monitoring. (The codes are the same in Fig. 4 and Tables 1 and 2).

In 1990, we started to find a relationship between caterpillar population density and the number of T. viridana and A. loeflingiana males captured by pheromone traps. In the outbreak area of these species in Slovakia we stabilised monitoring points (Fig.4). During the period 1990 - 1993, Tortrix viridana and Aleimma loeflingiana population density was measured on fixed plots. Caterpillars in the 3rd and 4th instar were counted on sample branches ( 1990: 5 plots for T. viridana only, 1991: 5 plots for both species, 1992: 11 plots for both species and 1993: 10 plots for both species). The sampling was done by using special shears from lower parts of the trees. Sample branches from high parts of trees were taken by cone-pickers. On the basis of variability Tortrix viridana caterpillars on sample branches we stated a statistically necessary sample branch set. By using this results we took 4 sample branches from the upper part (0.5 m long) of the crown. Necessary sample set is 10 - 20 trees - mainly depending on the population density. Caterpillars were determined on study plots, non determined specimens were reared in the laboratory and determined after adults emerged. The same method has been used for Aleimma loeflingiana.

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Fig.5: Scheme of pheromone traps on monitoring point

In 1990, study plots consisted of 9 pheromone trap sets (3 x 3 pheromone traps in square form). During the next years (when we involved results of variability analysis) we have used 16 pheromone trap sets (Fig.5). Distance 10 m between the neighbour traps was established as optimal. 3. Results

Comparison of caterpillar abundance and number of males captured by pheromone traps.

Average Tortrix viridana population density data and average number of males captured by pheromone traps on different areas are presented in Table 1. Population density was quite low during the study period. High population density was observed in 1991 and 1992 on study plot Cerenany only. Due to high population density medium - heavy (40 - 70 %) defoliation was caused by this pest mainly. During this period the number of Tortrix viridana males captured by pheromone traps was high on this plot as well. The relationship between population density assessed by using sampling branches corresponded with number of T. viridana captured males on some plots approximately (Fig. 6, 7, 8).

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Tab. 1: Tortrix viridana average population density and average number of males captured by pheromone traps during the period 1990 - 1993.

Year 1990 Year 1991 Year 1992 Year 1993 Trap BETA Trap BETA Trap Mushr. Trap BETA Place 1 2 1 2 1 2 1 2 Plastovce I (A7) 0.23 13.33 1.69 21.89 1.15 1.39 0.15 8.69 Radzovce (A16) 0.03 10.89 0.10 12.11 - - 0.00 0.88 Medovarce (A4) 1.23 15.11 0.78 23.22 - - - - Sudovce I (A5) 0.48 16.56 0.30 26.00 0.06 0.32 - - Hrusov (A15) 0.68 4.78 ------Cerenany II (A2) - - 9.56 53.22 11.05 16.50 1.20 23.44 Sturovo I (A12) - - - - 1.17 0.43 - - Sturovo II (A13) - - - - 2.05 3.25 0.15 30.31 Sturovo III (A14) - - - - 1.85 9.31 - - Plastovce II (A8) - - - - 0.63 1.06 0.05 6.69 Plastovce III (A9) - - - - 1.07 0.87 - - Cerenany I (A1) - - - - - 10.13 - 26.56 Cerenany III (A3) - - - - 14.55 12.63 - - Sudovce II (A6) - - - - 0.00 0.25 0.15 7.81 Sikenica I (A10) ------12.38 Sikenica II (A11) ------8,88 Soroska (A17) ------0.00 2.50 Legend: 1 - Average number of T. viridana caterpillars per 1 sample branch (100 leaves). 2 - Average number T. viridana males captured by 1 pheromone trap. Ax - Codes of study plots in Fig. 4.

During the study period (1991 - 1993), population densities of Aleimma loeflingiana may be characterised as low (Tab. 2). Population density over 1 A. loeflingiana caterpilar per 1 sample branch was observed very rarely (1991 - location Plastovce I, 1992 and 1993 - location Sturovo II). The trend of caterpillar population density corresponds with the trend of A. loeflingiana males captured by pheromone traps in all years. When population density increased, average number of captured A. loeflingiana males usually increased (Tab. 2, Fig. 9). The relationship does not exist in some plots across all years as well (Sudovce - B5). The data may be influenced by the short study period in the same place.

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Tab.2 Aleimma loeflingiana average population density and average number of males captured by pheromone traps during the period 1991 - 1993.

Year 1991 Year 1992 Year 1993 Trap BETA Trap BETA Trap BETA Place 1 2 1 2 1 2 Radzovce (B10) 0.23 0.22 - - - - Sudovce (B5) 0.44 3.89 0.02 3.31 0.00 5.13 Medovarce (B4) 0.64 3.44 - - - - Cerenany I (B1) 0.98 12.44 0.75 22.50 - - Plastovce I (B6) 1.08 11.67 0.80 13.73 - - Sturovo I (B8) - - 0.10 8.31 0.20 13.31 Sturovo II (B9) - - 1.09 31.56 1.88 61.00 Plastovce II (B7) - - 0.40 9.25 0.00 7.25 Cerenany II (B2) - - - 10.50 - - Cerenany III (B3) - - - - 0.20 22.44 Silica (B12) - - - - 0.05 8.00 Plesivec (B11) - - - - 0.00 5.25

Legend: 1 - Average number of A. loeflingiana caterpillars per 1 sample branch (100 leaves). 2 - Average number A. loeflingiana males captured by 1 pheromone trap. Bx - Codes of study plots in Fig. 4.

In the study plot Sudovce the caterpillar population density was 0.44 caterpillars / sample branch and number of captured males 3.89 males / trap. In next year, population density decreased rapidly, but number of males decreased a little bit only. In 1993, population density decreased again, but average number of captured males increased.

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25

21,89

20

15 13,33 Abundance 10 8,69

5

1,69 1,15 1,39 0,23 0,15 0 1990 1991 1992 1993 Years

Fig.6: Comparison of mean population density (caterpillars / sample branch - darker columns) and mean number of Tortrix viridana males caught in 1 pheromone trap (lighter columns) -plot Plastovce I. (In 1992, different types of traps were used).

30

26 25

20 16,56

15 Abundance 10

5

0,48 0,3 0,060,32 0 1990 1991 1992 Years

Fig.7: Comparison of mean population density (caterpillars / sample branch - darker columns) and mean number of Tortrix viridana males caught in 1 pheromone trap (lighter columns) -plot Sudovce I. (In 1992, different types of traps were used).

141

60 114

50

40

30 23,44 Abundance 20 16,5 11,05 9,56 10

1,2 0 1991 1992 1993 Years

Fig.8 Comparison of mean population density (caterpillars / sample branch - darker columns) and mean number of Tortrix viridana males caught in 1 pheromone trap (lighter columns) -plot Cerenany II. (In 1992, different types of traps were used).

70 61 60

50

40 Y 31,56 30

20

10 1,09 1,88 0 1992 1993 x - roky

Fig.9 Comparison of mean population density (caterpillars / sample branch - darker columns) and mean number of Aleimma loeflingiana males caught in 1 pheromone trap (lighter columns) -plot Sturovo II.

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Correlation analysis

Tortrix viridana We used selected data obtained from pheromone traps for correlation analysis (extreme data and data from damaged traps were excluded from data processing). For 1990 we obtained a coefficient of determination of 0.01, which indicated an absence of any correlation. The situation was entirely different in next year. It can be documented by the results, the coefficient of determination reached 0.88, which indicated high correlation. A similar situation was observed in 1992, when coefficient of determination decreased a little bit and reached 0.76. In 1993, coefficient of determination decreased rapidly and reached a value of 0.32, which is judged as a poor correlation. Aleimma loeflingiana In 1991, coefficient of determination reached a value of 0.92. This value indicates high correlation. During the next years (1992 and 1993) the situation was similar. The coefficient of determination reached values 0.83 in 1992 and 0.96 in 1993, which indicated high degrees of correlation. 4. Discussion

During the research period, we stated high numbers of the pheromones traps, which are necessary to use for statistical processing. It is caused by the high variability of nature conditions mainly. Some authors (Albert, Bogenschütz, König, 1989) consider sets of 3 - 6 pheromone traps as a sufficient number. Sanders (1988) created pheromone trap sets for pheromone monitoring from 3 - 5 specimens with regard to place conditions and population density. One pheromone trap was used by Luciano (1987) for gypsy moth pheromone monitoring research. It seems that all these values are too low for comparison of caterpillar population density and number of captured males. On the basis of my experiments I state a set of 16 (4x4) traps as a necessary number. Advantage of this set is the possibility to use an external row of pheromone traps as a barrier. It is possible to understand from this information, why some authors concluded, that no relationship existed between egg and caterpillar population density (estimated by using conventional methods) and number of captured males. We started the next step in pheromone monitoring development - comparison of real (relative) population densities with the number of T. viridana and A. loeflingiana males captured by pheromone traps, after the finishing optimisation of minimal necessary sets of sample branches and pheromone traps i. e. minimisation of some negative factors. On the basis of data from literature, information about the oak rollers pheromone monitoring are known rarely (for example T. viridana - Altenkirch 1989, Merle 1985). In spite of this fact Tortricidae group damaging coniferous species was studied more exactly (Bogenschütz, Mazur, Sierpinski (1982), Cornic, Geraud, Merle (1987). These authors studied Choristoneura murinana. Cwiklinski, Grodzki (1989) made experiments with Zeiraphera diniana. Sanders (1988) studied the species Choristoneura fumiferana. Moths from the family Tortricidae can be characterised as suitable for pheromone monitoring system development (they are weak fliers). On the basis of my results on research of T. viridana and A. loeflingiana I can state a usually high correlation between values of population density, which was estimated in caterpillars stage and number of males captured by using the pheromone traps.

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Of course, the relationships obtained may be influenced by caterpillar mortality during 4 - 5 instar and during pupation. This period is 2 - 3 weeks long, when population densities change expressively. This may mainly be due to the weather and parasitoids. High number of caterpillars and pupae may die in unsuitable weather periods due to starving or from different fungi and bacteriosis. The important factor, which may reduce population density of oak rollers are caterpillars of Noctuidae. If weather is cold and rainy, these species become predators and prey upon smaller species. In spite these defects of the pheromone monitoring, there are some advantages as well. The first one is the possibility to easily monitor large areas, the second one, that this method is not such a time consuming process as compared to conventional methods by counting non mature stages. Of course, all presented data are necessary to study during future experiments, which we are planning to do during the next years. 5. Conclusions

1. The attractant Z11 - 14Ac + E11 - 14 Ac (1:1) in dose 100 µg was determined as the optimum for Aleimma loeflingiana.

2. The attractant Z11 -14 Ac + Z11 - 14OH (9:1) in dose 50 µg was determined as the optimum for Tortrix viridana. 3. Traps BETA (modified Wing trap) and Mushroom with insecticide and dispenser "conical rubber cap" were determined as the best trap set. 4. Between population density T. viridana caterpillars and number of captured males by using pheromone traps was found close correlation relationship during some years (coefficient of determination -1990 - 0.01, 1991 - 0.88, 1992 - 0.76, 1993 - 0.32). 5. Between population density A. loeflingiana caterpillars and number of captured males by using pheromone traps was found close correlation relationship during all study period (coefficient of determination - 1991 - 0.92, 1992 - 0.83, 1993 - 0.96). 6. On the basis of the observed correlation, it is possible to state, that there is a possibility to estimate population densities approximately by using pheromones traps. 7. Further experiments are necessary to confirm this relationship. 6. References:

Albert, R., Bogenschütz, H., König, F. 1989: Untersuchungen zum Einsatz von Sexuallockstoff-Fallen zur Überwachung des Massenwechsels von Operophtera brumata. Zeistschr. für Angew. Ent. 98, 286 - 298. Altenkirch, W. 1989: Lockstoff-Fallen zur Überwachung forstlich wichtiger Schmetterlinge. Forst und Holz 44, 286 - 293. Bogenschütz, H., Mazur, K., Sierpinski, Z. 1982: Badania nad kontrola liczebnošci wygolówki jedlineczki (Choristoneura murinana Hb.) za pomoca pulapek feromonovych w Górach Swietokrzyskich. Sylwan 125, 121 - 131. Cornic, J.F., Geraud, D., Merle, P. Du 1987: Observations sur la distribution spatiale et sur la mesure des populations pre-imaginales de la tordeuse du sapin, Choristoneura murinana Hb. (Lep., Tortricidae). 2 Les populations larvaires et conclusions. Journal of Applied Entomology 103, 403 - 417. 144

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