ir. Gil Luypaert ir. hybrid simsii and its interactions with pot azalea, with its interactions and , latus Polyphagotarsonemus , broad The

The broad mite, Polyphagotarsonemus latus, and its ir. Gil Luypaert interactions with pot azalea, Rhododendron simsii hybrid 2015

Promotors:

Prof. dr. ir. Patrick De Clercq Department of Crop Protection, Faculty of Bioscience Engineering, Ghent University, Belgium

Dr. ir. Jan De Riek Sciences Unit, Department of Applied Genetics and Breeding, Institute for Agricultural and Fisheries Research, Belgium

Prof. Dr. Martine Maes Plant Sciences Unit, Department of Crop Protection, Institute for Agricultural and Fisheries Research, Belgium

Dean:

Prof. dr. ir. Guido Van Huylenbroeck

Rector:

Prof. dr. Anne De Paepe

The broad mite, Polyphagotarsonemus latus , and its interactions with pot azalea, Rhododendron simsii hybrid

by ir. Gil Luypaert

Thesis submitted in fulfilment of the requirements for the degree of Doctor (PhD) in Applied Biological Sciences Dutch translation:

De begoniamijt, Polyphagotarsonemus latus (: ), en zijn interacties met potazalea, Rhododendron simsii hybride

Please refer to his work as follows:

Luypaert G (2015) The broad mite, Polyphagotarsonemus latus (Acari: Tarsonemidae), and its interactions with pot azalea, Rhododendron simsii hybrid. PhD Thesis, Ghent University, Ghent, Belgium

Frontcover: Scanning electron image from a Polyphagotarsonemus latus female (photo: author)

Backcover: Top left, infested azalea flower; top right, infested azalea shoot tip; bottom left, azalea flowers with different colors and bottom right, scanning electron image from a Polyphagotarsonemus latus female (photos: author)

Cover design by Inge Coudron

ISBN-number: 978-90-5989-826-4

The research was conducted at the Institute for Agricultural and Fisheries Research, Plant Sciences Unit – Applied Genetics and Breeding, Caritasstraat 21, B-9090 Melle, Belgium

The author and promoters give the permission to use this study for consultation and to copy parts of it for personal use only. Every other use is subject to copyright laws. Permission to reproduce any material contained in this work should be obtained from the author. Acknowledgements

Finally, I can present my PhD, but sincerely this thesis reflects the work of many others who supported and guided me during four intense years of hard working. To express my gratitude to all the co-workers, however, it might not be enough, I would like to acknowledge them in their own language. Vooreerst wil ik mijn promotoren bedanken voor de vele steun in het onderzoek. Prof. dr. ir. Patrick De Clercq, jou wil ik speciaal bedanken om mij als doctoraatsonderzoeker op te nemen in de vakgroep Gewasbescherming. Ondanks de afstand tussen onze bureaus (jij in Gent en ik in Melle) was jij er altijd voor mij, van het lezen en verbeteren van papers, tot het uitstippelen en bediscussiëren van experimenten in de week, zelfs in het weekend en naar mijn vermoeden ook gedurende je vakanties. Ik wil je voor al je werk en aanmoedigingen nogmaals heel hard bedanken! Dr. ir. Jan De Riek en Prof. Dr. Martine Maes, mijn respectievelijke eerste en tweede evaluator op ILVO maar beide tevens ook co-promotor van deze thesis, jullie wil ik bedanken voor de vrijheid die ik kreeg binnen mijn onderzoek. Hierdoor kon ik naast het projectwerk ook dieper ingaan op bepaalde aspecten die geleid hebben tot deze thesis. Daarnaast wil ik ook Dr. ir. Johan Van Huylenbroeck speciaal bedanken voor de steun en de tijd die je steeds voor mij vrij maakte als ik weer eens langs kwam met een “vraagje”. Johan, naast het wetenschappelijke luik was jij ook op menselijk vlak een grote steun, dankzij jou heb ik mijn project en people management skills sterk kunnen ontwikkelen. Ing. Johan Witters, bedankt voor alle hulp tijdens het mijtenwerk. Het op bepaalde momenten 24 uur per dag klaar staan voor mijn experimenten, zal ik niet vergeten en ik ben je hiervoor enorm dankbaar. Verder wil ook uitdrukkelijk Dr. ir. Ellen De Keyser, Dr. ir. Emmy Dhooghe, Dr. ir. Tom Ruttink, Dr. apr. Christof Van Poucke en Dr. ir. Els Van Pamel bedanken voor de fijne samenwerkingen en hun hulp in het expressiewerk, RNAseq analyse en de hormoonanalyses. Robrecht, bijna drie jaar mijn bureaugenoot, bedankt voor de goede verstandhouding. Ik wens je veel succes met het afwerken van jouw doctoraat. Ik zou ook nog een speciale dank willen uitbrengen aan Magali, mijn teammaatje voor het labowerk binnen het project. Bedankt voor al het werk in het labo dat jij uit handen nam. Verder nog een grote dank aan Laurence, die met haar ervaring in het labo een echte rots in de branding was. Jorien, Veerle en Sophie, de hulp die jullie in het labo boden apprecieer ik erg hard, bedankt hiervoor! Evelien en Romain de azalea veredelaars bij uitstek, jullie bedank ik om mij de azaleateelt in de praktijk bij te leren. Dieter, Laurette en de andere medewerkers in de serre, bedankt voor het stekken, opgroeien en vooral het met veel liefde behandelen van mijn planten. Ook op het PCS, de projectpartner, wil ik nog enkele mensen bedanken. Bart en Els M mijn respectievelijk, vroegere en huidige onderzoekspartner en ten slotte azaleakenner Els P. Daarnaast wil ook nog alle andere collega’s en vrienden bedanken voor de fijne en aangename samenwerking op de verschillende departementen, afdelingen en/of eenheden waar ik gedurende vier jaar regelmatig langs kwam, zowel op ILVO, the Laboratory of Agrozoology, UGent als het PCS. Een woordje van dank gaat ook uit naar het IWT en de co-financiers van project die naast hun financiële steun ook hun volle vertrouwen en interesse gaven. Dr. ir. Kristiaan Van Laecke, bedankt om mij destijds de kans te geven om mijn wetenschappelijke carrière te laten starten met dit project. Tot slot nog een welgemeende dank-u-wel aan mijn ouders, broer, Anneliesje, (schoon)familie en vrienden. Vake en moeke, ik kan jullie echt niet genoeg bedanken voor alle steun, opofferingen, tijd en moeite die jullie in mij geïnvesteerd hebben en nog steeds doen.

Gil Luypaert, juni 2015

Table of contents

List of abbreviations ...... v

Chapter 1 Introduction, objectives and thesis outline ...... 1 1. Introduction, objectives and thesis outline ...... 2 1.1. Introduction ...... 2 1.2. Objectives ...... 2 1.3. Thesis outline ...... 3

Chapter 2 A literature review ...... 5 1. Introduction ...... 6 2. The broad mite, Polyphagotarsonemus latus ( Banks) ...... 6 2.1. and distribution ...... 6 2.2. Biology and life cycle ...... 8 2.3. Morphology ...... 11 2.4. Host spectrum and damage ...... 15 2.5. Control ...... 18 2.5.1. Plant resistance ...... 18 2.5.2. Chemical control ...... 19 2.5.3. Biological control ...... 21 2.5.4. Physical control ...... 22 3. The Belgian pot azalea, Rhododendron simsii hybrid (Planch) ...... 23 3.1. Taxonomy, origin and distribution ...... 23 3.2. Economic importance for Ghent region ...... 25 3.3. Cultivation process ...... 25 3.1. Breeding ...... 26 4. Plant defence regulated by the phytohormone jasmonic acid ...... 28 4.1. Plant defence ...... 28 4.2. Jasmonic acid biosynthesis and signalling ...... 29 4.2.1. Biosynthesis ...... 29 4.2.2. Signalling ...... 30

i 4.3. Functions of jasmonic acid in the plant ...... 32 4.4. Role of jasmonic acid in plant defence ...... 32 4.5. Crosstalk with other phytohormones in plant defence ...... 33 4.6. Salicylic acid biosynthesis and signalling ...... 34 4.6.1. Biosynthesis ...... 34 4.6.1. Signalling ...... 35

Chapter 3 Temperature-dependent development of the broad mite Polyphagotarsonemus latus (Acari: Tarsonemidae) on Rhododendron simsii ...... 39 1. Introduction ...... 40 2. Materials and methods ...... 41 2.1. Broad mite culture and plant material ...... 41 2.2. Broad mite development ...... 41 2.3. Mathematical models ...... 42 2.4. Temperature thresholds ...... 43 2.5. Model quality ...... 43 2.6. Statistical analysis ...... 43 3. Results ...... 44 3.1. Developmental time ...... 44 3.2. Model evaluation ...... 45 4. Discussion ...... 47

Chapter 4 Cold hardiness of the broad mite Polyphagotarsonemus latus (Acari: Tarsonemidae) ...... 51 1. Introduction ...... 52 2. Materials and methods ...... 53 2.1. Broad mite culture and plant material ...... 53 2.2. Survival and reproduction capacity following a cold exposure period ...... 53 2.3. Lower lethal times ...... 54 2.4. Supercooling points...... 55 2.5. Statistical analysis ...... 55 3. Results ...... 55 4. Discussion ...... 59

Chapter 5 Screening for broad mite susceptibility in Rhododendron simsii hybrids ...... 63 1. Introduction ...... 64 2. Materials and methods ...... 65 2.1. Plant material ...... 65 2.2. Broad mite development ...... 65 2.3. Scoring of cultivars and counting of broad ...... 66 2.4. Experiment 1 ...... 66 2.5. Experiment 2 ...... 67 2.6. Experiment 3 ...... 67 2.7. Scanning electron microscopy ...... 67 2.8. Statistical analysis ...... 68 3. Results ...... 68 4. Discussion ...... 76

Chapter 6 Selection and validation of plant defence related marker genes in Rhododendron simsii hybrids ...... 81 1. Introduction ...... 82 2. Materials and methods ...... 84 2.1. RNA-sequencing ...... 84 2.1.1. RNA isolation and RNA-sequencing ...... 84 2.1.2. De novo assembly ...... 84 2.1.3. Read mapping and gene expression analysis ...... 85 2.1.4. Functional categorization ...... 85 2.2. Cloning genes ...... 85 2.3. Experiments ...... 86 2.4. RNA extraction optimization ...... 87 2.5. RNA quality control ...... 88 2.6. DNase treatment and cDNA synthesis...... 88 2.7. Reference gene selection (geNorm) ...... 89 2.8. RT-qPCR ...... 89 2.9. Statistical analysis ...... 90

iii 3. Results ...... 91 3.1. RNA-sequencing ...... 91 3.2. Cloning genes ...... 92 3.3. RNA extraction optimization ...... 93 3.4. Reference gene selection (geNorm) ...... 96 3.5. Expression analysis ...... 96 4. Discussion ...... 103

Chapter 7 Hormonal changes and gene expression of jasmonic acid biosynthesis genes in response to Polyphagotarsonemus latus infestation in Rhododendron simsii hybrids ...... 109 1. Introduction ...... 110 2. Materials and methods ...... 111 2.1. Plant material and infestation ...... 111 2.2. Experiments ...... 111 2.3. RT-qPCR ...... 112 2.4. Isolation of phytohormones and analysis by means of LC-MS/MS ...... 112 2.5. Statistical analysis ...... 114 3. Results ...... 115 4. Discussion ...... 127

Chapter 8 Conclusion, general discussion and further prespectives ...... 131

Summary ...... 141 Samenvatting ...... 145 References ...... 149 Supplementary data ...... 179 Curriculum vitae – Gil Luypaert ...... 193

List of abbreviations a.i. active ingredient ABA Abscisic acid ACX acyl-CoA oxidase AOC Allene oxide cyclase AOS Allene oxide synthase BABA β-aminobutyric acid bp basepair BTH Acibenzolar-S-methyl ca. circa cDNA complementary DNA CDS Coding sequence CNRQ Calibrated Normalized Relative Quantity COI1 Coronatine Insensitive1 protein COR Coronatine CTAB Cetyl trimethylammonium bromide DAMP Damage-associated molecular pattern DGE Differential gene expression DMM Dimethoxymethane DNA Deoxyribonucleic acid dn-OPDA dinor-oxophytodienoic acid dpi Days post infestation DTT Dithiothreitol ESI Electrospray ionization ETI Effector triggered immunity EU European Union FC Fold change GA Gibberellin HAMP Herbivore-associated molecular pattern ICS Isochorismate synthase ILVO Instituut voor Landbouw- en Visserijonderzoek (Institute for Agricultural and Fisheries Research) IPM Integrated Pest Management JA Jasmonic acid JA-Ile Jasmonoyl-Isoleucine JAZ Jasmonate ZIM-domain-containing protein JMT Jasmonic acid carboxyl methyltransferase K Thermal constant KAT L-3-ketoacyl-CoA thiolase LC-MS/MS Liquid chromatography-tandem mass spectrometry LOX Lipoxygenase

v MeJA Methyl jasmonate MFP multifunctional protein MIQE Minimum Information for Publication of Quantitative Real-time PCR Experiments MRM Multiple reaction mode mRNA messenger RNA n number of samples NF Normalisation factor NGS Next-generation sequencing noRT no Reverse Transcriptase NTC No template control OPDA 12-oxophytodienoic acid OPR3 12-oxophytodienoate reductase 3 ORF Open reading frame PAL Phenylalanine ammonia-lyase PAMP Pathogen-associated molecular pattern PCR Polymerase Chain Reaction PPO Polyphenol oxidase PR Pathogenesis-related protein PTI Pathogen triggered immunity PVP Polyvinylpyrrolidone PVPP Polyvinylpolypyrrolidone R0 Net productive rate RH Relative humidity rm Intrinsic rate of increase RNA Ribonucleic Acid RPKM Reads per kilo-base per million reads mapped RQI RNA Quality Indicator rRNA ribosomal RNA RT-qPCR Reverse Transcriptase quantitative real-time PCR SA Salicylic acid SCP Supercooling Point SDS Sodium dodecyl sulfate SEM Scanning Electron Microscopy tRNA transfer RNA (U)HPLC (Ultra) High-Performance Liquid Chromatography WT Wild type

Chapter 1

Introduction, objectives and thesis outline

1. Introduction, objectives and thesis outline 1.1. Introduction In Belgium, Polyphagotarsonemus latus (Banks) (Acari: Tarsonemidae), is a key pest in ornamental plant production, particularly in pot azaleas, Rhododendron simsii (Planch) hybrids. Feeding by the pest on the youngest growth tips causes malformation of the flower buds and leads to down curling and browning of the terminal leaves (Labanowski and Soika, 2006). The pest status of P. latus significantly increased during last years in R. simsii , mainly due to restrictions on the use of broad spectrum pesticides, including the complete prohibition of the use of endosulfan (an active component in many plant protection products) from July 2007 onwards (EU directive 2005/864/EC). Integrated Pest Management (IPM) and its EU-wide implementation (EU directive 2009/128/EC) forces growers to develop sustainable strategies for the control of this pest (Gobin et al., 2011, 2013). In an IPM based strategy, preventative cultural practices are essential during the growing process. One of these measures includes developing and growing resistant R. simsii cultivars. Genotypes resistant to P. latus already have been identified in various crops (e.g. Gibson and Valencia, 1978; Ahmed et al., 2001; Kousik et al., 2007; Jana et al., 2007). Further, IPM is based on a multidisciplinary approach. Biological control, which is the use of a living organism to reduce the population density of another organism, provides another opportunity to control P. latus . Different phytoseiid mites are known to prey on P. latus, including swirskii (van Maanen et al., 2010; Onzo et al., 2012) and A. largoensis (Rodríguez et al., 2011a). Hence, the availability of basic biological information on P. latus is essential for optimizing the biological control of this pest in the production of R. simsii hybrids. Besides optimizing the biological control of P. latus , biological parameters such as the lower developmental thresholds and thermal constants are not only good predictors of the pest’s phenology (Honek and Kocourek, 1988), but are also very useful indicators for the potential distribution of the pest (Campbell et al., 1974). An alternative approach to control mite pests involves activating plant defences to reduce mite vigour and/or mite preference for a crop (Rohwer and Erwin, 2010). It has already been shown that the jasmonic acid (JA) pathway plays a central role in the resistance response against P. latus (Grinberg et al., 2007). Exogenous application of phytohormones successfully reduced infestations by another key pest mite, the two-spotted spider mite, Tetranychus urticae, on different plant (e.g. Omer et al., 2001; Rohwer and Erwin, 2010; Warabieda and Olszak, 2012).

1.2. Objectives The main objective of this thesis was to provide a better understanding of the biology of one of the most important pests in R. simsii hybrid production in Belgium, the broad mite P. latus and to investigate the role of defence responses in this ornamental plant against the pest. These objectives can be translated into the following research questions:

1. What is the impact of temperature on the development and survival of P. latus ? 2. Is there genetic variation in R. simsii hybrids towards susceptibility for P. latus ?

Chapter 1

3. Is there a role for JA induced defence responses in R. simsii hybrids?

1.3. Thesis outline A literature overview is presented in Chapter 2 , providing information on the basic biology of the broad mite, P. latus , with focus on taxonomy and distribution, biology and life cycle, morphology, host spectrum and damage, and control by plant resistance, or by chemical, biological or physical measures. This chapter also includes an introduction to R. simsii hybrids, one of the many host of P. latus and the main plant used in the presented research. A short introduction on the biosynthesis and signalling of JA, and the role of this phytohormone in plants also is given. In the next two research chapters, the impact of different temperatures on P. latus is evaluated. Chapter 3 investigates the temperature-dependent development of P. latus on R. simsii ‘Nordlicht’. Survival and development rates of P. latus juveniles are measured and the relationship between temperature and development of each life stage is modelled. In Chapter 4 the cold hardiness of the pest is evaluated. This chapter focuses on survival and reproductive performance of P. latus upon cold treatments that mimic cold storage of R. simsii plants during plant production. Chapter 5 reports the results of multiple screenings of R. simsii genotypes for broad mite susceptibility. Infestation of six genotypes is studied in detail and broad mite counts and damage rates at different time points are recorded. Leaf hair types of these plants are investigated by scanning electron microscopy. The final two research chapters focus on molecular aspects of the R. simsii plant defences and of the plant-mite interaction. In Chapter 6 a molecular toolbox is constructed and validated to measure gene expression in R. simsii hybrids. Validation is based on selecting a potent JA elicitor and the effect on JA biosynthesis genes in leaves is studied after local and systemic induction by this elicitor. Besides evaluation of local induction by a JA inducer, the effect of wounding is evaluated in a selection of six R. simsii genotypes. Chapter 7 includes molecular research on the induction of JA biosynthesis genes in R. simsii hybrids upon broad mite infestation. Whereas P. latus infestation of R. simsii ‘Nordlicht’ is monitored in detail, effects of its infestation are also evaluated in six R. simsii genotypes with a different susceptibility for the pest. Finally, in Chapter 8 a general discussion and further perspectives are given.

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Chapter 2

A literature review

1. Introduction Restrictions on the use of broad spectrum pesticides, including the complete prohibition of the use of endosulfan (an active component in many plant protection products) from July 2007 onwards (EU directive 2005/864/EC) increased growers’ awareness of different pests and diseases present in their crops. In recent years, the pest status of the broad mite, Polyphagotarsonemus latus (Banks), a key pest in numerous ornamental and vegetable crops, has significantly increased in the production process of the Belgian pot azalea, Rhododendron simsii hybrid (Audenaert et al., 2009). An overview of this pest and one of its host plants, R. simsii , is given in the following paragraphs.

2. The broad mite, Polyphagotarsonemus latus ( Banks) 2.1. Taxonomy and distribution The taxonomic classification of P. latus according to Krantz and Walter (2009):

KINGDOM Animalia PHYLUM Arthropoda SUBPHYLUM CLASS Arachnida SUBCLASS Acari SUPERORDER ORDER SUBORDER SUPERCOHORT Eleutherengonides COHORT SUPERFAMILY Tarsonemoidea FAMILY Tarsonemidae SUBFAMILY Pseudotarsonemoidinae GENUS Polyphagotarsonemus SPECIES Polyphagotarsonemus latus Banks

The broad mite, Polyphagotarsonemus latus (Banks) (Acari: Tarsonemidae) initially collected on tea in Sri Lanka (then Ceylon) was described and named Acarus translucens Green in 1890 (Gerson, 1992). Independently this mite was collected from mango shoots in Washington (DC) in 1904 and was named latus Banks (Banks, 1904). The later name became accepted upon the realization that A. translucens was preoccupied (Gerson, 1992). After having been placed in the genus Hemitarsonemus (Ewing, 1939) and the new genus Polyphagotarsonemus (Beer and Nucifora, 1965), Lindquist (1986) redefined and fully redescribed both Polyphagotarsonemus and latus and suggested that this taxon may actually be a species complex.

Chapter 2

P. latus is a pest of many commercial crops and has been reported from most parts of the world, including tropical and subtropical climates of Australia, Asia, Africa, North America, South America and the Pacific Islands (Fig 2.1). In temperate and subtropical areas the broad mite is a pest of greenhouse plants (Waterhouse and Norris, 1987). More recently, Vincent et al. (2010) suggested that P. latus can also be a pest in subtropical climates or controlled environments. According to Navajas et al. (2010), P. latus was first recorded in Europe (Italy) in 1961. Since then, the mite has been found in other European countries including Belgium, where it was reported for the first time on Rhododendron simsii hybrids or pot azaleas in the mid-1980s (Heungens, 1986).

(a)

(b)

Fig 2.1 (a) Distribution of Polyphagotarsonemus latus in the world. (b) Detailed distribution of P. latus within Europe. Both maps are adapted from CABI (www.cabi.org). As the broad mite is widespread many other common names are known. Most of these names refer to its colour, host plant or geographical origin; chilli mite, white mite, yellow tea mite, yellow jute mite, citrus silver mite, tropical mite or begonia mite (Gerson, 1992; Zhang, 2003; Kamruzzaman et al., 2013).

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2.2. Biology and life cycle Eggs are deposited by adult females mainly next to leaf veins on the abaxial side of leaves. These eggs hatch into larvae which then enter a quiescent stage, named the nymphal life stage. After passing three immature life stages, P. latus enters the adult stage. At this life stage sexual dimorphism exists, males or females emerge from the inactive nymphs (Fig 2.2). The adult sex ratio favours females (1:4, male:female) although it may fluctuate (Gerson, 1992). Under dry conditions many female immatures do not reach adulthood, and males become more abundant (Jones and Brown, 1983). The reproduction of P. latus occurs as in many other tarsonemid species by arrhenotoky (Gadd, 1946; Kabir, 1979; Jones and Brown, 1983). In this system, mated females are able to produce both males and females (ca. 40 eggs). In contrast, unmated females only produce males, with a considerable reduction in fecundity (on average 7.8 eggs) (Flechtmann and Flechtmann, 1984). To maintain the population, unmated females can mate with their sons, in that way they may produce female offspring (Karl, 1965).

Pharate females, which are females waiting to emerge from their old cuticle, are visited actively by males. Once located males carry the pharate females up to 24 hours using a large and conspicuous sucker-like organ. This structure is located on the posterior dorsal side surface of males and has a hollow centre. A sticky secretion which might be secreted in different slits on the outside of the sucker organs assists in adhesion and carriage of pharate females (Baker, 2012). Hind legs are not used to carry the pharate female but may be used for initial manipulation (Martin, 1991). These legs are also used for placing females at right angles to the male’s body for later mating (Peña and Campbell, 2005). Mating occurs immediately after female emergence, it lasts 15-120 seconds while the male and female face in opposite directions (Nucifora, 1963). At 25 °C, the net productive rate (R 0) is 16.53, 17.58 and 30.12 females/female on leaves of Capsicum annuum , Citrus aurantifolia and Vitis vinifera, respectively. The corresponding intrinsic rates of increase (r m) are 0.30, 0.43 and 0.31 females/female/day , respectively (Hugon, 1983; Silva et al., 1998; Ferreira et al., 2006).

There are no reports of the existence of diapause within the family of Tarsonemidae (Hoy, 2011). Total development of P. latus was estimated to require around 5 days at 25 °C (Karl, 1965; Hugon, 1983). At this temperature total development on C. annuum was completed in 2.8-4.3 days and 2.8-5.0 days for males and females, respectively (Sombatsiri, 1979). Further studies provided more detailed information on the suitability of temperatures for development of P. latus . But only a limited number of these studies evaluated mite development covering multiple temperatures, including Jones and Brown (1983), Li et al. (1985) and Ferreira et al. (2006). A linear model was used to study the relation between temperature and developmental rate on chilli pepper (Li et al., 1985) and V. vinifera (Ferreira et al., 2006). However, none of these studies determined this relationship by fitting experimental data to non-linear models. Nonetheless, several non-linear models, as summarized by Kontodimas et al. (2004), are able to estimate critical temperatures and thermal budgets for understanding the phenology of .

Chapter 2

Egg

Adult Larva female

Nymph

Adult male

Fig 2.2 Life cycle of Polyphagotarsonemus latus (photos: author). The total development on C. annuum at 15, 20, 22.5, 25 and 30 °C took 13.3, 6.3, 5.1, 4.2 and 2.7 days, respectively (Li et al., 1985). On V. vinifera , total development was completed after 7.3, 5.1, 4.4, 3.3 and 3.5 days at 18, 22, 25, 28 and 32 °C, respectively (Ferreira et al., 2006). Development on Carica papaya at 25 °C was somewhat slower, taking 4.9 days (De Coss et al., 2010). On cotton ( Gossypium hirsutum ) average developmental period was measured to be 4.1 days at 28.5 °C for both males and females (Vieira and Chiavegato, 1998), while on C. limon development took 3.6 days for males and 3.7 days for females at 27.5 °C (Vieira and Chiavegato, 1999). Differences in development times between both sexes have been observed. At 30 °C, the egg-adult period on C. annuum took 3.4 and 3.6 days for males and females, respectively (Silva et al., 1998). Male development on C. annuum was completed in 5.1 and 4.3 days while female development took 5.8 and 4.6 days at 22.5 and 27.5 °C, respectively (Singh and Dhooria, 2006). Under uncontrolled thermal conditions on mulberry ( Morus sp.), egg development took 27-32 hours, larval development averaged 36 hours and nymphal development took between 6 and 8 hours (Rajalakshmi et al., 2009). The latter development times appear to be shorter than those reported elsewhere in the literature and might be related to the selected host plant and the uncontrolled temperature regime in this study. In contrast, longest egg to adult developmental times were found on C. annuum and Cucumis sativus at 24 °C, averaging

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7.8 days for both sexes and 8.7 or 7.2 days for males or females, on the respective host plants (Montasser et al., 2011a).

Besides host plant, differences in reported developmental times of P. latus may be related to the relative humidity regime. This hypothesis is supported by the wide range of relative humidities (RH) used in the different studies (65% to 90%). Jones and Brown (1983) reported variation in theoretical population growth of P. latus on C. limon fruits at constant temperatures when subjected to different RH regimes. On C. papaya developmental times differed significantly upon RH regimes (De Coss et al., 2010). Temperatures above 30 °C coupled with low humidity appeared to be lethal to the egg and nymphal life stages (Jones and Brown, 1983). Variation in light conditions might also contribute to differences in developmental time as low light intensity was stated to be beneficial (Bassett, 1985).

The lower developmental threshold for P. latus feeding on V. vinifera, was estimated to be 9.7 °C (Ferreira et al., 2006). Using a multiple regression curve of development data, Jones and Brown (1983) extrapolated the lower and upper theoretical thresholds of P. latus feeding on fruits of C. limon to be between 12 and 14 °C, and 33 and 35 °C, respectively, depending on the humidity. Li et al. (1985) reported no complete development at 35 °C as all tested larvae died at this temperature. So far, only a few studies have attempted to estimate thermal budgets for P. latus development. The thermal constant (K), indicating the thermal budget (in degree-days) needed to complete development, was estimated to be 62.7 degree-days for total development of P. latus on V. vinifera (Ferreira et al., 2006). The thermal budget for development on chili pepper was predicted to be 64.1 degree- days (Li et al., 1985). In their calculations Li et al. (1985) included the preoviposition period (i.e. the period between adult emergence and the first egg laid).

Associations between P. latus and plant-feeding insects have been described, including green peach aphid, Myzus persicae (Sulzer) (Smith, 1935), sweet potato whitefly, Bemisia tabaci (Gennadius) = Bemisia gossypiperda (Misra and Lamba) (Gupta and Chaudhry, 1972; Natarajan, 1988; Flechtmann et al., 1990), silverleaf whitefly, Bemisia argentifolii (Bellows and Perring) = B strain of B. tabaci (Fan and Petit, 1998) and greenhouse whitefly, Trialeurodes vaporariorum (Westwood) (Parker and Gerson, 1994). From these studies it became clear that P. latus mites can attach themselves to the tibiae and tarsi of the insects and use them as a carrier for their dispersal. This process is called phoresy and has been described as a phenomenon in which one , the phoront, actively seeks out and attaches to an animal of another species, the phoretic host, for dispersal (Faryish and Axtell, 1971). Phoretic association may be non-specific, with the phoront potentially using a large range of phoretic hosts, or specific, with preference towards a family, genus or even a single species (Athias-Binche, 1991).

Parker and Gerson (1994) found the mite to be phoretic on T. vaporariorium but not on allate M. persicae . This was supported by Palevsky et al. (2001) who found that different

Chapter 2 genera of whiteflies ( Trialeurodes, Dialeurodes, Bemisia and Aleyrodes ) served as phoretic hosts for P. latus whereas other winged insects of the same host plants, namely thrips (Frankliniella occidentalis (Pergande)) and aphids ( M. persicae or Aphis gossypii (Glover)) appeared not to be a favourite carrier to P. latus . Palevsky et al. (2001) suggested that olfaction could be one of the main mechanisms that P. latus uses for seeking out and attaching to the phorectic hosts. This was confirmed by the fact that waxy particles of Aleyrodes singularis (Danzig) but not of the mealybug, Planococcus citri (Risso), caused a significant attraction of P. latus to wax treated patches within an hour after application (Soroker et al., 2003). Light conditions and sex of the host B. tabaci did not affect mite- vector recognition abilities (Soroker et al., 2003). Besides the use of a phoretic host, P. latus is able to reach new host plants by human transfer of infested plants (Gerson, 1992) or with winds (Aubert et al., 1981). However, passive wind dispersal is very limited (Soroker et al., 2003). Finally, P. latus is able to migrate actively. Smith (1935) reported that active mite individuals ‘crossed’ (presumably by walking) an 18-inch space between plants. Males carry pharate females upwards providing oviposition sites for females (Gadd, 1946). Phoretic mites as well as free-moving mites are capable of making active host choices but cues for host selection remain to be explored (Alagarmalai et al., 2009).

2.3. Morphology Eggs are elongated, oval and slightly elevated. According to Montasser et al. (2011b) eggs are on average 107 µm long and 64 µm wide. Martin (1991) reported a length of ca. 100 µm, Singh and Dhooria (2006) stated that eggs are 94 µm in length and 69 µm wide, while Baker (2012) reported that they are around 90 µm long and 60 µm wide. Eggs are covered with around 30 white dots (Baker, 2012) providing a reliable diagnosis as this trait is specific to eggs of P. latus (Martin, 1991). The dots on eggs of P. latus are referred to in the literature as “tubercles” (Martin, 1991; Singh and Dhooria, 2006), “scattered white tufts” (Faluso, 2010), “sacs” (Montasser et al., 2011b) or “discontinuous plastron” (Baker, 2012). These protuberances are ca. 10 µm long and 8.5 µm wide (Montasser et al., 2011b). Whereas Martin (1991) did not provide an explanation for the function of these structures, Montasser et al. (2011b) suggested a role in respiration. Baker (2012) describes this as plastron respiration. There is no evidence of a second prelarval cuticle inside the egg “shell” (Martin, 1991) (Fig 2.4a,f).

Eggs hatch into larvae of ca. 142 µm long and 72 µm wide (Montasser et al., 2011b), while they were measured to be 117.5 µm in length by Martin (1991). There is no sexual dimorphism in the larval stage. The larval body consists of three parts; propodosoma (ca. 42 µm long), metapodosoma (ca. 58 µm long) and opisthosoma (ca. 42 µm long) (Fig 2.3). On the dorsal and ventral surface eight and seven pairs of setae are present, respectively. Larvae possess three pairs of legs; two pairs situated more or less anterior and one pair posterior. Legs I, II and III are on average 34, 36 and 40 µm in length (Montasser et al., 2011b). The dorsal cuticle is marked with three dark lines like finger prints (Martin,

11

1991), the areas between these lines are formed by triangular or polygonal arrangements of tubercles (Montasser et al., 2011b) (Fig 2.4b).

Gnathosoma

Leg I

Propodosoma

Leg II

Metapodosoma

Leg III Opisthosoma

Fig 2.3 Schematic drawing of Polyphagotarsonemus latus larval body parts and indication of three bands in the cuticle of the dorsal side. Nymphs , the last life stage before becoming adult, show no sexual dimorphism. The nymph is elliptical in shape and is on average 120.5 µm long and 75 µm wide (Montasser et al., 2011b) or 165 µm long and 83 µm wide according to Singh and Dhooria (2006). Both ends of the nymph are more or less sharply pointed. Like in the larvae the body of nymphs consists of three well-defined parts; propodosoma (ca. 27.3 µm long), metapodosoma (ca. 56.8 µm long) and opisthosoma (ca. 36.4 µm long). Five pairs of setae are present on the ventral surface. Further, the nymph has four pairs of legs that adhere tightly to the nymphal body as long as it remains enclosed within the skin of the larva until the adult stage is formed. The dark lines dividing the dorsal larval cuticle disappear at the beginning of the molting by stretching of the cuticle (Montasser et al., 2011b) (Fig 2.4c).

Chapter 2

(a) (b)

(c) (d)

(e) (f)

Fig 2.4 Scanning electron microscopy images of Polyphagotarsonemus latus life stages. (a) egg, (b) larva, (c) nymph, (d) adult female, (e) adult male and (f) eggs (indicated by arrows) deposited by adult females on the abaxial side of Rhododendron simsii hybrid ‘Nordlicht’ (photos: author). The adult stage of P. latus is characterized by sexual dimorphism. Males measure 138.9 ± 7.8 µm in length and 74.9 ± 1.9 µm in width (Montasser et al., 2011b). Denmark 1980 measured the mite to be 168 µm in length and Perez-Otero et al. (2007) measured the length of P. latus to be 110 µm. According to Singh and Dhooria (2006) males are 175 µm long and 93 µm wide. The body of P. latus males can be divided in five parts. At the anterior proximity of the body the gnathosoma is located. It is ca. 34.2 µm long and 31.1 µm wide. The male main body is divided in the propodosoma, metadosoma and opisthosoma,

13

measuring 48.2 ± 1.9, 64.3 ± 1.4 and 26.3 ± 4.7 µm in length, respectively (Fig 2.5). Ventrally posterior to the opisthosoma the genital capsule is located. It consists of 2-3 triangular genital papillae and is on average 12.5 µm long and 15.6 µm wide. On the dorsal surface of males 10 pairs of setae are found while ventrally 6 pairs of setae are present. Polyphagotarsonemus latus males have four pairs of legs. The first three legs consists of five segments, i.e. coxa, femur, genu, tibia and tarsus. Leg I, II and III are 70.0 ± 2.4, 85.0 ± 1.4 and 107.5 ± 4.6 µm long, respectively. The fourth leg has only four segments, namely coxa (ca. 34.9 µm long), femur (ca. 17.4 µm long), genu (ca. 24.9 µm long) and tibiotarsus (ca. 17.4 µm long), and has a total length of 94.6 ± 4.4 µm. Leg IV is characterized by two long setae; a dorso-lateral seta of ca. 87.2 µm long and a ventro-lateral one of ca. 57.3 µm long. This leg terminates by a button-like claw and bends into a clasper-like organ. Leg I terminates in a single claw, while legs II and III terminate in a pair of claws and a membranous empodium (Montasser et al., 2011b) (Fig 2.4e).

Gnathosoma Leg I

Propodosoma Leg II

Metapodosoma

Opisthosoma Leg III

Genital capsule Leg IV

Fig 2.5 Schematic drawing of Polyphagotarsonemus latus male body parts. Females are 138.9 ± 5.5 µm long and 97.3 ± 5.6 µm wide according to Montasser et al. (2011b). However, Perez-Otero et al. (2007) reported the body length to be 160 µm while Singh and Dhooria (2006) mentioned an average body length of 200 µm. Compared to males, the body of P. latus females appears to be more oval. In general four different body parts can be distinguished. The gnathosoma is ca. 26.7 µm long and 22.7 µm wide (Montasser et al., 2011b). It contains two slender stylettiform chelicerae between non segmented triangular palps (Krantz, 1978; Zhang, 2003; Montasser et al., 2011b). Chelicerae are only slightly reversible (Jeppson et al., 1975) and approximate an extended length of 43 µm (Gui et al., 2001). Further, the body consists of a propodosoma (38.9 ± 3.3 µm long), metapodosoma (69.4 ± 1.6 µm long) and opisthosoma (33.3 ± 2.3 µm long) (Fig 2.6). Different structures are present on the female body. Among these, two stigmata are

Chapter 2 dorso-laterally located at the gnathosomal base. A pair of trichobatia (sensilla) appear at the base of clavate pseudostigmatic organs located dorsolaterally and distally between coxae I and II. On the ventral side a longitudinal median groove is seen. Females possess nine and seven pairs of setae on the dorsal and ventral surface, respectively. Like P. latus males, adult females have four pairs of legs. Leg I, II and III are on average 54.6, 54.3 and 72.4 µm long, respectively. Each of these legs consists of 5 segments: coxa, femur, genu, tibia and tarsus. Leg IV only has three segments, being the coxa (ca. 11.3 µm long), genu + femur (ca. 22.6 µm long) and tibiotarsus (ca. 11.3 µm long) and has a total length of ca. 54.3 µm. Like in males there are two long setae on the tibiotarsal segment. The dorso- lateral seta is ca. 70.2 µm long while the ventro-lateral seta is ca. 24.9 µm in length. Leg II, III and IV do not carry any claws (Montasser et al., 2010b) (Fig 2.4d).

Gnathosoma Leg I

Propodosoma Leg II

Metapodosoma

Leg III Opisthosoma

Leg IV

Fig 2.6 Schematic drawing of Polyphagotarsonemus latus female body parts. 2.4. Host spectrum and damage The Tarsonemidae family includes more than 500 mite species divided over more than 40 genera that are distributed worldwide. The family consists of three subfamilies: Pseudotarsonemoidinae, Acarapinae and Tarsoneminae. The majority of plant feeding species belong to a few genera in the subfamily of Tarsoneminae, except Polyphagotarsonemus , which is a member of the Pseudotarsonemoidinae. Among these many species different feeding patterns exist (Zhang, 2003).

Some members of the Tarsonemidae are predators of insects and mites. Acaronemus destructor (Smiley and Landwehr) feeds on the eggs of phytophagous mites infesting pines in California, USA (Tenuipalpidae and Tetranychidae) (Smiley and Landwehr, 1976). In the laboratory a Dendroptus sp. fed on the apple rust mite, Aculus schlechtendali (Nalepa) (Villannueava and Harmsen, 1996). In Japan Tarsonemus praedatorius (Lin,

15

Nakao & Saito) was found to feed on the eggs of Schizotetranychus langus (Saito) on Sasa senanensis (Lin et al., 2002). Another species, Tarsonemus acerbilis (Delfinado), has been noted to prey on maple spindle gall mite eggs ( Vasates aceriscrumena (Riley & Vasey)) in stands of sugar maple Acer saccharum in south-central Ontario, Canada (Patankar et al., 2012). Further, an unidentified tarsonemid mite was thought to feed on the grape phylloxera ( Daktulosphaira vitifoliae (Fitch)) (Forneck et al., 1998). Members of the Tarsonemidae can also be parasitoids . Especially Iponemus spp. that feed on eggs of Ips (Scolytidae) and other ipine bark beetles (Lindquist, 1969). Iponemus spp. are parasitoids because a single host individual suffices for the development of all progeny and only a single stage actually feeds on the host, which is thereby killed (Lindquist, 1983). Since 1921, it is known that at least one member of the Tarsonemidae lives on insects. The tracheal mite, woodi (Renne, White & Harvey), is a known parasite in bees (Rennie, 1921). Fungivory of tarsonemid mites is restricted to mycelia, like all members of the Heterostigmae (Wheeler and Blackwell, 1983). The observation of Tarsonemus fusarii in fruiting bodies of polypore Fomes fomentarius maintained under laboratory conditions was suggested to be the result of a laboratory contamination (Maathewman and Pielou, 1971). The latter hypothesis was confirmed by observations on field-collected polypore species where no members of the Tarsonemidae were found (Wheeler and Blackwell, 1983). Tarsonemus granarius is widespread in grain and hay storage (Sinha, 1964). In commercial mushroom cultivation other species of Tarsonemidae are known. Tarsonemus myceliophagus (Hussey) feeds on Agaricus bisporus vegetative mycelium and fruiting bodies (Hussey and Gurney, 1967). However, most of the species belonging to the Tarsonemidae feed on plants and several are pests of agricultural crops, particularly those grown in the greenhouse. Among these phytophagous mites, P. latus and the strawberry mite, pallidus (Banks), are undoubtly the most important pests of many crops and ornamentals in greenhouses worldwide (Zhang, 2003). A few other species of minor importance were also reported on Belgian pot azalea; Tarsonemus bilobatus (Suski), Tarsonemus confuses (Ewing), and mites belonging to genera Xenotarsonemus and Daidalotarsonemus . Tarsonemus floricolus (Canestrini & Fanzago) is also frequently found on azalea but is reported to be a fungivorous mite (Zhang, 2003).

Polyphagotarsonemus latus exhibits a polyphagous feeding behaviour as it is collected from almost 60 plant families (Gerson, 1992). The extremely wide range of host plants includes the entire dicotyledone spectrum. Among these dicotyledone families P. latus was reported on , a family of woody plants including the genus Rhododendron (Ewing, 1939). The mite also was observed on monocotyledons: Araceae (Jones, 1988) and Orchidaceae (Karl, 1965). Further, this pest was reported on the gymnosperm Cupressus sp. (Ochoa et al., 1991). The feeding behaviour of P. latus causes a variety of symptoms depending on the host plant (Gerson, 1992). Previously, many of these symptoms were misinterpreted and

Chapter 2 assigned to other plant pests, or to diseases or viruses. Since 1890, symptoms on cotton are known as ‘acariose’ in Africa. Further the symptoms have been known as ‘begonia rust’ on begonia in England (Fox Wilson, 1950), ‘murda’ (Jeppson et al., 1975) or ‘leaf curl disease’ (Amin, 1979) of chilli in India, ‘tambera’ of potatoes in India (Jeppson et al., 1975), while in Brazil infestation by the mite has been confused with papaya buchy top (Flechtmann, 1983). Some of these symptoms were formerly believed to be caused by viruses (Lavoipierre, 1940; Bar-Joseph and Gerson, 1977; Aubert et al., 1981; Jones, 1988), but this hypothesis was experimentally rejected (Higa and Namba, 1971; Aubert et al., 1981). By listing the different symptoms caused by viruses and P. latus , it became clear that a systemic infection on chilli which included vein-clearing and brownish streaks on stems and petioles was attributed to viruses while local damage, including rusty-red patches, growth of axillary shoots and necrosis of the growing points was suggested to be due to P. latus (Dhooria and Bindra, 1977). Papaya ringspot virus-p and P. latus showed similar effects on chilli plants but only mites were found on the younger leaves of chilli plants. As a result, chlorosis produced by mites on these leaves started in the central vein and developed towards the end of the leaves, whereas the damage by Papaya ringspot virus-p can be observed on any part of the leaves (Alcantara et al., 2011). Damage by P. latus has also been confused with herbicide toxicity (Taksdal, 1973; Bassett, 1981; Beattie and Gellatley, 1983), or micronutrient deficiency, such as magnesium (Cross and Bassett, 1982).

(a) (b)

Fig 2.7 Damage on Rhododendron simsii caused by Polyphagotarsonemus latus . (a) Browning and curling of apical leaves as the first symptoms of P. latus on R. simsii ‘Desiree’ ca. three weeks after infestation. (b) Darkening and curling of infested leaves on R. simsii ‘Nordlicht’ ca. seven weeks after infestation with P. latus (photos: author).

The mites are usually found on the upper parts of the plant, feeding on the apical shoots and the abaxial side of young leaves (Grinberg et al., 2005). In general, plant growth is inhibited (Peña and Bullock, 1994; Cho et al., 1996a,b). Usually, the newest growth is heavily damaged, apical leaves seem distorted, more rigid, severely stunted, and their edges curl downwards (Bassett, 1981; Cross and Bassett, 1982; Gerson, 1992; Cho et al., 1996b, Labanowski and Soika, 2006). If fruits appear, they may be cracked and sometimes reticulated (Bassett, 1981; Cross and Bassett, 1982; Gerson, 1992; Cho et al., 1996b).

17

When plants are severely attacked, they stop growing and die (Smith, 1939; Moutia, 1958). Typical symptoms of P. latus infestation on R. simsii are marked by browning, bronzing and curling of apical leaves. When the infestation continues older leaves start curling downwards and leaves become rigid and brittle. Infested flowers are distorted and discoloured. Development of a darker colour on leaves and plant growth inhibition may also follow mite attack. No leaf drop is reported on R. simsii infested with P. latus (Fig 2.7). Injury symptoms upon P. latus infestation may develop quite rapidly on certain hosts (Bassett, 1981). Furthermore, small initial numbers (ca. 20 mites) on cucumber (Grinberg et al. 2005), and even fewer mites on limes (Peña, 1990) and peppers (De Coss- Romero and Peña, 1998) are sufficient to invoke economic damage. On chilli as few as ten mites per plant could cause characteristic injury symptoms (Dhooria and Bindra, 1977). It is important to note that when P. latus has been eliminated by pesticide treatments injury symptoms may continue to develop (Gerson, 1992). This is evidenced by misshapen fruits and development of abnormal side shoots whose leaves had several fused and distorted veins on successfully treated cucumber plants (Bassett, 1981). Earlier, Dhooria and Bindra (1977) noted that chilli and potato plants which had recovered from P. latus attack grew leaves that were as broad as long. Taking into account the persistence of symptoms for some weeks post-treatment and the negative results on transmitting viruses by P. latus , Gerson (1992) suggested that symptoms are caused by toxins. Furthermore the effects of such putative toxins would also explain the rapid appearance of injuries shortly after the initiation of mite attack, and the occurrence of symptoms at some distance from the site of mite feeding (Gerson, 1992).

2.5. Control 2.5.1. Plant resistance Host plants resistant to P. latus were sought in different plant species but mainly in pepper (Capsicum annuum ) and cucumber ( Cucumus sativus ). These host plants belonging to the Solanaceae and Cucurbitaceae plant families, respectively, are important commercial vegetables. Based on natural infestations susceptible and resistant C. annuum cultivars were identified (Ahmed et al., 2001; Desai et al., 2006,2007; Kulkarni et al., 2011). Host plant screening has also occurred in other crops, e.g. potato species Solanum polyadenium , S. tarijense and S. berthaultii (Gibson and Valencia, 1978), eggplant ( Solanum melongena ) (Lianyou et al., 1999), watermelon ( Cirtullus lanatus var. lanatus ) (Kousik et al., 2007) and jute ( Corchorus spp.) (Jana et al., 2007). The cause for host plant resistance against P. latus has not been described yet, although many hypotheses have been put forward. The presence of trichomes may act as a limiting plant trait for the development of broad mites in Capsicum species, especially when foliar hairs are present in high densities (Thungrabeab and Boonlertnirun, 2002; Matos et al., 2009). This was also observed in S . polyadenium and in seedlings of S. tarijense and S. berthaultii , which had foliar hairs with a sticky tip (Gibson and Valencia, 1978). The examination of biochemical constituents among 15 C. annuum cultivars resulted in a negative correlation with infestation by P. latus for tannins, phenols, potassium, calcium, magnesium and chlorophyll. In contrast,

Chapter 2 total sugars, protein content and nitrogen content were correlated positively with a broad mite infestation while no correlation was found for the phosphorous content of the leaves (Ahmed et al., 2000). More recently, the negative correlation of chlorophyll content in C. annuum and mite numbers was confirmed (Qing et al., 2010). Furthermore, a negative correlation exists for the amount of soluble sugars and free serine and the density of leaf pubescence. The contents of total free amino acid and soluble protein was positively correlated with mite resistance in leaves of 11 pepper cultivars (Qing et al., 2010). Stomatal density of abaxial leaf surface was positively correlated on eggplant whereas no significant correlation was found with stomatal density at the adaxial leaf surface (Lianyou et al., 2001).

2.5.2. Chemical control Gerson (1992) reviewed chemical control of P. latus before 1992. Since then EU legislations became more and more strict, as shown by the revision and withdrawal of former Council Directives (79/117/EEG and 91/414/EEG) concerning authorized pesticides. Nowadays Regulation (EC) No. 1107/2009 lays down rules for authorization of plant protection products in commercial form and for their placing on the market, use and control within the community. One of the most far-reaching changes included the restriction on the use of broad spectrum pesticides, including the complete prohibition of the use of endosulfan (an active substance in many plant protection products) from July 2007 onwards (Commission Decision 2005/864/EC).

Polyphagotarsonemus latus appears to be insensitive to pyrethroids (Vaissayre, 1986). Many trial experiments were conducted to assess the effect of existing or novel pesticides against P. latus . Dicofol, chinomethionat, pyridaben, and pyraclofos were effective in controlling P. latus on pepper showing over 90% mortality 17 days after application (Cho et al., 1996a). Within Europe chinomethionat and pyraclofos are banned (Commission Regulation (EC) No. 2076/2002) and the use of dicofol is prohibited by Commission Decision 2008/764/EC. Abamectin and milbemectin resulted in nearly total mortality of P. latus on the ornamental plant Hedera helix (Audenaert et al., 2009). Cyhexatin reduced numbers of P. latus to zero on Paraguay-tea ( Ilex paraguariensis ) when monitored 90 days after application (Alves et al., 2010). Commission Decision 2008/296/EC prohibits the use of cyhexatin in Europe. On chilli, diafenthuron (prohibited within Europe by Commission Regulation (EC) No. 2076/2002) showed the best efficacy against P. latus (63.4-77.1% reduction, depending on the concentration). This acaricide was followed by milbemectin (62.2-74.6% reduction) and propargite (60.9-71.8% reduction). Lowest mortality of P. latus was recorded after treatment with spinosad (39.2-52.7% reduction) (Chakrabarti and Sarkar, 2014). By December 31, 2011 plant protection products containing the active substance propargite were withdrawn in Europe (Commission Implementing Regulation (EU) No. 943/2011).

An alternative for spray application of acaricides can be found in an acaricide- treated net. Polyphagotarsonemus latus was completely controlled by dicofol treated nets

19

on Solanum macrocarpon. A trial at a growers’ field in Cotonou (Benin) demonstrated the effect of this technique to control the pest when nets were placed over the plants every three nights (Martin et al., 2010). However, as stated before, dicofol is banned in the EU (Commission Decision 2008/764/EC).

Less toxic but efficient alternatives to conventional pesticides are sought. Among several alternative methods the use of botanical products is considered. Treatments with neem (Azadirachta indica ) seed extract (azadirachtin as primary active substance) at concentrations higher than 0.13 g a.i./L resulted in a negative population growth rate of P. latus on Capsicum frutescens (Venzon et al., 2008). Castor oil-based soaps ( Ricinus communis ) with potassium hydroxide and naturally available materials such as botanicals and preservatives were significantly effective against P. latus on chilli although they were inferior to avermectin in screenhouse experiments (soaps reduced mite population by 40.5 to 69.3% compared to control treatment). Under field conditions soft soaps were not comparable in efficacy to the acaricide avermectin (56.3% reduction), as reduction of population was between 9.4 and 31.3% compared to an untreated control (David et al., 2009). Under plastic, abamectin, liquid sulphur (calcium polysulfide) and a botanical product based on canola oil (2% erucic acid rapeseed oil) proved to be effective against adult P. latus mites on pepper. Efficacies on immature stages and eggs were highest for abamectin, followed by liquid sulphur and canola oil (Montasser et al., 2011a). In field experiments on potatoes in Egypt, mite populations were reduced by optimizing mineral nutrient levels using boric acid, ascorbic acid and potassium sorbate. Boric acid caused the highest reduction percentages (82.1 and 88.0%) during two successive seasons, 2011 and 2012, respectively. This was followed by ascorbic acid (79.3 and 86.3%) and potassium sorbate (53.7 and 56.9%). Ascorbic acid caused highest potato yield, while potassium sorbate caused the lowest one (Nour El-Deen et al., 2014). Although boric acid and potassium sorbate are described as environmentally safe materials by Nour El-Deen et al. (2014), their use within Europe is prohibited by Commission Decision 2004/129/EC.

Integrated pest management methods to control P. latus were evaluated on sweet pepper (Reddy and Kumar, 2006) and chilli (Gundannavar et al., 2007). On sweet pepper alternated spraying of different chemicals was most effective but in a third trial, all the revised methods (alternated chemicals or botanical products based on neem) were equal (Reddy and Kumar, 2006). Methods that comprised chemical spraying resulted in significantly lower amounts of pests per leaf on chilli and a significantly lower leaf curl index compared to a control method consisting of locally recommended plant protection by the chemicals dimethoate, dicofol and carbaryl, while this was not the case for the non- chemical intervention method that applied vermicompost, fertilizers and botanical products based on neem. However, no differences were found on plant height, dry chilli yield, quality and number of branches or green fruits between methods comprising chemicals and the method based on non-chemical interventions (Gundannavar et al., 2007). Dicofol and carbaryl are not authorized chemicals in Europe, based on Commission

Chapter 2

Decision 2008/764/EC and 2007/355/EC, respectively. However, the latter findings suggest the existence of potential non-chemical alternatives to effectively control P. latus .

2.5.3. Biological control Prior to 1992 no attempts to control P. latus biologically have been made and no specific natural enemies were known (Waterhouse and Norris, 1987), but first insights in the role of phytoseiids in suppressing the pest became clear (Gerson, 1992). The first reports of biological control of P. latus are summarized by Gerson (1992), who mentioned control by predatory mites belonging to Amblyseius, and Typhlodromus families on many crops in different parts of the world.

Research on the suitability of predatory mites to control plant pests started in the 1960’s. Presently about 230 species of invertebrate biological control agents are commercially available including 30 species belonging to the Acari (Van Lenteren, 2012). The main focus within the latter group was on phytoseiid mites, some of which are used as biological control agents against P. latus . These include the economically important species Amblyseius swirskii (Athias-Henriot) (van Manen et al., 2010; Onzo et al., 2012; Abou-Awad et al., 2014), cucumeris (Oudemans)(Li et al., 2003; Weintraub et al., 2003) and Neoseiulus californicus (McGregor)(Peña and Osborne, 1996; Jovicich et al., 2008). Most of these studies evaluated biological control of P. latus on pepper plants, but eggplant (Onzo et al., 2012) and bean and lime (Peña and Osborne, 1996) were also studied as host plants. Furthermore it has been shown that Amblyseius largoensis (Muma) is a biological alternative for the management of P. latus in the protected production of peppers in Brazil (Rodríguez et al., 2011a). Augmentative releases of Neoseiulus barkeri (Hughes) indicated an ecologically alternative method for control of P. latus on pepper (Fan and Petit, 1994). However the effect of this predator was erratic on lime and bean (Peña and Osborne, 1996). Screening trials by Audenaert et al. (2009) showed that A. swirskii has the potential of controlling P. latus on English ivy ( H. helix ). These results were confirmed on R. simsii (Gobin et al., 2011). Promising results for the control of P. latus on R. simsii were obtained for N. barkeri (Gobin et al., 2011). zulagai (Denmark & Muma) might be more efficient than Euseius concordis (Chant) in reducing populations of P. latus on Jatropha curcas , a promising plant species for biofuel production in Brazil (Sarmento et al., 2011a). It is more doubtful that the blattisociid mite Lasioseius floridensis (Berlese) is a potent predator of P. latus on gerbera in Brazil. Oviposition rates and life table parameters indicated that the predator performed much better on the soil nematode Rhabditella axei (Cobbold) than on P. latus (Britto et al., 2012). Amblyseius herbicolus (Chant), associated with P. latus in chilli pepper in Brazil, performed best (intrinsic growth rate) when fed on this pest compared to feeding on pollen of castor bean ( Ricinus communis ) or sunnhemp ( Crotalaria juncea ) (Rodríguez-Cruz et al., 2013).

Another method to control P. latus biologically would be the use of entomopathogenic fungi. Hirsutella nodulosa attacked P. latus in Cuba (Cabrera et al., 1987). Beauveria

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bassiana (Balsamo) proved to be the most effective fungal agent followed by fumosoroseus (Wize) and (Metchnikoff) against young adult females. Egg infection by these fungi was minimal, only 10% in the case of M. anisopliae while no infection was recorded by B. bassiana and P. fumosoroseus (Nugroho and Ibrahim, 2004). In 2008, it was found that among many other fungi, M. anisopliae strain CKM-048 was the most virulent in controlling both larvae and adults of P. latus on mulberry trees. There was, however, no ovicidal effect when tested against the eggs of the mite (Maketon et al., 2008). On chilli ‘Byadagi’ the overall mite population was suppressed effectively by Fusarium semitectum , either alone or in combination with the acaricide monocrotophos. The combination of both chemical and best suppressed P. latus , whereas it did not affect numbers of the predatory mite Amblyseius ovalis (Evans) (Mikunthan and Manjunatha, 2010). It is noteworthy that the use of monocrotophos within Europe is prohibited by Commission Regulation (EC) No. 2076/2002. Upon treatment with Hirsutella thompsonii isolate CG 541 moderate numbers of P. latus did not increase on Paraguay-tea plants during a 90-days period after application. Although there was no population increase, losses of 30% plant biomass were reported when compared to uninfested plots (Alves et al., 2010).

2.5.4. Physical control Hot water treatment of R. simsii cuttings might be an efficient method to reduce risks of P. latus infestation at the start of the plant production cycle. Submersion of R. simsii cuttings in warm water (46 °C) prior to planting resulted in good control of P. latus when submersion was longer than 6 minutes. However, rooting success of cuttings declined with increasing length of submersion at 46 °C, although the effect was inconsistent between trials (Gobin et al., 2001; Gobin et al., 2013). Higher temperatures, i.e. 48 °C, caused a significant drop-out in plant survival (Van Delsen et al., 2013).

Chapter 2

3. The Belgian pot azalea, Rhododendron simsii hybrid (Planch) 3.1. Taxonomy, origin and distribution Systematic studies that encompassed all sections and subgenera of Rhododendron were initiated by Sleumer (1949). Until recently a taxonomic system based on eight subgenera, proposed by Chamberlain et al. (1996), was generally accepted by Rhododendron specialists (Cox and Cox, 1997). Till then Rhododendron systematics was based on plant morphology. Molecular techniques made it possible to revise the Rhododendron classification into five subgenera including: Rhododendron , Hymenanthae , Azaleastrum , Choniastrum and Therorhodion . For this purpose, the nuclear gene RPB2-I, encoding a major RNA polymerase II subunit, was sequenced in 87 species and cladistics analysis was performed (Goetsch et al., 2005).

Taxonomic classification of R. simsii according to Goetsch et al. (2005):

KINGDOM Plantae ORDER FAMILY Ericaceae SUBFAMILY Ericoideae GENUS Rhododendron SUBGENUS Azaleastrum SECTION Tsutsusi SUBSECTION Tsutsusi SPECIES Rhododendron simsii Planch.

Approximately 800 Rhododendron species are described (Heursel, 1999; van Trier, 2012). They can be found in high densities in the Eastern Himalaya region. They appear in the northern hemisphere but spread to the south including Malaysia, Indonesia and New- Guinea. Only one species is described in Australia ( Rhododendron viriosum ). In the north they spread to Korea and Japan. Eastward, can be found in Northern America. While to the west of the Himalaya, Rhododendrons appear from Sino-Himalaya to Afghanistan and Caucasus, through Turkey and into Europe. No Rhododendrons have been found in Central- and South-America, Africa and Polynesia (van Trier, 2012).

Rhododendron subsection Tsutsusi , a subsection of the genus Rhododendron , in section Tsutsusi , subgenus Azaleastrum, includes some of the evergreen azaleas, among which R. simsii . At least four different species might have contributed to the modern R. simsii hybrids (Heursel, 1999; van Trier, 2012).

• Rhododendron simsii Planch. This species is accepted to be the main ancestor and originates from hilly areas in (Chang Jiang valley) (Heursel, 1999). The evergreen shrub is found at elevations of 500-1200 m and widely distributed in subtropical China (Li et al., 2012). It has also

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been found in Taiwan, Myanmar, Laos and Thailand (Heursel, 1999). R. simsii is not winter hardy since in its natural habitat temperatures vary between 8.6 and 20.2 °C. Flowers of R. simsii are typically red (Heursel, 1999). It is reported to be introduced in Belgium in 1818 (Scheerlinck et al., 1938).

• Rhododendron indicum (L.) Sweet R. indicum originates from the Japanese islands Honshu, Shikoku, Kyushu and Yakushima (Heursel, 1999). Formerly this species was confused with R. simsii but it can be distinguished based on the number of anthers. R. indicum has 5 anthers while 10 (sometimes 8) anthers can be found on flowers of R. simsii (Bean, 1980). Flowers are colored carmine red (Heursel, 1999).

• Rhododendron scabrum G.Don On the borders of the East China Sea and the Philippine Sea, R. scabrum can be found on the Ryukyu Islands (Japan). Originated in a subtropical climate this species is not winter hardy. Flowers can be colored purple, carmine red or red (Heursel, 1999).

• Rhododendron mucronatum G.Don This species is described on a cultivated white introduced to England from China. ‘Noordtiana’, providing white flowers, is probably used most for breeding as it also carries genetic information for purple colored flowers. R. mucronatum is winter hardy (Heursel, 1999).

R. simsii introduced the early flowering and the ability to produce sports into the modern pot azalea. A sport has been defined as “an individual exhibiting in whole or in parts a sudden spontaneous deviation beyond the normal limits of individual variation usually a result of mutation, especially of somatic tissue” (Pratt, 1980). R. scabrum and R. mucronatum have introduced purple flower colors and large flowers, the globular plant habitat and carmine red flower color are features coming from R. indicum (Heursel, 1999)

Table 2.1 Contributions of the three most important ancestors into Belgian pot azaleas. Horizontal arrows indicate mutual influences (Heursel, 1999).

Trait R. indi cum R. simsii R. scabrum Origin Japan China Japan Plant habit Globular ⟷ Upright Upright Flower size Small ⟷ Medium ⟵ Large Flower color Carmine red ⟷ Red ⟵ Purple Flowering time Late Early Medium early ↓ ↓ ↓ Hybrids Satsuki Pot azalea Hirado

Chapter 2

3.2. Economic importance for Ghent region Belgium, more specifically the Ghent region is known for the production of the modern pot azalea. Belgian production is located for 96.2% in East Flanders (EROV, 2012). In 2013, azalea production area in Belgium was estimated to be 260 ha in total, 111 ha in open air and 149 ha in greenhouses (VLAM, 2014). Within Europe the Belgian production accounts for 80%, averaging the production of 30 million plants annually (EROV, 2012). Belgian export value in 2013 was €32.2 million (including rhododendron) while the import value accounted for only €0.8 million, this resulted in a positive trade balance of €31.4 million. Ca. 80% of Belgian azaleas and rhododendrons are exported to France, The Netherlands, Italy, Germany and the UK. The other EU and non-EU countries import ca. 11 and 9% of these plants, respectively (VLAM, 2014).

In 2010, ‘Gentse azalea’ or Ghent azalea, was designated with a ‘Protected Geographical Indication’ (Commission Regulation (EU) 300/2010). This made it the first European ornamental plant product to be recognised. To obtain the label of Ghent azalea strict quality requirements have to be fulfilled. Plants should be cultivated in East Flanders and having 80% colored buds. Further the producer complies with ‘Project Azalea Kwaliteit’ and meets the strict quality standards including subjection to independent monitoring and participation in flower tests to control the pre-defined quality requirements (www.gentseazalea.be).

3.3. Cultivation process Today modern azalea cultivars are all propagated vegetatively. In the past some cultivars were multiplied by grafting on a rootstock. One to five cuttings, depending on the desired plant diameter, are placed directly in the final pot. Plant material is then covered with plastic providing constant conditions of high relative humidity and soil temperature around 23-25 °C. Under these conditions rooting is stimulated. After 8 to 10 weeks the plastic cover is removed and plants are pinched for the first time. Pinching results in branching as the removal of the apical meristem lifts the apical dominance, enabling the outgrowth of axillary buds. Successive rounds of pinching occur depending on the desired plant diameter and globular shape. After the second pinch plants are placed outdoors on container fields during summer. Plant growth and initiation of the generative phase is further regulated by treatments with plant growth regulators (Keever, 1990; Marosz and Matysiak, 2005; Meijón et al., 2009). Chlormequat chloride (Cycocel®) and paclobutrazol (Bonzi®) are the most commonly used growth retardants in azalea cultivation. Flower buds require a period of cold to break flower bud dormancy that inhibits fully open and functional flowers. Temperatures for dormancy breaking are generally between 2.5 and 9 °C (Richardson et al., 1974), and the optimum for R. simsii hybrids is 7 °C (Stuart, 1965). Depending on the length of chilling requirement, azaleas can be classified as very early flowering (from August 15), early flowering (from December 1), medium early flowering (from January 15) and late flowering (from February 15) (Heursel, 1999). Finally, azalea plants are forced to flower in heated greenhouses (20-21 °C). To speed up the uniform

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opening of flowers, additional light can be supplemented when natural light levels are low (Heursel, 1999; De Keyser, 2010; Christiaens, 2014a) (Fig 2.8).

Dormancy Rooting Vegetative outgrowth breaking Forcing

23 -25°C Field temperature, protected from freezing in winter 2-7°C 20 -21°C

8-10 weeks 8-14 months Up to 6 weeks 2-3 weeks

Fig 2.8 Scheme of pot azalea production cycle indicating the duration and temperature conditions in the main phases. 3.1. Breeding The first commercially released cultivar ‘Madame Vercruyssen’ was introduced in 1867. Since then breeding of azaleas gained more attention (van Trier, 2012). In Belgium there are two important breeding programs of azalea. They are led by a company, Hortinno®, and a public research institution, ILVO (Institute for Agricultural and Fisheries Research). In 2008, 21 azalea growers joined forces as a cooperative named AZANOVA and started investing in the breeding program of ILVO (www.azanova.be) (Fig 2.9). Besides these programs some Belgian growers still have their own small breeding programs. Historically, German breeders were most successful by introducing 47% of the cultivars on the Belgian market, whereas Belgian breeders only introduced 38% (Heursel, 1999). Today only two important German breeders are remaining, Rannacher and Stahnke- Dettmer.

After the commercial success of ‘Madame Vercruyssen’ in 1867, two other key cultivars were introduced. ‘Madame Petrick’ (1880) produces pink doubled-flowers. Blooming of this cultivar was possible before Christmas, unlike most cultivars which were late bloomers. The introduction of ‘H. Vogel’ in 1967 advanced flowering, making it already possible to flower in fall. Other advantages of this cultivar are the ability to grow on its own roots making grafting superfluous and the excellent ability to regulate flowering time (van Trier, 2012). ‘H. Vogel’ is known for its easy formation of sports. Together with its sports, it has a total market share of 65% (van Trier, 2012). Of all cultivars in pot azaleas, 52% are a result of sports (Horn, 2002).

Crossbreeding, the process of bringing pollen of a desired donor plant to the anthers of a selected host plant, is mainly used to select for new traits in azalea cultivars. Flower color is in accordance with other ornamentals the most important criterion for selection. Next to flower color, leaf shape and color, growth vigor, plant architecture, earliness of flowering, and post-production quality (longevity of flowers, absence of brown bud scales) are other important traits for breeding. Different factors can impede the process of crossbreeding. Large differences in flower time between cultivars or sterile azalea flowers, especially doubled-flowers which mostly lack stamens or have deformed carpels form serious problems. Azalea is a cross-pollinator; however, self-pollination is possible

Chapter 2 but will result in inbreeding depression as shown by lower seed yield and growth vigor (De Keyser, 2010; Christiaens, 2014a). Another way to introduce new cultivars can be achieved by bud sporting. It is the consequence of sudden variations in gene expression of somatic cells which leads to the occurrence of phenotypically altered shoots (De Schepper et al., 2004). This process occurs naturally but can also be induced by hormonal treatments of explants (Samyn et al., 2002) or by irradiation with soft X-rays (De Loose, 1979). In-vitro assisted breeding has been shown to be a useful tool to produce sports and polyploid plants (Eeckhaut et al., 2009, 2013). Other laboratory techniques can also assist in azalea breeding. Inheritance of color pigments in azalea was identified by Heursel (1999). After genes responsible for the biosynthesis of anthocyanins were identified (De Schepper, 2001; Nakatsuka et al., 2008; De Keyser, 2010), the first steps of molecular-assisted breeding have been taken (De Keyser, 2010). Agrobacterium -mediated or microprojectile bombardment transformation of azalea (Deroles et al., 2002), might introduce new flower colors.

YEAR 1 Making crosses

YEAR 3 First selection on seedlings

YEAR 5 Second selection on clones Three successive

years evaluating YEAR 7 Third selection on clones under disease resistance commercial growing conditions

YEAR 9 Release of new variety

Fig 2.9 Scheme of the commercial pot azalea breeding program of ILVO.

More recently breeding for biotic resistance in azalea gained attention as azalea production can be hampered by fungal diseases Phytophthora citricola Saw. and Cylindrocladium spp. (Backhaus, 1994a, 1994b) and the pest P. latus (Heungens, 1986). Supporting techniques for breeding against these fungal diseases have been developed by De Keyser et al. (2008) and are used in practice (Van Huylenbroeck et al., 2015). Screening for susceptibility against P. latus has been done in japonica azaleas (Luypaert and Mechant, 2014).

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4. Plant defence regulated by the phytohormone jasmonic acid 4.1. Plant defence As a first line of defence, plant pests encounter physical or structural barriers (e.g. lignified cell walls), as well as constitutive chemical defences (Gatehouse, 2002; Mysore and Ryu, 2004). These first line mechanisms are potent defences against a wide range of herbivores and pathogens except those that have suitable counter-adaptations like detoxification or excretion pathways (Gatehouse, 2002; Pedras and Ahiahonu, 2005). Besides these defences, more complicated induced immune defences protect plants from either pathogens or herbivores. The induced defence response to pathogens is a multi-layered process generally accepted as the four phased zigzag model by Jones and Dangl (2006). In this model induced defence responses are initiated by the recognition of conserved pathogen- associated molecular patterns (PAMPs) by the pattern recognition receptors. Upon the recognition of extracellular PAMPs, pattern recognition receptors induce PAMP-triggered plant immunity (PTI) that restricts pathogen propagation. Pathogens have evolutionary evolved effectors that suppress PAMP-triggered plant immunity. However, the specific recognition of these effectors by plant disease resistance proteins leads to effector triggered-immunity (ETI) and plant resistance (Jones and Dangl, 2006). As compared to the elaborate knowledge of plant-pathogen interactions, the understanding of plant-arthropod interactions is still rudimentary (Zhurov et al., 2014) and current insights suggest that plants perceive herbivore attack by recognition of general herbivore-associated molecular patterns (HAMPs), which are elicitors originating mainly from herbivore oral secretions (Bonaventure, 2012), and by damage-associated molecular patterns (DAMPs) that result from wounding or enzymatic reactions imposed by the herbivore (Pearce et al., 1991; McGurl et al., 1992; Schmelz et al., 2006; Yamaguchi and Huffaker, 2011). Jasmonic acid (JA) is associated in playing a major role in HAMP- and/or DAMP-induced defence responses against a range of herbivores (Browse, 2009a). In some instances defence responses can also be regulated by jasmonoyl-L-isoleucine (JA- Ile) and oxo-phytodienoic acid (OPDA) (Stintzi et al., 2001). However, phytohormones salicylic acid (SA) and ethylene have also been linked to herbivore responses since they are able to modulate JA metabolism and signalling (Robert-Seilaniantz et al., 2011) or induce responses independently from JA (Li et al., 2006). In 1962, the methyl ester of jasmonic acid (MeJA) was isolated as a floral scent compound from the aromatic oil of Jasminum grandiflorum (Demole et al., 1962). Approximately 20 years later, the first physiological effects were reported, i.e. a senescence-promoting effect of JA (Ueda and Kato, 1980) and growth inhibition activity in Vicia faba by MeJA (Dathe, 1981). Jasmonic acid is likely to occur ubiquitinously in the plant kingdom, and it has been found in some fungi (Schaller, 2001). The JA pathway plays a significant role in both monocots and dicots that diverged 200 million years ago. Ancestral plant forms such as contain a relatively primitive oxylipin signalling component but expansion of this hormone signalling pathway has likely occurred during the adaptation of plants into diverse land environments (Lyons et al., 2013). In this thesis

Chapter 2 we study the role of the phytohormone JA in defence against the mite pest, P. latus on R. simsii hybrids. A short overview on JA biosynthesis, signalling, function, and crosstalk with other phytohormones is given in the following paragraphs.

4.2. Jasmonic acid biosynthesis and signalling 4.2.1. Biosynthesis Jasmonic acid biosynthesis starting from free α-linolenic acid (LeA,18:3) as substrate, referred to as the Vick and Zimmermann pathway (also known as the octadecanoid pathway) (Schaller et al., 2005), is considered the major source of JA production in plants (Yan et al., 2013).

Biosynthesis of JA is initiated by the release of α-linolenic acid (18:3) from chloroplast membrane lipids (Ishiguro et al., 2001; Hyun et al., 2008) (Fig 2.10). Lipoxygenases (LOXs) incorporate molecular oxygen at either position 9 or 13 of α-linolenic acid (18:3) producing hydroperoxides, 9-/13-HPOT (Vick and Zimmermann, 1983). Allene oxide synthase (AOS) then dehydrates 9-/13-HPOT to unstable epoxides, either 9,10-EOT or 12,13-EOT (Laudert et al., 1996). AOS genes are strongly induced by JA and wounding (Laudert and Weiler, 1998). Another enzyme, allene oxide cyclase (AOC), catalyses the stereospecific cyclization to form the cis-(+) enantiomer OPDA (Ziegler et al., 2000). Arabidopsis and rice AOC genes are differentially regulated upon wounding, JA-treatment and environmental stresses (Agrawal et al., 2004). As all previous steps occurred in the chloroplast, the following conversions take place in the peroxisome. The mechanism for OPDA transport between chloroplast and peroxisome has not been completely understood. However, a peroxisomal ABC transporter protein COMATOSE (Footitt et al., 2002, 2007; Theodoulou et al., 2005), also known as PXA1 (Zolman et al., 2001) or PED3 (Hayashi et al., 2002) may be involved. In the peroxisome OPDA, a cyclopentenone, is oxidized to a cyclopentanone (OPC-8:0) by OPDA reductase (OPR) (Schaller and Weiler, 1997; Schaller et al., 1998). It has been shown that OPR3 (Müssig et al., 2000) is the only isoenzyme involved in JA biosynthesis (Schaller et al., 2000). Furthermore in Arabidopsis this gene is induced after wounding or MeJA treatment (Costa et al., 2000). In one of the alternative biosynthesis routes hexadecatrienoic acid (16:3) can be converted by the same enzymes forming dinor- oxophytodienoic acid (dn-OPDA) (Weber et al., 1997).

Finally, three rounds of β-oxidation shorten the carboxyl side chain from the precursor (Miersch and Wasternack, 2000). Before the final β-oxidation steps can take place, the carboxylic group of OPC-8:0 must be esterified forming OPC-8:0-CoA (Baker et al., 2006; Koo and Howe, 2007). Each β-oxidation step is catalysed by acyl-CoA oxidase (ACX) (Li et al., 2005), multifunctional protein (MFP) (Delker et al., 2007) and L-3-ketoacyl-CoA thiolase (KAT) (Castillo et al., 2004). Once synthesised, the (3R,7S)-JA is released in the cytoplasm from the peroxisome by an unknown mechanism (Santino et al., 2013).

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Jasmonic acid can then be converted to MeJA by JA carboxyl methyltransferase (JMT) (Seo et al., 2001). MeJA mediates diverse developmental processes and defence responses against biotic and abiotic stresses (Cheong and Choi, 2003). Jasmonoyl-L- isoleucine, the only bioactive ligand involved in JA signalling is formed by conjugation of isoleucine to JA via the enzyme JAR1 (Staswick and Tiryaki, 2004).

Fig 2.10 Biosynthesis pathway of jasmonic acid in Arabidopsis thaliana adapted after Dave and Graham (2012). DAD1, DEFECTIVE IN ANTHER DEHISCENCE1; DGL, DONGLE; 13-LOX, 13-lipoxygenase; 13-AOS, 13-allene oxide synthase; AOC, allene oxide cyclase; OPR3, 12- oxophytodienoate reductase3; OPCL1, OPC-8:CoA ligase1; CTS, COMATOSE; PXA1, PEROXISOMAL ABC TRANSPORTER1; ACX, acyl CoA oxidase; KAT, 3-l-ketoacyl-CoA- thiolase; MFP, multifunctional protein; JA, jasmonic acid; cis-OPDA, cis-(+)-12- oxophytodienoic acid; dn-OPDA, dinor-oxo-phytodienoic acid; JAR1, JASMONATE RESISTANT1; JA-Ile, jasmonoyl-L-isoleucine. Jasmonic acid biosynthesis and activities are regulated by three strategies; (1) expression of the enzymes in JA biosynthesis pathway, (2) controlling the substrate availability and (3) storing and reusing of intermediates or conjugations of JA (Yan et al., 2013).

4.2.2. Signalling Jasmonic acid signalling is extensively reviewed in literature (Wasternack, 2007; Balbi and Devoto, 2008; Chico et al., 2008; Katsir et al., 2008a; Kazan and Manners, 2008, 2012; Staswick, 2008; Browse, 2009a, 2009b; Chini et al., 2009; Chung et al., 2009; Fonseca et

Chapter 2 al., 2009; Memelink, 2009; Gfeller et al., 2010; Pauwels and Goossens, 2011; Wager and Browse, 2012; Lyons et al., 2013; Santino et al., 2013; Wasternack and Hause, 2013). In the absence of or at low levels of the hormonal signal, JAZ (Jasmonate ZIM- domain-containing protein), proteins actively repress the activity of basic-helix-loop- helix (bHLH) related transcript factors (e.g. MYC2) with help of adaptor protein NINJA that recruits co-repressor TOPLESS (TPL) and TPL-related proteins (Pauwels et al., 2010) (Fig 2.11a). In the presence of JA-Ile, produced as response upon external stimuli, JAZ proteins interact with COI1 (Coronatine Insensitive1 protein) (Chini et al., 2007; Thines et al., 2007; Yan et al., 2007). It was shown that COI1 encodes an F-box protein (Xie et al., 1998) which is known to associate with CUL1, Rbx1 and one of the SKP1 orthologues ASK1 or ASK2 forming an E3 ubiquitin ligase, known as the SCF COI1 complex (Xu et al., 2002). Interaction to the SCF COI1 complex leads to poly-ubiquitination and subsequent degradation of JAZs by the 26S proteasome releasing MYC2 and other transcription factors, permitting expression of jasmonate-responsive genes (Fig 2.11b).

Fig 2.11 Jasmonate perception in plants adapted after De Geyter et al. (2012). (a) In the absence of JA-isoleucine (JA–Ile). (b) Upon stress or developmental cues (i.e. when JA–Ile levels increase). Jas, Jas domain; COI1, CORONATINE INSENSITIVE 1; SCF, Skp–Cullin–F-box- type E3 ubiquitin ligase; NINJA, Novel Interactor of JAZ, JA-Ile, Jasmonic acid-isoleucine; JAZ, Jasmonic acid ZIM domain; 26S, 26S proteasome.

Jasmonic acid and MeJA have been considered for a long time to be the bioactive ligand because of their physiological effects and abundance in the plant (Creelman and Mullet,

31

1995). However JA-Ile is the only compound in JA signalling that promotes COI1-JAZ binding, indicating only JA-Ile is the direct bioactive JA signalling ligand in plants (Thines et al., 2007). The floral scent MeJA has now been identified as a vital cellular regulator that mediates diverse developmental processes and defence responses against biotic and abiotic stresses (Cheong and Choi, 2003). It is a key enzyme for airborne-jasmonate- regulated plant responses (Yan et al., 2013).

To terminate JA signalling in order to sense new environmental cues, plants must reset their signalling cascade. Besides the negative feedback loop mediated by COI1 (Thines et al., 2007), hydroxylation of JA (Miersch et al., 2008) and hydroxylation and carboxylation of JA-Ile (Kitaoka et al., 2011; Koo et al., 2011; Heitz et al., 2012) were reported to be efficient means of directly inactivating signalling molecules. Recently, an alternative pathway led by JIH1 is hypothesized to hydrolyse JA-Ile to JA (Woldemariam et al., 2012) and contributes to a termination of JA signalling in Nicotiana attenuata to regulate plant defence responses (Woldemariam et al., 2012, 2014).

4.3. Functions of jasmonic acid in the plant Jasmonic acid is involved in a large number of different physiological processes of plants. During plant development it plays a role in reproductive organ development in monocotyledonous and dicotyledonous plants. Besides inhibition of seed germination, root growth as well as the lateral root formation and adventitious rooting is inhibited. Growth of above-ground plant parts is also inhibited by JA. Furthermore it controls leaf senescence, trichome initiation and is involved in gravitropism and leaf movement. It is an essential signal in defence against herbivores and pathogens and regulates secondary metabolite biosynthesis. Other environmental cues mediated by JA are light, seasonal and circadian rhythms, cold stress, desiccation stress, salt stress and UV stress. Recent reviews by Wasternack and Hause (2013) and Yan et al. (2013) discuss the wide range of functions assigned to the phytohormone JA in plants.

4.4. Role of jasmonic acid in plant defence Defence mechanisms against insect herbivores have extensively been reviewed (Gatehouse, 2002; Howe and Jander, 2008; Wu and Baldwin, 2010; Fürstenberg-Hägg et al., 2013) while the chemical aspects of plant defence against herbivores are reviewed by Mithöfer and Boland (2012). Besides insects, a group of major ecological and agricultural importance among arthropods are the chelicerates, including phytophagous mites. Of these, the two-spotted spider mite, Tetranychus urticae (Koch), is the best characterized in terms of its interactions with plants (Zhurov et al., 2014). The use of JA-perception or –biosynthesis mutants has demonstrated that spider mite maximal reproductive performance is achieved in the absence of JA signalling (Li et al., 2002, 2004; Ament et al., 2004; Kant et al., 2008). Furthermore it has been shown that most genotypes of T. urticae simultaneously induce expression of JA- and SA-dependent marker genes (Li et al., 2002; Ament et al., 2004; Kant et al., 2004; Agut et al., 2014). Previous results suggest that JA defences are key anti-mite defences in tomato; it appears

Chapter 2 that some spider mites were found to suppress expression of these marker genes (Kant et al., 2008; Sarmento et al., 2011b). This was further evidenced by Alba et al. (2015), as two Tetranychus evansi (Baker & Pritchard) strains and one T. urticae strain showed to suppress plant defence downstream of JA and SA accumulation, independently from the JA-SA antagonism. A unique approach to mite control involves activating plant defences to reduce mite vigour and/or mite preference for a crop (Rohwer and Erwin, 2010). This can be achieved by exogenous application of phytohormones. The application of JA or one of its derivates (i.e. MeJA) successfully reduced T. urticae proliferation on different plant species (Omer et al., 2001; Li et al., 2002; Choh et al., 2004; Warabieda et al., 2005; Rohwer and Erwin, 2010; Warabieda and Olszak, 2012; Agut et al., 2014). As a consequence of the induced plant defence, a fitness cost may be the result for the plant (Baldwin, 1998; Redman et al., 2001). Horticultural and biotechnical applications of jasmonates and the impact on plant growth and physiology are reviewed by Rohwer and Erwin (2008) and Wasternack (2014). Despite the studies on plant defence against the two-spotted spider mite, literature concerning the molecular pathways involved in defence against P. latus remain scarce. Limited evidence for JA mediated defence responses upon P. latus infestation is summarized in the following paragraph.

It has been shown that P. latus infestation activates JA- and SA-dependent pathways, and possibly an oxidative stress response in cucumber leaves, Cucumis sativus (L.) (Grinberg et al., 2005). In order to evaluate the possible involvement of the JA signalling pathway in defence against this pest, Grinberg et al. (2007) compared feeding on the mite resistent wild type (WT) tomato, Lycopersicon esculentum (Mill) ‘Castlemart’ and the mite susceptible mutant def-1 that is impaired in JA biosynthesis. Broad mite feeding did not affect growth and leaf development in the WT plants, but did cause a 50-60% inhibition in def-1 plants. Progeny counts showed that mite populations developed only on def-1 plants. Furthermore, P. latus is able to actively discriminate between resistant and susceptible plants, preferring JA-defective mutants (Alagarmalai et al., 2007; Grinberg et al., 2007). In conclusion, the JA pathway appears to play a central role in the resistance response against P. latus (Grinberg et al., 2007).

4.5. Crosstalk with other phytohormones in plant defence While receptors and pathways for individual hormones are being illustrated, mounting evidence suggests that these signalling pathways are interconnected in a complex network, in which phytohormones not only orchestrate intrinsic developmental cues, but also convey environmental inputs by means of synergistic or antagonistic actions referred to as signalling crosstalk (Cui and Luan, 2012). Classic phytohormones are abscisic acid (ABA), auxins, cytokinins, ethylene, and gibberellins, but small signalling molecules such as brassinosteroids, jasmonates (JAs), and salicylic acid (SA) are recognized as phytohormones as well (Pieterse et al., 2012). Moreover, it is becoming clear that more than one phytohormonal pathway is induced by an attacker. Hormonal blends are

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produced in response to different types of attackers; they differ in composition, quantity and timing, and depend on the lifestyle and invasion strategy of the attacker (De Vos et al., 2005). SA signalling positively regulates plant defence against biotrophic pathogens, whereas ethylene/JA pathways are commonly required for resistance to necrotrophic pathogens and to herbivorous pests (Glazebrook, 2005). Interaction with or involvement of other phytohormones such as ABA, auxin, cytokinins, gibberellins and brassinosteroids, in plant defence is reviewed by Bari and Jones (2009), Pieterse et al. (2009, 2012) and Robert-Seilaniantz et al. (2011).

4.6. Salicylic acid biosynthesis and signalling 4.6.1. Biosynthesis Salicylic acid is synthesized through two different pathways; the phenylpropanoid pathway and the isochorismate pathway. Both pathways originate from chorismate (Dempsey et al., 2011) (Fig 2.12).

The chloroplasmatic isochorismate pathway (route 1). Conversion of chorismate to isochorismate (IC) is facilitated by the enzyme isochorismate synthase (ICS) (Wildermuth et al., 2001; Strawn et al., 2007; Garcion et al., 2008). ICS homologs have been identified in a wide variety of plant species and given their role in phylloquinon synthesis, it is highly likely that ICS homologs will be identified in all plant species (Dempsey et al., 2011). The mechanisms through which IC is converted is unclear, it is assumed to occur enzymatically via IC pyruvate-lyases (IPLs) (Dempsey et al., 2011). However, plant genomes encode no homologous genes to bacterial IPLs . Expression of bacterial enzymes catalyzing this conversion together with ICS in chloroplasts leads to constitutive accumulation of SA (Verberne et al., 2000; Mauch et al., 2001). Thus, it is conceivable that plants have yet-undetermined gene(s) whose product(s) possess IPL activity in chloroplasts (Seyfferth and Tsuda, 2014).

The cytoplasmic phenylpropanoid pathway (route 2). This biosynthesis pathway begins with the conversion of phenylalanine to trans -cinnamic acid ( t-CA) and NH 3 via a non-oxidative deamination reaction by the phenyl ammonia-lyase (PAL) enzyme (Raes et al., 2003; Rohde et al., 2004). t-CA is then converted via two possible intermediates: ortho - coumaric acid (route 2a) or benzoic acid (BA) (route 2b and 2c), depending on the plant species (Klämbt, 1962; El-Basyouni et al., 1964; Chadha and Brown, 1974). It was found that plants can potentially use three biosynthetic routes to BA. First, a beta-oxidation route from cinnomayl Co-A (route 2c-1). Secondly, a non-oxidative route from cinnamoyl CoA (route 2c-2). Thirdly, a non-oxidative route from t-CA to BA (route 2b) (Wildermuth, 2006). Finally, SA is derived from BA via hydroxylation and catalyzed by benzoic acid 2- hydroxylase (BA2H) (León et al., 1993; Yalpani et al., 1994; León et al., 1995; Ryals et al., 1996). The PAL enzyme is also the first enzyme involved in the phenylpropanoid derivative metabolism, and is one of the most extensively studied enzymes for its crucial function as branch point between primary and secondary metabolism (Hahlbrock and Scheel, 1989). Phenylpropanoid compounds are precursors to a wide range of phenolic

Chapter 2 compounds with many functions in plants. Lignin for instance is a major component of secondarily thickened cell walls in the plant vascular system essential for stem rigidity and for conducting water, minerals and photosynthetic products through the plant. Phenylpropanoids are also precursors to flavonoids, isoflavonoids, cumarins and stilbenes. These compounds have important functions in plant defence against pathogens and other biological threats, as UV light protectants and as regulatory molecules in signal transduction and communication with other organisms (Ferrer et al., 2008).

SA can then be converted to many side products including methyl salicylate (Chen et al., 2013). Most modifications render SA inactive, while at the same time they allow fine- tuning of its accumulation, function, and/or mobility (Dempsey et al., 2011). The biologically relevant chemical modifications including glycosylation, methylation, amino acid conjugation, sulfonation and hydroxylation are extensively reviewed by Dempsey at al. (2011).

4.6.1. Signalling NPR1 (NON-EXPRESSOR of PATHOGENESIS-RELATED GENES) is a master regulator of SA mediated transcriptional reprogramming and immunity, functioning as a transcriptional coactivator (Pajerowska-Mukhtar et al., 2013). It binds directly to SA, but not inactive structural analogs, through two key cysteine residus (Cys 521 and Cys 529 ) via the transition metal copper (Wu et al., 2012). However, Cys 521/529 are not conserved among plant species, raising an issue. Further research identified two homologs of NPR1, NPR3 and NPR4, as SA receptors (Fu et al., 2012). NPR1 is subjected to degradation via the 26S proteasome pathway in the absence of SA (Spoel et al., 2009). Fu et al. (2012) found that NPR3 and NPR4 interact with NPR1 and are required for NPR1 degradation. NPR3 and NPR4 bind SA with different affinities. NPR4 has higher binding affinity and NPR3 has lower binding affinity. In their model Fu et al. (2012) proposed that when SA levels are high enough NPR1 is degraded by NPR3 and NPR1 interaction. When the SA levels are low enough the NPR4-NPR1 interaction mediates NPR1 degradation (Fig 2.13).

35

acid s SCoA Lignan SCoA s, 2c-1 s droxybcnzoic y #' t ' 0 Lignin Flavonaids 4-H X Cinnamoyl-CoA Bcnzoyi-CoA ~ /~ ~ () 0 ~ ~ lglucosinolatc il y acid 2c-2 ozL ... Bcnzo H lvolatilcbcnzcnoidcstcrs acid AAo I #' ' (BA) I~ Bcnzaldchydc f U Bcnzoic SA 0 para-Coumaric \~ , to I OH C4H ~ ~ ac'd o OH OH , acid acid -CA) ·~\~~ #' 0 (I lic , ~ ~ / + O (SA) l pathway J: U trans-Cinnamic Salicy ortho-Coumaric PAL PAL ~ + NH3 OH aninc ) The lal Phc ( 0 2: L-Phcny - o, c 13 Route I acid COOH --../~ )i'ro"'"' 0 Prcphcnic I I ÖH , 3 ,,,, ~ I QY SA HOO to CM ~ COOH COOH 2 2 acid IC) ( CH CH x x OH pathway acid OH 0 OH 0 ,,,,,, acid Chorismic IC '"' ICS acid (SA) ooH OOH ~ ~ ~ OH OH 1~ ' ' ' ':C sochorismic 13C l u Salicylic ;: Ç" I ex The & CS Q Shikimic ' AI) "" 1 : 1 -....:..:.,: AS , HO ' ' ~~ acid Route I Phylloquinonc para-aminobcnzoatc ,3-Dihydroxybcnzoic ~ <;=JAnthranilicacid~ 2 atc l Fo Aux!n Camalcxm Tryplopban glucosinolatcs lndolc

Chapter 2

Fig 2.12 Biosynthesis pathways of salicylic acid in plants, adapted after Dempsey et al. (2011). Pathway products branching from precursors and intermediates in the proposed SA biosynthetic pathways are shown, with focus on Arabidopsis compounds. Open arrows indicate flux to these pathways, with larger arrows indicating greater flux. For the PAL pathway, there are a number of possible routes to SA (2a, 2b, 2c-1, 2c-2). Synthesis of SA from BA could also include glycosylated intermediates (not shown). Enzymes (red) are abbreviated as follows: aldehyde oxidase (AAO), 4-amino-4-deoxychorismate synthase (ADCS), anthranilate synthase (AS), benzoic acid 2-hydroxylase (BA2H), benzoyl-CoA ligase (BZL), cinnamate 4-hydroxylase (C4H), 4-coumarate: CoA ligase (4CL), chorismate mutase (CM), isochorismate synthase (ICS), isochorismate pyruvate lyase (IPL), and phenylalanine ammonia-lyase (PAL). Enzymes involved in modification of SA are not included.

Fig 2.13 Salicylic acid perception in plants, adapted after Yan and Dong (2014). NPR1 is a key positive regulator of SA-mediated responses. NPR3 and NPR4 are the adaptor proteins to mediate NPR1 degradation. NPR4 is a high affinity SA receptor and NPR3 is a low affinity SA receptor. SA blocks NPR4-NPR1 interaction and facilitates NPR3-NPR1 interaction. When SA level is very low, NPR1 level is low because NPR4 mediates its degradation. When SA level is very high, NPR1 level is also low because NPR3 mediates its degradation. At the medium SA level, NPR1 level is the highest because SA level is enough to disrupt NPR4- NPR1 interaction, but not enough to facilitate NPR3-NPR1 interaction.

37

Chapter 3

Temperature-dependent development of the broad mite Polyphagotarsonemus latus (Acari: Tarsonemidae) on Rhododendron simsii

This chapter is based on:

Luypaert G 1, Witters J 2, Van Huylenbroeck J 1, Maes M 2, De Riek J 1, De Clercq P 3 (2014) Temperature- dependent development of the broad mite Polyphagotarsonemus latus (Acari: Tarsonemidae) on Rhododendron simsii . Experimental and Applied Acarology 63(3):389-400

1Institute for Agricultural and Fisheries Research (ILVO), Plant Sciences Unit, Applied Genetics and Breeding, Caritasstraat 21, 9090 Melle, Belgium

2Institute for Agricultural and Fisheries Research (ILVO), Plant Sciences Unit, Crop Protection, Burg. Van Gansberghelaan 96, 9820 Merelbeke, Belgium

3Ghent University, Laboratory of Agrozoology, Department of Crop Protection, Coupure Links 653, 9000 Ghent, Belgium

1. Introduction The broad mite, Polyphagotarsonemus latus (Banks) (Acari: Tarsonemidae) is a cosmopolitan pest affecting numerous plants of more than 60 families (Gerson, 1992). Its highly polyphagous feeding behavior results in severe damage on many economically important crops and ornamentals worldwide (Jeppson et al., 1975; Gerson, 1992; Zhang, 2003). In Belgium, this mite is a key pest in ornamental plant production, particularly pot azalea, Rhododendron simsii Planch hybrids. Reports on the prevalence and biology of the broad mite on R. simsii are scarce in scientific literature. Ewing (1939) was the first to report the occurrence of broad mites on Ericaceae in North America. According to Navajas et al. (2010), P. latus was first recorded in Italy in 1961. Since then, the mite has been found in other European countries including Belgium, where it was reported for the first time on R. simsii in the mid-1980s (Heungens, 1986). The broad mite feeds on the youngest growth tips, causing malformation of the flower buds and the terminal leaves. The latter become severely stunted and hardened, curl down at the edges, and show brownish or reddish lower surfaces. As in many other ornamental crops, the mite’s feeding behavior on R. simsii results in severe economic damage (Labanowski & Soika, 2006). With an annual production of over 35 million plants, R. simsii hybrids are the most important flowering pot plants produced in Belgium. ‘Hellmut Vogel’ and its derived color sports account for approximately 60% of the Belgian azalea production. The culture is highly automated and flowering plants are available nearly year-round. Depending on the plant growth phase, different climate conditions are applied. The production process starts by rooting cuttings under a plastic cover during two months at a soil temperature of 22-25 °C. The plants are then grown vegetatively during several months in a greenhouse or in summer outside on container fields. Flower initiation takes place in June. In this stage temperature is less controlled. During winter, plants are protected from temperatures below 5 °C. To break flower bud dormancy, plants are placed in cold storage rooms at 7 °C for 4 to 7 weeks without assimilation light. The plants are then forced to flower in a heated greenhouse at a constant temperature of ± 21 °C for 10 to 21 days. To speed up this latter process additional light (± 40 µmol.m -2.s -1, daylength 16 to 20 h) can be supplied during dark periods of the year. During recent years, a significant increase in the pest status of P. latus in pot azalea has been observed due to restrictions on the use of broad spectrum acaricides in European horticulture. For this reason, biological control using predatory mites has gained attention (e.g., van Maanen et al., 2010; Rodríguez et al., 2011a; Onzo et al., 2012; Vangansbeke et al., 2013). However, the development of biological control programs against broad mites is hampered by a lack of insight into the basic biology of this cryptic pest. For instance, there is little information concerning thermal requirements of these mites. The relation between temperature and developmental rate of P. latus has only been studied based on a linear model using chili pepper (Li et al., 1985) and Vitis vinifera (Ferreira et al., 2006) as host plants.

Chapter 3

The objective of the present laboratory study was to estimate the thermal requirements and thresholds of P. latus on R. simsii ‘Nordlicht’. This cultivar is an important commercial sport of R. simsii ‘Hellmut Vogel’. Besides a linear model, two non- linear models (Lactin et al., 1995; Brière et al., 1999) were used to assess thermal budgets and thresholds of the different life stages of P. latus .

2. Materials and methods 2.1. Broad mite culture and plant material Broad mites were taken from a colony started by placing naturally infected R. simsii hybrids in a greenhouse. The minimum temperature in the greenhouse was set at 21 °C, ventilation was activated starting at 26 °C. The photoperiod was 16:8 h (L:D). When light intensity was below 100 W.m -2, additional assimilation light (50 µmol.m -2.s -1) was given. When light exceeded 200 W.m -2, shades were unrolled to reduce the light intensity below 200 W.m -2.

Young fresh leaves from newly emerging shoots from uninfested R. simsii ‘Nordlicht’ plants were used in the experiments to produce leaf discs. Clean plant material was kept in a separate greenhouse with a minimum temperature of 5 °C, ventilation temperature set at 20 °C and no supplementary assimilation light.

2.2. Broad mite development The duration of all immature stages of P. latus was measured in incubators (Vötsch Bio Line VB 1014, Germany) at 6 constant temperatures: 15, 17, 20, 25, 30 and 33 ± 1 °C. Temperature was logged using Testo 174T Loggers (Testo AG, Germany). Other parameter settings of the incubator were: relative humidity (RH) 80% ± 5%, photoperiod of 16:8 h (L:D) and light intensity 80-100 µmol.m -2.s -1 (lamps: TL-D 36W/33-640 1SL, Philips, the Netherlands). Leaf discs were placed with the adaxial side on water-saturated tissue paper and bordered with water-saturated tissue paper inside 10-cm diameter insect breeding dishes (SPL Life Sciences, Korea). The total leaf surface area available for the mites was 1.8 cm² (1 × 1.8 cm). The water-saturated tissue paper prevented mites from escaping. Inside the breeding dishes, light intensity varied between 50 and 60 µmol.m -2.s -1 (measured with a LI-250A Light Meter, Li-COR Biosciences, USA) and RH was 90% ± 5% (measured with a digital hygro-thermometer GFTH 200, Greisinger electronic GmbH, Germany). Using a fine brush, P. latus females were transferred from the stock colony to the leaf discs, at a density of 3 females per leaf disc. They were left to oviposit at each of the tested temperatures. Between 30 and 60 leaf discs were set up depending on the temperature. Females were removed from the leaf discs when at least 50 eggs were available for study at the different temperatures. Upon hatching, larvae were individually transferred to new leaf discs and developmental durations ( D) of all stages were monitored every 6 h until adult emergence. This resulted in between 59 (25 °C) and 110

41

(33 °C) individual leaf discs (or replications) at the different temperatures. Sex was determined in the adult stage. For calculation purposes, molting events were assumed to have occurred at the midpoint between two consecutive observations. All observations were made using a stereo microscope (Leica MZ6, Germany).

2.3. Mathematical models One linear and two non-linear models were used for estimating the temperature- dependent development of P. latus on R. simsii ‘Nordlicht’. Both types of models were fitted using the statistical package R 2.15.1 (R Development Team, Austria). The non- linear model fitting of the curves was done using the Gauss-Newton algorithm. The linear model was used for estimation of the development rate ( 1⁄ ), the lower temperature threshold ( ) and the thermal constant ( ) for each life stage. For correct estimation of using the linear model, the exclusion of the results at a temperature of 33 °C was deemed necessary, as the corresponding development rate was out of the linear portion of the curve (De Clercq and Degheele, 1992; Ikemoto and Takai, 2000). The linear model is as follows:

1 = × + where 1⁄ is the development rate (1/days) at temperature (°C), is the intercept and is the slope of the linear function (Arnold, 1959). Description of the relation between temperature and developmental rate based on a non-linear developmental model enables estimation of thermal thresholds , and . In the present study, the widely used Brière (Brière et al., 1999) and Lactin (Lactin et al., 1995) developmental models were used. The model by Brière et al. (1999) consists of a non-linear part at low and high temperatures and a linear part at moderate temperatures. The formula of the Brière model is as follows:

1 = . . − . − where 1⁄ is the development rate (1/days) at temperature (°C), is an empirical constant, (°C) is the lower development threshold, and (°C) is the upper development threshold. The Lactin model is an adaption of the model of Logan et al. (1976), who combined two exponentials to describe development rate at intermediate and high temperatures. Lactin et al. (1995) removed the parameter and added a new parameter ( ) that allows the curve to intersect the x-axis at low temperatures enabling estimation of . The Lactin model is described as:

1 . = . − +

Chapter 3 with 1⁄ the development rate (1/days) at temperature (°C), a constant defining the rate of optimal temperature, (°C) the lethal maximum temperature, and the ∆ temperature range over which physiological breakdown becomes the overriding influence.

2.4. Temperature thresholds Lower development threshold ( ): the temperature at which the rate of development reaches zero or there is no measurable development. The value was estimated by the linear model as −/, and for both non-linear models as the first intercept of the curve with the x-axis. Optimum development threshold ( ): the temperature at which the rate of development is maximal. The value was obtained only by the non-linear models from the value of at ⁄ = 0. Upper development threshold ( ): the temperature at which the rate of development reaches zero or life cannot be maintained for a prolonged period. The value was estimated only by the non-linear models as the second intercept of the curve with the x-axis. Thermal constant ( ): indicates the thermal budget (in degree-days) needed to complete development. It was estimated only by the linear model as 1/.

2.5. Model quality

The (coefficient of determination) and RSS (residual sum of squares) values can be used for describing the quality of fitting a model but are not sufficient to discriminate between models with different numbers of parameters. Hence, both non-linear models were evaluated for describing temperature-dependent development based on the adjusted ( (Kvalseth, 1985) and Akaike Information Criterion ( ) (Akaike 1974). These parameter-independent criteria are calculated as:

, = 1 − × 1 − and = × ln + 2 × where is the number of observations, the number of model parameters and RSS the residual sum of squares. Lowest and highest values indicate the best fitted model.

2.6. Statistical analysis Because the data were not normally distributed and heteroscedastic, a Kruskal-Wallis H test was conducted to evaluate the effect of temperature regime on development time of P. latus . Means were separated post hoc using Dunn’s test (Dunn, 1964) in SPSS (Version

43

22; IBM SPSS Statistics). Differences in total development time between the sexes was assessed using Mann-Whitney U tests in STATISTICA (version 11.0; StatSoft, Inc.). Survival rates were compared using a logistic regression in SPSS (Version 22; IBM SPSS Statistics). In this regression a generalized linear model was constructed using a probit (log odds) link and a binomial error function. For each test, a regression coefficient was calculated and tested for being significantly different from zero, for which p-values are presented (McCullagh and Nelder, 1989). Figures were prepared in SigmaPlot (Systat Software, San Jose, CA).

3. Results 3.1. Developmental time Development of P. latus to adulthood on leaf discs of R. simsii ‘Nordlicht’ was successful at all tested temperatures (Table 3.1). Significant differences in survivorship among the various temperature treatments for each life stage were found. The survivorship from egg to adult was between 23.6 and 96.9% at temperatures of 33 °C and 17 °C, respectively. Overall the percentage of hatched eggs was the highest (87.9-100%), while lowest survivorship was recorded at larval stage (33-96.9%). Total development time of females was significantly longer compared to males at temperatures of 15 °C (Mann-Whitney U = 13.0; p = 0.005), 17 °C (U = 323.5; p < 0.001), 20 °C (U = 6.5; p < 0.001) and 30 °C (U = 361.5; p = 0.005). No significant difference between sexes were observed at 25 °C (U = 112.0; p = 0.896) and 33 °C (U = 20.0; p = 0.732). For both males and females, the total development time decreased significantly when temperatures increased from 15 to 30 °C. Total development time tended to increase again at 33 °C (Table 3.2). Incubation period of female eggs ranged from 6.92 days at 15 °C to 1.67 days at 30 °C, whereas for males this period was between 6.34 and 1.68 days. Overall, egg incubation took about half of the total development time. The nymph stage was the shortest life stage at all tested temperatures for both males (2.85 days at 15 °C and 0.65 days at 30 °C) and females (2.85 days at 15 °C and 0.63 days at 30 °C).

Table 3.1 Survivorship of each life stage of Polyphagotarsonemus latus feeding on Rhododendron simsii ‘Nordlicht’ at different temperatures.

Temperature Egg Larva Nymph Total b na (°C) (%) (%) (%) (%) 15 66 87.9cc 36.2de 100.0a 31.8de 17 98 100.0a 96.9a 100.0a 96.9a 20 62 91.9c 49.1d 96.4b 43.5d 25 59 96.6bc 68.4c 94.9b 62.7c 30 104 100.0 a 88.5 b 95.7 b 84.7 b 33 110 99.1 ab 33.0 e 72.2 c 23.6 e aInitial number of eggs tested. bTotal percentage of eggs that reached adulthood. cMeans within a column followed by the same letters are not significantly different (P > 0.05, Wald-).

Chapter 3

Table 3.2 Mean development times (days ± SE) of the life stages of Polyphagotarsonemus latus at different constant temperatures on Rhododendron simsii ‘Nordlicht’ for males, females and both sexes combined.

Temperature na Egg Larva Nymph Total (°C) Both sexes combined 15 21 6.70 ± 0.16a b 3.86 ± 0.20a 2.76 ± 0.06a 13.32 ± 0.31a 17 95 5.28 ± 0.04a 2.96 ± 0.07a 2.24 ± 0.02a 10.49 ± 0.09a 20 27 3.29 ± 0.14b 1.87 ± 0.10b 1.39 ± 0.03b 6.55 ± 0.21b 25 37 1.95 ± 0.08c 1.34 ± 0.07bc 0.89 ± 0.03bc 4.18 ± 0.11b 30 88 1.67 ± 0.02c 1.15 ± 0.04c 0.63 ± 0.01d 3.46 ± 0.04c 33 26 1.77 ± 0.05c 1.57 ± 0.09bc 0.70 ± 0.05cd 4.04 ± 0.13bc Males 15 8 6.34 ± 0.32a 3.22 ± 0.23a 2.63 ± 0.07a 12.19 ± 0.47a 17 22 5.16 ± 0.13ab 2.49 ± 0.16a 2.17 ± 0.04ab 9.82 ± 0.15ab 20 9 2.75 ± 0.09bc 1.53 ± 0.09ab 1.33 ± 0.04bc 5.61 ± 0.06bc 25 8 2.13 ± 0.15c 1.06 ± 0.04b 0.94 ± 0.04c 4.13 ± 0.18c 30 18 1.68 ± 0.03c 0.89 ± 0.08b 0.65 ± 0.04c 3.22 ± 0.08c 33 2 1.88 ± 0.13 c 1.25 ± 0.25c 0.75 ± 0.00 c 3.88 ± 0.13 c Females 15 13 6.92 ± 0.14a 4.25 ± 0.23a 2.85 ± 0.08a 14.02 ± 0.26a 17 73 5.32 ± 0.04ab 3.10 ± 0.07a 2.27 ± 0.02ab 10.69 ± 0.09ab 20 18 3.56 ± 0.17c 2.04 ± 0.13b 1.42 ± 0.04bc 7.01 ± 0.25bc 25 29 1.90 ± 0.09c 1.41 ± 0.08bc 0.88 ± 0.03cd 4.19 ± 0.13cd 30 70 1.67 ± 0.03c 1.22 ± 0.04c 0.63 ± 0.02e 3.52 ± 0.04d 33 24 1.76 ± 0.06c 1.59 ± 0.09bc 0.70 ± 0.06de 4.05 ± 0.14cd aIndicates the number of observed individuals reaching adulthood. bMeans within a column and sex group (both sexes combined, males or females) followed by the same letter are not significantly different (P > 0.05, Dunn’s test). cNot included in the statistical analysis due to the low number of observations (n = 2).

3.2. Model evaluation The values of the parameters estimated by the linear, Brière and Lactin models for combined development rate data (males and females) for each life stage of P. latus on R. simsii ‘Nordlicht’ are shown in Table 3.3. Linear and non-linear regression of fitted development rate data resulted in low and high (above 0.97) values for all stages. The regressions of the three fitted models for all development stages are depicted in Fig. 1. Using the linear model, the estimated for the different life stages of P. latus varied from 8.3 to 11.2 °C. The number of degree-days (K) estimated by the linear model followed the pattern of egg > larva > nymph (Table 3.3). For total development of P. latus on azalea leaf discs, 66.7 degree-days were required. Both non-linear models allowed estimation of , and values for all developmental stages. Estimations of and by the Brière model were lower than values predicted by the Lactin model, except for the nymph stage (Table 3.3).

45

Fig 3.1 Linear (solid line), Brière (dotted line) and Lactin (dashed line) models fitted to the observed developmental rates (day -1) for egg (a), larval (b), nymph (c) and total (d) life cycle of Polyphagotarsonemus latus on Rhododendron simsii ‘Nordlicht’. The optimum development thresholds estimated by the Lactin model for all stages were similar to those predicted by the Brière model with exception of the nymph stage. Predictions of by the Brière model for the different life stages of the mite varied from 28.8 to 31.3 °C, whereas based on the Lactin model the predicted values ranged between 29.1 and 31.3 °C.

Chapter 3

Table 3.3 Estimated parameters (± SE) of the linear, Brière and Lactin model with values of their evaluation criteria for each life stage of Polyphagotarsonemus latus feeding on Rhododendron simsii ‘Nordlicht’.

Parameters/ Model evaluation Egg Larva Nymph Total criteria Linear 0.0321 ± 0.0028 0.0418 ± 0.0040 0.0825 ± 0.0031 0.0150 ± 0.0010 -0.3354 ± 0.0615 -0.3479 ± 0.0885 -0.9213 ± 0.0694 -0.1503 ± 0.0215 31.2 23.9 12.1 66.7 10.46 8.30 11.16 10.03 0.0035 0.0072 0.0044 0.0004 0.978 0.973 0.996 0.988 0.971 0.964 0.994 0.983

Brière 0.0004 ± 0.00003 0.0003 ± 0.00004 0.0009 ± 0.00016 0.0002 ± 0.00001 10.210 ± 0.715 9.023 ± 0.686 10.330 ± 1.452 10.025 ± 0.613 36.570 ± 0.516 34.730 ± 0.211 37.580 ± 1.401 35.813 ± 0.337 30.48 28.85 31.30 29.86 0.0009 0.0016 0.0190 0.0001 0.996 0.994 0.985 0.996 0.993 0.990 0.976 0.994 -47.16 -43.40 -28.52 -57.63

Lactin 0.0254 ± 0.0045 0.0312 ± 0.0028 0.0370 ± 0.0012 0.0134 ± 0.0009 41.133 ± 3.776 37.816 ± 1.002 35.546 ± 1.089 39.545 ± 1.608 4.268 ± 2.614 3.507 ± 0.918 1.358 ± 0.617 2.745 ± 0.783 -1.324 ± 0.094 -1.335 ± 0.074 -1.398 ± 0.049 -1.154 ± 0.021 11.15 9.29 9.04 10.64 37.73 35.53 34.85 36.01 30.49 29.10 31.27 30.14 0.0011 0.0011 0.0018 0.0001 0.995 0.996 0.999 0.998 0.987 0.990 0.996 0.994 -43.88 -43.49 -40.51 -58.63

4. Discussion The survival rates of P. latus feeding on R. simsii ‘Nordlicht’ were highest between temperatures of 17 °C and 30 °C (43.5-96.9% survival). The overall sex ratio of mites that reached adulthood in the present study was close to the expected ratio of 1:4 male:female (Gerson, 1992). The fastest population growth in practice may therefore be expected to be within this temperature range. Closer to the thermal thresholds a significant reduction in the percentage of total survival was measured, indicating that population growth of the mite will be hampered under these production conditions. The larval stage proved to be most sensitive to temperatures close to the thermal thresholds. Model predictions based on our data suggest that keeping plants at temperatures below 10 °C (i.e., during wintertime) would prevent development of P. latus in azalea production. There are very

47

few records of survival rates of the different life stages of P. latus as a function of temperature and host plant. Vieira and Chiavegato (1998) found a total (egg to adult) survivorship of 91.2% at 28.5 °C on cotton ( Gossypium hirsutum ). This value is higher than that at the closest temperatures in present study on pot azalea (62.7 and 84.7% at 25 and 30 °C, respectively). With the exception of Li et al. (1985), no other study has covered the full range of temperatures to test their suitability for the development of P. latus . The total development times measured by Li et al. (1985) for 15 °C, 20 °C and 25 °C were very similar to our values, but differed at 30 °C (2.67 versus 3.46 days). Values reported by Silva et al. (1998) for the egg-adult period of P. latus on Capsicum annuum at 30 °C (3.4 and 3.6 days for males and females, respectively) correspond with our findings, but those at the other temperatures tend to be lower than the values observed on R. simsii . Hugon (1983) reported ( Citrus sp.) a total development time of 4.08 days when reared on lime at 30 °C, which is somewhat higher than the value found in the present study. De Coss et al. (2010) observed a longer development of P. latus on Carica papaya at 25 °C averaging 4.93 days versus 4.18 days in our study. The total developmental times reported by Ferreira et al. (2006) using V. vinifera as a host plant correspond well with the data from the present study. Vieira and Chiavegato (1998) found an average developmental period of 4.1 days for both males and females on cotton ( Gossypium hirsutum ), measured at 28.5 °C. On C. limon development times were 3.6 days for males and 3.7 days for females at 27.5 °C (Vieira and Chiavegato, 1999). Rajalakshmi et al. (2009) reported on developmental values for P. latus on mulberry ( Morus sp.) in uncontrolled thermal conditions. In that study egg development took 27-32 hours, larval development averaged 36 hours and nymph development took between 6 and 8 hours. These development rates are significantly shorter than those reported elsewhere in the literature. This might be related to the selected host plant and uncontrolled temperature regime. Montasser et al. (2011) published the longest egg to adult developmental times on C. annuum and Cucumis sativus at 24 °C, averaging 7.83-7.84 (male-female) days and 8.71-7.18 (male-female) days, respectively. Besides temperature, relative humidity (RH) has another important influence on the development of P. latus . Although RH inside the incubators was set at a constant 80% (± 5%), the effective RH inside the insect breeding dishes was between 90 and 95%. Jones and Brown (1983) reported variation in theoretical population growth of P. latus on C. limon fruits at constant temperatures when subjected to different RH regimes using small humidity chambers. Temperatures above 30 °C coupled with low humidity appeared to be lethal to the egg and nymph stages (Jones and Brown, 1983). De Coss et al. (2010) found significant differences in development time for P. latus on C. papaya when subjected to various humidity regimes with constant temperatures. Besides host plant, the observed differences in development time of P. latus discussed above may be related to the relative humidity regime. This hypothesis is supported by the wide range of relative humidities used in the different experiments (65-90%).

Chapter 3

As indicated by low and high values, all selected models fitted the data of this study well. A common method for evaluating the accuracy of temperature thresholds is based on their comparison with experimental data (Kontodimas et al., 2004). Since P. latus developed at the lowest (15 °C) and highest (33 °C) tested temperatures and no lower or higher temperature values were included in the present study, we could not use this criterion. With the use of the parameter-independent criteria and (Table 3.3) we were able to select the most adequate model at each developmental stage. Nymph development was best described by the Lactin model (lowest and highest values), as the Brière model overestimated . For the egg stage, the Brière model fitted the development rate data best, indicating an overestimation of by the Lactin model. Minimal differences between the Brière and Lactin model were observed for larval and total development. When considering all life stages, the Lactin model performed best and is therefore preferred to estimate thermal thresholds. This is in agreement with the findings of Kontodimas et al. (2004) who showed that the Lactin model is more accurate in describing the influence of temperature on the development of many arthropods as compared with other models. Other models, like the Logan type III model (Hilbert and Logan, 1983), have been shown to approach more asymptotically to the point of zero development than the non-linear models used in the present work, which should yield more reliable estimates of lower thermal thresholds. Nonetheless, the estimates from the presently used models were corroborated by preliminary observations on the capacity of P. latus to develop at 12 °C. In the latter experiment, eggs collected at 25 °C were placed at 12 °C after gradual cooling. Very few of the latter eggs hatched (35.3%), and the very few larvae that did emerge were not able to survive. We emphasize that the developmental thersholds presented in this study are estimated based on extrapolation of models fitted to experimental data not incorporating these thresholds. Additional experiments in the lower or upper temperature range might provide a more accurate insight into the capacity of P. latus to survive and develop under cold or hotconditions. So far, only a few studies have attempted to estimate thermal budgets and thresholds of the broad mite, P. latus. The estimated thermal constant ( ) of 66.7 degree- days is similar to the value of 62.7 degree-days reported by Ferreira et al. (2006) for total development of P. latus on V. vinifera . The thermal budget for development on chili pepper was predicted by Li et al. (1985). In their calculations, the preoviposition period (i.e. the period between adult emergence and the first egg laid) was included. The 64.1 degree- days estimated for development of P. latus from egg to egg in the latter study thus predicts a lower thermal budget on immature development than does our study. In the present study both the linear and non-linear models yielded similar -values of 10.0-10.6 °C for total development on leaves of R. simsii ‘Nordlicht’. For P. latus feeding on V. vinifera, a comparable value of 9.7 °C was calculated (Ferreira et al., 2006). Using a multiple regression curve of development data, Jones and Brown (1983) extrapolated that the lower and upper theoretical thresholds of P. latus feeding on fruits of Citrus limon were between 12 and 14, and 33 and 35 °C, depending on the humidity. Li et al. (1985) reported no complete development at 35°C as all tested larvae died at this temperature. In the

49

present study, however, the mite successfully completed development at 33° C and most estimated upper thresholds were above 35 °C. Data and models from the present study provide a basis to predict the development of a P. latus population in practice at given climate conditions and to parameterize warning systems. For instance, at the end of the cultivation process pot azaleas are forced to flower. This involves heating the greenhouses to a constant temperature of 21 °C and providing supplementary assimilation light to 16:8 h (L:D). Based on the data of the present laboratory study, we can theoretically expect between 1 and 4 generations to be completed during a forcing period of 10-21 days. This value is obviously an overestimation since we did not take the pre-oviposition period of the mite into account, but it does indicate the potential risk for an outbreak of this pest at this temperature regime. When calculating the thermal budget based on long-term average temperature data for Merelbeke (Belgium) (51°0’0”N/3°45’0”E) we can theoretically expect 1.3 generations during the colder months (October-March) and 14.7 generations during the warmer months (April-September). Azalea plants are grown outside during the warmer period, thus they are faced with successive generations of the pest. During the winter plants are protected in greenhouses from freezing. Theoretically, more generations may develop in the warmer greenhouse environment than the number predicted in the field. The importance of critical temperatures and thermal budgets for understanding the phenology of an arthropod has long been recognized. Lower developmental thresholds and thermal constants are not only good predictors of the timing of life events (Honek and Kocourek, 1988), but are also very useful indicators for the potential distribution of the pest (Campbell et al., 1974). Combined with developmental data of different phytoseiid mites preying on P. latus, like Amblyseius swirskii (van Maanen et al., 2010; Onzo et al., 2012) and A. largoensis (Rodríguez et al., 2011a), this study provides valuable information for optimizing the biological control of this pest in the production of pot azalea and other ornamentals.

Chapter 4

Cold hardiness of the broad mite Polyphagotarsonemus latus (Acari: Tarsonemidae)

This chapter is based on:

Luypaert G 1, Witters J 2, Berkvens N 2, Van Huylenbroeck J 1, De Riek J 1, De Clercq P 3 (2015) Cold hardiness of the broad mite Polyphagotarsonemus latus (Acari: Tarsonemidae). Experimental and Applied Acarology 66(1):29-39

1Institute for Agricultural and Fisheries Research (ILVO), Plant Sciences Unit, Applied Genetics and Breeding, Caritasstraat 21, 9090 Melle, Belgium

2Institute for Agricultural and Fisheries Research (ILVO), Plant Sciences Unit, Crop Protection, Burg. Van Gansberghelaan 96, 9820 Merelbeke, Belgium

3Ghent University, Laboratory of Agrozoology, Department of Crop Protection, Coupure Links 653, 9000 Ghent, Belgium

1. Introduction The broad mite, Polyphagotarsonemus latus (Banks) (Acari: Tarsonemidae), is a pest of numerous crops in many parts of the world, including tropical and subtropical regions such as Australia, Asia, Africa, the Americas and the Pacific Islands (Gerson, 1992). In temperate and subtropical areas the broad mite is a pest of greenhouse crops (Waterhouse and Norris, 1987). According to Navajas et al. (2010), P. latus was first recorded in Europe (Italy) in 1961. Since then, the mite has also been found in a number of northwestern European countries, including Belgium, where it was reported for the first time on pot azalea, Rhododendron simsii, in the mid-1980s (Heungens, 1986). The broad mite feeds on the youngest growth tips of R. simsii , causing malformation of the flower buds and the terminal leaves. The latter become severely stunted and hardened, curl down at the edges, and show brownish or reddish lower surfaces. Like in many other ornamental crops, the mite’s feeding behavior on R. simsii results in severe economic damage (Labanowski and Soika, 2006).

Rhododendron simsii hybrids are the most important flowering pot plants produced in Belgium and are made available nearly year-round. Climatic conditions during the production process differ depending on the plant growth phase. The production process starts by rooting cuttings under a plastic cover during two months at a soil temperature of 22-25 °C. The plants are then grown vegetatively during several months in a greenhouse or in summer outside on container fields. Flower initiation takes place in June; at this stage temperature is less controlled. During winter, plants are protected from freezing temperatures. Exposure of the plants to the lower temperatures during winter is necessary to release the flowers from their dormant state. Unfulfilled chilling requirements lead to erratic bud break and low-quality flowering (Christiaens et al., 2014b). Temperatures for dormancy breaking are generally between 2.5 and 9 °C (Richardson et al., 1974), and the optimum for R. simsii hybrids is 7 °C (Stuart, 1965). In commercial plant production the material is placed in cold storage rooms at 7 °C during 4 to 6 weeks. The plants are then forced to flower in a heated greenhouse at a constant temperature of ca. 21 °C for 10 to 21 days. To speed up this process additional light (ca. 40 µmol.m -2.s -1, day length 16 to 20 h) can be supplied during dark periods of the year.

Luypaert et al. (2014) estimated the relationship between temperature and developmental rate of P. latus on R. simsii ‘Nordlicht’ using a linear and two non-linear models. Thresholds for development of P. latus were estimated to be 10 and 36 °C for the lower and upper developmental threshold, respectively. Earlier, this relationship was studied based on a linear model using chili pepper (Li et al., 1985) and Vitis vinifera (Ferreira et al., 2006) as host plants. Besides these studies, there is very little information concerning the relationship between temperature and life history of P. latus, and in particular the cold hardiness of the pest has not been studied in detail. Karl (1965, cited in Gerson, 1992) stated that P. latus could not survive outside of a tropical, subtropical or controlled greenhouse environment. More recently, Vincent et al. (2010) reconsidered the hypothesis that P. latus can also be a pest in subtropical climates or controlled

Chapter 4 environments, based on the fact that infestation on primocane-fruiting blackberry ( Rubus rubus ) continued during a 3-year period in the northern parts of Arkansas (USA).

The objective of the present laboratory study was to gain insight into the cold hardiness of P. latus , as a key pest of pot azalea in northwestern Europe. The egg hatchability, survival of larvae and female adults and reproduction capacity of females following a prolonged period of cold exposure was determined. The supercooling point (SCP) of female adult mites was measured and lower lethal times (LTime 10,50,90 ) were estimated when the females were exposed to -3 or 2 °C.

2. Materials and methods 2.1. Broad mite culture and plant material The broad mite culture and plant material were kept in the same environmental conditions as described in section 2.1 of Chapter 3.

2.2. Survival and reproduction capacity following a cold exposure period To ensure that all experimental mites developed under equal conditions, the following method was used. Using a fine brush, P. latus females were transferred from the stock colony in the greenhouse to the leaf discs, at a density of 1 female per leaf disc. Leaves were placed with the adaxial side on water-saturated tissue paper and bordered with water-saturated tissue paper inside 10-cm diameter insect breeding dishes (SPL Life Sciences Co. Ltd., Pocheon, Korea) to produce leaf discs. The total leaf surface area available for the mites was 1.8 cm² (1 × 1.8 cm). The water-saturated tissue paper prevented the mites from escaping. Adult females were given 24 hours for oviposition and were subsequently removed from the leaf discs. Leaf discs containing eggs were transferred to an incubator (Vötsch-Industrietechnik Bio Line model VB 1014, Balingen- Frommern, Germany) at a constant temperature of 25 ± 1 °C, 80 ± 5% relative humidity (RH), a 16 h photoperiod and a light intensity of 80-100 µmol.m -2.s -1 (lamps: TL-D 36W/33-640 1SL, Philips, Eindhoven, the Netherlands). Leaf discs were moistened daily to prevent the mites from escaping. Two-day-old adult females were transferred to new individual leaf discs at the beginning of the experiments. Experimental eggs and larvae were reared under the same conditions. Eggs and larvae less than 24h old were collected from leaf discs infested with adult mites.

Polyphagotarsonemus latus mites were exposed to a 7 °C cold treatment, corresponding with the optimum temperature for dormancy breaking of pot azalea (Stuart, 1965). The tested exposure periods for eggs were 1, 17, 31 and 52 days (corresponding with the 6-7 week timespan for cold storage of azalea plants). Larvae were exposed for periods of 14, 28 and 49 days, whereas two-day-old adult females were exposed to 7 °C for 14, 28 and 42 days. Hatchability of eggs returned to rearing conditions (25 °C, 80% RH and 16:8 h (L:D)) was monitored every 24 h for a six day period. The survivorship of exposed larvae was assessed 24 h and 7 days after the cold exposure. Likewise, survival of the exposed

53

adults and the percentage of reproducing females was monitored 24 h and 7 days after the end of the cold period. The oviposition rate of the surviving females was calculated over a 2 day-period following cold exposure.

The cyclamen mite, Phytonemus pallidus (Banks) (Acari: Tarsonemidae) , another plant feeding tarsonemid mite, has been noted to overwinter in the adult stage in temperate climates (Denmark, 1988). Therefore, adult P. latus females were also exposed to temperatures of 2 or -3 °C. At 2 °C the time periods chosen were 6, 13 or 26 days, whereas at -3 °C the mites were exposed for 24 or 72 h, or 7 days. As before, survival of exposed adults and the percentage of reproducing females was monitored 24 h and 7 days after having been returned to rearing conditions. Oviposition rates of females surviving the -3 or 2 °C cold treatments were recorded as described above and compared with those of non-exposed females taken from the stock colony.

The mites were exposed to the cold temperatures (-3, 2 or 7 °C) in a cooled incubator (MIR-254, Panasonic Healthcare Co., Ltd., Tokyo, Japan) without light. Humidity during cold exposure was not controlled, but given the fact that leaf discs were kept on water- saturated tissue paper relative humidity was assumed to be around 90% throughout the trials (Luypaert et al., 2014). Mites were allowed to acclimatize for 20 minutes at different intermediate temperatures (18 °C, 10 °C and, for exposure to -3°C, 2 °C) before and after exposure to the tested temperatures, in order to prevent cold and heat shock mortality, respectively. This type of acclimatization was also applied in cold tolerance studies on other mites or insects including, Amblyseius swirskii (Athias-Henriot), Phytoseiulus longipes (Evans) and Bombus terrestris (L.) (Allan, 2010; Owen, 2013). All observations were made using a stereo microscope (Leica MZ6, Leica, Wetzlar, Germany).

2.3. Lower lethal times

Lower lethal times (LTime 10,50,90 ) were estimated for two-day-old adult females by placing leaf discs with a single two-day-old adult female at -3 or 2 °C for a varying amount of time. Calculations are based on the averages of two independent experiments with 10 replicates each. The cold exposure period varied between 0-96 h and 0-17 days for -3 and 2 °C, respectively. To cover survival ranging from 100 to 0% at each temperature, the mites were exposed during 5 (0, 24, 48, 72 and 96 h) or 6 (0, 3, 7, 10, 14 and 17 days) different durations at temperatures of -3 or 2 °C, respectively. After the cold exposure period, the mites were returned to a temperature of 25 °C. Again, the mites were allowed to acclimatize by placing them at intermediate temperatures (18 °C, 10 °C and, for exposure to -3°C, 2 °C) for 20 min. Mortality of the mites was assessed 24 h after recovery. Mites that were unable to move, upon prodding with a fine brush, were scored dead and the durations corresponding to 10, 50 or 90% mortality (i.e., LTime 10,50,90 ) were estimated.

Chapter 4

2.4. Supercooling points Individual two-day-old P. latus females were attached to a type-T 30-gauge copper- constantan thermocouple after it was dipped in high vacuum grease (Silicone high vacuum grease medium, Merck KGaA, Darmstadt, Germany). The thermocouple was attached to a data logger (TC-08 Thermocouple Data Logger, Pico Technology Ltd., Eaton Socon, UK) which recorded the temperature of the probe at 100-ms intervals. The adults attached to the thermocouple were led through the small opening of a 1 mL pipet tip. A second pipet tip was placed over the lower end of the first pipet tip producing an experimental tube. These tubes were individually led in glass test tubes and placed in a Haake G50 alcohol bath (Thermo Fisher Scientific Inc., Waltham, MA, USA), with a cooling rate of -0.5 °C.min -1.The starting temperature was set at 20 °C. The SCP was defined as the lowest temperature recorded before the release of the latent heat of fusion (n = 30).

2.5. Statistical analysis Survival rates and reproduction capacity after cold exposure were compared using a logistic regression in SPSS (Version 22.0, IBM Corp., Armonk, NY, USA). In this regression a generalized linear model was constructed using a probit (log odds) link and a binomial error function. (McCullagh and Nelder, 1989) . The oviposition rates after cold exposure were compared with a control group by way of independent samples Kruskal-Wallis tests in SPSS. LTime 10,50,90 -values were estimated in SPSS using probit analysis. Descriptive data analysis was conducted in STATISTICA (Version 12.0, Statsoft Inc., Tulsa, OK, USA). Figures were prepared in SigmaPlot (Version 13.0, Systat Software, San Jose, CA, USA).

3. Results Eggs laid at 25 °C and then transferred to 7 °C did not survive an exposure period of either 17, 31 or 52 days (at each time period n = 20). When exposed to 7 °C for 24 h, all eggs (n = 23) hatched to larvae and successfully reached adulthood when returned to rearing conditions.

Exposure of P. latus larvae to 7 °C during periods of 14 and 28 days resulted in similar survival rates ranging from 40 to 53% 24 h after recovery (Fig 4.1). When the exposure period was extended to 49 days, survival dropped to 13%, which was significantly lower than after the 14-day exposure (Wald-χ2 = 5.26, df = 1, p = 0.022).

The survival of adult P. latus females was not significantly altered when exposed to 7 °C for 14 or 42 days (Wald-χ2 = 0.23, df = 1, p = 0.63 and Wald-χ2 = 0.62, df = 1, p = 0.43, 24 h and 7 days after ending the treatment, respectively) (Fig 4.2c). Rates of survival 24 h after exposure to 7 °C were 85, 100 and 90% for exposure periods of 14, 28 and 42 days, respectively. Monitoring survival 24 h or 7 days after ending each specific exposure period (14, 28 or 42 days) yielded similar results. The proportion of ovipositing females was not significantly affected by the duration of the cold exposure at 7 °C and ranged from 15-30% and 75-85%, 24 h and 7 days after recovery, respectively (24 h after recovery: Wald-χ2 = 1.28, df = 1, p = 0.26; Wald-χ2 = 0.53, df = 1, p = 0.47 and Wald-χ2 = 0.17, df = 1,

55

p = 0.68; 7 days after recovery: Wald-χ2 = 0.00; df = 1; p = 1, Wald-χ2 = 0.62; df = 1; p = 0.43 and Wald-χ2 = 0.62; df = 1; p = 0.43; for comparisons between periods of 14 and 28 days, 14 and 42 days and 28 and 42 days, respectively) (Fig 4.2f). The oviposition rate of females exposed to a temperature of 7 °C for 14 days did not differ from that of control mites maintained at rearing conditions (H = 0.48, df = 1, p = 0.49) (Table 4.1). A prolonged exposure of females at 7 °C significantly decreased the oviposition rate as compared to control mites (H = 15.44, df = 1, p < 0.001 and H = 10.29, df = 1, p = 0.001, for a 28- and 42-day cold treatment, respectively)(Table 4.1).

100 24 hours after treatment 7 days after treament

80

60 c

bc bc

Survival (%) Survival 40

abc

20 ab a

0 14 28 49 Cold exposure days at 7 °C

Fig 4.1 Survival of Polyphagotarsonemus latus after exposure of larvae to 7 °C for different durations, monitored 24 hours (black bars) and 7 days (gray bars) post treatment. Bars marked by the same letter are not significantly different (p > 0.05, Wald-χ2; n = 15).

When exposed to 2 °C, survival rates of adult females monitored 24 h or 7 days after recovery were similar for each exposure period (6, 13 and 26 days) (Fig 4.2b). Survival rates dropped significantly after an exposure period of 13 days as compared to a 6-day exposure (Wald-χ2 = 11.94, df = 1, p = 0.001 and Wald-χ2 = 8.42, df = 1, p = 0.004, 24 h and 7 days after ending the treatment, respectively). No survival was observed after a 26-day cold exposure period. Females were only able to oviposit within the first 24 h after a 6- day exposure at 2 °C (Fig 4.2e); at that time point 20% of the females had laid a single egg. One week after having been returned to rearing conditions, the percentage of ovipositing females increased to 50 and 15% after a cold exposure period of 6 or 13 days, respectively. Ovipositing females from both groups had similar oviposition rates as control mites (H = 3.49, df = 1, p = 0.062 and H = 0.35, df = 1, p = 0.56, for a 6- and 13-day cold treatment, respectively) (Table 4.1). Whereas in this experiment hatching of the deposited eggs was not monitored, the appearance of juveniles and new adults in the arenas during the recovery period indicated that at least part of these eggs were viable.

Chapter 4

(a) b 100 100 (b) 100 (c) ab d a a ab d 24 hours after treatment 80 80 a 7 days after treatment 80

cd 60 cd 60 60

40 40 40

Survival (%) bc bc

20 b 20 b 20

a a a a 0 0 0 24 72 168 6 13 26 14 28 42 100 (d) 100 (e) 100 (f) b b 80 80 80 b

60 60 60 b c

40 40 40 a b b a

Ovipositing females (%) 20 20 b 20 a

a a a a a a a 0 0 0 24 72 168 6 13 26 14 28 42 Cold exposure hours at -3 °C Cold exposure days at 2 °C Cold exposure days at 7 °C

Fig 4.2 Percentages of surviving (a, b and c) and ovipositing (d, e and f) adult female Polyphagotarsonemus latus mites after exposure to -3, 2 or 7 °C during different periods, monitored 24 hours (black bars) and 7 days (gray bars) post treatment. Bars within each graph marked by the same letter are not significantly different (p > 0.05, Wald-χ2; n = 15 at -3 °C and n = 20 at 2 and 7 °C).

Survival of adult P. latus females monitored 24 h or 7 days after recovery from cold exposure was not affected by the duration of the exposure period (24, 72 or 168 h) to -3 °C (Fig 4.2a). A significantly lower survival was noted after an exposure period of 72 h as compared to a 24 h exposure (Wald-χ2 = 8.35, df = 1, p = 0.004 and Wald-χ2 = 12.61, df = 1, p < 0.001, 24 h and 7 days after ending the treatment, respectively). Continuous exposure for 7 days to -3 °C resulted in no surviving females. None of the females was capable to oviposit within the first 24 h after exposure to -3°C during 24 or 72 h (Fig 4.2d). After 7 days of recovery, however, 53 and 20% of the females exposed during 24 and 72 h, respectively, oviposited. Their oviposition rates were significantly lower than those of non-exposed females (H = 17.85, df = 1, p < 0.001 and H = 4.88, df = 1, p = 0.027, for a cold exposure period of 24 and 72 h, respectively (Table 4.1).

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Table 4.1 Oviposition rates (means ± SE) of Polyphagotarsonemus latus females after exposure to -3, 2 or 7 °C during different periods. Data represent mean oviposition rates when monitored 48 hour after having been returned to rearing conditions (25 °C, 80% RH and 16:8 h (L:D)). Level of significance denotes statistical differences compared to a control group of mites maintained at rearing conditions.

Treatment Duration of n Oviposition rate Level of temperature treatment (eggs.female -1.day -1) significance (p-value) 25 °C Control 106 1.52 ± 0.11 -3 °C 24 h 10 0.10 ± 0.07 < 0.001 72 h 4 0.38 ± 0.13 0.027 7 days - - - 2 °C 6 days 15 0.97 ± 0.25 0.062 13 days 4 1.13 ± 0.38 0.56 26 days - - - 7 °C 14 days 19 1.29 ± 0.22 0.49 28 days 20 0.53 ± 0.16 < 0.001 42 days 16 0.63 ± 0.13 0.001

Lethal time values for adult females exposed to temperatures of -3 or 2 °C are shown in Fig 4.3. The LTime 50 -values were estimated to be 61.2 h and 9.3 days at -3 and 2 °C, respectively. The SCP for two-day-old adult females averaged -16.5 ± 0.2 °C (mean ± SE).

120 (a) 18 (b)

16 100 14

80 12

10 60 8

40 6 Exposure time (days) time Exposure Exposure time (hours) time Exposure 4 20 2

0 0 10 50 90 10 50 90 Mortality at -3 °C (%) Mortality at 2 °C (%)

Fig 4.3 LTime 10,50,90 (± 95% fiducial limits) of adult Polyphagotarsonemus latus females at (a) -3 and (b) 2 °C.

Chapter 4

4. Discussion In the production of pot azalea, P. latus mites are exposed to temperatures below the lower developmental threshold of 10 °C (Luypaert et al., 2014) during part of the growing cycle. Temperatures in the range of 2 to 7 °C are common when R. simsii hybrids are placed in cold storage rooms to break dormancy of flower buds. At an optimum temperature of 7 °C (Stuart, 1965) or even at temperatures of 2-3 °C plants can be stored for 4 to 6 weeks. Chilling of R. simsii plants is necessary to avoid erratic bud break and low-quality flowering (Christiaens et al., 2014b). Plant damage caused by P. latus is frequently recorded at the end of the production cycle when flowers are forced in heated greenhouses (ca. 21 °C), where also additional light can be supplied. It has been shown that these conditions are favorable for P. latus development on R. simsii (Luypaert et al., 2014), as well as on other crops (Li et al. 1985; Ferreira et al. 2006). Whereas several studies have focused on the relation of temperature and development of P. latus (e.g. Jones and Brown, 1983; Li et al., 1985; Ferreira et al., 2006; Luypaert et al., 2014) to our knowledge no workers have exposed the mite to temperatures below the lower developmental threshold. Furthermore, experimental evidence on the cold hardiness or overwintering capacity of P. latus is limited in the literature. Two studies discussed the climatic regions where P. latus might able to survive being the tropical, subtropical (Karl, 1965 (cited by Gerson, 1992)) and moderate temperate climatic regions (Vincent et al., 2010). Overwintering and cold tolerance is also poorly studied in other plant dwelling tarsonemid mites. Tarsonemid mites are generally believed to overwinter in the adult stage (e.g. Smith and Goldsmith, 1936; Wiesmann, 1941; Jeppson et al., 1975; Denmark, 1998). Jeppson et al. (1975) stated that the adult stage of tarsonemid mites is known to survive through prolonged exposure of freezing temperatures. Further, there are no reports of the existence of diapause within the family of Tarsonemidae (Hoy, 2011).

The present study shows that egg hatchability was zero after an exposure of 17 days to a temperature of 7 °C. This indicates a detrimental effect on embryogenesis of temperatures below the lower developmental threshold for eggs of P. latus , which was estimated to be 10.2 °C (Luypaert et al., 2014). However, eggs are capable to withstand a temperature of 7°C during a period of 24 h, without any effects on hatching or offspring performance. Further, our data also indicate that a substantial proportion of larvae was able to withstand exposure to 7 °C for much longer periods: about half of the larvae were still alive after an exposure period of 14 days, and survival only dropped below 20% after 49 days of cold exposure. The adult females were clearly more robust, showing limited detrimental effects from a 7 °C cold exposure up to 6 weeks, both in terms of survival rates and reproduction capability: nearly all females surviving an exposure to 7 °C were able to resume egg laying when returned to favorable conditions. However, their oviposition rates were affected by the duration of the cold treatment: whereas oviposition rates of females exposed to 7 °C for 14 days were similar to those of control females, they were 2.9 and 2.4 times lower after a 28- and 42-day exposure, respectively.

59

For temperatures far below the developmental threshold of 10 °C, i.e. 2 and -3 °C, the focus of the study was placed on the adult phase of P. latus, as this is reportedly the stage in which tarsonemid mites overwinter in temperate areas (e.g. Smith and Goldsmith, 1936; Wiesmann, 1941; Jeppson et al., 1975; Denmark, 1998). Our findings indicate the limited ability of P. latus to survive a prolonged exposure to subzero temperatures. None of the females survived an exposure of 26 days at 2 °C and of 7 days at -3 °C. Reproduction of females surviving shorter exposure periods at such low temperatures was also affected. Most of the females exposed to 2 or -3 °C required a recovery period longer than 24 h to resume egg laying. Whereas the oviposition rates of females exposed for either 6 or 13 days to 2 °C were not different from those of control females, all females surviving exposure to -3 °C had lower oviposition rates than in the control. Validation (over- or underestimation) of these laboratory results in the field is necessary to reliably evaluate the cold hardiness of P. latus . Practical experiments at the PCS Ornamental Plant Research Station confirmed our findings, showing that mites were able to survive cold storage on R. simsii . The survival rate depended on the storage temperature and the duration of the cold exposure (personal communication).

Whereas data in the literature on cold hardiness parameters of members of the Tarsonemidae family are scarce, more information is available on the overwintering capacity of phytoseiid mites, some of which are used as biological control agents against P. latus . These include the economically important species Amblyseius swirskii (van Mannen et al., 2010; Onzo et al., 2012; Abou-Awad et al., 2014), Neoseiulus cucumeris (Li et al., 2003; Weintraub et al., 2003) and Neoseiulus californicus (Peña and Osborne, 1996; Jovicich et al., 2008). The average SCP value of P. latus females (-16.5 °C) is somewhat higher than that reported for non-acclimated A. swirskii mites (-18.3 °C; Allen, 2010). Neoseiulus cucumeris and N. californicus showed to have a much lower SCP, with values of -20.7 (Morewood, 1992) and -21.6 °C (Hart et al., 2002), respectively. SCP data alone are not believed to be sufficiently reliable indicators of cold tolerance (Bale, 1987; Bale, 1996) because the vast majority of species are freeze avoiding and SCP temperatures are rarely experienced by individuals in their natural habitats. Lethal time data best represent naturally occurring cold stress because they not only test temperature but also exposure time (Allen, 2010). The lethal time experiments done in our study are the most closely related laboratory measurements to field survival because they take into account mortality over time at temperatures likely to be encountered by P. latus during the production process of R. simsii hybrids, with the exception of the -3 °C cold treatment. In our experiments, the microclimate and shelter provided by foliar trichomes and veins mimicking the natural environment was simulated using leaf discs. However, these leaf discs remain an artificial environment differing from real on-plant conditions where besides foliar trichomes and veins, shelters such as leaf axils, buds or flowers might provide a more favorable microclimate for the mite. The lethal times for 50% mortality in P. latus females estimated here (61.2 hours and 9.3 days for -3 and 2 °C, respectively) are higher than the values of 9.2 min, 1.6 days and 2.7 days for non-acclimated A. swirskii adults at -5, 0 and 5 °C, respectively (Allen, 2010), indicating better level of cold tolerance

Chapter 4 in P. latus than in A. swirskii . On the other hand, N. californicus appears to be more cold hardy, with much higher LTime 90 -values of 8 and 15 days for -5 and 0°C, respectively (Hart et al., 2002), versus 76.9 h and 12.1 days for -3 and 2 °C, respectively, for P. latus . Information on the thermal responses of P. latus and its phytoseiid predators may be relevant for biological control programs during cooler parts of the growing season.

In conclusion, the findings of our study suggest that P. latus is a freeze intolerant, chill susceptible species (Bale, 2002). However, females of the pest are expected to be able to withstand all temperatures encountered during the R. simsii hybrid production process in northwestern Europe. Problems caused by this pest during the forcing process are likely due to female mites easily surviving cold storage needed to break bud dormancy. Further, data from the present study suggest that under open field conditions Polyphagotarsonemus latus is insufficiently cold tolerant to withstand winters generally occurring in the temperate climate of northwestern Europe, as subzero temperatures dramatically decrease survival and reproduction capacity of the mite. Additional data on the plasticity and adaptation of P. latus to changing temperatures will provide more evidence to fully understand its potential for outdoor survival. More in-depth studies on the indoor overwintering of P. latus or other members of the tarsonemid family may help optimizing the integrated management of this pest in the production of R. simsii hybrids or other ornamentals and vegetable crops grown in the greenhouses of temperate climatic zones.

61

Chapter 5

Screening for broad mite susceptibility in Rhododendron simsii hybrids

This chapter is partly based on:

Luypaert G 1, Van Huylenbroeck J 1, De Riek J 1, De Clercq P 2 (2014) Screening for broad mite susceptibility in Rhododendron simsii . Journal of Plant Diseases and Protection 121(6):260-269

1Institute for Agricultural and Fisheries Research (ILVO), Plant Sciences Unit, Applied Genetics and Breeding, Caritasstraat 21, 9090 Melle, Belgium

2Ghent University, Laboratory of Agrozoology, Department of Crop Protection, Coupure Links 653, 9000 Ghent, Belgium

1. Introduction The broad mite, Polyphagotarsonemus latus (Banks) (Acari: Tarsonemidae), is a cosmopolitan pest affecting numerous plants. It causes severe damage to many economically important agricultural crops and ornamental plants worldwide (Jeppson et al., 1975; Gerson, 1992; Zhang, 2003). The broad mite is usually found on the lower surfaces of young apical leaves and in flowers, where it deposits its eggs (Gerson 1992). Although broad mites feed mainly on the epidermis of the plant parts, structural aberrations occur throughout the tissue (Grinberg et al., 2005). The pest feeds on the youngest growth tips, causing malformation of the flower buds and the terminal leaves. The latter become severely stunted and hardened, curl down at the edges, and show brownish or reddish lower surfaces (Labanowski and Soika, 2006). Ewing (1939) was the first to report the occurrence of broad mites on Ericaceae in North America. According to Navajas et al. (2010), broad mites were first recorded in Italy in 1961. Since then, the mite has been found in other European countries including Belgium, where it was reported for the first time on R. simsii in the mid-1980s (Heungens, 1986). During recent years, a significant increase in the pest status of broad mites in pot azalea has been observed, mainly due to restrictions on the use of broad spectrum pesticides, including the complete prohibition of the use of endosulfan (an active component in many plant protection products) from July 2007 onwards (EU directive 2005/864/EC). Integrated Pest Management (IPM) and its EU-wide implementation (EU directive 2009/128/EC) forces growers to develop sustainable strategies for the control of this pest (Gobin et al., 2011, 2013). In an IPM based strategy, preventative cultural practices are essential during the growing process. One of these measures could be developing and growing resistant R. simsii cultivars. Resistant genotypes to broad mite have been successfully selected in various crops. Most studies related to screening for susceptibility or to factors leading to resistance against broad mite have been performed on Capsicum spp. Matos et al. (2009) found that trichomes act as a limiting trait for the development of broad mites , especially when the trichomes occur in higher densities and are well-distributed on the leaf surface. According to Thungrabeab and Boonlertnirun (2002), trichome density and leaf thickness tend to be predominant factors related to resistance (few mites and low damage rates) to broad mite in Capsicum annuum L. (line P 3066-20-20 and line P 3067-17-2) and C. frutescens L. (line CHMC 1118). For both host species, Echer et al. (2002) concluded that symptom assessment was a more suitable method for determining resistance of genotypes than counting the number of adult mites present on the plant. This resulted from an indirect relation between the number of broad mites and the intensity of the damage symptoms observed on certain genotypes (Echer et al., 2002). Furthermore, according to Bassett (1981), the manifestation of symptoms can quickly evolve in certain host plants, where a small number of mites would be enough to cause economic damage. Susceptible as well as resistant accessions were identified in screenings of C. annuum cultivars using a natural infestation of broad mites (Ahmed et al., 2001; Desai et al., 2006, 2007; Kulkarni et al., 2011). The correlation of biochemical constituents in 15 C. annuum cultivars with mite incidence was assessed by Ahmed et al. (2000). Based on a study on

Chapter 5 the relationship of broad mites to host phenology and injury levels in C. annuum , de Coss- Romero and Peña (1998) concluded that damage caused by broad mites appears to depend on the stage of development of C. annuum . The plant is most vulnerable to infestation during the vegetative growth phase (Rodríguez et al., 2011b). Vichitbandha and Chandrapatya (2011) also studied the effect of broad mites on damage and yield in C. annuum ‘Tavi 64’ and showed significant yield reduction after a plant damage level of more than 50% curled shoots. Reports on screening for resistance against broad mites in other crops are also available, e.g. potato species Solanum polyadenium Greenm., S. tarijense Hawkes and S. berthaultii Hawkes (Gibson and Valencia, 1978), eggplant (Solanum melongena L.) (Lianyou et al., 1999), watermelon ( Cirtullus lanatus var. lanatus ) (Kousik et al., 2007) and jute ( Corchorus spp.) (Jana et al., 2007). To our knowledge no previous studies have assessed genetic variance in susceptibility to broad mite in ornamental plants. The main objective of the present study was to evaluate the genetic variation of a selection of R. simsii hybrids in terms of susceptibility to broad mites. The use of cultivars resistant to pests is sustainable and fits well into an overall IPM strategy for broad mite control. Furthermore, information presented in this article will be useful for breeders for the development of new resistant cultivars.

2. Materials and methods 2.1. Plant material A total of 32 Rhododendron cultivars were collected. Thirty belonged to the subgenus Tsutsusi , section Tsutsusi; two accessions ( R. burmanicum (Yellow) and ‘Fragrantissimum’) belong to the subgenus Rhododendron , section Rhododendron , subsection Maddenia (Table 5.1). Cuttings originated from a core collection of Rhododendron spp. kept at ILVO, Melle, Belgium. Plants were grown from rooted cuttings and pruned once. This material was used in experiment 1 or experiment 2, six or eight weeks after regrowth. For experiment 2, cultivars ‘Bergmann Feu’, ‘Doctor Bergmann Albus’ and ‘Madame Charles Gabert’ were grafted onto rootstock of R. scabrum hybrid ‘Concinna’. In experiment 3, plants used were cut three times before being used in the experiment. Before the experiments plants were grown in a standard greenhouse under natural light conditions with a minimum temperature of 5 °C and ventilation temperature set at 20 °C.

2.2. Broad mite development A stock culture of broad mites was maintained on Hedera helix L., initiated from naturally infested H. helix ‘Montgomory’ and ‘Chester’ plants. For this purpose the plants were grown in a greenhouse with night/day temperature set at 17/20 °C. The photoperiod was 16:8 h (L:D). When light intensity was below 150 W.m -2, additional assimilation light (50 µmol.m -2.s -1) was provided using Gavita GAN 600 AL fixtures (GAVITA Nederland B.V., the Netherlands) with 600W 230V lamps.

65

In each experiment, the Rhododendron cultivars were artificially infested by surrounding each individual plant with four H. helix plants containing a stock culture of broad mites. All H. helix plants were selected after visual inspection of mite damage. Five H. helix samples were examined to confirm pest presence, using an observation microscope (Leica MZ6, Leica, Wetzlar, Germany). Greenhouse conditions were set at 21 °C as a minimum temperature and ventilation was activated at 26 °C during all experiments, and the photoperiod was 16:8 h (L:D). When light intensity was below 100 W.m -2, additional assimilation light (100 µmol.m -2.s -1) was provided by a conventional fixture, HS2000 (Hortilux Schréder B.V., the Netherlands) using Master GreenPower 600W 400V lamps (Philips, Eindhoven, the Netherlands). Above 200 W.m -2, shades were used to reduce the light intensity. Light intensity was measured with LP02 pyranometer (Hukseflux Thermal Sensors B.V., the Netherlands) connected to a logging system.

2.3. Scoring of cultivars and counting of broad mites Broad mite effect on Rhododendron cultivars was rated according to the symptoms. A scoring system of five classes was used, where 0 = no injury observed; 1 = very low injury, very slight browning of the youngest leaves of one or more shoot tips, less than 25% of the shoot tips showing symptoms; 2 = slight injury, all shoot tips show browning and curling of youngest leaves; symptoms are visible on 26 – 50% of the plant; 3 = moderate injury, some shoot tips bronzed and deformed, mature leaves start curling, between 51 and 75% of the plant is injured; and 4 = severe injury, all shoot tips bronzed, hard and necrotic and mature leaves severely distorted, more than 75% of the plant shows damage symptoms. Quantification of broad mites was assayed on the apical shoot tips (cut off between the third and fourth free-standing leaf as counted, starting from the apex). Samples were collected in a plastic vial with screw cap (60 ml, diameter 33 mm) (Gosselin, France) filled with 20 ml ethanol 70%. The vial was shaken and its contents poured over a nylon sieve (mesh size 1.5 mm), on a reusable filter holder with receiver (Nalge Nunc International, NY, USA), equipped with a cellulose mixed ester type Machery-Nagel Porafil® CM filter (pore size 0.45 µm, diameter 47 mm, black and with white grid). Using a vacuum pump, the membrane filter was drained and then transferred into a 60 mm diameter petri dish (Greiner Bio One BVBA/SPRL, Belgium) for counting under an observation microscope. This was done after the protocol described by Mechant et al. (2015). In experiments 1 and 2, all life stages were counted as one, to facilitate the counting process. During experiment 3, eggs, larvae, nymphs, males and females were recorded separately in order to evaluate the population dynamics of the mite.

2.4. Experiment 1 Thirty cultivars (01-14-1 and NA40246 KURIO were excluded) were screened in four replicated plots from November 2012 till January 2013. Each of the four plots contained one entry of a cultivar. Three and seven weeks post infestation, a single plot

Chapter 5

(three shoot tips per plant) was used for quantitative detection. Visual scoring of each plant was done weekly for seven consecutive weeks.

2.5. Experiment 2 Three replicated plots were used to screen 30 cultivars (95-20-5 and ‘Rosa Belton’ were excluded). Each plot contained three entries of a single cultivar, resulting in a total of nine replications per cultivar. During the six-week period between March 2013 and April 2013, all plants were scored weekly for damage symptoms. Broad mites were quantified 2, 4 and 6 weeks post infestation by harvesting marked shoot tips. These tips were labelled prior to the start of the experiment in order to preclude bias during subsequent sampling. A single entry from each cultivar was sampled within each plot at every time interval.

2.6. Experiment 3 The cultivars, ‘Aiko Pink’, ‘Elien’, ‘Mevrouw Gerard Kint’, ‘Michelle Marie’, ‘Nordlicht’ and ‘Sachsenstern’ were screened in six individual plots. Each plot consisted of nine plants and was separated from the others by one meter of free space. Twelve days after the initial infestation was set up, re-infestation was done by placing new infested H. helix plants, because the first infestation was ineffective (no visual damage symptoms). Therefore plant damage symptoms were scored weekly during an eight-week period between October 2013 and November 2013. Broad mites were quantified every two weeks on each entry. Shoot tips used for quantitative detection were again labeled before broad mite infestation began.

2.7. Scanning electron microscopy Presence or absence of trichomes on leaves and stems of R. simsii hybrids ‘Aiko Pink’, ‘Nordlicht’, ‘Michelle Marie, ‘Sachsenstern’, ‘Mevrouw Gerard Kint’, ‘Elien’, ‘Mistral’ and ‘Mevrouw Marc van Eetvelde’ was investigated using scanning electron microscopy (SEM). Leaf material was dissected in 70% ethanol under a binocular (Leica MZ6, Leica, Wetzlar, Germany). To prepare the material for critical-point drying, it was washed with 70% ethanol for 5 min. Next it was placed in a mixture (1:1) of 70% ethanol and DMM (dimethoxymethane) for 5 min. The material was then transferred for 20 min to pure DMM. Critical-point drying was done using liquid CO2 with a CPD 030 critical-point dryer (BAL-TEC AG, Balzers, Liechtenstein). The dried samples were mounted on aluminium stubs using Leit-C. For SEM observation, the material was coated with gold via a SPI- Module™ Sputter Coater (SPI Supplies, West-Chester, PA, USA). SEM images were obtained with a JEOL JSM-6360 (JEOL Ltd, Tokyo, Japan) at the Laboratory of Plant Conservation and Population Biology (KU Leuven).

67

2.8. Statistical analysis Two-way analyses of variance (ANOVA) (IBM SPSS Statistics; version 22) were conducted to evaluate whether the cultivars had a different effect on broad mite counts, when sampled at different time intervals in experiment 1 or 2. Likewise, damage rates were analyzed using two-way-ANOVA. Pearson correlations were calculated between the average number of broad mites per shoot tip and the damage rate for each cultivar in experiment 1 and 2. A repeated measures ANOVA was used to evaluate broad mite counts or damage rates at different time intervals for each cultivar in experiment 3. Degrees of freedom were corrected using the Greenhouse-Geisser correction when Mauchly’s assumption of sphericity showed to be violated. In case of a significant effect, means were separated using Bonferroni (damage rate) or LSD correction (broad mite counts). Broad mite counts were log transformed [ln(x+1)] and square root transformations were applied for damage rates before the statistical analyses.

3. Results In experiment 1, broad mites were retrieved after three weeks on plants of all cultivars, except for ‘Elien’ (Table 5.2). Broad mites were recorded on all cultivars after two weeks in experiment 2 (Table 5.3). In experiment 1, the highest numbers of broad mites was found on ‘Roxette’ at 21 days post infestation (dpi) and on 95-20-5 at 49 dpi (Table 5.2). At the end of the experiment, the lowest average number of broad mites was counted on ‘Elien’. Both ‘Mistral’ and its bud sport ‘Elien’, had the lowest damage rates in experiment 1. At 49 dpi, the average rates were 0 and 0.3 for ‘Mistral’ and ‘Elien’, respectively. The highest damage rates were recorded for ‘Emil De Coninck’ and ‘Mont Blanc’ (Table 5.2). In experiment 2, the lowest numbers of broad mites were observed on ‘Mistral’ at 15 dpi and 29 dpi and on ‘Elien’ at 43 dpi. The highest numbers of broad mites were observed on ‘Lara’ at 15 dpi and 29 dpi and on ‘Emil De Coninck’ at 43 dpi (Table 5.3). In experiment 2, ‘Elien’, ‘Fragrantissimum’ and ‘Mistral’ had the lowest average damage rates (≤0.6). The highest damage rates were recorded for ‘Emil De Coninck’, ‘Mont Blanc’ and ‘Otto’, ranging between 3.3 and 3.7 (Table 5.3). For both experiments a significant interaction was found between time interval and cultivar for the number of broad mites and the given damage rates (Table 5.4). The correlations ( ) between the average damage rates and broad mite counts in experiment 1 where 0.21 and 0.36 at 21 and 49 dpi, respectively. In experiment 2, -values were 0.25, 0.61 and 0.14 at 15, 29 and 43 dpi, respectively. At every time of recording, these correlations were found to be significant (p < 0.05).

Chapter 5

Table 5.1 Selection of Rhododendron cultivars used in screening experiments for broad mite susceptibility. For each of the selected cultivars the species name and the parentage are given. This table also indicates in which of the experiments each cultivar was included.

X X X X X X Exp. 3 Exp.

X X X X X X X X X X X X X X X X X X X X X X X X X X X X X Exp. 2 Exp.

X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X Exp. 1 Exp. crossed

´ eriocarpum

cultivars

simsii simsii `Docteur Bergmann´ `Docteur series DocteurBergmann `DocteurBergmann Saumoneus´ `Glaser Nummer10´* `Hellmut Vogel´ `JamesBelton´

a fromHaerens´`Jean derivative heredataweretaken from Heursel (1999). rtfrom `Mistral´*

MemoriaSander´ (s) X `Heiwa-no-hikari´ PinkDream´ X `Heiwa-no-hikari´* Rosalie´X un-named seedling Osaka´X `Heiwa-no-hikari´ Tempérance´X `Ambrosius Superbus´ SanktValentin´ X `MevrouwGerard Kint´ Ambrosiana´X `Jubile´(Japanese azalea)* Ambrosiana´X `Jubile´(Japanese azalea)* Parentage - - - Reportedtobe derived from with various from Sport ` Selectionfrom of Sport the Spo Sport - ` from Sport from Sport ` ` ` - - from Sport ` from Sport - - - ` - ` ` - - simsii

hybrid hybrid hybrid hybrid hybrid hybrid hybrid hybrid hybrid hybrid hybrid hybrid hybrid hybrid hybrid hybrid hybrid hybrid hybrid hybrid hybrid hybrid hybrid hybrid hybrid hybrid hybrid hybrid hybrid hybrid

Species R.simsii R.simsii R.simsii R.simsii R.simsii R.simsii R.simsii R.simsii R.simsii R.simsii R.simsii R.simsii R.simsii R.simsii R.simsii R.simsii R.simsii R.simsii R.simsii R.simsii R.simsii R.simsii R.simsii R.simsii R.simsii R.simsii R.simsii R.obtusum R.obtusum Rhododendron R.burmanicum

(Yellow)

Data based Data Leslieon (2004) unlessindicated by *, w a

r r

-14-1 -20-5 rincess Mariarincess Pia Cultiva 01 95 AikoPink Antartica BergmannFeu ChristineMatton DocteurBergmann Albus Elien Emil DeConinck Kassandra Lara MadameCharles Gabert MevrouwGerard Kint MevrouwMarc VanEetvelde MichelleMarie Mistral MontBlanc Noralinde Nordlicht Otto P RosaBelton Roxette Sachsenstern Tamira Thesla Yankadi Desiree Nazarena Fragrantissimum R.burmanicum

69

Table 5.2 Average damage rates and broad mite counts of 30 selected Rhododendron cultivars at seven time intervals post infestation (dpi) in experiment 1. Average broad mite counts are given in absolute numbers. Data are means ± SE. n = 3 for broad mite counts, and n = 4 (7, 14 and 21 dpi) or n = 3 (28, 35, 42 and 49 dpi) for the damage rates.

a a a b

3.32.0 ± 7.33.0 ± 8.34.9 ± 9.00.6 ± 7.75.7 ± 6.33.2 ± 3.72.2 ± 4.70.9 ± 4.02.1 ±

9.0 ± 2.0 ± 9.0 11.6 ± 1.7 13.6 ± 4.9 14.0 ± 5.3 21.3 ± 5.8 18.3 ± 1.5 13.6 ± 5.7 10.0 ± 6.0 26.6 ± 3.3 10.6 ± 2.2 10.6 ± 4.8 25.0 ± 6.0 25.0 ± 5.1 61.6 ± 6.7 15.3 ± 3.2 49 dpi 49 16.6 ± 3.5 16.6 71.0 ±71.0 29.2 ±57.6 23.5 ±20.0 10.0 16.0 ± 12.0 9

a a a mites 0

Broad 7.3 ± 4.7 ± 7.3 0.3 ± 0.3 3.2 ± 6.7 2.9 ± 8.3 0.9 ± 1.3 0.3 ± 0.3 2.0 ± 9.7 2.0 ± 2.0 0.3 ± 0.3 2.3 ± 8.7 3.6 ± 9.0 2.0 ± 2.0 1.9 ± 3.3 2.3 ± 6.0 4.3 ± 8.7 2.1 ± 9.0 0.3 ± 5.3 3.50.5 ± 11.3 ± 2.4 19.6 ± 9.0 20.0 ± 3.6 10.0 ± 4.0 31.6 ± 7.5 13.3 ± 2.7 22.6 ± 3.2 42.5 ± 6.5 33.6 ± 11.1 20.3 ± 10.4 21dpi 67.5 ± 67.5 31.5

0.3

0 49 dpi 49 1.7 ±1.70.3 ±2.30.7 ±2.00.6 ± 2.7 ±2.30.3 ±2.70.3 ±0.30.3 ±3.00.6 ±2.00.6 ±0.70.3 ±2.70.3 ±1.30.3 ±0.70.3 ±2.30.7 ±2.30.7 ±2.30.7 ±2.30.7 ±1.30.3 ±2.00.6 ±2.30.7 ±1.30.3 ±2.00.6 ±1.30.3 ±2.30.7 ±2.00.6 ±1.30.3 ±0.70.3 ±1.30.3 3.0 ±3.00.6

0 42 dpi 42 1.3 ± 1.3 0.3 ± 2.3 0.7 ± 2.0 0.6 ± 2.3 0.3 ± 2.3 0.3 ± 2.3 0.3 ± 0.3 0.3 ± 2.3 0.3 ± 2.0 0.6 ± 0.3 0.3 ± 2.7 0.3 ± 1.3 0.3 ± 0.7 0.3 ± 2.3 0.7 ± 2.0 0.6 ± 2.3 0.7 ± 2.3 0.7 ± 1.0 0.0 ± 2.3 0.3 ± 2.0 0.6 ± 1.3 0.3 ± 2.0 0.6 ± 1.3 0.3 ± 2.3 0.7 ± 2.0 0.6 ± 1.0 0.0 ± 0.3 0.3 ± 1.3 0.3 3.0 ± 3.0 0.6

0 0 0 0 0 0

35dpi 1.0 ± 0.0 ± 1.0 0.6 ± 2.0 0.3 ± 1.7 0.0 ± 2.0 0.3 ± 2.3 0.3 ± 2.3 0.3 ± 1.7 0.3 ± 1.3 0.0 ± 2.0 0.3 ± 1.3 0.0 ± 1.0 0.3 ± 1.7 0.7 ± 1.7 0.6 ± 2.0 0.0 ± 1.0 0.3 ± 2.3 0.7 ± 1.7 0.0 ± 1.0 0.6 ± 2.0 0.3 ± 1.3 0.3 ± 1.7 0.3 ± 1.3 0.3 ± 0.7 0.3 ± 0.3 0.3 ± 1.7 2.0 ± 0.6 ± 2.0

0 0 0 0 0 0 0 28dpi 0.30.3 ± 2.00.6 ± 1.00.6 ± 1.70.3 ± 2.00.6 ± 1.30.3 ± 2.00.6 ± 1.30.3 ± 1.70.3 ± 1.30.3 ± 1.00.0 ± 1.30.3 ± 1.70.7 ± 2.00.6 ± 0.30.3 ± 1.30.3 ± 1.00.6 ± 1.30.3 ± 1.30.3 ± 1.00.0 ± 1.30.3 ± 1.30.3 ± 0.70.3 ± 1.30.3 ± 2.00.6 ± Damage rates

0 0 0 0 0 0 0 21dpi mple. ntwo samples. 0.5 ± 0.3 ± 0.5 0.3 ± 1.3 0.3 ± 0.5 0.4 ± 1.0 0.0 ± 2.0 0.3 ± 1.3 0.5 ± 0.8 0.5 ± 0.8 0.3 ± 0.3 0.5 ± 1.3 0.3 ± 0.5 0.3 ± 1.3 0.4 ± 1.0 0.3 ± 0.5 0.5 ± 1.5 0.4 ± 1.0 0.3 ± 0.5 0.4 ± 1.0 0.3 ± 0.3 0.4 ± 1.0 0.3 ± 0.5 1.3 ± 0.6 ± 1.3 0.0 ± 1.0 0.3 ± 0.3

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 14 dpi 14 0.5 ± 0.3 ± 0.5 0.3 ± 0.3 0.3 ± 0.3 0.3 ± 0.3 0.5 ± 0.3 ± 0.5 0.3 ± 0.8 0.3 ± 0.3 0.3 ± 0.5 0.3 ± 0.5 0.3 ± 0.5

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 7 dpi 7 0.30.3 ±

(Yellow)

-20-5 Number of Number broad mitesper shoottip basedon sa one Average number Average of broad mitespershoot tip based o b a Cultivar 95 AikoPink Antartica BergmannFeu ChristineMatton DoctorBergmann Albus Elien EmilDe Coninck Kassandra Lara MadameCharles Gabert MevrouwGerard Kint MevrouwMarc VanEetvelde MichelleMarie Mistral MontBlanc Noralinde Nordlicht Otto MariaPrinses Pia RosaBelton Roxette Sachsenstern Tamira Thesla Yankadi Desiree Nazarena Fragrantissimum R.burmanicum

Chapter 5

Table 5.3 Average damage rates and broad mite counts of 30 selected Rhododendron cultivars at six time intervals post infestation (dpi) in experiment 2. Average broad mite counts are given in absolute numbers. Data are means ± SE. n = 3 for broad mite counts, and n = 9 for the damage rates.

± 15.2 2.00.6 ± 7.34.3 ± 7.34.3 ± 16.0 ± 3.8 51.6 ± 5.4 19.0 ± 5.2 13.0 ± 4.7 42.6 ± 2.6 40.0 ± 6.1 40.0 ± 3.6 15.0 ± 7.2 11.3 ± 2.9 23.0 ± 7.2 11.6 ± 1.2 49.6 ± 2.9 18.0 ± 8.3 43 dpi 43 56.6 ±56.6 15.1 35.3 ±21.6 20.7 ±43.6 13.6 ±27.0 11.3 ±52.6 14.3 ±63.3 34.9 ±36.3 14.5 ±57.3 18.2 ±26.6 10.7 ±53.6 26.7 ±25.3 12.4 ±91.6 29.7 131.0 ±131.0 43.7

b 0

6 ± ± 6 5.2 9.7 ± 5.8 ± 9.7 4.0 ± 4.0 0.3 ± 9.7 7.5 ± 9.0 1.3 ± 2.3 6.73.3 ± 13.0 ± 4.0 29.0 ± 6.5 32.0 ± 6.4 27.0 ± 3.5 13.3 ± 2.3 39.6 ± 8.5 22.0 ± 3.8 29.6 ± 4.8 57.3 ± 9.8 44. 11.3 ± 4.9 25.0 ± 3.1 12.0 ± 5.1 15.0 ± 1.5 29dpi 25.6 ± 11.6 77.0 ± 22.6 35.0 ± 15.4 45.3 ± 18.8 49.3 ± 21.1 81.0 ± 16.6 30.6 ± 12.5 54.6 ± 21.5 130.0± 66.2 Broadmites

2

9.7 ± 8.2 ± 9.7 17.3 ± 5.0 15.0 ± 5.7 37.0 ± 4.2 37.0 ± 4.0 17.6 ± 4.7 21.3 ± 4.6 13.3 ± 5. 14.3 ± 0.9 32.0 ± 6.0 19.3 ± 1.2 21.3 ± 8.2 17.6 ± 2.9 17.6 ± 5.8 25.0 ± 9.2 22.6 ± 3.8 14.6 ± 6.3 16.0 ± 3.8 14.6 ± 2.9 16.6 ± 3.5 15dpi 13.0 ± 12.0 64.6 ± 46.3 22.0 ± 10.1 39.6 ± 12.3 50.3 ± 11.7 36.3 ± 17.0 38.6 ± 29.8 46.3 ± 21.0 34.3 ± 23.5 160.0± 30.0

a

2.5 ± 0.2 ± 2.5 0.0 ± 3.0 0.0 ± 3.0 0.2 ± 2.8 0.0 ± 3.0 0.0 ± 3.0 0.2 ± 0.2 0.2 ± 3.3 0.1 ± 3.1 0.2 ± 2.7 0.2 ± 2.8 0.2 ± 2.7 0.3 ± 2.2 0.2 ± 3.6 0.2 ± 0.6 0.1 ± 3.1 0.2 ± 3.3 0.0 ± 3.0 0.2 ± 3.7 0.0 ± 3.0 0.1 ± 3.1 0.1 ± 2.1 0.2 ± 3.2 0.0 ± 3.0 0.1 ± 2.9 0.1 ± 2.9 0.2 ± 2.7 0.2 ± 0.3 0.2 ± 1.8 2.10.2 ± 43dpi

a

2.00.2 ± 3.00.0 ± 2.90.1 ± 2.40.2 ± 2.90.1 ± 2.90.1 ± 0.10.1 ± 3.30.2 ± 3.00.0 ± 2.70.2 ± 2.40.2 ± 2.40.2 ± 2.20.2 ± 3.40.2 ± 0.80.2 ± 3.00.2 ± 3.00.0 ± 3.00.0 ± 3.10.2 ± 2.80.2 ± 3.00.0 ± 2.00.2 ± 3.00.2 ± 2.80.2 ± 2.80.2 ± 2.90.1 ± 2.80.2 ± 0.60.3 ± 1.80.2 ± 1.7 ± 1.7 0.2 36 dpi 36

a

0

1.5 ± 0.2 ± 1.5 0.1 ± 2.9 0.2 ± 2.6 0.2 ± 2.6 0.2 ± 2.8 0.1 ± 2.9 0.0 ± 3.0 0.2 ± 2.7 0.3 ± 2.6 0.2 ± 2.3 0.2 ± 1.8 0.3 ± 1.6 0.2 ± 3.0 0.2 ± 0.2 0.2 ± 2.8 0.1 ± 2.9 0.1 ± 2.9 0.0 ± 3.0 0.2 ± 2.7 0.0 ± 3.0 0.2 ± 1.0 0.2 ± 2.4 0.2 ± 2.3 0.2 ± 2.7 0.2 ± 2.6 0.2 ± 2.3 0.1 ± 0.1 0.2 ± 1.3 1.00.0 ± 29 dpi 29

a .3

0 0 0 Damage Damage rate 1.0 ± 0.0 ± 1.0 0.2 ± 2.6 0.2 ± 2.3 0.2 ± 2.0 0.2 ± 2.7 0.0 ± 2.0 0.1 ± 2.9 0.1 ± 2.1 0 ± 2.0 0.0 ± 2.0 0.1 ± 1.1 0.2 ± 1.2 0.2 ± 2.6 0.2 ± 2.7 0.2 ± 2.8 0.2 ± 2.2 0.2 ± 1.7 0.2 ± 2.8 0.2 ± 0.7 0.2 ± 1.4 0.2 ± 1.4 0.0 ± 2.0 0.2 ± 2.0 0.2 ± 2.3 0.2 ± 1.0 2.4 ± 0.2 ± 2.4 0.9 ± 0.2 ± 0.9 ntwo samples. 22dpi

a

.

0 0 0 0 0.9 ± 0.1 ± 0.9 0.2 ± 1.8 0.2 ± 1.2 0.2 ± 1.2 0.3 ± 1.9 0.2 ± 1.3 0.3 ± 1.9 0.2 ± 1.3 0.2 ± 1.3 0.2 ± 1.7 0.1 ± 0.9 0.2 ± 0.6 0.2 ± 1.6 0.1 ± 1.9 0.2 ± 1.4 0.2 ± 1.2 0.2 ± 1.3 0.2 ± 1.4 0.2 ± 0.2 0.2 ± 1.2 0.1 ± 1.1 0.1 ± 1.1 0.0 ± 1.0 0.2 ± 1.3 1.4 ± 0.2 ± 1.4 0.3 ± 0.2 ± 0.3 15dpi

a

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 ghtobservations 0.10.1 ± 0.20.2 ± 1.00.3 ± 0.20.2 ± 0.70.2 ± 0.40.2 ± 0.20.2 ± 0.60.3 ± 0.30.2 ± 0.10.1 ± 0.10.1 ± 0.30.2 ± ei 8 dpi 8

(Yellow)

1

- me Charlesme Gabert

14 - Averagenumber ofbroad mites shoot per tip based o Average damagebasedrate on a b Cultivar 01 AikoPink Antartica BergmannFeu Matton Christine Bergmann Doctor Albus Elien Emil DeConinck Kassandra Lara Mada Gerard Mevrouw Kint Marc Mevrouw VanEetvelde MichelleMarie Mistral Blanc Mont Noralinde Nordlicht Otto MariaPrinses Pia Roxette Sachsenstern Tamira Thesla Yankadi Desiree Nazarena Fragrantissimum R.burmanicum 40246 NA KURIO

71

Finally, a third experiment was conducted to evaluate broad mite infestation on six selected cultivars when the mite’s feeding choices were limited between a single selected cultivar and H. helix . Therefore each cultivar was screened in an individual plot (experiment 3). The selection of ‘Aiko Pink’, ‘Nordlicht’, ‘Michelle Marie’, ‘Sachsenstern’, ‘Mevrouw Gerard Kint’ and ‘Elien’ was based on the different characteristics and the differences in patterns of broad mite counts and damage rates in experiment 1 and 2. The outcome of this experiment was comparable with that of experiments 1 and 2. The final average damage rate for cultivars ‘Aiko Pink’, ‘Nordlicht’ and ‘Michelle Marie’ was higher than 2.4. ‘Elien’ had the lowest average damage rate (<0.5). The damage rates for ‘Sachsenstern’ and ‘Mevrouw Gerard Kint’ were around 1.5 at 56 dpi. The damage rates for ‘Elien’ did not differ significantly at the various time intervals (Fig 5.1 and Table 5.5). In contrast, damage rates varied significantly among ‘Aiko’, ‘Mevrouw Gerard Kint’, ‘Nordlicht’, ‘Sachsenstern’ and ‘Michelle Marie’ at the different time intervals (Fig 5.1 and Table 5.5). Also the number of broad mites counted at the different time intervals was significantly different for ‘Aiko’, ‘Mevrouw Gerard Kint’, ‘Nordlicht’, ‘Sachsenstern’ and ‘Michelle Marie’. At the same time intervals, broad mite counts did not differ significantly for ‘Elien’ (Fig 5.1 and Table 5.5). Approximately the same number of broad mites was counted on all six cultivars 14 and 28 days after the initial infestation, indicating that a homogenous spread of mites was obtained after the reinfestation at day 12. However, at the end of the experiment, mites were no longer detected on ‘Elien’. On ‘Michelle Marie’, ‘Sachsenstern’ and ‘Mevrouw Gerard Kint’ a comparable number of ca. 13 broad mites per shoot tip were counted, while the highest numbers of broad mites were observed on ‘Aiko Pink’ and ‘Nordlicht’ at the end of the experiment. On the latter cultivars, broad mite numbers approximately doubled during the last two time intervals, reaching around 35 broad mites per shoot tip. On ‘Aiko Pink’ and ‘Nordlicht’ a higher number of immature life stages was found than in the other cultivars. The number of eggs, larvae and nymphs increased after every sampling on these two cultivars (Fig 5.1). Among the six genotypes studied in detail in experiment 3, all had long non- glandular trichomes. On ‘Elien’, short sticky trichomes were found, while all other genotypes showed the presence of short non-sticky trichomes. A fourth type of thorn-like trichomes was present on leaves of ‘Michelle Marie’ and ‘Sachsenstern’ (Fig 5.2).

Chapter 5

Table 5.4 Two-way-ANOVA indicating the effect of different time intervals and Rhododendron cultivars on the given damage rates and the average counted number of broad mites in experiments 1 and 2. In experiment 1 the time intervals for damage rate were 7, 14, 21, 28, 35, 42 and 49 dpi. The time intervals for broad mite counts were 21 and 49 dpi. Time intervals for experiment 2 were 8, 15, 22, 29, 36 and 43 dpi and 15, 29 and 43 dpi, for damage rate and broad mite counts, respectively.

Source Experiment 1 Experiment 2 Broad mites Damage rate Broad mites Damage rate Time interval F 21.51 235.82 2.47 1245.41 df 1 6 2 5 p <0.001 <0.001 0.088 <0.001 Cultivar F 7.24 21.06 6.58 126.21 df 29 29 29 29 p <0.001 <0.001 <0.001 <0.001 Time interval x cultivar F 2.74 1.85 1.5 5 5.74 df 29 174 58 145 p <0.001 <0.001 0.015 <0.001 Error df 112 510 179 1434

Table 5.5 Repeated measures ANOVA indicating the effect of time intervals on the given damage rates and the average counted number of broad mites on ‘Aiko’, ‘Mevrouw Gerard Kint’, ‘Nordlicht’, ‘Sachsenstern’, ‘Elien’ or ‘Michelle Marie’ in experiment 3. Time intervals for broad mite counts and damage rates were 7, 14, 21, 28, 35, 42, 49 and 56 dpi.

Source ‘Aiko’ ‘Mevrouw Gerard Kint’ ‘Nordlicht’ ‘Sachsenstern’ ‘Elien’ ‘Michelle Marie’ Broad mites F 6.07 6.07 14.11 17.97 2.14 16.62 df 3 3 3 3 3 3 p 0.003 0.003 <0.001 <0.001 0.122 <0.001 Error df 24 24 24 24 24 24

Damage rates a F 67.31 31.47 39.42 27.86 1.27 53.19 df 2.31 2.73 3.33 2.81 2.06 2.34 p <0.001 <0.001 <0.001 <0.001 0.309 <0.001 Error df 18.45 21.83 26.64 22.47 16.50 26.74 aMauchly’s test of sphericity indicated that the assumption had been violated for each cultivar (p <0.05). Degrees of freedom (df) were corrected using the Greenhouse-Geisser correction.

73

(a) (b) 3.5 60 3.5 60 c 3.0 C 3.0 d 50 C 50 2.5 bc 2.5 40 40 2.0 bc 2.0 c c b 30 B 30 1.5 1.5

Average damage rate damage Average 20 rate damage Average bc 20 a 1.0 BC 1.0 a AB a ab 0.5 A 10 0.5 AB ab A 10 a a a tip shoot mites per broad of number Average tip shoot mites per broad of number Average

(c) (d) 3.5 60 3.5 60

3.0 c 3.0 50 50 c 2.5 2.5 40 40 2.0 2.0 c 30 30 1.5 b 1.5 bc

Average damage rate damage Average 20 rate damage Average ab 20 1.0 ab C 1.0 C B BC a a a 0.5 10 0.5 10 A a B a a B a a tip shoot mites per broad of number Average a A tip shoot mites per broad of number Average

(e) 3.5 (f) 3.5 60 60

3.0 3.0 50 50 2.5 2.5 40 40 2.0 2.0 c 30 30 1.5 1.5 bc Average damage rate damage Average Average damage rate damage Average abc 20 20 1.0 A B 1.0 a a 10 10 0.5 A A ab 0.5 Average number of broad mites per shoot tip shoot mites per broad of number Average a a tip shoot mites per broad of number Average

10 20 30 40 50 10 20 30 40 50 dpi dpi

Fig 5.1 Average damage rates (lines) and number of broad mites per shoot tip (bars) observed for Rhododendron simsii hybrids (a) ‘Aiko Pink’, (b) ‘Nordlicht’, (c) ‘Michelle Marie’, (d) ‘Sachsenstern’, (e) ‘Mevrouw Gerard Kint’ and (f) ‘Elien’ during experiment 3. Female broad mites (shaded bars), male broad mites (black bars), nymphs (dark gray bars), larvae (light gray bars) and eggs (open bars) are shown. Data are means ± SE, n = 9 for damage rates and broad mite counts. Different lower case (damage rates) and upper case (broad mite counts) letters indicate significant differences between the time intervals for each cultivar.

Chapter 5

(a) (b) 1

2

1 2

(c) (d)

1 4 2

2 1 4

(e) (f) 1

1 2 3

Fig 5.2 Scanning electron microscopy images of the abaxial leaf side of Rhododendron simsii hybrids (a) ‘Aiko Pink’, (b) ‘Nordlicht’, (c) ‘Michelle Marie’, (d) ‘Sachsenstern’, (e) ‘Mevrouw Gerard Kint’ and (f) ‘Elien’. Four trichome types can be distinguished (1) long trichome, (2)

short trichome, (3) short trichome with sticky end and (4) thorn-like trichome.

75

4. Discussion In the present study we applied artificial infestation by placing infested H. helix plants around the plants of the tested cultivars. From preliminary experiments it was clear that the number of broad mites remains stable on the infestation source at different time intervals. This continually-present artificial infestation source resulted in an infestation of all cultivars at the start of each experiment (Table 5.2, Table 5.3 and Fig 5.1). During the experiments, we observed much variation between the cultivars in the average broad mite counts and damage rates. In both experiment 1 and 2, ‘Mistral’ and its color sport ‘Elien’ were resistant to broad mite. Hence, few broad mites were found and damage rates were low. ‘Bergmann Feu’, ‘Doctor Bergmann Albus’ and ‘Madame Charles Gabert’, which are all bud sports of the ‘Doctor Bergmann’ series (Table 5.1), responded in the same way and were all susceptible to broad mite infestations, as shown by high damage rates and large numbers of broad mites. The screening of cultivars randomly placed in a plot, as done in experiment 1 and 2, might lead to a bias when these cultivars with an unknown susceptibility for broad mites have a different attractiveness to the mites. Therefore, in experiment 3 each of the six selected cultivars was screened in an individual plot. Regarding the mite numbers and damage rates, ‘Elien’ was selected because few mites were found on this cultivar, while in contrast high numbers of P. latus mites were counted on ‘Nordlicht’ and ‘Aiko Pink’. Latter two cultivars also showed high damages rates compared to ‘Elien’. Cultivars ‘Sachsenstern’ and ‘Mevrouw Gerard Kint’ showed moderate rates of damage and mite counts. ‘Michelle Marie’ showed high damage rates (experiment 1 and 2). ‘Elien’ is a very vigorously growing cultivar with sticky stem and leaves. ‘Nordlicht’ is a sport of ‘H. Vogel’. The latter cultivar has together with all of its other sports a market share of 65% (van Trier, 2012). Flower characteristics included time of flowering and flower color. ‘Nordlicht’ is an early flowering type and ‘Michelle Marie’ is a semi-early flowering type. ‘Mevrouw Gerard Kint’ and ‘Sachsenstern’ are categorized as late flowering types. The flower color of ‘Michelle Marie’, ‘Aiko Pink’ and ‘Elien’ is pink whereas ‘Nordlicht’ has red flowers. ‘Mevrouw Gerard Kint’ has pink flowers with a white edge while ‘Sachsenstern’ has white flowers with a red edge. Nonetheless, the patterns observed for both the average damage rates and the mite counts of the six cultivars tested in experiment 3 were comparable with the results obtained for the same cultivars in the first two screening experiments. This suggests that placing cultivars randomly in a plot does not affect the outcome of the experiments and indicates that mites have no preference for H. helix over any of the azalea cultivars. Furthermore, these findings suggest that the artificial infestation using H. helix as a carrier for the mites is an appropriate method for conducting screenings on R. simsii hybrids in the greenhouse.

The collection of 32 Rhododendron cultivars, mainly containing R. simsii hybrids, showed a wide genetic variation in susceptibility against broad mites, as evidenced by the observed differences in mite counts and damage rates. Although significant correlations between damage rates and broad mite counts were found, a direct link between both observations was not obvious when checked at the level of individual cultivars. In a previous study, no significant correlations between broad mite counts and levels of

Chapter 5 damage were observed on cucumber, Cucumis sativus L. (Grinberg et al., 2005). In contrast, significant positive correlations between ratings of outgrowing terminals and broad mite counts in watermelon were reported by Kousik et al. (2007). A positive correlation between broad mite growth rate and injury was found on physic nut, Jatropha curcas L. (Evaristo et al., 2013). On Capsicum (Echer et al., 2002) and watermelon (Kousik et al., 2007) broad mites have a tendency to aggregate, which leads to a high spatial variability of mite populations on one plant. This tendency might be responsible for the unclear relationship between broad mite counts and damage rates in the R. simsii hybrids. To account for this we transformed broad mite counts by taking the natural logarithm of the actual broad mite count plus one. Kousik et al. (2007) applied a log-transformation of the mite counts before further statistical analysis. Another way to tackle this problem would be to increase the number of samples taken on the plant, but this might interfere with the damage rating process of monitored plants in the long term. Indeed, the more samples taken on one entry, the less shoots that remain, inhibiting proper evaluation of this entry. A second factor that could distort the correlation between broad mite counts and the damage rate is the difference among cultivars in the emergence of symptoms caused by a given density of broad mites. It has been reported that injury symptoms can appear very rapidly on some hosts with only a few mites, resulting in symptoms that are severe enough to cause economic damage (Dhooria and Bhindra, 1977; Basset, 1981; De Coss-Romero and Peña, 1998). Results from the present study indicate different types of responses to a broad mite infestation. At 43 dpi in experiment 2, ‘Elien’ showed nearly no symptoms (average damage rate of 0.6) and no broad mites were detected (Table 5.3). On the other hand, ‘Aiko Pink’ and ‘Nordlicht’ showed high damage rates (3.0 for both cultivars) and high mite counts (>50 mites on average), at the same time interval as in experiment 2 (Table 5.3). In contrast, a high damage rate (3.6) was noted for ‘Michelle Marie’ but only a few mites (seven on average) were found on that azalea cultivar under the same conditions (Table 5.3). However, on the latter cultivar, on average 22 broad mites were found two weeks earlier at 29 dpi in experiment 2 (Table 5.3). In Capsicum genotypes, the assessment of symptoms caused by broad mites was shown to be more suitable for separating the accessions in resistant or susceptible types (Echer et al., 2002). For ornamental plants, however, the entire plant has economic value; and only undamaged or uninfested plants are marketable. Evaluation of the susceptibility of an accession of pot azalea should therefore initially be based on the damage rate. At a constant temperature of 20 °C, broad mite eggs are able to reach adulthood in 6.6 days on R. simsii hybrid ‘Nordlicht’ (Luypaert et al., 2014). Development at higher temperatures is achieved even more quickly, e.g. in 4.2 days at 25 °C and 3.5 days at 30 °C (Luypaert et al. 2014). The present experiments were conducted at a minimum temperature of 21 °C. Broad mites could thus have completed two to three generations during the two consecutive recording times during experiment 3. Nevertheless, on ‘Elien’, which was a resistant cultivar based on the damage rates, no mites were found at the end of the experiment (Fig 5.1). This re-confirmed the resistant character of that cultivar. On ‘Aiko Pink’ and ‘Nordlicht’, two very susceptible cultivars, the mite population rose over

77

time. Total mite counts increased at every subsequent sampling interval (Fig 5.1). In addition to the number of adult males and females, the number of eggs, larvae and nymphs increased, demonstrating the mite’s affinity for these cultivars. Adult females were found more frequently than males, which is in line with the natural adult male:female ratio averaging 1:4 (Gerson, 1992) (Fig 5.1). Due to only two recording times for broad mite counting it is not appropriate to discuss the mite's population development in experiment 1. In experiment 2, total mite populations remained either stable or increased over time in susceptible cultivars. However, on ‘Lara’, a highly sensitive cultivar, a rapid decline of the mite population was observed between 29 and 43 dpi. Under optimal conditions, broad mite populations may increase to large numbers and then rapidly decline due to the deterioration of food quality and of weather conditions or to the presence of natural enemies (Gerson, 1992). Since weather conditions in our study did not change and no predatory mites were present during the experiments, it is suggested that the mite population declined as a result of lowered host plant quality, as evidenced by bronzed growing shoot tips, hard and necrotic leaf tissue and severe curling of leaves. The factors underlying resistance against broad mites in the above-evaluated cultivars of pot azalea are unknown. Thungrabeab and Boonlertnirun (2002) and Matos et al. (2009) reported on the effect of trichomes as a limiting trait for the development of broad mites in Capsicum species. Resistance to damage caused by broad mites was observed in S. polyadenium and in seedlings of S. tarijense and S. berthaultii , which had foliar hairs with a sticky tip (Gibson and Valencia, 1978). The resistant characteristics of cultivars ‘Mistral’ and ‘Elien’ in our study might be related to the presence of this type of foliar glandular hairs (data not shown for ‘Mistral’ and Fig 5.2 for ‘Elien’). Resistance in ‘Mistral’ and ‘Elien’ could be attributed to the defensive mechanism of antixenosis, which appears for P. latus to be based on the presence of glandular trichomes. However, ‘Marc Van Eetvelde’ and ‘Lara’, two R. simsii hybrids that also have foliar glandular hairs (data not shown), were susceptible to broad mites. Regarding the parentage of these four cultivars, it is remarkable that ‘Mistral’, ‘Marc Van Eetvelde’ and ‘Lara’ resulted from a cross with ‘Heiwa-no-hikari’ as one of the parents (Table 5.1). Since ‘Elien’ is a sport from ‘Mistral’ it is also related to ‘Heiwa-no-hikari’. Based on broad mite counts and damage rates from all three screening experiments it is suggested that thorn-like trichomes found on ‘Sachsenstern’ and ‘Michelle Marie’ are not of major importance in plant defense against P. latus . Ahmed et al. (2000) examined numerous biochemical constituents in 15 C. annuum cultivars. Tannins, phenols, potassium, calcium, magnesium and chlorophyll had a negative correlation with broad mite incidence. In contrast, total sugars, protein content and nitrogen content were correlated positively with a broad mite infestation. The authors found no correlation with the phosphorous content of the leaves. The assessment of several of these compounds in a selection of susceptible versus resistant R. simsii hybrid cultivars might provide extra information regarding the traits that account for resistance in azalea against broad mites. In conclusion, the present study has demonstrated that the use of a continually present artificial infestation source is an appropriate method to screen Rhododendron spp. for susceptibility to broad mites. Significant genetic variation was found within a

Chapter 5 selection of R. simsii hybrids. Among the evaluated cultivars, ‘Emil De Coninck’ and ‘Mont Blanc’ were the most susceptible, whereas ‘Mistral’ and its sport ‘Elien’ proved to be resistant towards broad mites possibly due to antixenosis mechanisms (e.g. glandular trichomes with a sticky end). These findings may be useful to develop further breeding for resistance against these mites in R. simsii hybrids. Further research will be necessary to understand factors underlying the observed resistance.

79

Chapter 6

Selection and validation of plant defence related marker genes in Rhododendron simsii hybrids

1. Introduction Polyphagotarsonemus latus causes malformations of flower buds and terminal leaves in azalea, Rhododendron simsii hybrids. Generally, plant pests encounter physical or structural barriers as well as constitutive chemical defences (Gatehouse, 2002; Mysore and Ryu, 2004). Induced defence responses against herbivores can be associated with jasmonic acid (JA) (Browse, 2009a) but might also be regulated by jasmonyl-isoleucine (JA-Ile) and 12-oxophytodienoic acid (OPDA) (Stintzi et al., 2001). Literature on induced defence responses by or upon P. latus infestation remains scarce. It has been shown that P. latus infestation activates JA- and salicylic acid (SA)-dependent pathways in cucumber leaves, Cucumis sativus (Grinberg et al., 2005). Recently, Grinberg-Yaari et al. (2015) showed the role of jasmonic acid in signalling in tomato ( Solanum lycopersicum ) defence against P. latus . Molecular work in the ornamental plant R. simsii mainly focussed on flower color. It was possible to identify genes responsible for the biosynthesis of flavonoids (e.g. De Schepper, 2001; Nakatsuka et al., 2008; De Keyser et al., 2013).

Data from Grinberg et al. (2005) and Grinberg-Yaari et al. (2015), were based on northern blotting data and semi-quantitative PCR. The blotting technique is time-consuming, requires a large quantity of RNA (Chelly and Kahn, 1994) and most crucial it is not quantitative. Reverse transcriptase quantitative real-time PCR (RT-qPCR) is a rapid and sensitive technique and is far more specific than northern blot analysis (Dean et al., 2002). It provides meaningful quantitative results in contrast to semi-quantitative PCR (Ferré, 1992). Furthermore, quantification of messenger RNA (mRNA) by means of RT-qPCR has a good reproducibility and wide quantification range (Bustin, 2000; Pfaffl and Hageleit, 2001) when handled thoughtfully (Bustin et al., 2009; Thellin et al., 2009). MIQE- guidelines (Minimum Information for Publication of Quantitative Real-time PCR Experiments) were introduced in 2009 to uniformly quantify gene expression in an accurate manner and to provide essential data regarding quality control (Bustin et al., 2009). De Keyser et al. (2013) presented a protocol to assure accurate quantification of gene expression in plants in accordance with these guidelines.

One of the most critical points in the RT-qPCR workflow is to ensure that only RNA of high purity and high integrity is used (Taylor et al., 2010). Obtaining RNA from recalcitrant plant tissues is often confronted with major problems. First, disruption of the rigid can be a challenge. Grinding samples with pestle and mortar is laborious and might introduce contamination when not cleaned properly. A fast and homogenous pulverization of samples can be achieved when grinding with beads (e.g. Borgen et al., 2010; Asada et al., 2013). Secondly, tissues from woody and perennial plant species can contain high concentrations of polysaccharides, polyphenols (Zeng and Yang, 2002; Vasanthaiah et al., 2008, Rubio-Piña and Zapata-Pérez, 2011) and other secondary metabolites (Ghangal et al., 2009) that impede RNA extraction. Phenolic compounds are able to bind proteins and nucleic acids and can form high molecular weight complexes. Polysaccharides tend to co-precipitate with the RNA in the presence of alcohols, as such remaining as contaminants (Salzman et al., 1999). Several chemical substances exist to

Chapter 6 avoid oxidation, inhibition and detrimental influence on downstream applications of RNA caused by the presence of these components. The influence of polysaccharides can be reduced by using a cationic detergent like CTAB and reducing the ratio sample/extraction buffer. Polyphenols and their side effects are reduced by detergents (CTAB and SDS), antioxidants (β-mercaptoethanol, ascorbic acid or DTT) or polymers (PVP and PVPP) (Newbury and Possingham, 1977; Salzman et al., 1999). These difficulties in RNA extraction from recalcitrant plant tissues has led to the development of tissue-specific isolation protocols by modifying common methods like the guanidinium-phenol-chloroform (Vareli and Frangou-Lazaridis, 1996) and the hot borate method (Wan and Wilkins, 1994; Yao et al., 2005; Pang et al., 2011). Multiple modifications to a CTAB-based extraction method are published for working with woody plants (e.g. Zeng and Yang, 2002; Liao et al., 2004; Gambino et al., 2008; Ghangal et al., 2009; Rubio-Pina and Zapata-Pérez, 2011; Morante-Carriel et al., 2014). However, many of these methods are technically complex and require expensive lab equipment or reagents. Some methods are time consuming and result in low RNA yield.

The objective of the present study was to construct and validate a molecular toolbox to estimate activity of JA and SA biosynthesis related genes in R. simsii hybrids. Hereto, the extraction of leaf RNA from R. simsii was optimized and quality was assessed via various ways. RNA sequencing was performed to assemble the first transcriptome of R. simsii . At the moment of this study, no genomic or transcriptomic data for R. simsii was available. A preliminary RNAseq experiment was conducted to evaluate hormonal responses upon infestation or methyl jasmonate treatment. Gene activity of JA and SA biosynthesis related genes was quantified in R. simsii genotypes with a different susceptibility for P. latus . Lipoxygenase ( RsLOX ) (Vick & Zimmerman, 1983), allene oxide synthase (RsAOS ) (Laudert et al ., 1996), allene oxide cyclase ( RsAOC ) (Ziegler et al ., 2000), 12- oxophytodienoate reductase 3 ( RsOPR3 ) (Müssig et al ., 2000) and jamonate methyltransferase ( RsJMT ) (Seo et al ., 2001) were selected as marker genes for the JA biosynthesis pathway. In the SA biosynthetic pathway the homologs of PAL and ICS (Howe, 2005) were selected. The PAL enzyme is also the first enzyme involved in the phenylpropanoid derivative metabolism (Hahlbrock and Scheel, 1989). Phenylpropanoid compounds are precursors to a wide range of phenolic compounds with many functions in plants e.g. lignin, flavonoids, isoflavonoids, cumarins and stilbenes (Ferrer et al., 2008). RsPPO (polyphenol oxidase) served as a marker gene for oxidative stress responses (Bergery et al ., 1996). Aiko Pink’ and ‘Nordlicht’ are very susceptible genotypes, ‘Michelle Marie’, ‘Mevrouw Gerard Kint’ and ‘Sachsenstern’ are susceptible genotypes, whereas ‘Elien’ is a resistant genotype (Chapter 5). Besides JA inducing compounds, wounding of leaves was applied to study the role of these genes.

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2. Materials and methods 2.1. RNA-sequencing 2.1.1. RNA isolation and RNA-sequencing Plant material of different tissues (flower, leaf and buds) from R. simsii genotypes was harvested after specific treatments. This resulted in 16 tissue samples for comprehensive transcriptome assembly. Of this set, six samples were related to plant defence and were used for expression analysis: leaf samples of a mock treatment were compared to a 100 µM foliar application of MeJA (6 h after treatment) or P. latus infestation (12 days post infestation) treatment. Each of these three treatments was applied to genotypes ‘Nordlicht’ and ‘Elien’. Leaf samples consisted of six leaf punches taken at random positions on one plant. Samples were flash frozen in liquid nitrogen immediately after harvest and stored at -80 °C. RNA was isolated using a modified CTAB protocol, as described in paragraph 2.4. Quality and quantity of RNA samples was measured using a NanoDrop (ND-1000) spectrophotometer (Isogen Life Science Belgium, Temse, Belgium). RNA quality was further assessed with the Experion™ Automated Electrophoresis system and RNA StdSens Chips (Bio-Rad Laboratories N.V., Temse, Belgium). Three biological replicates were pooled to construct one RNA-seq library. Poly-A selected RNA-seq libraries were prepared by the sequence provider (SeqMatic, Fremont, USA) using the Illumina TruSeq Stranded protocol. Sequencing was performed using Illumina HiSeq™ High Output v3, with PE-100 cycles. 16 barcoded libraries were pooled and sequenced on two parallel lanes in the same run, yielding two technical replicate datasets for each sample. Quality trimming was performed with NGS Core Tools feature Trim Sequences in CLCbio Genomics Workbench (GW) v8.0 (quality limit 0.01 and min 25 bp). An additional four RNA samples of various stages of R. simsii bud development had previously been sequenced using 454 technology (De Keyser et al., unpublished data), and were included to complement transcriptome assembly.

2.1.2. De novo assembly De novo assembly was done according to the Orthology Guided Assembly routine described by Ruttink et al. (2013). In brief, sequence reads were assembled per RNA-seq library using the de novo assembly algorithm of CLCbio GW based on De Bruijn Graphs (DBG), with default settings and minimum contig length of 200 bp. The Arabidopsis thaliana proteome available from PLAZA 3.0 (Proost et al., 2015) was used as a reference for assembly and annotation. For each individual A. thaliana protein the following steps were performed: a tBLASTn against the DBG R. simsii 893k contig set was performed and all matching contigs were extracted (e-value 1e -5 and up to 250 hits allowed). Next, overlap-layout-consensus clustering (OLC) was performed using CAP3 (Huang and Madan, 1999), with default CAP3 parameters (minimal overlap length of 40 bases and 90% identity). All CAP3 contigs resulting from the CAP3 assembly were renamed according to the name of the founding A. thaliana protein. Next, a BLASTx against all A. thaliana proteins was performed and only those CAP3 contigs that had a best hit (e-value < 1e -10 ) against the original A. thaliana protein that initiated the clustering were retained,

Chapter 6 as these represent the most likely orthologous pairs. Subsequently, the longest open reading frame (ORF) was selected and corresponding coding sequence (CDS) extracted. A second round of CAP3 further clustered allelic sequences. Finally, the longest R. simsii CDS ortholog per founding A. thaliana gene was selected to remove the last remaining redundancy. This method assembles a comprehensive set of R. simsii transcripts, and annotates them according to their closest orthologue in A. thaliana .

2.1.3. Read mapping and gene expression analysis For differential gene expression (DGE) analysis of the six samples related to plant defence, non-deduplicated reads were mapped to the R. simsii transcriptome with 15k non- redundant transcripts using RNA-seq analysis utility of CLCbio GW. Maximum number of hits for a read was set at one. Unique read counts per gene for each sample were normalized by calculating the reads per kilo-base transcript per million reads mapped (RPKM) for each transcript. Prior to empirical analysis of DGE, two-group comparison (mock versus MeJA treatment or P. latus infestation) for both genotypes ( R. simsii ‘Nordlicht’ and ‘Elien’) was done in CLCbio GW. The empirical analysis is an ‘Exact Test’ for two-group comparisons developed by Robinson and Smyth (2008). For each sample a single library was made, split and sequenced in two independent lanes, yielding two subsets of sequencing data per sample which were considered (technical) replicates in statistical analysis. A p-value cut off of p < 0.001 and an absolute 1.5- or 3-fold change in gene expression was used to identify differentially expressed transcripts.

2.1.4. Functional categorization Gene ontology (GO) enrichment was analysed using the BiNGO plugin (version 3.0.3) for Cytoscape (version 3.2.1) (Shannon et al., 2003; Maere et al., 2005). A hypergeometric test for statistical analysis in combination with the gene list of the R. simsii transcriptome as reference was used. Annotations were based on the default A. thaliana annotation. For p- value correction, the Benjamini and Hochberg false discovery rate (FDR) correction was employed. The cut off p-value after correction was 0.05. For functional interpretation of differentially expressed gene sets, the GO-terms and functions of orthologous A. thaliana proteins are projected onto R. simsii transcripts. Conversely, for GO enrichment analysis, the respective A. thaliana gene identifiers are used as a substitute gene list in BiNGO.

2.2. Cloning genes Sequence information of putative marker genes were retrieved from the R. simsii transcriptome. Hereto, homologous sequences from literature were locally blasted against the R. simsii transcriptome in CLCbio GW. Blast results were visually interpreted for selecting the putative homologous fragments in R. simsii . Primers were developed for the isolation of candidate gene fragments for lipoxygenase ( RsLOX ), allene oxide synthase (RsAOS ), allene oxide cyclase ( RsAOC ), 12-oxophytodienoate reductase 3 ( RsOPR3 ), jasmonate methyltransferase ( RsJMT ), isochorismate mutase ( RsICS ), phenylalanine ammonia-lyase ( RsPAL ) and polyphenol oxidase ( RsPPO ). This was done using Primer3Plus software (Untergasser et al., 2007). Amplicons (cDNA) of RsLOX and RsAOS

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were cloned in pCR™II-TOPO® vector using TOPF10’ competent cells (Invitrogen™, Life Technologies Europe B.V., Gent, Belgium ) while other cDNA amplicons were cloned in pGEM®-T vector using JM109 competent cells (Promega Benelux BV, Leiden, the Netherlands). Subsequently fragments were sequenced from both sides according to the protocol of the BigDye® Terminator v1.1 Cycle Sequencing Kit on an ABI Prism 3130 xl Genetic Analyzer (Applied Biosystems®, Life Technologies Europe B.V., Gent, Belgium). BLASTx (Altschul et al., 1997) with default settings was used to confirm fragment identity. Putative reference genes based on Czechowski et al. (2005) were identified and cloned from R. simsii using the same routine (Table 6.1 and Supplementary Table 6.1).

2.3. Experiments Rhododendron simsii ‘Nordlicht’, ‘Elien’, ‘Michelle Marie’, ‘Aiko Pink’, ‘Sachsenstern’ and ‘Mevrouw Gerard Kint’ plantlets were rooted and grown as described in section 2.1 of Chapter 5 Methyl jasmonate (MeJA), β-aminobutyric acid (BABA) and coronatine (COR) were purchased from Sigma-Aldrich BVBA (Diegem, Belgium). Bion® containing 50% acibenzolar-S-methyl (BTH) was a kind gift from Syngenta Belgium (Oosterzele, Belgium). Solutions were prepared in distilled water containing 0.02% (v/v) Tween20. Distilled water containing 0.02% (v/v) Tween20 was used as mock treatment. Samples were taken at different time points after different treatments or mechanical wounding. Each sample consisted of six leaf punches (1 cm diameter) randomly divided over one plant (ca. 60 mg). The samples were flash frozen in liquid nitrogen immediately after harvest and stored at -80 °C until analysis.

Experiment 1 Rhododendron simsii ‘Nordlicht’ plants were treated with four different elicitors: 100 µM MeJA, 150 µM BABA, 0.1 µM COR and 300 µM BTH. A mock treatment was included as a reference. Plants were sprayed with vaporizers with a fine mist until run off. Ten biological replicates for gene expression analysis were taken from each treatment 6 hours after application.

Experiment 2 After selecting MeJA as a potent elicitor of JA related biosynthesis genes in R. simsii ‘Nordlicht’ (experiment 1), the effect on gene expression of foliar application (spray until run off) at a concentration of 100 µM was evaluated 0, 4 and 6 hours after treatment. MeJA samples were compared to mock treated samples. At each time point eight biological replicates were harvested from each treatment.

Experiment 3 In experiment 3, the systemic response on gene expression in leaves was assessed after root application of 100 µM MeJA. Samples were taken 0, 4, 6 and 24 hours after application. The root balls were submerged for 1 min in 100 µM MeJA or mock solution. The samples of MeJA treated plants were compared to samples taken from mock treated plants. At each time point eight biological replicates were taken from each treatment.

Experiment 4 Gene expression after leaf application of 100 µM MeJA was assessed in genotypes with a different response towards P. latus (Chapter 5): ‘Nordlicht’, ‘Elien’,

Chapter 6

‘Michelle Marie’, ‘Aiko Pink’, ‘Sachsenstern’ and ‘Mevrouw Gerard Kint’. A mock treatment was used as a reference for each genotype. Six biological replicates were harvested 6 hours after application for every treatment and genotype.

Experiment 5 The effect of wounding on gene expression was evaluated in R. simsii ‘Nordlicht’, ‘Elien’, ‘Michelle Marie’, ‘Aiko Pink’, ‘Sachsenstern’ and ‘Mevrouw Gerard Kint’ after 6 hours. Wound response in plants was assessed by mechanically wounding (squeezing with forceps) multiple leaves. Clean undamaged plants were used as mock treatment. For each treatment and genotype, six biological replicates were harvested.

2.4. RNA extraction optimization Three protocols were compared for extracting high quality RNA from R. simsii leaves. Apart from a modified CTAB protocol (protocol I), the TRIzol® Reagent (protocol II) and a routinely used CTAB protocol for bud and meristem tissue of R. simsii based on Wang and Stegemann (2010) (protocol III) were evaluated.

The optimized protocol (protocol I) to extract leaf RNA from R. simsii plants consists of three steps:

Grinding step Tubes containing tissue samples (60 mg for leaves) were placed in pre- cooled (-80 °C) Retsch Tissuelyser II (Qiagen Benelux B.V., Venlo, the Netherlands) blocks and grinded two times for 90 s at 30 Hz using three zirconium beads (3 mm). Between two grinding steps blocks were dipped in liquid nitrogen and orientation was switched.

Isolation step This step was initially described by Yu et al. (2012), but several modifications were included: altered centrifugation conditions, addition of vortex and rotation steps, and a re-centrifugation step. After a short spin in a pre-cooled centrifuge (4 °C), pre-heated (65 °C) CTAB extraction buffer (700 µl per sample) was added [extraction buffer: 3% (w/v) CTAB; 100 mM Tris-HCl (pH 8.0); 1.4 M NaCl; 20 mM Na 2EDTA; 5% (w/v) PVP and 1% (v/v) β-mercaptoethanol (added just before use)]. The mixture was vortexed for 1 min and then incubated at 65 °C, 800 rpm for 10 min using an Eppendorf Thermomixer. Afterwards the mixture was centrifuged at 15,800 g for 5 min at room temperature. The supernatant was transferred into a new 1.5-ml microfuge tube, followed by the addition of an equal volume of chloroform. The mixture was vortexed and shaken vigorously for 1 min. After being on a rotator for 5 min, the mixture was centrifuged at 15,800 g for 10 min at room temperature. The upper aqueous phase (without disturbing the interface) was transferred to a new 1.5-ml microfuge tube. To enhance purity of final extracted RNA, the sample was re-centrifuged at the same conditions.

Purification step The supernatant was again transferred to a new 1.5-ml microfuge tube and an equal volume of 4 M LiCl was added (Ambion®, Life Technologies Europe B.V., Gent, Belgium). RNA was allowed to precipitate overnight at 4 °C, followed by centrifuging at 15,800 g for 15 min at 4 °C. The resulting pellets were washed twice with 75% ethanol.

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Centrifugation during wash steps occurred at 15,800 g for 15 min at 4 °C. Finally, ethanol was allowed to evaporate and the pellets were re-suspended in 50 µl of RNase free water.

2.5. RNA quality control Quality and quantity of the obtained RNA was assessed in three ways. First, absorbance ratios and yield were measured using the NanoDrop (ND-1000) spectrophotometer (Isogen Life Science Belgium, Temse, Belgium). Absorbance ratios near or above 2.5 and 2 are generally accepted as excellent purity ratios, for A 260 /A 230 and A260 /A 280 respectively (Schultz et al., 1994; Baelde et al., 2001). Second, RNA quality was further determined with the Experion™ Automated Electrophoresis System and RNA StdSens Chips (Bio-Rad Laboratories N.V., Temse, Belgium). However, RNA Quality Indicator (RQI values) as well as 28 S to 18 S ratios were not suitable parameters for integrity assessment in plants (De Keyser et al., 2013). Ribosomal RNA (rRNA) bands in plants differ from or humans since plants have 25 S rRNA instead of 28 S rRNA. Chloroplast containing plant tissue introduces two extra peaks, 16 S and 23 S (Krupp, 2005) which will be recognized as degradation peaks by the software, leading to a miscalculation of the RQI value and an underestimation of the true integrity of the material in plants (De Keyser et al., 2013). The integrity was manually rated on the brightness and sharpness of the 25 S and 18 S rRNA bands. In each experiment a random selection of 10% of the samples were analysed as such. Third, the presence of potentially inhibiting substances in the extract was assessed with a SPUD assay (Nolan et al., 2006a). Heparin was used as a potential inhibitor to mimic the effect of inhibiting substances (Sigma-Aldrich BVBA, Diegem, Belgium) (0.04, 0.4, 4 and 40 U.ml -1). Samples were measured in duplicate on a LightCycler480 (Roche Diagnostics, Vilvoorde, Belgium). The SPUD protocol was performed as described in De Keyser et al. (2013).

2.6. DNase treatment and cDNA synthesis The obtained RNA was treated with DNase I according to manufacturer’s recommendations (DNA-free™ , Ambion®, Life Technologies Europe B.V., Gent, Belgium) to remove residual DNA contamination. The amount of starting RNA was 300, 250, 200, 150 and 200 ng for experiment 1, 2, 3, 4 and 5, respectively. First strand cDNA synthesis was performed using a commercial cDNA Synthesis Kit (iScript™ cDNA Synthesis Kit, Bio- Rad Laboratories N.V., Temse, Belgium). The no reverse-transcriptase controls (noRTs) were made using a mixture of DNase treated RNA and nuclease free water (1:1). Prior to use in qPCR reactions the cDNA and noRTs were diluted (1:5).

Chapter 6

2.7. Reference gene selection (geNorm) A geNorm pilot study to find stable and validated reference genes was conducted as recommended by Vandesompele et al. (2002). Twelve samples and fifteen reference genes (Table 6.1 and Suppl. Table 6.1) were used for optimal reference gene selection. The sample set consisted of six P. latus infested samples and six clean control samples. The qPCR reaction was performed as described under section 2.8 RT-qPCR. Cq-values were calculated by Lightcycler480 software (Roche Diagnostics, Vilvoorde, Belgium) and exported to Microsoft Excel. The data were used for reference gene selection using the geNorm module in qbase+ (Hellemans et al., 2007).

2.8. RT-qPCR RT-qPCR primers were developed using Primer3Plus software (Untergasser et al., 2007) (Table 6.1). SYBR Green based PCR analyses were performed using the LightCycler® 480 (Roche Diagnostics, Vilvoorde, Belgium). The qPCR reactions were performed in a final volume of 10 µl, containing 2 µl cDNA, 0.3 µl forward primer (10 µM), 0.3 µl reverse primer (10 µM), 2.4 µl nuclease free water and 5 µl of SensiFAST™ SYBR® No-ROX (Bioline France, Paris, France). RT-qPCR used the following amplification parameters for all genes: 5 min activation at 95 °C followed by 45 cycles of PCR at 95 °C for 5 s, 60 °C for 10 s and 72 °C for 20 s followed by melting curve analysis. Hereto temperature was held at 65 °C for 1 min, followed by ramping from 65 to 97 °C with a rate of 0.06 °C.s -1. No technical replicates were included in experiment 1 to 5. Gene specific PCR efficiencies (E = 10 (-1/slope) -1) were calculated by means of standard curves. These standard curves were prepared as described by De Keyser et al. (2013). In brief, plasmid DNA for each target gene fragment was purified using the PureLink® HQ Mini Plasmid Purification Kit (Invitrogen™, Life Technologies Europe B.V., Gent, Belgium) and linearized using a restriction enzyme ( Bam H I for RsICS , RsLOX and RsAOS ; Eco R V for RsAOC , RsPAL and RsOPR3 ; Apa I for RsPPO and RsJMT ). All enzymes were purchased from Invitrogen™ (Life Technologies Europe B.V., Gent, Belgium). The stock concentration of plasmids was diluted to a working solution of 1 ng.µl -1 in 50 ng.µl -1 yeast tRNA (Invitrogen™, Life Technologies Europe B.V., Gent, Belgium). Standard curves were constructed as six log10 dilutions of this working solution in yeast tRNA (50 ng.µl -1). For each experiment aliquots of the standard curve dilution series were prepared fresh from the stock of plasmid DNA stored at -20 °C. As described by De Keyser et al. (2013), data from each experiment were analysed using LightCycler480 software 1.5 (Roche Diagnostics, Vilvoorde, Belgium). No template controls (NTCs) were included in the experiments. Furthermore, in each experiment a noRT control for every sample was included. The 2 nd derivative maximisation method of Luu-The et al. (2005) was selected for Cq determination in every run. These values were exported to Microsoft Excel. All calculations of RT-qPCR data were done by means of qbase+ software from Biogazelle N.V. (Zwijnaarde, Belgium) (Hellemans et al., 2007). The normalization factors were based on two validated reference genes ( RsRG7 and RsRG14 ). Relative data were scaled to the

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maximum relative expression units of each experiment to calculate calibrated normalized relative quantities (CNRQs).

Table 6.1 List of RT-qPCR primer sequences and product size for Rhododendron simsii target gene fragments and reference genes during experiments 1-5.

Product Gene Acc. No. F or R Primer sequence 5’-3’ size F GCATCA CCTTCTTCGGGAATT RsLOX LN829618 97 bp R AACATGCCGCCCTCAACTT F AACCTCAATCTCGAACGAATCG RsAOS LN829619 86 bp R CGTCACGTCGTTGAAGAGAG F CGCCTAGATGCTTCAACACC RsAOC LN829620 126 bp R GAGATCGCCGAGAGAATTGA F TTGGTCTTGCGGTTCAAACT RsOPR3 LN829621 86 bp R GTGTACCCAACAACAGGATCG F GAGATCGCCGAGAGAATTGA RsJMT LN829622 100 bp R CATTTCTCCCTCCCCTTAGC F GGCACGGATTCTAATTGCAG RsICS LN829623 80 bp R CTCCTCCTCCAAACCAACCT F TCAAGTGGCCAAGAAAGTCC RsPAL LN829624 96 bp R TCGCGATCGACAACTTGA F GTTCCATGGC TGAGAAATCG RsPPO LN829625 126 bp R GTCGAGTTTTGCGGGAAATA F TGCGATGTGATGTGACTG RsRG7 a LN829626 132 bp R CGCTCTTAGTGGGACGTGAC F GGTATAGGATTGACAATCCCAAGG RsRG14 b AM932894 151 bp R CATTCAATCTCCGTCCCTATCG aSelected from Czechowski et al. (2005) (encodes a clathrin adaptor complex). bDescribed by De Keyser et al. (2013) as HK47 (encodes a nucleosome assembly protein).

2.9. Statistical analysis Significant differences in gene expression between mock and treatment were assessed using Mann-Whitney U tests in STATISTICA (Version 12.0, Statsoft Inc., Tulsa, OK, USA). This was done on log-transformed data. Figures based on non-log-transformed data were prepared in SigmaPlot 13.0 (Systat Software, San Jose, CA, USA).

Chapter 6

3. Results 3.1. RNA-sequencing In total 711M Illumina reads (split over 16 libraries) and 1.3M 454 expressed sequence tags (from 4 libraries, De Keyser et al., unpublished data) from 20 RNA-seq libraries were used for de novo assembly of the R. simsii transcriptome, yielding a total of 893k contigs for all 20 samples together. After Orthology Guided Assembly, the transcriptome contains a total of 32,367 transcript fragments with an average length of 953 bp, representing the orthologs of 14,860 unique A. thaliana genes. Sequence lengths ranged from a defined minimum of 102 up to 11190 bp. For expression analysis, we selected the longest transcript fragment per gene as non-redundant reference for read mapping. 8,012 out of these 14,860 CDS presumably represent full-length proteins.

In R. simsii ‘Nordlicht’, more than 1,400 genes of the R. simsii transcriptome were differentially expressed in response to either MeJA treatment or P. latus infestation. Both treatments had more than 500 DEGs in common when compared to the mock treatment (absolute FC 1.5 or greater and p < 0.001) (Fig 6.1). The top 25 (down- or upregulation) of DEGs for each treatment compared to the mock in ‘Nordlicht’ and ‘Elien’ is shown in Suppl. Table 6.2 to 6.9. When the absolute FC cut off was set to be greater than 3, the number of DEGs expressed in response to either MeJA treatment or P. latus infestation decreased to approx. 500. The number of common DEGs reduced to 150. The majority of the DEGs upon P. latus infestation in ‘Nordlicht’ caused an upregulation compared to the uninfested control. For the mite resistant genotype ‘Elien’ more than 2400 DEGs were reported upon mite infestation when compared to the mock treatment when the absolute FC cut off was set to be at least 1.5. In contrast only 1342 DEGs were reported upon MeJA treatment. Both treatments had 851 DEGs in common when compared to the mock treatment. As a consequence of the increase in the absolute FC cut off, greater than 3, the number of DEGs decreased to 285 and 881 for the 100 µM MeJA treatment and P. latus infestation, respectively. In three out of the four mock-treatment comparisons (absolute FC > 1.5), the direct evidence of JA involved in response to the treatment was clear. Hence, one or more of the following biological GO process terms were enriched: ‘response to jasmonic acid stimulus’, ‘jasmonic acid biosynthetic process’ and ‘jasmonic acid metabolic process’. Only when comparing the mock to P. latus infestation in ‘Elien’ no GO terms indicating the direct involvement of JA were enriched. No underrepresentation of SA terms was found for any of the treatments. The MeJA treatment in ‘Nordlicht’ caused an overrepresentation of GO terms directly linked to SA: ‘cellular response to salicylic acid stimulus’ and ‘salicylic acid mediated signalling pathway’. More stringent conditions on the FC (absolute FC > 3) resulted in a significant enrichment of one GO term directly linked to JA: ‘response to jasmonic acid stimulus’. The latter term was induced by MeJA treatment in ‘Elien’ and P. latus infestation in ‘Nordlicht’.

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The RNA-sequencing provided data to construct the first transcriptome of R. simsii. This transcriptome was used to find DEGs. The results provide evidence for the involvement of hormone pathways in reponse to P. latus infestation.

‘Nordlicht’ ‘Elien’

DEG DEG DEG DEG MeJA to mock P. latus to mock MeJA to mock P. latus to mock Fold change = 1.5 Fold 1.5 change =

Fold 3 change =

DEG DEG DEG DEG MeJA to mock P. latus to mock MeJA to mock P. latus to mock

Fig 6.1 Analysis of differentially expressed genes (DEG) detected in Rhododendron simsii ‘Nordlicht’ and ‘Elien’ transcriptional responses upon 100 µM methyl jasmonate treatment (orange) or Polyphagotarsonemus latus infestation (green) compared to uninfested control plants (p < 0.001). Results are presented for fold change cut off values 1.5 and 3. Numbers represent the amounts of DEG, (▲) upregulation in the treatment compared to the unifested control and (▼) downregulation in the treatment compared to the uninfested control. 3.2. Cloning genes Candidate genes for biosynthesis pathways of JA: LOX , AOS , AOC , OPR3 , JMT ; or salicylic acid (SA): ICS and PAL were successfully isolated. A candidate marker gene for oxidative stress response, PPO , was isolated as well. Homology of these genes was confirmed with orthologous genes from other plant species by means of BLASTx search (Table 6.2). Among the 15 possible reference genes included in the geNorm pilot study (section 3.4), four genes were previously validated by De Keyser et al. (2013) (Table 6.1 and Suppl. Table 6.1). Eleven new potential reference genes were cloned and identified after homology search with a list of stably expressed reference genes (Chechowski et al., 2005). These genes were cloned from R. simsii (Table 6.1 and Suppl. Table 6.1).

Chapter 6

3.3. RNA extraction optimization Initially, an evaluation was made of RNA extraction protocols for R. simsii leaves based on commercially available kits. Among the kits were the SV Total RNA Isolation System (Promega Benelux BV, Leiden, the Netherlands), PowerPlant® RNA Isolation Kit (MO BIO Laboratories, Inc., Carlsbad, CA, USA) and BcMaq™ Quick RNA/Animal or Plant Tissue Purification Kit (Bioclone Inc., San Diego, CA, USA). They all suffered from the same problem: columns clogged as samples were very viscous. Reducing the initial amount of tissue did not solve this problem.

Extractions using a modified CTAB protocol (protocol I), the TRIzol® Reagent (protocol II) and the protocol of Wang and Stegemann (2010) (protocol III) resulted in a successful extraction of leaf RNA. Regarding the difficulties in extracting leaf RNA from R. simsii , the quality of RNA obtained with these three protocols was further assessed. Extractions of raw leaf RNA using protocol I yielded RNA with an A 260 /A 230 ratio being 2.4-2.5 and a A 260 /A 280 ratio being 2.0-2.1. The absorbance ratios for leaf RNA obtained with protocol II and protocol III resulted in low A 260 /A 230 ratios (< 1). The corresponding A260 /A 280 ratios were suboptimal averaging 1.5 and 1.7 for protocol II and III, respectively (Fig 6.2). Extremely low A260 /A 230 ratios in RNA extracts from protocols II and III indicated a contamination of polysaccharides and polyphenols (Schultz et al., 1994; Loulakakis et al., 1996).

RNA integrity was evaluated with the Experion Automated Electrophoresis Station from Bio-Rad. The extraction based on protocol I resulted in RNA with excellent integrity compared to protocol II and III (Fig 6.3). The virtual gel-views indicated clean ribosomal bands only for protocol I. Samples obtained with protocol II were unsuitable for further analysis, since ribosomal bands were unrecognizable. Ribosomal bands from samples extracted with protocol III were somewhat more abundant but lanes 13, 15 and 16 showed a severe degree of degradation. The degradation was also noticed in the electropherogram (data not show) by a decrease of the ribosomal peaks and a shift to the so called fast region (Schroeder et al., 2006).

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Table 6.2 Sequence homology (BLASTx, default settings) of the isolated Rhododendron simsii gene fragments with orthologues in other species.

0 0 0 0 -126 -126 -122 -121 -81 -78 -77 -50 -50 -50 -37 -37 -36 -90 -89 -86 -66 -64 -64 -69 -58 -57 E-value E-value 2.00E 8.00E 4.00E 5.00E 5.00E 3.00E 3.00E 4.00E 5.00E 7.00E 8.00E 1.00E 1.00E 1.00E 3.00E 3.00E 1.00E 2.00E 2.00E 8.00E 3.00E

Max Max ident ident 81% 80% 80% 89% 84% 86% 87% 89% 92% 75% 79% 81% 68% 70% 64% 82% 83% 81% 91% 91% 89% 77% 65% 66%

Query Coverage Coverage 95% 95% 95% 73% 73% 73% 96% 96% 90% 99% 99% 92% 99% 99% 99% 97% 94% 94% 100% 100% 100% 99% 99% 99% -lyase 1 1 -lyase

-methyltransferase -methyltransferase -methyltransferase-like -lyase -lyase

-oxophytodienoate reductase 3 3 reductase -oxophytodienoate ase

-methyltransferase, putative putative -methyltransferase,

oxide synthase synthase oxide

-oxophytodienoate reductase 3 reductase 3 -oxophytodienoate reductase 3 -oxophytodienoate Description putative lipoxygenase, 1 lipoxygenase 3 lipoxygenase synthase allene oxide allene synthase allene oxide cyclase 2 allene oxide cyclase allene oxide cyclase allene oxide 12 12 12 PREDICTED: O jasmonate O jasmonate PREDICTED: O jasmonate PREDICTED: synthase isochorismate synthase isochorismate synthase Isochorismate PAL2 ammonia phenylalanine PREDICTED: ammonia phenylalanine polyphenoloxid oxidase polyphenol (chloroplast) oxidase polyphenol

nifera nifera Species Ricinus communis cinerariifolium Tanacetum cacao Theobroma Camellia sinensis max Glycine notabilis Morus max Glycine Camellia sinensis roseus Catharanthus miltiorrhiza Salvia vi Vitis indicum Sesamum Ricinus communis mume Prunus domestica Malus domestica Malus angustifoli Populus x fremontii Populus notabilis Morus Pyrus pyrifolia domestica Malus Camellia japonica Camellia nitidissima notabilis Morus officinale Taraxacum

23820.1 23820.1 Acc. No. RsLOX XP_002534368.1 AGO03785.1 XP_007024646.1 RsAOS AHY03308.1 NP_001236445.1 XP_010106073.1 RsAOC AEE99197.1 ADY38579.1 AFV68275.1 RsOPR3 AGW27205.1 NP_001267975.1 XP_011093821.1 RsJMT XP_002522163.1 XP_008241717.1 XP_008354653.1 RsICS AIL ACZ50508.1 XP_010086571.1 RsPAL AIZ76768.1 XP_008387584.1 AFJ80777.1 RsPPO ACM43505.1 XP_010100570.1 CAQ76694.1

Chapter 6

(a) (b)

2.5 2.0

2.0 1.5 280 230

/A /A 1.5 260 260 A

1.0 A 1.0

0.5 0.5

I II III I II III

Protocol Protocol

Fig 6.2 Absorbance ratios (a) A 260 /A 280 and (b) A 260 /A 230 of raw RNA extracted from leaf tissue of Rhododendron simsii with three different protocols; protocol I, protocol II, and protocol III. For all extraction protocols, n = 6.

25 S rRNA 23 S rRNA 18 S rR NA 16 S rRNA

Small RNA

Loading marker

M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 Fig 6.3 Virtual gel-view of raw RNA extracted from Rhododendron simsii leaves. RNA extracted with protocol I (lane 1 to 6), protocol II (lane 7 to 12) and protocol III (lane 13 to 18). Discriminating bands under normal conditions (large to small): 25 S rRNA, 23 S rRNA, 18 S rRNA, 16 S rRNA, small RNA and loading marker. Marker lane is represented as M. Estimated sizes are presented in kilodaltons (kDa).

In a SPUD assay the effect of potential inhibiting substances in the RNA extracts of protocol I and III were compared to control samples (average Cq-value ± SE, 25.01 ± 0.02). Based on the extremely bad RNA integrity results for samples from protocol II, this protocol was not evaluated in the SPUD assay. Concentrations of 40 U.ml -1 and 4 U.ml -1 heparin inhibited the RT-qPCR completely (Cq-values > 40), while a concentration of 0.04 U.ml -1 heparin caused no inhibition (average Cq-value 25.16 ± 0.02). The RT-qPCR was

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partly inhibited at a concentration of 0.4 U.ml -1 heparin, with an average Cq-value of 26.68 ± 0.01. There was no indication of inhibition in RNA from leaf tissue obtained with protocols I (24.99 ± 0.01) and III (24.95 ± 0.03) since Cq-values of RNA samples were within the range of 1 Cq of the control samples.

Finally, protocol I was validated on a set of 26 leaf samples of R. simsii . Samples yielded on average (± SE) 154.59 ± 12.24 ng RNA.µl -1. Average (± SE) absorbance ratios were 1.94 ± 0.01 and 2.29 ± 0.05 for A 260 /A 230 and A 260 /A 280 , respectively. Based on these results, we concluded that the protocol was sufficient for further use in our experiments.

3.4. Reference gene selection (geNorm) In a geNorm pilot experiment the optimal combination of a set of reference genes was evaluated. This was done by comparing pairwise variation between sequential normalisation factors (NFs). Results showed that the inclusion of a third gene would only have a minimal effect (V 2/3 = 0.031). The value was much lower than the proposed cut off value of 0.15 (Hellemans et al., 2007). As such the optimal NF in each experiment was calculated as the geometric mean of two reference genes, RsRG7 and RsRG14. The corresponding M-value of 0.089 indicated that these reference targets were very stably expressed in R. simsii , since it was far below the threshold of 0.5 (Hellemans et al., 2007). Furthermore, both genes belong to different functional classes and are as such likely not to be co-regulated. This makes the combination of both reference genes trustworthy for calculating a NF (Vandesompele et al., 2002).

3.5. Expression analysis In all experiments noRTs, reference gene stability and PCR efficiencies were checked prior to analysis. In case of amplification of noRTs, contamination was considered to be negligible when the difference in Cq between noRT and the sample was above 5 cycles. If there was contamination, the particular sample was discarded from the data set before further calculations. In all experiments the validated reference genes RsRG7 and RsRG14 were stably expressed. The M- and CV-values were below their respective thresholds of 0.5 and 0.15 (Suppl. Table 6.10). Run specific efficiencies were used to calculate standard curves. For all genes except for RsRG14 and RsPPO, PCR efficiencies were close to the optimum of 2 (Suppl. Table 6.11, Suppl. Table 6.12, Suppl. Table 6.13). The efficiencies of RsRG14 and RsPPO were a bit lower than the optimal value of 2, but these results were consistent throughout all experiments.

In experiment 1, the effect of different elicitors on gene expression in R. simsii was evaluated. Jasmonic acid biosynthesis genes ( RsLOX , RsAOS , RsAOC , RsOPR3 and RsJMT ) as well as SA biosynthesis gene RsPAL and RsPPO (a marker gene for oxidative stress response) were significantly up-regulated in leaves of R. simsii ‘Nordlicht’ 6 hours after treatment with 100 µM MeJA or 0.1 µM COR when compared to basal expression levels (Fig 6.4). The highest induction was observed for RsAOC as FCs (Fold Changes, calculated on non-log-transformed data as the proportion of treatment over mock) were 7.7 and 9.5 for MeJA and COR treatment, respectively. In contrast, a significant down-regulation for

Chapter 6 the SA biosynthesis gene RsICS was observed upon MeJA (FC = -1.5) and COR (FC = -1.3) treatment. Foliar treatment of R. simsii ‘Nordlicht’ with either 150 µM BABA or 300 µM BTH did not cause a significant induction of the selected marker genes 6h after application compared to basal expression levels (Fig 6.4). Methyl jasmonate was retained as an elicitor in R. simsii during subsequent experiments since it was able to induce the entire set of marker genes and it was much cheaper than COR.

10 *** *** 100 µM MeJA 150 µM BABA 0.1 µM COR 300 µM BTH

***

*** *** *** ** ns *** *** *** *** *** ns *** ns ns ** Fold changes Fold ns ns ns ns ns ns 1 ns ns ns ns ns ns * **

0.1 RsLOX RsAOS RsAOC RsOPR3 RsJMT RsICS RsPAL RsPPO

Fig 6.4 Fold changes (proportion of treatment over mock) of marker genes for jasmonic acid ( RsLOX , RsAOS , RsAOC , RsOPR3 , RsJMT ); salicylic acid ( RsICS and RsPAL ) and oxidative stress response ( RsPPO ) in Rhododendron simsii ‘Nordlicht’ leaves 6 hours after application of 100 µM methyl jasmonate (MeJA), 150 µM β-aminobutyric acid (BABA), 0.1 µM coronatine (COR) or 300 µM acibenzolar-S-methyl (BTH) (experiment 1). Significant differences compared to the respective mock treatment: ns = not significant; * p < 0.05; ** p < 0.01 and *** p < 0.001. In experiment 2, the effect of foliar application of 100 µM MeJA was assessed in a time course experiment. Immediately after treatment there was no significant effect on the gene expression of the marker genes except for RsAOS and RsICS (Fig 6.5). Both genes were significantly down-regulated with an FC of -1.5. Jasmonic acid biosynthesis genes RsLOX , RsAOS , RsAOC , RsOPR3 and RsJMT were significantly up-regulated at 4 and 6 hours upon treatment. Besides a significant down-regulation of RsICS immediately after foliar spraying of MeJA, also a significant down-regulation was observed 4 (FC = -1.6) and 6 (FC = -2.3) hours after application. On the contrary, the RsPAL gene, another SA biosynthesis marker gene, was only significantly up-regulated 4 hours after treatment (FC = 1.4). Foliar application of MeJA caused a significant up-regulation of the PPO gene. Fold changes were significantly different with values of 1.5 and 1.8, 4 and 6 hours after foliar application, respectively. Taking samples 6 hours after foliar application seems to provide relevant information for gene expression analysis in R. simsii using our set of marker genes.

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10 0h after treatment 4h after treatment 6h after treatment ***

*** *** ** *** *** *** ** ** * * * * ns ns ns ns

Fold changes Fold 1 ns ns ns * * ***

***

0.1 RsLOX RsAOS RsAOC RsOPR3 RsJMT RsICS RsPAL RsPPO Fig 6.5 Fold changes (proportion of treatment over mock) of marker genes for jasmonic acid ( RsLOX , RsAOS , RsAOC , RsOPR3 , RsJMT ), salicylic acid ( RsICS and RsPAL ) and oxidative stress response ( RsPPO ) in Rhododendron simsii ‘Nordlicht’ leaves at 0, 4 and 6 hours after foliar application of 100 µM methyl jasmonate (MeJA) (experiment 2). Significant differences compared to the respective mock treatment: ns = not significant; * p < 0.05; ** p < 0.01 and *** p < 0.001. The systemic effect on gene expression in R. simsii leaves after MeJA root application was evaluated in experiment 3. Only RsJMT was significantly up-regulated (FC = 1.6) 4 hours after application. The first significant induction for other JA biosynthesis genes was measured 6 hours after treatment and resulted in FCs of 3.2, 3.3, 2.8 and 1.4 for RsLOX , RsAOS , RsAOC and RsOPR3 , respectively (Fig 6.6). One day after dipping R. simsii ‘Nordlicht’ roots in MeJA none of the evaluated JA biosynthesis marker genes was still significantly induced. A significant down- and up-regulation was measured 6 hours upon treatment for SA biosynthesis marker genes RsICS and RsPAL, respectively. RsPAL remained significantly induced (FC = 1.6) one day after application whereas for RsICS this was not the case. A root application of MeJA was able to significantly up-regulate the expression of RsPPO in leaves only 6 hours after application.

Methyl jasmonate root application induced a similar response in ‘Nordlicht’ leaves as did foliar application (Fig 6.5 and Fig 6.6). However, the time frame of gene induction in leaves after root application was delayed when compared to foliar application. Hence, foliar application was preferred for further experiments.

Chapter 6

10

0h after treatment 4h after treatment 6h after treatment 24h after treatment *** *** *

*** **** ns ns ns ns * ns ns ns * ns ns ns ns ns Fold changes Fold ns ns ns ns ns 1 ns ns ns ns ns ns **

0.1 RsLOX RsAOS RsAOC RsOPR3 RsJMT RsICS RsPAL RsPPO

Fig 6.6 Fold changes (proportion of treatment over mock) of marker genes for jasmonic acid ( RsLOX , RsAOS , RsAOC , RsOPR3 , RsJMT ), salicylic acid ( RsICS and RsPAL ) and oxidative stress response ( RsPPO ) in Rhododendron simsii ‘Nordlicht’ leaves at 0, 4, 6 and 24 hours after root application of 100 µM methyl jasmonate (MeJA) (experiment 3). Significant differences compared to the respective mock treatment: ns = not significant; * p < 0.05; ** p < 0.01 and *** p < 0.001. In experiment 4, gene expression 6 hours upon MeJA treatment (100 µM) was evaluated in six different R. simsii genotypes: ‘Nordlicht’, ‘Elien’, ‘Michelle Marie’, ‘Aiko Pink’, ‘Sachsenstern’ and ‘Mevrouw Gerard Kint’. The purpose of this experiment was to determine genotype differences after MeJA treatment. The application of MeJA caused significant induction of the marker gene RsLOX in leaves of all six genotypes when compared basal levels (Fig 6.7). ‘Aiko Pink’ had the lowest FC (1.6) while ‘Sachsenstern’ showed the highest FC (4.2). Fold changes of RsAOS showed a significant up-regulation in leaves of all six genotypes when comparing to basal levels versus MeJA treatment and varied between 2.8-5.8 times. In contrast to the other genotypes, ‘Aiko Pink’ resulted in no significant induction of RsAOC upon MeJA treatment compared to the basal levels. Fold changes in ‘Elien’ and ‘Michelle Marie’ were low but significant (2.6 and 5.8, respectively). However, this was more than two times lower compared to the other genotypes with FCs of 10.9, 18.9 and 20.9 for ‘Nordlicht’, ‘Sachsenstern’ and ‘Mevrouw Gerard Kint’, respectively. The application of MeJA caused a comparable and significant induction of RsOPR3 in all genotypes, fold changes were 2.1-4.2. Interestingly, no significant induction of RsJMT was observed in ‘Aiko Pink’ upon MeJA treatment. This was also the case for RsAOC . Furthermore, significance levels of RsLOX , RsICS and RsPAL in ‘Aiko Pink’ were lower compared to other genotypes. Gene expression of RsICS decreased significantly after treatment with FCs between -1.7 and -3.8 among the six genotypes. RsPAL was significantly up-regulated by MeJA treatment (FC = 1.9-3.1) in all genotypes except for ‘Sachsenstern’ where no significant up-regulation was detected. Basal expression levels

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of RsPPO ‘Elien’ were much lower compared to the other genotypes, but it had the highest fold change (FC = 8.1). The mock treatment of ‘Elien’ showed only limited expression when compared to the other genotypes. The treatment caused in the other genotypes a significant up-regulation of RsPPO compared to basal expression levels and FCs varied between 1.6-4.2.

Wounding of leaves (experiment 5) caused a significant and strong up-regulation of JA biosynthesis marker genes RsLOX , RsAOS and RsAOC in all six genotypes when comparing to their respective basal expression levels (Fig 6.8). Fold changes for RsLOX varied between 8.1 and 29.5. RsAOS was strongest up-regulated 6 hours after wounding in ‘Mevrouw Gerard Kint’ (FC = 58.6), lowest up-regulation was observed in ‘Aiko Pink’ (FC = 27.9). Wounding resulted in a significant up-regulation of RsAOC as FCs varied between 23.0-216.2. The following gene in the JA biosynthesis cascade, RsOPR3 was not significantly induced in ‘Nordlicht’ while in the other genotypes FCs were 1.4-3.9. Although significant compared to the previous three genes, these FCs were negligible. For RsJMT none of the six genotypes showed to have altered gene expression levels upon wounding. In the SA pathway leaf wounding caused a significant down-regulation (FC = - 1.7 to -8.4) of RsICS in all genotypes. On contrary, for RsPAL wounding caused a significant up-regulation (FC = 1.6-4.9) in all genotypes except for ‘Michelle Marie’. The latter genotype also showed no significant induction of RsPPO . The highest up-regulation of RsPPO was observed in ‘Elien’ (FC = 3.3).

Chapter 6

Mock RsLOX 2.9 RsAOS 100 µM MeJA 4.1 0.8 ** ** 0.8 2.8 2.6 2.6 3.3 ** ** 0.6 ** 0.6 ** 3.5 1.6 CNRQs 0.4 4.2 5.8 ** 0.4 CNRQs * 3.6 ** ** 2.8 ** ** 0.2 0.2

** RsAOC RsOPR3 18.9 0.8 0.8 20.9 3.4 4.2 ** ** ** 0.6 2.6 2.3 0.6 ** ** 3.1 2.1 10.9 **

CNRQs ** 0.4 ** 2.2 0.4 CNRQs ** 5.8 0.2 ** ns 0.2

** 1.5 ns RsJMT RsICS

0.8 -3.0 0.8 1.8 -2.0 2.2 ** ** ** -2.3 0.6 ** 0.6 1.5 -2.5 -1.7 ** 1.6 -3.8 ** ** *

CNRQs ** 0.4 ** 0.4 CNRQs

0.2 0.2

2.9 RsPAL ** RsPPO 2.4 ** 4.2 0.8 ** 0.8

3.1 0.6 1.9 0.6 ** 2.9 ** ** CNRQs 0.4 2.3 ns 0.4 CNRQs ** 2.1 2.4 * 1.6 0.2 ** 0.2 8.1 ** **

t e e en i int i rn lich ar cht ar te d Eli K dli Elien or or ko Pink ns N le M iko Pink lle M se ard Kint el A N Ai h r ich che Sachsenstern Sac M uw Gerard Mi o

Mevr Mevrouw Ge

Fig 6.7 Gene expression profiles (CNRQ, non-log-transformed) of marker genes for jasmonic acid ( RsLOX , RsAOS , RsAOC , RsOPR3 , RsJMT ), salicylic acid ( RsICS and RsPAL ) and oxidative stress response ( RsPPO ) in Rhododendron simsii ‘Nordlicht’, ‘Elien’, ‘Michelle Marie’, ‘Aiko Pink’, ‘Sachsenstern’ and ‘Mevrouw Gerard Kint’ leaves 6 hours after foliar application of 100 µM methyl jasmonate (MeJA) or mock treatment (experiment 4). Numbers above the bars denote fold changes of treatment compared to mock. Significant differences compared to the respective mock treatment: ns = not significant; * p < 0.05; ** p < 0.01 and *** p < 0.001.

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Mock RsLOX 43.5 RsAOS Wounding 0.8 29.5 ** 0.8 ** 26.1 12.4 0.6 ** 0.6 ** 58.6 42.8 ** 11.1 ** CNRQs 0.4 0.4 CNRQs ** 8.1 33.6 46.6 8.1 ** ** 27.9 ** ** ** 0.2 0.2

RsAOC RsOPR3 93.8 175.0 2.7 ** 0.8 ** 3.9 0.8 216.2 ** 2.5 ** ** ** 0.6 0.6 1.8 1.4 28.3 ns ** * CNRQs 0.4 ** 77.8 23.0 0.4 CNRQs ** **

0.2 0.2

RsJMT RsICS -8.4 -4.2 0.8 ** ** 0.8 -7.8 ns -3.4 ** 0.6 ** -1.7 0.6 -3.8 * ** ns CNRQs CNRQs 0.4 ns 0.4 ns ns ns 0.2 0.2

2.3 RsPAL 2.4 ** RsPPO ** 2.5 1.6 ** 0.8 ** 0.8 4.9

1.6 ** 0.6 3.0 0.6 * 2.2 ** ** CNRQs 0.4 0.4 CNRQs

ns 1.9 0.2 ns 0.2 3.3 ** **

t n k t t in h rie rn in lich lie lic lien a d E P E ste rd Kint rd M lle Marie iko ra o le Nor e A N el Aiko Pink rard K h h Ge ic M Sachsensternuw Ge Mic Sachsen vro e M Mevrouw

Fig 6.8 Gene expression profiles (CNRQ, non-log-transformed) of marker genes for jasmonic acid ( RsLOX , RsAOS , RsAOC , RsOPR3 , RsJMT ), salicylic acid ( RsICS and RsPAL ) and oxidative stress response ( RsPPO ) in Rhododendron simsii ‘Nordlicht’, ‘Elien’, ‘Michelle Marie’, ‘Aiko Pink’, ‘Sachsenstern’ and ‘Mevrouw Gerard Kint’ leaves 6 hours after wounding leaves or mock treatment (experiment 5). Numbers above the bars denote fold changes of treatment compared to mock. Significant differences compared to the respective mock treatment: ns = not significant; * p < 0.05; ** p < 0.01 and *** p < 0.001.

Chapter 6

4. Discussion Next-generation sequencing of R. simsii RNA resulted in the first R. simsii transcriptome. Circa 14.9k unique orthologs from A. thaliana genes were identified. This represents about half of the genes described in model species A. thaliana (Lamesch et al., 2012). From RNAseq experiments GO enrichment indicated the involvement of different biological processes upon MeJA treatment in ‘Nordlicht’ and ‘Elien’, a susceptible and resistant genotype, respectively. Various biological processes show the involvement of JA. In ‘Nordlicht’, a similar response was found upon P. latus infestation. This might suggest JA playing a role in plant defence against P. latus . Molecular data on the interaction between plant and mite are limited to cucumber and tomato but stressed the importance of the JA pathway (Grinberg et al., 2005; Grinberg-Yaari et al. 2015). In order to confirm RNAseq results genes in the biosynthesis pathways of JA and SA were cloned for gene expression analysis. For JA biosynthetic pathway LOX (Vick and Zimmermann, 1983), AOS (Laudert et al., 1996), AOC (Ziegler et al. 2000), OPR3 (Müssig et al., 2000) and JMT (Seo et al., 2001) were successfully cloned. In the SA biosynthetic pathway PAL and ICS (Howe et al. 2005) were isolated in R. simsii . Finally a putative marker gene for oxidative stress response was cloned as PPO (Bergery et al., 1996)

For making a complete study on the role of JA in response to P. latus in R. simsii hybrids the set of putative maker genes was validated. Gene expression measurements in specific tissues necessitate the availability of high quality RNA in order to perform reliable experiments and to obtain consistent results (Bustin et al., 2009). Extremely low A260 /A 230 ratios in extracts from protocols II and III indicated contamination with polysaccharides and polyphenols (Schultz et al., 1994; Loulakakis et al., 1996). It is suggested that the loss of RNA which might form a complex to residual proteins and polyphenols was limited in our protocol. This was most likely due to the use of pure chloroform instead of chloroform:isoamyl alcohol (24:1) or phenol:chloroform:isoamyl alcohol (25:24:1) in other protocols (e.g. Liao et al., 2004; Wang and Stegemann, 2010; Yu et al., 2012). Furthermore, in most protocols RNA precipitation is carried out with isopropanol and followed by additional precipitation steps with NaAOc for low concentrated samples (Shahrokhabadi et al., 2008). Here lithium chloride (Zeng and Yang, 2002) was used, which resulted in higher RNA concentrations. In downstream applications such as the sensitive RT-qPCR technique, inhibiting substances in RNA extracts are unwanted. From the SPUD assay (Nolan et al., 2006a, 2006b) it was concluded that substances inhibiting RT-qPCR were not present in extracts resulting from the presented protocol I. Hence, the protocol I was used for further experiments.

Our experiments show a significant induction of the putative marker genes in R. simsii after MeJA application when compared to the basal expression levels. This up-regulation of JA biosynthesis genes upon MeJA treatment is in agreement with results obtained in Arabidopsis by Jung et al. (2007). The present study shows that transcript levels of the marker genes increased over time in MeJA treated R. simsii plants. Macroarray analysis showed up-regulation of LOX2 , AOS and OPR3 genes in Arabidopsis rosette leaves 24 hours

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after treatment with MeJA (100 µM). A time course assay (northern blotting) showed that in Arabidopsis LOX and AOS genes were induced early in response to MeJA, and the maximum accumulation of transcripts was seen 12 hours after treatment (Jung et al., 2007). RT-PCR amplification and southern blot analysis also suggested a significant gene transcription of JMT in Arabidopsis upon MeJA treatment (Jung et al., 2007). Sasaki et al. (2001) stated that in Arabidopsis MeJA positively induces the expression of JA biosynthesis genes such as LOX2 , AOS , AOC and OPR3 . AOC is also induced in other dicotyledonous plant species such as tomato by MeJA application (Stenzel et al., 2003). Furthermore JA application evidenced induction of this gene in monocotyledonous plant species e.g. rice (Agrawal et al., 2004). Expression patterns of JA biosynthesis related marker genes were in general the same in six different R. simsii genotypes. But for ‘Aiko Pink’ the expression patterns were less significant and genes RsAOC and RsJMT were not significantly induced 6 hours after application compared to the other genotypes. In wheat (Triticum aestivum ), MeJA application showed to result in genotype specific gene expression, including LOX and PAL genes (Motallebi et al., 2015). These differences in induction may be due to the different backgrounds of the genotypes. Another hypothesis might be that genotypes react in producing signalling molecules upon a stimulus received. A correct assessment of this hypothesis necessitates samples taken at different time points upon treatment. Compared to what is known about local MeJA induced responses in shoots, relatively little knowledge is available about JA signalling and induction of JA biosynthesis genes in shoots as a systemic response induced by root application. In present study, similar expression patterns were observed in R. simsii leaves after a foliar or root treatment with MeJA. However, timing of gene expression induction in shoots differed depending on the site of application in R. simsii ‘Nordlicht’. Local application resulted in a much earlier (approx. 2 hours) significant induction of these genes compared to the systemic (root) application. From Tytgat et al. (2013) it is clear that the JA biosynthesis pathway in Brassica oleracea is differently regulated in roots and shoots. This may in turn cause differential responses in both organs depending on where the initial JA signal was first perceived. It is suggested that a different OPDA/JA ratio depending on the site of JA induction might be one of the mechanisms causing the observed differential gene expression in B. oleracea (Tytgat et al., 2013). In R. simsii , expression of the PAL gene, the key enzyme for SA biosynthesis via the phenylpropanoid pathway (Howe et al., 2005), was significantly induced upon application of MeJA. On the contrary, expression of RsICS , the key enzyme responsible for biosynthesis of SA via the isochorismate pathway (Howe et al., 2005), was down-regulated in MeJA treated R. simsii plants compared to basal expression levels. Król et al. (2015) suggested that the phenylalanine ammonia-lyase pathway but not the isochorismate pathway is the primary route for SA production in tomato upon treatment with MeJA. This is concluded from gene expression analysis (e.g. upregulation of PAL5 and downregulation of ICS ), determination of endogenous phytohormone levels and amounts of quercetin and kaempferol flavonols related to the expression of PAL .

Chapter 6

Induction by MeJA application of the PPO gene, an oxidative stress-tolerance gene, which has an anti-herbivore role (Constabel et al., 1995), was observed in ‘Nordlicht’ but also in five other R. simsii genotypes; ‘Elien’, ‘Michelle Marie’, ‘Mevrouw Gerard Kint’, ‘Sachsenstern’ and ‘Aiko Pink’. The same effect was observed in many other plant species such as tomato (Constabel et al., 1995), tobacco (Ren and Lu, 2006), several poplar species (Constabel et al., 2000; Haruta et al., 2001) and also in other herbaceous crops and trees (Constabel and Ryan, 1998). Furthermore, it was found that PPO mRNA was strongly induced in the growing leaves of MeJA treated plants, but only slightly induced in older leaves (Constabel et al., 2000). In tomato, MeJA and the octadecanoid pathway induce PPO in addition to a battery of anti-herbivore proteins that help regulate the defence response itself (Bergey et al., 1996).

Plants treated with COR, a potent agonist of the JA receptor, had similar expression patterns of the set of marker genes as compared to the MeJA treatment. Since COR shares structural and functional similarities with JA and related signalling compounds such as MeJA and JA-Ile (Feys et al., 1994; Weiler et al., 1994; Staswick and Tiryaki, 2004; Uppalapati et al., 2005), the effect of COR on JA or SA biosynthesis genes was as expected comparable to that of MeJA application. In R. simsii ‘Nordlicht’, COR strongly induced expression of all the key enzymes in the JA biosynthesis pathway. Application of COR to Arabidopsis rosette leaves resulted in the accumulation of OPR3 (Katsir et al., 2008b; Melotto et al., 2008). This induction was reported in tomato as well (Uppalapati et al., 2005). It is shown in experiment 1, that 6 hours after application of BABA on ‘Nordlicht’ no significant induction of the selected marker genes was observed. β-aminobutyric acid was selected as a potential inducer since Zimmerli et al. (2000) observed a protective effect of BABA in Arabidopsis when used at comparable concentrations. This was due to the potentiation of natural defence mechanisms against biotic stresses, a phenomenon later referred to as priming (Conrath et al., 2002). It might be that the concentration of BABA used in R. simsii was not appropriate to induce a relevant expression of the marker genes in R. simsii or that samples were not taken in the correct time window. Hamiduzzaman et al. (2005) observed no induction of LOX-9 in grapevine ( Vitis vinifera ) upon application of BABA (1 mM), supporting our findings, but in that study samples were taken one or more days after application. The application of BTH, a synthetic analogue of SA (Friedrich et al., 1996; Lawton et al., 1996), caused no significant induction of the SA biosynthesis marker genes in R. simsii ‘Nordlicht’. The JA biosynthesis marker genes were also not induced. In Arabidopsis leaves expression of LOX2 and PAL1 was not markedly influenced 72 hours upon BTH treatment (Moran and Thompson, 2001) nor was the LOX-9 gene in grapevine 1-4 days after BTH treatment (Hamiduzzaman et al., 2005). In Arabidopsis BTH was not, or only slightly active at the immediate induction of the PAL gene (Kohler et al., 2002). Here again, one can discuss concentration or the time points when taking samples after application. But BTH concentration used in R. simsii was the same as in Arabidopsis and tomato. Van Wees et al. (1999) showed that BTH application can suppress JA-dependent responses in

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Arabidopsis . This might explain the non-induction of JA related biosynthesis genes in R. simsii ‘Nordlicht’ upon BTH application.

Another well-known inducer of JA-dependent responses is mechanical wounding (Reymond and Farmer, 1998). Our results indicate that the set of marker genes showed generally the same response upon wounding when compared to the external application of MeJA. No significant accumulation of RsJMT mRNA was observed in neither of the six R. simsii genotypes. In contrast, JMT expression was induced by wounding in Arabidopsis (Seo et al., 2001). Transport of OPDA, the JA precursor, into peroxisomes via ATP-binding cassette transporter COMATOSE (Theodoulou et al., 2005) represents a potential point of post-translational regulation of the OPDA metabolism as stated by Koo et al. (2009). Furthermore OPR3 , as well as downstream enzymes in the β-oxidation pathway (Schaller et al., 2005; Browse et al., 2009), are potential control points for the wound-induced post- translational regulation of JA synthesis (Koo et al., 2009) or the subsequent conversion to MeJA by JMT . In Capsicum annuum expression levels for OPR3 and JMT increased immediately after wounding (10 min), and were maintained for 1 to 4 hours (Song et al., 2005). This might suggest our samples in R. simsii were probably taken when transcript levels already were decreasing, resulting in lower fold changes between MeJA induced and basal expression levels. Fold changes of RsOPR3 were relatively lower compared to those of JA biosynthesis genes RsLOX , RsAOS and RsAOC 6 hours upon wounding. RsOPR3 was even not significantly induced in ‘Nordlicht’ by wounding. However, in Arabidopsis , OPR3 was induced after wounding (Costa et al., 2000). Low FCs measured 6 hours after wounding, may be the result of a rapid accumulation (within 15 min) of transcript levels, attaining peak levels 30-60 min after wounding, followed by a decline at later time points, as was also observed in Arabidopsis (Koo et al., 2009). The latter results suggest samples were probably taken when transcript levels already were decreasing. On contrary, RsLOX , RsAOS and RsAOC genes that are known to be located in the chloroplast and supposed to be active earlier in the JA biosynthesis pathway, were highly induced in all genotypes. Resulting FCs were between 8.1-29.5, 27.9-58.6 and 23.0-216.2 for RsLOX , RsAOS and RsAOC, respectively. This indicates that in R. simsii wounding results in a much later response of JA biosynthesis genes as compared to C. annuum and Arabidopsis . Furthermore it is very plausible that samples were taken too early in order to detect differences in gene expression for RsOPR3 and RsJMT . Despite the strong induction of RsLOX (FC = 8.1) and RsAOS (FC = 27.9) in ‘Aiko Pink’ no significant induction was found for RsPAL . In all other genotypes the induction of RsLOX , RsAOS and RsPAL upon wounding is in accordance with Reymond et al. (2000) who showed induction of LOX2 , AOS and PAL2 , a group of COI1-dependent genes for which induction by wounding depends strictly on the ability of the plant to respond to JA. PAL1 , a COI1-independent gene also was induced by wounding in Arabidopsis (Reymond et al., 2000). Leaves that had been wounded and adjacent leaves on the same plant showed to accumulate LOX2 mRNA in Arabidopsis (Bell and Mullet, 1993). Induction of this gene is required for the wound- induced synthesis of the plant growth regulator JA in leaves (Bell et al., 1995). In tomato also an increase of LOX , AOS , and AOC upon wounding was reported (Heitz et al., 1997;

Chapter 6

Sivasankar et al., 2000; Ziegler et al., 2000). The homolog of the PPO gene in R. simsii showed an increase in transcript levels in all of the evaluated genotypes except for ‘Michelle Marie’. Constabel et al. (2000) observed an accumulation of PPO transcripts upon wounding in hybrid poplar ( Populus trichocarpa × Populus deltoides ). When comparing all genotypes it is remarkable that ‘Elien’ had the highest FCs upon MeJA treatment and wounding. But when comparing the basal expression levels among the other genotypes ‘Elien’ showed extremely low expression of RsPPO.

This work highlights the expression patterns of marker genes related to JA and SA biosynthesis pathways and an oxidative-stress response gene. In future studies this set of validated marker genes can be used to identify their role in plant physiological processes in R. simsii hybrids, e.g. as related to plant defence against the mite pest P. latus .

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Chapter 7

Hormonal changes and gene expression of jasmonic acid biosynthesis genes in response to Polyphagotarsonemus latus infestation in Rhododendron simsii hybrids

1. Introduction Insects are able to feed on plants in various ways. Chewing insects such as caterpillars and beetles consume significant proportions of plant tissue. Sucking-type feeding insects like thrips combine sucking and rasping to feed. Phloem-feeding is done by aphids and whiteflies who use a feeding tube and cause minimal tissue disruption and rarely mechanical damage to the host plant (Abe et al., 2009; Zhurov et al., 2014). Another major class of insect feeding includes mining-type feeding in which leafminers feed within leaves and stems forming tunnels (Abe et al., 2009). Chelicerates which include phytophagous mites, pierce plant tissue to feed on cell contents (Tanigoshi & Davis, 1978; Campbell et al., 1990). Of these, the two-spotted spider mite, Tetranychus urticae , is the best characterized in terms of its interaction with the plant (Zhurov et al., 2014). Another plant feeding mite, the broad mite Polyphagotarsonemus latus (Banks) (Acari: Tarsonemidae), has a polyphagous feeding behaviour (Gerson, 1992; Zhang, 2003) and feeds on the youngest growth tips of a wide range of plants, including Rhododendron simsii. This causes malformation of flower buds and terminal leaves curling down at the edges and becoming brownish (Labanowski & Soika, 2006). Not all R. simsii genotypes are susceptible to P. latus. ‘Elien’ is resistant, whereas ‘Aiko Pink’ and ‘Nordlicht’ are very susceptible genotypes and ‘Michelle Marie’, ‘Mevrouw Gerard Kint’ and ‘Sachsenstern’ are considered susceptible genotypes (Chapter 5). It is clear that plant defence responses can be attributed to the mechanical feeding damage provoked (Mithöfer et al., 2005), but some responses only can be attributed to certain herbivory-derived signals (Howe and Jander, 2008). Most of these emanate from herbivore saliva or regurgitant and result in defined herbivory-induced changes, such as phytohormone accumulation, induction of defence genes and the emission of volatiles (Wu and Baldwin, 2010). The phytohormone jasmonic acid (JA) plays an important role in plant defences against pests and insects in horticultural crops (Rohwer and Erwin, 2008). Studies on mutant plants demonstrated the effect of JA on caterpillars and aphids in Arabidopsis (Ellis et al., 2002; Reymond et al., 2004). But also plant defences to other insects such as thrips are shown to be jasmonate-dependent (De Vos et al., 2005; Abe et al., 2008a, 2008b, 2009). Spider mites ( Tetranychus sp.) are known to induce JA-regulated gene-expression when feeding on plants (e.g. Arimura et al., 2000; Zheng et al., 2007; Kant et al., 2008; Sarmento et al., 2011b; Agut et al., 2014; Zhurov et al., 2014; Alba et al., 2015). However, some strains of the spider mites Tetranychus urticae and Tetranychus evansi were found to suppress expression of JA and/or SA marker genes (Kant et al., 2008; Sarmento et al., 2011b; Alba et al., 2015). This phenomenon is well known from plant pathogens (e.g. Abramovitch et al., 2006; Kamoun, 2006; De Jonge et al., 2011) and nematodes (Haegeman et al., 2012). Also insects are capable to manipulate plant defences (Musser et al., 2002, 2005; Will et al., 2007; Zarate et al., 2007; Weech et al., 2008; Zhang et al., 2009, 2011; Bos et al., 2010; Consales et al., 2012; Stuart et al., 2012; Wu et al., 2012). In 2005 it was shown that P. latus infestation activates JA- and salicylic acid (SA)-dependent pathways, and possibly an oxidative stress response in cucumber leaves ( Cucumis sativus ) (Grinberg et al., 2005). Salicylic acid is essential for defence against diseases (Vlot et al., 2009). In tomato, ( Lycopersicon esculentum ) mite infestation induced high levels of proteinase

Chapter 7 inhibitor transcripts. These proteinase inhibitors are expressed during the late stage of the JA pathway. It has been reported that P. latus feeding on def-1 plants, mutants of tomato ‘Castlemart’ impaired in JA biosynthesis, caused a ca. 40 % reduction in leaf number and ca. 55 % reduction in plant height whereas the wild type was resistant to P. latus infestation (Grinberg-Yaari et al., 2015).

In the present study, the role of JA and/or SA induced defences in response to P. latus infestation on R. simsii was clarified. Gene expression of marker genes for the JA and SA biosynthesis pathway in R. simsii hybrids was studied, while phytohormone quantification was done by LC-MS/MS. Finally, the effect of JA or SA induction on P. latus population growth rate was evaluated in a bio-assay.

2. Materials and methods 2.1. Plant material and infestation Rhododendron simsii ‘Nordlicht’, ‘Elien’, ‘Michelle Marie’, ‘Aiko Pink’, ‘Sachsenstern’ and ‘Mevrouw Gerard Kint’ plantlets were rooted and grown as described in section 2.1 of Chapter 5. Plants were infested by surrounding them with P. latus infested Hedera helix plants. The infestation technique and rearing of the mites are described in section 2.2 of Chapter 5. Plant damage caused by P. latus was rated on a scale from 0 to 4 as described in section 2.3 of Chapter 5. The corresponding number of mites present per shoot tip was determined using the protocol described in section 2.3 of Chapter 5.

2.2. Experiments Experiment 1 Polyphagotarsonemus latus infested R. simsii ‘Nordlicht’ was compared to an uninfested control. Leaf samples were taken at 0, 4, 6, 8, 12, 15, 20 and 25 days post infestation (dpi) in infested plants and uninfested control plants. Gene expression analysis at each time point was based on 8 replicates for each treatment. Mite counts per shoot tip and damage rates were calculated as the average of 10 replicates.

Experiment 2 A comparison was made between R. simsii ‘Nordlicht’, ‘Elien’, ‘Michelle Marie’, ‘Aiko Pink’, ‘Sachsenstern’ and ‘Mevrouw Gerard Kint’ plants infested with P. latus mites and their uninfested controls. For each treatment the six genotypes were randomly placed in one block. At 7, 14 and 21 dpi, 6 replicate leaf samples were collected for gene expression analysis from the infested as well as the uninfested control plants. The average damage rate and number of mites present per shoot tip was calculated on 8 replicates taken at 0, 7, 14 and 21 dpi. Replicates for phytohormone analysis (n = 8) were taken at 7 dpi for both treatments on ‘Nordlicht’ and ‘Elien’.

Experiment 3 Phytohormones were compared between P. latus infested and uninfested R. simsii ‘Nordlicht’ and ‘Elien’. Samples were taken at 4, 6, 8 and 11 dpi (n = 5) from the infested as well as the uninfested control plants. Corresponding damage rates and numbers of mites present per shoot tip were determined at each time point (n = 5).

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Experiment 4 Population growth of P. latus on R. simsii leaf discs exposed to phytohormones or a mock solution was studied. In a 24-well plate leaf discs (diameter 12 mm) of R. simsii ‘Nordlicht’ were floated on hormone solutions. Solutions were prepared in distilled water containing 0.02% (v/v) Tween20 and 50, 100 µM MeJA or 50, 100 µM SA. Distilled water containing 0.02% (v/v) Tween20 was used as mock treatment. Methyl jasmonate and SA were purchased from Sigma-Aldrich BVBA (Diegem, Belgium). On each leaf disc one two-day old adult female broad mite was placed. In total 24 replications of each treatment were prepared. Plates were placed in an incubator (Vötsch- Industrietechnik Bio Line model VB 1014, Balingen-Frommern, Germany) at a constant temperature of 25 ± 1 °C, 80% relative humidity and a photoperiod of 16:8 h (L:D). Population growth was calculated as the rate of increase; R i = ln(N f/N i)/Δt, were N f is the population at a certain time point f, N i is the initial population and Δt is the time period between f and i expressed in days. All observations were made using a stereo microscope (Leica MZ6, Leica, Wetzlar, Germany).

2.3. RT-qPCR Each sample consisted of six leaf punches (1 cm diameter) on three randomly chosen shoots (ca. 60 mg). From each shoot one leaf punch was taken from a young leaf and from an older leaf. In order to overcome variability related to the fact that hormone accumulation and gene expression follow circadian rhythms, sampling time was standarized for each experiment (e.g. Novakova et al., 2005; Mizuno and Yamashino 2008; Goodspeed et al., 2012). Five marker genes for JA biosynthesis were selected; RsLOX , RsAOS , RsAOC , RsOPR3 and RsJMT . For monitoring the SA biosynthesis pathway RsICS and RsPAL were used. The RsPPO gene was used as a marker gene for measuring the oxidative stress-response. Leaf RNA was extracted as described in section in section 2.4 of Chapter 6. The amount of RNA used was 100 and 160 ng for experiment 1 and 2, respectively. The extracted RNA was treated with DNase and cDNA was synthesised according to section 2.6 of Chapter 6. RT-qPCR analysis was conducted according to the descriptions in section 2.8 of Chapter 6. GeNorm M-value and coefficient of variation (CV) for reference genes RsRG7 and RsRG14 expressing reference gene stability in experiments 1 and 2 are shown in Suppl. Table 7.1. Run specific amplification efficiencies are shown in Suppl. Table 7.2 and Suppl. Table 7.3 for experiment 1 and 2, respectively.

2.4. Isolation of phytohormones and analysis by means of LC-MS/MS For phytohormone analysis whole leaves were harvested. The samples were flash frozen in liquid nitrogen immediately after harvest and stored at -80 °C until analysis. The amounts of SA, JA and its precursor 12-oxophytodienoic acid (OPDA) were determined using LC-MS/MS analysis. Phytohormone isolation and analysis was done as described by Bosco et al. (2014). In brief, frozen leaf samples were ground with mortar and pestle. From each sample 20 mg homogenized material was extracted with 2 ml solvent mixture methanol:water:acetic acid (10:89:1). This was spiked with deuterium labelled 2H4-SA and 2H6-JA as internal

Chapter 7 standards with a final concentration of 800 ng/ml and 40 ng/ml, respectively. Samples were extracted at 4 °C for 16 h on a shaker at 210 rpm. Subsequently samples were vortexed and filtrated through a 0.22 µm Millex-GV filter (Millipore, Billerica, MA, USA). Extracts were collected in glass tubes and transferred (1 ml) onto Oasis MCX 1 cm³ cartridges (Waters, Milford, MA, USA) that were preconditioned with 1 ml pure methanol and 1 ml milliQ water. After the samples were loaded, the cartridges were washed with 0.5 ml milliQ water. The flow through was discarded and the columns were eluted by applying 1 ml methanol under vacuum. Samples were evaporated to dryness in a warm water bad at a constant temperature of 35 °C under a nitrogen atmosphere. The dried residues were re-suspended in 0.5 ml 75% methanol. The final extract was filtrated through a 0.22 µm Millex-GV filter (Millipore, Billerica, MA, USA) and transferred to a microvial prior to injection (10 µl) into the LC-MS/MS. A serial dilution of pure standards of OPDA (0.125-7.5 ng; 8 dilutions) and JA (1-15 ng; 6 dilutions), was run as external calibration curve to calculate final concentrations of the samples. The amounts of SA were compared relatively based on area internal standard/area analyte. Analytical determination of the target compounds was achieved using an Acquity UHPLC system coupled to a Xevo TQ-S triple quadrupole MS detector (Waters, Milford, MA, USA). Chromatographic separation was achieved using an Acquity UHPLC BEH C 18 column (100 x 2.1 mm, 1.7 µm) (Waters, Milford, MA, USA) at 30 °C. The mobile phase consisted of 0.3 % acetic acid in water (A) and 0.3 % acetic acid in acetonitrile (B). The flow rate was set at 0.3 ml.min -1. The gradient was as follows: 0 min, 2 % B; 1.0-2.5 min, 2-40 % B; 2.5-4.0 min, 40-50 % B; 4.0-5.0 min, 50-80 % B; held for 2 min; 7.0-7.1 min, 80- 100 % B; held for 0.9 min; and 8.0-8.01 min 100-2 % B. Total runtime was 10 minutes. The mass spectrometer was operated in the ESI negative mode using the multiple reaction monitoring (MRM) mode. Desolvation gas was set at 1200 l.h -1 and desolvation and source temperature were set at 500 and 120 °C, respectively. The MRM settings in the MS/MS function with corresponding cone voltage and collision energy are found in Table 7.1. Data were acquired using MassLynx, version 4.1 (Waters, Milford, MA, USA). Calculations were done in Microsoft Excel. System sensitivity and reproducibility was checked for a P. latus infested and a control sample.

Table 7.1 ESI-MS/MS conditions with indication of the precursor ion (m/z), cone voltage (V), product ions (m/z), collision energy (eV) and chromatographic retention time (min) of salicylic acid (SA), 12-oxophytodienoic acid (OPDA), (-)-jasmonic acid (JA), and deuterium- labeled internal standards [ 2H4]-salicylic acid and [ 2H6]-(±)-jasmonic acid.

Cone Precursor Collision Retention Compound voltage Product ions (m/z) ion (m/z) energy (eV) time (min) (V) SA 137.00 40 64.95/92.96 22/14 3.64 OPDA 291.33 18 165.16/247.23/273.14 20/18/18 5.87 JA 209.15 25 58.77/165.00 12/17 4.24 2H4-SA 140.74 35 96.84 17 3.63 2H6-JA 215.11 30 58.77 12 4.22

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2.5. Statistical analysis For each genotype, mite counts and damage rates at different time points were analysed by means of a Kruskal–Wallis H test. Means were separated post hoc using Dunn’s test (Dunn, 1964) in SPSS (Version 22.0, IBM Corp., Armonk, NY, USA). Broad mite counts were log-transformed [ln(x+1)] and square root transformations were applied for damage rates before the statistical analyses. Differences in gene expression profiles or phytohormone contents of P. latus infested and mock plants were assessed at each time point using Mann-Whitney U tests in STATISTICA (Version 12.0, Statsoft Inc., Tulsa, OK, USA). Population growth data were statistically analysed with Mann-Whitney U tests (each hormone treatment versus mock treatment) using SPSS (Version 22.0, IBM Corp., Armonk, NY, USA).

Chapter 7

3. Results In all experiments amplification of noRT’s was checked. Contamination was considered to be negligible when the difference in Cq between noRT and the sample was above 5 cycles. In case of contamination, the particular sample was discarded from the data set before further calculations. Reference gene stability (Suppl. Table 7.1) and PCR efficiencies (Suppl. Table 7.2 and Suppl. Table 7.3) were checked prior to analysis. No problems for reference gene stability and PCR efficiency were observed. System sensitivity and reproducibility of JA, OPDA and SA measurements was good (Suppl. Table 7.4).

In experiment 1, gene expression in R. simsii ‘Nordlicht’ was studied upon P. latus infestation. Mite numbers increased significantly over time. First P. latus mites were counted on samples taken 4 dpi and averaged 1.9 mites per shoot tip (Fig 7.1). At 8 and 25 dpi on average 10.3 and 14.6 mites per shoot tip were counted, respectively. The first damage symptoms became visible at 12 dpi. At the end of the experiment (25 dpi), the average damage rate reached 1 on a scale of 0 to 4. The corresponding mite-specific induction of JA and SA biosynthesis marker genes during a time course of 25 days is shown in Fig 7.2. Jasmonic acid biosynthesis marker genes RsAOC and RsJMT were significantly up-regulated starting from 6 dpi. RsLOX was only significantly up-regulated at 20 and 25 dpi. RsOPR3 showed to have significantly higher transcript levels at 6, 8 and 12 dpi. Transcript levels were not significantly different when comparing both treatments for marker gene RsAOS . Salicylic acid biosynthesis marker gene RsICS was not significantly induced upon P. latus infestation in R. simsii ‘Nordlicht’. In contrast, RsPAL , another marker gene for SA biosynthesis, was induced significantly starting from 6 dpi. Expression analysis showed a significant up-regulation of RsPPO , a marker gene for oxidative stress-response, starting from 6 dpi. To conclude, taking samples from 6 dpi on appears to provide relevant information on the induction of our set of marker genes; earlier sampling is not needed.

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4.0 Broad mite counts C 3.5 Damage rate BC C BC 3.0 15 ABC 2.5

2.0 10

1.5 b AB Average damage rate damage Average 1.0 ab 5 AB 0.5 ab ab A a a a a tip per shoot mites of broad number Average 0.0 0 5 10 15 20 25 dpi Fig 7.1 Average damage rates (line) and number of broad mites, Polyphagotarsonemus latus (bars), per shoot tip observed for Rhododendron simsii ‘Nordlicht’ in experiment 1. Different letters in lower case (damage rates) and in upper case (broad mite counts) indicate significant differences between the time intervals. Data are means ± SE, n = 10.

In experiment 2, gene induction upon P. latus infestation was compared in six R. simsii genotypes. At the start of the experiment, no mites were present on all genotypes (Fig 7.3). During subsequent samplings, P. latus mites were found on each genotype at every time point (7, 14 and 21 dpi). Highest counts of P. latus were observed for ‘Nordlicht’ and ‘Aiko Pink’ (on average > 50 mites). On ‘Michelle Marie’ and ‘Sachsenstern’ the average mite number remained constant at 7, 14 and 21 dpi and varied between 12.1-18.6 and 10.4-13.0 mites, respectively. On ‘Mevrouw Gerard Kint’, mite numbers increased till 25.5 at 21 dpi. In contrast to these genotypes, very low numbers of P. latus mites were found at 7 dpi (1.0 ± 0.7 mites) on ‘Elien’. The mite numbers increased at 14 dpi (21.6 mites), but declined again one week later at 21 dpi (17.8 mites). However, no damage symptoms were observed on ‘Elien’. In contrast, the five other genotypes showed damage by P. latus from 7 dpi on. Damage rates increased over time and the highest damage rates were seen on ‘Nordlicht’ (Fig 7.3).

Chapter 7

Mock RsLOX RsAOS P. latus infestation

0.6 0.6

0.4 ns ns 0.4 ns CNRQs * CNRQs ns ** ns ns ns ns ns ns ns 0.2 ns * 0.2 ns

RsAOC ** RsOPR3 ns ** * 0.6 0.6 ns ns ns ** ns 0.4 ** 0.4 CNRQs * CNRQs ** * * ns 0.2 0.2 ns

* RsJMT ** ns RsICS ns ns 0.6 0.6 ns ns ns * ** ** ns ** ** 0.4 ns 0.4 CNRQs CNRQs ns

0.2 0.2

RsPAL ** RsPPO ** ns 0.6 *** * 0.6 ** *** * ***

0.4 ** ns 0.4 CNRQs ** ns CNRQs * ns 0.2 ns 0.2

0 5 10 15 20 25 0 5 10 15 20 25 Fig 7.2 Gene expression profiles (CNRQ, non-log-transformed) of jasmonic acid marker genes: RsLOX , RsAOS , RsAOC , RsOPR3 and RsJMT ; salicylic acid marker genes: RsICS and RsPAL and oxidative stress response marker gene: RsPPO , in Rhododendron simsii ‘Nordlicht’ leaves at different time points upon infestation by Polyphagotarsonemus latus or mock treatment in experiment 1. Significant differences compared to the respective mock treatments: ns = not significant; * p < 0.05; ** p < 0.01; *** p < 0.001. Data are means ± SE, n = 8.

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One week after surrounding R. simsii plants with P. latus infested H. helix plants, JA biosynthesis genes RsLOX , RsAOS and RsJMT showed significantly induced fold changes (FCs) in all genotypes except for ‘Elien’ (Fig 7.4). Fold changes were calculated from non- log-transformed data as the proportion of P. latus infestation over basal gene expression. RsAOC showed to be significantly up-regulated at 7 dpi in ‘Nordlicht’ and ‘Mevrouw Gerard Kint’ and down-regulated in ‘Aiko Pink’. However, at 14 and 21 dpi no significant up-regulation in JA biosynthesis marker genes ( RsLOX , RsAOS and RsAOC ) was observed in ‘Mevrouw Gerard Kint’ and ‘Aiko Pink’. RsJMT transcript levels remained up-regulated in ‘Nordlicht’ and ‘Aiko Pink’ at 14 and 21 dpi, while this was not the case in the other genotypes. In ‘Elien’ RsJMT was significantly up-regulated at 21 dpi, whereas the other genotypes were already significantly up-regulated at 7 dpi. The homologue of OPR3 in R. simsii , RsOPR3 , was not positively induced upon broad mite infestation. A significant down regulation was observed in ‘Elien’ (7 dpi), ‘Aiko Pink’ (21 dpi), ‘Sachsenstern’ (14 and 21 dpi) and ‘Mevrouw Gerard Kint’ (14 dpi). Expression profiles showed a significant suppression of RsPAL in most genotypes at 14 and/or 21 dpi (Fig 7.5). However, in ‘Nordlicht’ and ‘Aiko Pink’ this gene was significantly up-regulated at 7 dpi. The other marker gene for SA biosynthesis, RsICS was significantly down-regulated in ‘Elien’, ‘Michelle Marie’, ‘Aiko Pink’ and ‘Mevrouw Gerard Kint’ at 7 and/or 14 dpi. On ‘Sachsenstern’ transcript levels were significantly higher in P. latus infested samples at 21 dpi. The presence of P. latus on ‘Nordlicht’, ‘Aiko Pink’, ‘Sachsenstern’ and ‘Mevrouw Gerard Kint’ led to a significant increase of mRNA levels of RsPPO when compared to the basal expression levels at 7 dpi (Fig 7.6). In ‘Elien’ and ‘Michelle Marie’ no such effect was detected. During subsequent samplings, this gene was induced again at 21 dpi in ‘Nordlicht’ only. To monitor changes in hormone accumulation, SA, OPDA and JA levels in leaves were measured in R. simsii ‘Elien’ and ‘Nordlicht’ at 7 dpi. Polyphagotarsonemus latus did not induce the increase of OPDA, the JA precursor, in ‘Nordlicht’ or ‘Elien’ at 7 dpi (Fig 7.7). The phytohormone JA accumulated significantly in infested ‘Nordlicht’ plants at 7 dpi. The concentration almost doubled upon infestation in ‘Elien’ but was not significantly different compared to the basal expression levels at 7 dpi. In both genotypes the amount of free SA significantly increased upon infestation (Table 7.2). The amounts of free SA were significantly higher in R. simsii ‘Elien’ and ‘Nordlicht’, by 2 and 56 times, respectively. However these significant levels, there is a marked difference between the amounts of free SA in ‘Elien’ and ‘Nordlicht’. The amounts of SA were compared relatively based on area internal standard/area analyte. No concentrations were calculated as the standard curve suffered from problems with the internal standard.

Chapter 7

(a) Broad mite counts (b) Damage rate 70 70 B 3 60 3 60 b 50 50

2 b 40 2 40

B C 30 B 30 BC Average damage rate damage Average Average damage rate damage Average 1 20 1 20 ab A 10 10 a A AB Average number of broad mites per shoot tip per shoot of mites broad number Average Average number of broad mites per shoot tip per shoot of mites broad number Average

B (c) (d) 70 70

3 60 3 60

50 50 b b 2 b 40 2 b 40

30 30 B B B

Average damage rate damage Average AB Average damage rate damage Average 1 20 1 B 20 ab ab 10 A 10 A a a Average number of broad mites per shoot tip per shoot of mites broad number Average Average number of broad mites per shoot tip per shoot of mites broad number Average

(e) (f) 70 70

3 60 3 60

50 50

2 40 2 40 b b b 30 b 30 B Average damage rate damage Average Average damage rate damage Average 1 20 1 B 20 B B B B 10 10 A a A a a a Average number of broad mites per shoot tip per shoot of mites broad number Average Average number of broad mites per shoot tip per shoot of mites broad number Average 0 5 10 15 20 0 5 10 15 20

dpi dpi

Fig 7.3 Average damage rates (line) and number of broad mites, Polyphagotarsonemus latus , per shoot tip (bars) observed for Rhododendron simsii (a) ‘Nordlicht’, (b) ‘Elien’, (c) ‘Michelle Marie’, (d) ‘Aiko Pink’, (e) ‘Sachsenstern’ or (f) ‘Mevrouw Gerard Kint’ in experiment 2. Different letters in lower case (damage rates) and in upper case (broad mite counts) indicate significant differences between the time intervals. Error bars indicate SE, n = 10.

119

7 dpi 10 14 dpi 21 dpi (a) RsLOX

** ** ** ** ** ** * *

ns ns ns ns ns ns

1 *

Fold changes Fold ns ns

ns ns

0.1

10 (b) RsAOS

** ** ** ** *

ns ns ns ns ns 1 * ns

Fold changes Fold ns ns ns ns ns ns

ns

0.1

10 (c) RsAOC ** ** ** ns ns ns ns * ns ns 1 * Fold changes Fold ns ns ns ns ns ns * ns 0.1 n ie lie E Nordlicht Aiko Pink ichelle Mar M Sachsenstern rouw Gerard Kint Mev

Chapter 7

10 (d) RsOPR3

ns ns ns ns ns ns ns ns ns 1 ns ns ns Fold changes Fold ns * * * * **

0.1

10 (e) RsJMT

** ** ** ** ** ** ** ** * * ns ns ns ns ns ns ns * 1 * Fold changes Fold

ns

0.1 t k t h n n ic Elien arie ster M o Pi n k se Nordl Ai h rard Kin c ichelle a M S w Ge vrou Me

Fig 7.4 Fold changes (proportion of treatment over mock) of marker genes for jasmonic acid: (a) RsLOX , (b) RsAOS , (c) RsAOC , (d) RsOPR3 and (e) RsJMT in Rhododendron simsii ‘Nordlicht’, ‘Elien’, ‘Michelle Marie’, ‘Aiko Pink’, ‘Sachsenstern’ or ‘Mevrouw Gerard Kint’ leaves at different time points upon infestation by Polyphagotarsonemus latus (experiment 2). Significant differences compared to the respective mock treatments: ns = not significant; * p < 0.05; ** p < 0.01; *** p < 0.001.

121

10 7 dpi 14 dpi 21 dpi (a) RsICS

* ns ns ns ns 1

Fold changes Fold ns ns ns ns * * ns * ** ** * ** ns

0.1

10

(b) RsPAL

**

* ns ns * ns 1 * ns Fold changes Fold ns ns ns ** * ns * * ** ** ** *

0.1 t n e n h e ri r int Eli K rdlic nste o Ma ko Pink e lle i rard N e A e h Sachs w G Mic u vro Me

Fig 7.5 Fold changes (proportion of treatment over mock) of marker genes for salicylic acid: (a) RsICS and (b) RsPAL , in Rhododendron simsii ‘Nordlicht’, ‘Elien’, ‘Michelle Marie’, ‘Aiko Pink’, ‘Sachsenstern’ or ‘Mevrouw Gerard Kint’ leaves at different time points upon infestation by Polyphagotarsonemus latus (experiment 2). Significant differences compared to the respective mock treatments: ns = not significant; * p < 0.05; ** p < 0.01; *** p < 0.001.

Chapter 7

7 dpi 10 14 dpi 21 dpi RsPPO

** ** ** ** ** ns ns ns ns ns ns 1 *

Fold changes Fold ns ns ns ns ns ns ns

0.1

rn int icht lien rie E a Pink K rdl nste d lle M se No Aiko erar iche ach G M S ouw evr M

Fig 7.6 Fold changes (proportion of treatment over mock) of the marker gene for oxidative stress-response: RsPPO , in Rhododendron simsii ‘Nordlicht’, ‘Elien’, ‘Michelle Marie’, ‘Aiko Pink’, ‘Sachsenstern’ or ‘Mevrouw Gerard Kint’ leaves at different time points upon infestation by Polyphagotarsonemus latus (experiment 2). Significant differences compared to the respective mock treatments: ns = not significant; * p < 0.05; ** p < 0.01; *** p < 0.001. Table 7.2 The relative difference of salicylic acid content in Rhododendron simsii ‘Nordlicht’ and ‘Elien’ leaflets at 7 days post infestation with Polyphagotarsonemus latus or mock treatment (experiment 2). Relative differences are calculated after comparing area internal standard/area analyte of both treatments (n = 8).

Relative Level of Area internal Genotype Treatment difference of significance standard/area analyte SA content (p-value) ‘Nordlicht’ Mock 0.03 55.7 <0.001 P. latus infestation 1.67 ‘Elien’ Mock 0.04 2.0 <0.001 P. latus infestation 0.08

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In Mock P. latus infestation *

ns 400 300

300

ns 200

200

Concentration JA fw) (ng/g Concentration ns

Concentration OPDA fw) (ng/g OPDA Concentration 100 100

Nordlicht Elien Nordlicht Elien

Fig 7. 7 The amounts of 12 -oxo phytodienoic acid (OPDA) and jasmonic acid (JA) in Rhododendron simsii ‘Nordlicht’ and ‘Elien’ leaflets at 7 days post infestation (dpi) with Polyphagotarsonemus latus or mock treatment (experiment 2). The figure shows the average (+ SE) amounts of OPDA and JA (n = 8). Significant differences compared to the respective mock treatments are marked: ns = not significant; * p < 0.05; ** p < 0.01; *** p < 0.001. experiment 3, the phytohormone content was determined in R. simsii ‘Nordlicht’ and ‘Elien’ at four time points upon infestation. On ‘Nordlicht’ mites were observed from 6 dpi on while on ‘Elien’ this was at 8 dpi. Average mite numbers on ‘Nordlicht’ increased to 12.4 ± 4.4, at 11 dpi. At the same time point 1.4 ± 1.2 mites were counted on ‘Elien’. The infestation of R. simsii ‘Nordlicht’ had no effect on the amount of JA and its precursor, OPDA (Fig 7.8). In contrast, ‘Elien’ had a significantly higher concentration of OPDA at 6 and 8 dpi. The amount of JA was also significantly higher in P. latus infested plants compared to the control at 6 dpi. In ‘Nordlicht’ the amounts of free SA were increased by 1.6, 10.4, 6.3 and 18.7 times at 4, 6, 8 and 11 dpi, respectively (Table 7.3). But only at 6 and 11 dpi these differences were statistically significant. In contrast to ‘Nordlicht’, in ‘Elien’ the amount of free SA remained relatively constant. Over all time points the increase of the amount free SA varied between 1-1.6 times upon P. latus infestation and were not statistically different.

Chapter 7

Mock (a) (b) P. latus infestation 700 ns ns 600 ns * ** 500 ns ns ns 400

300

Concentration OPDA (ng/gConcentration fw) 200

100

(c) ns (d) ns 200 **

150 ns

ns ns 100 ns ns Concentration JAConcentration (ng/g fw)

50

4 6 8 10 12 4 6 8 10 12 dpi dpi

Fig 7.8 The amounts of 12-oxophytodienoic acid (OPDA) and jasmonic acid (JA) in Rhododendron simsii (a, c) ‘Nordlicht’ and (b, d) ‘Elien’, mock or Polyphagotarsonemus latus infested leaflets during a time course of 11 days in experiment 3. The figure shows the average (+ SE) amounts of (a, b) OPDA and (c, d) JA at 4, 6, 8 and 11 days post infestation (dpi) or mock treatment (n = 5). Significant differences compared to the respective mock treatments are marked: ns = not significant; * p < 0.05; ** p < 0.01; *** p < 0.001.

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Table 7.3 The relative difference of salicylic acid content in Rhododendron simsii ‘Nordlicht’ and ‘Elien’ leaflets at 4, 6, 8 and 11 days post infestation (dpi) with Polyphagotarsonemus latus or mock treatment (experiment 3). Relative differences are calculated after comparing area internal standard/area analyte of both treatments (n = 5).

Area internal Relative Level of Time Genotype Treatment standard/area difference of significance point analyte SA content (p-value) ‘Nordlicht’ 4 dpi Mock 0.11 1.6 0.09 P. latus infestation 0.16 6 dpi Mock 0.08 10.4 0.01 P. latus infestation 0.83 8 dpi Mock 0.10 6.3 0.09 P. latus infestation 0.60 11 dpi Mock 0.12 18.7 0.01 P. latus infestation 2.17 ‘Elien’ 4 dpi Mock 0.10 1.48 0.19 P. latus infestation 0.15 6 dpi Mock 0.16 1.21 0.69 P. latus infestation 0.19 8 dpi Mock 0.15 0.99 1.00 P. latus infestation 0.14 11 dp i Mock 0.13 1.62 0.31 P. latus infestation 0.22

In experiment 4, a bio-assay was used to study the effect of MeJA and SA treatment on P. latus population growth. Methyl jasmonate applied at a concentration of 50 or 100 µM resulted in a significant reduction of P. latus population growth in ‘Nordlicht’ when compared to a mock treatment (Fig 7.9). Application of SA at a concentration of 50 or 100 µM SA had no significant effect on P. latus population growth when compared to the mock treatment.

Chapter 7

mock 50 µM MeJA 0.12 100 µM MeJA 50 µM SA 100 µM SA ns

0.10

0.08

0.06 ns *

0.04 ** Average rate of population increase ofrate population Average

0.02

Fig 7.9 Average rate of population increase (+ SE) of Polyphagotarsonemus latus on Rhododendron simsii ‘Nordlicht’ leaf discs when floated on 50 or 100 µM methyl jasmonate (MeJA), 50 or 100 µM salicylic acid (SA) or mock solution for one week in experiment 4. Significant differences compared to the mock treatment: ns = not significant; * p < 0.05 and ** p < 0.01.

4. Discussion Gene expression analysis on JA biosynthesis marker genes showed an increase of RsLOX , RsAOS , RsAOC and RsJMT transcript levels upon P. latus infestation in R. simsii . Grinberg et al. (2005) showed the induction of LOX1 and LOX2 genes in P. latus infested leaves of cucumber ( Cucumis sativus ). In tomato ( Solanum lycopersicum ), P. latus infestation strongly induced the expression of protease inhibitors PI-I and PI-II in a def-1 mutant (impaired in JA biosynthesis) and its wild type (Grinberg-Yaari et al., 2015). We were not able to clone protease inhibitors from R. simsii . The protease inhibitors are marker genes for JA-mediated plant defence and are induced by JA, wounding and herbivory, as was the case for the two-spotted spider mite (Li et al., 2004; Ament et al., 2004, 2006; Kant, 2006). They cause retardation of insect growth and development. Expression of these protease inhibitors in def-1 mutants would not be expected but is in agreement with previous reports. The def-1 mutation only delays JA biosynthesis, and partial induction of the pathway still occurs (Howe et al., 1996; Ament et al., 2004). Our study showed the involvement of RsJMT in all R. simsii genotypes in response to P. latus infestation. However, mechanical wounding (Chapter 6) resulted in no significant induction 6 hours upon treatment in all of the genotypes. This might suggest that other signals than mechanical wounding, presumably the insertion of the mite’s mouth parts, mediate the response. Alternatively, the wounding treatment may have been milder than the

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mechanical damage caused by P. latus mites. This hypothesis was also put forward by Grinberg et al. (2005). RsOPR3 , expected to be active in the peroxisome, was not involved in the defence response upon P. latus infestation. This is in contrast to the observed response after MeJA treatment or wounding (chapter 6). In accordance to the induction of JA marker genes upon P. latus infestation, the amount of JA increased in the mite susceptible genotype ‘Nordlicht’. In ‘Elien’, the amount of JA increased significantly upon infestation in experiment 3, but not in experiment 2. We suggest that timing of sampling is an essential aspect in the measurement of hormonal changes. The infestation rate at the time of sampling also plays a role in the obeserved responses. Probably sampling on ‘Elien’ at 7 dpi in experiment 2, was too early since the RsLOX gene was significantly up-regulated only at 14 dpi. In other plant species such as tomato and Arabidopsis spider mite infestation caused an increase in the amount of JA as well (Zhurov et al., 2014; Alba et al., 2015). Agut et al. (2014) found that spider mite attack induced strong changes in the JA biosynthesis pathway in the resistant cultivar, sour orange ( Citrus aurantium ), showing higher levels of OPDA and JA. Herbivore-induced plant volatiles released from sour orange plants following T. urticae infestation induced resistance in susceptible rootstock Cleopatra mandarin ( Citrus reshni ), thereby inducing LOX2 gene expression and increasing OPDA content (Agut et al., 2015). In the present study a significant increase of OPDA was observed at 6 and 8 dpi in the mite resistant genotype ‘Elien’ (experiment 3). We hypothesize again that timing of sampling was not optimal to detect significant differences of the OPDA metabolite in ‘Elien’ in experiment 2. Correspondingly, phytohormone analysis of JA precursor OPDA in the mite susceptible genotype ‘Nordlicht’ showed no different metabolite concentration upon infestation. This might explain why the RsOPR3 gene, which plays a role in the conversion of OPDA, was not induced by P. latus infestation. Spider mite infestation on A. thaliana ecotypes Bla-2, Col-O and Kon had no effect on OPDA concentration (Zhurov et al., 2014). Significantly higher amounts of OPDA were found in tomato at 7 dpi by spider mite strains DeLier-1 (Tetranychus urticae ) and Santpoort-2 ( T. urticae ), a putative suppressor and a non- suppressor strain of plant defence, respectively. Two putative suppressor strains of T. evansi , Viçosa-1 and Algarrobo-1 had no effect on OPDA concentration in tomato (Alba et al., 2015). The amounts of OPDA and JA in R. simsii are somewhat higher when compared to tomato (Alba et al., 2015). The concentration of JA in R. simsii is approximately the same as in A. thaliana ecotypes Bla-2 and Kon. Hormone levels of JA are higher in ecotype Col- 0 when compared to R. simsii (Zhurov et al., 2014). In rose, another ornamental plant the concentration of JA in leaves was somewhat higher when compared to R. simsii and averaged 170 ng/g fresh weight (Bosco et al., 2014). Besides timing of sampling, another explantion for hormonal differences between experiments 2 and 3 might be found in the infestation pressure. The infestation pressure was much higher on both ‘Nordlicht’ and ‘Elien’ during experiment 2, e.g. on ‘Nordlicht’ mite numbers were 2 to 3 times higher in experiment 2 compared to experiment 3.

Polyphagotarsonemus latus infestation activates JA-dependent pathways in R. simsii hybrids, most strongly at the earlier time points (5-12 dpi). At later time points it is clear

Chapter 7 that the SA biosynthesis pathway led by either RsPAL or RsICS was suppressed in response to P. latus infestation in R. simsii. Remarkably, the amount of free SA increased upon infestation in the mite susceptible genotype ‘Nordlicht’. The content of free SA was only slightly higher in the mite resistant genotype ‘Elien’ upon mite infestation. It might be that P. latus suppresses SA marker genes to manipulate plant defences. This phenomenon is well known from plant pathogens (e.g. Abramovitch et al., 2006; Kamoun, 2006; De Jonge et al., 2011) and nematodes (Haegeman et al., 2012). But also some strains of the spider mites Tetranychus urticae and Tetranychus evansi were found to suppress expression of JA and/or SA marker genes (Kant et al., 2008; Sarmento et al., 2011b; Alba et al., 2015). Alternatively, SA suppression could be the effect of an antagonistic crosstalk through a strong JA induction (Pieterse et al., 2012). Grinberg et al. (2005) found transcripts of BGL2 , encoding a PR protein (PR2, basic β-1,3-glucanase), to be expressed in cucumber upon infestation by P. latus . This protein is generally accepted to be a marker gene for the SA pathway. For other arthropods, a simultaneous induction of several pathways is reported. Moran and Thopmson (2001) found higher transcript levels of JA and SA marker genes upon aphid ( Myzus persicae ) infestation in A. thaliana . In tomato, a role for both JA and SA biosynthetic pathways regulating response to two spotted spider mite was implicated (Ament et al., 2004, 2006; Li et al., 2004). This is also the case in citrus (Agut et al., 2014). Gene expression analysis upon spider mite infestation showed the involvement of genes related to JA, SA and ethylene in tomato (Kant et al., 2004). An explanation for the simultaneous induction of JA and SA responses upon infestation is given by Alba et al. (2015). First, responses might appear heterogeneous in space. By harvesting samples from different leaves JA and/or SA responses are mixed. Alternatively it might be that simultaneous JA and SA responses actually do antagonize each other and what is observed are intermediate responses.

From the findings in the present study it is clear that MeJA had a negative effect on the fitness of P. latus in R. simsii ‘Nordlicht’ when compared to a mock treatment (experiment 4). This agrees with reports that spider mite proliferation was decreased by MeJA application (Omer et al., 2001; Warabieda et al., 2005; Rohwer and Erwin, 2010; Warabieda and Olszak, 2012; Agut et al., 2014). Grinberg-Yaari et al. (2015) demonstrated that free-moving mites significantly preferred tomato def-1 mutant over its wild type, ‘Castlemart’. Phoretic mites were able to discriminate between isogenic varieties that differed in JA-mediated defence. This was concluded from higher detachment percentages on def-1 mutants compared to ‘Castlemart’. The application of JA on def-1 mutants reduced the detachment rate to a level comparable to that of the wild type and thus restored the defence response in def-1 plants. Furthermore, the number of progeny per female was significantly higher on def-1 mutants. Pre-treatment of def-1 mutant plants with JA significantly affected progeny development on those plants, which was similar to that on ‘Castlemart’ plants (Grinberg-Yaari et al., 2015). The exogenous application of MeJA had about the same effect on the spider mite T. urticae infesting tomato (Li et al., 2002). In our study, the effect on the population increase of P. latus likely depended on the concentration applied as the rate of population increase was reduced by 3.2 and 2.3 times

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for 0.05 and 0.1 mM MeJA, respectively. These results suggest that the defence response in R. simsii mediated by JA may be suboptimal at a concentration of 0.1 mM as compared to the 0.05 mM concentration. A similar effect was reported before by Choh et al. (2004), who showed that the number of eggs produced by T. urticae after JA application on lima beans ( Phaseolus lunatus ) differed as a function of the concentration applied. Unlike MeJA, the application of SA to leaf disks of ‘Nordlicht’ resulted in no significantly different rate of population increase. There was only a small tendency of reduction when comparing the 0.05 mM SA to the mock treatment. These results are comparable to those of an SA treatment in Cleopatra mandarin and sour orange. Salicylic acid had no effect on the oviposition of T. urticae, whereas MeJA treatment only significantly reduced the number of eggs produced by the spider mite in the T. urticae -sensitive Cleopatra mandarin when compared to a control treatment (Agut et al., 2014). When working with leaf discs mechanical damage might induce defence responses in the plant that interfere with mite proliferation. However, no adverse effects on the rate of population increase of the mite were observed in the mock treatment. Furthermore, leaf discs of all treatments (mock, SA and MeJA) were exposed to the same mechanical damage. Thus, we believe that any effect of mechanical damage caused from preparing the leaf discs can be dismissed. Our study on R. simsii provides further evidence for a role of JA in induced plant defence responses upon P. latus infestation.

Browning of the youngest leaves, the first symptoms of P. latus infestation observed on R. simsii , might be linked to enhanced transcript levels of RsPPO . This enzyme is involved in the production of o-quinones and o-diphenols (Steffens et al., 1994). It is responsible for the typical browning of plant extracts and damaged tissues caused by spontaneous polymerization and crosslinking of the o-quinones (Constabel et al., 2000). Furthermore, a role of foliar PPO in the defence against leaf-eating insects has been proposed and documented (Felton et al., 1989; Duffey and Felton, 1991). Polyphagotarsonemus latus infestation caused no visible symptoms and thus no browning on leaves of ‘Elien’ during experiment 2. Concordantly, gene expression analysis showed no increase of RsPPO transcripts. In contrast, damage rates differing from zero were assigned to all other genotypes. Likewise, RsPPO was induced in these genotypes except for ‘Michelle Marie’. This provides evidence for the involvement of an oxidative stress response upon infestation by P. latus in susceptible R. simsii genotypes. In tomato it was proposed that P. latus infestation might activate an oxidative stress response (Grinberg et al., 2005). Polyphenol oxidases were induced in tomato leaves by different spider mite strains (Alba et al., 2015).

In conclusion, we demonstrated that in R. simsii genotypes JA responses were induced upon infestation, most strongly at the earlier time points (5-12 dpi) and that MeJA has a negative effect on the fitness of P. latus . Gene expression analysis also showed the suppression of SA biosynthesis at later time points. Further studies are needed to unravel the effects of both pathways on the fitness of P. latus and to deepen our understanding of the role of both pathways in plant defence.

Chapter 8

Conclusion, general discussion and further prespectives

In our research several key aspects of the bio-ecology of the broad mite Polyphagotarsonemus latus and its interactions with Rhododendron simsii were investigated. Thermal thresholds and the thermal constant for development were estimated for this pest. Detailed knowledge on the cold hardiness of P. latus was presented for the first time. The susceptibility for P. latus infestation of 30 R. simsii accessions was characterized. Trichomes producing sticky exudates were observed on the mite resistant genotypes. We provided molecular knowledge for an ornamental crop of local importance, R. simsii , on its plant-herbivore interactions with a focus on the broad mite P. latus . The phytohormone jasmonic acid (JA) was induced in R. simsii upon the infestation by P. latus . The levels of salicylic acid (SA) were significantly higher upon infestation with P. latus in the mite susceptible genotype ‘Nordlicht’ but not in the mite resistant genotype ‘Elien’. The generated knowledge of this work may be useful for the development of an Integrated Pest Management (IPM) strategy against P. latus . It entails the manipulation of plant-herbivore interactions with the goal of regulating populations of the herbivore at levels below those at which economic losses occur (Stout and Davies, 2009). According to the literature, eight general principles for IPM can be identified (Malavolta et al., 2005; Joas and Cotillon, 2009). In our research we contributed directly or indirectly to five of these principles, i.e. “measures for prevention and/or suppression of harmful organisms”, “tools for monitoring”, “threshold values as a basis for decision-making”, “preference for non-chemical control methods”, and “application of pesticide anti-resistance strategies”

Temperatures and Polyphagotarsonemus latus

Temperature conditions for the production of R. simsii depend on the growth phase of the plant. Vegetative cuttings are rooted under a plastic cover for 8-10 weeks at high relative humidity and a temperature of 23-25 °C. During the vegetative growth phase plants are grown either outside on a container field or in a greenhouse. Besides protecting the plants from freezing, the temperature is not controlled in this phase. At the end of the production process flowering is induced by breaking the bud dormancy with a cold period at temperatures generally between 2.5 and 9 °C. Finally, plants are forced to flower in heated greenhouses (20-21°C). Being an ectothermic organism, the development of P. latus is temperature- dependent. Thermal development thresholds and thermal constants were estimated for each life stage of P. latus . The lower and upper development thresholds of adult mites were estimated to be around 10 and 36 °C, respectively. Development of the different life stages of the pest in the greenhouse or outside on container fields is thus determined by

environmental temperatures. These temperatures can easily be monitored. Calculations on the long-term average temperature data for Merelbeke (Belgium) (51°0’0”N/3°45’0”E) indicate that during warmer months (April-September) 14.7 theoretical generations could be expected. Peak populations of the pest are expected in mid-summer since for July and August each month 3.7 successive generations are expected to develop. Given the fact that these are theoretical calculations based on average temperatures and a model estimated thermal constant of P. latus , additional fundamental knowledge (e.g. on the role of relative humidity) may facilitate the construction of process-based models of P. latus development and phenology in ornamental plant production and the development of early warning systems. A warning system using temperature as the main predictor may assist growers in controlling the pest. The system might provide essential information for timing the scouting. Scouting reports help growers to keep the mite populations below economically damaging thresholds by choosing an appropriate management technique (e.g. the use of biological control agents, spray application of a selective pesticide). Validation of these warning systems is essential. To optimize any system it should be compared with field observations over multiple years. Validation of the warning system for the Phomopsis viticola disease in grape ( Vitis spp.) confirmed that it can provide a similar degree of control as calendar-based fungicide applications but the former resulted in less sprays (Nita et al., 2006). An excessive number of applications needlessly increases growers costs and is detrimental for the environment. Cost saving can be seen in terms of reduced pesticide applications, as well as labour and equipment costs. Even without cost saving a warning system might provide essential information to define critical periods in plant production. Tarsonemid mites are generally believed to overwinter in the adult stage (e.g. Smith and Goldsmith, 1936; Wiesmann, 1941; Jeppson et al., 1975; Denmark, 1998). We were the first to provide detailed information on the cold hardiness of a tarsonemid mite. Survival of adult P. latus mites was dependent on the temperature and the duration of the cold exposure period i.e. median lethal times were 61.2 hours and 9.3 days at -3 and 2 °C, respectively. In addition, it was shown that survival of immature life stages such as eggs and larvae significantly decreased upon an increased cold exposure period at 7 °C. Adult females were more robust, suffering limited detrimental effects on the survival capacity from a 7 °C cold exposure up to 6 weeks. Practical trials at the PCS Ornamental Plant Research (personal communication) confirmed our findings, showing that mites were able to survive a cold storage period on R. simsii but survival depended on the temperature and the duration of the cold exposure. Mite populations were significantly decreased by 71, 83 and 90% upon cold storage of complete plants at 3 °C for 2, 3 or 4 weeks, respectively. Cold treatments of 2 to 3 weeks led to an increase of the population two weeks after being returned to optimal conditions for mite development. This indicated that mites still were able to oviposit after a prolonged cold exposure. But two weeks after returning the infested plants from a 4-week cold treatment to the greenhouse, the initial mite population was reduced by 93% (PCS Ornamental Plant Research, personal communication). Here a serious effect on the oviposition rate is assumed, which is in accordance with our laboratory findings.

Chapter 8

Besides the essential biological knowledge provided in our study, directly applicable information for R. simsii growers was obtained. Cold storage or dormancy breaking of R. simsii plants at temperatures in the range of 2-3 °C has the advantage to kill the mites or at least significantly reduce the oviposition rate of surviving P. latus mites. In that way risks on damaged flowers and consequent economic losses for the growers are reduced when plants are placed in heated greenhouses to force flowering after a cold period in the fridge. Today, augmentation biological control is applied on a commercial scale in relatively few agricultural systems (van Lenteren, 2012). Timing of releases is often critical for successful biological control. Our data help to identify optimal periods for augmentative releases of beneficials to control P. latus . The pest is proven to be a prey for a number of commercially available phytoseiid mites, including Amblyseius swirskii (van Maanen et al., 2010; Onzo et al., 2012: Abou-Awad et al., 2014), A. largoensis (Rodríguez et al., 2011), Neoseiulus cucumeris (Li et al., 2003; Weintraub et al., 2003), N. californicus (Peña and Osborne, 1996; Jovicich et al., 2008), and N. barkeri (Fan and Petit, 1994). Besides predatory mites, entomopathogenic fungi controlling the pest were reported (Cabrera et al., 1987; Nugroho and Ibrahim, 2004; Maketon et al., 2008; Mikunthan and Manjunatha, 2010; Alves et al., 2010). We suggest it would not be necessary to continuously re-release augmentative biological control agents or to provide supplemental foods supporting the predators throughout the complete R. simsii production cycle. When temperatures during production are above 10 °C the use of predatory mites can be useful. First, we showed that temperatures above this lower developmental threshold of P. latus result in development of the pest. At lower temperatures the population increase of the pest is impeded as the mites do not oviposit. Second, most of the predators of P. latus originate from the same geographic region and they consequently have similar developmental thresholds. Therefore it is better to release new predatory mites on R. simsii plants during the warmer phases of plant production that follow after a cold period. The predatory mites are mostly not active at colder temperatures. A cold exposure of the predatory mites might hamper the control of P. latus when plants containing these mites are returned at optimal temperatures for development (e.g. reduced survival or lowered oviposition rate of the predatory mites). The latter is, however, not always the case: Ghazy et al. (2012) concluded from laboratory experiments that the population growth of N. californicus was not affected after more than 20 days at 10 °C and 0.1 kPa (ca. 92% RH). By selecting optimal periods for the augmentative release of beneficials we tackle one of the main reasons for the relatively low adoption of this pest management strategy (van Lenteren, 2012); augmentative releases are usually more expensive to growers than chemical pesticides (Collier and Van Steenwyck, 2004). In further research it would be necessary to focus on life table parameters such as the intrinsic rate of increase (r m). The r m is defined as the rate of increase per head under specified physical conditions, in an unlimited environment where the effects of increasing density do not need to be considered (Birch, 1948). Although values for this parameter have been estimated in different crops for P. latus, they might not reflect the populations

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growth rates in R. simsii . Indeed, between Capsicum annuum , Citrus aurantifolia and Vitis vinifera corresponding life table parameters markedly differed at similar temperatures (Hugon, 1983; Silva et al., 1998; Ferreira et al., 2006). Obtaining values for this parameter on R. simsii genotypes that respond in a different way on the presence of P. latus optimize the development of prediction models. Furthermore it might be warranted to determine the economic thresholds for P. latus on pot azalea. This may, however, be a challenge as on ornamental plants no cosmetic damage is accepted by the consumer and consequently threshold limits would likely be close to zero.

Plant traits for the management of Polyphagotarsonemus latus

Different types of plant responses were observed in R. simsii after infestation by P. latus . The majority of the screened genotypes showed severe damage symptoms upon P. latus infestation. The mite caused browning and curling of the youngest growth tips at an early stage. Subsequently, older leaves and flowers were attacked by the pest resulting in severe economic damage to the ornamental plant. Rhododendron simsii genotypes ‘Elien’ and ‘Mistral’ were most resistant to the pest. These resistant characteristics were confirmed throughout multiple experiments in this thesis. Host plant resistance to P. latus was discovered in many non-ornamental crops such as Capsicum annuum (Ahmed et al., 2001; Desai et al., 2006; Desai et al., 2007; Kulkarni et al., 2011), potato species Solanum polyadenium , S. tarijense and S. berthaultii (Gibson and Valencia, 1978), eggplant ( Solanum melongena ) (Lianyou et al., 1999), watermelon ( Cirtullus lanatus var. lanatus ) (Kousik et al., 2007) and jute ( Corchorus spp.) (Jana et al., 2007). Several chemical constituent have been found to have a negative or positive correlation with mite numbers (Ahmed et al., 2000; Qing et al., 2010). Our research showed the presence of trichomes with a sticky end on mite resistant genotypes ‘Mistral’ and ‘Elien’. The results suggest that defence against P. latus might be regulated via antixenosis in R. simsii . Crop resistance to the pest was found in S. polyadenium and in seedlings of S. tarijense and S. berthaultii , which also had foliar hairs with a sticky tip (Gibson and Valencia, 1978). Trichomes can contribute to plant defence in different ways. Non-glandular trichomes can physically obstruct the movement of herbivorous arthropods over the plant surface or prevent herbivores to reach the surface with their mouthparts (Cardoso, 2008; Pott et al., 2012). Moreover, arthropods may become entrapped in sticky and/or toxic exudates produced by glandular trichomes (Glas et al., 2012). The entrapped herbivores usually die as a result of starvation or of ingested toxins (Simmons et al., 2004). Trichomes acted as a limiting trait for the development of P. latus , especially when foliar hairs are present in high densities as was seen in Capsicum (Thungrabeab and Boonlertnirun, 2002; Matos et al., 2009). As reviewed by Glas et al. (2012) it would be possible to develop a strong grip on the protection of crops against herbivores via breeding for/or genetic engineering of trichomes. They can be seen easily with a binocular and therefore trichomes are an excellent characteristic for breeding. They allow a fast selection of the desired plant traits after crossing since they are already present during the vegetative growth phase. Screening of additional sources of R. simsii

Chapter 8 plants might reveal more mite resistant genotypes usable for breeding. In ornamentals a disadvantage of trichomes is that they produce sticky exudates, an unwanted characteristic by the market. This makes it less attractive to breed plants with high densities of glandular trichomes. In other crops such as tomato or peppers this strategy would be helpful for controlling P. latus . However, here the incompatibility of such physical plant resistance traits with other IPM tactics such as augmentative biological control should be taken in account. In future research metabolite profiling analyses would definitely provide more insights into factors causing enhanced resistance and/or susceptibility for P. latus . Comparing the metabolite profiles of susceptible and resistant genotypes after feeding by P. latus is one way to identify causal factors. Botanga et al. (2012) demonstrated via metabolite profiling that ascorbate is an important component of resistance to the pathogenic ascomycete Alternaria brassicola in Arabidopsis thaliana . Metabolite profiling in maize (Zea mays ) provided insights in the interactions between the plant and the generalist herbivore Spodoptera littoralis (Marti et al., 2014). Alternatively, metabolite profiling of the pest organism after feeding on susceptible and resistant plants also helps revealing plant-pest interactions. This was done for the foxglove aphid ( Aulacorthum solani ) feeding on soybean ( Glycine max ) (Sato et al., 2014). In our study we used RNAseq experiments to reveal transcriptomic differences between a mite infested susceptible and mite resistant R. simsii genotype and their respective uninfested controls.

Molecular insights in the interaction between Polyphagotarsonemus latus and Rhododendron simsii

We assembled the first transcriptome of R. simsii that contained ca. 15k orthologs from unique Arabidopsis thaliana genes. This is about half of the genes described in the model plant A. thaliana . Transcriptomic differences were obtained from a preliminary RNAseq experiment comparing P. latus infestation in R. simsii ‘Elien’ and ‘Nordlicht’ to their respective uninfested controls. Gene onthology (GO) enrichment showed an overrepresentation of JA induced biological processes upon infestation by P. latus . Our results are in accordance with Grinberg et al. (2005) and Grinberg-Yaari et al. (2015) who showed a role for jasmonic acid signalling in defence against this pest in cucumber and tomato. Next-generation RNA-sequencing provided, besides essential insights into the regulation of plant defence, molecular sequence data in a minor crop, R. simsii . As we lacked biological replicates in RNAseq experiments these data needed to be confirmed. The role of phytohormone pathways of JA and SA upon P. latus infestation was assessed after gene expression analysis and phytohormone measurements. To guarantee accurate quantification of gene expression in R. simsii we followed the protocol by De Keyser et al. (2013) assuring correct handling according to the MIQE-guidelines (Bustin et al., 2009). RNA extraction from recalcitrant plant tissues has led to the development of multiple CTAB-based extraction methods in woody ornamentals (e.g. Zeng and Yang, 2002; Liao et al., 2004; Gambino et al., 2008; Ghangal et al., 2009; Rubio-Piña and Zapata-Pérez, 2011; Morante-Carriel et al., 2014), therefore we optimized a CTAB extraction protocol of RNA

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from leaves of R. simsii . Quality control measures and selection of multiple stable expressed reference genes was included as recommended by Vandesompele et al. (2002) and Hellemans et al. (2007). Since we worked in a non-model plant, homologous genes described in literature had to be cloned and their function needed to be validated. Methyl jasmonate (MeJA) application and wounding of leaves was used to assess the role of putative marker genes. Foliar application of MeJA resulted in a significant up-regulation of JA biosynthesis marker genes RsLOX , RsAOS , RsAOC , RsOPR3 and RsJMT . Our results were in accordance with findings in other plant species, mostly model the plant Arabidopsis thaliana (Sasaki et al., 2001; Jung et al., 2007). Wounding of leaves resulted in generally the same response when compared to the external application of MeJA. However, no significant induction of the JA biosynthesis marker gene RsJMT was measured. Gene expression analysis of marker genes for the opposing defence pathway regulated by SA showed a significant induction of the phenylalanine ammonia-lyase pathway ( RsPAL ) but not the isochorismate pathway (RsICS ). The oxidative stress-tolerance gene, RsPPO , had the highest fold changes upon wounding or MeJA treatment in R. simsii ‘Elien’. However, basal expression levels for RsPPO were very lown in ‘Elien’ compared to the other genotypes. Our results provide insights into the molecular mechanisms behind the interaction between P. latus and R. simsii . Transcript levels of JA biosynthesis marker genes RsLOX , RsAOS , RsAOC and RsJMT were significantly induced upon P. latus infestation in R. simsii . This was in agreement with Grinberg et al. (2005) and Grinberg-Yaari et al. (2015) who showed LOX1 and LOX2 genes were induced in cucumber while other JA-related marker genes were induced in tomato. In accordance with gene expression, the amount of jasmonic acid increased upon infestation. As in ‘Nordlicht’ no significant changes were measured for OPDA concentration, this might explain why the RsOPR3 gene, which plays a role in the conversion of OPDA, was not induced upon infestation. The expression of SA marker genes upon infestation was not clear but the amounts of free SA increased significantly in the mite susceptible genotype ‘Nordlicht’ while this was not the case in the mite resistant genotype ‘Elien’. It was shown that MeJA application significantly decreased the population growth of P. latus on R. simsii ‘Nordlicht’. This suggests an antibiosis defence mechanism against P. latus in R. simsii . Salicylic acid had no significant effect on the rate of population increase. As there was no direct effect on P. latus , SA still might be involved in indirect plant defence. In literature it is shown that SA might be involved in herbivore induced plant volatiles that produce a repellent for arthropods (Dicke, 1986; Bernasconi et al., 1998) whereas other studies have demonstrated the opposite effect (Bolter et al., 1997; Pallini et al., 1997;Halitschke et al., 2008). The application of JA or MeJA successfully reduced T. urticae proliferation on different plant species (Omer et al., 2001; Li et al., 2002; Choy et al., 2004; Warabieda et al., 2005; Rhower and Erwin, 2010; Warabieda and Olszak, 2012; Agut et al., 2014). In Fig 8.1 a schematic drawing is given of the effect of P. latus infestation on biosynthesis genes in the JA and SA pathway in R. simsii . The RsPPO gene was significantly induced in the mite susceptible R. simsii genotypes. This gene is responsible for the typical browning of plant extracts and damaged tissues caused by spontaneous polymerization and crosslinking of o-quinones (Constabel et al., 2000).

Chapter 8

The first symptoms of P. latus infestation observed on susceptible R. simsii genotypes can be explained after this gene. In tomato it was proposed that P. latus infestation might activate an oxidative stress response (Grinberg et al., 2005). Polyphenol oxidases were induced in tomato leaves by different spider mite strains (Alba et al., 2015).

Further research is needed to unravel the exact role of the JA and SA defence pathway upon P. latus infestation and to study the antibiosis defence mechanism. It would be interesting to compare the responses of P. latus feeding on ‘Elien’ upon MeJA or SA treatment. When the plant defence response is fully understood in the laboratory, evaluating these effects in field trials is a next step. For instance, thrips and aphid populations were reduced in field plots of tomatoes treated with JA (Thaler et al., 2001). When studying the defence responses upon application of elicitors, attention should also be given to possible undesired effects of their application on non-target organisms and on plant physiology and morphological responses. Morphological responses are observed in flowering, including anther development and dehiscence, female organ development, chemical defence production, colour production, and the attraction of pollinators (Wasternack, 2006). Jasmonates (JA) have also been shown to enhance leaf senescence in barley (Herrmann et al., 1992) and flower senescence in petunia and dendrobium (Porat et al., 1993). In contrast, in grapevines JA application did not induce senescence (Omer et al., 2000). Constitutive or chemical defences may be inducible in plants. Inducible chemical defences include a wide variety of compounds that are toxic, anti-nutritive, or injurious to attacking organisms, including alkaloids, phenolic compounds, chitinases, and protease inhibitors (Rohwer and Erwin, 2008). A well-known structural defence induced by JA application is the increase in trichome number and density (Traw and Bergelson, 2003; Boughton et al., 2005). Some researchers have noted undesirable effects on non-target organisms, including predatory or parasitic arthropods, following JA- induced plant defences (Rohwer and Erwin, 2008). Malone and Burgess (2000) proposed the accumulation of toxic compounds in the herbivorous prey or host as a mechanism for these effects. The platform to measure transcript level changes in R. simsii upon P. latus infestation, can be useful to study the molecular interactions of other pests and diseases occurring on this ornamental plant. The main two fungal diseases during R. simsii production are Phytophthora citricola and Cylindrocladium spp . (Backhaus, 1994a, 1994b). Among the pests occurring on R. simsii , thrips and aphids cause serious damage to leaves and flowers. In future research it would also be interesting to transfer the insights on P. latus infestation to other crops, thus unveiling the defence mechanisms in these crops. Measuring the effect on gene expression using the developed tools in R. simsii can be an advantage to screen for the mode of application of new or existing elicitors that induce plant defence upon attack by a pest or a disease. This molecular tool can be useful in the screening for genetic variation in crops in terms of gene expression or the accumulation of phytohormones such as JA and SA upon infestation.

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Fig 8.1 Schematic drawing of the effect of Polyhagotarsonemus latus infestation on biosynthesis genes in the jasmonic acid (JA) and salicylic acid (SA) pathway in Rhododendron simsii . The effect of wounding and methyl jasmonate (MeJA) or coronatine (COR) 6 hours after treatment is also shown. At early time points after infestation, 5-12 days post infestation (dpi), JA biosynthesis genes are induced while no induction is oberserved for SA related biosynthesis genes. At later time points after infestation (over 12 dpi), JA biosynthesis genes are not induced but SA biosynthesis genes a suppressed. Arrows showing upwards indicate gene upregulation. Arrows showing downwards indicate gene suppression. Squared boxes indicate no induction of gene expression.

Chapter 8

Wounding

MeJA or COR

P. latus

Between 5-12 dpi Over 12 dpi

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Summary

The broad mite Polyphagotarsonemus latus (Banks) (Acari: Tarsonemidae) is an economically important pest in greenhouses and field crops worldwide. In Belgium it is a key pest in pot azalea production ( Rhododendron simsii Planch hybrids). Damage symptoms are seen as malformation of the youngest growth tips and flowers, and down- curling of leaves. Limited availability of chemicals and recent restrictions on the use of broad spectrum chemicals necessitate the use of Integrated Pest Management (IPM) strategies to control this pest. This comprises a multidisciplinary approach to lower risks of infestation or to reduce the pest population levels in the crop. Measures include the breeding and growing of tolerant or resistant genotypes. When the pest is reported in the crop it is recommended to combine different strategies which might include chemical but also biological measures. Nowadays, different phytoseiid mites feeding on P. latus, such as Amblyseius swirskii, are commercially available. An alternative approach to control mite pests might involve inducing plant defences to reduce mite vigour and/or mite preference for a crop. The availability of basic biological information on the pest could considerably improve the efficacy of several of these control measures, which should result in a better control of P. latus in ornamental crops like R. simsii . The overall objective of this study was to provide a better understanding of the bio-ecology of the broad mite P. latus as one of the most important pests in R. simsii hybrid production in Belgium, and to investigate the role of defence responses in this ornamental plant against the pest. In Chapter 3 , the effect of temperature on the development of P. latus on R. simsii leaves was studied. In combination with a photoperiod of 16:8 h (L:D) and a relative humidity of 80 ± 5%, six constant temperatures (15, 17, 20, 25, 30 and 33 ± 1 °C), were studied. Total development times of 13.3, 10.5, 6.6, 4.2, 3.5 and 4.0 days were measured at the respective temperatures. Development of females took significantly longer than that of males at 15, 17, 20 and 30 °C. Survival rates observed between 17 and 30 °C varied between 43.5 and 96.9%. Lower survival rates were found at 15 and 33 °C, i.e. 31.8 and 23.6%, respectively. The lower, optimal and upper developmental threshold ( tmin , topt and tmax , respectively) and thermal constant ( K) of the pest were estimated for each life stage by a linear and two non-linear models. Based on measurements of total development of P. latus thermal thresholds of 10.0, 30.1 and 36.0 °C were calculated for tmin , topt and tmax , respectively. The number of degree-days needed to complete immature development when feeding on R. simsii was 66.7. The exposure of R. simsii plants to temperatures generally between 2.5 and 9 °C is necessary to release flowers from their dormant state as unfulfilled chilling requirements lead to erratic bud break and lower quality. The optimum temperature for dormancy breaking in R. simsii is 7 °C. In Chapter 4 , survival of eggs, larvae, and female adults and reproduction capacity of female P. latus were evaluated following cold exposure at 7 °C. Adult females were also exposed to temperatures of 2 and -3 °C. Further, the supercooling point and lower lethal times of adult females were determined. No eggs survived exposure

to 7 °C for 17 or more days. Larval survival upon the cold treatment decreased from 53 to 13% when exposed to 7 °C for 14 and 49 days, respectively. Two-day-old adult females exposed to 7 °C for up to 42 days did not suffer significant mortality, but when returned to 25 °C their oviposition rates were lower than those of mites maintained at 25 °C. Less than 40% of females exposed for 13 days to 2 °C survived; only 20% of these females was able to reproduce upon recovery. Subzero temperatures dramatically decreased survival and reproduction capacity of adult females. The supercooling point of adult females was -16.5 °C. Median lethal times averaged 61.2 h and 9.3 days at -3 and 2 °C, respectively. A long term exposure (up to 6 weeks) of R. simsii plants infested with P. latus to a temperature of 7 °C, which is required for breaking dormancy of the flowers, is not expected to have detrimental effects on the survival and reproductive performance of the female mites. In Chapter 5 , a selection of 32 Rhododendron cultivars, mainly R. simsii hybrids, were evaluated for their susceptibility to P. latus . The plants were artificially infested in a greenhouse by surrounding each azalea with four P. latus infested English ivy plants (Hedera helix ). Infestation by P. latus was evaluated by counting the number of mites per shoot tip and assigning a damage rate. A comparable infestation rate was found, this was expressed by the number of mites on all cultivars at the initial stage of the experiments. Correlations between the average damage rate and the number of mites per shoot tip on all cultivars at different time intervals were significantly positive, although low, in each experiment, with R² -values varying between 0.14 and 0.61. At the end of the experiments significant differences in susceptibility between the evaluated cultivars were observed. The cultivars, ‘Emil De Coninck’ and ‘Mont Blanc’ were rated as the most susceptible, whereas ‘Mistral’ and its budsport ‘Elien’ were resistant towards P. latus , as damage rates were low and very few broad mites were found on these plants. Six genotypes with a different susceptibility for P. latus were studied in detail: ‘Nordlicht’, ‘Elien’, ‘Aiko Pink’, Michelle Marie’, ‘Mevrouw Gerard Kint’ and ‘Sachsenstern’. Of these six genotypes ‘Elien’ had glandular trichomes with a sticky end as observed with scanning electron microscopy. On ‘Mistral’ this type of trichomes also was found. Therefore it might be suggested that this type of trichomes contributes to the resistant characteristics of ‘Elien’ and ‘Mistral’. In the next two chapters the effect of induced responses in R. simsii upon P. latus infestation was studied. In Chapter 6 , the first transcriptome of R. simsii , containing ca. 15k orthologs from unique Arabidopsis thaliana genes, was assembled after next- generation RNA-sequencing. RNAseq experiments upon treatment with the elicitor methyl jasmonate (MeJA) or P. latus infestation suggested a role for jasmonic acid (JA) induced responses in R. simsii . A set of putative marker genes to study plant defence responses was selected and validated in R. simsii . Genes belonged to the biosynthetic pathway of phytohormones JA ( RsLOX , RsAOS , RsAOC , RsOPR3 and RsJMT ) and salicylic acid (SA) ( RsPAL and RsICS ). Furthermore, RsPPO , a putative marker gene for oxidative stress response was successfully cloned from R. simsii . A CTAB-based extraction protocol was optimized to assure excellent RNA quality for subsequent RT-qPCR analysis. The RT- qPCR protocol was extensively tested and RsRG7 and RsRG14 were selected as reference

genes from a geNorm pilot study. Validation of the marker genes was done after application with elicitors (MeJA, coronatine, β-aminobutyric acid and acibenzolar-S- methyl) or wounding. Both 100 µM MeJA and 0.1 µM coronatine had a significant effect on the expression of all marker genes. Foliar application of MeJA on the shoots resulted in a significantly earlier response when compared to root application and subsequent sampling of the shoots. Expression patterns after MeJA treatment were generally the same in six R. simsii genotypes: ‘Nordlicht’, ‘Elien’, ‘Aiko Pink’, Michelle Marie’, ‘Mevrouw Gerard Kint’ and ‘Sachsenstern’. Wounding resulted in the same expression patterns as MeJA treatment except for RsJMT . None of the genotypes showed a significant induction of the latter gene 6 hours upon wounding. Findings of these experiments indicated that ‘Elien’ have low basal expression levels of RsPPO . Finally in Chapter 7, the set of validated marker genes for plant defence responses was used to investigate the infestation of P. latus on R. simsii . Gene expression profiles were measured by means of RT-qPCR and phytohormone measurements of SA, JA and its precursor 12-oxophytodienoic acid (OPDA) were conducted using LC-MS/MS. In a bio- assay the direct effect of MeJA and SA was evaluated on the mite population increase. Polyphagotarsonemus latus infestation on R. simsii ‘Nordlicht’, ‘Aiko Pink’, Michelle Marie’, ‘Mevrouw Gerard Kint’ and ‘Sachsenstern’ resulted in a significant induction of JA marker genes starting from ca. one week after infestation. No clear involvement of the JA defence pathway was shown in the mite resistant genotype ‘Elien’. However, the amount of OPDA and JA increased significantly in ‘Elien’ while this was not the case for ‘Nordlicht’. In contrast, the amount of free SA increased significantly in ‘Nordlicht’. In all genotypes except for ‘Elien’ RsPPO, a marker gene for oxidative stress response, was significantly up- regulated. This went along with damage symptoms seen as brown colouring of youngest growth tips in susceptible genotypes. Methyl jasmonate application had a significant negative effect on P. latus population increase in the mite susceptible genotype R. simsii ‘Nordlicht’, but application of SA had no marked effect on the population increase of the pest. In Chapter 8 , a general discussion of the findings is presented and future prospects are discussed. It is concluded that the pest is able to survive the whole spectrum of temperatures encountered during R. simsii production, indicating its potential to spread and build up a population very easily when conditions are favourable. Our findings also showed the existence of resistant genotypes in the R. simsii gene pool. Glandular trichomes with a sticky end are thought to be involved in the resistant characteristics of these genotypes. Confirmation of this hypothesis requires additional research to determine the composition of the sticky exudates and to assess their effect on P. latus physiology. We found that JA-mediated responses might contribute to resistance against this pest. Further research is warranted to study the role of JA and SA defence pathways upon P. latus infestation and to assess the effect of external application of elicitors inducing defence responses in the plant.

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Samenvatting

De weekhuidmijt Polyphagotarsonemus latus (Banks) (Acari: Tarsonemidae) is een economisch belangrijke plaag in de kas en van gewassen wereldwijd in het veld. In België is het voornamelijk een sleutelplaag in de azalea productie (Rhododendron simsii Planch hybride). Symptomen van schade worden herkend als misvormingen van de jongste groeipunten en bloemen, en het naar beneden krullen van de bladeren. De beperkte hoeveelheid chemische middelen en recente restricties in het gebruik van breed werkende chemische middelen maakt het noodzakelijk om deze plaag te beheersen gebruikmakend van het principe van de geïntegreerde gewasbescherming (Integrated Pest Management, IPM). Dit omvat een multidisciplinaire aanpak om de risico’s op besmetting of om de plaagpopulatie in het gewas te reduceren. Maatregelen omvatten het kweken en telen van tolerante of resistent genotypen. Wanneer de plaag in het gewas is waargenomen, is het aangeraden om verschillende strategieën te combineren, dit kunnen chemische maar ook biologische behandelingen zijn. Tegenwoordig zijn er verschillende mijten behorende tot de familie van de die commercieel beschikbaar zijn en die zich voeden met P. latus zoals Amblyseius swirskii . Een alternatieve strategie om de plaag te beheersen kan het induceren van de plantdefensie zijn om zo de vitaliteit van de mijt en/of haar waardplantvoorkeur te beïnvloeden. De beschikbaarheid van essentiële informatie over de biologie van de plaag kan de bestrijding van P. latus aanzienlijk verbeteren in siergewassen zoals R. simsii . De algemene doelstelling van deze studie was om een beter inzicht te krijgen in de bio-ecologie van de weekhuidmijt P. latus , één van de meest belangrijke plagen in R. simsii hybride productie in België, en om de rol van defensiemechanismen in de sierplant tegen deze mijt te onderzoeken. In hoofdstuk 3 werd het effect van temperatuur op de ontwikkeling van P. latus op R. simsii bestudeerd. In combinatie met een fotoperiode van 16:8 u (L:D) en een relatieve vochtigheid van 80 ± 5%, werden zes constante temperaturen (15, 17, 20, 25, 30 en 33 °C) onderzocht. Totale ontwikkelingstijden van 13,3; 10,5; 6,6; 4,2; 3,5 en 4,0 dagen werden gemeten bij de respectievelijke temperaturen. De ontwikkeling van wijfjes duurde significant langer dan die van mannetjes bij 15, 17, 20 en 30 °C. Overlevingspercentages bij temperaturen tussen 17 en 30 °C varieerden tussen 43.5 en 96.9%. Lagere overlevingspercentages werden gemeten bij 15 en 33 °C, 31.8 en 23.6%, respectievelijk. De minimale, optimale en maximale ontwikkelingsgrens ( tmin , topt en tmax , respectievelijk) en thermische constante ( K) van de plaag werden voor elke levensfase geschat door middel van een lineair en twee niet-lineaire modellen. De thermische limieten van 10,0; 30,1 en 36,0 °C werden berekend op basis van de metingen van de totale ontwikkeling van P. latus , dit respectievelijk voor tmin , topt en tmax . Het aantal daggraden noodzakelijk om de juveniele ontwikkeling te volmaken wanneer de mijt zich voedt op R. simsii was 66.7. De blootstelling van planten aan temperaturen algemeen gelegen tussen 2,5 en 9 °C is noodzakelijk om bloemen uit hun dormant stadium te halen, dit omdat niet

nagekomen koeleisen leiden tot onberekenbare doorbraak van de knop en lagere kwaliteit. De optimale temperatuur om de bloemknopdormantie te doorbreken in R. simsii bedraagt 7 °C. In hoofdstuk 4 werd de overleving van eitjes, larven en vrouwelijke adulten, en de voortplantingscapaciteit van vrouwelijke P. latus mijten geëvalueerd na een blootstelling bij 7 °C. Adulte wijfjes werden ook blootgesteld aan temperaturen van 2 en -3 °C. Daarnaast werd het onderkoelingspunt en de letale tijden voor koude van adulte wijfjes vastgesteld. Geen van de eitjes overleefde de blootstelling aan 7 °C voor een periode van 17 of meer dagen. De larvale ontwikkeling na een koude behandeling van 7 °C gedurende 14 en 49 dagen daalde respectievelijk van 53 tot 13%. Twee-dagen-oude adulte wijfjes die tot 42 dagen werden blootgesteld aan 7 °C vertoonden geen significante sterfte, maar wanneer deze werden teruggeplaatst bij 25 °C waren de ovipositiesnelheden lager dan die van mijten gekweekt bij 25 °C. Minder dan 40% van de wijfjes die voor 13 dagen werden blootgesteld aan 2 °C overleefden; enkel 20% van deze wijfjes was in staat om zich voort te planten na herstel. Temperaturen onder het vriespunt verminderden drastisch de overlevings- en voortplantingscapaciteit van adulte wijfjes. Het onderkoelingspunt van adulte wijfjes was -16,5 °C. Mediaan letale tijden bedroegen 61,2 h en 9,3 dagen respectievelijk bij -3 en 2 °C. Een langdurige blootstelling (tot 6 weken) aan een temperatuur van 7 °C, noodzakelijk voor het doorbreken van de dormantie van de bloemen, van R. simsii planten besmet met P. latus wordt niet verwacht om nadelige effecten op de overlevings-en voortplantingsprestaties van vrouwelijke mijten te hebben. In hoofdstuk 5 werd een selectie van 32 Rhododendron cultivars, hoofdzakelijk R. simsii hybriden, gescreend voor hun gevoeligheid naar P. latus . De planten werden in de kas op kunstmatige wijze besmet door het omringen van elke azaleaplant met vier door P. latus geïnfecteerde klimopplanten ( Hedera helix ). De besmetting met P. latus werd geëvalueerd door het tellen van het aantal mijten per scheuttip en door beoordeling van de schade. Een vergelijkbare besmettingsgraad werd vastgesteld aan het begin van de verschillende experimenten. Correlatie tussen de gemiddelde beoordeling van de schade en het aantal mijten per scheuttip op alle cultivars was significant positief, hoewel laag, op elk tijdstip. In elk experiment varieerden de R² -waarden tussen 0,14 en 0,61. Op het einde van de experimenten werden significante verschillen in gevoeligheid tussen de geëvalueerde cultivars geobserveerd. De cultivars ‘Emil De Coninck’ en ‘Mont Blanc’ werden gecatalogeerd als heel gevoelig. Daarentegen werden ‘Mistral’ en zijn knopsport ‘Elien’ als resistent beschouwd voor P. latus , dit omdat de schade beperkt was en heel weinig weekhuidmijten werden teruggevonden op deze planten. Zes genotypen met een verschillende gevoeligheid voor P. latus werden in detail bestudeerd: ‘Nordlicht’, ‘Elien’, ‘Aiko Pink’, ‘Michelle Marie’, ‘Mervouw Gerard Kint’ en ‘Sachsenstern’. Na observatie met scanning elektronen microscopie bleek dat van deze zes genotypen ‘Elien’ klierharen met een kleverig uiteinde hadden. Dit type van beharing werd ook terug gevonden op ‘Mistral’. Op basis hiervan kan men suggereren dat dit type beharing bijdraagt aan de resistente eigenschappen van ‘Elien’ en ‘Mistral’. In de volgende twee hoofdstukken werd het effect van geïnduceerde responsen in R. simsii bij besmetting met P. latus bestudeerd. In hoofdstuk 6 werd het eerste transcriptoom van R. simsii , dat ca. 15k orthologen van unieke Arabidopsis thaliana genen

bevat, geassembleerd na ‘next-generation RNA-sequencing’. RNAseq experimenten na benadeling met de elicitor methyljasmijnzuur (MeJA) of besmetting met P. latus suggereerden een rol voor jasmijnzuur (JA) geïnduceerde responsen in R. simsii . Om plant defensie te bestuderen werd een set van vermeende merkergenen geselecteerd en gevalideerd in R. simsii . De genen behoorden tot de biosynthese pathways van de fytohormonen JA ( RsLOX , RsAOS , RsAOC , RsOPR3 en RsJMT ) en salicylzuur (SA) ( RsPAL en RsICS ). Bovendien werd RsPPO , een vermeend merkergen voor oxidatieve stressrespons succesvol gekloneerd in R. simsii . Een CTAB-gebaseerd extractie protocol werd geoptimaliseerd om uitstekende RNA kwaliteit te garanderen in de navolgende RT-qPCR analyse. Het RT-qPCR protocol werd uitvoerig getest en RsRG7 en RsRG14 werden geselecteerd als referentiegenen na een geNorm pilootstudie. De merkergenen werden gevalideerd na behandeling met elicitoren (MeJA, coronatine, β-aminoboterzuur en acidbenzolar-S-methyl) of verwonding. Zowel 100 µM MeJA als 0,1 µM coronatine hadden een significant effect op de expressie van alle merkergenen. Behandeling van bladeren op de scheuten met MeJA resulteerde in significant eerdere responsen vergeleken met behandeling van de wortels en vervolgens staalnames in de scheuten. De expressiepatronen na MeJA behandeling waren over het algemeen gelijkaardig in zes R. simsii genotypen: ‘Nordlicht’, ‘Elien’, ‘Aiko Pink’, ‘Michelle Marie’, ‘Mervouw Gerard Kint’ en ‘Sachsenstern’. Verwonding resulteerde in dezelfde expressiepatronen als MeJA behandeling met uitzondering voor RsJMT . Geen van deze genotypen vertoonde een significante inductie van het laatstgenoemde gen 6 uur na verwonding. De bevindingen van deze experimenten tonen aan dat ‘Elien’ erg lage basale expressie levels heeft voor RsPPO . Finaal werd in hoofdstuk 7 de set van gevalideerde merkergenen voor plant defensie responsen gebruikt om de besmetting op R. simsii door P. latus te bestuderen. Genexpressie profielen werden gemeten door middel van RT-qPCR en fytohormoon metingen van SA, JA en zijn precursor 12-oxofytodieenzuur (OPDA) werden uitgevoerd gebruikmakend van LC-MS/MS. In een bio-assay werd het directe effect van MeJA en SA op de groei van de mijtpopulatie geëvalueerd. Polyphagotarsonemus latus besmetting in R. simsii ‘Nordlicht’, ‘Aiko Pink’, ‘Michelle Marie’, ‘Mervouw Gerard Kint’ en ‘Sachsenstern’ resulteerde in een significante inductie van JA merkergenen startende vanaf ongeveer één week na de besmetting. Er kon geen duidelijke betrokkenheid van de JA defensie pathway worden aangetoond in het mijt resistente genotype ‘Elien’. Hoewel de hoeveelheid OPDA en JA significant toenam in ‘Elien’ was dit niet het geval voor ‘Nordlicht’. In tegenstelling, de hoeveelheid SA nam significant toe in ‘Nordlicht’. In alle genotypen met uitzondering van ‘Elien’ was RsPPO , een merkergen voor oxidatieve stressrespons, significant opgereguleerd. Dit ging samen met de symptomen van schade, die gezien werden als bruinkleuring van de jongste groeipunten in gevoelige genotypen. De behandeling met methyljasmijnzuur had een significant negatief effect op de toename van de populatiegroei van P. latus in het mijt gevoelige genotype ‘Nordlicht’. De toediening van SA had geen significant effect op de populatiegroei van de plaag. In hoofdstuk 8 wordt een algemene discussie van de resultaten gepresenteerd en worden toekomstperspectieven besproken. Als algemene conclusie kan men stellen dat

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de plaag in staat is om te overleven bij alle temperaturen die in de productie van R. simsii voorkomen. Dit geeft aan dat de plaag zich gemakkelijk kan verspreiden en een populatie kan opbouwen onder gunstige condities. De bevindingen geven ook aan dat er resistente genotypen bestaan in de R. simsii genenpool. Van klierharen met een kleverig uiteinde wordt gedacht dat deze betrokken zijn in de resistentie-eigenschappen van deze genotypen. Bevestiging van deze hypothese noodzaakt bijkomstig onderzoek om de samenstelling van de kleverige exudaten te bepalen en om de fysiologische effecten ervan op P. latus na te gaan. We vonden dat JA afhankelijke responsen kunnen bijdragen tot resistentie tegen deze plaag. Verder onderzoek is noodzakelijk om de rol van de JA en SA defensie pathways bij besmetting met P. latus te bestuderen en om het effect na te gaan van de externe applicatie van elicitoren die plant defensie responsen induceren.

References

Abe H, Ohnishi J, Narusaka M, Seo S, Narusaka Y, Tsuda S, Kobayashi M (2008a) Function of jasmonate in response and tolerance of Arabidopsis to thrips feeding. Plant Cell Physiol. 49:68-80

Abe H, Ohnishi J, Narusaka M, Seo S, Narusaka Y, Tsuda S, Kobayashi M (2008b) Arabidopsis-thrips system for analysis of plant response to insect feeding. Plant Signaling and Behav. 3:1-2

Abe H, Shimoda T, Ohnishi J, Kugimiya S, Narusaka M, Seo S, Narusaka Y, Tsuda S, Kobayashi M (2009) Jasmonate-dependent plant defense restricts thrips performance and preference. BMC Plant Biology 9:97 doi:10.1186/1471-2229-9-97

Abou-Awad BA, Hafez SM, Farhat BM (2014) Biological studies of the predacious mite Amblyseius swirskii , a predator of the broad mite Polyphagotarsonemus latus on pepper plants (Acari: Phytoseiidae: Tarsonemidae). Arch. Plant Pathol. Plant Protec. 47(3):349-354

Abramovitch RB, Anderson JC, Martin GB (2006) Bacterial elicitation and evasion of plant innate immunity. Nature reviews. Molecular Cell Biology 7:601-611

Agrawal GK, Tamogami S, Han O, Iwahashi H, Rakwal R (2004) Rice octadecanoid pathway. Biochem. Biophys. Res. Commun. 317:1-15

Agut B, Gamir J, Jacas JA, Mónica Hurtado, Victor Flors (2014) Different metabolic and genetic responses in citrus may explain relative susceptibility to Tetranychus urticae . Pest Manag. Sci. 70:1728-1741

Agut B, Gamir J, Jaques JA, Flors V (2015 ) Tetranychus urticae -triggered responses promote genotype dependent conspecific repellence or attractiveness in citrus. New Phytologist doi: 10.1111/nph.13357

Ahmed K, Rao PP, Kumari AL (2000) Biochemical aspects of host plant resistance to yellow mite in chilli. Agric. Sci. Digest 20(4):238-240

Ahmed K, Rao VH, Rao PP (2001) Resistance in chilli cultivars to yellow mite Polyphagotarsonemus latus Banks. Indian J. Agric. Res. 35(2):95-99

Akaike H (1974) A new look at the statistical model identification. IEEE Transactions on Automatic Control 19:716-723

Alagarmalai J, Grinberg M, Perl-Treves R, Soroker V (2009) Host selection by the herbivorous mite Polyphagotarsonemus latus (Acari: Tarsonemidae). J. Insect. Behav. 22:375-387

Alagarmalai J, Grinberg M, Reneh S, Sharon L, Perl-Treves R, Soroker V (2007) Host selection behavior of broad mite, Polyphagotarsonemus latus (Acari: Tarsonemidae). IOBC-WPRS Bulletin 30(5):1-7

Alba JM, Schimmel BCJ, Glas JJ, Ataide LMS, Pappas ML, Villarroel CA, Schuurink RC, Sabelis MW, Kant MR (2015) Spider mies suppress tomoato defences downstream of jasmonate and salicylate independently of hormonal crosstalk. New Phytol. 205(2):828-840

Alcántara JJA, Santillán-Galicia MT, Otero-Colina G, Mora AA, Gutiérrez EMA, Hernández CE (2011) Relación entre Polyphagotarsonemus latus (Acari: Tarsonemidae) y el virus de la mancha anular del papayo (PRSV-p). Revista Colombiana de Entomología 37(2): 228-233

Allen, CM (2010) Thermal biology and behaviour of two predatory Phytoseiid mites: Amblyseius swirskii (Athias-Henriot) (Acari:Phytoseiidae) and Phytoseiulus longipes (Evans) (Acari:Phytoseiidae). Ph.D. thesis, University of Birmingham.

Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local alignment search tool. J. Mol. Biol. 215:403-410

Alves LFA, Santana DLQ, Andrade DP (2010) Damage characterization and control tactics to broad mite (Polyphagotarsonemus latus Banks) in Paraguay-tea plants ( Ilex paraguariensis A. St.-Hil.). R. Bras. Bioci. 8(2):208-212

Ament K, Kant MR, Sabelis MW, Haring MA, Schuurink RC (2004) Jasmonic acid is a key regulator of spider mite-induced volatile terpenoid and methyl salicylate emission in tomato. Plant Physiol. 135:2025-2037

Ament K, Van Schie CC, Bouwmeester HJ, Haring MA, Schuurink RC (2006) Induction of a leaf specific geranylgeranyl pyrophosphate synthase and emission of ( E,E )-4,8,12-trimethyltridec-1,3,7,11-tetraene in tomato are dependent on both jasmonic acid and salicylic acid signaling pathways. Plant 224:1197- 1208

Amin PW (1979) Leaf curl disease of chilli peppers in Maharashtra, India. International Journal of Pest Management. 25(2):131-134

Arimura G, Ozawa R, Shimoda T, Nishioka T, Boland W, Takabayashi J (2000) Herbivory-induced volatiles elicit defence genes in lima bean leaves. Nature 406:512-515

Arnold CY (1959) The determination and significance of the base temperature in a linear heat unit system. Proc. Am. Soc. Hort. Sci. 74:430–445

Asada K, Salim V, Masada-Atsumi S, Edmunds E, Nagatoshi M, Terasaka K, Mizukami H, De Luca V (2013) A 7-deoxyloganetic acid glucosyltransferase contributes a key step in secologanin biosynthesis in Madagascar periwinkle. The Plant Cell 25:4123-4134

Athias-Binche F (1991) Evolutionary ecology of dispersal in mites. In: Dusbabek F, Bukva V (Eds.) Modern acarology. Academia and SPB Prague and The Hague: Academic Publishing, pp 27-41

Aubert B, Lossois P, Marchal J (1981) Mise en evidence des dégâts causes par Polyphagotarsonemus latus (Banks) sur papayers à l’ile de la Réunion. Fruits 36:9-24

Audenaert J, Vissers M, Haleydt B, Verhoeven R, Goossens F, Gobin B (2009) Acaricides and predatory mites against the begonia mite, Polyphagotarsonemus latus (Acari: Tarsonemidae), on Hedera helix . Comm. Appl. Biol. Sci., Ghent University, 74(1):217-224

Backhaus GF (1994a) Cylindrocladium scoparium causing wilt disease in Rhododendron and azalea. Acta Hort. 364:163-166

Backhaus GF (1994b) Phytophthora citricola (Sawada) - cause of an important shoot rot of Rhododendron and azalea. Acta Hort. 364:145-154

Baelde HJ, Cleton-Jansen AM, Van Beerendonck H, Namba K (2001) High quality RNA isolation from tumors with low cellularity and high extra-cellular matrix component for cDNA microarrays. Application to chondrosarcoma. J. Clin. Pathol. 54:778-782

Baker A, Graham IA, Holdsworth M, Smith SM, Theodoulou FL (2006) Chewing the fat: beta-oxidation in signaling an development. Trends Plant Sci. 11:124-132

Baker RA (2012) ‘Plastrons and adhesive organs’ – The functional morphology of structures in the broad mite, Polyphagotarsonemus latus (Banks, 1904). Acta Biologica 19:89-96

Baldi V, Devoto A (2008) Jasmonate signaling network in Arabidopsis thaliana: crucial regulatory nodes and new physiological scenarios. New Phytol. 177:301-318

Baldwin IT (1998) Jasmonate-induced responses are costly but benefit plants under attack in native populations. Proc. Natl. Acad. Sci. USA 95:8113-8118

Bale JS (1987) Insect cold hardiness - freezing and supercooling - an ecophysiological perspective. J Insect Physiol 33(12):899-908

Bale JS (2002) Insects and low temperatures: from molecular biology to distribution and abundance. Philos Trans R Soc Lond B Biol Sci 357(1423):849-862

Bale JS, Jones TH, Gibbons D (1996) Impacts of climate change. J Zool 240:593-597

Banks N (1904) Class 111, Arachnida, Order 1, Acarina, four new species of injurious mites. J. New York Entomol. S. 12:53-56

Bari R, Jones JDG (2009) Role of plant hormones in plant defence responses. Plant Mol. Biol. 69:473-488

Bar-Joseph M, Gerson U (1977) Virus-like injuries of avocado induced by arthropods. Hassadeh 57:1070- 1072 (in Hebrew)

Bassett P (1981) Observations on broad mite ( Polyphagotarsonemus latus ) (Acarina: Tarsonemidae) attacking cucumber. Crop Protection Conference Pest and Diseases 1:99-103

Bassett P (1985) Tarsonemid mites: In: Biological Pest Control, The Glasshouse Experience (Ed. By NW Hussey and N Scopes), pp 93-96, Blandford Press

Bean WJ (1980) Trees and shrubs, hardy in the British Isles. Vol. III, p. 689

Beattie GAC, Gellatley JG (1983) Mites pests of citrus. Agfacts H2, AE 3, Dept. Agriculture, New South Wales, 6 pp.

Beer RE, Nucifora A (1965) Revisione dei generi della famiglia Tarsonemidae (Acarina). Boll. Zool. Agrar. Bachic. 2(7):19-43

Bell E, Creelman RA, Mullet JE (1995) A chloroplast lipoxygenase is required for wound-induced jasmonic acid accumulation in Arabidopsis . Proc. Natl. Acad. Sci. USA 92:8675-8679

Bell E, Mullet JE (1993) Characterization of an Arabidopsis lipoxygenase gene responsive to methyl jasmonate and wounding. Plant Physiol. 130:1133-1137

Bergey DR, Howe GA, Ryan CA (1996) Polypeptide signalling for plant defensive genes exhibits analogies to defense signalling in animals. Proc. Natl. Acad. Sci. USA 93:12053-12058

Bernasconi ML, Turlings TCJ, Ambrossetti L, Bassetti P, Dorn S (1998) Herbivore-induced emissions of maize volatiles repel the corn-leaf aphid, Rhopalosiphum maidis . Entomologia Experimentalis et Applicata 87:133-142

Bolter CJ, Dicke M, van Loon JJA, Visser JH, Posthumus MA (1997) Attraction of Colorado potato beetle to herbivore damaged plants during herbivory and after its termination. Journal of Chemical Ecology 23:1003-1023

Bonaventure G (2012) Perception of insect feeding by plants. Plant Biol. (Stuttg.) 14:872-880

151

Borgen BH, Thangstad OP, Ahuja I, Rossiter JT, Bones AM (2010) Removing the mustard oil bomb from seeds: transgenic ablation of myrosin cells in oilseed rape ( Brassica napus ) produces MINELESSS seeds. Journal of Experimental Botany 61(6) 1683-1697

Bos JIB, Prince D, Pitino M, Maffei Me, Win J, Hogenhout SA (2010) A functional genomics approach identifies candidate effectors from the aphid species Myzus persicae (Green Peach aphid). PLoS Genetics 6:e1001216

Bosco R, Daeseleire E, Van Pamel E, Scariot V, Leus L (2014) Development of an ultrahigh-performance liquid chromatography-electrospray ionization-tandem mass spectrometry method for the simultaneous determination of salicylic acid, jasmonic acid, and abscisic acid in rose leaves. J. Agric. Food Chem. 62(27):6278-6284

Botanga CJ, Benthke G, Chen Z, Gallie DR, Fiehn O, Glazebrook J (2012) Metabolite profiling of Arabidopsis inoculated with Alternaria brassicola reveals that ascorbate reduces disease severity. MPMI 25(12):1628-1638

Boughton AJ, Hoover K, Felton GW (2005) Methyl jasmonate application induces increased densities of glandular trichomes on tomato, Lycopersicon esculentum . Journal of Chemical Ecology 31:2211-2216

Brich LC (1948) The intrinsic rate of natural increase of an insect population. J. Anim. Ecol. 17:15-26

Brière JF, Pracros P, le Roux AY, Pierre JS (1999) A novel rate model of temperature dependent development for arthropods. Environ. Entomol. 28:22-29

Britto EP, Gago E, de Moraes GJ (2012) How promising is Lasioseius floridensis as a control agent of Polyphagotarsonemus latus ? Exp. Appl. Acarol. 56(3):221-231

Browse J (2009a) Jasmonate passes muster: A receptor and targets for the defence hormone. Annu. Rev. Plant Biol. 60:183-205

Browse J (2009b) The powder of mutants for investigating jasmonate biosynthesis and signaling. Phytochemistry 70:1539-1546

Bustin SA (2000) Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays. J. Mol. Endocrinol. 25:169-193

Bustin SA, Benes V, Garson JA, Hellemans J, Huggett J, Kubista M, Mueller R, Nolan T, Pfaffl MW, Shipley GL, Vandesompele J, Wittwer CT (2009) The MIQE guidelines: Minimum Information for publication of Quantitative real-time PCR experiments. Clin. Chemistry 55:611-622

Cabrera RI, Dominguez D, Blanco JJ (1987) Informe de Hirsutella nodulosa , enemigo natural de acaro blanco Polyphagotarsonemus latus . Comun Cienc Techn. Agric. Cirt. Frut. 10:135-138

Campbell A, Frazer BD, Gilbert N, Gutierrez AP, Mackauer M (1974) Temperature requirements of some aphids and their parasites. J. Appl. Ecol. 11:431-438

Campbell RJ, Grayson RL, Marini RP (1990) Surface and ultrastructural feeding injury to strawberry leaves by the two spotted spider mite. HortScience 25:948-951

Cardoso MZ (2008) Herbivore handling of a plant’s trichome: The case of Heliconius charithonia (L.) (Lepidoptera: Nymphalidae) and Passiflora lobate (Killip) Hutch. (Passifloraceae). Neotrop. Entomol. 37:247-252

Castillo MC, Martínez C, Buchala A, Métraux JP, León J (2004) Gene-specific involvement of β-oxidation in wound-activated responses in Arabidopsis . Plant Physiol. 135: 85-94

Ceyfferth C, Tsuda K (2014) Salicylic acid signal transduction: the initiation of biosynthesis, perception and transcriptional reprogramming. Front Plant Sci. 5:679

Chadha KC, Brown SA (1974) Biosynthesis of phenolic acids in tomato plants infected with Agrobacterium tumefaciens . Can. J. Bot. 52:2041-2046

Chakrabarti S, Sarkar PK (2014) Studies on efficacy of some acaricidal molecules for the management of Polyphagotarsonemus latus (Banks) (Acari: Tarsonemidae) infecting chilli ( Capsicum annuum L.) in West Bengal. Current Biotica 7(4):299-305

Chamberlain DF, Hyam R, Argent G, Fairweather G, Walter KS (1996) The genus Rhododendron its classification and synonymy. Royal Botanic Garden, Edinburgh

Chelly J, Kahn A (1994) RT-PCR and mRNA quantification. In: Mullis DB, Ferre F, Gibbs RA (eds), The Polymerase Chain Reaction, pp 77-109, Birkhauser, Boston, MA, USA

Chen F, D’auria JC, Tholl D, Ross JR, Gershenzon J, Noel JP, Pichersky E (2003) An Arabidopsis thaliana gene for methylsalicylate biosynthesis, identified by a biochemical genomics approach, has a role in defense. Plant J. 36:557-588.

Cheong JJ, Choi YD (2003) Methyl jasmonate as a vital substance in plants. Trends Genet. 19(7):409-413

Chico JM, Chini A, Fonseca S, Solano R (2008) JAZ repressors set the rhythm in jasmonate signaling. Curr Opin Plant Biol 11:486-494

Chini A, Boter M, Solano R (2009) Plant oxylipins: COI1/JAZs/MYC2 as the core jasmonic acid-signalling module. FEBS J. 276:4682-4692

Chini A, Fonseca S, Fernandez G, Adie B, Chico JM, Lorenzo O, Garcia-Casado G, Lopez-Vidriero I, Lozano FM, Ponce MR, Micol JL, Solano R (2007) The JAZ family of repressors is the missing link in jasmonate signaling. Nature 448:666-671

Cho MR, Jeon HY, Kim DS, Chung BS, Yiem MS, Kim SB (1996b) Host plant and damage of broad mite (Polyphagotarsonemus latus ) on horticultural crops. Rural Development Administration Journal of Agricultural Sciences 38:516-525

Cho MR, Jeon HY, La SY, Kim DS, Yiem MS (1996a) Damage of broad mite, Polyphagotarsonemus latus (Banks), on pepper growth and yield and its chemical control. Korean Journal of Applied Entomology 35:326-331

Choh Y, Ozawa R, Takabayashi J (2004) Effects of exogenous jasmonic acid and benzo (1,2,3) thiadiazole- 7-carbothioic acid S-methyl ester (BTH), a functional analogue of salicylic acid, on the egg production of a herbivorous mite Tetranychus urticae (Acari: Tertanychidae). Appl. Entomol. Zool. 39(2):311-314

Christiaens A (2014a) Factors affecting flower development and quality in Rhododendron simsii . PhD Thesis, Ghent University, Ghent, Belgium

Christiaens A, De Keyser E, Lootens P, Pauwels E, Roldán-Ruiz I, De Riek J, Gobin B, Van Labeke M-C (2014b) Cold storage to overcome dormancy affects the carbohydrate status and photosynthetic capacity of Rhododendron simsii. Plant Biology doi:10.1111/pbl.12195

Chung HS, Niu Y, Browse J, Howe GA (2009) Top hits in contemporary JAZ: An update on jasmonate signaling. Pyhochemistry 70:1547-1559

Collier T, Van Steenwyk R (2004) A critical evaluation of augmentative biological control. Biological Control 31:245-256

153

Conrath U, Pieterse CMJ, Mauch-Mani B (2002) Priming in plant-pathogen interactions. Trends Plant Sci. 7:210-216

Consales F, Schweizer F, Erb M, Gouhier-Darimont C, Bodenhausen N, Bruessow F, Sobhy I, Reymond P (2012) Insect oral secretions suppress wound-induced responses in Arabidopsis . Journal of Experimental Botany 63:727-737

Constabel CP, Bergey DR, Ryan CA (1995) Systemin activates synthesis of wound-inducible tomato leaf polyphenol oxidase via the octadecanoid defense signaling pathway. Proc. Natl. Acad. Sci. USA 92:407- 411

Constabel CP, Ryan CA (1998) A survey of wound- and methyl jasmonate-induced leaf polyphenol oxidase in crop plants. Phytochemistry 47:507-511

Constabel CP, Yip L Patton JJ, Christopher ME (2000) Polyphenol oxidase from hybrid popular. Cloning and expression in response to wounding and herbivory. Plant Physiology 124:285-295

Costa CL, Arruda P, Benedetti CE (2000) An Arabidopsis gene induced by wounding functionally homologous to flavoprotein oxidoreductases. Plant Mol. Biol. 44(1):61-71

Cox PA, Cox KNE (1997) The encyclopedia of Rhododendron species. Glendoick Publishing, Perth

Creelman RA, Mullet JE (1995) Jasmonic acid distribution and action in plants: regulation during development and response to biotic and abiotic stress. Proc. Natl. Acad. Sci. USA 92:4114-4119

Cross JV, Bassett P (1982) Damage to tomato and aubergine by broad mite, Polyphagotarsonemus latus (Banks). Plant Pathol. 31:391-393

Cui X, Luan S (2012) A new wave of hormone research: crosstalk mechanisms. Mol. Plant 5(5):959-960

Czechowski T, Stitt M, Altmann T, Udvardi MK, Scheible W-R (2005) Genome-wide identification and testing of superior reference genes for transcript normalization in Arabidopsis . Plant Physiology 139:5- 17

Dathe W (1981) Endogenous plant hormones of the broad bean, Vicia faba L.(-)-jasmonic acid, a plant growth inhibitor in pericarp. Planta 153:530

Dave A, Graham IA (2012) Oxylipin signalling: a distinct role for the jasmonic acid precursor cis-(+)-12- oxophytodienoic acid (cis-OPDA). Front Plant Sci. doi:10.3389/fpls.2012.00042

David PMM, Rajkumar K, Elanchezhyan K, Razak TA, Nelson SJ, Nainar P, Murali Baskaran RK (2009) Effect of soft soaps on broad mite, Polyphagotarsonemus latus (Banks) on chilli. Karnataka J. Agric. Sci. 22(3):554-556

De Clercq P, Degheele D (1992) Development and survival of Podisus maculiventris (Say) and Podisus sagittal (Fab.) (Het.: Pentatomidae) at various constant temperatures. Can. Entomol. 124:125-133

De Coss M, Oter-Colina G, Pena JE, Magallanes R (2010) Demographic and reproductive parameters of Polyphagotarsonemus latus in Carica papaya . In: Trends in acarology: Proceedings of the 12 th international congress (Ed. by MW Sabelis and J Bruin), pp. 357-360. Springer, the Netherlands

De Coss-Romero M, Peña JE (1998) Relationship of broad mite (Acari: Tarsonemidae) to host phenology and injury levels in Capsicum annuum. Florida Entomologist 81(4):515-526

De Geyter N, Gholami A, Goormachtig S, Goossens A (2012) Transcriptional machineries in jasmonate- elicited plant secondary metabolism. Trends in Plant Science 17(6):349-359

De Jonge R, Bolton M, Thomma BPHJ (2011) How filamentous pathogens co-opt plants: the ins and outs of fungal effectors. Current Opinion in Plant Biology 14:400-406

De Keyser E (2010) Integration of phenotype, genotype and gene expression to unraveal flower colour biosynthesis and complex plant quality traits in azalea ( Rhododendron simsii hybrids). PhD Thesis, Ghent University, Ghent, Belgium

De Keyser E, De Riek J, Heungens K (2008) Development of supporting techniques for pot azalea (Rhododendron simsii hybrids) breeding focused on plant quality, disease resistance and enlargement of the assortment. Acta Hort. 766:361-366

De Keyser E, Desmet L, Van Bockstaele E, De Riek J (2013) How to perform RT-qPCR accurately in plant species? A case study on flower colour gene expression in an azalea ( Rhododendron simsii hybrids) mapping population. BMC Mol Biol 14:13, doi:10.1186/1471-2199-14-13

De Loose R (1979) Radiation induced chimeric rearrangement in flower structure of Rhododendron simsii Planch. ( Azalea indica L.). Use of recurrent irradiation. Euphytica 28(1):105-113

De Schepper S (2001) Molecular analysis of the induction of sports in azalea ( Rhododendron simsii hybrids). PhD Thesis, Ghent University, Ghent, Belgium

De Schepper S, Leus L, Eeckhaut T, Van Bockstaele E, Debergh P, De Loose M (2004) Somatic polyploidy petals: regeneration offers new roads for breeding Belgian pot azalea. Plant Cell, Tissue and Organ Culture 76:183-188

De Vos M, Van Oosten VR, Van Poecke RMP, Van Pelt JA, Pozo MJ, Mueller MJ, Buchale AJ, Métraux J- P, Van Loon LC, Dicke M, Pieterse CMJ (2005) Signal signature and transcriptome changes of Arabidopsis during pathogen and insect attack. Molecular Plant-Microbe Interactions 18(9):923-937

Dean JD, Goodwin PH, Hsiang T (2002) Comparison of relative RT-qPCR and northern blot analyses to measure expression of beta-1,3-glucanase in Nicotiana benthamiana infected with Colltotrichum destructivum . Plant Mo. Biol. Rep. 20:347-356

Delker Z, Zolman BK, Miersch O, Wasternack C (2007) Jasmonate biosynthesis in Arabidopsis thaliana requires peroxisomal beta-oxidation enzymes-additional proof by properties of pex6 and ami1 . Phytochemistry 68:1642-1650

Demole E, Lederer E, Mercier D (1962) Isolement et détermination de la structure du jasmonate de méthyle, contituant odorant charactéristique de l’essence de jasmin. Helvetica Chimica Acta 45:675-685

Dempsey D’MA, Vlot AC, Wildermuth MC, Klessig DF (2011) Salicylic acid biosynthesis and metabolism. Arabidopsis Book 9:e0156

Denmark HA (1980) Broad mite Polyphagotarsonemus latus (Banks). FDACS-DPI Bureau of Entomology Circular No. 213

Deroles S, Boase M, Lee C, Peters T (2002) Gene transfer to plants. In: Vainstein A (Ed.) Breeding for ornamentals: classical and molecular approaches. Kluwer Academic Publishers, Dordrecht, The Netherlands pp. 155-196

Desai HR, Bandhania KA, Patel AJ, Patel MB, Rai AB (2006) Screening of chilli varieties/germplasms for resistance to yellow mite, Polyphagotarsonemus latus Banks in South Gujarat. Pest Management in Horticultural Ecosystems 12(1):55-62

155

Desai HR, Bandhania KA, Rai AB, Patel AJ, Patel MB (2007) Assessment of yield loss and resistance to yellow mite Polyphagotarsonemus latus Banks in chilli. Veg. Sci. 31(1):46-50

Dhooria MS, Bhindra OS (1977) Polyphagotarsonemus latus (Banks) a mite pest of chilli and potato in Punjab. Acarol. Newsl. 4:7-9

Dicke M (1986) Volatile spider-mite pheromone and host-plant kairomone, involved in spaced-out gregariousness in the spider mite Tetranychus urticae . Physiological Entomology 11:251-262

Duffey SS, Felton GW (1991) Enzymatic antinutritive defenses of the tomato against plant insects. In Hedin PA (Ed.) Naturally occurring pest bioregulators. ACS Press, Washington, DC, USA, pp. 167-197

Dunn OJ (1964) Multiple comparisons using rank sums. Technometrics 6:241-252

Echer MM, Fernandes MCA, Ribeiro RLD, Peracchi AL (2002) Avaliação de genótipos de Capsicum para resistência ao ácaro branco. Horticultura Brasileira 20(2):217-221

Eeckhaut T, Calsyn E, Van Huylenbroeck J (2013) Intersubgeneric hybridization of Belgian pot azaleas (Rhododendron simsii ) with tropical Vireya genotypes. Acta Horticulturae 990:259-263

Eeckhaut T, De Keyser E, Van Huylenbroeck J (2009) In vitro assisted breeding of evergreen azaleas as a tool for assortment extension. Acta Horticulturae 829:55-60

El-Basyouni S, Chen D, Ibrahim RK, Neish AC, Towers GHN (1964) The biosynthesis of hydroxybenzoic acids in higher plants. Phytochemistry 3:485-492

Ellis C, Karafyllidis I, Turner JG (2002) Constitutive activation of jasmonate signaling in an Arabidopsis mutant correlates with enhanced resistance to Erysiphe cichoracearum , Pseudomonas syringae , and Myzus persicae . Mol. Plant Microbe Interact. 15:1025-1030

EROV (2012) Oost-Vlaamse sierteelt in een Vlaamse context, edition 2012

Evaristo AB, Venzon M, Matos FS, de Freitas RG, Kuki KN, dos Santos DL (2013) Susceptibility and physiological responses of Jatropha curcas accessions to broad mite infestation. Exp. Appl. Acarol. 60(4):485-496

Ewing HE (1939) A revision of the mites of the subfamily Tarsoneminae of North America, the West Indies and the Hawaiian Islands. USDA Tech. Bull. 653, USA, p 63

Faluso TR (2010) Broad mite, Polyphagotarsonemus latus (Banks) (Arachnida: Acari: Tarsonemidae). EENY-183 (IN340) Entomology and Nematology Department, Cooperative Extension Service, IFAS, University of Florida

Fan Y, Petit FL (1998) Dispersal of the broad mite, Polyphagotarsonemus latus (Acari: Tarsonemidae) on Bemisia argentifolii (Homoptera: Aleyrodidae). Exp. Appl. Acarol. 22:411-415

Fan YQ, Petit FL (1994) Biological control of broad mite, Polyphagotarsonemus latus (Banks), by Neoseiulus barkeri Hughes on pepper. Biological control 4(4):390-395

Faryish DJ, Axtell RC (1971) Phoresy redefined and examined in Macrocheles muscaedomestica (Acarina: Macrochelidae). Acarologia 13:16-29

Felton GW, Donato KK, Del Vecchio RJ, Duffey SS (1989) Activation of foliar oxidases by insect feeding reduces nutritive quality of dietary protein of foliage for noctuid herbivores. J. Chem. Ecol. 15:2667-2693

Ferré F (1992) Quantitative or semi-quantitative PCR: reality versus myth. PCR Methods Appl. 2(1):1-9

Ferreira RCF, de Oliveira JV, Haji FNP, Gondim Jr MG (2006) Biologia, exigências térmicas e tabela de vida de fertilidade do ácaro-branco Polyphagotarsonemus latus (Banks) (Acari: Tarsonemidae) em videira ( Vitis vinifera L.) cv. Itália. Neotrop. Entomol. 35:126-132

Ferrer JL, Austin MB, Stewart C Jr, Noel JP (2008) Structure and function of enzymes involved in the biosynthesis of phenylpropanoids. Plant Physiology and Biochemistry 46:356-370

Feys BJF, Benedetti CE, Penfold CN, Turner JG (1994) Arabidopsis mutants selected for restance to the phytotoxin coronatine are male sterile, insensitive to methyl jasmonate, and resistant to a bacterial pathogen. Plant Cell 6:751-759

Flechtmann CHW (1983) Acaros de importancia Agricola. Livraria Nobel, SA São Paulo, Brazil pp. 112-114

Flechtmann CHW, Flechtmann CAH (1984) Reproduction and chromosomes in the broad mite, Polyphagotarsonemus latus (Banks, 1904) (Acari: Prostigmata, Tarsonemidae). In Griffiths DA, Bowman CE (Eds.), Acarology VI. Ellis Horwood, Chichester, UK pp. 455-456

Flechtmann CHW, Guerrero BJM, Arroyave JA, Constantino CHLM (1990) A little known mode of dispersal of Polyphagotarsonemus latus (Banks). Int. J. Acarol. 16:181-182

Fonseca S, Chico JM, Solano R (2009) The jasmonate patway: The ligand, the receptor and the core signaling module. Curr. Opin. Plant Biol. 12:539-547

Footitt S, Dietrich D, Fait A, Fernie AR, Holdsworth MJ, Baker A, Thedoulou FL (2007) The COMATOSE ATP-binding cassette transporter is required for full fertility in Arabidopsis . Plant Physiol. 146:1467- 1480

Footitt S, Slocombe SP, Larner V, Kurup S, Wu Y, Larson T, Graham I, Baker A, Holdsworth M (2002) Control of germination and lipid mobilization by COMATOSE, the Arabidopsis homologue of human ALPD. EMBO J. 21:2912-2922

Forneck A, Merkt N, Blaich R (1998) A tripartite aseptic culture system for grape ( Vitis spp.) phylloxera (Daktulosphaera vitifoliae ) and mites ( Tarsonemus sp.). Vitis 37:95-96

Fox Wilson G (1950) The broad mite. J. R. Hort. Soc. 75:69-74

Friedrich L, Lawton K, Ruess W, Masner P, Specker N, Gut-Rella M, Meier B, Dincher S, Staub T, Uknes S (1996) A benzothiadiazole derivative induces systemic acquired resistance in tobacco. Plant J. 10:61- 70

Fu ZQ, Yan SP, Saleh A, Wang W, Ruble J, Oka N, Mohan R, Spoel SH, Tada Y, Zheng N, Dong X (2012) NPR3 and NPR1 are receptors for the immune signal salicylic acid in plants. Nature 486:228-232

Fürstenberg-Hägg J, Zagrobelny M, Bak S (2013) Plant defence against insect herbivores. Int. J. Mol. Sci. 14:10242-10297

Gadd CH (1946) Observations on the yellow tea mite Hemitarsonemus latus (Banks) Ewing. Bull. Entomol. Res. 37:157-162

Gambino G, Perrone I, Gribaudo I (2008) A rapid and effective method for RNA extraction from different tissues of grapevine and other woody plants. Phytochem. Anal. 19:520-525

Garcion C, Lohmann A, Lamodière E, Catinot J, Buchala A, Doermann P, Métraux J-P (2008) Characterization and biological function of the ISOCHORISMATE SYNTHASE2 gene of Arabidopsis . Plant Physiol. 147:1279-1287

157

Gatehouse JA (2002) Plant resistance towards insect herbivores: a dynamic interaction. New Phytol. 156:145-169

Gerson U (1992) Biology and control of the broad mite, Polyphagotarsonemus latus (Banks) (Acari: Tarsonemidae). Exp. Appl. Acarol. 13:163-178

Gfeller A, Dubugnon L, Liechti R, Farmer EE (2010) Jasmonate biochemical pathway. Sci. Signal 16:3(109):cm3. doi:10.1126/scisignal.3109cm3

Ghangal R, Raghuvanshi S, Sharma PC (2009) Isolation of good quality RNA from medicinal plant seabuckthorn, rich in secondary metabolites. Plant Physiology and Biochemistry 47:1113-1115

Ghanzy NA, Suzuki T, Amano H, Ohyama K (2012) Effects of air temperature and water vapor pressure deficit on storage of the predatory mite Neoseiulus californicus (Acari: Phytoseiidae). Exp. Appl. Acarol. 58:111-120

Gibson RW, Valencia L (1978) A survey of potato species for resistance to the mite Polyphagotarsonemus latus , with particular reference to the protection of Solanum berthaultii and S. tarijense by glandular hairs. Potato Res. 21:217-223

Glas JJ, Schimmel BCJ, Alba JM, Escobar-Bravo R, Schuurink RC, Kant MR (2012) Plant glandular trichomes as targets for breeding or engineering of resistance to herbivores. Int. J. Mol. Sci. 13:17077- 17103

Glazebrook J (2005) Contrasting mechanisms of defence against biotrophic and necrotrophic pathogens. Annu. Rev. Phytopathol. 43:205-227

Gobin B, Audenaert J, Vissers M, Van Delsen B, Vlaeminck M, Pauwels E (2013) Broad mite control in woody ornamentals: developing an Integrated Pest Management solution. In: J. Van Huylenbroeck et al. (Eds.) Proc. 2 nd international symposium on woody ornamentals of the moderate zone. Acta Hort. 990:47-51

Gobin B, Vissers M, Pauwels E (2011) Integrated Pest Management solutions for the control of Polyphagotarsonemus latus in ornamentals: from trial to practice. IOBC-WPRS Bulletin 68:47-50

Goetsch L, Eckert AJ, Hall BD (2005) The molecular systematics of Rhododendron (Ericaceae): a phylogenic based upon RBP2 gene sequences. Systematic Botany 30(3):616-626

Goodspeed D, Chehab EW, Min-Venditti A, Braam J, Covington MF (2012) Arabidopsis synchronizes jasmonate-mediated defense with insect circadian behaviour. PNAS 109(12):4674-4677

Grinberg M, Alagarmalai J, Sharon L, Perl-Treves R, Soroker V (2007). The effect of jasmonic acid signalling pathway on tomato defence to broad mite behaviour. Isr. J. Entomol. 37:389-390

Grinberg M, Perl-Treves R, Palevsky E, Shomer I, Soroker V (2005) Interaction between cucumber plants and the broad mite , Polyphagotarsonemus latus : from damage to defence gene expression. Entomologia Experimentalis et Applicata 115:135-144

Grinberg-Yaari M, Alagarmalai J, Lewinsohn E, Perl-Terves R, Soroker V (2015) Role of jasmonic acid signaling in tomato defense against broad mite, Polyphagotarsonemus latus (Acari: Tarsonemidae). Arthropod-Plant Interactions doi: 10.1007/s11829-015-9372-1

Gui L, Gong X, Meng G, Gui LY, Gong XW, Meng GL (2001) On the relationship between eggplant leaf structure and its resistance to broad mite. Acta Phytophylacica Sinica 28:213-217

Gundannavar KP, Giraddi RS, Kulkarni KA, Awaknavar JS (2007) Development of integrated pest management modules for chilli pests. Karnataka J. Agric. Sci. 20(4):757-760

Gupta PC, Chaundhry HS (1972) New record of Hemitarsonemus latus Banks (Tarsonemidae) as a parasite of Bemisia gossypiperda M. & L. Indian J. Entomol. 33:476

Haegeman A, Mentelin S, Jones JT, Gheysen G (2012) Functional roles of effectors of plant-parasitic nematodes. Gene 492:19-31

Hahlbrock K, Scheel D (1989) Physiology and molecular biology of phenylpropanoid metabolism. Annu. Rev. Plant Physiol. Plant Mol. Biol. 40:347-369

Halitschke R, Stenberg JA, Kessler D, Kessler A, Baldwin IT (2008) Shared signals – ‘alarm calls’ from plants increase apparency to herbivores and their enemies in nature. Ecol Lett 11:24-34.

Hamiduzzaman M Md, Jakab G, Barnavon L, Neuhaus J-M, Mauch-Mani B (2005) Beta-aminobutyric acid-induced resistance against downy mildew in grapevine acts through the potentiation of callose formation and jasmonic acid signalling. MPMI 18(8):819-829

Hart AJ, Bale JS, Tullet AG, Worland MR, Walters KFA (2002) Effects of temperature on the establishment potential of the predatory mite Amblyseius californicus McGregor (Acari: Phyoseiidae) in the UK. J Insect Physiol 48:593-599

Haruta M, Pedersen JA, Constabel CP (2001) Polyphenol oxidase and herbivore defense in trembling aspen ( Populus tremuloides ): cDNA cloning, expression, and potential substrates. Physiol. Plant 112:552- 558

Hayashi M, Nito K, Takei-Hoshi R, Yagi M, Kondo M, Suenaga A, Yamaya T, Nishimura M (2002) Ped3p is a peroxisomal ATP-binding cassette transporter that might supply substrates for fatty acid beta- oxidation. Plant Cell Physiol. 43:1-11

Heitz T, Bergey DR, Ryan CA (1997) A gene encoding a chloroplast-targeted lipoxygenase in tomato leaves is transiently induced by wounding, systemin, and methyl jasmonate. Plannat Physiol. 114:1085-1093

Heitz T, Wildemann E, Lugan R, Miesch L, Ullmann P, Désaubry L, Holder E, Grausem B, Kandel S, Miesch M, Werck-Reichhart D, Pinot F (2012) Cytochromes P450 CYP94C1 and CYP94B3 catalyze two successive oxidation steps of plant hormone jasmonoyl-L-isoleucine for catabolic turnover. J. Biol. Chem. 287:6296-6306

Hellemans J, Mortier G, De Paepe A, Speleman F, Vandesompele J (2007) qBase relative quantification framework and software for management and automated analysis of real-time quantitative PCR data. Genome Biol. 8:R19

Herrmann G, Lehmann J, Parthier B, Sembdner G (1992) Interaction of cytokinins and jasmonic acid in Barley leaf senescence. In: Kaminek M, Mok DWS, Zazimolova E (Eds.). Physiology and biochemistty of cytokinins in plants. SPB Academic, The Hague, the Netherlands. pp 277-281

Heungens A (1986) Soft-skinned mites in azalea culture and comparable control results on other host plants. Verbondsnieuws voor de Belgische Sierteelt 30:257-269 (in Dutch)

Heursel J (1999) Azalea’s oorsprong, veredeling en cultivars. Lannoo-Terra, Tielt, Belgium, 192 pp

Higa SY, Namba R (1971) Vectors of the papaya mosaic virus in Hawaii. Proc. Hawaiian Entomol. Soc. 21:93-96

159

Hilbert DW, Logan JA (1983) Empirical model of nymphal development for the migratory grasshopper, Lobesia botrana (Orthoptera: acrididae). Environ. Entomol. 12:1-5

Honek A, Kocourek F (1988) Thermal requirements for development of aphidophagous Coccinellidae (Coleoptera), Chrysopidae, Hemerobiidae (Neuroptera), and Syrphidae (Diptera): some general trends. Oecologia 76:455-460

Horn W (2002) Breeding methods and breeding research. In: Vainstein A (Ed.) Breeding for ornamentals: classical and molecular approaches. Kluwer Academic Publishers, Dordrecht, The Netherlands pp. 47- 84

Howe GA (2005) Jasmonates. In: Davies PJ (Ed) Plant hormones: biosynthesis signal transduction and action! Kluwer Academic Publishers pp. 610-634

Howe GA, Jander G (2008) Plant immunity to insect herbivores. Annu. Rev. Plant Biol. 59:41-66

Howe GA, Lightner J, Browse J, Ryan CA (1996) An octadecanoid pathway mutant (JL5) of tomato is compromised in signaling for defense against insect attack. Plant Cell 8:2067-2077

Hoy M (2011) Agricultural acarology: introduction to integrated mite management. Chapter 7: The Tarsonemidae. CRC Press, Boca Raton, FL, USA pp 103-108

Huang X, Madan A (1999) CAP3: A DNA sequence assembly program. Genome Res. 9(9):868-877

Hugon R (1983) Biologie et écologie de Polyphagotarsonemus latus Banks, ravageur sur agrumes aux Antilles. Fruits 38:635-646

Hussey NW, Gurney B (1967) Bionomics and control of Tarsonemus myceliophagus (Acarina: Tarsonemidae) in mushroom composts. Entomologia Experimentalis et Applicata 10(3-4):287-294

Hyun, Y, Choi S, Hwang HJ, Yu J, Nam SJ, Ko P, Park JY, Seo YS, Kim EY, Ryu SB, Kim WT, Lee YH, Kang H, Lee I (2008) Cooperation and functional diversification of two closely related galactolipase genes for jasmonate biosynthesis. Dev. Cell 14:183-192

Ikemoto T, Takai K (2000) A new linearized formula for the law of total effective temperature and the evaluation of line-fitting methods with both variables subject to error. Environ. Entomol. 29:671-682

Ishiguro S, Kawai-Oda A, Ueda J, Nishida I, Okada K (2001) The DEFECTIVE IN ANTHER DEHISCENCE gene encodes a novel phospholipase A1 catalyzing the initial step if jasmonic acid biosynthesis, which synchronizes pollen maturation, anther dehiscence, and flower opening in Arabidopsis . Plant Cell 13: 2119-2009

Jana SK, Nair N, Sarkar PK, Somchoudhury AK (2007) Varietal susceptibility of jute varieties against yellow mite, Polyphagotarsonemus latus Banks (Acari: Tarsonemidae). Journal of Entomological Research 31(1):29-32

Jeppson LR, Keifer HH, Baker EW (1975) Mites injurious to economic plants. University of California Press, Berkeley, xxiv + 614pp

Joas R, Cotillon A-C. (2009) Development of guidance for establishing Integrated Pest Management (IPM) principles. Final report, European Commission, Munich, Germany

Jones JD, Dangl JL (2006) The plant immune system. Nature 444:323-329

Jones MT (1988) Status of the broad mite (Polyphagotarsonemus latus) (Banks) – a threat to the vegetable, fruit and ornamental industry of Trinidat. Proc. 2 nd Annual NIHERST Seminar, Trinidad.

Jones VP, Brown RD (1983) Reproductive responses of the broad mite Polyphagotarsonemus latus (Acari: Tarsonemidae), to constant temperature–humidity regimes. Ann. Entomol. Soc. Am. 76:466–469

Jovicich E, Cantliffe DJ, Osbore LS, Stoffella PJ, Simonne EH (2008) Release of Neoseiulus californicus on pepper transplants to protect greenhouse-grown crops from early broad mite ( Polyphagotarsonemus latus ) infestations. In: Mason PG, Gillespie DR, Vincent C (Eds.) Proceedings of the Third International Symposium on Biological Control of Arthropods, Christchurch, New Zealand, pp 347-353

Jung C, Lyou SH, Yeu SY, Kim MA, Rhee S, Kim M, Lee JS, Choi YD, Cheong J-J (2007). Microarray-based screening of jasmonate-responsive genes in Arabidopsis thaliana . Plant Cell. Rep. 26:1053-1063

Kabir AKMF (1979) Bioecology and behaviour of yellow jute mite. In Rodriguez JG (Ed.) Recent Advances in Acarology, Vol. 1. Academic Press, New York, pp. 519-523

Kamoun S (2006) A catalogue of the effector secretome of plant pathogenic oomycetes. Annual Review of Phytopathology 44:41-60

Kamruzzaman ASM, Alam MZ, Miah MRU (2013) Bionomics and fertility life table of the yellow mite, Polyphagotarsonemus latus (Banks) (Acari: Tarsonemidae) in jute ( Corchorus olitorius L.) at different temperature-humidity. Mun. Ent. Zool. 8(1):223-235

Kant M (2006) Variable herbivores, variable plant defenses. The consequences of herbivore variability for direct and indirect defenses of plants. University of Amsterdam, Amsterdam, The Netherlands

Kant MR, Ament K, Sabelis MW, Haring MA, Schuurink RC (2004) Differential timing of spider mite- induced direct and indirect defences in tomato plants. Plant Physiol. 135:483-495

Kant MR, Sabelis MW, Haring MA, Schuurink RC (2008) Intraspecific variation in a generalist herbivore accounts for differential induction and impact of host plant defences. Proceedings. Biological Sciences/The Royal Scociety 275:443-452

Karl E (1965) Untersuhungen zur Morphologie und Ökologie von Tarsonemiden gärtnerischer Kulturpflanzen. II Hemitarsonemus latus (Banks), Tarsonemus confusus Ewing, T. talpae Schaarschmidt, T. setifer Ewing, T. smithi Ewing und Tarsonemoides belemnitoides Weis-Fogh. Biol. Zentralbl. 84:331- 357 (in German)

Katsir L, Chung HS, Koo AJK, Howe GA (2008a) Jasmonate signaling: a conserved mechanism of hormone sensing. Curr. Opn. Plant Biol. 11:428-435

Katsir L, Schilmiller AL, Staswick PE, He SY, Howe GA (2008b) COI1 is a critical component of a receptor for jasmonate and the bacterial virulence factor coronatine. Proc. Natl Acad. Sci. USA 105:7100-7105

Kazan K, Manners JM (2008) Jasmonate signaling: toward an integrated view. Plant Physiol. 146: 1459- 1468

Kazan K, Manners JM (2012) JAZ repressors and the orchestration of phytohormone crosstalk. Trends Plant Sci. 17:22-31

Keever GJ (1990) Response of two forcing azalea cultivars to Bonzi and B-nine applications. Journal of Environmental Horticulture 8:182-184

Kitaoka N, Matsubara T, Sato M, Takahashi K, Wakuta S, Kawaide H, Matsui H, Nabete K, Matsuura H (2011) Arabidopsis CYP94B3 encodes jasmonyl-L-isoleucine 12-hydroxylase, a key enzyme in the oxidative catabolism of jasmonate. Plant Cell Physiol. 52:1757-1765

161

Klämbt HD (1962) Conversion in plants of benzoic acid to salicylic acid and is beta-D-glucoside. Nature 196:491

Kohler A, Schwindling S, Conrath U (2002) Benzothiadiazole-induced priming for potentiated responses to pathogen infection, wounding, and infiltration of water into leaves requires the NPR1/NIM1 gene in Arabidopsis . Plant Physiol. 128:1046-1056

Kontodimas DC, Eliopoulos PA, Stathas GJ, Economou LP (2004) Comparative temperature-dependent development of Nephus includens (Kirsch) and Nephus bisignatus (Boheman) (Coleoptera: Coccinellidae), preying on Planococcus citri (Risso) (Homoptera: Pseudococcidae): evaluation of a linear and various non-linear models using specific criteria. Environ. Entomol. 33:1-11

Koo AJ, Howe GA (2007) Role of peroxisomal beta-oxidation in the production of plant signaling compounds. Plant Signal. Behav. 2:20-22

Koo AJK, Cooke TF, Howe GA (2011) Cytochrome P450 CYP94B3 mediates catabolism and inactivation of the plant hormone jasmonoyl-L-isoleucine. Proc. Natl. Acad. Sci. USA 108:9298-9303

Koo AJK, Gao X, Jones AD, Howe GA (2009) A rapid wound signal activates the systemic synthesis of bioactive jasmonates in Arabidopsis . The plant Journal 59:974-986

Kousik CS, Shepard BM, Hassell R, Levi A, Simmons AM (2007) Potential sources of resistance to broad mites ( Polyphagotarsonemus latus ) in watermelon germplasm. HortScience 42(7):1539-1544

Krantz GW (1978) A manual of acarology. 2 nd ed. Oregan State University Book Stores, Corvalis, OR, USA

Król P, Igielski R, Pollmann S, Kepczynska E (2015) Priming of seeds with methyl jasmonate induced resistance to hemi-biotroph Fusarium oxysporum f.sp. lycopersici in tomato via 12-oxophytodienoic acid, salicylic acid, and flavonol accumulation. Journal of Plant Physiology doi: 10.1016/j.jplph.2015.01.018

Krupp G (2005) Stringent RNA quality control using the Agilent 2100 bioanalyzer. Agilent Technologies, application note

Kulkarni SK, Gasti VD, Mulge R, Madalageri MB, Kulkarni MS, Shirol AM (2011) Reaction of chilli genotypes against mites, [ Polyphagotarsonemus latus (Banks)] and thrips, [ Scirthothrips dorsalis (Hood)] under natural conditions. Karnataka J. Agric. Sci. 24(2):258-259

Kvalseth TO (1985) Cautionary note about R². Am. Statistician 9:279-285

Labanowski G, Soika G (2006) Tarsonemid mites on ornamental plants in Poland: new data and an overview. Biological Lett. 43(2):341-346

Lactin DJ, Holliday NJ, Johnson DL, Craigen R (1995) Improved rate model of temperature-dependent development by arthropods. Environ. Entomol. 24:68-75

Lamesch P, Berardini TZ, Li D, Swarbreck D, Wilks C, Sasidharan R, Muller R, Dreher K, Alexander DL, Garcia-Hernandez M, Karthikeyan AS, Lee CH, Nelson WD, Ploetz L, Singh S, Wensel A, Huala E (2012) The Arabidopsis Information Resource (TAIR): improved gene annotation and new tools. Nucleic Acids Res. 40:D1202-1210

Laudert D, Pfannschmidt U, Lottspeich F, Holländer-Czytko H, Weiler EW (1996) Cloning, molecular and functional characterization of Arabidopsis thaliana allene oxide synthase (CYP74), the first enzyme of the octadecanoid pathway to jasmonates. Plant. Mol. Biol. 31:323-335

Laudert D, Weiler EW (1998) Allene oxide synthase: a major control point in Arabidopsis thaliana octadecanoid signalling. Plant J. 15:675-684

Lavoipierre MMJ (1940) Hemitarsonemus latus (Banks) (Acarina), a mite of economic importance new to South Africa. J. Entomol. Soc. South Afr. 3:116-123

Lawton KA, Friedrich L, Hunt M, Weymann K, Delaney T, Kessmann H, Staub T, Ryals J (1996) Benzothiadiazole induces disease resistance in Arabidopsis by activation, of the systemic acquired resistance signal transduction pathway. Plant J. 10:71-82

León J, Lawton MA, Raskin I (1995) Hydrogen peroxide stimulates salicylic acid biosynthesis in tobacco. Plant Physiol. 108:1673-1678

León J, Yalpini N, Raskin I, Lawton MA (1993) Induction of benzoic acid 2-hydroxylase in virus-inoculated tobacco. Plant Physiol. 103:323-328

Leslie CA (2004) The international Rhododendron register & checklist. Second Edition. Royal Horticultural Society, London, UK, 1544 pp

Li C, Schilmiller AL, Liu G, Lee GI, Jayanty S, Sagema, C, Vrebalov J, Giovannoni JJ, Yagi K, Kobayashi Y, Howe GA (2005) Role of β-oxidation in jasmonate biosynthesis and systemic wound signalling in tomato. Plant Cell 17:971

Li C, Williams MM, Loh YT, Lee GI, Howe GA (2002) Resistance of cultivated tomato to cell content-feeding herbivores is regulated by the octadecanoid-signaling pathway. Plant Physiol. 130(1):494-503

Li J, Yanyun Y, Yunfang Q, Qianhong W (2003) Experimental population life table of Amblyseius cucumeris with Polyphagotarsonemus latus as prey. Acta Phytophylacica Sin. 30(4):389-395 (in Chinese)

Li L, Zhao Y, McCaig BC, Wingerd BA, Wang J, Whalon WE, Pichersky E, Howe GA (2004) The tomato homolog of coronatine-insensitive-1 (COI1) is required for the maternal control of seed maturation, jasmonate-signaled defence responses, and glandular trichome development. Plant Cell 16:126-143

Li LS, Li YR, Bu GS (1985) The effect of temperature and humidity on the growth and development of the broad mite, Polyphagotarsonemus latus . Acta Entomol. Sinica 28:181–187 (in Chinese)

Li Q, Xie QG, Smith-Becker J, Navarre DA, Kaloshian I (2006) Mi-1-mediated aphid resistance involves salicylic acid and mitogen activated protein kinase signalling cascades. Mol. Plant-Microbe Interact. 19:655-664

Li Y, Yan HF, Ge XJ (2012) Phylogeographic analysis and environmental niche modelling of widespread shrub Rhododendron simsii in China reveals multiple glacial refugia during the last glacial maximum. Journal of Systematics and Evolution 50:362-373

Lianyou G, Guoling M, Xinwen G, Sanhao X (1999) Field resistance of eggplant varieties of Polyphagotarsonemus latus . Journal of Hubei Agricultural College 19(4):307-309

Lianyou G, Xinwen G, Gouling M, Sanhao X (2001) The relationship between the stoma density of eggplant leaves and their resistance to Polyphagotarsonemus latus . Acta Horticulturae Sinica 28(2):170-172 (in Chinese)

Liao Z, Chen M, Guo L, Gong Y, Tang F, Sun X, Tang K (2004) Rapid isolation of high-quality total RNA from Taxus and Ginkgo. Prep. Biochem. Biotechnol. 34:209-214

Lin J, Nakao H, Saito Y (2002) A new species of Tarsonemus (Acari: Tarsonemidae), preying on eggs of Schizotetranychus longus (Acari: Tarsonemidae). J. Acarol. Soc. Jnp. 11(2):85-89

Lindquist EE (1969) Review of Holarctic tarsonemid mites (Acarina: Prostigmata) parasitizing eggs of pine bark beetles. Mem. Entomol. Soc. Can. 60:1-111

163

Lindquist EE (1983) Some thoughts on the potential for use of mites in biological control, including a modified concept of ‘parasitoids’. In: Biological Control of Pests by Mites (Ed. by MA Hoy, GL Cunningham and L Knutson), pp. 12-20. University of California, Special Publ. No. 3304, Berkeley, CA

Lindquist EE (1986) The world genera of Tarsonemidae (Acari: Heterostigmata): morphological, phylogenetic, and systematic revision, with a reclassification of family-group taxa in the Heterostigmata. Mem. Entomol. Soc. Can. 136:1-517

Logan JA, Wollkind DJ, Hoyt SC, Tanigoshi LK (1976) An analytical model for description of temperature dependent rate phenomena in arthropods. Environ. Entomol. 5:1133-1140

Loulakakis KA, Roubelakis-Angelakis KA, Kanellis AK (1996) Isolation of functional RNA from grapevine tissues poor in nucleic acid content. Am. J. Eneol. Vitic 47:181-185

Luu-The V, Paquet N, Calvo E, Cumps J (2005) Improved real-time RT-PCR method for high-throughput measurements using second derivative calculation and double correction. Biotechniques 2005 38:287- 293

Luypaert G, Mechant E (2014) Inzicht in gevoeligheid voor weekhuidmijten bij azalea cultivars. Sierteelt&Groenvoorziening 16:22-23

Luypaert G, Witters J, Van Huylenbroeck J, Maes M, De Riek J, De Clercq P (2014) Temperature- dependent development of the broad mite Polyphagotarsonemus latus (Acari: Tarsonemidae) on Rhododendron simsii . Exp Appl Acarol 6(3):389-400

Lyons R, Manners JM, Kazan (2013) Jasmonate biosynthesis and signaling in monocots: a comparative overview. Plant Cell Rep. 32(6):815-827

Maere S, Heymans K, Kuiper M (2005) BiNGO: a Cytoscape plugin to access overrepresentation of gene ontology categories in biological networks. Bioinformatics 21(16):3448-3449

Maketon M, Orosz-Coghlan P, Sinprasert J (2008) Evaluation of Metarhizium anisopliae (Deuteromycota: Hyphomycetes) for control of broad mite Polyphagotarsonemus latus (Acari: Tarsonemidae) in mulberry. Exp. Appl. Acarol. 46(1-4):157-167

Malavolta C, Boller EF, Wijnands FG (2005) Guidelines for integrated production of field grown vegetables. IOBC/WPRS bulletin 28:1-24

Malon LA, Burgess EPJ (2000) Interference of protease inhibitors on non-target organisms. In: Michaud D (Ed.) Recombinant protease inhibitors in plants. Landes Bioscience, Georgetown, TX, USA. pp 89-106

Marosz A, Matysiak B (2005) Influence of growth retardants on growth and flower bud formation in rhododendron and azalea. Dendrobiology 54:35-40

Marti G, Erb M, Boccard J, Glausier G, Doyen GR, Villard N, Robert CAM, Turlings TCJ, Rudaz S, Wolfender J-L (2013) Metabolomics reveals herbivore-induced metabolites of resistance and susceptibility in maize leaves and roots. Plant, Cell and Environment 36(3):621-639

Martin NA (1991) Scanning electron micrographs and notes on broad mite Polyphagotarsonemus latus (Banks), (Acari: Heterostigmata: Tarsonemidae). New Zealand Journal of Zoology 18:353-35

Martin T, Assogba-Komlan F, Sidick I, Ahle V, Chandre F (2010) An acaricide-treated net to control phytophagous mites. Crop Protection 29:470-475

Matos CHC, Pallini A, Venzon M, De Freitas RCP, Rezende DDM, Schoereder JH (2009) Os Tricomas de Capsicum spp. interferem nos aspectos biológicos do ácaro-branco, Polyphagotarsonemus latus Banks (Acari: Tarsonemidae)? Neotropical Entomology 38(5):589-594

Matthewman WG, Pielou DP (1971) Arthropods inhabiting the sporophore of Fomes fomentarius (Polyporaceae) in Gatineau Park, Quebec. Canadian Entomologist 103:775-847

Mauch F, Mauch-Mani B, Gaille C, Kull B, Haas D, Reimmann C (2001) Manipulation of salicylate content in Arabidopsis thaliana by the expression of an engineered bacterial salicylate synthase. Plant J. 25:67- 77

McCullagh P, Nelder JA (1989) Generalized linear models. Chapman & Hall, New York, p 511

McGurl B, Pearce G, Orozco-Cardenas M, Ryan CA (1992) Structure, expression, and antisense inhibition of the systemin precursor gene. Science 255:1570-1573

Mechant E, Luypaert G, Van Delsen B, Pauwels E, Witters J, Van Huylenbroeck J, Gobin B (2015) Development and validation of a three-step detection protocol for broad mites ( Polyphagotarsonemus latus ) on pot azalea ( Rhododendron simsii hybrids). Entomologia Experimentalis et Applicata DOI: 10.1111/eea.12315

Meijón M, Rodríguez R, Jesús Cañal M, Feito T (2009) Improvement of compactness and floral quality in azalea by means of application of plant growth regulators. Scientia Horticulturae 119:169-176

Melotto M, Mecey C, Niu Y, Chung HS, Katsir L, Yao J, Zeng W, Thines B, Staswick P, Browse J, Howe G, He SY (2008) The bacterial toxin coronatine and the plant hormone jasmonoyl isoleucine target the physical interaction between the Arabidopsis COI1 F-box protein and the Jas domain of JAZ repressor proteins. Plant J. 55:979-988

Memelink J (2009) Regulation of gene expression by jasmonate hormones. Phytochem. 70:1560-1570

Miersch O, Neumerkel J, Dippe M, Stenzel I, Wasternack C (2008) Hydroxylated jasmonates are commonly occurring metabolites of jasmonic acid and contribute to a partial switch-off in jasmonate signaling. New Phytol. 177:114-127

Miersch O, Wasternack C (2000) Octadecanoid and jasmonate signaling in tomato ( Lycopersicon esculentum Mill.) leaves: endogenous jasmonates do not induce jasmonate biosynthesis. Biol. Chem. 381(8):715-722

Mikunthan G, Manjunatha M (2010) Effect of monocrotophos and the acaropathogen, Fusarium semitectum , on the broad mite, Polyphagotarsonemus latus , and its predator, Amblyseius ovalis in the field. In: Trends in acarology: Proceedings of the 12 th international congress (Ed. by MW Sabelis and J Bruin), pp. 489-492. Springer, the Netherlands

Mithöfer A, Boland W (2012) Plant defence against herbivores: chemical aspects. Annu. Rev. Plant Biol. 63:431-450

Mithöfer A, Wanner G, Boland W (2005) Effect of feeding Spodoptera littoralis on lima bean leaves. II. Continuous mechanical wounding resembling insect feeding is sufficient to elicit herbivory-related volatile emission. Plant Physiol. 137:1160-1168

Mizuno T, Yamashino T (2008) Comparative transcriptome of diurnally oscillating genes and hormone- responsive genes in Arabidopsis thaliana : insight into circadian clock-controlled daily responses to common ambient stresses in plants. Plant Cell Physiol. 49(3):481-487

165

Montasser AA, Hanafy ARI, Marzouk AS, Hassan GM (2011b) Description of life cycle stages of the broad mite Polyphagotarsonemus latus (Banks, 1904), (Acari: Tarsonemidae) based on light and scanning electron microscopy. Int. J. Environ. Sci. Eng. 1:59-72

Montasser AA, Taha AM, Hanafy ARI, Hassan GM (2011a) Biology and control of the broad mite Polyphagotarsonemus latus (Banks, 1904) (Acari: Tarsonemidae). Int. J. Environ. Sci. Eng. 1:26-34

Moran PJ, Thompson GA (2001) Molecular responses to aphid feeding in Arabidopsis in relation to plant defense pathways. Plant Physiol. 125:1074-1085

Morante-Carriel J, Sellés-Marchant S, Martínez-Márquez A, Martínez-Esteso MJ, Luque I, Bru- Martínez R (2014) RNA isolation from loquat and other recalcitrant woody plants with high quality an yield. Anal. Biochem. 452:46-53

Morewood WD (1992) Cold hardiness of Phyoseiulus perisimilis Athias-Henriot and Amblyseius cucumeris (Oudemans) (Acarina: Phytoseiidae). Can Entomol 124(6):1015-1025

Motallebi P, Niknam V, Ebrahimzadeh H, Hashemi M, Pisi A, Prodi A, Tonti S, Nipoti P (2015) Methyl jasmonate strengthens wheat plants against root and crown rot pathogen Fusarium culmorum infection. J. Plant Growth Regul. DOI: 10.107/s00344-015-9496-7

Moutia LA (1958) Contribution to the study of some phytophagous Acarina and their predators in Mauritius. Bull. Entomol. Res. 49:59-75

Musser RA, Cipollini DF, Hum-Musser SM, Williams SA, Brown JK, Felton GW (2005) Evidence that the caterpillar salivary enzyme glucose oxidase provides herbivore offense in Solanaceous plants. Archives of Insect Biochemistry and Physiology 58:128-137

Musser RO, Hum-Musser SM, Eichenseer H, Peiffer M, Ervin G, Murphy JB, Felton GW (2002) Caterpillar saliva beats plant defences. Nature 416:599-600

Müssig C, Biesgen C, Lisso J, Uwer U, Weiler EW, Altmann T (2000) A novel stress-inducible 12- oxophytodienoate reductase from Arabidopsis thaliana provides a potential link between brassinosteroid-action and jasmonic acid synthesis. J. Plant Physiol. 157:143-152

Mysore KS, Ryu CM (2004) Nonhost resistance: how much do we know? Trends Plant Sci. 9:97-104

Nakatsuka A, Mizuta D, Kii Y, Myajima I, Kobayashi N (2008) Isolation and expression analysis of flavonoid biosynthesis genes in evergreen azalea. Sci. Hort. 118:314-320

Nantarajan K (1988) Transport of yellow mite Polyphagotarsonemus latus by cotton whitefly. Curr. Sci. 57:1142-1143

Navajas M, Migeon A, Estrada-Pena A, Mailleux AC, Servigne P, Petanovic R (2010) Chapter 7.4: The Acari. In: Roques A et al. (ed) Invasive terrestrial invertebrates in Europe. BioRisk (special issue) pp 149- 192

Newbury HJ, Possingham JV (1977) Factors affecting the extraction of intact ribonucleic acid from plant tissues containing interfering phenolic compounds. Plant Physiol. 60:543-547

Nita M, Ellis MA, Wilson LL, Madden LV (2006) Evaluation of a disease warning model for Phomopsis cane and leaf spot of grape: a field study. Plant Disease 90(9):1239-1246

Nolan T, Hands RE, Bustin SA (2006b) Quantification of mRNA using real-time RT-PCR. Nat. Protoc. 1:1559-1582

Nolan T, Hands RE, Ogunkolade BW, Bustin SA (2006a) SPUD: a qPCR assay for the detection of inhibitors in nucleic acid preparations. Anal. Biochem. 351:308-310

Nour El-Deen MA, Abo-Zid AE, Azouz HA (2014) Use of some environmentally safe materials as alternatives to the chemical pesticides in controlling Polyphagotarsonemus latus (Banks) mite & Myzus persica (Koch) aphid which attack potatoes crop. Middle East Journal of Agriculture Research 3(1):32- 41

Novakova M, Motyka V, Dobrev PI, Malbeck J, Gaudinova A, Vankova R (2005) Diurnal variation of cytokinin, auxin, and abscisic acid levels in tobacco leaves. J. Exp. Bot. 56:2887-2883

Nucifora A (1963) Osservaziono sulla riproduzioni de Hemitarsonemus latus (Banks) (Acarina Tarsonemidae). Atti Accad. Ital. Entomol. 10:142-153

Nugroho I, Ibrahim YB (2004) Laboratory study of some entomopathogenic fungi against broad mite (Polyphagotarsonemus latus Banks). International Journal of Agriculture & Biology 6(2):223-225

Ochoa R, Smiley RL, Saunders JL (1991) The family Tarsonemidae in Costa Rica (Acari: Heterostigmata). Int. J. Acarol. 10:142-153

Omer AD, Granett J, Kerban R, Villa EM (2001) Chemically-induced resistance against multiple pests in cotton. Int. J. Pest Man. 47:49-54

Onzo A, Houedokoho AF, Hanna R (2012) Potential of the predatory mite, Amblyseius swirskii to suppress the broad mite, Polyphagotarsonemus latus , on the gboma eggplant, Solanum macrocarpon . J. Insect. Sci. 12:7 doi: 10.1673/031.012.0701

Owen EL, Bale JS, Hayward SAL (2013) Can winter-active bumblebees survive the cold? Assessing the cold tolerance of Bombus terrestris audax and the effects of pollen feeding. PLoS ONE 8(11):e80061.

Pajerowska-Mukhtar KM, Emerine DK, Mukhtar MS (2013) Tell me more: roles of NPRsin plant immunity. Trents Plant Sci. 18:402-411

Palevsky E, Soroker V, Weintraub P, Mansour F, Abo-Moch F, Gerson U (2001) How species-specific is the phoretic relationship between the broad mite, Polyphagotarsonemus latus (Acari: Tarsonemidae), and its insect hosts? Exp. Appl. Acarol. 25:217-224

Pallini A, Janssen A, Sabelis MW (1997) Odour-mediated responses of phytophagous mites to conspecific and heterospecific competitors. Oecologia 100:179-185

Pang M, Stewart J McD, Zhang J (2011) A mini-scale hot borate method for the isolation of total RNA from a large number of cotton tissue samples. African Journal of Biotechnology 10(68):15430-15437

Parker R, Gerson U (1994) Dispersal of the broad mite, Polyphagotarsonemus latus (Banks) (Heterostigmata: Tarsonemidae), by the greenhouse whitefly, Trialeurodes vaporariorum (Westwood) (Homoptera: Aleyrodidae). Exp. Appl. Acarol. 18:581-585

Patankar R, Beaulieu F, Smith SM, Thomas SC (2012) The life history of gall-inducing mite: summer phenology, predation and influence of gall morphology in a sugar maple canopy. Agricultural and Forest Entomology 14:251-259

Pauwels L, Barbero GF, Geerinck J, Tilleman S, Grunewald W, Pérez AC, Chico JM, Bossche RV, Sewell J, Gil E, García-Casado G, Witters E, Inzé D, Long JA, De Jaeger G, Solano R, Goossens A (2010) NINJA connects the co-repressor TOPLESS to jasmonate signaling. Nature 464:788-791

167

Pauwels L, Goossens A (2011) The JAZ proteins: a crucial interface in the jasmonate signaling cascade. Plant Cell 23:3089-3100

Pearce G, Strydom D, Johnson S, Ryan CA (1991) A polypeptide from tomato leaves induces wound- inducible proteinase inhibitor proteins. Science 253:895-897

Pedras MSC, Ahiahonu PWK (2005) Metabolism and detoxification of phytoalexins and analogs by phytopathogenic fungi. Phytochemistry 66:391-411

Peña JE (1990) Relationships of broad mite (Acari: Tarsonemidae) density to lime damage. Journal of Economic Entomology 83:2008-2015

Peña JE, Bullock RC (1994) Effects of feeding of broad mite (Acari: Tarsonemidae) on vegetative plant growth. Florida Entomologist 77(1):180-184

Peña JE, Campbell CW (2005) Broad mite. Download from DIS. http://edis.ifas.ufl.edu/CH020

Peña JE, Osborne L (1996) Biological control of Polyphagotarsonemus latus (Acarina: Tarsonemidae) in greenhouse and field trials using introductions of predacious mites (Acarina: Phytoseiidae). Entomophaga 41(2):279-285

Perez-Otero R, Mansilla-Vazquez JP, Saliero-Corral MC (2007) First report of the broad mite Polyphagotarsonemus latus (Banks) on Camellia japonica in Spain. American Camellia Yearbook, pp. 52- 56

Pfaffl MW, Hageleit M (2001) Validities of mRNA quantification using recombinant RNA and recombinant DNA external calibration curves in real-time RT-PCR. Biotechnol. Lett. 23:275-282

Pieterse CM, Van Der Does D, Zamioudis C, Leon-Reyes A, Van Wees SC (2012) Hormonal modulation of plant immunity. Annu. Rev. Cell Dev. Biol. 28:489-521

Pieterse CMJ, Leon-Reyes A, Van der Ent S, Van Wees SCM (2009) Networking by small-molecule hormones in plant immunity. Nature Chemical Biology 5(5):308-316

Porat R, Borochov A, Halevy AH (1993) Enhancement of Petunia and Dendrobium flower senescence by jasmonic acid methyl ester via the promotion of ethylene production. Plant Growth Regulation 13:297- 301

Pott C, McLoughmin S, Shunqiung W, Friis EM (2012) Trichomes on the leaves of Anomozamites villosus sp. Nov. (Bennettiales) from the Daohugou beds (Middle Jurassic), Inner Mongolia, China: Mechanical defence against herbivorous arthropods. Rev. Paleobot. Palyno. 169:48-60

Pratt C (1980) Somatic selection & chimeras. In: Moore JN and Janick J (Eds.) Methods in fruit breeding. Purdue University Press, West Lafayette, Indiana, USA pp. 172-185

Proost S, Van Bel M, Vaneechoutte D, Van de Peer Y, Inzé D, Mueller-Roeber B, Vandepoele K (2015) PLAZA 3.0: an access point for plant comparative genomics. Nucleic Acids Res. 43:D974-981

Qing L, Ni Z, Qunfang Y, Haijian W, Chunxian J (2010) Relationship among the nutritional components and pubescence characteristics of pepper cultivars and their resistance to Polyphagotarsonemus latus (Bank). Acta Phytophylacica Sinica 38(1):15-19

Raes J, Rhode A, Christensen JH, Van de Peer Y, Boerjan W (2003) Genome-wide characterization of the lignification toolbox in Arabidopsis . Plant Physiol. 133(3):1051-1071

Rajalakshmi E, Sankaranarayanan P, Pandya RK (2009) The Yellow Mite, Polyphagotarsonemus latus a serious pest of Mulberry under Nilgiris hill conditions. Indian J. Seric. 48:187-190

Reddy SGE, Kumar NKK (2006) Integrated management of the yellow mite, Polyphagotarsonemus latus (Banks), on sweet pepper grown under polyhouse. Journal of Horticultural Sciences 1(2):120-123

Redman A, Cipollini DF, Schultz JC (2001) Fitness costs of jasmonic acid induced defence in tomato, Lycopersicon esculentum . Oecologia 126:380-385

Ren F, Lu YT (2006) Overexpressing of tobacco hydroxyproline-rich glycopeptide systemin precursor a gene in transgenic tobacco enhances against Helicoverpa armigera larvae. Plant Sci. 171:286-292

Rennie J (1921) Isle of Wight disease in hive bees - Acarine disease: The organism associated with the disease Tarsonemus woodi , n. sp. Transactions of the Royal Society of Edinburgh 52:768-779

Reymond P, Bodenhausen N, Van Poecke RM, Krishnamurthy V, Dicke M, Farmer EE (2004) A conserved transcript pattern in response to a specialist and a generalist herbivore. Plant Cell 16:3132- 3147

Reymond P, Farmer EE (1998) Jasmonate and salicylate as global signals for defense gene expression. Curr. Opin. Plant Biol. 1:404-411

Reymond P, Weber H, Damond M, Farmer EE (2000) Differential gene expression in response to mechanical wounding and insect feeding in Arabidopsis . The Plant Cell 12:707-719

Richardson E, Seeley S, Walker D (1974) A model for estimating the completion of rest for Redhaven Elberta peach trees. HortScience 9:331-332

Robert-Seilaniantz A, Grant M, Jones JD (2011) Hormone crosstalk in plant disease and defence: more than just jasmonate-salicylate antagonism. Annu. Rev. Phytopathol. 49:317-343

Robinson MD, Smyth GK (2008) Small-sample estimation of negative binomial dispersion, with applications to SAGE data. Biostatistics 9(2):321-332

Rodríguez H, Montoya A, Miranda I, Rodríguez Y, Ramos M (2011b) Influence of the phenological phase of two pepper cultivars on the behavior of Polyphagotarsonemus latus (Banks). Rev. Proteccíon Veg. 26(2):73-79

Rodríguez H, Ramos M, Montoya A, Rodríguez Y, Chico R, Miranda I, Depestre TL (2011a) Development of Amblyseius largoensis as biological control agent of the broad mites ( Polyphagotarsonemus latus ). Biotecnol. Apl. 28:171-175

Rodríguez-Cruz FA, Venzon M, Pinto CMF (2013) Performance of Amblyseius herbicolus on broad mites and on castor bean and sunnhemp pollen. Exp. Appl. Acarol. 60(4):497-507

Rohde A, Morreel K, Ralph J, Goeminne G, Hostyn V, De Rycke R, Kushnir S, Van Doorsselaere J, Joseleau J-P, Vuylsteke M, Van Driessche G, Van Beeumen J, Messens E, Boerjan W (2004) Molecular phenotyping of the pal1 and pal2 mutants of Arabidopsis thaliana reveals far-reaching consequences on phenylpropanoid, amino acid, and carbohydrate metabolism. The Plant Cell 16(10):2749-2771

Rohwer CL, Erwin JE (2008) Horticultural applications of jasmonates: a review. Journal of Horticultural Science and Biotechnology. 83:283-304

Rohwer CL, Erwin JE (2010) Spider mites ( Tetranychus utricae ) perform poorly on and disperse from plants exposed to methyl jasmonate. Entomologia Experimentalis et Applicata 137(2):143-152

169

Rubio-Piña JA, Zapata-Pérez O (2011) Isolation of total RNA from tissues rich in polyphenols and polysaccharides of mangrove plants. Electronic Journal of Biotechnology 14(5), doi:10.2225/vol15- issue5-fulltext-10

Ruttink T, Sterck L, Rohde A, Bendixen C, Rouzé P, Asp T, Van de Peer Y, Roldan-Ruiz I (2013) Orthology Guided Assembly in highly heterozygous crops: creating a reference transcriptome to uncover genetic diversity in Lolium perenne . Plant Biotechnol. J. 11(5):605-617

Ryals JA, Neuenschwander UH, Willits MG, Molina A, Steiner H-Y, Hunt MD (1996) Systemic acquired resistance. The Plant Cell 8:1809-1819

Salzman RA, Fujita T, Zhu-Salzman K, Hasegawa PM, Bressan RA (1999) An improved RNA isolation method for plant tissues containing high levels of phenolic compounds or carbohydrates. Plant Mol. Biol. Rep. 17:11-17

Samyn G, De Schepper S, Van Bockstaele E (2002) Adventitious shoot regeneration and appearance of sports in several azalea cultivars. Plant Cell, Tissue and Organ Culture 70(2):223-227

Santino A, Taurino M, De Domenico S, Bonsegna S, Poltronieri P, Pastor V, Flors V (2013) Jasmonate signaling in plant development and defence response to multiple (a)biotic stresses. Plant Cell Rep. 32:1085-1098

Sarmento RA, Lemos F, Bleeker PM, Schuurink RC, Pallini A, Oliveira MGA, Lima E, Kant M, Sabelis MW, Janssen A (2011b) A herbivore that manipulates plant defence. Ecology Letters 14:229-236

Sarmento RA, Rodrigues DM, Faraji F, Erasmo EAL, Lemos F, Teodoro AV, Kikuchi WT, dos Santos GR, Pallini A (2011a) Suitability of the predatory mites and Euseius concordis in controlling Polyphagotarsonemus latus and Tetranychus bastosi on Jatropha curcas plants in Brazil. Exp. Appl. Acarol. 53(3):203-214

Sasaki Y, Asamizu E, Shibata D, Nakaura Y, Kaneko T, Axai K, Amagai M, Kuwata C, Tsugane T, Masuda T, Shimada H, Takamiya K, Ohta H, Tabata S (2001) Monotoring of methyl jasmonate-responsive genes in Arabidopsis by cDNA macroarray: self-activation of jasmonic acid biosynthesis and crosstalk with other phytohormone signaling pathways. DNA Res. 8:153-161

Sato D, Sugimoto M, Akashi H, Tomita M, Soga T (2014) Comparative metabolite profiling of foxglove aphids ( Aulacorthum solani Kaltenbach) on leaves of resistant and susceptible soybean strains. Mol. Biosyst. 10(4):909-915

Schaller F (2001) Enzymes of the biosynthesis of octadecanoid-derived signalling molecules. J. Exp. Bot. 52(354):11-23

Schaller F, Biesgen C, Müssig C, Altmann T, Weiler EW (2000) 12-Oxophytodienoate reductase 3 (OPR3) is the isoenzyme invloved in jasmonate biosynthesis. Planta 210(6):979-984

Schaller F, Hennig P, Weiler EW (1998) 12-Oxophytodienoate-10,11reductase: occurrence of two isoenzymes of different specificity against stereoisomers of 12-oxophytodienoic acid. Plant Physiol. 118:1345-1351

Schaller F, Schaller A, Stintzi A (2005) Biosynthesis and metabolism of jasmonates. J. Plant Growth Regul. 23:179-199

Schaller F, Weiler EW (1997) Molecular cloning and characterization of 12-oxophytodienoate reductase, an enzyme of the octadecanoid signalling pathway from Arabidopsis thaliana . Structural and functional relationship to yeast old yellow enzyme. J. Biol. Chem. 272:28066-28072

Scheerlinck H, Baumann J, De Somer F, Haerens A, Van Laere A (1938). Tuinbouwencyclopedie I. De azalea indica L. De Sikkel pp 527

Schmelz EA, Carrol MJ, LeClere S, Phipps SM, Meredith J, Chourey PS, Alborn HT, Teal PEA (2006) Fragments of ATP synthase mediate plant perception of insect attack. Proc. Natl. Acad. Sci. USA 103:8894-8899

Schroeder A, Mueller O, Stocker S, Salowsky R, Leiber M, Gassmann M, Lightfoot S, Menzel W, Granzow M, Ragg T (2006) The RIN: an RNA integrity number for assigning integrity values to RNA measurements. BMC Mol. Biol. 7:3

Schultz DJ, Craig R, Cox-Foster DL, Mumma RO, Medford JI (1994) RNA isolation from recalcitrant plant tissue. Plant Mol. Biol. Rep. 12:310-316

Seo HS, Song JT, Cheong JJ, Lee YW, Hwang I, Lee JS, Choi YD (2001) Jasmonic acid carboxyl methyltransferase: a key enzyme for jasmonate-regulated plant responses. Proc. Natl. Acad. Sci. USA 98:4788-4793

Shahrokhabadi K, Afshari RT, Alizade H, Afshari JT, Javadi GR (2008) Compared two methods for isolating RNA from freezing and nonfreezing bread wheat ( Triticum aestivum L.) plant tissues. Asian Journal of Plant Sciences 7:505-509

Shannon P, Markiel A, Ozier O, Baliga NS, Wang JT, Ramage D, Amin N, Schwikowski B, Ideker T (2003) Cytoscape: a software environment for integrated models of biomolecular interaction networks. Genome Res. 13(11):2498-2504

Silva EA, Oliveira JV, Gondim Jr MGC, Menezes D (1998) Biologia de Polyphagotarsonemus latus (Banks) (Acari : Tarsonemidae) em pimentão. An. Soc. Entomol. Bras. 27:223-228

Simmons AT, Gurr GM, McGrath D, Martin PM, Nicol HI (2004) Entrapment of Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) on glandular trichomes of Lycopersicon species. Austr. J. Entomol. 43:196-200

Singh M, Dhooria MS (2006) Biology, host range and population dynamics of Tarsonemid mite, Polyphagotarsonemus latus (Banks) on chilli ( Capsicum annum ) in Punjab. J. Appl. Zool. Res. 17(1):18-23

Sinha RN (1964) Mites of stored grain in western Canada, ecology and survey. Proc. Entomol. Soc. Manit. 20:19-33

Sivasankar S, Sheldrick B, Rothstein SJ (2000) Expression of allene oxide synthase determines defense gene activation in tomato. Plant Physiol. 122:1335-1342

Sleumer H (1949) Ein system der gattung Rhododendron L. Botanische Jahrbuch Systematics 74:511-553

Smiley RL, Landwehr VR (1976) A new species of Tarsonemus (Acarina: Tarsonemidae), predaceous on tetranychid mite eggs. Ann. Entomol. Soc. Am. 69:1065–1072

Smith FF (1935) Control experiments on certain Tarsonemus mites on ornamentals. J. Econ. Entomol. 28:91- 98

Smith FF (1939) Control of cyclamen and broad mite in gerbera. USDA Cir. No. 516, 14 pp.

Smith LM, Goldsmith EV (1936) The cyclamen mite, Tarsonemus pallidus , and its control on field strawberries. Hilgardia 10(3):53-94

171

Sombatsiri K (1979) Ecological study on insect pests of chilli, tomato and their control. Annual Report, Thailand pp 40-41

Song MK, Kim DG, Lee SH (2005) Isolation and characterization of a jasmonic acid carboxyl methyltransferase gene from hot pepper ( Capsicum annuum L.). Journal of Plant Biology, 48(3):292-297

Soroker V, Nelson RD, Bahar O, Reneh S, Yablonski S, Palevsky E (2003) Whitefly wax as a cue for phoresy in the broad mite, Polyphagotarsonemus latus (Banks) (Acari: Tarsonemidae). Chemoecology 13:163-168

Spoel SH, Mou ZL, Tada Y, Spivey NW, Genschik P, Dong XN (2009) Proteasome-mediated turnover of the transcription coactivator NPR1 plays dual roles in regulating plant immunity. Cell 137:860-872

Staswick PE (2008) JAZing up jasmonate signaling. Trends Plant Sci. 13:66-71

Staswick PE, Tiryaki I (2004) The oxylipin signal jasmonic acid is activated by an enzyme that conjugates it to isoleucine in Arabidopsis . Plant Cell 16:2117-2127

Steffens J, Harel E, Hunt M (1994) Polyphenol oxidase. In Ellis BE, Kuroki GW, Stafford HA (Eds.) Genetic engineering of plant secondary metabolism. Plenum Press, NY, USA, pp. 276-304

Stenzel I, Hause B, Maucher H, Pitzschke A, Miersch O, Ziegler J, Ryan CA, Wasternack C (2003) Allene oxide cyclase dependence of the wound response and vascular bundle-specific generation of jasmonates in tomato – amplification in wound signalling. The Plant journal 33:577-589

Stintzi A, Weber H, Reymond P, Browse J, Farmer EE (2001) Plant defense in the absence of jamonic acid: the role of cyclopentenones. Proc. Natl. Acad. Sci. USA. 98(22):12837-12842

Stout M, Davis J (2009) Chapter 7: Keys to the increased use of host plant resistance in Integrated Pest Management. In Peshin R, Dhawan AK (Eds.) Integrated Pest Management; innovation-development process. Springer, Dordrecht, the Netherlands. pp 163-181

Strawn MA, Marr SK, Inoue K, Inada N, Zubieta C, Wildermuth MC (2007) Arabidopsis isochorismate synthase functional in pathogen-induced salicylate biosynthesis exhibits properties consistent with a role in diverse stress responses. J. Biol. Chem. 282:5919-5933

Stuart JJ, Chen M-S, Shukle R, Harris MO (2012) Gall midges (Hessian flies) as plant pathogens. Annual Review of Phytopathology 50:339-357

Stuart NW (1965) Growth retardants, storage temperature and length of storage for controlling the flowering of greenhouse azaleas. Florists Review 136:14-15

Taksdal G (1973) Interactions between pest and host plant in an attack by broad mite, Hemitarsonemus latus (Banks) (Acarina, Tarsonemidae), on passion fruit, Passiflora edulis Sims. Nor. Entomol. Tidsskr. 20:301-304

Tanigoshi LK, Davis RW (1978) An ultrastructural study of Tetranychus mcdanieli feeding injury to leaves of ‘Red Delicious’ apple (Acari: Tetranychidae). Int. J. Acarol. 4:47-51

Taylor S, Wakem M, Dijkman G, Alsarraj M, Nguyen M (2010) A practical approach to RT-qPCR- publishing data that conform to the MIQE guidelines. Methods 50:S1-S5

Thaler JS, Stout MJ, Karban R, Duffey SS (2001) Jasmonate-mediated induced plant resistance affects a community of herbivores. Ecological Entomolgy 26:312-324

Thellin O, Elmoualij B, Heinen E, Zorzi W (2009) A decade of improvements in quantification of gene expression and internal standard selection. Biotechnol. Adv. 4:323-333

Theodoulou FL, Job K, Slocombe SP, Footitt S, Holdsworth M, Baker A, Larson TR, Graham IA (2005) Jasmonoic acid levels are reduced in COMATOSE ATP-binding cassette transporter mutants. Implications for transport of jasmonate precursors into peroxisomes. Plant Physiol. 137:835-840

Thines B, Katsir L, Melotto M, Niu Y, Mandaokar A, Liu G, Normura K, He SY, Howe GA, Browse J (2007) JAZ repressor proteins are targets of the SCF(COI1) complex during jasmonate signaling. Nature 448:661-665

Thungrabeab M, Boonlertnirun K (2002) Selection for resistant varieties of pepper ( Capsicum spp.) to Polyphagotarsonemus latus . Proceedings of the 19th Rajamangala University of Technology conference. pp. 157-158

Traw MB, Bergelson J (2003) Interactive effects of jasmonic acid, salicylic acid, and gibberellin on induction of trichomes in Arabidopsis . Plant physiology 133:1367-1375

Tytgat TOG, Verhoeven KJF, Jansen JJ, Raaijmakers CE, Bakx-Schotman T, McIntyre LM, van der Putten WH, Biere A, van Damn NM (2013) Plant know where it hurts: root and shoot jasmonic acid induction elicit differential responses in Brassica oleracea . PLoS ONE 8(6):e65502

Ueda J, Kato J (1980) Isolation and identification of a senescence-promoting substance from wormwood (Artemisia absinthium L.). Plant Physiol 66:246-249

Untergasser A, Nijveen H, Rao X, Bisseling T, Geurts R, Leunissen JAM (2007) Primer3Plus, an enhanced web interface to Primer3 Nucleic Acids Research 35: W71-W74, doi:10.1093/nar/gkm306

Uppalapati SR, Ayoubi P, Weng H, Palmer DA, Mitchell RE, Jones W, Bender CL (2005) The phytotoxin coronatine and methyl jasmonate impact multiple phytohormone pathways in tomato. The Plant Journal 42:201-217

Vaissayre M (1986) Chemical control of the mite Polyphagotarsonemus latus (Banks) in cotton fields. Coton Fibres Trop. Eng. Ed. 41:31-44

Van Delsen B, Van Hecke S, Luypaert G, Pauwels E, De Boodt E, Gobin B (2013) Temperature treating azalea cuttings as IPM strategy for broad mite ( P. latus ) control. Comm. Appl. Biol. Sc., Ghent University, 78(2):279

Van Huylenbroeck J, Calsyn E, De Keyser E, Luypaert G (2015) Breeding for biotic stress resistance in Rhododendron simsii . Accepted for publication in Acta Hort.

Van Lenteren JC (2012) The state of commercial augmentative biological control: plenty of natural enemies, but a frustrating lack of uptake. BioControl 57:1-20 van Maanen R, Vila E, Sabelis MW, Janssen A (2010) Biological control of broad mites (Polyphagotarsonemus latus ) with the generalist predator Amblyseius swirskii . Exp. Appl. Acarol. 52:29- 34 van Trier H (2012) Gentse Azalea. Stichting Kunstboek, Oostkamp

Van Wees SCM, Luijendijk M, Smoorenburg I, van Loon LC, Pieterse CMJ (1999) Rhizobacteria- mediated induced systemic resistance (ISR) in Arabidopsis is nog associated with a direct effect on expression of known defense-related genes but stimulates the expression of the jasmonate-inducible gene AtVsp upon challenge. Plant Mol. Biol. 41:537-549

173

Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, De Paepe A, Speleman F (2002) Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol. 3(7):research0034.1-research0034.11

Vangansbeke D, De Schrijver L, Spranghers T, Audenaert J, Verhoeven R, Nguyen DT, Gobin B, Tirry L, De Clercq P (2013) Alternating temperatures affect life table parameters of Phytoseiulus persimilis , Neoseiulus californicus (Acari: Phytoseiidae) and their prey Tetranychus urticae (Acari: Tetranychidae). Exp. Appl. Acarol. 61(3):285-298

Vareli K, Frangou-Lazaridis M (1996) Modification of the acid guanidinium thiocyanate-phenol- chlorofrom method for nuclear RNA isolation. BioTechniques 21:236-237

Vasanthaiah HKN, Katam R, Sheikh MB (2008) Efficient protocol for isolation of functional RNA from different grape tissue rich in polyphenols and polysaccharides for gene expression studies. Electronic Journal of Biotechnology 11(3), doi:10.2225/vol11-issue3-fulltext-5

Venzon M, Rosado MC, Molina-Rugama AJ, Duarte VS, Dias R, Pallini A (2008) Acaricidal efficacy of neem against Polyphagotarsonemus latus (Banks) (Acari: Tarsonemidae) Crop Prot. 27:869–872

Verberne MC, Verpoorte R, Bol JF, Mercado-Blanco J, Linthorst HJM (2000) Overproduction of salicylic acid in plants by bacterial transgenes enhances pathogen resistance. Nature Biotech. 18:779-783

Vichitbandha P, Chandrapatya A (2011) Broad mite effects on chilli schoot damage and yields. Pakistan J. Zool. 43(4):637-649

Vick BA, Zimmerman DC (1983) The biosynthesis of jasmonic acid: a physiological role for plant lipoxygenase. Biochem. Biophys. Res. Commun. 111:470-477

Vieira MR, Chiavegato LG (1998) Biologia de Polyphagotarsonemus latus (Banks, 1904) (Acari: Tarsonemidae) em algodoeiro. Res. Agric. Bra. 33:1437-1442

Vieira MR, Chiavegato LG (1999) Biologia de Polyphagotarsonemus latus (Banks) (Acari: Tarsonemidae) em limão siciliano ( Citrus limon Burm). An. Soc. Entomol. Bra. 28:27-33

Villanueva R, Harmsen R (1996) Biological interactions of tarsonemid mites in apple orchards: predation of apple rust mite and use of Phyllonorycter blancardella mines. Proc. Entomol. Soc. Ontario 127:99-106

Vincent CI, García ME, Johnson DT, Rom CR (2010) Broad mite on primocane-fruiting blackberry in organic production in Arkansas. HortTechnology 20(4):718-723

VLAM (2014) Sierteeltbarometer 2014. Download from http://www.vlam.be

Vlot AC, Dempsey DA, Klessig DF (2009) Salicylic acid, a multifaceted hormone to combat disease. Annual Review of Phytopathology 47:177-206

Wager A, Browse J (2012) Social network: JAZ protein interactions expand our knowledge of jasmonate signaling. Front. Plant Sci. 3:41 doi:10.3389/fpls.2012.00041

Wan C-Y, Wilkins TA (1994) A modified hot borate method significantly enhances the yield of high-quality RNA from cotton ( Gossypium hirsutum L.). Anal. Biochem. 223:7-12

Wang L, Stegemann JP (2010) Extraction of high quality RNA from polysaccharide matrices using cetyltrimethylammonium bromide. Biomaterials 31:1612-1618

Warabieda W, Miszczak A, Olszak RW (2005) The influence of methyl jasmonate (JA-Me) and β- glucosidase on introduction of resistance mechanisms of strawberry against two-spotted spider mite (Tetranychus urticae Koch). Commun. Agric. Appl. Biol. Sci. 70(4):829-836

Warabieda W, Olszak RW (2012) Aspects of the influence of exogenous methyl jasmonate on strawberry plants and on the population size of the two-spotted spider mite ( Tetranychus urticae Koch). IOBC-WPRS Bulletin 83:7-13

Wasternack (2014) Action of jasmonates in plant stress responses and development – applied aspects. Biotechnol. Adv. 32(1):31-39

Wasternack C (2006) Oxylipins: biosynthesis, signal transduction and action. In: Hedden P, Thomas SG (Eds.) Plant hormone signaling. Blackwell Publishing, Oxford, UK. pp 185-228

Wasternack C (2007) Jasmonates: an update on biosynthesis, signal transduction and action in plant stress response, growth and development. Ann. Bot. 100:681-697

Wasternack C, Hause B (2013) Jasmonates: biosynthesis, perception, signal transduction and action in plant stress response, growth and development. An update of the 2007 review in Annals of Botany . Ann. Bot. 111:1021-1058

Waterhouse DF, Norris KR (1987) Biological control: Pacific prospects. Inkata Press, Melbourne, Australia

Weber H, Vick BA, Farmer EE (1997) Dinor-oxo-phytodienoic acid: a new hexadecanoid signal in the jasmonate family. Proc. Natl. Acad. Sci. USA 94:10473-10478

Weech MH, Chapleau M, Pan L, Ide C, Bede JC (2008) Caterpillar saliva interferes with induced Arabidopsis thaliana defence responses via the systemic acquired resistance pathway. Journal of Experimental Botany 59:2437-2448

Weiler EW, Kutchan TM, Gorba T, Brodschelm W, Neisel U, Bublitz F (1994) The Pseudomonas phytotoxin coronatine mimics octadecanoid signalling molecules of higher plants. FEBS Lett. 345:9-13

Weintraub PG, Kleitman S, Mori R, Shapira N, Palevsky E (2003) Control of the broad mite (Polyphagotarsonemus latus (Banks)) on organic greenhouse sweet peppers ( Capsicum annuum L.) with the predatory mite, Neoseiulus cucumeris (Oudemans). Biol. Control 27:300-309

Wheeler Q, Blackwell M (1984) Fungus-insect relationships: Perspectives in ecology and evolution. Columbia University Press, NY

Wiesmann R (1941) Investigations on the biology and control of the strawberry mite, Tarsonemus pallidus (fragariae Z.) Banks. Landwirtschaftliches Jahrbuch der Schweiz 55(3):259-329

Wildermuth MC (2006) Variations on a thema: synthesis and modification of plant benzoic acids. Current Opinion in Plant Biology 9:288-296.

Wildermuth MC, Dewdney J, Wu G, Ausubel FM (2001) Isochorismate synthase is required to synthesize salicylic acid for plant defence. Nature 414:562-565

Will T, Tjallingii WF, Thonnessen A, Van Bel AJE (2007) Molecular sabotage of plant defense by aphid saliva. Proceedings of the National Academy of Sciences, USA 104:10536-10541

Woldemariam MG, Gális I, Baldwin IT (2014) Jamonoyl-L-isoleucine hydrolase 1 (JIH1) contributes to a termination of jasmonate signaling in N. attenuata . Plant Signalling & Behaviour 9, e28973

175

Woldemariam MG, Onkokesung N, Baldwin IT, Gális I (2012) Jamonoyl-L-isoleucine hydrolase 1 (JIH1) regulates jasmonoyl-L-isoleucine levels and attenuates plant defences against herbivores. The plant Journal 72:758-767

Wu J, Baldwin IT (2010) New insights into plant responses to attack from insect herbivores. Annual Review of Genetics 44:1-24

Wu S, Peiffer M, Luthe DS, Felton GW (2012) ATP hydrolysing salivary enzymes of caterpillars suppress plant defences. PLoS ONE 7:e41947

Wu Y, Zhang G, Chu JY, Boyle P, Wang Y, Brindle ID, De Luca V, Despres C (2012) The Arabidopsis NPR1 protein is a receptor for the plant defense hormone salicylic acid. Cell Reports 1:639-647

Xie DX, Feys BF, James S, Nieto-Rostro M, Turner JG (1998) COI1: an Arabidopsis gene required for jasmonate-regulated defence and fertility. Science 280:1091-1094

Xu LH, Liu FQ, Lechner E, Genschik P, Crosby WY, Ma H, Peng W, Huang DF, Xie DX (2002) The SCF COI1 ubiquitin-ligase complexes are required for jasmonate response in Arabidopsis . Plant Cell 14:1919-1935

Yalpani N, Enyedi AJ, León J, Raskin I (1994) UV light and ozone stimulate accumulation of salicylic acid, pathogenesis-related proteins and virus resistance in tobacco. Planta 193:372-376

Yamaguchi Y, Huffaker A (2011) Endogenous peptide elicitors in higher plants. Curr. Opin. Plant Biol. 14:351-357

Yan S, Dong X (2012) Perception of the plant immune signal salicylic acid. Current Opinion in Plant biology 20:64-68

Yan Y, Borrego E, Kolomiets MV (2013) Jasmonate biosynthesis, perception and function in plant development and stress responses. In: Baez RV (Ed.) Lipid metabolism. ImTech, Rijeka, pp 393-442, Chapter16

Yan Y, Stolz S, Chételat A, Reymond P, Pagni M, Dubugnon L, Farmer EE (2007) A downstream mediator in the growth repression limb of the jasmonate pathway. Plant Cell 19:2470-2483

Yao YX, Zhao LL, Hao YJ, Zhai H (2005) A modified borate method significantly enhances the yield of high- quality RNA from apple pulp. Journal of Fruit Science 22(6):737-740

Yu D, Tang H, Zhang Y, Du Z, Yu H, Chen Q (2012) Comparison and improvement of different methods of RNA isolation from strawberry ( Fragria × ananassa ). Journal of Agricultural Science 4(7):51-56

Zarate SI, Kempema LA, Walling LL (2007) Silverleaf whitefly induces salicylic acid defences and suppresses effectual jasmonic acid defences. Plant physiology 143:866-875

Zeng Y, Yang T (2002) RNA isolation from highly viscous samples rich in polyphenols and polysaccharides. Plant Mol. Biol. Rep. 20:417a-417e

Zhang P, Zhu X, Huang F, Liu Y, Zhang J, Lu Y, Ruan Y (2011) Suppression of jasmonic acid-dependent defense in cotton plant by the mealybug Phenacoccus solenopsis . PLoS ONE 6:e22378

Zhang PJ, Zheng SJ, Van Loon JJA, Boland W, David A, Mumm R, Dicke M (2009) Whiteflies interfere with indirect plant defense against spider mites in Lima bean. Proceedings of the National Academy of Sciences, USA 106: 21202-21207

Zhang Z-Q (2003) Mites of Greenhouses: Identification, Biology and Control. CABI Publishing, Wallingford, UK, xii + p 244

Zheng SJ, van Dijk JP, Bruinsma M, Dicke M (2007) Sensitivity and speed of induced defense of cabbage (Brassica oleracea L.): Dynamics of BoLOX expression patterns during insect and pathogen attack. Mol. Plant Microbe Interact. 20:1332-1345

Zhurov V, Navarro M, Bruinsma KA, Arbona V, Santamaria ME, Cazaux M, Wybouw N, Osborne EJ, Ens C, Rioja C, Vermeirssen V, Rubio-Somoza I, Krishna P, Diaz I, Schmid M, Gómez-Cadenas A, Van de Peer K, Grbic M, Clarck RM, Van Leeuwen T, Grbic V (2014) Reciprocal responses in the interaction between Arabidopsis and the cell-content feeding chelicerate herbivore spider mite. Plant Physiol. 164:384-399

Ziegler J, Stenzel I, Hause B, Maucher H, Hemberg M, Grimm R, Ganal M, Wasternack C (2000) Molecular cloning of allene oxide cyclase. The enzyme establishing the stereochemistry of octadecanoids and jasmonates. J. Biol. Chem. 275:19132-19138

Zimmerli L, Jakab G, Métraux J-P, Mauch-Mani B (2000) Potentiation of pathogen-specific defense mechanisms in Arabidopsis by beta-aminobutyric acid. Proc. Natl. Acad. Sci. USA 97:12920-12925

Zolman BK, Silva ID, Bartel B (2001) The Arabidopsis pxa1 mutant is defective in an ATP-binding cassette transporter-like protein required for peroxisomal fatty acid beta-oxidation. Plant Physiol. 127:1266- 1278

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Supplementary data

Suppl. Table 6.1 List of RT-qPCR primer sequences and product size for Rhododendron simsii reference genes used in geNorm pilot study but not withheld for calculating the normalisation factor in experiments 1-5.

bp

Productsize bp 86 bp 96 bp 102 105 bp 133 bp 91 bp 81 bp 74 bp 80 bp 78 bp 153 bp 165 bp 151

3' GGTGTA - CACCTTGGAGAT Primersequence 5' GGCAGTGAAGGATCTCAAGC CCTGGGATGTGAAATTTGCT GTCCTTGGTGGCCCAGATA CTGGGAGTACTTTCAGGCACA CGCTGTTGGACAAGTGACTG AGCTTTGCACGCAGTGTATG GACACCTTTGCTTGGAGGAA GCAAGGGGAAGACA AGTTCCTGGTGCTGGACTTG CTCCGCAGTCTCAGTGTGGT TCTCAGGTGGAAAACATGATATTGA TTAACGGTCCGACTACCTTCAGATT CCCCATTTTGGCTATGAAACTG CCCTACAAATGCATAATCACCCTTA AGCCCTCCTTTATGATGTGGATAA GGCTGTTGCAGGACTGAAGAG GATGAAGGTAGCAGGCAATCTGA CCATTCTCGCGTAAAATCTTTCC ATTGACAGCAA CAATGGCAGTATTTGCCTTGATAT GAAACTCCCATTCCAGAGGCT GCATGGATGGCACAGAGGTT TGCAAAGATCGAATGCACGA CCTGCAAACGGAACTCGAGA TCGGAATCAACGGTTTTGGAA CACTTGACCGTGAACACTGT

F or R F R F R F R F R F R F R F R F R F R F R F R F R F R

Acc. Acc. No. LN829627 LN829628 LN829629 LN829630 LN829631 LN852375 LN852376 LN852377 LN852378 LN852379 AM932886 AM932909 FN552706

bindingprotein -

proteinligase UPL7 repeatprotein

- -

conjugating conjugating enzyme -

dependentRNA helicase - box/kelch - Function Elongationfactor 1 alpha Expressedprotein Clathrinadaptor complex family SAND Expressedprotein ubiquitin E3 Polypyrimidinetract F ATP Ubiquitin H3 Histone Proteinphosphatase GAPDH

Gene RsRG1 RsRG2 RsRG3 RsRG4 RsRG5 RsRG8 RsRG9 RsRG10 RsRG11 RsRG12 HK5 HK129 GAPDH

179

Suppl. Table 6.2 Top 25 of the most upregulated accessions detected in Rhododendron simsii ‘Nordlicht’ upon 100 µM methyl jasmonate treatment compared to the mock treatment. The table shows the accession, gene description and fold change. Level of significance was set at p <0.001.

R. simsii accession Gene description FC Rs1G33750_CAP3sORF_M2_893k Terpenoid cyclases/Protein prenyltransferases 292.9 superfamily protein Rs3G14540_CAP3sORF_M2_893k Terpenoid cyclases/Protein prenyltransferases 118.1 superfamily protein Rs4G10490_CAP3sORF_M2_893k 2-oxoglutarate (2OG) and Fe(II)-dependent 105.4 oxygenase superfamily protein Rs5G28237_CAP3cORF_M2_893k Pyridoxal-5'-phosphate-dependent enzyme family 105.0 protein Rs5G23960_CAP3sORF_M2_893k terpene synthase 21 92.2 Rs2G37980_CAP3sORF_M2_893k O-fucosyltransferase family protein 65.7 Rs5G64810_CAP3sORF_M2_893k WRKY DNA-binding protein 51 61.7 Rs1G74950_CAP3sORF_M2_893k TIFY domain/Divergent CCT motif family protein 55.0 Rs1G30135_CAP3cORF_M2_893k jasmonate-zim-domain protein 8 48.4 Rs4G27410_CAP3cORF_M2_893k NAC (No Apical Meristem) domain transcriptional 47.0 regulator superfamily protein Rs3G14520_CAP3sORF_M2_893k Terpenoid cyclases/Protein prenyltransferases 45.9 superfamily protein Rs2G24210_CAP3sORF_M2_893k terpene synthase 10 44.5 Rs2G14610_CAP3sORF_M2_893k pathogenesis-related gene 1 42.7 Rs5G54300_CAP3sORF_M2_893k Protein of unknown function (DUF761) 36.7 Rs1G61680_CAP3cORF_M2_893k terpene synthase 14 36.5 Rs1G21440_CAP3cORF_M2_893k Phosphoenolpyruvate carboxylase family protein 36.3 Rs5G54240_CAP3sORF_M2_893k Protein of unknown function (DUF1223) 36.1 Rs1G05300_CAP3sORF_M2_893k zinc transporter 5 precursor 34.9 Rs1G23550_CAP3sORF_M2_893k similar to RCD one 2 32.1 Rs5G62310_CAP3sORF_M2_893k AGC (cAMP-dependent, cGMP-dependent and protein 31.8 kinase C) kinase family protein Rs3G50310_CAP3sORF_M2_893k mitogen-activated protein kinase kinase kinase 20 29.1 Rs3G03600_CAP3sORF_M2_893k ribosomal protein S2 28.5 Rs4G29140_CAP3sORF_M2_893k MATE efflux family protein 27.1 Rs5G17350_CAP3sORF_M2_893k unknown protein 26.1 Rs5G39950_CAP3sORF_M2_893k thioredoxin 2 25.5

Suppl. Table 6.3 Top 25 of the most downregulated accessions detected in Rhododendron simsii ‘Nordlicht’ upon 100 µM methyl jasmonate treatment compared to the mock treatment. The table shows the accession, gene description and fold change. Level of significance was set at p < 0.001.

R. simsii accession Gene description FC Rs1G55365_CAP3cORF_M2_893k unknown protein 0.000 Rs1G76770_CAP3cORF_M2_893k HSP20-like chaperones superfamily protein 0.000 Rs2G18060_CAP3sORF_M2_893k vascular related NAC-domain protein 1 0.000 Rs2G38170_CAP3sORF_M2_893k cation exchanger 1 0.000 Rs3G05430_CAP3sORF_M2_893k Tudor/PWWP/MBT superfamily protein 0.000 Rs3G44990_CAP3sORF_M2_893k xyloglucan endo-transglycosylase-related 8 0.000 Rs3G52450_CAP3cORF_M2_893k plant U-box 22 0.000 Rs4G08620_CAP3sORF_M2_893k sulphate transporter 1 0.000 Rs4G32840_CAP3sORF_M2_893k phosphofructokinase 6 0.000 Rs4G33700_CAP3sORF_M2_893k CBS domain-containing protein with a domain of 0.000 unknown function (DUF21) Rs5G17450_CAP3sORF_M2_893k Heavy metal transport/detoxification superfamily 0.000 protein Rs5G19875_CAP3sORF_M2_893k unknown protein 0.000 Rs5G52290_CAP3sORF_M2_893k shortage in chiasmata 1 0.000 Rs5G53200_CAP3sORF_M2_893k Homeodomain-like superfamily protein 0.000 Rs1G29500_CAP3sORF_M2_893k SAUR-like auxin-responsive protein family 0.012 Rs5G49350_CAP3sORF_M2_893k Glycine-rich protein family 0.022 Rs2G30395_CAP3sORF_M2_893k ovate family protein 17 0.023 Rs4G25970_CAP3sORF_M2_893k phosphatidylserine decarboxylase 3 0.024 Rs5G62850_CAP3sORF_M2_893k Nodulin MtN3 family protein 0.024 Rs2G34060_CAP3sORF_M2_893k Peroxidase superfamily protein 0.024 Rs1G43630_CAP3cORF_M2_893k Protein of unknown function (DUF793) 0.025 Rs5G56800_CAP3sORF_M2_893k Protein with RNI-like/FBD-like domains 0.028 Rs2G31865_CAP3sORF_M2_893k poly(ADP-ribose) glycohydrolase 2 0.032 Rs2G02850_CAP3sORF_M2_893k plantacyanin 0.032 Rs5G10890_CAP3sORF_M2_893k myosin heavy chain-related 0.035

181

Suppl. Table 6.4 Top 25 of the most upregulated accessions detected in Rhododendron simsii ‘Elien’ upon Polyphagotarsonemus latus infestation compared to the mock treatment. The table shows the accession, gene description and fold change. Level of significance was set at p < 0.001.

R. simsii accession Gene description FC Rs3G06120_CAP3sORF_M2_893k basic helix-loop-helix (bHLH) DNA-binding 436.1 superfamily protein Rs5G01910_CAP3sORF_M2_893k unknown protein 269.0 Rs1G03180_CAP3sORF_M2_893k unknown protein 195.2 Rs1G18370_CAP3sORF_M2_893k ATP binding microtubule motor family protein 132.1 Rs2G42840_CAP3sORF_M2_893k protodermal factor 1 108.0 Rs2G42800_CAP3cORF_M2_893k receptor like protein 29 104.0 Rs1G53140_CAP3sORF_M2_893k Dynamin related protein 5A 97.4 Rs2G28610_CAP3cORF_M2_893k Homeodomain-like superfamily protein 84.0 Rs4G39770_CAP3sORF_M2_893k Haloacid dehalogenase-like hydrolase (HAD) 79.0 superfamily protein Rs1G11600_CAP3cORF_M2_893k cytochrome P450, family 77, subfamily B, polypeptide 70.6 1 Rs5G45890_CAP3sORF_M2_893k senescence-associated gene 12 70.1 Rs4G35930_CAP3cORF_M2_893k F-box family protein 60.2 Rs3G16300_CAP3sORF_M2_893k Uncharacterised protein family (UPF0497) 59.8 Rs1G07270_CAP3sORF_M2_893k Cell division control, Cdc6 57.9 Rs5G60930_CAP3sORF_M2_893k P-loop containing nucleoside triphosphate hydrolases 56.8 superfamily protein Rs5G48485_CAP3cORF_M2_893k Bifunctional inhibitor/lipid-transfer protein/seed 52.3 storage 2S albumin superfamily protein Rs3G10320_CAP3sORF_M2_893k Glycosyltransferase family 61 protein 46.8 Rs3G17152_CAP3sORF_M2_893k Plant invertase/pectin methylesterase inhibitor 39.6 superfamily protein Rs1G64080_CAP3sORF_M2_893k unknown protein 39.4 Rs2G20515_CAP3cORF_M2_893k unknown protein 39.0 Rs1G29630_CAP3sORF_M2_893k 5'-3' exonuclease family protein 37.9 Rs3G02120_CAP3cORF_M2_893k hydroxyproline-rich glycoprotein family protein 37.0 Rs1G67630_CAP3cORF_M2_893k DNA polymerase alpha 2 35.6 Rs3G12870_CAP3cORF_M2_893k unknown protein 33.0 Rs4G02800_CAP3sORF_M2_893k unknown protein 32.7

Suppl. Table 6.5 Top 25 of the most downregulated accessions detected in Rhododendron simsii ‘Elien’ upon Polyphagotarsonemus latus infestation compared to the mock treatment. The table shows the accession, gene description and fold change. Level of significance was set at p < 0.001.

R. simsii accession Gene description FC Rs1G10900_CAP3sORF_M2_893k Phosphatidylinositol-4-phosphate 5-kinase family 0.000 protein Rs1G47580_CAP3sORF_M2_893k Pentatricopeptide repeat (PPR) superfamily protein 0.000 Rs1G49405_CAP3sORF_M2_893k Uncharacterised protein family (UPF0497) 0.000 Rs1G55590_CAP3sORF_M2_893k RNI-like superfamily protein 0.000 Rs1G67970_CAP3sORF_M2_893k heat shock transcription factor A8 0.000 Rs1G76100_CAP3sORF_M2_893k plastocyanin 1 0.000 Rs2G01910_CAP3sORF_M2_893k Microtubule associated protein (MAP65/ASE1) family 0.000 protein Rs2G47770_CAP3sORF_M2_893k TSPO(outer membrane tryptophan-rich sensory 0.000 protein)-related Rs3G30775_CAP3sORF_M2_893k Methylenetetrahydrofolate reductase family protein 0.000 Rs3G44700_CAP3sORF_M2_893k Plant protein of unknown function 0.000 Rs3G63060_CAP3cORF_M2_893k EID1-like 3 0.000 Rs4G25850_CAP3sORF_M2_893k OSBP(oxysterol binding protein)-related protein 4B 0.000 Rs4G36600_CAP3sORF_M2_893k Late embryogenesis abundant (LEA) protein 0.000 Rs4G38420_CAP3sORF_M2_893k SKU5 similar 9 0.000 Rs5G01770_CAP3sORF_M2_893k HEAT repeat 0.000 Rs5G10230_CAP3sORF_M2_893k annexin 7 0.000 Rs5G13440_CAP3sORF_M2_893k Ubiquinol-cytochrome C reductase iron-sulfur subunit 0.000 Rs5G40170_CAP3sORF_M2_893k receptor like protein 54 0.000 Rs5G45770_CAP3sORF_M2_893k receptor like protein 55 0.000 Rs5G52390_CAP3cORF_M2_893k PAR1 protein 0.000 Rs5G59220_CAP3sORF_M2_893k highly ABA-induced PP2C gene 1 0.000 Rs1G15410_CAP3cORF_M2_893k aspartate-glutamate racemase family 0.007 Rs5G38710_CAP3cORF_M2_893k Methylenetetrahydrofolate reductase family protein 0.008 Rs1G67090_CAP3sORF_M2_893k ribulose bisphosphate carboxylase small chain 1A 0.010 Rs3G03341_CAP3cORF_M2_893k unknown protein 0.010

183

Suppl. Table 6.6 Top 25 of the most upregulated accessions detected in Rhododendron simsii ‘Elien’ upon 100 µM methyl jasmonate treatment compared to the mock treatment. The table shows the accession, gene description and fold change. Level of significance was set at p < 0.001.

R. simsii accession Gene description FC Rs1G09155_CAP3sORF_M2_893k phloem protein 2-B15 84.1 Rs2G25000_CAP3sORF_M2_893k WRKY DNA-binding protein 60 40.4 Rs1G30670_CAP3sORF_M2_893k basic helix-loop-helix (bHLH) DNA-binding 32.5 superfamily protein Rs3G16460_CAP3sORF_M2_893k Mannose-binding lectin superfamily protein 28.6 Rs5G61890_CAP3sORF_M2_893k Integrase-type DNA-binding superfamily protein 26.8 Rs1G19260_CAP3sORF_M2_893k TTF-type zinc finger protein with HAT dimerisation 24.7 domain Rs2G30660_CAP3sORF_M2_893k ATP-dependent caseinolytic (Clp) protease/crotonase 24.6 family protein Rs5G56450_CAP3sORF_M2_893k Mitochondrial substrate carrier family protein 21.7 Rs4G29930_CAP3sORF_M2_893k basic helix-loop-helix (bHLH) DNA-binding 20.6 superfamily protein Rs4G18970_CAP3sORF_M2_893k GDSL-like Lipase/Acylhydrolase superfamily protein 19.7 Rs5G13080_CAP3cORF_M2_893k WRKY DNA-binding protein 75 19.4 Rs4G04630_CAP3sORF_M2_893k Protein of unknown function, DUF584 16.8 Rs5G05020_CAP3sORF_M2_893k Pollen Ole e 1 allergen and extensin family protein 15.7 Rs1G61680_CAP3cORF_M2_893k terpene synthase 14 14.4 Rs3G16300_CAP3sORF_M2_893k Uncharacterised protein family (UPF0497) 14.4 Rs4G26740_CAP3sORF_M2_893k seed gene 1 14.1 Rs5G28237_CAP3cORF_M2_893k Pyridoxal-5'-phosphate-dependent enzyme family 14.0 protein Rs3G25810_CAP3cORF_M2_893k Terpenoid cyclases/Protein prenyltransferases 12.6 superfamily protein Rs5G27920_CAP3sORF_M2_893k F-box family protein 11.2 Rs1G72230_CAP3sORF_M2_893k Cupredoxin superfamily protein 10.8 Rs1G80160_CAP3cORF_M2_893k Lactoylglutathione lyase / glyoxalase I family protein 10.8 Rs4G18550_CAP3cORF_M2_893k alpha/beta-Hydrolases superfamily protein 10.5 Rs4G03270_CAP3sORF_M2_893k Cyclin D6 10.4 Rs5G13220_CAP3cORF_M2_893k jasmonate-zim-domain protein 10 9.6 Rs1G74950_CAP3sORF_M2_893k TIFY domain/Divergent CCT motif family protein 9.1

Suppl. Table 6.7 Top 25 of the most downregulated accessions detected in Rhododendron simsii ‘Elien’ upon 100 µM methyl jasmonate treatment compared to the mock treatment. The table shows the accession, gene description and fold change. Level of significance was set at p < 0.001.

R. simsii accession Gene description FC Rs1G09155_CAP3sORF_M2_893k phloem protein 2-B15 84.1 Rs2G25000_CAP3sORF_M2_893k WRKY DNA-binding protein 60 40.4 Rs1G30670_CAP3sORF_M2_893k basic helix-loop-helix (bHLH) DNA-binding 32.5 superfamily protein Rs3G16460_CAP3sORF_M2_893k Mannose-binding lectin superfamily protein 28.6 Rs5G61890_CAP3sORF_M2_893k Integrase-type DNA-binding superfamily protein 26.8 Rs1G19260_CAP3sORF_M2_893k TTF-type zinc finger protein with HAT dimerisation 24.7 domain Rs2G30660_CAP3sORF_M2_893k ATP-dependent caseinolytic (Clp) protease/crotonase 24.6 family protein Rs5G56450_CAP3sORF_M2_893k Mitochondrial substrate carrier family protein 21.7 Rs4G29930_CAP3sORF_M2_893k basic helix-loop-helix (bHLH) DNA-binding 20.6 superfamily protein Rs4G18970_CAP3sORF_M2_893k GDSL-like Lipase/Acylhydrolase superfamily protein 19.7 Rs5G13080_CAP3cORF_M2_893k WRKY DNA-binding protein 75 19.4 Rs4G04630_CAP3sORF_M2_893k Protein of unknown function, DUF584 16.8 Rs5G05020_CAP3sORF_M2_893k Pollen Ole e 1 allergen and extensin family protein 15.7 Rs1G61680_CAP3cORF_M2_893k terpene synthase 14 14.4 Rs3G16300_CAP3sORF_M2_893k Uncharacterised protein family (UPF0497) 14.4 Rs4G26740_CAP3sORF_M2_893k seed gene 1 14.1 Rs5G28237_CAP3cORF_M2_893k Pyridoxal-5'-phosphate-dependent enzyme family 14.0 protein Rs3G25810_CAP3cORF_M2_893k Terpenoid cyclases/Protein prenyltransferases 12.6 superfamily protein Rs5G27920_CAP3sORF_M2_893k F-box family protein 11.2 Rs1G72230_CAP3sORF_M2_893k Cupredoxin superfamily protein 10.8 Rs1G80160_CAP3cORF_M2_893k Lactoylglutathione lyase / glyoxalase I family protein 10.8 Rs4G18550_CAP3cORF_M2_893k alpha/beta-Hydrolases superfamily protein 10.5 Rs4G03270_CAP3sORF_M2_893k Cyclin D6 10.4 Rs5G13220_CAP3cORF_M2_893k jasmonate-zim-domain protein 10 9.6 Rs1G74950_CAP3sORF_M2_893k TIFY domain/Divergent CCT motif family protein 9.1

185

Suppl. Table 6.8 Top 25 of the most upregulated accessions detected in Rhododendron simsii ‘Nordlicht’ upon Polyphagotarsonemus latus infestation compared to the mock treatment. The table shows the accession, gene description and fold change. Level of significance was set at p < 0.001.

R. simsii accession Gene description FC At4G10490_CAP3sORF_M2_893k 2-oxoglutarate (2OG) and Fe(II)-dependent 610.4 oxygenase superfamily protein At5G35830_CAP3cORF_M2_893k Ankyrin repeat family protein 342.7 At5G01380_CAP3sORF_M2_893k Homeodomain-like superfamily protein 243.4 At1G14480_CAP3sORF_M2_893k Ankyrin repeat family protein 210.5 At2G47610_CAP3sORF_M2_893k Ribosomal protein L7Ae/L30e/S12e/Gadd45 family 191.0 protein At5G09510_CAP3sORF_M2_893k Ribosomal protein S19 family protein 187.6 AtMG01190_CAP3sORF_M2_893k ATP synthase subunit 1 165.7 At5G04690_CAP3sORF_M2_893k Ankyrin repeat family protein 115.8 At2G19750_CAP3sORF_M2_893k Ribosomal protein S30 family protein 115.0 At1G33750_CAP3sORF_M2_893k Terpenoid cyclases/Protein prenyltransferases 98.5 superfamily protein At4G39200_CAP3sORF_M2_893k Ribosomal protein S25 family protein 93.8 At1G20560_CAP3cORF_M2_893k acyl activating enzyme 1 93.0 At3G50160_CAP3sORF_M2_893k Plant protein of unknown function (DUF247) 79.5 At5G59290_CAP3sORF_M2_893k UDP-glucuronic acid decarboxylase 3 72.7 At1G74950_CAP3sORF_M2_893k TIFY domain/Divergent CCT motif family protein 63.3 At3G26320_CAP3sORF_M2_893k cytochrome P450, family 71, subfamily B, 59.6 polypeptide 36 At3G14540_CAP3sORF_M2_893k Terpenoid cyclases/Protein prenyltransferases 58.9 superfamily protein At1G30135_CAP3cORF_M2_893k jasmonate-zim-domain protein 8 57.9 At2G21540_CAP3sORF_M2_893k SEC14-like 3 54.9 At3G11090_CAP3sORF_M2_893k LOB domain-containing protein 21 54.1 At3G47910_CAP3sORF_M2_893k Ubiquitin carboxyl-terminal hydrolase-related 50.9 protein At5G24550_CAP3sORF_M2_893k beta glucosidase 32 47.1 At1G49640_CAP3sORF_M2_893k alpha/beta-Hydrolases superfamily protein 46.0 At5G64810_CAP3sORF_M2_893k WRKY DNA-binding protein 51 45.6 At3G05740_CAP3sORF_M2_893k RECQ helicase l1 45.2

Suppl. Table 6.9 Top 25 of the most downregulated accessions detected in Rhododendron simsii ‘Nordlicht’ upon Polyphagotarsonemus latus infestation compared to the mock treatment. The table shows the accession, gene description and fold change. Level of significance was set at p < 0.001.

R. simsii accession Gene description FC Rs1G09794_CAP3sORF_M2_893k Cox19 family protein (CHCH motif) 0.000 Rs1G21110_CAP3sORF_M2_893k O-methyltransferase family protein 0.000 Rs2G07674_CAP3sORF_M2_893k Unknown conserved protein 0.000 Rs2G33500_CAP3sORF_M2_893k B-box type zinc finger protein with CCT domain 0.000 Rs2G38170_CAP3sORF_M2_893k cation exchanger 1 0.000 Rs3G02980_CAP3sORF_M2_893k MEIOTIC CONTROL OF CROSSOVERS1 0.000 Rs3G05430_CAP3sORF_M2_893k Tudor/PWWP/MBT superfamily protein 0.000 Rs3G24780_CAP3sORF_M2_893k Uncharacterised conserved protein UCP015417, vWA 0.000 Rs4G33700_CAP3sORF_M2_893k CBS domain-containing protein with a domain of 0.000 unknown function (DUF21) Rs5G06670_CAP3sORF_M2_893k P-loop containing nucleoside triphosphate hydrolases 0.000 superfamily protein Rs5G17450_CAP3sORF_M2_893k Heavy metal transport/detoxification superfamily 0.000 protein Rs1G54740_CAP3sORF_M2_893k Protein of unknown function (DUF3049) 0.007 Rs5G15130_CAP3sORF_M2_893k WRKY DNA-binding protein 72 0.010 Rs5G52290_CAP3sORF_M2_893k shortage in chiasmata 1 0.022 Rs5G53200_CAP3sORF_M2_893k Homeodomain-like superfamily protein 0.026 Rs1G13470_CAP3sORF_M2_893k Protein of unknown function (DUF1262) 0.026 Rs2G22410_CAP3sORF_M2_893k SLOW GROWTH 1 0.032 Rs1G13800_CAP3sORF_M2_893k Tetratricopeptide repeat (TPR)-like superfamily 0.034 protein Rs5G59720_CAP3cORF_M2_893k heat shock protein 18.2 0.046 Rs4G34419_CAP3sORF_M2_893k unknown protein 0.067 Rs2G37530_CAP3sORF_M2_893k unknown protein 0.081 Rs3G53680_CAP3sORF_M2_893k Acyl-CoA N-acyltransferase with RING/FYVE/PHD- 0.082 type zinc finger domain Rs1G28050_CAP3sORF_M2_893k B-box type zinc finger protein with CCT domain 0.082 Rs3G18200_CAP3sORF_M2_893k nodulin MtN21 /EamA-like transporter family protein 0.092 Rs1G51800_CAP3sORF_M2_893k Leucine-rich repeat protein kinase family protein 0.093

187

Suppl. Table 6.10 GeNorm M and Coefficient of Variation (CV) values for reference genes RsRG7 and RsRG14 expressing reference gene stability in experiment 1-5. Values are calculated using qbase+ software (Hellemans et al., 2007).

Parameter Exp. 1 Exp. 2 Exp. 3 Exp. 4 Exp. 5 M value 0.151 0.226 0.184 0.271 0.292 CV value 0.052 0.078 0.064 0.094 0.101

Suppl. Table 6.11 Run specific amplification efficiency (E) (± SE), R², slope (± SE) and intercept (± SE) for marker genes and reference genes in RT-qPCR for experiment 1.

Gene E R² Slope Intercept RsAOC 1.98 ± 0.02 0.998 -3.36 ± 0.05 37.66 ± 0.20 RsAOS 2.03 ± 0.03 0.996 -3.26 ± 0.06 38.55 ± 0.25 RsICS 2.06 ± 0.04 0.992 -3.18 ± 0.09 38.01 ± 0.36 RsJMT 2.00 ± 0.01 1 -3.32 ± 0.01 35.08 ± 0.05 RsLOX 2.04 ± 0.03 0.996 -3.23 ± 0.06 38.10 ± 0.26 RsOPR3 1.97 ± 0.02 0.999 -3.39 ± 0.05 39.27 ± 0.20 RsPAL 2.01 ± 0.01 1 -3.31 ± 0.02 32.69 ± 0.08 RsPPO 1.88 ± 0.00 1 -3.64 ± 0.02 36.89 ± 0.06

RsRG14 1.83 ± 0.00 1 -3.83 ± 0.01 35.95 ± 0.03 RsRG7 2.02 ± 0.01 1 -3.28 ± 0.02 35.48 ± 0.0 7

Suppl. Table 6.12 Run specific amplification efficiency (E) (± SE), R², slope (± SE) and intercept (± SE) for marker genes and reference genes in RT-qPCR for experiment 2.

Gene E R² Slope Intercept RsAOC 1.95 ± 0.03 0.996 -3.44 ± 0.07 38.45 ± 0.26 RsAOS 2.08 ± 0.02 0.999 - 3.15 ± 0.04 38.16 ± 0.15 RsICS 1.93 ± 0.03 0.994 - 3.52 ± 0.09 39.68 ± 0.35 RsJMT 2.02 ± 0.02 0.998 - 3.26 ± 0.05 38.11 ± 0.18 RsLOX 2 .00 ± 0.02 0.998 - 3.33 ± 0.05 38.94 ± 0.19 RsOPR3 2.01 ± 0.01 1 - 3.3 0 ± 0.02 32.81 ± 0.09 RsPAL 1.98 ± 0.01 0.999 - 3.36 ± 0.03 33.48 ± 0.12 RsPPO 1.88 ± 0.01 1 -3.64 ± 0.02 33.86 ± 0.07 RsRG14 1.82 ± 0.01 1 -3.84 ± 0.03 36.61 ± 0.11 RsRG7 2.02 ± 0.02 0.998 -3.28 ± 0.05 35.14 ± 0.18

Suppl. Table 6.13 Run specific amplification efficiency (E) (± SE), R², slope (± SE) and intercept (± SE) for marker genes and reference genes in RT-qPCR for experiment 3.

Gene E R² Slope Intercept RsAOC 1.96 ± 0.03 0.994 -3.42 ± 0.08 37.58 ± 0.32 RsAOS 2.22 ± 0.17 0.915 -2.89 ± 0.28 36.64 ± 1.08 RsICS 1.99 ± 0.02 0.998 -3.33 ± 0.05 39.01 ± 0.2 0 RsJMT 1.97 ± 0.01 1 -3.39 ± 0.02 34.93 ± 0.06 RsLOX 2.00 ± 0.02 0.999 -3.32 ± 0.04 38.56 ± 0.17 RsOPR3 2.00 ± 0.02 0.998 -3.33 ± 0.05 38.98 ± 0.2 0 RsPAL 2.00 ± 0.01 1 -3.33 ± 0.01 32.66 ± 0.05 RsPPO 1.91 ± 0.01 1 -3.57 ± 0.01 36.69 ± 0.06 RsRG14 1.84 ± 0.00 1 -3.77 ± 0.01 35.36 ± 0.05 RsRG7 2.02 ± 0.01 1 -3.27 ± 0.02 34.85 ± 0.08

Suppl. Table 6.14 Run specific amplification efficiency (E) (± SE), R², slope (± SE) and intercept (± SE) for marker genes and reference genes in RT-qPCR for experiment 4 and 5.

Gene E R² Slope Intercept RsAOC 2.06 ± 0.04 0.994 -3.18 ± 0.08 36.42 ± 0.3 0 RsAOS 2.05 ± 0.02 0.999 -3.21 ± 0.04 38.29 ± 0.14

RsICS 2.03 ± 0.03 0.998 -3.24 ± 0.06 38.67 ± 0.24 RsJMT 2.01 ± 0.01 1 -3.31 ± 0.02 34.58 ± 0.1 0 RsLOX 1.94 ± 0.02 0.998 -3.49 ± 0.05 39.52 ± 0.19 RsOPR3 2.01 ± 0.02 0.997 -3.31 ± 0.05 39.07 ± 0.20 RsPAL 1.96 ± 0.01 1 -3.43 ± 0.02 33.14 ± 0.06 RsPPO 1.87 ± 0.01 0.999 -3.67 ± 0.03 37.65 ± 0.11 RsRG14 1.76 ± 0 .00 1 -4.09 ± 0.01 36.53 ± 0.03 RsRG7 1.98 ± 0.01 0.999 -3.38 ± 0.03 35.35 ± 0.13

189

Suppl. Table 7.1 GeNorm M and Coefficient of Variation (CV) values for reference genes RsRG7 and RsRG14 expressing reference gene stability in experiment 1-2. Values are calculated using qbase+ software (Hellemans et al., 2007).

Parameter Exp. 1 Exp. 2 M value 0.308 0.277 CV value 0.107 0.096

Suppl. Table 7.2 Run specific amplification efficiency (E) (± SE), R², slope (± SE) and intercept (± SE) for marker genes and reference genes in RT-qPCR for experiment 1.

Gene E R² Slope Intercept RsAOC 2.01 ± 0.02 0.999 -3.29 ± 0.04 36.98 ± 0.16 RsAOS 1.97 ± 0.02 0.997 -3.41 ± 0.06 39.38 ± 0.25 RsICS 2.07 ± 0.02 0.998 -3.18 ± 0.05 38.54 ± 0.19 RsJMT 2.07 ± 0.06 0.985 -3.17 ± 0.14 33.79 ± 0.58 RsLOX 1.99 ± 0.03 0.996 -3.35 ± 0.07 38.94 ± 0.27 RsOPR3 2.05 ± 0.02 0.998 -3.2 0 ± 0.05 38.79 ± 0.19 RsPAL 1.96 ± 0.01 1 -3.42 ± 0.02 33.26 ± 0.07 RsPPO 1.87 ± 0.01 0.999 -3.68 ± 0.04 38.21 ± 0.15 RsRG14 1.77 ± 0.01 1 -4.04 ± 0.02 37.62 ± 0.08 RsRG7 2.00 ± 0.01 0.999 -3.33 ± 0.03 35.26 ± 0.11

Suppl. Table 7.3 Run specific amplification efficiency (E) (± SE), R², slope (± SE) and intercept (± SE) for marker genes and reference genes in RT-qPCR for experiment 2.

Gene E R² Slope Intercept RsAOC 2.09 ± 0.04 0.994 -3.12 ± 0.08 36.19 ± 0.30 Rs AOS 2.03 ± 0.04 0.994 -3.26 ± 0.09 38.69 ± 0.36 Rs ICS 2.04 ± 0.02 0.997 -3.22 ± 0.04 38.5 0 ± 0.15 Rs JMT 2.08 ± 0.05 0.99 0 -3.15 ± 0.11 33.78 ± 0.48 RsLOX 1.97 ± 0.01 0.999 -3.4 0 ± 0.04 39.09 ± 0.14 Rs OPR3 2.05 ± 0.03 0.997 -3.21 ± 0.06 38.77 ± 0.23 RsPAL 2.04 ± 0.03 0.996 -3.23 ± 0.06 36.11 ± 0.24 Rs PPO 1.87 ± 0.01 0.999 -3.68 ± 0.04 37.87 ± 0.14 RsRG14 1.78 ± 0.01 0.999 -4.01 ± 0.04 40.80 ± 0.16 RsRG7 2.01 ± 0.02 0.997 -3.31 ± 0.06 34.78 ± 0.23

Suppl. Table 7.4 Repeatability and reproducibility of jasmonic acid (JA), 12- oxophytodienoic acid (OPDA) and salicylic acid (SA) measurements of Polyphagotarsonemus latus infested and mock treated leaf samples of Rhododendron simsii hybrids.

Phytohormone Treatment Repeatability (%) Reproducibility (%) OPDA Mock 17.55 23.43 P. latus infestation 15.68 16.22 JA Mock 15.9 0 19.65 P. latus infestati on 7.4 0 9.92 SA Mock 18.02 18.93 P. latus infestation 8.16 20.65

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Curriculum vitae – Gil Luypaert

PERSONALIA

Name: Gil Luypaert Date of birth: April 3, 1988 Address: Drielindenbaan 140 1785 Merchtem Nationality: Belgian E-mail: [email protected] Telephone: +32 474 24 74 70

EDUCATION

2011 – present Ph.D. in Bioscience Engineering Faculty of Bioscience Engineering, Ghent University

Thesis: The broad mite, Polyphagotarsonemus latus , and its interactions with pot azalea, Rhododendron simsii hybrid Promotors: Prof. dr. ir. P. De Clercq, Prof. dr. M. Maes and Dr. ir. J. De Riek

2009 – 2011 M.Sc. in Bioscience Engineering: Cell and Gene Biotechnology Major: Molecular Plant Breeding Faculty of Bioscience Engineering, Ghent University

Thesis: Moleculaire analyse van potentiële parasitismegenen bij de plantenparasitaire nematode Hirschmanniella oryzae Promotor: Prof. dr. G. Gheysen

2006 – 2010 B.Sc. in Bioscience Engineering Main subject: Cell and Gene Biotechnology Faculty of Bioscience Engineering, Ghent University

ADDITIONAL EDUCATION

° Day course: Statistical Training, INBO (2014) ° English proficiency for presentations, University Language Centre, Ghent (2014) ° Workshop: Clear and Engaging Writing (English), ILVO (2013)

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PROFESSIONAL CAREER

2011 – present Research associate Institute for Agricultural and Fisheries Research (ILVO), Plant Sciences Unit, Melle

IWT-project No. 100859: Plantresistentie tegen Polyphagotarsonemus latus (Acari: Tarsonemidae) in de sierteelt

PUBLICATIONS

Peer-reviewed journal articles:

Luypaert G, Witters J, Van Huylenbroeck J, Maes M, De Riek J & De Clercq P (2014) Temperature- dependent development of the broad mite Polyphagotarsonemus latus (Acari: Tarsonemidae) on Rhododendron simsii. Experimental and Applied Acarology 63(3):389-400

Luypaert G, Van Huylenbroeck J, De Riek J & De Clercq P (2014) Screening for broad mite susceptibility in Rhododendron simsii hybrids. Journal of Plant Diseases and Protection 121(6):260- 269

Luypaert G, Witters J, Berkvens N, Van Huylenbroeck J, De Riek J & De Clercq P (2015) Cold tolerance of the broad mite, Polyphagotarsonemus latus (Acari: Tarsonemidae). Experimental and Applied Acarology 66(1):29-39

Mechant E, Luypaert G, Van Delsen B, Pauwels E, Witters J, Van Huylenbroeck J, Gobin B (2015) Development and validation of a three-step detection protocol for broad mites (Polyphagotarsonemus latus ) in pot azalea ( Rhododendron simsii hybrids). Entomologia Experimentalis et Applicata DOI: 10.1111/eea.12315

Published abstracts:

Luypaert G, Van Delsen B, Witters J, Van Huylenbroeck J, Maes M, De Riek J & De Clercq P (2013) Screening Rhododendron spp. for broad mite ( Polyphagotarsonemus latus ) resistance. Communications in Agricultural and Applied Biological Sciences 78(2):229-232

Luypaert G, De Keyser E, Van Huylenbroeck J, Witters J, Maes M, De Riek J & De Clercq P (2013) Is there a role for jasmonic acid in induced resistance against broad mites in pot azalea? IOBC-WPRS Bulletin 89:63-66

Luypaert G, De Keyser E, Van Huylenbroeck J, De Riek J & De Clercq P (2014) Broad mites activate jasmonic acid biosynthesis genes in azalea. IOBC-WPRS Bulletin 102:133-136

Mechant E, Pauwels E, Luypaert G & Gobin B (2014) Three-step broad mite detection protocol as an IPM-tool in pot azalea. IOBC-WPRS Bulletin 102:137-142

Van Huylenbroeck J, Calsyn E, De Keyser E, Luypaert G (2015) Breeding for biotic stress resistance in Rhododendron simsii . Accepted for publication in Acta Horticulturae

Luypaert G, Mechant E, Pauwels E, Van Huylenbroeck J, De Riek J, De Clercq P (2015) Opportunities to breed for broad mite resistance in Rhododendron simsii hybrids. Accepted for publication in Acta Horticulturae

Other publications (popular articles):

Luypaert G & Sluydts V (2011) IWT steunt nieuw onderzoeksproject weekhuidmijten. Verbondsnieuws 15:20-21

Luypaert G & Van Delsen B (2012) IWT-onderzoeksproject naar weekhuidmijten bij azalea. Sierteelt&Groenvoorziening 8:23-24

Luypaert G & Van Delsen B (2012) Onderzoeksproject naar weekhuidmijten bij azalea. Sierteelt&Groenvoorziening 20:21-23

Van Delsen B, Luypaert G & Witters J (2013) Stand van zaken in het weekhuidmijtenproject. Azalea Informatief nr. 2

Van Delsen B & Luypaert G (2013) IWT-onderzoeksproject naar weekhuidmijten bij azalea. Sierteelt&Groenvoorziening 16:25-28

Pauwels E, Audenaert J, Mechant E, Witters J & Luypaert G (2014) Geïntegreerde bestrijding van weekhuidmijten bij azalea. Sierteelt&Groenvoorziening 6:24-27

Luypaert G & Mechant E (2014) Inzicht in gevoeligheid voor weekhuidmijten bij azalea cultivars. Sierteelt&Groenvoorziening 16:22-23

Luypaert G & Mechant E (2015) Hoe koud heeft een weekhuidmijt het in de frigo? Sierteelt&Groenvoorziening 8:16-17

PRESENTATIONS & PARTICIPATIONS

International meetings:

64 th International Symposium on Crop Protection, Ghent - May 22, 2012

Gordon Research Seminar - Plant Molecular Biology, Holderness, NH, USA - July 15-20, 2012 (poster presentation) “Isolation of jasmonic acid related gene fragments and validation of qPCR primers for gene expression analysis in Rhododendron simsii hybrids upon broad mite infection”

BeNeLuxSHS Symposium: Integrated Pest Management in Horticulture: Research for Practice, Melle - March 7, 2013 (oral presentation) ”Towards Integrated Pest Management in azalea to control broad mite”

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65 th International Symposium on Crop Protection, Ghent - May 21, 2013 (oral presentation) “Screening Rhododendron spp. for broad mite ( Polyphagotarsonemus latus ) resistance”

IOBC-WPRS: 6 th Meeting of the WG "Induced Resistance in Plants Against Insects and Diseases", Avignon, France - June 10-13, 2013 (oral presentation) ”Is there a role for jasmonic acid induced resistance against broad mites in azalea?”

66 th International Symposium on Crop Protection, Ghent - May 20, 2014 (oral presentation) “Genetic variance against broad mites ( Polyphagotarsonemus latus ) in a Rhododendron simsii hybrid gene pool”

IOBC-WPRS: ”Integrated Control in Protected Crops, Temperate Climate”, Ghent - September 14-18, 2014 (oral presentation) “Broad mites activate jasmonic acid biosynthesis genes in azalea”

National meetings:

Studiedag PCS, Destelbergen - September 15, 2011

20 ste Studiedag Azalea, Destelbergen – March 1, 2012

21 ste Studiedag Azalea, Destelbergen - February 21, 2013

Mini-symposium on quantitative PCR “Improving the quality of the results”, Ghent – February 11, 2014

22 ste Studiedag Azalea, Destelbergen - February 20, 2014 (oral presentation) “Weekhuidmijten bij azalea: beter begrijpen van de plaag…”

23 ste Studiedag Azalea, Destelbergen - February 26, 2015 (oral presentation) “Hoe koud heeft een weekhuidmijt het in de frigo?”

OTHER ACTIVITIES

Lecturer:

Modulaire Wintercursus “Monitoring onder de loep” Destelbergen - January 7-28, 2015 Part: “Zelf weekhuidmijten detecteren op je bedrijf met een microscoop” (January 28, 2015)