UNIVERSITY OF AGRICULTURAL SCIENCES AND VETERINARY MEDICINE CLUJ NAPOCA DOCTORAL SCHOOL ANCA DAFINA COVACI SUMMARY. PhD THESIS

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1 UNIVERSITY OF AGRICULTURAL SCIENCES AND VETERINARY MEDICINE CLUJ NAPOCA DOCTORAL SCHOOL ANCA DAFINA COVACI SUMMARY OF PhD THESIS Research on biology, ecology and integrated management of Thysanoptera species in greenhouses SCIENTIFIC SUPERVISORS: Univ. prof. dr. ION OLTEAN Researcher prof. dr. Rocco ADDANTE CLUJ-NAPOCA

2 Key words: Frankliniella occidentalis Pergande, monitoring, morfology, biology, ecology, colore traps, pheromones, functional response, chemical control. INTRODUCTION The species of this order are among the major pests of the protected areas, causing major damage to both vegetable crops and flowers. Except the damages in greenhouses and solariums, during hot periods of summer they cause damage in field crops also, by sticking and sucking plant juice. The plants that were attacked by thrips suffer different morpho-physhiological and biochemical changes. Assimilation, transpiration and chlorophyll content are reduced and this aspect may lead to debilitation and finally the death of the plant.these aspects are seen in terms of damages and loss. Often thrips are considered vector pests, which, in addition to the damages they bring, they transmit a series of highly dangerous pathogens (viruses, bacteriosis, fungi) that increase the amount of damage. Thus, many species of thrips (Thrips tabaci, Aphis fabae, Cerosipha gossypii, Myzodes persicae etc.) transmit different tospoviruses (Bunyaviridae) to other solanaceous plants and to other agricultural crops. Due to a control system based mainly on chemical products, the grand majority of these species became immune to insecticides; the latter reduced the number of parasites and pests up to the start of intensive chemicalization. ECOLOGICAL CONDITIONS FOR RESEARCHES Anorganic or abiotic factors, represented primarily by climatic factors, are the most important in the development, geographical distribution and insect activity. In greenhouses this group includes the influence of temperature, humidity, light, radiation and airflow. The studies were conducted both in flower and vegetable greenhouses. Researches in flower greenhouses were conducted in Molfetta, a city located 35 km away from Bari, Apuglia Region (Italy), during a period of three months in the experimental year 2011 and the experiments from vegetable greenhouses were conducted in Cluj. OBIECTIVES, MATERIALS AND METHODS According to data provided by the Directorate of Agriculture of Cluj County, on its area there are 10 greenhouses and 6 solariums, a total of 10 protected areas, private properties with varied surfaces, whose main objective is the production and marketing of various flower 2

3 and vegetable plants. After contacting the owners on the phone, we got to the conclusion that Thysanoptera proved to be a common pest of protected areas in Cluj County, most of the producers confirming their presence on grown plants. The study of this paper aims at the western flower thrips, Frankliniella occidentals Pergande, a species identified in two of the greenhouses in Cluj County and in a floricultural greenhouse in Molfetta (Italy) and the main objectives of this thesis include: Frankliniella occidentalis Pergande morphology; Frankliniella occidentals Pergande biology; monitoring of Frankliniella occidentalis Pergande by different methods (population dynamics, establishing a correlation between monitoring methods, sex ratio, vertical distribution of the species) testing the efficacy of coloured traps for capturing the Frankliniella occidentalis Pergande individuals; testing the efficacy of pheromone lures; evaluation of the type of functional response on the juvenile spiders from Philodromus genus (Araneae: Philodromidae) when the increasing number of western flower thrips individuals, given as source of food; testing the efficacy of a range of insecticides; The study of morphologic elements is an essential criterion in recognizing and identifying the Western flower thrips. The morphologic elements underlie the distinction between this species and other pests in protected areas. Tackling some biological control methods (with coloured traps, pheromone traps or natural enemies) greatly contributes to reducing pesticide, obtaining healthier products and, at the same time, protecting the environment. By monitoring Frankliniella occidentalis Pergande population using various techniques for the development cycle stages and therefore for the relationship between individuals of the opposite sex, consistent data are obtained representing a solid support in establishing effective integrated control strategies. Materials and methods In Romania, identification to species level for samples collected from UASVM Cluj-Napoca greenhouse and from the private greenhouse (S.C. SERIMPEX S.R.L.) was conducted in November 2010 by PhD Liliana Vasiliu-Oromulu. The biological material was collected from both greenhouses, from cucumbers, using the shaking method; afterwards the insects were introduced into vials which contained preservative solution, previously prepared in the laboratory. The samples were sent to Bucharest and analysed by PhD Liliana Vasiliu-Oromulu at the Institute of Biology. Both in the case of UASVM Cluj Napoca greenhouse and in the case of private greenhouse, the only species identified was the western flower thrips, Frankliniella occidentalis Pergande. In Italy, the biological material was collected from Rosa sp. (Rosaceae) reared in one of the flower greenhouses in Molfetta, in September 2011, and species 3

4 determination was made by Professor Researcher Rocco Addante, using Olympus optical microscope (model U-DO) and identification keys L.A. Mound and Kibby, 1998 and zur Strassen, As in greenhouses in Romania, western flower thrips was found. Research on Frankliniella occidentalis Pergande morphology The main recognition morphostructural elements of the western flower thrips, Frankliniella occidentalis Pergande, were revealed using the electron microscope and the optical microscope. Research on Frankliniella occidentalis Pergande biology Aspects of the biology of the Frankliniella occidentalis Pergande were investigated in the Entomology Department laboratory of University of Bari, Italy. To highlight the life cycle of the Frankliniella occidentalis Pergande two types of cages were used: a cage of oviposition and several rearing cages. Pods of bean, Phaseolus vulgaris (Solanaceae) were both the trophic basis (in rearing cages) and the substrate for laying eggs (in oviposition cages). Throughout the research on Frankliniella occidentalis Pergande bioecology the temperature conditions were constant, in incubator, of 25 C and a photoperiod of 16 hours of light and 8 hours of darkness. Research on monitoring Frankliniella occidentalis Pergande Using various methods, by monitoring the western flower thrips on different host plants, the following were considered: aspects regarding species evolution during the study (population dynamics); aspects regarding correlation between different monitoring methods; aspects regarding sex ratio; aspects regarding vertical distribution. Research on testing the efficacy of coloured traps Experiments on testing the efficacy of colour in capturing Frankliniella occidentalis Pergande adults were conducted in the UASVM Cluj Napoca and the private greenhouses from Cluj county (over three experimental years) and in the rose greenhouse in Italy (over a period of three months). In the UASVM Cluj Napoca greenhouse, the studies were conducted on cucumbers, lettuce, beans, eggplants and tomatoes, and in the private greenhouse, the efficacy of coloured traps in capturing the western flower thrips was conducted on lettuce, tomatoes and beans. Both in the vegetable and flower greenhouses plastic traps of different colours (blue, yellow, red, white and clear) were used with a curative purpose, as mechanical 4

5 method of control. All these are commercially available. Blue, yellow and white traps, are especially produced for plant protection, and the red and transparent ones are stationery products (plastic sheets) and are part of the A4 accessories (covers). Research on testing the effectiveness of pheromone lures Pheromone lures for attracting Frankliniella occidentalis Pergande, available under the trade name, AtraTHRIPS, are products of the Raluca Ripan Institute of Chemistry - Cluj-Napoca. The pheromone used, includes, in a mixture ratio of 1:1, two volatile substances (R) - lavandulyl acetate and Neryle (S)-2-methylbutanoate. AtraTHRIPS biological activity testing was achieved within 4 weeks from 04/27/12 to 05/25/12, in the cucumbers crop from UASVM Cluj Napoca greenhouse. Research on evaluation of the functional response type of Philodromus spider juveniles (Araneae: Philodromidae) when increasing the number of prey individuals belonging to Frankliniella occidentalis Pergande The study was conducted in Italy, during October-November 2011, in collaboration with Pamela Loverre, PhD student in Entomology, Faculty of Agriculture, University of Bari. The spiders were collected from the tree branches in the campus of the University of Bari, using the frappage method. In order to determine the type of functional response in various food conditions, four different densities of prey populations were used: 1, 5, 15, 30, 60 western flower thrips individuals. For most densities, 15 replications were performed (except for density of 30 thrips which was replicated 14 times and the case of the density of 60 thrips, which was only replicated 6 times). Thus, a total number of 66 spiders and 1125 thrips were used. Throughout the study, the experiment was performed in the laboratory microclimatic conditions at an average temperature of 23 C and 42% relative humidity (UR%). Research on testing a range of insecticide Testing the efficacy of five chemical products commercially available was conducted on tomatoes grown in the UASVM Cluj Napoca glass solarium. The tested insecticides were: Pyrinex 25 NE, Oscar 10 CE, Vertimec 1.8 CE, Karat-Ezeon, Actara 25 WG. They also represented the five experimental variants. Each variant was tested on a single line, therefore five rows of tomatoes were treated. 5

6 RESULTS ON FRANKLINIELLA OCCIDENTALIS PERGANDE BIOLOGY The protocol was taken from Espinosa et al., Mean period of Frankliniella occidentalis Pergande development (T= 25 C, U=75% ± 5%) Stage Average period (days) Preoviposition period 2.8 Incubation 3.3 Larva 4.9 Nymph 6.7 One generation duaration 17.7 RESULTS ON FRANKLINIELLA OCCIDENTALIS PERGANDE MONITORING Results of western flower thrips population dynamics In both greenhouses in Cluj County, it was observed that the average number of captures on coloured traps was significantly different from one year to another, during the experimental period (H = 6.95, df = 2, P <0.05, for the UASVM Cluj-Napoca greenhouse and H = 21.49, df = 2, P <0.05, for the private greenhouse). In the rose greenhouse the variation of mean number of insects on the trap, was not significantly different from one week to another, over the three months of monitoring, according to Kruskal-Wallis test results (H = 13.5, df = 10, P> 0.05). Results on the relationship between the monitoring methods The correlation coefficient R 2 value, showed no positive relationship between the coloured traps method and the shaking method and between the direct method and the coloured traps method in all crops analysed in the three greenhouses. Results on Frankliniella occidentalis Pergande sex ratio Subsequent to the studies on cucumbers reared in the UASVM Cluj-Napoca greenhouse a statistically insignificant difference was noticed in the sex ratio determined from leaves compared to the sex ratio determined from flowers (t = 0.09, df = 12, P> 0.05 for studies conducted in 2010; t = 0.41, df = 18, P> 0.05 for studies conducted in 2012). In the private greenhouse the studies were conducted in 2010 and statistical results showed a significant difference in the sex ratio determined on flowers from the sex ratio determined on leaves (t = 2.43, df = 14, P <0.05). 6

7 Regarding the floricultural greenhouse in Italy, the results highlighted a significant difference in terms of gender structure of the western flower thrips population monitored on two rose varieties (A and B) (t = 0.16, df = 17, P> 0.05). Results on the vertical distribution By analysing the data from UASVM greenhouse, a statistically insignificant difference was revealed regarding the larval distribution (F = 2.24, df = 2, P> 0.05, for research in 2010, F = 0.86, df = 2, P> 0.05, for research in 2012). A statistically insignificant difference was found in terms of adult dispersal on plant levels for both the experimental years (F = 1.63, df = 2, P> 0.05, in 2010 (F = 0.79, df = 2, P> 0.05, in 2012). Moreover, by examining pooled data (adults + larvae), the results showed that between the number of individuals found on the different levels of the plant, there is no statistically significant difference. (F = 2.71, df = 2, P > 0.05), for researches conducted in 2010; F = 1,37, df = 2, P > 0.05), for researches conducted in By statistical analysis of data obtained in 2010, in the private greenhouse, the results were similar to those obtained at the UASVM Cluj-Napoca greenhouse, showing an insignificant difference in terms of vertical distribution of larvae (F = 0.58, df = 2, P > 0.05), and of adults (F = 1.72, df = 2, P> 0.05). As for the UASVM Cluj-Napoca greenhouse, in the private greenhouse, pooled data (adults + larvae) result demonstrated insignificant statistical differences (F = 2,18, df = 2, P > 0.05) regarding the number of individuals shaken down from the leaves of cucumber located on different parts of the plant. Research on population density monitoring using coloured traps placed on beans at a distance of 1.5 m from the ground (in the private greenhouse) and at 2.5 m above ground level (in the UASVM Cluj-Napoca greenhouse) provided data regarding spatial distribution. Statistical analysis of these data revealed results contrasting to those obtained using the shaking method. In other words, the number of insects caught on traps at 2.5 m was significantly statistically different compared to the number of insects recorded on traps placed at 1.5 m above ground level (t = 3.88, df = 73, P <0.05). RESULTS ON THE EFFICACY OF THE COLOURED TRAPS Becouse Frankliniella occidentalis Pergande species is strongly influenced by the colour of the traps, wavelength measurement was also carried out. Overall, the largest number of captures has been reported on the yellow and blue traps and in comparison with them, on the white, red and clear traps the capture density was much lower. By variance analysis a significant difference was determined in the number of insects caught on different coloured traps set on the two tomato hybrids in the private greenhouse (F = 9.88, df = 4, P <0.05 for Rally hybrid, F = 14.94, df = 4, P <0.05 for Norallee hybrid). 7

8 It was also determined a significant difference in the number of captures on the coloured traps set on three tomato hybrids in the UASVM Cluj-Napoca greenhouse (F = 9.47, df = 4, P <0.05 for Ballet hybrid F = 13.90, df = 4, P <0.05 for Cristal hybrid, F = 7.79, df = 4, P <0.05 for Tamaris hybrid). Both in the UASVM Cluj-Napoca and in the private greenhouse within the last experiments of the last experimental year (2012) compared to previous years, individuals belonging to the Aeolothripidae family were observed on the coloured traps, the latter being reported as predatory species that feed on other species of thrips (Knechtel, 1951). In the private greenhouse other Thysanoptera species individuals, belonging to the Tubulifera suborder, and one individual of the Chirothrips genus were also reported (according to PhD Liliana Oromulu Vasiliu). These individuals were observed especially on yellow traps, but also on the white blue and clear ones; still their number was too small (less than 10 individuals) to lead to an analysis of the percentage abundance of the species population RESULTS ON TESTING THE PHEROMONE LURE TRAPS FOR FRANKLINIELLA OCCIDENTALIS PERGANDE The F test established a statistically significant difference between the number of males captured on three traps (with and pheromone free) (F = 6.04, df = 2, P <0.05). After comparing the number of males on trap 1, with the number of males on the control trap using Dunnett's test, there was a statistically significant difference, however, by comparing trap 2 to the control trap, the difference was statistically insignificant. A possible explanation for this discrepancy could be the distance between them, the pheromonal stimulus could have reached close to the control trap, thus influencing the captures reported on it (Hamilton et al., 2005). Also, the results showed an insignificant difference in the average number of females captured on traps, with or without pheromones (F = 0.15, df = 2, P> 0.05). RESULTS ON EVALUATING THE FUNCTIONAL RESPONSE TYPE OF PHILODROMUS SP. SPIDERS After introducing the spiders in the vials with insects, they were immediately seen feeding so in the case of one trips per vial density in all replications, they were consumed in less than 30 minutes (moment of the first control). In one of the 15 replications with 5 of thrips per vial, they were consumed before the first control. In the rest of the replications, the 5 thrips were killed after 24 hours (time of the third control). As the prey density was increased, the rate of prey killing increased as well, and these observations were confirmed statistically (F 4,64 = , P <0.0001), as a result, the 8

9 experiment conducted in the laboratory by exposing Philodromus juveniles to increasing food amount conditions, from 1 to 60 insects per vial, led to obtaining a type II functional response. RESULTS ON TESTING A RANGE OF INSECTICIDES FOR FRANKLINIELLA OCCIDENTALIS PERGANDE CONTROL Efficacy of five commercially available insecticides for chemical control of western flower thrips on tomato crop in the UASVM Cluj Napoca solarium ( ) Variant Product Mean no. of thrips/plant (24 h.b.t.) Mean no. of thrips/plant (24 h.a.t.) E% Mean no. of thrips/plant (7 d.a.t.) 1 Pirinex 25 CE Oscar 10 CE Vertimec 1.8 CE Karate-Zeon Actara 25 WG h.b.t. = 24 hours before treatment 24 h.a.t. = 24 hours after treatment 7 d.a.t. = 7 days after treatment E% 9