1 Living on an unfriendly plant host: impact of tomato on the predatory mite Amblyseius swirskii ANGELIKI PASPATI Supervisors Alberto Urbaneja Joel González-Cabrera November 2019 2 3 Doctoral Programme in Sciences Universitat Jaume I Doctoral School Living on an unfriendly plant host: impact of tomato on the predatory mite Amblyseius swirskii Report submitted by ANGELIKI PASPATI in order to be eligible for a doctoral degree awarded by the Universitat Jaume I Angeliki Paspati Supervisors Alberto Urbaneja Joel González-Cabrera Castelló de la Plana, November 2019 4 Funding This work was funded by the European Union‟s Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie grant agreement No 641456. 5 To the ones that inspire us 6 7 Acknowledgments First of all, I would like to express my gratitude to my supervisors Alberto Urbaneja and Joel González-Cabrera for giving me the opportunity to do this PhD and guiding me and encouraging me during this long journey. Many thanks to Dr. Antonio Granell, Dr. José Luis Rambla and Dr. María Pilar Lopez Gresa from the Institute for Plant Molecular and Cellular Biology “IBMCP” for their support during our collaboration on the identification of acyl sugars. Also, I am very thankful to Jóse, Miquel, Carolina, Maria and Giorgos for their technical support and their valuable help with experiments. Also, I am very grateful that I had the opportunity to be part of the Entomology Group of IVIA, a group of wonderful colleagues to make research with, discuss ideas, receive advice and gain knowledge. Special thanks to Milena Chinchilla, a very good companion and friend the past four years, with whom, I was more than lucky to participate in the same project and share the most exciting experiences of this journey. I would like to thank my colleagues and good friends, Jessi, Alice, Azu, Paco, Francesc, Juan Pedro, Marta, Miguel, Omar, Sara, Carlos, Carolina and Maria that now I feel like my family in Valencia, for bringing joy, laughters, true companionship, affection and lots of support in my daily life at work, all those years. Many thanks to Angelos for his art, that decorates the cover of this book. Last, my grateful thanks to all my good friends and family for their moral support, encouragement and infinite love. 8 9 Summary Integrated pest management programs based on the augmentation and conservation of phytoseiid predators, natural enemies of agricultural pests, are available for many crops worldwide. A valuable player in biological control of key greenhouse pests, such as the whiteflies Bemisia tabaci Gennadius (Hemiptera: Aleyrodidae), is the predatory mite Amblyseius swirskii Athias-Henriot (Acari: Phytoseiidae). However, on tomato crops, the release of A. swirskii does not provided effective control of the pests, because it cannot become established. The impact of the plant traits on A. swirskii biological parameters has not been explored in depth and the tomato metabolites affecting the phytoseiid establishment are not known. The first objective of this PhD was to investigate the biological parameters of A. swirskii which are mostly affected on tomato plants and the tomato trichomes‟s secondary metabolites with the highest negative impact on the establishment of this predatory mite. It was found, that the survival of adult mites attempting to disperse on the tomato plant was significantly reduced, due to the impact of trichome secondary metabolites, present in high concentration on the stems. Among the secondary metabolites detected in tomato trichomes, the acyl sugars were most likely involved in mite mortality. Moreover, this study showed the attachment and allocation of the acyl sugars on the body of the phytoseiids, after walking on the plants. Altogether, these results open perspectives to further investigate the mechanisms underlining the detrimental effect of tomato acyl sugars on phytoseiids and their prey, the herbivores. Currently, cultivars used for tomato production are often selected for high density of trichomes, since they show increased tolerance to herbivore attacks; however, they can be harmful to predatory mites, as shown in this study. The second objective of this PhD was to investigate the dispersal ability of A. swirskii on three tomato mutants with different trichome composition: plants without type I trichomes (hairs absent), plants with excess of type I 10 trichomes (woolly), and plants with all types of trichomes distorted (hairless). Moreover, in the second objective of this study, the efficiency of A. swirskii and Phytoseiulus persimilis Athias-Henriot (Acari: Phytoseiidae) to control respectively, two tomato key pests, the whiteflies and the spider mites on hairless tomato plants was addressed as well. On the hairless plants, the dispersal ability of A. swirskii was higher than on wild-type plants, however, the negative effect of the trichome exudates was still present. The herbivore pests, whiteflies and spider mites were found to perform better on hairless tomato plants than on wild- type plants. The efficiency of the predatory mites, A. swirskii and P. persimilis on hairless tomato plants was poor, since they were not able to suppress the pest. The findings highlight the effect of the plant physiology on biological control based on phytoseiids, and also, the importance of taking this effect into account by tomato plant-breeding programs and addressing new lines of research for selecting more „friendly‟ plants to phytoseiids and other natural enemies. It is known that the tomato defences induce gene responses to herbivores; however, the responses of phytoseiids to tomato trichomes remain unknown. As third objective of this PhD, the gene response of A. swirskii to the tomato trichome exudates and also, three major detoxification gene sets, the cytochromes P450 (CYPs), the glutathione S-transferases (GSTs) and the carboxyl/cholinesterases (CCEs) were identified and characterized. Thirty-nine of A. swirskii genes were found differentially expressed after transfer to tomato leaves, when compared to pepper leaves, a favourable host plant for A. swirskii, and some of the expressed genes were associated with the metabolism of tomato exudates. Moreover, it was shown that many members of the detoxification gene sets CYPs, GSTs and CCEs are present in A. swirskii, but do not play a significant role when in contact with the tomato exudates. In addition, more than 45,000 unigenes of A. swirskii were successfully annotated, based on known genes, after the de novo transcriptome assembly was conducted. These results suggest 11 that the gene responses of the phytoseiid mites are very different from what has been found for the herbivore mites. The impact of the conditions in mass rearing facilities, on the genetic diversity of commercial biocontrol agents, such as the phytoseiids, is not known. The forth objective of this PhD was to investigate the effect of long-term mass rearing on the genetic diversity and differentiation of commercial A. swirskii. Eight wild and one commercial population of A. swirskii were compared and the results indicated that the commercial population is less heterozygous and has higher genetic differentiation than their wild counterparts. The implications of reduced genetic variation in this biocontrol agent in their performance when released to control pests were discussed within this chapter. In summary, in this study it is concluded that the acyl sugars produced by the tomato trichomes are detrimental for the phytoseiid A. swirskii, and this information can serve as the basis for future research in plant-breeding programs, aiming to successful application of phytoseiids for tomato crop protection. Also, the demonstration of the impact of current mass rearing practices on the genetic variation of A. swirskii, suggest the revision and innovation of these practices, in order to achieve utmost performance of biocontol agents in pest management. 12 Resumen Los programas integrados de manejo de plagas basados en el aumento y la conservación de depredadores fitoseidos, enemigos naturales de las plagas agrícolas, están disponibles para muchos cultivos en todo el mundo. Un enemigo natural clave en el control biológico de plagas en cultivos de invernadero, como la mosca blanca Bemisia tabaci Gennadius (Hemiptera: Aleyrodidae), es el ácaro depredador Amblyseius swirskii Athias-Henriot (Acari: Phytoseiidae). Sin embargo, en los cultivos de tomate, la liberación de A. swirskii no proporciona un control efectivo de las plagas, ya que no puede establecerse. El impacto de las defensas de la planta en los parámetros biológicos de A. swirskii no se ha explorado en profundidad y se desconocen los metabolitos del tomate que afectan el establecimiento de los fitoseidos. El primer objetivo de este doctorado fue investigar los parámetros biológicos de A. swirskii que se ven afectados principalmente en las plantas de tomate, y encontrar los metabolitos secundarios de los tricomas del tomate con el mayor impacto negativo en el establecimiento de este ácaro depredador. Se descubrió que la supervivencia de los ácaros adultos que intentaban dispersarse en la planta de tomate se redujo significativamente, debido al impacto de los metabolitos secundarios de los tricomas, presentes en altas concentraciones en los tallos. Entre los metabolitos secundarios detectados en los tricomas de tomate, los acil azúcares probablemente estuvieron involucrados en la mortalidad de los ácaros. Además, en este estudio pudimos mostrar como los acil azúcares se pegan y distribuyen en el cuerpo de los fitoseidos, después de caminar sobre las plantas. En conjunto, estos resultados ofrecen perspectivas para investigar más a fondo los mecanismos que subyacen en el efecto perjudicial de los acil azúcares del tomate en los fitoseidos y sus presas, los herbívoros. 13 Actualmente, los cultivares utilizados para la producción de tomate se seleccionan a menudo para la alta densidad de tricomas, ya que muestran una mayor tolerancia a los ataques de herbívoros; sin embargo, esta selección puede ser dañina para los ácaros depredadores, como se muestra en este estudio. El segundo objetivo de este doctorado fue investigar la capacidad de dispersión de A.