Validation of a new method for monitoring eggs of the Grape Leafhopper (Empoasca vitis) in grapevine leaves

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1 Bayerische Landesanstalt für Weinbau und Gartenbau Validation of a new method for monitoring eggs of the Grape Leafhopper (Empoasca vitis) in grapevine leaves IOBC-WPRS Bulletin (24) 7, 2001 Herrmann, J.V., Böll, S. Sachgebiet Rebschutz Bayerische Landesanstalt für Weinbau und Gartenbau Herrnstr. 8, Veitshöchheim Tel. 0931/ josef.herrmann@lwg.bayern.de

2 The Grape Leafhopper (Empoasca vitis) deposits its eggs in the veins and leaf stalks of grapevine leaves. Under blue light excitation ( nm) the eggs fluoresce strongly and can thus be monitored quantitatively in intact leaves. In a laboratory experiment it was verified that only leaf hopper larvae hatched from these eggs. The eggs are preferentially deposited in veins of 1 st or 2 nd order or in leaf stalks, while small veins are avoided. Parasitized eggs do not fluoresce during advanced stages of development. Key words: egg monitoring, autofluorescence, Empoasca vitis Introduction Population dynamics of larvae as well as of adults of the Grape Leafhopper are well studied (e.g. Herrmann et al. 1999, Schruft & Wegner-Kiß 1999). In contrast, quantitative data on deposited eggs of this pest species are scarce as their monitoring is rather difficult. Females deposit their eggs in the veins and leaf stalks of grapevine leaves, unfortunately invisible to the observer. However, an easy quantitative sampling method would be of great use to get a complete picture of the population dynamics of this pest species to get a fair population estimate, time ahead of a potential outbreak to be able to study parasitation rates of relevant mymarids, small wasp species that parasitize efficiently the eggs of the Grape Leafhopper. Vidano et al. (1987) used a method bleaching the leaves before dying the eggs with fuschine. In vegetable cultures as cucumber, for example, leafhopper eggs can be made visible by lighting the leaves from behind or from the side. In our study, however, both techniques failed. Materials and Methods We developed a new and simple technique that allows to monitor eggs in intact grapevine leaves. Various natural compounds fluoresce under UV light, some also under blue light excitation. We found that eggs, larvae and adults of the Grape Leafhopper show an intense green fluorescensce under blue light, but not under UV excitation (Böll & Hermann 2001). To validate our method we conducted the following hatching experiment from week 19 to week 25, 2000: we took 25 grapevine leaves per week from two vineyards. In the lab, the major and minor veins as well as the leaf stalks were scanned with a direct light fluorescence microscope under blue light excitation ranging from 450 to

3 490 nm. The magnification rate was 50 times. Fluorescent spots were marked on the leaves. Careful scanning and marking took 15 to 30 minutes per leaf. To monitor larvae hatching from the eggs, the leaves were put into water-filled glass vessels that were sealed in plastic bags. These miniature glass houses were kept for three weeks in a green house. This time is known to be sufficient to allow hatching of arthropod eggs under the observed temperature regime. Thereafter, the leaves and bags were searched carefully for hatched leafhopper larvae, egg parasitoids and other occuring arthropods. Results and Discussion In a preliminary study in 1999, we found that fluorescent spots contained eggs that were identical in size and shape to eggs from dissected female leafhoppers, which in turn showed the same intense green fluorescence. Furthermore, we got direct evidence during a hot weather period of the hatching experiment that leafhopper larvae were hatching from marked fluorescent sites as some of the larvae got stuck while hatching and died. However, other arthropod species could as well have deposited fluorescent eggs in the veins of grapevine leaves. In Veitshöchheim, the study site with the higher leafhopper abundance, a total of 811 fluorescent eggs was found in the 175 leaves examined. Three weeks later, leafhopper larvae and leafhopper skins were observed on the leaves, but no adults. In addition, adult thrips as well as thrips larvae were present, besides varying numbers of mites. Mites, however, are too small to be of any relevance. Whereas no relationship was found between egg number and thrips larvae, the number of leafhopper larvae was significantly correlated to the number of fluorescent eggs (Fig.1, t=3.28, p<0.05). This agrees with what is known about the biology of the grape thrips: this species deposits its eggs not in the veins but under the epidermis of the intercostal lobes. No fluorescence was ever observed in these areas. The above results demonstrate quite clearly that only Grape Leafhopper hatched from the fluorescent eggs, however the hatching rate in the greenhouse was rather low with 35%. This was probably due to the unnatural high humidity in the sealed bags, leading most likely to reduced survival rates. Dead larvae, however, shrivel to an extent that they cannot be identified anymore. Furthermore, as mentioned before, an unusually hot weather period during the latter part of the study reduced hatching rates dramatically.

4 Hatching experiment Veitshöchheim 2000 number of detected animals leafhopper larvae leafhopper skins thrips larvae R 2 = 0,68 R 2 = 0,23 R 2 = 0, number of eggs/ 25 leaves Fig.1: Relationship between number of fluorescent eggs in leaf veins and the corresponding number of larvae of different arthropod species. Eggs from leaf stalks were not considered as they had no chance to hatch under water. % 100 Veitshöchheim n = 811 eggs major leaf vein minor leaf vein leaf stalk Fig.2: Percentage of leafhopper eggs in different leaf tissues.

5 Grape Leafhoppers deposit their eggs only in veins and leaf stalks of the grapevine leaves (Fig.2). Major veins are clearly prefered, while minor veins are avoided. About one fifth of the eggs was found in leaf stalks. This percentage is probably even higher as leaf stalks are not easily scanned under the microscope due to their round structure and large surface area. Besides, circular holes were found on leaf veins caused by hatched egg parasitoids of the Grape Leafhopper. In most cases, these sites had not shown fluorescence during egg monitoring. This indicates that parasitized eggs do not fluoresce during advanced stages of development. References BÖLL, S. & HERRMANN, J. V. 2001: Eine neue Untersuchungsmethode zur Bonitur der Eier der Rebzikade (Empoasca vitis) in Rebblättern. Journal of Plant Diseases and Protection 108 (in press). HERRMANN, J.V., EICHLER P. & GUEDOVA, K. 1999: Grüne Rebzikade - Natürlichen Feinden auf der Spur. Das Deutsche Weinmagazin 11: SCHRUFT, G. & WEGNER-KIß, G. 1999: Untersuchungen zum Auftreten der Grünen Rebzikade Empoasca vitis. Deutsches Weinbau-Jahrbuch 50: VIDANO, C., ARNO, C. & ALMA, A. 1987: On the Empoasca vitis intervention threshold on vine (Rhynchota, Auchenorrhyncha). In: Proceedings of the 6th Auchenorrhyncha Meeting, CNR-OPRA, Torino, eds. Vidano & Arzone: