WAGENINGEN UNIVERSITY LABORATORY OF ENTOMOLOGY

Thrips resistance in cabbage

Plant Research International

Supervised by: Dr. R. Voorrips G. Steenhuis-Broers

No.: 06.18 A.B. Allema Apr–nov 2006 1e Examinator: Prof. Dr. J.C. van Lenteren 2e Examinator: Dr. Ir. J.J.A. van Loon

WAGENINGEN UNIVERSITY LABORATORY OF ENTOMOLOGY

Thrips resistance in cabbage

Plant Research International

Supervised by: Dr. R. Voorrips G. Steenhuis-Broers

No.: 06.18 A.B. Allema Apr–nov 2006 1e Examinator: Prof. Dr. J.C. van Lenteren 2e Examinator: Dr. Ir. J.J.A. van Loon

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DANKWOORD Dit afstudeerverslag is het resultaat van acht maanden onderzoek aan trips resistentie in kool dat ik heb uitgevoerd bij Plant Research International (PRI, Wageningen). Door de opzet heb ik dit onderzoek uitgevoerd met veel begeleiders, en dat heeft in het voordeel van het onderzoek gewerkt. Ik bedank Greet Steenhuis (PRI) voor de plezierige opstart en voor haar hulp en begeleiding bij het lab experiment en het veldwerk en ik bedank Roeland Voorrips (PRI) voor zijn begeleiding van het onderzoek en hulp bij het verwerken van de gegevens. Ik bedank Joop van Lenteren en Joop van Loon van het Laboratorium voor Entomologie (Wageningen UR) voor hun begeleiding bij de voorbereidingen en de voortgangsgesprekken daarna. Rob van Tol en Willem-Jan de Kogel (PRI) bedank ik voor de begeleiding van het experiment met de plakvallen en Gerrie Wiegers en Antje de Bruin (PRI) bedank ik voor de hulp op het lab. Ik bedank Bert Vierbergen en Antoon Loomans van de Plantenziektekundige Dienst (PD) voor de instructies bij het labexperiment. Bert Vierbergen bedank ik ook voor zijn begeleiding en medewerking bij het determineren van de trips. Tot slot bedank ik Marion Walraven voor het ondersteunen van mijn opleiding en voor de mogelijkheden die ik in de Vredebergtherapie heb ontwikkeld. Op de Middelbare School stotterde ik. Dit had een grote invloed op mijn leven. De belangrijke beslissingen liet ik aan een ander over. Mijn studieresultaten waren onvoldoende en het zag er naar uit dat ik zou overstappen naar de Middelbare Tuinbouwschool. Toen kwam ik in contact met Marion Walraven en de principes van de Vredebergtherapie. Met het werken aan mijn spreken leerde ik mijn talenten te gebruiken. Ik leerde studeren en onderzoeken en merkte dat ik een hoger opleidingsniveau kon bereiken. Ik leerde me uit te drukken, ik leerde doelen te stellen en ik leerde werken. Zo werd mijn spreekprobleem de springplank voor mijn mogelijkheden. Marion loodste mij door de verschillende kruispunten in mijn studie heen. Bovendien kon ik in de Vredebergstichting alle vaardigheden oefenen die ik nodig heb: communiceren, organiseren en samenwerken. Met deze basis was ik in staat de informatie in mijn studie op de juiste wijze te verwerken

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ACKNOWLEDGEMENT This thesis report is the result of eighth month research on thrips resistance in cabbage that I carried out at Plant Research International (PRI, Wageningen). Through the set-up I carried out this research with many supervisors, and that has worked in advance of the research. I thank Greet Steenhuis (PRI) for the pleasant start up and for her help with the laboratory- and field study and I thank Roeland Voorrips (PRI) for his supervision of the research and his help with the analysis of the data. I thank Joop van Lenteren and Joop van Loon from the Laboratory of Entomology (Wageningen UR) for their supervision during the preparations and for the progress meetings thereafter. Rob van Tol and Willem-Jan de Kogel (PRI) I thank for their supervision of the experiment with the traps and I thank Gerrie Wiegers and Antje de Bruin (PRI) for their help on the lab. I thank Bert Vierbergen and Antoon Loomans from the Plant Protection Service (PD) for their instructions for the life history study and I thank Bert also for his cooperation with the determination of thrips. At last I thank Marion Walraven for supporting my education and for the abilities that I developed within the Vredeberg therapy. At Secondary School I stuttered. This had a large influence on my life. I let the important decisions to be made by some else. My study results were insufficient and it was likely that I had to switch to the College for Horticultural Studies. Then I got in contact with Marion Walraven and the principles of the Vredeberg therapy. While working on my speech I learned to use my talents. I learned to study and to investigate, and saw that I could reach a higher level of education. I learned to express myself, I learned to set goals and I learned to work. In this way my speech problem became the stepping-stone for developing my abilities. Marion directed me through the several crossroads in my study. Moreover, within the Vredeberg foundation I could practice all the competences I need: communicating, organizing and cooperating. This basis enabled me to utilize the information in my study in a good manner.

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SUMMARY Thrips tabaci Lindeman (Thysanoptera: Thripidae) is one of the most severe pests on onion, leek and cabbage, in Northern Europe. Chemical control of thrips is difficult, especially when they have entered the protective environment of the head. There is a large variation in the level of resistance among cabbage cultivars and breeding for host plant resistance might therefore to be a good option to reduce yield losses caused by thrips. The aims of this study were to get more information about the mechanism that is causing resistance and about the plant parts (frame leaf and cabbage head) in which the resistance mechanism is operational and about the life stages of thrips (adult and juvenile) that are affected by resistance. This information will help to identify the plant traits that are involved in thrips resistance. Possible mechanisms causing thrips resistance in cabbage are: antibiosis, non-preference, tolerance (these are true resistance mechanisms) and pseudo- and indirect resistance. The role of antibiosis has not been studied before and will be investigated in this study. In a laboratory life history study the mortality percentage and development time of thrips on leaf disks were compared between the highly resistant (Galaxy) and susceptible (Bartolo) cultivar. The mortality percentage was not significantly different between the two cultivars. The higher level of resistance of the Galaxy cultivar also did not result in a longer development time. This life history study could not detect a difference in resistance between fully extended (frame) leaves of a highly resistant and susceptible cultivar. In a field experiment the population size and dynamics of adults and juveniles feeding on the outer frame leaves and of adults and juveniles feeding inside the cabbage head were compared between the Galaxy and Bartolo cultivar. The adult and juvenile population size on the frame leaves was not significantly different between the two cultivars. This result is in line with the life history study on fully extended (frame) leaves. The adult and juvenile population in the heads, however, was significantly different between the two cultivars. Heads of the susceptible (Bartolo) cultivar contained significantly more adults and much more juveniles than the heads of the resistant (Galaxy) cultivar. The results of the life history- and field study indicate that thrips resistance is probably not operational in frame leaves. Moreover the results of the field study show that antibiosis is probably the main mechanism causing resistance in the head and that the juvenile population is more affected by this mechanism than the adult population. Within the field experiment the flight activity was monitored with blue sticky traps above the vegetation. To half of the traps a volatile thrips attractant was added. There were two distinct peaks in flight activity of T. tabaci observed by the traps with attractant. These to peaks correspond to a population increase on the plants. The amount of T. tabaci caught in the traps, however, did not correlate with the actual thrips numbers on the plants. The flight activity monitored with traps without attractant was slightly increased compared to the traps with attractant and did not correlate well with the actual number of thrips on the plants. The number of thrips caught in traps with attractant was significantly larger (two to six times) than the number of thrips caught in traps without attractant. The flight activity was expected to be larger above the plots with susceptible plants if thrips have a preference for these plants based on colours and/or odours. But the flight activity was not significantly different between the plots of the highly resistant (Galaxy) and susceptible (Bartolo) cultivar. 3

CONTENTS DANKWOORD .

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ACKNOWLEDGEMENT.

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SUMMARY

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Chapter 1: GENERAL INTRODUCTION .

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§ 4.1.1 COMPARISON BETWEEN CULTIVARS .

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§ 4.1.2 COMPARISON BETWEEN GENERATIONS

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§ 4.1.3 COMPARISON BETWEEN POPULATIONS

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THRIPS DEVELOPMENT TIME .

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§ 4.2.1 COMPARISON BETWEEN CULTIVARS .

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§ 4.2.2 COMPARISON BETWEEN GENERATIONS

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§ 4.2.3 COMPARISON BETWEEN POPULATIONS

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LABORATORY LIFE HISTORY STUDY Chapter 2: INTRODUCTION

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Chapter 3: MATERIALS AND METHODS Chapter 4: RESULTS § 4.1

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THRIPS MORTALITY

Chapter 5: CONCLUSIONS AND DISCUSSION

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Chapter 7: MATERIALS AND METHODS .

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Chapter 8: RESULTS

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POPULATIONS DYNAMICS ON BARTOLO AND GALAXY PLANTS

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§ 8.1.1 ADULT AND JUVENILE POPULATION ON FRAME LEAVES.

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§ 8.1.2 ADULT AND JUVENILE POPULATION IN THE HEAD .

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FLIGHT ACTIVITY ABOVE THE PLANTS

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§ 8.2.2 COMPARISON BETWEEN TRAPS WITH AND WITHOUT ATTRACTANT

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FIELD POPULATION STUDY Chapter 6: INTRODUCTION

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§ 8.2.1 COMPARISON BETWEEN BARTOLO AND GALAXY PLOTS

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RELATING FLIGHT ACTIVITY WITH POPULATION DYNAMICS ON THE PLANT 26

Chapter 9: CONCLUSIONS AND DISCUSSION

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Chapter 10: GENERAL CONCLUSIONS AND RECOMMENDATIONS FOR FURTHER RESEARCH .

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REFERENCES .

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APPENDICES

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RELATING THE LIFE HISTORY STUDY WITH THE FIELD STUDY

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CHAPTER 1

GENERAL INTRODUCTION Thrips tabaci Lindeman (Thysanoptera: Thripidae) is one of the most severe pests on onion, leek and cabbage, in Northern Europe (Pobozniak and Wiech 2005). Cabbage that has been damaged by thrips feeding is unattractive for consumption (see figure 1.1) and therefore unmarketable unless the damaged leaves are removed. Especially cabbage that is used for storage and is harvested late in the season may have severe feeding damage. Chemical control of thrips is difficult due to the continuous invasion of adult thrips (Pobozniak and Wiech, 2005). Once thrips have entered the protective environment of the head, chemical control becomes even more difficult. There is a large variation in the level of resistance among cabbage cultivars and breeding for host plant resistance might therefore to be a good option to reduce yield losses caused by thrips (Shelton et al. 1983; Ellis et al. 1994).

Figure 1.1: Thrips damage on a cabbage head

Since 2004, Plant Research International and the Louis Bolk Institute investigate different cultivars in field experiments to identify the plant traits and genes that are involved in thrips resistance. This knowledge will help to improve selection methods for purposes of plant breeding. The aims of this study were to get more information about the mechanism that is causing resistance and about the plant parts (frame leaf and cabbage head) in which the resistance mechanism is operational and about the life stages of thrips (adult and juvenile) that are affected by resistance. This information will help to identify the plant traits that are involved in thrips resistance. Hereafter the possibilities of each mechanism that may cause resistance in cabbage are briefly described.

Painter (1951) describe three main mechanisms that may cause resistance in plants. These are antibiosis, non-preference and tolerance. These are true resistance mechanisms in contrast to pseudo-resistance and indirect resistance that I will describe next. A plant may receive less damage by the pest insect if it is able to avoid the period with the largest density of pest insects. Cabbage plants, for example, are most vulnerable to thrips damage during head formation. If the cabbage head is forming before or after the period with the largest thrips densities it receives less damage and seems to be resistant, but this is not true resistance but pseudo-resistance. If a plant is better accessible for natural enemies or if it attracts more parasitoids of the pest insect, the plant may also receive less damage. Because this resistance is caused by another trophic level it is revered to as indirect resistance. ANTIBIOSIS

Antibiosis is the mechanism in which the host plant has an adverse effect on the survival, reproduction and/or development of an insect that is feeding on the plant (Painter 1951). The plant may, for example, have a lower nutritional quality or it produces growth inhibitors or toxins. Little or no attention have been paid to the role of antibiosis in thrips resistance in cabbage. About this subject no literature was found. The role of antibiosis in thrips resistance in cabbage will be investigated in this study.

NON PREFERENCE

Non-preference refers to the mechanism by which plants repel a pest species by chemical or physical plant traits. Stoner and Shelton (1988) showed in their field 5

experiments that initially clean plants from a susceptible cultivar had more thrips in their head after one week exposure to thrips than plants from a resistant cultivar. Since the time span of one week was too short for the thrips to reproduce, this difference in thrips number between cultivars was probably caused by non-preference. Tolerance is the ability of the plant to grow and reproduce itself or to repair TOLERANCE injury to a marked degree in spite of supporting a population approximately equal to that damaging a susceptible host (Painter, 1951). In the study of Voorrips et al. (2006) the size of the thrips population and the amount of damage were highly correlated. None of the ten cabbage cultivars had a remarkable low damage rating in relation to the number of thrips. The difference in damage between cultivars was thus probably not caused by tolerance. PSEUDO RESISTANCE

Stoner and Shelton (1988) studied whether a difference in timing of maturity could explain differences in level of resistance between cabbage cultivars, by comparing damage ratings of plants with different planting dates. Their experiments show that cabbage cultivars remain resistant or susceptible to damage by T. tabaci regardless of changes in timing of maturity. Voorrips et al. (2006) also tested the effect of planting date on the amount of damage in the head. They found, in contrast to Stoner and Shelton (1988) that a later planting date resulted in a reduction of damage in the heads of two susceptible, earlier varieties (Slawdena and Bartolo) but not in a resistant, later variety (Galaxy).

INDIRECT RESISTANCE

A large spectrum of natural enemies is known to attack thrips. They basically fall apart in two groups: arthropod predators and parasitoids and microbial entomopathogens (fungi and nematodes) (Loomans 2003). Anthocorid predatory bugs that belong to the genus Orius, and polyphagous predatory mites from the Phytoseiidae family are considered the most effective predators of thrips (Loomans 2003). The most common thrips parasitoid is Ceranisus menus (Eulophidae), but this species is only found occasionally in the Netherlands (Loomans et al. 1992). The occurrence of natural enemies of T. tabaci has been investigated on leek by Vierbergen and Ester (2000). The most common predators of T. tabaci observed were Heteroptera (Orius), Phytoseiidae, whereas hymenopterous parasitoids were absent. Theunissen (1997) concluded that the effect of natural enemies on populations of thrips is probably marginal and that their numbers are usually low.

RESEARCH METHODOLOGIES An in vitro life history study was done to see if this method could detect a difference in resistance between cultivars and to see whether resistance in cabbage is caused by antibiosis. The results of this experiment are presented in chapter 2.3. In a field experiment the thrips population was evaluated on both frame leaves and in the cabbage head to see which plant parts are involved in resistance. And to see which life stages (adults or juveniles) are affected by resistance both stages were evaluated. The results of this experiment are presented in chapter 3.3.1. In the field experiment the flight activity was also evaluated above the plants of a susceptible and highly resistant cultivar to get information about a possible difference in preference of thrips for the two cultivars. The traps were used with and without a specific thrips attractant that have been shown to increase the number of caught T. tabaci in a greenhouse culture and in the open field. To see if the traps with attractant would also attract more thrips in a cabbage field these traps were compared with the number of thrips caught on traps without attractant. The results of these experiments are presented in chapter 3.3.2. At last, the flight activity above the plants was related with the population size on the plants to see if the flight activity can be used to predict the population size on the plants. These results are presented in chapter 3.3.3.

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CHAPTER 2

LABORATORY LIFE HISTORY STUDY

INTRODUCTION The aim of a life history study is to investigate the growth, development and reproduction rate of a species in detail. The biology of insects can be studied on leaf disks in Petri dishes on wet cotton plugs or water agar. In this way the experimental conditions can be controlled and good observations on the insect’s development are possible. This introduction summarizes knowledge about handling and rearing thrips and about the biology of T. tabaci that is needed before a life history is started. At the end the aim and research questions are formulated. Table 2.1: Duration of the developmental stages of Thrips tabaci at 25°C on cucumber leaf disks (van Rijn et al. 1995). Life stage Egg Larva 1 Larva 2 Prepupa Pupa

N 100 78 62 60 58

Duration (days) 3.92 2.13 3.17 1.09 2.43

Egg to adult Pre-oviposition

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12.9 1.9

Egg to Egg

14.8 Figure 2.1: Life cycle of T. tabaci from egg to first and second instar larvae to prepupa, pupa and the adult.

Thrips tabaci life cycle consists of six stages: LIFE CYCLE AND REPRODUCTION STATEGY egg – two larval stages – two pupal stages and the adult stage (see figure 2.1). The eggs are laid inside the plant tissue by the ovipositor of the female. When the larvae hatch they immediately start to feed and get a green appearance due to ingested chlorophyll. The first instar moult into the second instar larvae, which may at first be smaller than the first instar just before moulding. The second instars grow fast and are more mobile than the first instars. When they are totally fed and have emptied their lumen, they seek a suitable place to moult into the first pupal stage and later into the second pupal stage. In both pupal stages thrips do not feed and hardly move. When the second pupae moult into an adult it first has a white-grey colour that later turns into a darker brownish colour. Most populations of T. tabaci consist only of females which reproduce by parthogenesis, however males have been found in the Netherlands in the field on garlic, leek, onion and shallot (Vierbergen and Ester, 2000). Because the development time of T. tabaci is relative short (see table 2.1) and because T. tabaci can reproduce by parthenogenis, it can fast build-up a large population in the field. LIFE HISTORY PARAMETERS Life history parameters of T. tabaci have been studied on several plant species including castor oil seedlings (Gawaad and Shazli, 1969), onion cataphyll leaves (Salas, 1994), and Emilia sagittata (Asteraceae) (Sakimura, 1932). Van Rijn et al. (1995) studied life history parameters of T. tabaci on cucumber leaf disks of which the

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developmental time of the life stages are presented in table 2.1. There is no previous work that studied thrips life history parameters on cabbage leaves. In the study of van Rijn et al. (1995) the mortality percentage for the development from egg hatching to adulthood was 19% and occurred mainly during the larval period. Salas (1994) found a mortality percentage of more than 58% on onion cataphyll leaves. Both authors mention that manipulation of the larval stages was a probable cause of the mortality. The number of hatched eggs per day per female of T. tabaci is at its maximum shortly after the start of the oviposition period (Lewis, 1973). Van Rijn et al. (1995) report an average rate of hatched eggs per day per female for the first two days of oviposition of 5.5 at 25 °C and a decline thereafter. In total, females produce 70 to 100 eggs during their oviposition period (Malais and Ravensberg, 1991). ADAPTATION OF THRIPS TO HOST PLANT

When insects are transferred from one host plant species to another host plant species, natural selection on the population and adaptation of insects may take place. Life history parameters for the first generations on the new host plant may therefore differ from the life history parameters of later generations. Transfer of an insect from one host plant species to another host plant species may give unreliable results concerning host plant resistance (Thomas, 1993). AIMS AND RESEARCH QUESTIONS LIFE HISTORY STUDY If antibiosis would be the dominant defense mechanism this would result in a higher mortality and/or longer development time of thrips feeding on leaves of resistant plants. Main aim of the life history study was to see if thrips resistance of cabbage can be detected in an in vitro experiment by measuring the mortality percentage and development time of thrips. Questions are: (1) Do thrips have a higher percentage of mortality and (2) a longer development time on leaf disks of a highly resistant cultivar compared to a susceptible cultivar?

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CHAPTER 3

LABORATORY LIFE HISTORY STUDY

MATERIALS AND METHODS The mortality percentage of larvae and the development time from egg to adult were determined for three generations of thrips on leaf disks from fully extended (frame) leaves from young greenhouse-reared cabbage plants of the thrips-susceptible cultivar Bartolo and the highly thrips-resistant cultivar Galaxy. The method that is used for the life history study was modified from van Rijn et al. (1995). The experiment was carried out in a climatized room (25±1ºC and ca. 70% RH) under long day conditions (L16:D8). All observations were done with a binocular, provided with a cold light source. HANDLING AND REARING THRIPS Thrips are not easily reared and manipulated in the laboratory. Especially the first instar larvae are very small (0.34 mm; Salas, 1994) and fragile to handle and may die as a result of the handlings. Adult thrips are 1.2 mm long (Salas, 1994) and less fragile, but are very active and need to be anesthetized to handle. There are several methods to rear thrips, for example in vials or Petri dishes or in larger cages. A cage may give problems with escaping thrips when the window does not close perfectly well. But in a cage thrips can be reared on living plants which will give fewer problems with fungi than vials or Petri dishes in which thrips are reared on excised plant materials. Gawaad and Shazli (1969) describe a method to rear Thrips tabaci in specially constructed boxes on castor oil seedlings. Temperature and humidity are important conditions that determine the success of the culture. At higher temperatures T. tabaci females produce more eggs and the percentage of eggs that hatch is higher (Lewis, 1973). Moreover, the development time of thrips is shorter at higher temperatures (16.1 days at 25 °C; 11.2 days at 30 °C, Harris et al., 1936). Thrips tabaci are best reared at a high relative humidity (> 60%, pers. comm. Loomans) and it is important to rear thrips in an environment with constant temperature and humidity to prevent the formation of condense to which the thrips may stick and die (Lewis, 1973). COLLECTING THRIPS

Thrips were collected from a population on onion plants and

transferred to cabbage plants to let the thrips adapt to its new host plant species. Females that emerged after development on cabbage were used for the life history study. Despite the many adults that were added (>600) to this culture, only about 40 adults could be collected after four weeks for the experiment. These adults were allowed to oviposit on leaf disks to obtain the first cohort of larvae. This population is referred to as ‘brassica’ population hereafter. Additionally to this population about two hundred adults, colleted from onion plants, were also allowed to oviposit on leaf disks. This population is referred to as ‘onion’ population. Both populations were kept separate during the experiment. Thrips collected from the onion plants for transfer to cabbage plants were anesthetized with carbon dioxide and were put in a small container through which a constant flow of carbon dioxide was blown. In this way, the thrips could be observed by a binocular. Thrips that did not look similar to T. tabaci (Malais and Ravensburg, 1991) were removed. PREPARATION OF LEAF DISKS The thrips were allowed to oviposit for six days. Each day the adults were put onto a new leaf disk. The disks, 40 mm in diameter, were taken from randomly chosen leaves from four to five weeks old plants. The dishes were covered with a cling film (Dutch: vershoudfolie) (King Nederland) that allowed some respiration, so that condensation was minimized. The leaf disks for the first generation larvae were put in 0.5% water agar (Agar no. 3) that was still warm when the leaves were added to make the bottom side of the leaf attach to the agar. Because the survival of the first generation larvae was very low, the leaf disks for the second and third generation were put on top of 1.5% water agar that

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was at room temperature. The concentration of agar was increased from 0.5% to 1.5% to prevent that the larvae would stick in the agar. MEASUREMENT OF MORTALITY AND DEVELOPMENT TIME

The larvae hatched four to nine days after oviposition and were transferred to new leaf disks (day 0). The larvae of the first generation were put with a maximum of ten larvae per disk. The larvae of the second and third generation were put with a maximum of five larvae per disk. Larvae were transferred with a one-hair paint brush under a binocular. For the first generation, developmental stage and mortality were determined on day five and ten. For the second and third generation developmental stage and mortality were determined daily from day eight onwards. Pre-pupae could be distinguished from larvae by there lighter colour, short wing sheaths and erect antennae. Pupae could be distinguished by longer wing sheaths and antennae that are bent backwards along the head. Both pre-pupae and pupae do not feed and hardly move. Adult thrips can be distinguished from pupae by their wings and darker skin colour. STATISTICAL ANALYSIS

The mortality percentage was calculated as (dead larvae, pupae and adults)*100 / (dead larvae, pupae and adults plus living adults). Juveniles (larvae + pupae) that were transferred to the leaf disk but were not found back were excluded from the calculation of the mortality percentage. See table B in the appendices for the number of missing juveniles. The proportion living adults to dead juveniles plus dead adults was compared between the two cultivars with a contingency table and a Chi-square test with continuity correction. With the same analysis the mortality percentage was compared between the three generations of one population and for each generation between the two populations. The development time was compared between the two cultivars with a t-test. The number of replicates was too low (N