Poinsettia (Euphorbia Pulcherrima Willd. Ex Klotzsch: Euphorbiacea)

Poinsettia (Euphorbia pulcherrima Willd. ex Klotzsch: Euphorbiacea) Resistance Mechanisms against the Silverleaf Whitefly, Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae) Biotype B

DISSERTATION

Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University

Karla J. Medina-Ortega, M.S.

Graduate Program in Entomology

The Ohio State University

2011

Dissertation Committee:

Dr. Luis Cañas, Advisor

Dr. Daniel A. Herms

Dr. P. Larry Phelan

Dr. Claudio Pasian

Karla Jacqueline Medina Ortega

2011

Abstract

The silverleaf whitefly (Bemisia tabaci biotype B) is the main above-ground insect pest of poinsettias (Euphorbia pulcherrima). The ornamental industry is important to the economy of the U.S. and among potted plants, poinsettias rank first in sales across the nation. Control of whiteflies in greenhouses is primarily with insecticides; therefore, mechanisms of resistance in poinsettias are not well understood and have not been thoroughly studied. In an effort to elucidate defense mechanisms in poinsettias, both physical and chemical traits of the plant were studied. The goal of this research was to determine if poinsettia cultivars possess factors/traits affecting the behavior and physiology of the silverleaf whitefly and to some extent evaluate tritrophic interactions.

The objectives were to (1) determine silverleaf whitefly behavioral preference for, and performance on different cultivars through choice and no-choice assays (Chapter 2);

(2) evaluate physical traits possibly mediating resistance, including visual cues by excluding light, leaf thickness, and leaf color indirectly via chlorophyll content using a

SPAD meter, and with the use of a color reader (Chapter 2); (3) determine free amino acids and phenolic compounds potentially involved in mediating resistance to B. tabaci

(Chapter 3); (4) evaluate tritrophic interactions by comparing Eretmocerus mundus

ii parasitism preference between B. tabaci nymphs from a more and a less susceptible poinsettia cultivars (Chapter 4).

Behavioral preference of B. tabaci varied among cultivars, but cultivars with light green leaves were preferred than cultivars with dark green leaves. Leaf color appeared to be influenced by chlorophyll content and leaf thickness. Cultivars with light green leaves had less chlorophyll and had thinner leaves. Trichome density was not significantly different among the cultivars. While whiteflies were capable of recognizing a more susceptible host in the presence of light, this was not the case when in the absence of it indicating leaf color of poinsettia plants plays a key role in recognition by B. tabaci.

Phloem chemistry revealed that cultivars were similar in the composition and concentration of amino acids, and cultivars with light green leaves averaged a higher concentration of amino acids. No differences, although, were observed in concentration of essential amino acids. While concentration of individual amino acids differed in ways that did not help to explain patterns of resistance, concentration of individual phenolic compounds, in both leaf and petioles, did correspond with patterns of resistance to B. tabaci. Total concentration of phenolics were higher in a less susceptible cultivar (cv

[‗Freedom Red‘]) with dark green leaves, and where fertility of B. tabaci was lower, than on a more susceptible cultivar (‗Monet Twilight‘) with light green leaves where fertility of B. tabaci was higher. Concentrations of over 45 % of individual phenolics shared between the two cultivars were higher in ‗Freedom Red‘. While ‗Freedom Red‘ had four unique compounds, only one compound was unique to ‗Monet Twilight‘. Compounds unique to, and in higher amount in ‗Freedom Red‘ included galloyl quinic acids, digalloyl

iii glucose, apigenin diglucoside, apigenin glucoside, kaempferol hexoside, and quercetin pentose rutinoside, and rutin. These compounds have been associated with resistance to herbivores, prolonging developmental times, and acting as antifeedants and toxins.

Higher parasitism of B. tabaci nymphs on cv ‗Monet Twilight‘ was observed than on cv ‗Freedom Red‘. More studies are needed to determine if preferences of parasitism are due to the host plant itself or the nymphs to understand the dynamic interactions of bottom-up and top-down factors; also more in-depth analysis comparing the ratio of amino acids and phenolic compounds in more and less susceptible poinsettia cultivars in relation to the behavior and physiology of both whiteflies and its parasitoids needs to be conducted to better understand their ecological role in this system.

Dedication

To my parents and sisters, my pillars! And to my dear love Justin Whitehill for giving sense to my life!

Acknowledgments

I thank Justin Whitehill first of all for all his support and guidance in most of this journey—without you this journey would have not been the same.

I thank my committee members Luis A. Cañas, Larry Phelan, Daniel Herms, and

Claudio Pasian for their guidance through all my degree. Luis A. Cañas was more than an advisor to me, thank you for your friendship. Larry Phelan, your valuable mentorship and critical thinking will always be with me. Daniel Herms, thank you for your constructive feedback and critical thinking principles as well. Claudio Pasian, thank you for your support and help. I thank also Pierluigi Bonello for the use of his HPLC and an office in Plant Pathology my last months‘ writing this dissertation.

I thank Claudia Kuniyoshi and Nuris Acosta for their invaluable friendship, so many entertaining afternoons and help throughout my degree. I thank my dear friend

Ronald Batallas and Rodrigo Chorbadjian for being a great moral support. I thank all my friends from Entomology including Brenda Franks, who was so special and kind always, all of you made better my living at Wooster.

I thank the help of Wilmer Rodríguez, Jim Hacker, Mark Belcher, Alejandro Paz,

Eliana Rosales, Alejandra Claure, Samuel Discua, Diego Rincón, Theodore Derksen,

Gabriel Abud, and David Abud for help in collecting data.

Vita

November 30th, 1979 ...... Tegucigalpa, Honduras

2000...... B.S. Agriculture, Escuela Agrícola Panamericana, El Zamorano, Honduras

2001...... Research Scholar, Illinois Natural History Survey, University of Illinois at Urbana- Champaign

2002...... Research Scholar, CABI, Switzerland

2003...... Field and Laboratory Research Scientist, Escuela Agrícola Panamericana, El Zamorano, Honduras

2005...... M.S. Entomology, University of Idaho

Apr-Sept 2006 ...... Graduate Research Associate, Department of Entomology, The Ohio State University

Oct 2006-Jun 2007 ...... Graduate Teaching Associate, Department of Entomology, The Ohio State University

Jul 2007-Mar 2011 ...... Graduate Research Associate, Department of Entomology, The Ohio State University

Apr 2011-present ...... Graduate Teaching Associate, Department of Entomology, The Ohio State University

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Publications

Medina-Ortega, K.J., Bosque-Pérez, N.A., Ngumbi, E., Jiménez-Martínez, E.S., and Eigenbrode, S.D. 2009. Rhopalosiphum padi (Hemiptera: Aphididae) responses to volatiles cues from barley yellow dwarf virus-infected wheat. Environ. Entomol. 38(3): 836-845.

Fields of Study

Major Field: Entomology

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Table of Contents

Abstract ...... ii Dedication ...... v Acknowledgments...... vi Vita ...... vii Publications ...... viii Fields of Study ...... viii Table of Contents ...... ix List of Tables ...... xi List of Figures ...... xii Chapter 1 Literature Review ...... 1 General background ...... 1 Statement of the problem ...... 3 Research foci ...... 4 Poinsettias ...... 5 Bemisia tabaci biotype B ...... 6 Host-plant resistance to Bemisia tabaci biotype B ...... 9 Biological control of whiteflies ...... 19 Research objectives ...... 21 Chapter 2 Behavioral preferences and performance of silverleaf whitefly among poinsettia cultivars: physical traits associated with resistance ...... 23 Abstract ...... 23 Introduction ...... 25 Materials and Methods ...... 27 Results ...... 40 Discussion ...... 59 Chapter 3 Constitutive phloem chemistry of poinsettia cultivars: nutritional and defensive traits associated with resistance to the silverleaf whitefly ...... 64 Abstract ...... 64 ix

Introduction ...... 66 Material and Methods ...... 68 Results ...... 74 Discussion ...... 87 Chapter 4 Eretmocerus mundus Mercet (Hymenoptera: Aphelinidae) response to whitefly infestations on a resistant and a susceptible poinsettia cultivar ...... 92 Abstract ...... 92 Introduction ...... 94 Materials and Methods ...... 96 Results ...... 98 Discussion ...... 100 Chapter 5 Summary and future work ...... 104 Behavior and physiology of B. tabaci in poinsettias ...... 105 Poinsettias physical defense mechanisms affecting B. tabaci ...... 106 Poinsettias chemical defense mechanisms affecting B. tabaci ...... 106 Poinsettia cultivar-mediated differences in behavioral preference of a parasitoid of B. tabaci...... 108 Appendix A: Screening for whitefly resistance in poinsettias, summer 2006 ...... 111 Appendix B: Screening for whitefly resistance in poinsettias, summer 2007 ...... 117 Bibliography ...... 124

List of Tables

Table 2.1 Cultivars screened for resistance and susceptibility...... 30 Table 2.2 Mean number (± SE) of Bemisia tabaci biotype B on multiple poinsettia (Euphorbia pulcherrima) cultivars (choice test experiment 1)...... 41 Table 2.3 Mean number (± SE) of Bemisia tabaci biotype B settling on multiple poinsettia (Euphorbia pulcherrima) ...... 42 Table 2.4 No-choice experiment 1: Average reproductive rate ‗Ro‘, intrinsic rate of increase ‗r‘, and cohort generational time ‗Tc‘ in DD1 of Bemisia tabaci biotype B reared in different poinsettia (Euphorbia pulcherrima) cultivars and the number of eggs per female...... 45 Table 2.5 No-choice experiment 2: Average reproductive rate ‗Ro‘, intrinsic rate of increase ‗r‘, and cohort generational time ‗Tc‘ in DD1 of Bemisia tabaci biotype B reared in different poinsettia (Euphorbia pulcherrima) cultivars and the number of eggs per female...... 47 Table 2.6 No-choice experiment 3: Average reproductive rate ‗Ro‘, intrinsic rate of increase ‗r‘, and cohort generational time ‗Tc‘ in DD1 of Bemisia tabaci biotype B reared in a less and a more susceptible poinsettia (Euphorbia pulcherrima) cultivar...... 48 Table 2.7 Effect of light or dark conditions (visual) and combination of cultivars on the host recognition capabilities of Bemisia tabaci biotype B. Results of a nested ANOVA...... 53 Table 2.8 Mean1 (and SE) of leaf physical traits of five poinsettia (Euphorbia pulcherrima) cultivars...... 56 Table 2.9 Color reading coordinates from the Minolta Reader CR-10 for five poinsettia cultivars...... 57 Table 3.1 Concentrations (nmol/mg fresh weight ± SE) of free amino acids detected in petiole tissues of poinsettia cultivars (Euphorbia pulcherrima). Cultivars with dark green leaves are less preferred than light green leaf cultivars by Bemisia tabaci...... 75 Table 3.2 Characterization of constitutive phenolics in leaves and petioles of poinsettia (Euphorbia pulcherrima) cultivars ‗Freedom Red‘ (FR) and ‗Monet Twilight‘ (MT) using UV and MSn spectrometry...... 79 -4 Table 3.3 Mean ± SE UV280 peak area per microliter (x 10 ) of individual and total phenolic compounds in leaf and petiole ...... 85 xi

List of Figures

Figure 2.1 Clip cages used for infesting plants with whiteflies...... 33 Figure 2.2 Visual cues experiment set-up. Cages are in a randomized complete block design...... 36 Figure 2.3 Experiment 3. Mean whitefly numbers (± SE) after 48 h on a light green leaf cultivar ‗Monet Twilight‘ and a dark green leaf cultivar ‗Freedom Red; the asterisks indicates a significant difference at p = 0.0007...... 43 Figure 2.4 Experiment 3. Rate of emergence of B. tabaci biotype B on poinsettia (Euphorbia pulcherrima) cultivars ‗Freedom Red‘ and ‗Monet Twilight‘...... 50 Figure 2.5 (A) Bemisia tabaci biotype B females reared on a less susceptible poinsettia cultivar ‗Freedom Red‘ and a more susceptible cultivar ‗Monet Twilight‘.(B) Representation of the cumulative number of eggs laid per female per day. The asterisk indicates that, on day 10 after emergence, mean total number of eggs laid per female in ‗Freedom Red‘ and ‗Monet Twilight‘ was significantly different at p = 0.0029...... 52 Figure 2.6 Effect of visual cues on Bemisia tabaci biotype B host recognition capabilities. Average number of whiteflies on each plant (±SE) in dark and light condition (see for layout Figure 2.2); (A) when offered two plants of the same cultivar ‗Freedom Red‘; (B) when offered two plants of the same cultivar ‗Monet Twilight‘; and (C) when offered one plant of each cultivar. Asterisk indicates a significant difference in preferential response by whiteflies between the two cultivars (p = 0.0079; see also Table 2.7)...... 54 Figure 2.7 3D-bubble size representation of color coordinates from Minolta Reader CR- 10. The dot represents the lightness of the color L* = 0 yields black and L* = 100 indicates diffuse white; a* negative values indicate green; b* positive values indicate yellow...... 58 Figure 3.1 Concentration of (A) total amino acids (± SE) and (B) essential amino acids (± SE) in petiole tissue of several poinsettia (Euphorbia pulcherrima) cultivars. Bars with different letters are significantly different using protected Fisher‘s LSD at α = 0.05. Orthogonal contrast with overall total (± SE) (C) and essential (± SE) (D) amino acid concentrations between cultivars with dark and light green leaves...... 76 Figure 3.2 Biplot of principal component analysis of free amino acids (significant and not significantly different) profiles among poinsettia (Euphorbia pulcherrima) xii

cultivars. PC1 and PC2 axes explained 95.7% of total amino acid variance. Cultivars: EFr=‘Early Freedom Red‘, EPr=‘Early Prestige Red‘...... 78 Figure 4.1 Percentage (± SE) of Bemisia tabaci biotype B nymphs parasitized by Eretmocerus mundus in a less susceptible (‗Freedom Red‘) and a more susceptible (‗Monet Twilight‘) poinsettia (Euphorbia pulcherrima) cultivar. Asterisk means a significant difference from ANOVA p = 0.05...... 99 Figure 4.2 Percentage (± SE) of Eretmocerus mundus emerged from whitefly nymphs parasitized in a less susceptible (‗Freedom Red‘) and a more susceptible (‗Monet Twilight‘) poinsettia (Euphorbia pulcherrima) cultivar. ANOVA p = 0.42. .... 101

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Chapter 1 Literature Review

General background

Poinsettias, Euphorbia pulcherrima Willd. ex Klotzsch. (Euphorbiacea) are popular ornamentals and have been considered the single most valuable flowering plant crop in the U.S. and the most popular potted flowering or foliage plant for the Christmas season (Gast 2009). Poinsettia plants also rank as the nation‘s top-selling potted ornamental and as a crop are valued at $180 million (NASS 2008). In the U.S., greenhouse and nursery products generate the second highest net value added per dollar of gross income (Jinkins and Ahearn 1991). Furthermore, poinsettia‘s demand continues to grow (Prince and Godfrey 2010).

Poinsettias are colonized by many insects, but whiteflies are the most dominant pest that severely reduces the value of poinsettia plants. Whiteflies are severe pests of many other floricultural and vegetable crops worldwide (Bethke et al. 1991, Byrne and

Bellows 1991, McAuslane 1996, Van Driesche and Lyon 2003, Perring and Symmes

2006a). Among whiteflies biotypes, the silverleaf whitefly, Bemisia tabaci (Gennadius) biotype B (Hemiptera: Aleyrodidae) is the major whitefly found colonizing poinsettias

(Van Driesche and Lyon 2003, Ecke et al. 2004).

Major pests colonizing poinsettias

Poinsettias are colonized by many insect species, including thrips, fungus gnats, mealybugs, and whiteflies (Chen et al. 2004). However, the major pests are whiteflies and fungus gnats (Ecke et al. 2004). The two common types of whiteflies that feed on poinsettias are: common greenhouse whitefly (Trialeurodes vaporariorum Westwood

(Hemiptera: Aleyrodidae), and the silverleaf whitefly (SLWF) (Ecke et al. 2004).

Whitefly adults and nymphs feed from the phloem sap of leaves and stems, which can result in wilted plants with chlorotic leaves and mottled or discolored bracts (Van

Driesche and Lyon 2003, Ecke et al. 2004).

Poinsettia cultivars and preference by the silverleaf whitefly

The commercially available poinsettia cultivars differ physically by color of green leaf foliage and bract coloration. Two types of green foliage are dominant: light and dark green leaves (Ecke et al. 2004). There are also variegated leaf cultivars but those are not examined in this study. Studies suggests that light green leaf cultivars and those with white and light colored bract poinsettias are preferred by the silverleaf whitefly

(Sanderson 1992, Petro and Redak 2000, Rice and Crane 2000). Preference for light colored bract poinsettias was documented for the greenhouse whitefly, Trialeurodes vaporariorium (Fischer and Shanks 1974), which was the dominant type of whitefly found in poinsettias prior to the 1990‘s. Preference for cultivars with light colored green

2 leaves by the silverleaf whitefly has also been reported in Hibiscus rosa sinensis

(Malvaceae) (Liu and Stansly 1998a).

Among poinsettia cultivars, ‗Freedom Red‘, ‗Red Velvet‘, and ‗Pepride‘ are described as less susceptible in two independent studies (Petro and Redak 2000, Rice and

Crane 2000, Petro et al. 2002). More susceptible cultivars included ‗Peterstar‘ and

‗Success‘ (Petro and Redak 2000, Petro et al. 2002). Altogether, few studies have evaluated poinsettia cultivars susceptibility to B. tabaci, and no studies have documented susceptibility of current cultivars in the market to B. tabaci, even more, factors that may be underlying it.

Statement of the problem

Bemisia tabaci biotype B is a concern among poinsettia growers because of its high reproductive capacity, feeding damage, and ability to transmit geminiviruses

(Oliveira et al. 2001) . Continual feeding on the phloem sap of leaves and stems by the nymphs and adults weakens the plant and reduces aesthetic value. Additionally, excreted phloem sap, known as honeydew, promotes the growth of mold on the surface of the leaves. Control of whiteflies in greenhouses by conventional methods has been challenging due to its rapid development of resistance to insecticides (Palumbo et al.

2001). Whitefly resistance to different classes of insecticides, such as organophosphates, carbamates, pyrethroids, and organochlorines, has been well documented (Dittrich et al.

1990, Cahill et al. 1995). One recent study documented whitefly resistance in 3 greenhouses to imidacloprid, a neonicotinoid insecticide widely used in Guatemala

(Byrne et al. 2003). Despite B. tabaci‘s fast development of resistance to insecticides, growers still rely heavily on them for its management because of the important role aesthetics appearance plays in the ornamental market, which makes B. tabaci control very difficult (Heinz and Parrella 1994).

Because of the heavy reliance on insecticides and the ability of whiteflies to develop insecticide resistance, emphasis on other management tactics is needed. The use of resistant cultivars of poinsettias would provide a valuable alternative strategy to whitefly management in poinsettia production by reducing reliance on insecticides. To develop such cultivars, it is necessary to obtain information about whitefly preferences and to identify plant resistant characteristics; none of which have been thoroughly investigated for the poinsettia-whitefly system.

Previous research has demonstrated that whiteflies and specifically, B. tabaci biotype B, shows some preference for light green colored foliage over dark green foliage on plants (Liu and Stansly 1998a). This trend has continued. Anecdotal observations by greenhouse growers in Northwest Ohio observed that B. tabaci biotype B prefers poinsettias of light green leaves over dark green leaf ones (Personal communication, Luis

A. Cañas). Investigations and documentation has not been pursued, which is important to gives us insight into potential whitefly-resistant characteristics.

Research foci

Identification of less preferred cultivars could be useful for future incorporation into pest management programs against whiteflies. In the short run, growers could be able to adjust their management practices like focusing early sampling efforts on preferred cultivars. In the long run, growers could also benefit from cultivars bearing resistant traits since addition of specific traits into poinsettia breeding programs could reduce the reliance on pesticides and lead to the use of alternative management tactics, like biological control. Therefore, the overall goals of this research were to identify resistant and susceptible cultivars of poinsettias to B. tabaci biotype B and then to characterize traits (i.e. resistance mechanisms) of poinsettia cultivars that negatively impact B. tabaci behavior and physiology.

Poinsettias

Poinsettia, Euphorbia pulcherrima Willd. ex Klotzsch (Euphorbiacea), is native to

Mexico and was introduced into the U.S. (NC) by Joel Poinsett in 1825 (Ecke et al.

2004). In 1963 the first commercial-quality cultivars were available (Parks and Moyer

2004). Today more than 250 cultivars are available and distributed by the major poinsettia breeder companies: Paul Ecke Ranch, Dummen USA, Fischer USA, Oglevee

Ltd., and Select First Class.

Selection of cultivars is driven by value market share, and breeders are under constant pressure to develop new cultivars that are pleasing to consumers but still profitable for the growers. For example, bract color (the part that changes in color) is an important aesthetic trait to consumers, but it is the growth habit and time of bract change coloration that are important to growers. Poinsettias are vegetatively propagated, and thus annual selection is needed to maintain crop uniformity (Ecke et al. 2004). Desired cultivars or desirable traits are derived by natural or induced mutations (Ecke et al. 2004,

Parks and Moyer 2004).

Bemisia tabaci biotype B

Whiteflies are classified in the order Hemiptera, family Aleyrodidae, and subfamily Aleyrodinae (Byrne and Bellows 1991). Bemisia tabaci has received many common names such as the tobacco whitefly, sweetpotato whitefly biotype B, and the B. tabaci poinsettia strain. Bemisia tabaci biotype B is also known as Bemisia argentifolii

Bellows and Perring (Bellows et al. 1994), but, due to insufficient molecular or biological data available to support the distinction of B. tabaci and B. argentifolii (De Barro et al.

2005), in this study research I will refer always to the former name. It is common to refer to the whitefly using the species name and biotype when reporting research. The common name used today for B. tabaci biotype B is the silverleaf whitefly, because when

6 this insect feeds on squash a silvering on the leaves occurs (Cohen 1993, Costa et al.

1993b, Perring et al. 1993).

Currently, B. tabaci is believed to be a species complex comprised of many biotypes. Biotype is a term used to designate populations, morphologically indistinguishable, that possess a range of biological characteristics (eg., host range, geographical location, behavior, ability to transmit viruses, resistance to insecticides, and interaction with natural enemies) that set them apart from each other (Oliveira et al.

2001), (Perring 2001). There are 41 populations of B. tabaci that have been studied around the world, of which 24 have been designated with a biotype label (Bedford et al.

1994, Brown et al. 1995, Brown et al. 2000, Perring 2001). Of these biotypes, the most widespread worldwide and common in association with ornamentals, especially poinsettias, is biotype B (Perring et al. 1992, Beitia et al. 1997, De Barro et al. 1998,

Abdullahi et al. 2003, Wu et al. 2003).

Bemisia tabaci has been in the U.S. since 1897 (Russell 1957), but primarily as biotype A. Biotype B was first reported in Florida, U.S. in 1986 when high whitefly infestations not experienced before in greenhouse-grown ornamentals were observed

(Price et al. 1987, Costa et al. 1993a). Surveys conducted in the Caribbean Basin provide supporting evidence that introduction of B. tabaci B-biotype was by way of poinsettia production facilities (Brown et al. 1995). Consequently, whiteflies infested poinsettia nurseries and expanded into other vegetables crops, like tomato, cotton, melon, okra, pepper, squash, bean, tobacco, and cassava (Berlinger 1986, Chalfant and Summer 1993,

Brown 1994).

Early in the 90‘s biotype A was displaced from poinsettias by biotype B, and consequently, this whitefly was found in the Sunbelt states, prominently in Arizona,

California, Florida, and Texas (Costa and Brown 1991, Perring et al. 1992). Today biotype B is found across all of the United States. Although the precise origins of this whitefly remain to be determined, it is believed to have come from India and/or Pakistan due to the abundance of natural enemies found in the region (Brown et al. 1995).

Biology of Bemisia tabaci

Bemisia tabaci is multivoltine and may have up to 11-15 generations a year under optimal conditions. It is a polyphagous pest species that feeds exclusively from the phloem sap excreting excess sugar as honeydew (Walker and Perring 1994). Its stylets penetrate between intercellular spaces, minimizing the rupture of adjacent plant cells, until reaching the sieve element cells (Kaloshian and Walling 2005). Hence, the damage to host plants comes from directly feeding on sap, causing weakening and early wilting of the host plant, thus, reducing yield (Avidov 1956). Indirect damage is also caused by the excretion of honeydew on the foliage, which favors the growth of sooty mold causing leaves to look dirty and feel sticky, thereby, affecting aesthetic commercial value of the plants. The presence of sooty mold also reduces photosynthesis (Avidov 1956). Another indirect consequence of whitefly feeding is the transmission of viruses that may render plants unmarketable (Cohen and Berlinger 1986).

The whitefly life cycle is temperature dependent and consists of an egg, four nymphal instars, and winged adults (Bellows et al. 1994). Fecundity will depend on the host plant females were reared on and it can range from 48 to 394 eggs per female over their life span (Byrne and Bellows 1991). For example, in cotton female B. tabaci can lay an average of 160.4 eggs over their life span (Gameel 1974, Byrne and Bellows

1991). In poinsettias, the mean reproductive capacity is approximately 85 eggs per female (Bethke et al. 1991), which peaks in the first week after adult emergence.

Enkegaard (1993a) found a similar pattern on poinsettias, where the highest fecundity occurred between 5-12 days after adult emergence, with an average maximum number of eggs per female per day of 1.9, 5.8 and 8.4 at 16°C, 22°C and 28°C, respectively. The average developmental time from eggs to adults and average adult longevity on poinsettias was of 168.1 and 50.8 days at 16°C, and 29.9 and 16 days at 28°C, respectively (Enkegaard 1993a). Overall, developmental time, adult longevity, and female reproductive time appear to be optimal in the temperature range of 22-28 ºC (Tsai and Wang 1996, Wang and Tsai 1996).

Host-plant resistance to Bemisia tabaci biotype B

Plant resistance to insects can be expressed and defined in many ways. A resistant plant, in the broadest sense, is one that possesses traits that makes it less preferred by and has detrimental effects on insects upon feeding, consequently being less

9 damaged compared to a susceptible plant. These traits may be expressed as physical

(morphological) and/or chemical [plant primary (nutrient quality) and secondary compounds (defensive toxins)] defense characteristics that affect negatively the preference and/or performance of the insect pest. In integrated pest management, plant resistance refers to the use of resistant cultivars/varieties that reduce pest abundance or damage and is used in conjunction with other cultural control methods.

Resistance of plants to a particular insect species is a relative term because the designation ―resistant‖ depends on the comparison with more susceptible plants.

Consequently, resistance could be visualized as a continuum, where complete resistance is on one end and complete susceptibility on the other (Berlinger 1986). Types of resistance against B. tabaci have been commonly reported as antixenosis, antibiosis, or tolerance. Painter (1951) defined these as the following: antixenosis affects the behavior of the insect and typically is expressed as non-preference for resistant plants; antibiosis affects the biology of the insect, usually increasing mortality or reducing longevity and reproduction; tolerance is a mechanism in which a plant is able to withstand damage but maintain yield as a plant without any insect pests.

My research has focused on studying resistance mechanisms that affect the insects‘ response to plants; therefore, tolerance will not be discussed. Specifically, this study addresses the role of constitutive defenses of poinsettias and not induced against B. tabaci biotype B. Constitutive defenses are always present or expressed in plants regardless of having been challenged with insects and/or pathogens. Conversely, induced defenses, which were not investigated in this research, are defenses that are triggered by

10 an external elicitor (substances, insects, pathogens, or any other form of attack) and are usually expressed in higher amounts than constitutive levels.

Whiteflies are one of the major worldwide pests affecting a wide range of cultivated and non-cultivated plants, which are comprised mostly in plant families such as Fabaceae, Malvaceae, Euphorbiaceae, Solanaceae, and Asteraceae (Mound and Halsey

1978, Berlinger 1986, Oliveira et al. 2001). Surprisingly, complete host plant resistance

(HPR) to whiteflies is rare in cultivated crops (Bellotti and Arias 2001). This, however, may be due to insufficient screening of germplasm (De Ponti et al. 1990, Bellotti and

Arias 2001). In poinsettias, very few studies have evaluated cultivar resistance to B. tabaci despite being a serious pest in poinsettia productions since the mid-80‘s (Brown et al. 1995). Since then, few studies have focused on elucidating the mechanisms driving natural selection of whiteflies for certain poinsettia cultivars (Petro and Redak 2000, Rice and Crane 2000, Petro et al. 2002).

Olfactory and physical traits associated with resistance to whiteflies

One component of host finding/recognition used by insects is the use of olfactory cues known as volatile organic compounds (VOCs). Little is known of the role, if any,

VOCs may play in host finding/recognition by B. tabaci among poinsettia cultivars.

What is known is that B. tabaci is capable of responding to olfactory cues. For example, a study evaluated responses of B. tabaci biotype B to headspace volatiles of different intact poinsettia cultivars, and showed that whiteflies had no particular preference for any

11 cultivar evaluated (Brilliant Diamond, Celebrate 2, Lilo and Red Sails) (Heinz et al.

1993). All the cultivars used in the study were dark green leaves. Using a Y-tube olfactometer, (Jing et al. 2003) measured significant response to odors of five host plants

(poinsettias, chinese cabbage, sweet potato, cabbage, and tomato; varieties or cultivars used were not specified) compared to a blank control. In earlier experimental evaluations, B. tabaci biotype B showed no preferential olfactory responses to old and young crushed cassava or tobacco leaves (Mound 1962).

From these studies, it is unclear the role olfactory cues from specific hosts play in

B. tabaci host finding/recognition. For Trialeurodes vaporariorum, the greenhouse whitefly, olfactory cues play a minor role in orientation and landing on hosts like beans and poinsettias (Vaishampayan et al. 1975). The authors determined that responses were largely driven by visual cues (color in the range of 500-600 nm). For the studies supporting B. tabaci’s response to olfactory cues, exclusion of visual cues was not performed; therefore, the role of visual cues, such as color, could have confounded responses to VOC‘s (Heinz et al. 1993, Jing et al. 2003). In addition, qualitative and/or quantitative analyses of VOC‘s have not been evaluated on poinsettia plants.

Whiteflies use visual cues such as leaf color to find potential plant hosts (Gerling and

Horowitz 1983). For T. vaporariorum, Fischer and Shanks (1974) studied poinsettia bract color effect on whitefly preference, and observed that whiteflies showed strong preferences towards poinsettia cultivars with light-colored leaves. A total of 68 cultivars were evaluated, with 8 pink, 14 white, and 46 red bract cultivars respectively. Of these, red bracts were less heavily infested than pink, and pink less than white bracts. Cultivars

12 used in that study are not available today, but the trend of cultivating dark and light green leaves is still present in the market (Barret et al. 2003). Therefore, studies to determine whiteflies‘ behavior to plant hosts with different green leaf colors are necessary.

For B. tabaci, it has been demonstrated that color is an important factor in whitefly host recognition (Mound 1962, Berlinger 1980a). Bemisia tabaci biotype B showed a positive response to yellow-green light (520-620 nm). A greater number of adults settled on this wavelength stimulus, and to a lesser extent to blue ultraviolet light

(Mound 1962). More recently, the ornamental Hibiscus rosa-sinensis (Malvaceae) showed resistance to B. tabaci biotype B associated with dark green leaf color cultivar

(Liu and Stansly 1998a). In a choice test, hibiscus plants with red bloom flowers, dark green and thicker foliage were less preferred by whitefly adults for oviposition compared to plants with pink bloom flowers and light green foliage (Liu and Stansly 1998a).

Apparently, these plants are not only less preferred because of their leaf color, but a more complex suite of traits may be associated to their resistance, since development and survivorship of B. tabaci biotype B were also affected on these cultivars. It is possible that whiteflies use color to discriminate among optimal and suboptimal hosts.

It has been documented that some insects find or recognize hosts based on a combination of visual and olfactory cues (Visser 1988). For example, Lygus hesperus

(Heteroptera: Miridae) has positive responses to volatiles emitted from alfalfa, and these responses are significantly enhanced by visual cues (530 nm green light emitting diode)

(Blackmer and Cañas 2005). Similarly, Myzus persicae (Hemiptera: Aphididae) response to hosts was enhanced when both olfactory and visual cues were present (Srinivasan et al.

2009). From previous studies, responses to VOCs by B. tabaci appear to be specific to the plant host being evaluated (Cao et al. 2008).

Odors from tomato and cabbage, but not from pepper, have been reported to attract B. tabaci. Tomato and cabbage were also preferred based on their light green leaf color, in conjunction with VOCs (Cao et al. 2008). Pepper, despite not eliciting responses to olfactory cues, was attractive to whiteflies due to lighter green leaf color

(Cao et al. 2008). More recently, researchers have identified specific volatiles in tomatoes that may be repellent or attractive to B. tabaci biotype B (Bleeker et al. 2009).

The evaluation of whitefly responses to visual and olfactory cues from poinsettias would provide useful information about whitefly host selection.

Trichome density can impact B. tabaci biotype B populations. Several studies have evaluated the preference of B. tabaci biotype B among cultivars of cotton, hibiscus, and tomato and found that whiteflies show preference for cultivars with moderately hairy leaves (Butler et al. 1986, Liu and Stansly 1998a, Fancelli and Vendramim 2002,

Toscano et al. 2003). For example, more whitefly adults and eggs were observed on cotton (Butler et al. 1986) and soybean (McAuslane 1996) cultivars with hairy leaves than on semi-smooth or smooth-leaf isolines.

Another plant physical trait that can affect whiteflies is leaf thickness. Newly hatched crawlers have a stylet length of 113.8 ± 4.2 µm (Freeman et al. 2001). Chu et al.

(1995b) demonstrated that the distance of vascular bundle sheaths to the lower surface of leaves, where phloem is accessed by the stylets of whiteflies, influences host preference, oviposition, and feeding of B. tabaci (Chu et al. 1995b). For example, in cotton the

14 distance from lower leaf surface to vascular bundle sheaths was of 60 µm compared to

131 µm from the upper leaf surface. Cotton cultivars with greater leaf thickness have a greater distance from lower leaf surface to phloem sieve elements, and thus, were less preferred for oviposition and feeding by whiteflies (Chu et al. 1999a). Similarly, hibiscus cultivars with thicker leaves had less oviposition and lower B. tabaci survival than thinner leaf cultivars (Liu and Stansly 1998a).

Chemical traits associated with resistance to whiteflies

Plant suitability to insects is determined in great part by the plant‘s chemical composition. Both primary and secondary metabolites play a role in defense against insects (Haukioja et al. 1991, Berenbaum 1995). Primary metabolites usually refer to the nutritional components of the plants used for growth and maintenance, while secondary compounds usually refer to compounds not directly involved in development, growth, or reproduction but known to be involved in defense against pathogens and herbivores.

Variation of plant host nutrition levels has profound effects on B. tabaci populations. Poinsettia leaf nitrogen content and the type of nitrogen used for fertilization of plants perceived during probing influences host selection and oviposition preferences of the silverleaf whitefly (Bentz et al. 1995b, a). For example, oviposition and adult emergence was higher on fertilized plants than on non-fertilized, and higher on ammonium nitrate treated poinsettia plants than on calcium nitrate treated plants. In other experiments, higher number of adults and immature whiteflies found in cotton were

15 associated with increased nitrogen levels in the leaves (Bi et al. 2001). In a similar study,

B. tabaci biotype B time to adult emergence in cotton decreased with increased levels of leaf nitrogen (Blua and Toscano 1994). Most studies to date have only focused on the evaluation of one poinsettia or cotton variety. Therefore, it is unknown if the type of poinsettia cultivar subjected to the same fertilization conditions would have an effect on whitefly host selection.

Research focuses on nitrogen because this nutritional element has been proven to be a limiting factor for insects in general (Mattson 1980). Nitrogen is available to sap sucking insects in the form of free amino acids and proteins (Mattson 1980). Plants with greater nitrogen content have been shown to support higher oviposition and survival of whiteflies (Bentz et al. 1995c). Other components of phloem sap include carbohydrates, which are found in high concentrations compared to free amino acids (Byrne and Miller

1990). Previous studies have shown minimal or no effect of amino acids on whitefly populations. For instance, (Bi et al. 2003) found that soluble protein and free amino acid levels in cotton petioles were not correlated with the number of whitefly immature nymph and adult populations in the field. Similar studies, evaluating amino acid levels associated with performance or abundance of insects, have found no effect of amino acids on glassy-winged sharpshooter (Homalodisca vitripennis) and mealybugs (Phenacoccus manihoti), which are both sap-sucking insects (Tertuliano and Leru 1992, Bi et al. 2007).

Other studies have shown that whiteflies can compensate or modify their phloem consumption rate based on dietary component levels. For example, Byrne and Miller

(1990) found that whiteflies increase their phloem consumption rate (measured by an

16 increase in honeydew excretion) when feeding on diets low in nitrogen. Because free amino acids are found in low concentrations in the phloem sap of plants, compared to carbohydrates, and their amount is correlated with nitrogen in plants, whiteflies feeding on plants with low nitrogen will need to increase consumption to obtain more amino acids (Crafts-Brandner 2002, Bi et al. 2003, Kuniyoshi 2007).

While the effects of amino acid levels on whiteflies have been subtle, there is evidence that amino acid profiles might change depending on the interaction with other herbivores or the type of plant variety (Byrne and Miller 1990, Tertuliano and Leru 1992,

Kuniyoshi 2007). Kuniyoshi (2007) showed that herbivore presence can affect the concentration of amino acids in poinsettias. Free amino acids levels were lower on poinsettias cv. ‗Freedom red‘ when exposed to both B. tabaci biotype B and Bradysia impatiens, compared to plants free of insects (Kuniyoshi 2007). Amino acid concentrations may also vary among varieties of the same plant species. Tertuliano and

Leru (1992) found that amino acid concentrations varied greatly among five cassava varieties.

Poinsettia nutritional quality and resistance to insects are impacted by primary and secondary compounds. Secondary compounds have negative effects on herbivores, playing a critical role in plant resistance to insects (Fraenkel 1959, Mattson 1980, Scriber and Slansky 1981, Berenbaum 1995). Chemical studies of poinsettias revealed that the latex collected from stems and bracts contained sterols, phenols, and triterpenols

(Dominguez and et al. 1967). Although latex could potentially trap whiteflies, as in lettuce (Dussourd 1995), in poinsettias this is not the case. The only secondary

17 compounds found in phloem of poinsettias are phenolics (Calatayud et al. 1994a).

Phenolics have been shown to reduce whitefly preference and number of adult whiteflies on tomatoes (Inbar et al. 2001). Another study, with cotton plants, also showed a negative relationship of B. tabaci density with phenolic levels (Butter et al. 1992).

Other insects with similar feeding habits (aphids, mealybugs, and psyllids) have been affected by the presence of phenolic compounds. Latex-producing plants, like lettuce (Lactuca sativa L.), have been shown to have organic acids, phenolics and a triterpene alcohol (Crosby 1963). High levels of phenolic acids have being associated with resistance to lettuce root aphids among lettuce cultivars (Cole 1984). Mealybugs

(unarmored scale insects) feeding from cassava (Euphorbiaecea) diets containing rutin, a phenolic compound, had longer developmental time (Calatayud et al. 1994b). Another study evaluated the effect of screening methods on the expression of antibiosis of two romaine lettuce cultivars to Diabrotica balteata LeConte (Coleoptera: Chrysomelidae) and found that regardless of the screening methods, there were significant differences in

D. balteata behavioral preferences between the two lettuce cultivars (Huang et al. 2003).

The authors suggested feeding preferences may be due to differences in the concentration of secondary compounds between these varieties.

Phenolic composition among Euphorbia spp. cultivars has been evaluated in a few studies. Some authors suggest that related cultivars show similar phenolic profiles

(Biesboer et al. 1982, Warnaar 1987). Eight poinsettia cultivars, six closely related on the basis of presumed ancestry and two unrelated, were evaluated for phenolic composition (Werner and Sink 1977). Analyses of the six closely related cultivars (‗Paul

Mikkelsen‘, Annette Heg‘, ‗Mikkel Improved Rochford‘, 7006, ‗Eckespoint C-1 Red‘, and ‗Eckespoint C-1 White‘) showed they were similar if not identical in phenolic compound composition. The two unrelated cultivars (‗Truly Pink‘, ‗Eckes Flaming

Sphere‘) presented minor differences in phenolic profiles. Qualitative differences were found in phenolic composition, but these differences were minor according to the authors.

Recently, it was determined that the site of resistance against B. tabaci in alfalfa is in the phloem sieve elements (Jiang and Walker 2007). Most of the phenolic identification in previous studies for poinsettias has been done using whole leaf extracts. Therefore, determining the composition and concentration of phenolic compounds from dark and light green leaf poinsettia cultivars from a more concentrated pool of phloem sap is necessary to determine if differences in quantity/quality of this defense compound are associated with resistance to B. tabaci biotype B.

Biological control of whiteflies

When it comes to biological control of whiteflies many studies have been done

(Enkegaard 1993b, Drost et al. 1999, De Barro et al. 2000, Ellis et al. 2001, Gerling et al.

2001, Bogran and Heinz 2002, Antony et al. 2003, Ardeh et al. 2005a, b), but only one study has evaluated the effect of two poinsettia cultivars, ‗Annette Hegg Brilliant

Diamond‘ and ‗Lilo‘, on the performance of one predator, Delphastus pusillus LeConte, and four parasitoids, Encarsia formosa Gahan, Encarsia luteola Howard, Encarsia pergandiella Howard, and Encarsia transvena (Timberlake), of B. tabaci biotype B

(Heinz and Parrella 1994). Adult longevity of the biological control agents was evaluated and no significant differences were observed between the two cultivars, although it did varied significantly between natural enemies. Prey consumption and oviposition by the predator was favored on ‗Annette Hegg Brilliant Diamond‘ poinsettia, which has 15 % less trichome densities than ‗Lilo‘ poinsettias. Host feeding and parasitism among the four parasitoids was similar among the two cultivars, but consistently the four parasitoids performed better on the 15 % less trichome density cultivar (‗Annette Hegg Brilliant

Diamond‘). It is possible cultivars with fewer leaf trichomes are more favorable to parasitoids and predators of B. tabaci biotype B (Heinz and Parrella 1994).

One new predatory mite, Amblyseius swirskii (Athias-Henriot), has been introduced for the control of thrips and whiteflies (Messelink and de Groot 2005) on eggplants and also in ornamentals (Piron et al. 2006). This predatory mite has shown to be promising for biological control programs that include parasitic wasps, like

Eretmocerus mundus Mercet, which has proven to be the most effective against B. tabaci biotype B in commercial greenhouses (De Barro et al. 2000). To date there are no studies evaluating the effect of physical or chemical characteristics from different poinsettia cultivars on E. mundus. The effect of poinsettia cultivars influencing preference of whiteflies and its natural enemies has not been studied thoroughly. Evaluating tritrophic interactions can broaden our knowledge of the factors that affect the responses of whiteflies and its natural enemies. This knowledge is critical to implement effective biological control practices in greenhouse ornamental systems.

Research objectives

The goals of this research were to characterize mechanisms of resistance in poinsettias to the silverleaf whitefly Bemisia tabaci biotype B. This was accomplished by comparing behavioral responses and performance of B. tabaci biotype B among resistance and susceptible poinsettia cultivars as well as by comparing physical and chemical traits of resistant and susceptible poinsettia cultivars. My specific objectives were to:

1. Evaluate whitefly preferences for poinsettia cultivars and identify physical factors

associated with preferred cultivars. My specific sub-objectives were to:

i. study whitefly adult behavioral settling and ovipositional preference among

poinsettia cultivars (Chapter 2 and appendix A,-B),

ii. determine nymphs performance and oviposition by F1 females among

poinsettia cultivars (Chapter 2 and Appendix A,-B),

iii. determine the role of visual cues in adult whitefly preference between a

resistant and a susceptible poinsettia cultivar (Chapter 2), and

iv. compare trichome density, leaf thickness, chlorophyll content, and color of

leaves among resistant and susceptible poinsettia cultivars (Chapter 2)

2. Evaluate poinsettia chemical characteristics associated with whitefly preference and

performance. My specific sub-objectives were to:

i. characterize and compare the composition and profile of total free amino acids

in petioles between resistant and susceptible poinsettia cultivars (Chapter 3)

and

ii. characterize and compare total phenolics in leaves and petioles between a

resistant and a susceptible poinsettia cultivar (Chapter 3).

3. Observe a whitefly‘s parasitoid preference between nymphs reared in a resistant and a

susceptible poinsettia cultivar (Chapter 4).

Chapter 2 Behavioral preferences and performance of silverleaf whitefly among poinsettia cultivars: physical traits associated with resistance

Abstract

The objective of this research was to determine silverleaf whitefly preference and performance on poinsettia cultivars through the use of choice and no-choice tests. Adult settling/feeding after 48 h, oviposition, developmental time, emergence rates, survival of nymphs, F1 oviposition, reproductive rate, cohort generational times, and intrinsic rate of increase were measured. To determine the factors underlying whitefly preference, leaf thickness, trichome density, chlorophyll content, and leaf color of poinsettia cultivars were compared. To better understand plant visual cues and to determine if they play a role in whitefly host plant recognition on poinsettias a dual choice assay with and without light was conducted.

Bemisia tabaci had a distinct preference to feed and oviposit in light green leaf cultivars, such as ‗Monet Twilight‘, compared to dark green leaf cultivars, such as

‗Freedom Red‘. On ‗Monet Twilight,‘ B. tabaci developed faster and offspring performed better compared to whiteflies on ‗Freedom Red‘. Physical traits that could underline the preference of whiteflies for susceptible cultivars are leaf thickness, color, and other visual cues. Trichome density was not significantly different among the

23 poinsettia cultivars evaluated. Leaf thickness and greenness were significantly different among poinsettia cultivars. Cultivars with higher number of adults and eggs/cm2 had thinner leaves and lighter green leaf color. Visual cues, particularly leaf color, appear to play a key role in host recognition by B. tabaci.

Introduction

The silverleaf whitefly (Bemisia tabaci biotype B), also known as B. argentifolii

Bellows and Perring, colonizes a variety of ornamentals and crops worldwide (Bethke and Paine 1991). It is one of the most prevalent and important insect pests attacking poinsettias (Ecke et al. 2004). Poinsettias are considered the single most valuable flowering crop in the U.S. and it is ranked as the nation‘s top selling potted ornamental

(NASS 2008). Bemisia tabaci adults and nymphs feed from the phloem, continually weakening stems and leaves causing chlorotic wilted looking plants. Such damage reduces the aesthetic and economic value of this crop ultimately affecting the ornamental industry (Van Driesche and Lyon 2003, Ecke et al. 2004).

Control of the silverleaf whitefly has been challenging primarily because of its rapid development of resistance to insecticides (Palumbo et al. 2001) and its high reproductive capacity (Oliveira et al. 2001). The main method of control today is still chemical, but host plant resistance may offer an alternative that is compatible with other practices, and may help reduce insecticidal applications. The use of cultivars resistant to whiteflies has been documented in crops like alfalfa, cotton, cassava, and hibiscus. The use of resistant cultivars has resulted in less whitefly feeding, reduced oviposition, longer developmental times, and higher nymph mortality (Butler et al. 1986, Chu et al. 1995a,

Teuber et al. 1997, Chu et al. 1998, Liu and Stansly 1998b, Bellotti and Arias 2001, Jiang et al. 2003). Physical factors shown to be involved in resistance include but are not limited to trichome density, leaf thickness, and leaf greenness. In cotton, for example,

25 cultivars with hairier leaves had more B. tabaci adults and eggs compared to normal/smooth cotton plants (Butler et al. 1986). In hibiscus, cultivars with thicker and darker green leaves supported less oviposition and were less preferred by whiteflies (Liu and Stansly 1998a). There are no recent studies evaluating poinsettia cultivar resistance by means of whitefly preference or performance. Moreover, limited studies have been conducted to determine the specific physical and/or chemical factors involved in poinsettia defense mechanisms against the silverleaf whitefly, despite being a serious pest in poinsettia productions since the mid 1980‘s (Brown et al. 1995).

There are many poinsettia cultivars today available in the market with different physical characteristics. One characteristic I focused on is the green color of the foliage.

Two types of foliage are dominant in poinsettias based on the greenness of the leaves— light and dark green leaves (Ecke et al. 2004). Earlier studies have shown that poinsettia cultivars with light green leaves appear to be preferred by the silverleaf whitefly

(Sanderson 1992, Petro and Redak 2000, Rice and Crane 2000). Conversely, poinsettia cultivars such as ‗Freedom Red‘, ‗Red Velvet‘, and ‗Pepride‘, all with dark green leaves, were shown to be less preferred by B. tabaci (Petro and Redak 2000, Rice and Crane

2000, Petro et al. 2002). More recently, growers in Ohio noticed increased whitefly attack on current cultivars with light green leaves (personal communication, Luis A.

Cañas), for which no studies has been reported. In two of my preliminary choice tests we found ‗Freedom Red,‘ with dark green leaves, to be less preferred by whitefly adults and

‗Monet Twilight‘, ‗Snowcap‘, and ‗Early Prestige Red,‘ with light green leaves, more preferred (Appendix A and B). This research reports for the first time on the possible

26 factors that result in poinsettias with light green leaves being more susceptible or attractive to whiteflies.

The objectives of this study were to determine the preference and performance of

B. tabaci biotype B on poinsettia cultivars, to determine the role visual stimuli play in host plant resistance, and to compare trichome density, leaf thickness, and color

(greenness) of the leaves. Anecdotal evidence suggests that light green leaf cultivars will be more attractive to B. tabaci, therefore, I will test and document such pattern and in addition investigate if this behavioral preference is mediated primarily by visual cues including leaf color and physical cues including trichomes and specific leaf mass. I also hypothesized that on more susceptible poinsettia cultivars whitefly developmental performance will be better than on less susceptible cultivars. Identifying behavioral preferences of B. tabaci could be instrumental to improve management methods, such as scouting, and to develop resistant poinsettia cultivars.

Materials and Methods

All experiments were conducted at the Entomology Department facilities at the

Ohio State University, OARDC campus, Wooster, OH.

Experimental plants

Test plants were obtained as rooted cuttings from the company Paul Ecke Ranch®,

Encinitas, CA. Upon arrival, cuttings were transplanted to 15.5 x 11 x 11.5 cm pots

(Dillen Products, Middlefield, OH) with soilless media Pro-Mix ‗BX‘ /Mycorise ® PRO

(General purpose: Canadian sphagnum peat moss, 75-85% /vol.; perlite, horticultural grade; vermiculite; and dolomitic and calcitic limestone; Premier Horticultural Inc.,

Quakertown, PA, USA) and placed in a greenhouse. Greenhouse setting were set at

23°C, 50% relative humidity, and a 16:8 hours light: dark (L:D) regime. Watering and fertilization of the plants was done using a drip irrigation system and a Dosatron injector, set at a dilution of 1/64 that delivered N at a rate of 250 mg·L-1. Plants were fertilized using 20-10-20 (Scotts-Sierra Horticultural Products Company, Marysville, OH). Plants used in both choice and no-choice test were infested with whiteflies 10 days after transplant.

Whitefly source: Bemisia tabaci biotype B

The whitefly colony was maintained on poinsettias (cultivars ‗Freedom Red‘ and

‗Monet Twilight‘) with greenhouse settings at 24°C. Plants used for the colony were also fertilized at a rate of 250 mg·L-1 of nitrogen (20-10-20) with every watering event.

Whiteflies from the colonies were used to evaluate adult settling and ovipositional

28 preference among poinsettia cultivars in a set of choice tests and also to determine developmental times of nymphs and oviposition by F1 females using no-choice tests.

Choice tests

Host choice tests were performed to screen for B. tabaci preference for poinsettia cultivars. A total of three choice-test experiments were conducted over a two-year span, each with varying number of cultivars. A total of 12 cultivars were screened for whitefly preference (Table 3.1). These cultivars were chosen based on evidence provided by growers (personal communication, Luis A. Cañas) of low and high incidence of whiteflies on these plants and also these cultivars were of particular interest to Paul Ecke

Ranch because they are good sellers for the company (Personal communication, Rebecca

Siemonsma). Therefore, we selected poinsettia cultivars with varying leaf color types and one with curly leaves (see Table 3.1 for detail on leaf color).

Choice tests: Experimental design and Infestation

All choice test experiments were arranged each in a randomized complete block design (RCBD) and conducted in a walk-in growth chamber set at 24 ° C, 50 % relative humidity and 16:8 h (L:D). The first experiment had five blocks represented each by a cage containing nine plants; each plant was of one cultivar; nine cultivars were used as treatments and were screened for whitefly preference.

Table 2.1 Cultivars screened for resistance and susceptibility. Cultivars Visual green leaf color type ‗Freedom Red‘ Dark green ‗Freedom White‘ Dark green ‗Early Freedom Red‘ Dark green ‗Prestige Red‘ Dark green ‗Winter Rose Red‘ (curly leaves) Dark green ‗Early Prestige Red‘ Medium/dark green ‗Enduring Red‘ Medium/dark green ‗Enduring White‘ Medium/dark green ‗Peterstar Red‘ Medium/dark green ‗Peterstar White‘ Medium/dark green ‗Zapoteca‘ (wild type) Light green ‗Snowcap White‘ Light green ‗Monet Twilight‘ Light green

Plants were arranged randomly in a circle, distanced equally from each other, inside a 70 x 70 x 40 cm mesh cage, which represented each block as mentioned before.

In each cage, 180 pairs of 3-day-old whiteflies were released, for a total of 900 pairs observed. Whiteflies were collected and placed into a plastic container inside each cage before being released. The release point was the center inside the circle formed by the different cultivars. The behavioral preferential response of whiteflies for cultivars was recorded as number of whiteflies settling/feeding on each plant after a period of 48h because whiteflies settle after such period of time—the movement among plants is negligent thereafter (Bird and Kruger 2006). The second experiment had five blocks as well. Seven cultivars, including four initially tested in experiment 1, were evaluated.

Insects were released as in experiment 1. Additionally, in experiment 2, the total number of eggs per cm2 oviposited by the adults settled on each plant were counted. For experiment 3, nine blocks were used. Each of the nine cages had one ‗Monet Twilight‘ and one ‗Freedom Red‘ poinsettia and was placed into a walk-in growth environmental chamber at 26°C and 16:8 h (L/D). These two cultivars were selected because ‗Monet

Twilight‘ is the best cultivar with light green leaves that sells in the market and ‗Freedom

Red‘ is the best with dark green leaves that sells in the market. In each cage, 60 pairs of

3-day-old whiteflies were released near the opening of the cage, to give the whiteflies a choice of cultivars, for a total of 540 pairs observed. The two cultivars were distanced equally from each other, inside a 70 x 70 x 40 cm mesh cage that represented the replicate. Preferential response was recorded as number of whiteflies settling on each

31 plant cultivar after a period of 48 h. All experiments were conducted with plants that had no previous exposure to whiteflies.

No-choice tests

For every choice test a no-choice test was conducted simultaneously to evaluate whitefly performance.

No-choice tests: Experimental design and Infestation

Plants were arranged in a RCBD in a greenhouse set at 21-24 °C and 16:8 h

(L:D). The no-choice experiments had the same subset of cultivars and replicates as experiments in the choice tests except for experiment number 3, which had six blocks and each block two cultivars, ‗Freedom Red‘ and ‗Monet Twilight‘. For all experiments, three leaves, the fourth, the fifth, and the sixth from the apex of the plant, were selected for infestation. Each leaf was infested with six female: male pairs of whiteflies using clip cages (Figure 2.1). Whiteflies were left undisturbed for 48-72 hours to get approximately

30 eggs per leaf. Egg emergence was recorded for experiments 1 and 2 (Appendix A and

B). When eggs hatched, ca. 7-10 days after infestation, an average of 18-24 first instar nymphs per leaf were followed by drawing a circle on the leaf around them with an ultrafine permanent marker. Nymphs are sessile, only crawlers and adults are mobile.

Developmental time of nymphs was recorded three times per week in experiments 1 and 32

Figure 2.1 Clip cages used for infesting plants with whiteflies.

2 and every day in experiment 3. The number of nymphs that emerged into adults were counted in every experiment. Additionally, female adults (F1) emerged from each cultivar were caged to record oviposition within the first 24 h after emergence in experiment 1 and 24 h and seven days for experiment 2. Whiteflies reach their peak of oviposition between 5-12 days after emergence. For an average of 6 females per plant in experiments 1 and 2 was oviposition recorded. In experiment 3, F1 females‘ oviposition was recorded daily for an average of 10 days. A life-table analysis was developed for each experiment, including reproductive rate (Ro) and intrinsic rate of increase (r) for experiments 1-3 using degree days (DD) of cohort generational time (Tc) at two time points: 1) when 50% of nymph emergence had occurred and 2) until last day of emergence. The formulas used for calculating DD, ‗Ro‘, and ‗r‘ were the following

(taken from (Begon 2006):

 DD = (average daily temperature – 10 º C)

 Ro = lxmx (this is a measurement of reproductive success)

 r = log (Ro)/Tc (this measurement takes into account cohort time)

Where: lx = proportion of nymphs surviving to adults mx = birth rate

 mx = fx/ax

. fx = (number of reproducing females)*(eggs/female)

. number of reproducing females = ax*(0.50), which is

the assumed sex ratio

. eggs/female = calculated based on 24 h for experiment

1, seven days for experiment 2, and ten days for

experiment 3.

. ax = number of individuals starting in the population

Tc = Degree days it took to develop from egg emergence until adult emergence.

Visual cues

To determine if visual cues drive attraction and play a role in host plant recognition–therefore susceptibility to whiteflies–dual choice assays conducted using the mesh cages with a less susceptible cv ‗Freedom Red‘ and a more susceptible cv ‗Monet

Twilight‘ was conducted in dark conditions, to preclude the use of visual cues, and with light conditions (Figure 2.2). These two cultivars were selected because (1) they are the best sellers within their category of light and dark green leaf cultivars and (2) they were among the least and more susceptible cultivars from the choice preference tests. Controls with a set of only ‗Freedom Red‘ and another with only ‗Monet Twilight‘ were also offered to whiteflies. The experiments were conducted using a single leaf from each cultivar that was placed in a four inch anchor aquapic waterpick with stopper (SYND50-

97, Lynn Eggett & Associates, Inc., Seeley Lake, MT). The aquapics were placed in a foam platform to keep them upright. All plants used in the experiments had no previous exposure to whiteflies and the observations were conducted in a walk-in growth chamber with temperature set at 24°C and a 50% relative humidity. Eighty whitefly female: male

Freedom Freedom Cage 5 Monet Cage 6 Freedom

Monet

Cage 4 Monet

Monet Cage 7 Monet

Monet

Cage 3 Freedom Freedom

Cage 8 Freedom Mone Cage 2 t Freedom

Freedom

Monet Cage 9

Cage 1 Freedom Monet DOOR Figure 2.2 Visual cues experiment set-up. Cages are in a randomized complete block design.

36 pairs were released in the mesh cages, 70 x70 x40 cm, in the middle of the arena and equally distanced from each leaf. Release and observation of whiteflies for the experiment precluding visual cues was performed in the dark with the help of a flashlight covered with red cellophane paper. Settling of adults was recorded 48 h later, using the flashlight for the dark conditions experiment to avoid any source of light eliciting movement of the whiteflies.

Leaf thickness, trichome density, indirect chlorophyll content and color of leaves

Leaf area was measured using a leaf area meter (Model LI-3100 Area Meter, LI-

COR, Inc., Lincoln, Nebraska, USA). Leaf area of individual leaves and also of whole plants was recorded for a total of ten replications from each cultivar. Leaf thickness was estimated based on methodology described by Vile et al. (2005). The aim of their study was to demonstrate, which they did, that leaf thickness can be safely deduced from the following two leaf traits (Vile et al. 2005). I calculated specific leaf area (SLA) of young-fully expanded leaves (10 leaves per cultivar), usually the 4th leaf from the apex of the plant, and leaf dry matter content (LDMC). For dry weight measurements, plants were oven dried at 60 °C for 48 h. The following formulas were employed:

- SLA (cm2/g), ratio of leaf area to leaf dry mass

- LDMC (g), ratio of leaf dry mass to saturated fresh mass = 1 – leaf water content

- Leaf thickness (µm) = (SLA x LDMC)-1

For trichome density, 25 mm2 leaf disks were excised using a core borer # 5 from the left and right side of the fifth leaf counting from the apex of the plant; the number of trichomes was counted under a scope. We used eight plants to count trichomes from each cultivar.

Indirect chlorophyll content or greenness of the leaves of cultivars was calculated with a Minolta SPAD 502 Chlorophyll Meter (Konica MINOLTA Sensing, INC. Made in

Japan) (Loh et al. 2002, Wang et al. 2005). Triplicate SPAD readings were collected from one young fully expanded (usually the 4th leaf from the apex of the plant) leaf from ten plants per cultivar. Color was recorded with a Minolta Color Reader CR-10 (Konica

MINOLTA Sensing, INC. Made in Japan) from one young fully expanded (usually the 4th leaf from the apex of the plant) leaf from 15 plants per cultivar. Color readings are expressed by CIE (International Commission on Illumination) coordinates (L*, a*, b*), which are based on a chromaticity diagram with wavelength in nanometers. The three coordinates of CIELAB represent the lightness of the color (L* = 0 yields black and L* =

100 indicates diffuse white), its position between red/magenta and green (a*, negative values indicate green while positive values indicate magenta) and its position between yellow and blue (b*, negative values indicate blue and positive values indicate yellow).

The asterisk (*) after L, a and b are part of the full name, since they represent L*, a* and b*, to distinguish them from Hunter's L, a and b, described below. L indicates whiteness/darkness relative to the light being transmitted through the leaf; a, the hue of redness/greenness of the leaf; and b, the hue of blueness/yellowness of the leaf.

Statistical analysis

All data from choice tests and no-choice tests were analyzed via RCB ANOVA and with Fisher‘s LSD (least significant difference) means separations with α = 0.05 using SAS 9.2 (SAS 2010). A priori planned contrasts were conducted between cultivars with dark and medium green leaves from experiment choice test 1; and between cultivars with dark and light green leaves from experiment choice test 2. Color readings

(coordinates) were analyzed with a MANOVA multivariate analysis. The visual cues dual-choice tests were analyzed using a nested design. The experiments conducted in the dark and the experiments conducted in light conditions were both nested within growth chamber as they had to be run separately. Data were checked for normality and homogeneity of variances and data transformations were performed as needed.

Results

Insect focus

Choice tests: adult preference. The number of adult whiteflies settled/feeding after 48 h was not significantly different among cultivars (F(8, 32) = 2.05; p = 0.0711)

(Table 2.2). Whiteflies though showed a preference overall for medium green foliage cultivars than for cultivars with dark green leaves (38.8 ± 15 and 17.8 ± 6.5 adult whiteflies, respectively, with contrast F(1, 32) = 8.04) (Table 2.2, Appendix A.1). In choice experiment 2, no significant difference were observed in number of adults settled among the cultivars (F(6, 24) = 1.89; p = 0.1246) (Table 2.3). However, a priori planned contrast shows that overall as in experiment 1, cultivars with light green leaves were preferred by adults for feeding (52 ± 12.6 and 31.5 ± 7.0 adults, respectively, with contrast F(1,24) =

6.52). Based on the preferences of adult whiteflies observed in experiment 1 and 2,

‗Freedom Red‘ was among the cultivars with a consistent low number of adults and from experiment 2 ‗Monet Twilight‘ was among the cultivars with a high number of adults.

Not only are these cultivars good representatives of susceptible and less susceptible cultivars, but these are also among the best sellers from Paul Ecke Ranch, thus, these cultivars were selected to conduct a dual-choice test (choice experiment 3) to evaluate adult feeding preference. A significant preference was observed by adult whiteflies for

‗Monet Twilight‘ when compared to ‗Freedom Red‘ (F(1,8) = 29.02, p = 0.0007; Figure

2.3).

Table 2.2 Mean number (± SE) of Bemisia tabaci biotype B on multiple poinsettia (Euphorbia pulcherrima) cultivars (choice test experiment 1). Cultivar (green leaf color) Adults settling after 48 h1 ‗Freedom Red‘ (dark) 16.6 ± 10.6 ‗Freedom White‘ (dark) 23.0 ± 6.50 ‗Prestige Red‘ (dark) 16.8 ± 4.40 ‗Winter Rose Red‘ (dark) 15.0 ± 4.80 ‗Early Prestige Red‘ (medium) 65.6 ± 9.9 ‗Enduring Red‘ (medium) 27.0 ± 10.1 ‗Enduring White‘ (medium) 32.2 ± 9.10 ‗Peterstar Red‘ (medium) 41.4 ± 22.2 ‗Peterstar White‘ (medium) 28.2 ± 8.90 Contrast ‘Dark vs Medium green foliage’ p = 0.0079 1 Data had square root transformation for ANOVA analysis to comply with homogeneity of variances, but original means are presented. No significant differences were observed (p = 0.071). A priori contrast was conducted.

Table 2.3 Mean number (± SE) of Bemisia tabaci biotype B settling on multiple poinsettia (Euphorbia pulcherrima) cultivars and number of eggs laid after 48 h (choice test experiment 2). Cultivar (green leaf color) Adults settling1 Eggs Mean leaf area (cm2) Mean1 no. eggs/cm2 ‗Freedom Red‘ (dark) 19.0 ± 4.4 a 73.4 a 462.51 0.16 ± 0.050 b ‗Early Freedom Red‘ (dark) 33.6 ± 7.7 a 122.6 a 392.52 0.30 ± 0.086 ab ‗Prestige Red‘ (dark) 41.8 ± 9.0 a 141.8 a 454.36 0.32 ± 0.047 ab ‗Early Prestige Red‘ (medium) 51.4 ± 11.2 a 165.6 a 279.31 0.58 ± 0.176 a ‗Zapoteca‘ (light) 47.2 ± 11.9 a 167.4 a 875.47 0.19 ± 0.041 b ‗Snowcap White‘ (light) 52.0 ± 13.5 a 234.6 a 466.36 0.50 ± 0.155 a 42 ‗Monet Twilight‘ (light) 57.2 ± 13.7 a 145.0 a 263.40 0.56 ± 0.115 a Contrast ‘Dark vs Med/Light green’ p = 0.0174 p = 0.0499 p = 0.0291 1 Means within the same column followed by the same letter are not significantly different using Fisher‘s LSD (α = 0.05), n=5. Data: for Eggs and Eggs/cm2 were transformed using Log (x) for analysis, but original data is presented.

30 *

No. adults settling/feeding adults No.

0 Susceptible 'Monet' Resistant 'Freedom' Figure 2.3 Experiment 3. Mean whitefly numbers (± SE) after 48 h on a light green leaf cultivar ‗Monet Twilight‘ and a dark green leaf cultivar ‗Freedom Red; the asterisks indicates a significant difference at p = 0.0007.

Choice tests: oviposition preference. In experiment 2, whitefly oviposition was not significantly different among poinsettia cultivars (F(6,24) = 1.47; p = 0.2309), but a significant difference was observed when all dark and light green leaf cultivars were grouped and analyzed with an a priori contrast (112.6 and 178.1, respectively, with an

2 F(1, 24) = 4.26) (Table 2.3). The number of eggs/cm per plant was different among the cultivars (F(6, 24) = 3.40; p = 0.0143), but overall was highest on cultivars with light green leaves than on dark green leaves (0.46 ± 0.12 and 0.26 ± 0.06, respectively,with an F(1, 24)

= 5.39) (Table 2.3). Therefore, we selected ‗Freedom Red‘ as a less suceptible cultivar and ‗Monet Twilight‘ as a susceptible cultivar. Although ‗Zapoteca‘ appears to be less affected by whiteflies based on eggs per cm2, this cultivar is a wild type and is not representative of normal growth and appearance with commercial cultivars, therefore, was not selected for further evaluations.

No-choice tests: survivorship. Nymph survival was not significantly different among cultivars in any of the experiments: experiment 1 (F (8, 32) = 1.03, p = 0.4374;

Appendix A); experiment 2 (F (6, 24) = 1.17, p = 0.3523; Appendix B); and experiment 3

(F (1, 5) = 0.30, p = 0.6081; data not shown).

No-choice tests: reproductive rate (Ro). In all three no-choice experiments, Ro differed significantly among cultivars. In experiment 1, whiteflies growing on cv

‗Freedom Red‘ were among the ones with the highest Ro, and among the cultivars with the lowest were on ‗Enduring White‘ (Table 2.4)

Table 2.4 No-choice experiment 1: Average reproductive rate ‗Ro‘, intrinsic rate of increase ‗r‘, and cohort generational time ‗Tc‘ in DD1 of Bemisia tabaci biotype B reared in different poinsettia (Euphorbia pulcherrima) cultivars and the number of eggs per female. 50% nymph Until last nymph F1 eggs/female Cultivar (green leaf color) emergence emerged (24h) ‗Ro‘2 ‗r‘ ‗Tc‘ ‗r‘ ‗Tc‘ ‗Freedom Red‘ (dark) 4.338 a 0.00306 198.36 0.00227 270.84 b 10.46 a ‗Freedom White‘ (dark) 4.336 a 0.00242 223.42 0.00192 288.37 ab 10.20 ab ‗Prestige Red‘ (dark) 2.440 ab 0.00136 216.64 0.00096 293.09 ab 5.38 c ‗Winter Rose Red‘ (dark) 3.268 ab 0.00223 223.79 0.00167 302.39 a 7.42 abc ‗Early Prestige Red‘ (medium) 3.683 ab 0.00237 216.64 0.00173 290.89 ab 10.08 ab 45 ‗Enduring Red‘ (light) 2.490 ab 0.00115 234.58 0.00091 281.79 ab 5.36 c ‗Enduring White‘ (light) 2.039 b 0.00071 215.31 0.00055 241.06 c 6.52 bc ‗Peterstar Red‘ (light) 3.714 ab 0.00244 227.80 0.00184 293.09 ab 9.08 abc ‗Peterstar White‘ (light) 3.928 ab 0.00254 225.48 0.00198 293.09 ab 9.88 ab F 2.94 1.62 1.86 1.38 2.72 2.76 Df(N,D) 8, 32 8, 32 8, 32 8, 32 8, 32 8, 30 P 0.0364 0.1597 0.1036 0.2452 0.0222 0.0207 1 DD = degree days when 50% of nymphs per replicate had emerged and until last nymph of the replicate emerged. 2 Reproductive rate is independent of DD. Means within the same column followed by the same letter are not significantly different using LSD (α = 0.05), n=5.

In experiment 2, ‗Freedom Red‘ and ‗Prestige Red‘ were among the cultivars where whiteflies had the lowest ‗Ro‘ and were significantly different from ‗Zapoteca‘ and ‗Snowcap White‘ which were among the cultivars on which whiteflies reproduced better (Table 2.5). Results for ‗Ro‘ changes from experiment 1 and 2, this may be explained by the amount of days that I measured F1 oviposition. For experiment 1, it was only 24h and for experiment 2 it was 7 days. F1 oviposition for 7 days is more representative, since adult whiteflies‘ oviposition peaks around 5-12 days after emergence (Enkegaard 1993a). In experiment 3, whiteflies growing on ‗Monet Twilight‘ had a higher Ro than those growing on ‗Freedom Red‘ (Table 2.6).

No-choice tests: intrinsic rate of increase (r). No significant differences in ‗r‘ were observed among whiteflies growing on the cultivars in experiment 1 (Table 2.4). In experiment 2, ‗Zapoteca‘ and ‗Snowcap White‘ were among the cultivars where whiteflies had a higher r, while ‗Prestige Red‘ and ‗Freedom Red‘ were among the cultivars with the lowest r (Table 2.5). Although ‗r‘ varied among cultivars in experiment 2, the pattern of differences was not in a way that could explain susceptibility or resistance. In experiment 3, whiteflies growing on ‗Monet Twilight‘ had a higher r compared to those growing on ‗Freedom Red‘ (Table 2.6). The rate of increase in

‗Freedom Red‘ and ‗Monet Twilight‘ clearly indicates these two cultivars are perceive differently by B. tabaci.

Table 2.5 No-choice experiment 2: Average reproductive rate ‗Ro‘, intrinsic rate of increase ‗r‘, and cohort generational time ‗Tc‘ in DD1 of Bemisia tabaci biotype B reared in different poinsettia (Euphorbia pulcherrima) cultivars and the number of eggs per female. Until last nymph Cultivar (green leaf color) 50% nymph emergence emerged F1 eggs/female: ‗Ro‘2 ‗r‘ ‗Tc‘ ‗r‘ ‗Tc‘ 24 h 7 d ‗Freedom Red‘ (dark) 6.424 c 0.00414 bc 194.51 c 0.00274 bc 285.14 3.7 2.5 bcd ‗Early Freedom Red‘ (dark) 7.020 bc 0.00407 c 201.37 bc 0.00311 ab 267.70 3.6 2.5 cd ‗Prestige Red‘ (dark) 4.432 c 0.00301 d 214.49 ab 0.00225 c 292.05 2.6 1.8 d ‗Early Prestige Red‘ (medium) 7.931 bc 0.00409 bc 218.86 a 0.00312 ab 288.02 3.2 2.9 bc 47 ‗Zapoteca‘ (light) 14.239 a 0.00520 a 214.49 ab 0.00368 a 303.24 1.6 5.5 a ‗Snowcap White‘ (light) 11.214 ab 0.00488 ab 214.49 ab 0.00384 a 280.19 2.7 3.8 ab ‗Monet Twilight‘ (light) 8.758 bc 0.00405 c 219.44 a 0.00292 bc 304.59 3.0 3.0 bc F 4.40 6.40 2.86 4.56 0.46 0.93 6.17 Df(N,D) 6, 24 6, 24 6, 24 6, 24 6, 24 6, 24 6, 24 P 0.0039 0.0004 0.0302 0.0032 0.8339 0.4912 0.0005 1 DD = degree days when 50% of nymphs per replicate had emerged and until last nymph of the replicate emerged. 2 Reproductive rates are independent of DD. Means within the same column followed by the same letter are not significantly different using LSD (α = 0.05), n=5.

Table 2.6 No-choice experiment 3: Average reproductive rate ‗Ro‘, intrinsic rate of increase ‗r‘, and cohort generational time ‗Tc‘ in DD1 of Bemisia tabaci biotype B reared in a less and a more susceptible poinsettia (Euphorbia pulcherrima) cultivar. Cultivar (green leaf color) 50% nymph emergence Until last nymph emerged ‗Ro‘2 ‗r‘ ‗Tc‘ ‗r‘ ‗Tc‘

‗Freedom Red‘ (dark) 14.927 b 0.00410 b 265.91 a 0.00318 b 335.91 a ‗Monet Twilight‘ (light) 33.501 a 0.00620 a 248.35 a 0.00511 a 304.57 a F 9.68 8.96 0.67 16.62 0.99 Df(N,D) 1, 6 1, 6 1, 6 1, 6 1, 6 P 0.0265 0.0304 0.4509 0.0096 0.3648 48 1 DD = degree days when 50% of nymphs per replicate had emerged and until last nymph of the replicate emerged. 2 Reproductive rates are independent of DD. Means within the same column followed by the same letter are not significantly different using a LSD (α = 0.05), n=6.

No-choice tests: cohort generational time (Tc). While nymphs‘ cohort generational time at 50 % emergence was not significantly different, cohort generational time (Tc)of nymphs was different at the time when all nymphs had emerged into adults.

In experiment 1, whiteflies reared on ‗Winter Rose Red‘ were among the ones that took considerably longer to develop into adults compared to ‗Enduring White‘, which was the cultivar on which they took less time (Table 2.4). In experiment 2, cohort generational time of nymphs‘ at 50 % emergence was significantly different, but by the time all nymphs emerged no significant different was observed in cohort generational time among all cultivars (Table 2.5). The fact that I observed differences in cohort generational time by the time all nymphs had emerged in experiment 1 and not in experiment 2 can be the artifact of how the data was collected. Emergence was recorded three times a week for both experiments, which could have under or overestimated the differences in nymphs‘ emergence times. For this reason in experiment 3, developmental times were recorded every day until emergence and I found no significant effects between nymphs grown on

‗Freedom Red‘, which is a less susceptible cultivar, and ‗Monet Twilight‘, which is a more susceptible cultivar (Table 2.6); a tendency, however, to emerge at a rate much slower was observed on ‗Freedom Red‘ than on ‗Monet Twilight‘ (Figure 2.4).

No-choice tests: offspring performance. Oviposition by F1 females—females reared on the cultivars—was evaluated in all three experiments. Among cultivars with the lowest number of eggs per female in 24 h after emergence were ‗Enduring Red‘ and

‗Prestige Red‘ and among cultivars with the highest were ‗Freedom Red‘, ‗Freedom

0.35

0.30

0.25

0.20

0.15 Freedom Monet 0.10

0.05

Emergence rates (proportion whiteflies) (proportion rates Emergence 0.00

100 150 200 250 300 350 400 450 Degree days Figure 2.4 Experiment 3. Rate of emergence of B. tabaci biotype B on poinsettia (Euphorbia pulcherrima) cultivars ‗Freedom Red‘ and ‗Monet Twilight‘.

White‘, Early Prestige Red‘, ‗Peterstar White‘ and ‗Peterstar Red‘. The data shows a variation that does not help explain resistance or susceptibility of the cultivars (

Table 2.4, Appendix A). In experiment 2, oviposition of F1 at 24 h did not vary among the cultivars, while at 7 days variation was observed among all cultivars (Table 2.5).

Variation among the cultivars at 7 days did not explain variation in susceptibility or resistance against B. tabaci. The results varied between experiment 1 and 2, presumably because data was measured only three times a week. In experiment 3, data was recorded daily and oviposition of females reared on ‗Freedom Red‘ and ‗Monet Twilight‘ were monitored for 10 days after emergence and a significantly higher number of eggs laid per female were observed on ‗Monet Twilight‘ than on ‗Freedom Red‘ (F(1,23) = 11.13, p =

0.0029; Figure 2.5).

Visual Cues. The effect of visual cues played a key role on Bemisia tabaci biotype B host recognition between a more susceptible, ‗Monet Twilight‘, and a less susceptible poinsettia cultivar, ‗Freedom Red‘. Whiteflies tested on dark conditions did not differentiate between plants of any cultivar (Table 2.7, Figure 2.6). In conditions where light was present, no differences in whitefly preference were observed on cages that contained plants of the same cultivar (Table 2.7, Figure 2.6). Significant differences, however, were observed when whiteflies were offered two cultivars (‗Monet Twilight‘ and ‗Freedom Red‘), consistently selecting more ‗Monet Twilight‘ (Table 2.7, Figure

2.6).

12 A Freedom Red 10 Monet Twilight

Eggs/female/day 4

0 80 * B

Cumulative no. eggs/female/day no. Cumulative 0

0 2 4 6 8 10 12 Days Figure 2.5 (A) Bemisia tabaci biotype B females reared on a less susceptible poinsettia cultivar ‗Freedom Red‘ and a more susceptible cultivar ‗Monet Twilight‘.(B) Representation of the cumulative number of eggs laid per female per day. The asterisk indicates that, on day 10 after emergence, mean total number of eggs laid per female in ‗Freedom Red‘ and ‗Monet Twilight‘ was significantly different at p = 0.0029.

Table 2.7 Effect of light or dark conditions (visual) and combination of cultivars on the host recognition capabilities of Bemisia tabaci biotype B. Results of a nested ANOVA. Source df F P Only ‗Freedom Red‘ Block 2 0.03 0.9737 Visual 1 4.31 0.1737 Cultivar(Visual) 2 3.01 0.1242 Error 6 Only ‗Monet Twilight‘ Block 2 0.01 0.9913 Visual 1 15.00 0.0607 Cultivar(Visual) 2 0.69 0.5394 Error 6 Mix: ‗Freedom‘:‗Monet‘ Block 2 0.04 0.9597 Visual 1 2.08 0.2857 Cultivar(Visual) 2 12.08 0.0079 Error 6

140 120 DARK LIGHTS ON A 100 80 60 40 20 0 Freedom Freedom Freedom Freedom 140 120 B DARK LIGHTS ON 100 80 60 40 20 0 Monet Monet Monet Monet 140 120 DARK LIGHTS ON * Number of adult whiteflies settling/feeding after 48 h 48 after settling/feeding whiteflies adult of Number C 100 80 60 40 20 0 Freedom Monet Freedom Monet

Figure 2.6 Effect of visual cues on Bemisia tabaci biotype B host recognition capabilities. Average number of whiteflies on each plant (±SE) in dark and light condition (see for layout Figure 2.2); (A) when offered two plants of the same cultivar ‗Freedom Red‘; (B) when offered two plants of the same cultivar ‗Monet Twilight‘; and (C) when offered one plant of each cultivar. Asterisk indicates a significant difference in preferential response by whiteflies between the two cultivars (p = 0.0079; see also Table 2.7).

Plant focus

Leaf thickness, trichomes, chlorophyll content, and color of leaves. The SLA was significantly different among poinsettia cultivars (F (4, 36) = 5.05, p = 0.0025; Table 2.8) and therefore, leaf thickness was also statistically different (F(4,36) = 13.00, p < 0.0001;

Table 2.8). Cultivar ‗Monet Twilight‘ consistently had thinner leaves compare to all other cultivars, while ‗Freedom Red‘ was among the cultivars with the thickest leaves.

The average trichome density of leaves was not significantly different among the poinsettia cultivars studied (F(4, 28) = 1.63 p = 0.1934 ) (Table 2.8). Indirect chlorophyll content was measured using a SPAD reader, which yields a unit-less number and also indicates the greenness of the leaves. SPAD readings were compared among the poinsettia cultivars and were significantly different, with the cultivar ‗Monet Twilight‘ having the lowest values (F(4,36) = 30.23, P < 0.0001; Table 2.8).

Color readings of the cultivars had a similar lay out to cultivars when comparing parameters of SLA, LT, and SPAD, except for ‗Snowcap White‘ which came after

‗Monet Twilight‘ (Table 2.9). Cultivars differed significantly (Wilks' Lambda, F =25.67,

P < 0.0001), meaning they had unique green color tones, which can be observed when plotting their color coordinates (Figure 2.7).

Table 2.8 Mean1 (and SE) of leaf physical traits of five poinsettia (Euphorbia pulcherrima) cultivars. Trichome density Cultivar (green leaf color) SLA2 (cm2/g) Leaf Thickness (µm) SPAD3 (in 25 mm2) ‗Freedom Red‘ (dark) 325.87 ± 53.73 a 269.7 ± 4.92 a 188.95 ± 3.05 a 38.9 ± 2.25 a ‗Prestige Red‘ (dark) 388.81 ± 26.91 a 278.8 ± 9.55 ab 184.96 ± 5.03 ab 40.4 ± 1.31 a ‗Early Prestige Red‘ (medium) 485.25 ± 68.45 a 291.3 ± 9.60 ab 177.98 ± 4.36 b 39.1 ± 0.97 a ‗Snowcap White‘ (light) 326.40 ± 42.61 a 294.7 ± 10.53 b 177.94 ± 3.65 b 34.3 ± 0.80 b ‗Monet Twilight‘ (light) 311.06 ± 92.59 a 320.7 ± 10.61 c 154.58 ± 4.49 c 22.1 ± 1.26 c 1 Means within the same column followed by the same letter are not significantly different using a LSD (α = 0.05), N=10, except for trichomes where n=8. 2 Specific leaf area (SLA), the more area per gram of weight means a thinner leaf. 56 3 SPAD is a unitless number that also can be used to indirectly determine chlorophyll content in the leaves.

Table 2.9 Color reading coordinates from the Minolta Reader CR-10 for five poinsettia cultivars. Cultivars (green leaf color) Color L Color a Color b ‗Freedom Red‘ (dark) 46.44 -5.91 9.7 ‗Prestige Red‘ (dark) 47.07 -8.04 11.21 ‗Early Prestige Red‘ (medium) 48.09 -8.04 12.79 ‗Snowcap White‘ (light) 52.99 -10.28 20.27 ‗Monet Twilight‘ (light) 50.63 -8.71 16.35 1 L, indicates whiteness/darkness relative to the light being transmitted through the leaf; a, the hue of redness/greenness of the leaf; and b, the hue of blueness/yellowness of the leaf.

54 'Snowcap' 53

52 'Monet' 51

L* 50

49 'Early Prestige' 48 -5 'Prestige' 'Freedom' -6 47 -7 a* 46 -8 20 -9 18 16 14 -10 12 10 -11 b* 8

Figure 2.7 3D-bubble size representation of color coordinates from Minolta Reader CR-10. The dot represents the lightness of the color L* = 0 yields black and L* = 100 indicates diffuse white; a* negative values indicate green; b* positive values indicate yellow.

Discussion

Bemisia tabaci biotype B was attracted to and preferred to feed and oviposit on light green leaf (LL) poinsettia cultivars. In addition, nymphs and offspring of B. tabaci performed better on light green leaf cultivars. Our results are similar to those of Sanderson,

Petro and Redak, and Petro et al. (1992, 2000, 2002), who showed that some LL poinsettia cultivars are more susceptible to B. tabaci than are some cultivars with dark green leaves

(DL). This is the first report addressing preference and performance with current poinsettia cultivars and documenting a pattern of cultivars with LL being more susceptible B. tabaci that before was mainly anecdotal and/or with cultivars no longer in the market. Similar findings by Liu and Stansly (1998a) reported of B. tabaci’s preference for LL cultivars in

Hibiscus rosa-sinensis (Malvaceae) (Liu and Stansly 1998a), which support the same pattern observed in poinsettias. Cultivars with DL of H. rosa sinensis showed resistance to B. tabaci based on behavioral preference, development, and survivorship. In our studies I found no differences in survivorship among all cultivars.

Visual cues were shown to play an important role in recognition of preferred cultivars. In the absence of light, whiteflies were unable to discriminate between a less and a more susceptible cultivar, suggesting visual cues are a major component playing an important role in whitefly host recognition. The inability of whiteflies to find the preferred host indicates: 1) the two cultivars are similar with respect to chemical and tactile cues or 2) that whiteflies do not use these differences to discriminate between a less and a more susceptible cultivar in poinsettias. It has been reported that B. tabaci responds to olfactory

59 cues in poinsettias, (Heinz et al. 1993, Jing et al. 2003), although it was not mentioned if visual cues were excluded from the olfactometer set-up. Jing et al. (2003), found that poinsettias were preferred in a Y-tube olfactometer over Chinese cabbage> sweet potato> cabbage> tomato. In tomato plants, total and specific volatile organic compounds (VOCs) have been shown to influence whiteflies‘ ability to discriminate among different varieties

(Bleeker et al. 2009). In tomato and cabbage, visual cues, more specifically the light green leaf color, in conjunction with VOCs, were preferred by B. tabaci. Pepper, despite not eliciting responses to olfactory cues, was attractive to whiteflies based on having lighter green leaf color (Cao et al. 2008). Depending on the host, therefore, is possible that whiteflies can discriminate among preferred and less-preferred hosts based on olfactory cues, although this appears to not occur with the current poinsettias cultivars evaluated in this study.

It has been proposed that polyphagous insects, like B. tabaci, could have difficulty in choosing a host for oviposition and feeding sites when offered many hosts simultaneously

(Bird and Krüger 2006). However, it has been shown that B. tabaci is capable of selecting hosts when presented with multiple choices (Bird and Kruger 2006). For this research, I offered similar hosts, i.e., different cultivars pertaining to one species (poinsettias), which in studies on tomatoes has been shown to affect decision making of B. tabaci, resulting in reduced fecundity and increased movement (Bird and Kruger 2006). In my study, B. tabaci did not behave the same with cultivars of poinsettias; in fact when offered the LL and DL cultivars it clearly showed a distinct preference for LL cultivars. This study shows how B. tabaci behavior can be affected by different cultivars of the same species. The survivorship,

60 fecundity, and development of insects have been shown to be positively or negatively correlated with the ovipositional preference of insect females (Thompson 1988, Gripenberg et al. 2010). Cultivars, therefore, were further investigated through no-choice tests to evaluate effects on the physiology of B. tabaci and determine if in poinsettias there was a positive or negative relationship with the host chosen by the adults. My research data shows that a less susceptible cultivar with DL (‗Freedom‘) was indeed a suboptimal host compared to a susceptible cultivar with LL (‗Monet Twilight‘). On a more susceptible cultivar of LL

B. tabaci laid more eggs, and the numbers observed corresponds to those found by Bethke et al. (1991), where the average number of eggs per female per day in poinsettias was 5.8 at 22

ºC and the mean reproductive capacity was of 85 eggs/female with the peak observed at the first week after adult emergence between 21-24 ºC (Bethke et al. 1991). I found similar results with 7.4 and 6.0 eggs/fem/day and 74.4 and 60.9 eggs/female in ‗Monet Twilight‘ and ‗Freedom Red‘, respectively. Enkegaard (1993) found that in poinsettias the highest fecundity occurred between 5-12 days after emergence, which is what I observed in 10 days of fecundity.

Cultivars with DL had thicker leaves, is a physical trait that could possibly be mediating resistance to whiteflies; consequently, B. tabaci might be using color to determine among less and more suitable hosts. Cultivars were shown to have distinct physical characteristics including SLA, LT, and SPAD that could possibly be influencing host finding and recognition among poinsettias cultivars. The SPAD and color readings from the

Minolta Color Reader corresponded with the preferential of B. tabaci adult response, which indicates the important role in whitefly host recognition. Color has been shown to be a key

61 factor in host recognition for other species as well (Mound 1962, Berlinger 1980b, Gerling

1996).

The distance of vascular bundle sheaths to the lower surface of leaves, where phloem is accessed by the stylets of whiteflies, influences host preference, oviposition, and feeding habit of B. tabaci biotype B (Chu et al. 1995a). Moreover, cotton cultivars with greater leaf thickness also have a greater distance from lower leaf surface to phloem sieve elements, and were also less preferred for oviposition and feeding by B. tabaci (Chu et al. 1999b). The poinsettia cultivars‘ leaf thickness ranged from 154-189 µm. Although we did not measure the distance of leaf surface to phloem sieve elements directly, it could be infer that DL cultivars would have a greater distance to the vascular bundle sheaths compared to LL cultivars. To illustrate, in cotton the distance from lower leaf surface to vascular bundle sheaths was of 60 µm compared to 131 µm from the upper leaf surface. Newly hatched crawlers of B. tabaci can reach a stylet length of 113.8 ± 4.2 µm (Freeman et al, 2001 in

(Chu et al. 1995a)); therefore, a shorter distance, or thinner leaves, appears to facilitate feeding.

In this study, trichome density may not play a key role in resistance, since no significant differences were observed among the poinsettia cultivars. Bemisia tabaci prefers plants with nonglandular trichomes over glabrous plants (Guershon and Gerling 2006), presumably because (1) crawlers, one of the mobile stages of B. tabaci, develop setae as a result of the tactile experience with trichomes, eggs, or exuvia; and as a consequence setose nymphs are smaller and develop faster—the cost of producing those setae—escaping natural enemies and (2) because trichomes may offers a microclimate.

Acknowledgments

I thank Nuris Acosta, Mark Belcher, Ronald Batallas, Alejandra Claure, Jim Hacker,

Claudia Kuniyoshi, Henry Paz, Wilmer Rodríguez, and Eliana Rosales for their assistance in the collection of data. I thank Ron Hammond for the use of the LI-3100 Area meter and for his assistance in reviewing the AMTs (Appendix A and B). I thank Wilmar Morjan, for the use of the color reader.

Research support provided by state and federal funds appropriated to the The Ohio

State University (OSU), OARDC, SEEDS grant # 2008-110. Additional funding provided by the American Floral Endowment grant # 20020754, and from the Entomology department of OSU. Plant material was provided by Paul Ecke Ranch, Encinitas, CA.

Chapter 3 Constitutive phloem chemistry of poinsettia cultivars: nutritional and defensive traits associated with resistance to the silverleaf whitefly

Abstract

Poinsettia cultivars with light green leaves support more Bemisia tabaci (Gennadius) biotype B populations compared to cultivars with dark green leaves. From previous research (Chapter 2), I determined whiteflies on a less susceptible dark green leaf cultivar

(‗Freedom Red‘) had less adult feeding and oviposition, reduced offspring performance, and slower emergence rates compared to whiteflies on a susceptible light green leaf cultivar

(‗Monet Twilight‘). Plant chemical mechanisms are potentially mediating the behavior and physiology of B. tabaci, but these mechanisms have not been studied in poinsettias. Host quality is affected by the amount of nutritional and defensive chemical compounds constitutively expressed in the phloem of the plants. The goals of this study were to: 1) compare the concentrations of free amino acids in phloem tissue among poinsettia cultivars in relation to feeding and ovipositional preference of B. tabaci; 2) compare concentrations of phenolic compounds in leaves and petioles between a less and a more susceptible poinsettia cultivar in relation to performance of B. tabaci. Significant quantitative, but no qualitative, differences were present in the concentration of total, essential, and individual amino acids

64 among poinsettia cultivars. The concentration of total amino acids was higher overall on light green leaf cultivars than on dark green leaf cultivars. Concentrations of essential amino acids varied among cultivars, but not in a way that explains B. tabaci behavioral preferences. I suggest that, based on total amino acids, the nutritional quality of light green leaf cultivars may be superior to that of dark green leaf cultivars and may be influencing B. tabaci host preferences.

Qualitative and quantitative differences were detected in phenolic composition between ‗Freedom Red‘ and ‗Monet Twilight‘. A total of 28 compounds were detected in poinsettias, of which 22 were shared between the two cultivars. Putatively identified: six compounds were detected only in ‗Freedom Red‘, including digalloyl glucose (11) present in leaves, one unknown (compound 6) and kaempferol hexoside (19) present in petioles, and present in both petioles and leaves were one unknown (compound 9), apigenin diglucoside

(15), and apigenin glucoside A (20); Catechin B (10) was detected only in ‗Monet Twilight‘ in both tissues. In addition, putatively identified galloyl quinic B and C (3 and 4), catechin

C (13), quercetin pentose rutinoside (22), apigenin glucoside B (23), tetragalloyl glucose

(24), quercetin rutinoside (25), hexagalloyl glucose, and unknown (compound 12), were in higher concentrations in ‗Freedom Red‘ than on ‗Monet Twilight‘. In summary, concentrations of total and 10 individual phenolic compounds were higher in ‗Freedom

Red‘, while pentagalloyl glucose (26) and monogalloyl glucose (1) were higher in ‗Monet

Twilight‘ leaves and petioles, respectively, and also only one compound was unique to

‗Monet Twilight‘. Both amino acids and phenolic compounds concentrations may be contributing to the chemical resistance of poinsettias to B. tabaci biotype B.

Introduction

The silverleaf whitefly Bemisia tabaci (Gennadius) biotype B (Hemiptera:

Aleyrodidae) is an insect pest on many agronomic, horticultural, and floricultural crops in the U.S. (Byrne and Bellows 1991, Gerling 1996, McAuslane 1996, Van Driesche and Lyon

2003, Perring and Symmes 2006b). In poinsettias, B. tabaci is the main pest feeding from phloem sap in the leaves and reducing the value of the plants. Whitefly feeding causes weakening and wilting of the plants, ultimately affecting aesthetic quality that leads to reduced marketability (Heinz and Parrella 1994, Ecke et al. 2004). Poinsettia is the number one potted ornamental plant in the U.S. and its popularity continues to grow (NASS 2008,

Prince and Godfrey 2010), but whitefly control is getting more challenging. In commercial greenhouse poinsettia production, whiteflies are managed primarily with insecticides, to which resistance has been developed widely (Dittrich et al. 1990, Cahill et al. 1995,

Palumbo et al. 2001).

In the preceding chapter, it was determined that leaf color and/or thickness mediate

B. tabaci host preference, and that cultivar preference corresponded to higher physiological performance: whiteflies showed lower reproductive rate and intrinsic rate of increase on the cultivar ‗Freedom Red‘ compared to whiteflies developing on a susceptible cultivar ‗Monet

Twilight‘ (Chapter 2). Physical traits alone cannot explain the resistance mechanisms affecting B. tabaci performance and therefore, chemical mechanisms should be explored and studied. However, little, if any, is understood of the potential biochemical mechanisms in poinsettia plants affecting the physiology of whiteflies. Conversely, it is widely known that

66 physiological performance of insects is determined, in general, by food quality (Scriber and

Slansky 1981, Slansky and Scriber 1982, Berenbaum 1995) and food quality, in large part, is determined by the amount and composition of nutritional (primary metabolites) and defensive (secondary metabolites) compounds.

Among primary metabolites associated with nutrition, nitrogen is regarded as a key factor for performance of insects, particularly phloem feeders, because the percentage of dietary nitrogen in phloem is usually low (Auclair et al. 1957b, Dixon 1970, Mattson 1980,

Weibull 1987). The poinsettia cultivars used in this study averaged leaf nitrogen of 4.5 % d.w., when fertilized according to commercial greenhouse guidelines. Heavy fertilization of poinsettia stimulates higher egg production by B. tabaci (Bentz et al. 1995c). Phloem nitrogen is present primarily as free amino acids and their concentration is positively correlated with fertilization practices (Bi et al. 2001, Bi et al. 2003, Bi et al. 2007). It is hypothesized that variation in the composition and abundance of amino acids is a determining factor of insect abundance and performance (Mattson 1980, Cockfield 1988,

Haukioja et al. 1991, Bi et al. 2003). Moreover, the nutritional status of a plant can vary among different phenotypes of the same genotype (Mattson 1980). The first objective in this chapter was to investigate the relationship between amino acid concentration and composition in poinsettia cultivars and B. tabaci behavioral preferences. I hypothesize that cultivars that stimulate higher oviposition and adult preference will have higher concentrations of total and/or individual amino acids.

Secondary plant compounds are other determinants of plant host quality, known to affect insect performance (Dethier 1941, Fraenkel 1959, Ehrlich and Raven 1964,

Berenbaum 1983, Butter et al. 1992, Berenbaum 1995). Among compounds found in poinsettia phloem known to impact B. tabaci are phenolics (Calatayud et al. 1994), which can reduce whitefly fecundity (Butter et al. 1992) and host acceptance (Inbar et al. 2001).

Based on previous research (Chapter 2), B. tabaci fecundity was significantly reduced on the resistant cultivar ‗Freedom Red‘ compared to ‗Monet Twilight;‘ I hypothesize that concentration of phenolics affects these interactions. In an effort to better understand the dynamic resistant mechanisms of poinsettias against B. tabaci, in this chapter the composition and concentration of phenolic compounds was determined in the cultivars

‗Freedom Red‘ and ‗Monet twilight‘ and evaluated in relation to B. tabaci performance.

Material and Methods

Plant material

Test plants were obtained as rooted cuttings from the company Paul Ecke Ranch®,

Encinitas, CA. Upon arrival, cuttings were transplanted in 15.5 x 11 x 11.5 cm pots (Dillen

Products, Middlefield, OH) with soilless media Pro-Mix ‗BX‘ /Mycorise ® PRO (General purpose: Canadian sphagnum peat moss, 75-85% /vol.; perlite, horticultural grade; vermiculite; and dolomitic and calcitic limestone) (Premier Horticultural Inc., Quakertown,

PA, USA) and placed in the greenhouse with settings set at 23°C, 50% relative humidity, and a 16:8 hours (L:D) regime at the Entomology Department facilities at OARDC,

Wooster, OH. Watering and fertilization of the plants was done using a drip irrigation system and a Dosatron injector, set at a dilution of 1/64 that delivered N at 250 mg·L-1.

Plants were fertilized using 20-10-20 (Scotts-Sierra Horticultural Products Company,

Marysville, OH).

Experimental design

From previous research, in an experimental choice test seven poinsettia cultivars

(‗Freedom Red‘, ‗Early Freedom Red‘, ‗Prestige Red‘, ‗Early Prestige Red‘, ‗Zapoteca‘,

‗Snowcap White‘, and ‗Monet Twilight‘) were evaluated in relation to B. tabaci adult feeding and ovipositional preference (Chapter 2: experiment choice test 2). Each cultivar was arranged in a randomized complete block design (RCBD) with five blocks conducted in an environmental walk-in growth chamber at 24 ° C, 50% relative humidity and 16:8 h

(L:D). Amino acid analysis was performed on all plants at the end of the behavioral evaluations of B. tabaci.

Phenolic analysis was conducted on the less susceptible cultivar ‗Freedom Red‘ and the susceptible ‗Monet Twilight‘ from no-choice test 3 (Chapter 2) after completion of B. tabaci bioassays. These two cultivars were arranged in a RCBD with six blocks conducted in the greenhouse with settings at 23°C, 50% relative humidity, and a 16:8 L:D, at the

Entomology Department facilities at OARDC, Wooster, OH.

Analysis of amino acids

Petioles consist mainly of vascular tissue, contain a higher proportion of phloem sap compared to leaves, and their composition mirrors those of phloem contents (Madore and

Webb 1981). Petioles in cotton have been analyzed for free amino acids as a representation of amino acids present in the phloem (Bi et al. 2003). From petioles of young fully developed leaves, 200 mg of fresh tissue were macerated in solution with 2 ml of 0.01 N hydrochloric acid (Fisher Scientific, Pittsburgh, PA) using a mortar and pestle. The extracted amino acids were derivatized using the EZ:faast® GC-MS physiological amino acid analysis kit (Phenomenex Inc., Torrence, CA). Then 2 µl of derivatized solution was injected in split mode (1:15) into the GC-MS using an Agilent 6890 Series GC System equipped with a flame ionization detector, using a Zebron ZB-AAA-GC column, a 7683B

Series autoinjector and a 7683 Series autosampler. Amino acids were quantified using calibration curves from standards of each amino acid. Concentrations of amino acids were expressed as nmol per mg fresh weight (each 100 µL sample contained 10 mg of fresh weight, since, 200 mg/2 ml = 0.1 mg/µL → 0.1 mg/µL × 100 µL = 10 mg).

Tissue collection and Phenolic Extraction

Petioles were collected between 15:00-16:00 hr, immediately submerged in liquid nitrogen, and stored at -80 ºC. Samples were freeze dried and ground with liquid nitrogen.

Next, 0.500 g of fresh petiole tissue was extracted twice overnight in the dark at 4º C with

500 μl of 100% HPLC grade MeOH (Fisher, Pittsburgh, PA) (Bonello and Blodgett 2003).

Between each extraction, sample extracts were centrifuged (12,000xg for 5 min) to remove solids. Samples were then stored at -20ºC and used in subsequent HPLC-UV and HPLC-

ESI-MS-PDA analyses.

Quantitation of Phenolics with HPLC-UV

HPLC-UV analyses were performed on an Alliance 2690 separation module (Waters,

Milford, MA, USA) equipped with an autosampler, and a 996 Photodiode Array Detector

(Waters). The autosampler and column temperatures were set to 4 and 30ºC, respectively.

The binary mobile phase consisted of water/acetic acid (A) (98:2, v/v) and methanol/acetic acid (B) (98:2, v/v), with a flow rate of 1 ml/min. Water was obtained from a Milli-Q water system (Millipore Ltd, Bedford, MA, USA) and HPLC grade methanol and acetic acid were purchased from Fisher (Pittsburgh, PA, USA). Fifteen μl were injected on a Waters

Xterra™ RP18, 5 μm, 4.6 x 150 mm column coupled with a Waters Xterra™ RP18, 3.9 μm,

3.0 x 20 mm guard column. All six samples per cultivar were passed through a Photodiode

Array Detector (PDA) (scanning range, 200-400 nm). The gradient elution program was as follows (in relation to eluent B): 0-5%, in 4 min; 4-10%, in 16 min; 10-30%, in 20 min; 30-

40%, in 10 min; 40-90%, in 5 min; 90-100%, in 10 min; 100-0%, in 2 min. Individual peaks from samples were quantified using peak area at 280nm if they were detected in more than half of the replicates.

Identification of Phenolics with HPLC-PDA-ESI-MS-PDA

HPLC-ESI-MSn (Varian 500 MS; Palo Alto, CA, USA) with an attached PDA

(Varian ProStar 335; Palo Alto, CA, USA), where the injection was split between the two, were used to identify phenolic compounds. A 500-MS ion-trap mass spectrometer

(equipped with an integrated HPLC system), a Prostar 335 Diode Array Detector, and Turbo

DDS software was used to identify phenolic compounds from extracts. Chromatographic separation of phenolic compounds was carried using the same column and gradient elution program as with the HPLC-UV. The binary mobile phase consisted of water/acetic acid (A)

(99.9:0.1 v/v) and methanol/acetic acid (99.9:0.1 v/v) with a flow rate of 300 μl/min. The injection volume for all samples was 5 μl. The ESI ionization was operated in negative ion mode. The parameters for in negative ion mode were: capillary voltage, -80 V; needle voltage, -5 kV, nebulizing gas (air), 25 psi; drying gas (nitrogen) 15 psi at 350° C, using a survey scan range of 50-1000 m/z. MSn scans were triggered on-the-fly by Turbo DDS to detect daughter ions, using trigger thresholds of 20000, 2000, and 200 counts for MS2, MS3, and MS4 respectively.

Phenolic compounds were identified based on the congruence of parent and daughter ions to the published literature and to external standards, when available. Data acquisition and processing was performed using MS Workstation 6 (Varian, Palo Alto, CA, USA).

HPLC-UV chromatograms of all samples were used to gather the retention time and UV- maxima of the individual compounds from each cultivar and match them to those identified using the HPLC-ESI-MS-PDA, were the injection was split into the PDA and the MS. After

72 identification, comparisons among individual compounds were done using total peak areas at 280 nm.

Statistical Analysis

Data of individual concentrations of free amino acids were analyzed by multivariate analyses by MANOVA using SAS 9.2 (SAS 2010), if significant, univariate ANOVA analyses were conducted, and when F-values were significant with an alpha at 0.05

(ANOVA with PROC GLM) for individual amino acids, then mean separation was conducted using Fisher‘s least significant difference (LSD) with an alpha at 0.05. Planned a priori contrasts were conducted between cultivars with light and dark green leaves. Data were checked for assumptions of normality and homogeneity of variances. Multivariate analyses were also conducted using principal component analysis (PCA) with R (R

Development Core Team, 2010) for individual amino acids. Analysis of variance

(ANOVA) was conducted with data of peak areas of phenolic compounds.

Results

Amino acid profiles

The concentrations of total, essential, and individual amino acids (except serine, phenylalanine, glutamine, and tyrosine) were different among poinsettia cultivars (Wilks‘

Lambda F (52, 48.587) = 4.69; P < 0.0001) (Error! Reference source not found.), although proportions were similar (Table 3.1). Four essential amino acids (leucine, isoleucine, lysine, and tryptophan) were detected in all cultivars, but inconsistently across all replicates (less than half), thus, were not considered in the analysis of total and essential amino acids.

Alanine, serine, aspartic acid, glutamic acid, and glutamine were the most abundant individual amino acids constituting over 90% of total concentrations (Table 3.1). The cultivar ‗Zapoteca‘ had higher concentrations of total amino acids than ‗Prestige‘, ‗Early

Freedom‘ and ‗Early Prestige‘ (Figure 3.1). Essential amino acid concentrations were higher on ‗Snowcap‘ and ‗Freedom‘ than on ‗Zapoteca‘ and ‗Early Freedom‘ (Figure 3.1).

Light green leaf cultivars had higher concentrations of total amino acids than dark green leaf cultivars (10.10 ± 0.72 and 7.71 ± 1.01 nmol/mg, respectively) (F(1, 24) = 13.64, P =

0.001)(Figure 3.1.). Concentrations of essential amino acids, however, were not different between light and dark green leaf poinsettia cultivars (0.444 ± 0.06 and 0.437 ± 0.08 nmol/mg, respectively) (F(1, 24) = 0.04, P = 0.8407) (Figure3.1).

Table 3.1 Concentrations (nmol/mg fresh weight ± SE) of free amino acids detected in petiole tissues of poinsettia cultivars (Euphorbia pulcherrima). Cultivars with dark green leaves are less preferred than light green leaf cultivars by Bemisia tabaci. Dark leaves Light leaves ‗Freedom ‗Early ‗Prestige Red‘ ‗Early ‗Zapoteca‘ ‗Snowcap ‗Monet Leaf color Red‘ Freedom Prestige Red‘ White‘ Twilight‘ Red‘ F p Amino acids alanine 0.80 ± 0.1 a 0.43 ± 0.0 bc 0.72 ± 0.2 ab 0.35 ± 0.1 c 0.82 ± 0.2 a 0.69 ± 0.1 ab 0.58 ± 0.1 abc 2.80 0.0329 glycine 0.16 ± 0.0 ab 0.11 ± 0.0 c 0.17 ± 0.0 ab 0.12 ± 0.0 bc 0.17 ± 0.0 ab 0.21 ± 0.0 a 0.14 ± 0.0 bc 3.64 0.0104 valine* 0.14 ± 0.0 ab 0.07 ± 0.0 c 0.09 ± 0.0 bc 0.06 ± 0.0 c 0.06 ± 0.0 c 0.15 ± 0.0 a 0.09 ± 0.0 bc 3.56 0.0115 threonine 0.16 ± 0.0 ab 0.11 ± 0.0 bc 0.14 ± 0.0 ab 0.03 ± 0.0 c 0.12 ± 0.0 abc 0.23 ± 0.0 a 0.11 ± 0.0 bc 2.55 0.0473 serine 2.38 ± 1.1 a 2.31 ± 0.9 a 2.21 ± 0.6 a 2.12 ± 0.4 a 3.41 ± 0.4 a 1.36 ± 0.8 a 3.84 ± 0.3 a 1.61 0.1867 proline 0.05 ± 0.0 b 0.03 ± 0.0 b 0.03 ± 0.0 b 0.02 ± 0.0 b 0.31 ± 0.1 a 0.09 ± 0.0 b 0.02 ± 0.0 b 5.63 0.0009 aspartic acid 0.65 ± 0.1 abc 0.53 ± 0.0 bc 0.41 ± 0.1 c 0.41 ± 0.1 c 0.72 ± 0.1 ab 0.85 ± 0.1 a 0.54 ± 0.0 bc 3.77 0.0087 methionine* 0.02 ± 0.0 bc 0.03 ± 0.0 ab 0.03 ± 0.0 bc 0.05 ± 0.0 a 0.02 ± 0.0 bc 0.01 ± 0.0 c 0.05 ± 0.0 a 5.22 0.0015 glutamic acid 2.71 ± 0.3 abc 1.84 ± 0.2 c 2.27 ± 0.2 bc 1.86 ± 0.3 c 3.27 ± 0.3 a 2.97 ± 0.4 ab 2.25 ± 0.2 bc 3.19 0.0192 * 75 phenylalanine 0.06 ± 0.0 a 0.02 ± 0.0 a 0.04 ± 0.0 a 0.03 ± 0.0 a 0.03 ± 0.0 a 0.05 ± 0.0 a 0.04 ± 0.0 a 2.00 0.1046 glutamine 1.80 ± 0.4 a 1.16 ± 0.3a 1.79 ± 0.5 a 0.99 ± 0.1 a 1.94 ± 0.5 a 2.99 ± 0.9 a 1.24 ± 0.2 a 2.37 0.0609 histidine* 0.21 ± 0.0 ab 0.13 ± 0.0 bc 0.19 ± 0.0 abc 0.18 ± 0.0 abc 0.11 ± 0.0 c 0.25 ± 0.0 a 0.18 ± 0.0 abc 2.46 0.0534 tyrosine* 0.12 ± 0.0 a 0.07 ± 0.0 a 0.10 ± 0.0 a 0.10 ± 0.0 a 0.11 ± 0.0 a 0.09 ± 0.0 a 0.09 ± 0.0 a 1.51 0.2179

essential 0.55 ± 0.1 a 0.32 ± 0.0 b 0.46 ± 0.0 ab 0.42 ± 0.0 ab 0.33 ± 0.1 b 0.56 ± 0.0 a 0.44 ± 0.0 ab 3.58 0.0112 total 9.37 ± 1.1 ab 6.84 ± 1.2 cd 8.26 ± 0.8 bcd 6.35 ± 0.9 d 11.12 ± 0.9 a 9.97 ± 0.7 ab 9.22 ± 0.4 abc 4.01 0.0064 Means within a row followed by different letters are significantly different with Fisher‘s LSD mean separation α = 0.05, n=10 * Essential amino acids

Light green leaf Dark green leaf 14 P = 0.0011 A a C * 12 ab ab 10 abc bcd cd 8 d

Total Amino Acids nmol/mg Acids Amino Total 2

Freedom Freedom Early Prestige Prestige Early Zapoteca Monet

Snowcap 0 B D 0.7 a a P = 0.8407 0.6 ab 0.5 ab ab b b 0.4

0.3

0.2

0.1

Essential Amino Acids nmol/mg Acids Amino Essential

Freedom Freedom Early Prestige Prestige Early Zapoteca Snowcap Monet 0.0 Poinsettia cultivars Dark Light

Figure 3.1 Concentration of (A) total amino acids (± SE) and (B) essential amino acids (± SE) in petiole tissue of several poinsettia (Euphorbia pulcherrima) cultivars. Bars with different letters are significantly different using protected Fisher‘s LSD at α = 0.05. Orthogonal contrast with overall total (± SE) (C) and essential (± SE) (D) amino acid concentrations between cultivars with dark and light green leaves.

Principal component analysis explained >95% of amino acid variance by PC1 (60%) and

PC2 (35.7%) (Figure 3.2). Cultivars ‗Monet Twilight‘, ‗Snowcap‘, and ‗Zapoteca‘ were along positive PC2, while ‗Freedom Red‘ still in positive PC2, ‗Prestige Red‘ and both ‗Early Freedom and Prestige Red‘ were along negative PC2 (Figure 3.2). Along PC2, light green leaf cultivars

‗Monet Twilight‘, ‗Zapoteca‘, and ‗Snowcap‘ were separated from dark green leaf cultivars primarily by concentration of the individual amino acids serine, glutamic acid, and glutamine, respectively (Figure 3.2).

Characterization of Phenolics

A total of 28 compounds were detected by HPLC from leaves and petioles of the two poinsettia cultivars (Table 3.2).

Compound 1 gave a [M –H]- at m/z 331. Further fragmentation of the molecular ion gave a dominant MS2 at m/z 169 corresponding to the loss of a hexose moiety [M – H – 162]- .

The aglycone m/z 169 is consistent with that of a gallic acid, and MS3 at m/z 125 corresponds to the decarboxylation of the gallic acid moiety [M – H – 44]- . This compound was tentatively identified as monogalloylglucose (Sandhu and Gu 2010).

Compound 2, 3, and 5 gave a [M – H]- at m/z 343. Compounds 2 and 3 each had a dominant MS2 fragment of m/z 191 corresponding to the loss of a galloyl group [M –152]- and the secondary dominant ion in the MS2 spectra of m/z 169 corresponds to the galloyl group.

Further fragmentation of m/z 191 is consistent with a quinic acid. Compound 5 yielded an MS2 base peak at m/z 169 accompanied by fragment ions at m/z 173 and 191. Further fragmentation of m/z 169 is consistent with that of a galloyl group.

Figure 3.2 Biplot of principal component analysis of free amino acids (significant and not significantly different) profiles among poinsettia (Euphorbia pulcherrima) cultivars. PC1 and PC2 axes explained 95.7% of total amino acid variance. Cultivars: EFr=‘Early Freedom Red‘, EPr=‘Early Prestige Red‘.

Table 3.2 Characterization of constitutive phenolics in leaves and petioles of poinsettia (Euphorbia pulcherrima) cultivars ‗Freedom Red‘ (FR) and ‗Monet Twilight‘ (MT) using UV and MSn spectrometry. peak Rt [M-H]- MS2 Ions a MS3 Ions MS4 Ions λ nm) Leaf b Petiole b Putative ID c References No. (min) (m/z) max ( 1 2.81 331 169, 271, 211 125 81, 97, 107, 79 278.9 FR, MT FR, MT monogalloyl glucose Sandhu & Gu, 2010 2 3.87 343 191, 169 127, 85, 93, 81, 97, 107, 69, 272.3 FR, MT ND galloyl quinic acid A Clifford et al 79 2007 3 5.02 343 191, 169 85, 127, 173, 274.1 FR, MT FR, MT galloyl quinic acid B Clifford et al 93, 111 2007 4 7.17 343 275.3 ND FR, MT galloyl quinic acid C Clifford et al 2007 5 7.22 343 169, 173, 191 125 81, 97, 107, 79 278.8, FR, MT ND galloyl quinic acid D Clifford et al 335sh 2007 6 9.47 325 275.3 ND FR, MT unknown 1 7 10.68 183 124, 168 78, 106 271.8 FR, MT MT methyl gallate A Kane et al 1988 79 8 14.18 289 245, 205 203, 227, 175, 188 278.9 FR, MT ND catechin A External 187, 161 standard 9 18.11 611 491, 371, 401, 371, 401, 209 295sh, FR FR unknown 2 473, 503 209, 329, 325.8 239 10 22.42 289 245, 205 203, 187, 175, 188, 161 278.9 MT MT catechin B Sandhu & 227 Gu 2010 11 22.48 483 271, 313, 331, 211, 169 168, 124 277.7, FR ND digalloyl glucose A Sandhu & 211 305sh Gu 2010 12 25.86 729 191 155 67 300sh, FR, MT ND unknown 3 328.7 13 28.18 289 245, 205 203 188, 161 280.1 ND FR, MT catechin C Sandhu & Gu 2010 14 29.07 183 124, 168 78, 106 310.9 FR, MT ND methyl gallate B Kane et al 1988 15 31.53 593 473, 353, 383, 353 325, 297 271.2, FR FR apigenin diglucoside Ferreres et al 503 335.9 2003 16 33.33 635 465 313, 169 169, 253 314.5 FR, MT ND trigalloyl glucose Sandhu & Gu 2010 continued…

Table 3.3 Continued - 2 a 3 4 b b c peak Rt [M-H] MS Ions MS Ions MS Ions λmax (nm) Leaf Petiole Putative ID References No. (min) (m/z) 17 34.57 493 331 169 125 263.5, ND FR, MT monogalloyldiglucose Sandhu & 295sh Gu 2010 18 36.87 447 327, 357 299, 284 255, 213, 175, 268.1, FR, MT FR, MT luteolin hexoside Marin et al 231, 257, 133 295sh, derivative 2004 350 19 37.38 447 285 199, 175, 171, 169 268.2, ND FR kaempferol hexoside Sandhu & 241, 151 290sh, Gu 2010 347.8 20 37.95 431 311 283 239, 163, 117, 268.5, FR FR apigenin glucoside A Waridel et al 197 295sh, 2001 344.2 21 39.11 483 271, 313, 211, 211, 169 168, 124 277.1 FR, MT ND digalloyl glucose B Sandhu & 331 Gu 2010 22 39.92 741 609 301 271, 179, 151, 257.2, FR, MT FR, MT quercetin pentose Mullen et al 255 355.3 rutinoside 2007 80 23 41.70 431 311, 341 283 239, 183, 211, 270, FR, MT FR, MT apigenin glucoside B Waridel et al 117, 163 295sh, 2001 338.3 24 42.63 787 617, 635 465, 573, 313, 295 278.9 FR, MT ND tetragalloyl glucose Sandhu & 447, 403 Gu 2010 25 45.69 609 301 271, 151, 243, 227, 215, 256.9, FR, MT FR, MT rutin (quercetin External 179, 255 199 295sh, rutinoside) standard 355.6 26 46.43 939 769 617, 447, 465, 447 281.2 FR, MT MT pentagalloyl glucose Sandhu & 601, 599 Gu 2010 27 47.14 1091 939 769 617, 601, 447, 277.7, FR, MT ND hexagalloyl glucose Sandhu & 599 346.6 Gu 2010 28 47.81 593 285 257, 229, 229, 163, 211 266.1, FR, MT FR, MT kaempferol rutinoside Sandhu & 255 295sh, Gu 2010 349 a Bold in MS2 and MS3 columns indicates those ion fragments that underwent further fragmentation. b Bold in the leaf and petiole columns indicates that the cultivar has significantly higher concentrations of such compound. c Bold in the putative ID column indicates that compound was detected only in that cultivar.

Although MS2 base peak from compound 5 differs from that of compound 2 and 3, it has been suggested that all three compounds are consistent with galloyl quinic acid (Clifford et al.

2007). Different elution times but same [M – H]- may indicate that these are isomers of galloyl quinic acid (Clifford et al. 2007). Therefore, these compounds were putatively identified as galloyl quinic acid A (2), galloyl quinic acid B (3), galloyl quinic acid D (5).

Compound 4 had the same [M – H]- at m/z 343 as compound 2, 3, and 5. Based on similar retention times and UV spectra, it is tentatively named as galloyl quinic acid C (4)

(Clifford et al. 2007).

Compound 6 had a [M – H]- at m/z 325 with no further fragmentation. This compound was labeled unknown 1.

Compound 7 and 14 had a [M – H]- at m/z 183, consistent with that of methyl gallate and were ,therefore, tentatively identified as methyl gallate A (7) and methyl gallate B (14)

(Kane et al. 1988).

Compound 8, 10, and 13 had a [M – H]- at m/z 289, with a main MS2 ions at m/z 245 and 205, and MS3 yielding a dominant base ion at m/z 203. On the basis of spectral characteristics and an external standard (catechin: Extrasynthese, B.P.62-69730 Genay, France) matching UV and retention time, compound 8, 10, and 13 were identified as catechin. Different retention times on these compounds may indicate they are isomers of catechin (Sandhu and Gu

2010). Therefore, these compounds were tentatively identified as catechin A (8) catechin B (10), and catechin C (13).

Compound 9 had a [M – H]- at m/z 611, yielded a major MS2 fragment at m/z 491, which underwent MS3 fragmentation at m/z 401, and this underwent further fragmentation yielding MS4 at m/z 209. This compound was labeled unknown 2.

Compound 11 and 21 had a [M – H]- at m/z 483 and the same MS2, MS3, and MS4 fragmentation pattern. The dominant MS2 at m/z 271 [M – H – 212]-, indicates the loss of a galloyl group with cross ring fragmentation of glucose, and m/z 331 [M – H – 152]-, which indicates the loss of another galloyl group. The aglycone m/z 169 in MS3 is consistent with gallic acid, suggesting this compound is tentatively digalloylglucose A. Compound 21 was tentatively identified as an isomer of digalloylglucose on the basis of fragmentation data and previous literature reports (Salminen et al. 1999, Barry et al. 2001, Meyers et al. 2006, Sandhu and Gu 2010). Therefore, compound 21 was identified tentatively as digalloylglucose B.

Compound 12 had a [M – H]- at m/z 729, with MS2 yielding a single fragment at m/z 191, which underwent further fragmentation with MS3 at m/z 155, and a MS4 at m/z 67. This compound was labeled unknown 3.

Compound 15 had a [M – H]- at m/z 593, it underwent further fragmentation with MS2 at m/z 503 [M – H – 90]-; 473 [M – H –120]-; and 383 [M – H – 210 (apigenin [270, aglycone of apigenin] + 113); this compound is consistent with the fragmentation pattern of 6,8-di-C- glucosyl apigenin (Ferreres et al. 2003). Therefore, it was tentatively identified as apigenin diglucoside.

Compound 16 had a [M – H]- at m/z 635, fragmentation pattern was consistent with, and therefore tentatively identified as trigalloyl glucose (Sandhu and Gu 2010).

Compound 17 had a [M – H]- at m/z 493, with MS2 yielding a m/z 331 and MS3 yielding a m/z 169, indicating the loss of two hexosyl (glucose) groups. The aglycone m/z 169 is consistent with gallic acid, suggesting this compound is tentatively monogalloyl diglucose

(Sandhu and Gu 2010).

Compound 18 had a [M – H]- at m/z 447, underwent further fragmentation with dominant

MS2 at m/z 327 [M – H – 120]-; 357 [M – H –90]-, indicating the sequential loss of hexose residues. Fragmentation pattern is consistent of that of luteolin-8-C-hexoside derivatives (Marín et al. 2004). Therefore, it was tentatively identified as luteolin hexoside derivative.

Compound 19 had a [M – H]- at m/z 447, with MS2 yielding a single m/z 285, indicating the loss of a hexosyl group [M – H – 162]-. The aglycone m/z 285 is consistent with kaempferol, therefore, this compound was tentatively identified as kaempferol hexoside (Sandhu and Gu

2010).

Compound 20 and 23 had a [M – H]- at m/z 431 and same MS2, MS3, and MS4 dominant ions. Fragmentation pattern is consistent with 8-C-glucosyl apigenin (Waridel et al. 2001).

Therefore, this compound was identified tentatively as apigenin glucoside. Compounds were designated as apigenin glucoside A (20) and apigenin glucoside B (23) as putative isomers.

Compound 22 had a [M – H]- at m/z 741, with MS2 yielding a m/z 609, MS3 yielding a fragment at m/z 301, and with MS4 yielding m/z 271, 179, 151, and 255. The loss of m/z 132 [M

– H – 609]- indicates a pentose and the fragmentation pattern from MS2-MS4 is consistent with rutin, suggesting this compound is tentatively quercetin pentose rutinoside (Mullen et al. 2007).

Compound 24 had a [M – H]- at m/z 787, which further fragmentation pattern was consistent with, and therefore, tentatively identified as tetragalloyl glucose (Sandhu and Gu

2010).

Compound 25 had a [M – H]- at m/z 609, with MS2 yielding a fragment at m/z 301.

Further fragmentation of m/z 301 yielded MS3 fragments at m/z 271, 151, 179, and 255; m/z 271 underwent MS4 yielding m/z 243, 227, 215, 199. The spectral characteristics, UV max and

83 retention time of this compound matches identically to an external standard of rutin (Sigma

Chemical Co, P.O. Box 14508, St. Louis, MO 63178).

Compound 26 had a [M – H]- at m/z 939, which further fragmentation pattern was consistent with, and therefore, tentatively identified as pentagalloyl glucose (Sandhu and Gu

2010).

Compound 27 had a [M – H]- at m/z 1091, which further fragmentation pattern was consistent with, and therefore, tentatively identifies as hexagalloyl glucose (Sandhu and Gu

2010).

Compound 28 had a [M – H]- at m/z 593 which fragmented to produce a MS2 fragment at m/z 285 [M – H – 308]-, indicating the loss of a hexosyl-rhamnosyl group. The aglycone m/z

285 is consistent with kaempferol, therefore, this compound was tentatively identified as kaempferol rutinoside (Sandhu and Gu 2010).

Composition and concentration of phenolic compounds

Both qualitative and quantitative variations were observed in expression of phenolic metabolites in leaf and petioles between a less susceptible ‗Freedom Red‘ and a more susceptible

‗Monet Twilight‘ poinsettias. In leaf extracts, ‗Freedom Red‘ contained four compounds not detected in ‗Monet Twilight‘, including digalloyl glucose A (11), apigenin diglucoside (15), apigenin glucoside A (20), and compound 9 (unknown 2), while catechin B (10) was found only in ‗Monet Twilight‘ (Table 3.3).

-4 Table 3.3 Mean ± SE UV280 peak area per microliter (x 10 ) of individual and total phenolic compounds in leaf and petiole extracts from poinsettia (Euphorbia pulcherrima) cultivars ‗Freedom Red‘ and ‗Monet Twilight‘ using HPLC-PDA. ND = not detected. Compounds (Peak No.) Leaf Tissue Petiole tissue ‗Freedom Red‘ ‗Monet Twilight‘ F(1,7) P-value ‗Freedom ‗Monet F(1, 4) P-value value Red‘ Twilight‘ value monogalloyl glucose (1) 25.25 ± 7.80 19.80 ± 4.52 0.51 0.4990 1.61 ± 0.26 3.22 ± 0.47 8.81 0.0412 galloyl quinic acid A (2) 0.71 ± 0.22 0.59 ± 0.12 1.17 0.3160 ND ND galloyl quinic acid B (3) 12.96 ± 0.65 9.32 ± 0.93 13.83 0.0075 4.07 ± 0.30 1.66 ± 0.11 77.06 0.0009 galloyl quinic acid C (4) ND ND 0.70 ± 0.07 0.27 ± 0.02 37.06 0.0037 galloyl quinic acid D (5) 1.98 ± 0.14 2.43± 0.25 2.20 0.1813 ND ND unknown 1 (6) ND ND 2.26 ± 0.24 0.56 ± 0.04 48.00 0.0023 methyl gallate A (7) 28.67 ± 3.88 27.04 ± 2.52 0.25 0.6295 ND 0.37 ± 0.05 catechin A (8) 1.46 ± 0.28 2.04 ± 0.36 0.97 0.3586 ND ND unknown 2 (9) 8.00 ± 0.42 ND 1.67 ± 0.17 ND catechin B (10) ND 3.75 ± 0.29 ND 0.75 ± 0.09 digalloyl glucose A (11) 4.01 ± 0.83 ND ND ND 85 unknown 3 (12) 11.76 ± 0.90 9.10 ± 0.49 6.68 0.0362 ND ND catechin C (13) ND ND 1.96 ± 0.13 0.51 ± 0.05 57.80 0.0169 methyl gallate B (14) 2.98 ± 0.46 3.85 ± 0.37 1.59 0.2477 ND ND apigenin diglucoside (15) 2.09 ± 0.16 ND 1.92 ± 0.28 ND trigalloyl glucose (16) 6.03 ± 1.16 5.06 ± 0.44 0.50 0.5031 ND ND monogalloyl diglucose (17) ND ND 1.12 ± 0.45 0.68 ± 0.08 0.67 0.4583 luteolin hexoside derivative (18) 2.65 ± 0.71 2.82 ± 0.31 0.05 0.8230 0.88 ± 0.21 0.93 ± 0.03 0.05 0.8341 kaempferol hexoside (19) ND ND 2.23 ± 0.27 ND apigenin glucoside A (20) 5.57 ± 0.74 ND 1.21 ± 0.14 ND digalloyl glucose B (21) 18.14 ± 2.38 16.75 ± 2.58 0.35 0.5710 ND ND quercetin pentose rutinoside (22) 1.65 ± 0.07 1.10 ± 0.10 16.47 0.0048 0.26 ± 0.02 0.18 ± 0.01 14.42 0.0191 apigenin glucoside B (23) 3.91 ± 0.21 1.06 ± 0.13 11.44 0.0277 2.48 ± 0.22 0.20 ±0.02 67.70 0.0038 tetragalloyl glucose (24) 12.25 ± 1.25 5.57 ± 0.59 16.24 0.0069 ND ND quercetin rutinoside (25) 59.00 ± 3.83 36.55 ± 3.87 12.33 0.0098 2.79 ± 0.37 1.66 ± 0.18 3.87 0.1205 pentagalloyl glucose (26) 40.97 ± 3.69 53.22 ± 4.56 7.11 0.0322 ND 0.33 ± 0.02 hexagalloyl glucose (27) 8.53 ± 0.91 4.82 ± 0.45 18.64 0.0035 ND ND kaempferol rutinoside (28) 61.81 ± 6.53 48.17 ± 4.99 3.83 0.0981 0.63 ± 0.03 0.50 ± 0.08 0.58 0.4900

total phenolics 319.43 ± 28.40 246.63 ± 18.46 8.66 0.0216 25.78 ± 2.29 11.65 ± 1.00 32.95 0.0046 Bold within the columns indicates compounds significantly higher in concentration.

Of the eighteen compounds shared in leaf extracts between the cultivars, seven

[galloyl quinic acid B (3), compound 12 (unknown 3), quercetin pentose rutinoside (22), apigenin glycoside B (23), tetragalloyl glucose (24), rutin/quercetin rutinoside (25), and hexagalloyl glucose (27)] were higher in ‗Freedom Red‘, while only one, pentagalloyl glucose, was higher in ‗Monet Twilight‘ (Table 3.3). In petiole extracts, four compounds, including apigenin diglucoside (15), apigenin glucoside A (20), kaempferol hexoside (19), and compound 9 (unknown 2) were found only in ‗Freedom Red‘, while three compounds were detected only in ‗Monet Twilight‘, methyl gallate A (7), catechin

B (10), and pentagalloyl glucose (26), although methyl gallate A (7) and pentagalloyl glucose (10) were in very low concentrations (Table 3.3). ‗Freedom Red‘ also had higher concentrations in six of the 11 compounds shared, including galloyl quinic acid B (3), galloyl quinic acid C (4), catechin C (13), quercetin pentose rutinoside (22), apigenin glucoside B (23), and compound 6 (unknown1), than ‗Monet Twilight‘. Total concentration of soluble phenolics (the addition of all individual phenolics identified) were higher in ―Freedom Red‘ than in ‗Monet Twilight‘ for both leaf and petiole tissues

(Table 3.3).

Discussion

Concentrations of total amino acids were different among cultivars and based on results, susceptible cultivars appear to have a higher nutritional quality and this may be playing a role in attracting B. tabaci. In previous research, light green leaf cultivars were preferred by B. tabaci adults for feeding and for oviposition (Chapter 2). Concentrations of essential amino acids also varied among cultivars, but not in relation to plant resistance as the means and ranges of concentrations for dark- and light-green-leafed cultivars were almost identical. Amino acids were designated as essential based on general insect nutrition studies (Scriber and Slansky 1981), but this designation may be less relevant for phloem-feeding Homoptera like whiteflies. Insect-essential amino acids only constituted ca. 5% of the total amino acids in poinsettia petioles, and Homoptera commonly harbor obligatory endosymbionts that provide the essential amino acids that the insect cannot synthesize or obtain from the plant (Houk and Griffiths 1980). Additionally, phloem feeders can compensate for low amino acid content by increasing consumption (Prosser et al. 1992), but feeding rates were not measured in this study. Testing of the relative importance of individual amino acids to whitefly nutrition is still inconclusive because of the formulation of effective diets (Davidson et al. 2000, Davidson et al. 2002).

Qualitatively, all amino acids detected are confirmed by findings of others studies with poinsettias (Byrne and Miller 1990, Tertuliano and Leru 1992, Kuniyoshi 2007). It is important to consider that the concentration of total or individual free soluble amino acids may fluctuate depending on stress, tissue, season, ontogeny, and time of the day (Bi et al.

2003, Bi et al. 2007). This is among the first studies characterizing amino acids in more than one current poinsettia cultivars.

Cultivar ‗Freedom Red‘, with which B. tabaci show lower preference and F1 oviposition performance (Chapter 2), has a higher concentration of phenolic metabolites than the more susceptible ‗Monet Twilight‘. The majority of the shared compounds were higher in ‗Freedom Red‘ and there were a greater number of compounds detected only in

‗Freedom Red‘ compared to ‗Monet Twilight‘. These qualitative and quantitative differences suggest that phenolics may play a significant role in mediating poinsettia resistance to whiteflies. To the best of my knowledge, this is the first study characterizing and identifying individual phenolic compounds in Euphorbia pulcherrima, and will be useful for more focused studies evaluating individual compounds in artificial diets or bioassays.

Phenolic compounds have been correlated with resistance against whiteflies in other plants, like cotton, where B. tabaci is also a major pest (Butter et al. 1992, Abdallah et al. 2001, Balakrishnan 2006, Acharya and Singh 2008). Specifically, the total concentration of phenolics has been negatively correlated with B. tabaci population

(Butter et al. 1992, Balakrishnan 2006, Acharya and Singh 2008), oviposition preference(Abdallah et al. 2001), and fecundity (Butter et al. 1992). In mung beans,

Vigna mungo (L.) Hepper, high phenolic concentration reduce B. tabaci attacks (Chhabra et al. 1993). Interestingly, V. mungo origins are in India/Pakistan, which is also the area from where B. tabaci is believed to have originated (Brown et al. 1995). Phenolic compounds have also been well documented against pathogens and other insects,

88 affecting insect growth and reproductive capacity (Bennett and Wallsgrove 1994,

Lattanzio et al. 2006). Aphids, for instance, have similar feeding habits as whiteflies and wheat cultivars with high total concentration of phenolics were shown to be behaviorally less preferred by Rhopalosiphum padi (Leszczynki 1985).

The main classes of phenolic compounds represented in this study have been shown to have active biological activity. The two unknown compounds may be playing a role in resistance, but further testing needs to be conducted to determine the identity of such. Galloyl quinic acid is related to chlorogenic acid, and Chrysanthemums spp. with higher amounts of chlorogenic acid have been shown to affect survival and be less preferred by thrips (Leiss et al. 2009). Chlorogenic acid is oxidized to chlorogenoquinone, which binds to proteins and amino acids (Leiss et al. 2009), potentially reducing the availability of amino acids. It is possible that galloyl quinic acids function in a similar way in poinsettias against B. tabaci.

Quercetin pentose rutinoside, kaempferol hexoside, catechin C, apigenin diglucoside and glucoside A and B, belong to the class of flavonoids. It is widely accepted that secondary metabolites and among them flavonoids evolved as a defense against herbivores (Treutter 2006). Flavonoids can act as antifeedants, as digestibility reducers, and as toxins (Treutter 2006). Quercetin pentose rutinoside is also found in cassava, another member of the Euphorbiacea, where it has been shown to slow developmental time of Phenaccocus manihoti (Calatayud 2000). Similarly, quercetin and its glycoside rutin are associated with higher larval mortality of Spodoptera liture

(Mallikarjuna et al. 2004). Catechins can act as feeding deterrents in vertebrate

89 herbivores (Tahvanainen et al. 1985), so it is possible they can affect the B. tabaci in a similar manner. Compound 6 (unknown 1) has the same UV absorption as a catechin; it is possible this compound relates to a flavonol.

In summary, both amino acids and phenolic compounds affect plant quality.

Resistant and susceptible poinsettia cultivars were similar in nutritional and defensive profiles, nevertheless, quantitative and qualitative differences were observed. It is possible that these differences determined how resistant or susceptible plants are, although resistant mechanisms I believed are multidimensional and less likely to be dictated by a few traits, but rather by a suite of biotic influenced by abiotic factors.

Phenolics exhibited a wider difference of expression and singularity, therefore, may be more influential in affecting the patterns of choice and performance of B. tabaci. Further studies are recommended to determine the effect of individual compounds on the behavior and physiology of whiteflies, and this study provides good candidates for study as a start.

In this study, amino acids and phenolics were analyzed each on different plants and from different experiments, therefore, regression analyses were not possible to conduct. However, I could hypothesized from the data that less susceptible cultivars had lower nutritional quality since they had both lower levels of nutrients and higher levels of defensive compounds, which could have a synergistic effect affecting B. tabaci negatively.

Acknowledgments

I thank Theodore Derksen and Wilmer Rodríguez for assistance in the preparation of samples. I thank Larry Phelan for his guidance, technical assistance and use of the

GC-MS in the amino acid analysis. I thank Pierlugui Bonello for use of his HPLC and

Justin Whitehill for his guidance and technical assistance in the phenolic analysis. I thank Stephen Opiyo for his PCA statistical consultation.

Research support provided by state and federal funds appropriated to the The

Ohio State University (OSU), OARDC, SEEDS grant # 2008-110. Additional funding provided by the American Floral Endowment grant # 20020754, and from the

Entomology department of OSU. Plant material was provided by Paul Ecke Ranch,

Encinitas, CA.

Chapter 4 Eretmocerus mundus Mercet (Hymenoptera: Aphelinidae) response to whitefly infestations on a resistant and a susceptible poinsettia cultivar

Abstract

When it comes to biological control of whiteflies many studies have evaluated the efficacy of different parasitoids and predators (Enkegaard 1993b, Drost et al. 1999, De

Barro et al. 2000, Ellis et al. 2001, Gerling et al. 2001, Bogran and Heinz 2002, Antony et al. 2003, Ardeh et al. 2005a, b). Only one study though has evaluated the performance of parasitoids (Encarsia spp., Hymenoptera: Aphelinidae) of Bemisia tabaci biotype B

(Hemiptera: Aleyrodidae) in relation to two poinsettia cultivars (Heinz and Parrella

1994). In this study, the authors found that the cultivar ‗Annette Hegg Brilliant

Diamond‘ with fewer leaf trichomes was more favorable to the parasitoids than ‗Lilo‘.

The cultivars evaluated in the present research had similar trichome density (Chapter 2); however, cultivars with light green foliage were better hosts to B. tabaci than cultivars with dark green foliage. B. tabaci adults preferentially fed and oviposited more on light green leaf cultivar ‗Monet Twilight‘ than on dark green leaf cultivar ‗Freedom Red‘.

Additionally, whitefly fecundity was enhanced on ‗Monet Twilight‘. In terms of host quality, ‗Monet Twilight‘ had a lower concentration of defensive phenolic compounds

92 than ‗Freedom Red‘ (Chapter 3). Thus, I hypothesized that B. tabaci nymphs reared in a susceptible host will be preferred by natural enemies as these nymphs are, from the perspective of host quality, feeding from a better food source. In an effort to evaluate how B. tabaci preference and performance on a resistant and a susceptible poinsettia cultivar could affect the response of parasitoids, I selected Eretmocerus mundus Mercet

(Hymenoptera: Aphenilidae), a well-established natural enemy of B. tabaci biotype B, to assess its parasitism rate on two poinsettia cultivars. The objective of this study was to determine the behavioral preference of E. mundus between whitefly nymphs feeding from a resistant and a susceptible poinsettia cultivar. Results indicate that parasitism was higher and emergence was faster on the light green leaf cultivar ‗Monet Twilight‘ than on the dark green leaf cultivar ‗Freedom Red‘. These observations may indicate nymphs of susceptible plants are better hosts for E. mundus, but further studies need to be conducted to understand the implications for E. mundus feeding on nymphs from a susceptible or a resistant plant.

Introduction

The silverleaf whitefly Bemisia tabaci (Gennadius) biotype B (Hemiptera:

Aleyrodidae) is the most prevalent insect pest of poinsettias (Ecke et al. 2004) and poinsettias are the U.S. top selling potted ornamental (NASS 2008). Previous studies evaluating poinsettia cultivars in relation to B. tabaci preference and performance showed that light green leaf cultivars are more susceptible and that dark green leaf cultivars are more resistant (Chapter 2). Dark green leaf cultivars higher phenolic content and thickness of the leaves may be resistance mechanisms playing a major role in relation to

B. tabaci performance and behavioral preferences (Chapter 3). Phenolics are compounds that can affect growth, survivorship, and fitness of insects. Hence, whitefly nymphs feeding from a resistant cultivar (‗Freedom Red‘) can turn out to be a suboptimal host for a parasitoid. A preliminary study was conducted to evaluated the behavioral preference of Eretmocerus mundus Mercet (Hymenoptera: Aphelinidae), a well-known parasitoid that deposits its eggs on second to early third instar B. tabaci nymphs. I hypothesized that nymphs feeding from resistant poinsettia cultivars will be less preferred by parasitoids. Parasitoids are often closely evolved with their hosts (Rausher 1996), therefore, it is possible that whitefly nymphs feeding on suboptimal plant hosts might be suboptimal host for parasitoids. Therefore, I sought to test this hypothesis using E. mundus, which has proven to be effective against B. tabaci biotype B in commercial greenhouses (De Barro et al. 2000).

When it comes to biological control of whiteflies many studies have been done

(Enkegaard 1993b, Drost et al. 1999, De Barro et al. 2000, Ellis et al. 2001, Gerling et al.

2001, Bogran and Heinz 2002, Antony et al. 2003, Ardeh et al. 2005a, b), but only one study evaluated the effect of two poinsettia cultivars, ‗Annette Hegg Brilliant Diamond‘ and ‗Lilo‘, on the performance of one predator, Delphastus pusillus LeConte, and four parasitoids, Encarsia formosa Gahan, E. luteola Howard, E. pergandiella Howard, and E. transvena (Timberlake), of B. tabaci biotype B (Heinz and Parrella 1994). Adult longevity of the biological control agents was evaluated and no significant differences were observed between the two cultivars, although it did vary significantly between natural enemies. Prey consumption and oviposition by the predator was favored on

‗Annette Hegg Brilliant Diamond‘ poinsettia, which has 15% less trichome density than

‗Lilo‘. Host feeding and parasitism among the four parasitoids was similar among the two cultivars, but consistently the four parasitoids performed better on the cultivar with less trichomes (‗Annette Hegg Brilliant Diamond‘). The cultivars (‗Freedom Red‘ and

‗Monet Twilight‘) evaluated in our study do not have significant differences in trichome density (Chapter 2), so it is not a factor expected to play a significant role related to host feeding and parasitism.

The main objective of this study is to compare parasitism of B. tabaci nymphs by

E. mundus on a resistant and a susceptible poinsettia cultivar.

Materials and Methods

The experiment was conducted in a walk-in environmental chamber with settings at 26°C and 16:8 h (L/D) at the Entomology Department facilities at the Ohio State

University, OARDC campus, Wooster, OH.

Experimental plants

Test plants were obtained as rooted cuttings from the company Paul Ecke Ranch®,

Encinitas, CA. Upon arrival, cuttings were transplanted to 15.5 x 11 x 11.5 cm pots

(Dillen Products, Middlefield, OH) with soilless media Pro-Mix ‗BX‘ /Mycorise ® PRO

(General purpose: Canadian sphagnum peat moss, 75-85% /vol.; perlite, horticultural grade; vermiculite; and dolomitic and calcitic limestone) (Premier Horticultural Inc.,

Quakertown, PA, USA) and placed in a greenhouse. Greenhouse settings were set at

23°C, 50% relative humidity, and a 16:8 hour (L:D) regime until they were used for the experiments in the chambers. Watering and fertilization of the plants was done using a drip irrigation system and a Dosatron injector, set at a dilution of 1/64 that delivered N at

250 mg·L-1. Plants were fertilized using 20-10-20 (Scotts-Sierra Horticultural Products

Company, Marysville, OH).

Whitefly source: Bemisia tabaci biotype B

The whitefly colony was maintained on poinsettias (cultivars ‗Freedom Red‘ and

‗Monet Twilight‘) at 24°C. Plants used for the colony were also fertilized at a rate of 250 mg·L-1 of nitrogen (20-10-20) with every watering event.

Choice test: Experimental design and Infestation

Plants used in the experiment had on average 8-12 leaves and were infested each with a minimum of 30 third instar whiteflies nymphs (following similar methodology to that of Heinz and Parella (1994).

A less suceptible and a more susceptible poinsettia cultivar, ‗Freedom Red‘ and

‗Monet Twilight,‘ respectively, were randomized in a complete block design (RCBD).

Nine blocks were used. Each block comprised a cage containing one ‗Monet Twilight‘ and one ‗Freedom Red‘ poinsettia. The two cultivars were inside the 70 x 70 x 40 cm mesh cage. In each cage, 60 female:male pairs of 3-day-old whiteflies were released near the opening of the cage for a period of 72 h to give the whiteflies enough time to settle and lay an adequate number of eggs. Whiteflies were gently blown and tapped into the container using Pasteur pipettes. After 72 h, whitefly adults were collected and removed and plants were held for 14 days, about the time for whitefly eggs to hatch and for nymphs to reach second to early third instar, which are the instars the parasitoid E retmocerus mundus attacks. The number of nymphs offered per plant was counted.

Twenty five E. mundus (IPM Labs, Locke, New York) were then placed into small petri dishes and one petri dish per cage. After 24 h, parasitoids were removed from the cages. Twelve days after the infestation of the E. mundus, the number of exuvias parasitized and not parasitized and fourth instar nymphs parasitized and not parasitized on each plant were counted to determine parasitism rate and also percentage of emergence. Parasitism was then calculated as percentage to provide uniform comparisons between the two cultivars.

Statistical analysis

Data of parasitism and emergence were analyzed by analysis of variance

(ANOVA; PROC GLM) with SAS 9.2 (SAS 2010). Data were checked for normality and homogeneity of variances previous to the analysis.

Results

Percentage of parasitism by E. mundus was ca. 132% percent higher on nymphs from ‗Monet Twilight‘ than on ‗Freedom Red‘ (F (1, 8) = 5.23, p = 0.05) (Figure 4.1).

25 * 20

Percentage nymphs parasitized nymphs Percentage 5

0 Freedom Red Monet Twilight Figure 4.1 Percentage (± SE) of Bemisia tabaci biotype B nymphs parasitized by Eretmocerus mundus in a less susceptible (‗Freedom Red‘) and a more susceptible (‗Monet Twilight‘) poinsettia (Euphorbia pulcherrima) cultivar. Asterisk means a significant difference from ANOVA p = 0.05.

Although not all parasitoids had emerged by day 12 after nymphs being parasitized, already for every E. mundus emerged from nymphs on ‗Freedom Red,‘ 1.5 had emerged from nymphs on ‗Monet Twilight‘ (F(1, 8) = 0.70, p = 0.42) (Figure 4.2).

The delayed emergence observed from ‗Freedom Red‘, although not statistically significant, may indicate developmental time of E. mundus may be affected by the host quality of the nymphs.

Discussion

Higher E. mundus parasitism preference for B. tabaci nymphs from a susceptible poinsettia cultivar supports the hypothesis that herbivores on resistant plants may be less attractive or of less quality to predators, parasitoids, or parasites (Campbell and Duffey

1979, Kauffman and Kennedy 1989, Barbosa et al. 1991, Barbour et al. 1993, Stamp et al. 1997). A tendency for delayed emergence was observed, although complete emergence of all parasitoids was not recorded, might indicate that E. mundus developmental time was affected by the quality of the prey.

100

Percentage parasitoids emerged parasitoids Percentage 5

Freedom Red Monet Twilight Figure 4.2 Percentage (± SE) of Eretmocerus mundus emerged from whitefly nymphs parasitized in a less susceptible (‗Freedom Red‘) and a more susceptible (‗Monet Twilight‘) poinsettia (Euphorbia pulcherrima) cultivar. ANOVA p = 0.42.

101

This has been reported in other systems (Barbosa et al. 1991). Barbosa et al

(1991) evaluated developmental rate, size, and survival of the parasitoid Cotesia congregate (Say), when reared from Manduca sexta (L.) fed on diets containing either nicotine (alkaloid), rutin (phenolic), or hordenine.(alkaloid) and found, in general, the effects of these defensive compounds on the parasitoid paralleled those on the unparasitized M. sexta, although rutin had a significant effect on developmental times of both herbivore and parasitoid.

Phenolics compounds have been detected in leaf and petiole of poinsettias and a significant higher concentration are present in the resistant cultivar ‗Freedom Red‘ than on the susceptible ‗Monet Twilight‘ (Chapter 3). Flavonoid rutin is present in the suite of phenolic defenses present in poinsettias (Chapter 3) and further studies should be conducted to determine the specific effects on B. tabaci and E. mundus. The defensive compounds found in poinsettia (Chapter 3) are postulated to play a major role in mediating the behavior of B. tabaci and the parasitoid E. mundus. This is the first study evaluating the choice of E. mundus between nymphs from a resistant and a susceptible poinsettia cultivar. Moreover, this is the first work postulating phenolic compounds in poinsettias cultivars as a factor affecting host quality of the prey —B. tabaci, and the tritrophic implications in host recognition of the parasitoid—E. mundus.

It is possible that phenolics and amino acids are playing a role in the quality whiteflies, but how exactly this is affecting the nutritional status of nymphs and further affecting E. mundus will need further investigation. In tomato plants rutin and chlorogenic acid have been shown to prolong developmental time of a generalist insect

102 predator (Podisus maculiventris: Pentatomidae) fed Manduca sexta (Sphingidae) caterpillars feeding from these allelochemicals (Stamp et al. 1997). Determining the phenolic content in silverleaf whiteflies and E. mundus and testing specific compounds with diets could be a plausible route to follow and compare between the cultivars in a future.

Acknowledgments

I thank Ronald Batallas and Theodore Derksen for their assistance in the collection of data.

Research support provided by state and federal funds appropriated to the The

Ohio State University (OSU), OARDC, SEEDS grant # 2008-110. Additional funding provided by the American Floral Endowment grant # 20020754, and from the

Entomology department of OSU. Plant material was provided by Paul Ecke Ranch,

Encinitas, CA.

103

Chapter 5 Summary and future work

In summary, light green leaf cultivars were preferred by B. tabaci and to some extent whiteflies performed better on them (Chapter 2). Physical factors potentially involved in affecting behavioral preferences of B. tabaci included leaf thickness, and indirectly, chlorophyll content, and overall color of the leaves (Chapter 2). Other physical factors, especially those contributing to physical properties of the leaves, for example wax content, may be involved, but were not evaluated in this research. It was demonstrated that color of the leaves is a visual cue used by B. tabaci to recognize susceptible and resistant poinsettia cultivars (Chapter 2). To what extent color may be associated with defense mechanisms needs to be address in future work.

Chemical factors investigated in Chapter 3 that possibly impacts the behavior and physiology of B. tabaci included free amino acids and soluble phenolics. Cultivars with light green leaves had higher concentration of amino acids. Concentrations of total and individual phenolic compounds varied between less and more susceptible cultivars.

Concentrations of total phenolics were higher in the less whitefly-susceptible ‗Freedom

Red‘ than in the more susceptible ‗Monet Twilight‘. These qualitative and quantitative differences may be playing a major role effecting the behavior and physiology of B. tabaci. The lower concentration of nutrients and higher concentration of phenolics in the 104 less susceptible cultivars may be very important in determining B. tabaci‘s behavior and performance.

In Chapter 4, I hypothesized that a higher parasitism would be observed in nymphs‘ feeding from ‗Monet Twilight‘ than in ‗Freedom Red‘ on the basis of nymphs being a suboptimal host when feeding from a resistant cultivar. Higher parasitism by

Eretmocerus mundus was observed in ‗Monet Twilight‘. This tritrophic interaction should be investigated more in depth to better understand how nymph quality is affecting host selection by natural enemies.

Behavior and physiology of B. tabaci in poinsettias

On average less than 30% of B. tabaci preferred to settle and oviposit on cultivars with dark green leaves. Conversely, light green leaf cultivars were three times more attractive to whiteflies than dark green leaf cultivars for oviposition. For future work, I would recommend growers to grow darker green leaf cultivars because they are inherently less preferred compared to lighter green leaf foliage and appear to be less optimal hosts to the silverleaf whitefly. If growers still need to grow light green leaf cultivars because of the public preference for them, I would recommend them to adjust their pest management methods so they prevent whitefly outbreaks. One such adjustment is to start sampling procedures early on susceptible cultivars and to take corrective measurements earlier in the season.

105

Poinsettias physical defense mechanisms affecting B. tabaci

Poinsettia cultivars with dark green foliage had significantly thicker leaves and it is suggested that this physical trait is contributing to resistance against the silverleaf whitefly. While trichome density may be a mechanism of defense in other systems, in poinsettia cultivars evaluated in this research no significant differences were observed in trichome density. Indirect measurements were made for chlorophyll content in the leaves of poinsettia cultivars and I found that cultivars with dark green foliage and thicker leaves had higher amounts of chlorophyll content than cultivars with light green foliage and thinner leaves. The role of chlorophyll content in resistance against whiteflies needs to be addressed in more depth.

Poinsettias chemical defense mechanisms affecting B. tabaci

Quality of plants to insects can be influenced by the relative amount of dietary nitrogen in the form of free amino acids and by the relative amount of defensive compounds potentially encountered by such insects. Concentrations of amino acids in poinsettia cultivars with light green leaves were ca. 30% higher than cultivars with dark green leaves indicating it may be driving variation in behavior and/or physiology of B. tabaci.

I would suggest studying the relative amount of amino acids over the growth of poinsettias to determine susceptible stages in the crop. In this study amino acids were

106 determined only at one point, which gives us some information about the suite of amino acids whiteflies may be encountering, but by no means should this be representative of the whole nutritional dynamics of poinsettia-whiteflies. The concentration of sugars could also be studied in poinsettias in relation to amino acids and the association with whiteflies, but it has been shown with mealybugs that the ratio of sugars: amino acids did not explain different levels of resistance in cassava varieties and poinsettia leaf extracts

(Tertuliano and Leru 1992).

Quality of poinsettia plants could have potentially influence whiteflies through the presence of soluble defensive compounds, such as phenolics. In this study, concentrations of soluble phenolics were higher on resistant than on susceptible cultivars, therefore, differences in phenolic compounds are suggested to be potentially influencing the behavior and physiology of B. tabaci. A suite of individual and unique phenolic compounds were found in higher levels in resistant than on susceptible cultivars, indicating these compounds are possibly involved in resistance to B. tabaci. More detailed analyses are needed to determine the role these phenolic compounds may be playing in resistance against the silverleaf whitefly.

Previous studies have associated the concentration of phenolics with whitefly populations, but specific experiments testing their biological activity on whiteflies have not been conducted. Many ecological outcomes may be proposed for individual phenolic compounds, including feeding deterrents, feeding stimulants, digestibility reducers, and/or toxins, but the most important aspect to consider is the overall interaction among insect and plant species, and the chemical context (Appel 1993, Duffey and Stout 1996).

107

I suggest testing the biological activity of individual and suite of individual phenolic compounds in combination with diets varying amino acids against B. tabaci with improved artificial diets (Davidson et al. 2000, Davidson et al. 2002). Studying the relative amount of amino acids to defensive compounds might elucidate better the quality of poinsettia cultivars to B. tabaci. This analysis could not be conducted with my data since analysis of amino acids were done from different plants from which phenolics were analyzed. Another interesting future research would be to evaluate the fluctuation of amino acids and phenolics over the growth of poinsettias to determine susceptible stages in the crop in relation to whitefly colonization. Preliminary studies have been conducted to determine the phenolic compounds in young and mature leaves of poinsettia over three time periods to study how phenolics fluctuate over time. Implications of this study will be further investigated in the future.

Poinsettia cultivar-mediated differences in behavioral preference of a parasitoid of

B. tabaci

Nymphs from the susceptible poinsettia cultivar were preferred than nymphs from the resistant plants, although any preference for the host plant rather than the prey could be confounded in this study. Phenolics compounds present in greater concentrations in resistant plants may be affecting indirectly the behavioral choices of Eretmocerus mundus (Dicke 2000), but it has been shown that parasitoids are also attracted to lighter green leaf foliage (Romeis and Zebitz 1997). Studies evaluating the phenolic content in

108 both nymphs of B. tabaci, from less and more susceptible cultivars, and in the parasitoids should be conducted to determine if phenolics are being sequestered, metabolized, and/or excreted. Also, to pursue the hypothesis that parasitoids prefer nymphs feeding from optimal hosts, life history traits should be measured on parasitoids.

109

APPENDIXES

110

Appendix A: Screening for whitefly resistance in poinsettias, summer 2006

111

POINSETTIA: Euphorbia pulcherrima Willd ex Klotzsch

SCREENING FOR WHITEFLY RESISTANCE IN POINSETTIAS, SUMMER

2006

Karla J. Medina-Ortega

Thorne Hall, Dept. of Entomology

OARDC/OSU

1680 Madison Ave.

Wooster, OH 44691

Phone: (330) 263-3935

Fax: (330) 263-3686

E-mail: [email protected]

Luis A. Cañas

Phone: (330) 263 3818

E-mail: [email protected]

Silverleaf whitefly (SLWF): Bemisia tabaci (Gennadius) biotype B

112

Nine commercially available poinsettia cultivars, provided as rooted cuttings by the company Paul Ecke Ranch, Encinitas, CA, were evaluated for whitefly preference and performance. Choice and no-choice tests were conducted in a growth chamber and in a greenhouse, respectively, at the Entomology Department, OARDC, Wooster, OH, with conditions at 24ºC, 45% RH and a 16:8 h (L:D), in the summer of 2006. Rooted cuttings were transplanted and ten days later a plant of each cultivar was arranged in a RCB design with five replications for both the choice and the no-choice tests. In the choice tests plants were arranged in a circle, distanced equally from each other, inside a 70 x 70 x 40 cm mesh cage that represented the block. In each cage 180 pairs of 3-day-old whiteflies were released, for a total of 900 pairs observed. The behavioral preferential response of whiteflies for cultivars was recorded as number of whiteflies settling/feeding on each plant after a period of 48h. In the no-choice tests, an average of 20 nymphs per plant was evaluated for nymphs‘ survival. Oviposition of F1females that emerged from these plants was observed for the first 24 h after emergence. Data were evaluated for normality and homogeneity of variances and were transformed as needed. Data from choice test was transformed using square root. Data were analyzed with ANOVA and if necessary treatment means were separated using Fisher‘s LSD at the 5% level.

More than 73% of male and female whiteflies responded in the choice tests to poinsettia cultivars. The number of adult whiteflies settled/feeding after 48 h was not significantly different among cultivars (F(8, 32) = 2.05; P = 0.0711). Whiteflies, although, showed preference for light green foliage cultivars having a higher number of whiteflies on ‗Early

113

Prestige Red‘ and less on ‗Freedom Red‘ (Table 1). Nymphs‘ survival was not significantly different among cultivars when confined to each plant in the no-choice tests

(Table 2). Oviposition within a day of emergence of F1 females, however, was significantly lower in ‗Prestige Red‘ and ‗Enduring Red‘ when compared to ‗Freedom

Red‘, ‗Freedom White‘, ‗Early Prestige Red‘, and ‗Peterstar White‘ (Table 2). There was not a clear connection that cultivars less preferred by adults in the choice test were also the less optimal hosts for the survival of nymphs. The cultivar ‗Prestige Red‘, although, consistently showed lower whitefly preference and oviposition, while the cultivar ‗Early

Prestige Red‘ consistently had higher whitefly preference and higher oviposition. From these tests, it appears that whitefly host preference rather than performance plays a more influential role in poinsettia cultivar resistance. This study is part of a project funded by the America Floral Endowment grant No. 20020754.

Table A. 1. Mean number of adults settling/feeding on each plant after 48 h Cultivars Males Females Total ‗Freedom Red‘ 5.8 10.8 16.6 ‗Freedom White‘ 10.0 13.0 23.0 ‗Prestige Red‘ 7.6 9.2 16.8 ‗Winter Rose Red‘ 7.8 7.2 15.0 ‗Early Prestige Red‘ 35.2 30.4 65.6 ‗Enduring Red‘ 12.8 14.2 27.0 ‗Enduring White‘ 14.6 17.6 32.2 ‗Peterstar Red‘ 21.8 19.6 41.4 ‗Peterstar White‘ 12.4 15.8 28.2

114

Table A. 2. Cultivars Mean nymph Mean no. of eggs/SLWF F1 survivorship (%) female 24h after emergence ‗Freedom Red‘ 89.4 a 10.5 a ‗Freedom White‘ 88.9 a 10.2 ab ‗Early Prestige Red‘ 90.8 a 10.1 ab ‗Peterstar White‘ 89.3 a 9.9 ab ‗Peterstar Red‘ 92.3 a 9.1 abc ‗Winter Rose Red‘ 94.4 a 7.4 abc ‗Enduring White‘ 74.0 a 6.5 bc ‗Prestige Red‘ 93.7 a 5.4 c ‗Enduring Red‘ 84.7 a 5.4 c Means on the same column followed by the same letter are not significantly different using Fisher‘s LSD (P = .05).

115

Part II. Materials Tested for Arthropod Management

Karla J. Medina-Ortega

Thorne Hall, Dept. of Entomology

OARDC/OSU

1680 Madison Ave.

Wooster, OH 44691

Phone: (330) 263-3935

Fax: (330) 263-3686

E-mail: [email protected]

POINSETTIA: Euphorbia pulcherrima Willd ex Klotzsch

SCREENING FOR WHITEFLY RESISTANCE IN POINSETTIAS, SUMMER

2006

Cultivars: 1) ‗Early Prestige Red‘, 2) ‗Enduring Red‘, 3) ‗Enduring White‘, 4) ‗Freedom Red‘, 5) ‗Freedom White‘, 6) ‗Peterstar Red‘, 7) ‗Peterstar White‘, 8) ‗Prestige Red‘, and 9) ‗Winter Rose Red‘ were provided by:

Paul Ecke Ranch

800 Ecke Ranch Road

Encinitas, CA 92024

Phone: (760) 753-1134

Fax: (760) 944-4002

E-mail: [email protected]

116

Appendix B: Screening for whitefly resistance in poinsettias, summer 2007

117

POINSETTIA: Euphorbia pulcherrima Willd ex Klotzsch

SCREENING FOR WHITEFLY RESISTANCE IN POINSETTIAS, SUMMER

2007

Karla J. Medina-Ortega

Thorne Hall, Dept. of Entomology

OARDC/OSU

1680 Madison Ave.

Wooster, OH 44691

Phone: (330) 263-3935

Fax: (330) 263-3686

E-mail: [email protected]

Luis A. Cañas

Phone: (330) 263 3818

E-mail: [email protected]

Silverleaf whitefly (SLWF): Bemisia tabaci (Gennadius) biotype B

118

Seven commercially available poinsettia cultivars, provided as rooted cuttings by the company Paul Ecke Ranch, Encinitas, CA, were evaluated for whitefly preference and performance. Choice and no-choice tests were conducted in a growth chamber and in a greenhouse, respectively, at the Entomology Department, OARDC, Wooster, OH, with conditions at 24ºC, 45% RH and a 16:8 h (L:D), in the summer of 2007. Rooted cuttings were transplanted and ten days later a plant of each cultivar was arranged in a RCB design with five replications for both the choice and the no-choice tests. In the choice tests plants were arranged in a circle, distanced equally from each other, inside a 70 x 70 x 40 cm mesh cage that represented the block. In each cage 180 pairs of 3-day-old whiteflies were released, for a total of 900 pairs observed. The behavioral preferential response of whiteflies for cultivars was recorded as number of whiteflies settling/feeding on each plant after a period of 48h. In the no-choice tests, an average of 20 nymphs per plant was evaluated for nymphal survival (a total of 100 nymphs per cultivar).

Oviposition of F1females that emerged from these plants was observed for the first 24 h and seven days after emergence. Data were evaluated for normality and homogeneity of variances and were transformed as needed. Data were analyzed with ANOVA and treatment means were separated using Fisher‘s LSD at the 5% level.

More than 80% of whiteflies responded in the choice tests to poinsettia cultivars. The number of adult whiteflies settled/feeding on ‗Freedom Red‘ was significantly lower compared to all other cultivars, except from ‗Early Freedom Red‘ (Table 1). ‗Freedom

Red‘ is considered resistant based on whitefly host preference (Table 1). Nymphs‘

119 survival was not significantly different among cultivars when confined to each plant in the no-choice tests (Table 2). Oviposition within a day of emergence of F1 females was relatively similar among the cultivars (Table 2), but at seven days it differed significantly

(Table 2). ‗Prestige Red‘ had significantly fewer eggs per female compared to ‗Early

Prestige Red‘, ‗Monet Twilight‘, Snowcap White‘, and ‗Zapoteca‘ (Table 2). In summary, the cultivar ‗Prestige Red‘, ‗Early Freedom Red‘, and ‗Freedom‘ were less preferred and had fewer eggs per female over time exhibiting some resistance to whiteflies. This study is part of a project funded by the America Floral Endowment grant

No. 20020754.

Table B. 1. Mean number of adults settling/feeding on each plant after 48 h Cultivars Males Females Total ‗Freedom Red‘ 7.6 11.4 19.0 b ‗Early Freedom Red‘ 15.0 18.6 33.6 ab ‗Prestige Red‘ 17.6 24.2 41.8 a ‗Early Prestige Red‘ 25.2 26.2 51.4 a ‗Zapoteca‘ wild type 20.4 26.8 47.2 a ‗Snowcap White‘ 24.8 27.2 52.0 a ‗Monet Twilight‘ 29.2 28 57.2 a Means within the same column followed by the same letter are not significantly different using Fisher‘s LSD (P = .05).

120

Table B. 2. Mean nymph Mean no. of eggs/SLWF F1 female after survivorship (%) emergence: Cultivars 24 h 7 d ‗Freedom Red‘ 86.5 a 3.7 a 2.5 bcd ‗Early Freedom Red‘ 96.8 a 3.6 a 2.5 cd ‗Prestige Red‘ 90.7 a 2.6 a 1.8 d ‗Early Prestige Red‘ 89.0 a 3.2 a 2.9 bc ‗Zapoteca‘ wild type 86.4 a 1.6 a 5.5 a ‗Snowcap White‘ 91.4 a 2.7 a 3.8 ab ‗Monet Twilight‘ 92.8 a 3.0 a 3.0 bc Means on the same column followed by the same letter are not significantly different using a Fisher‘s LSD (P = .05).

121

Part II. Materials Tested for Arthropod Management

Karla J. Medina-Ortega

Thorne Hall, Dept. of Entomology

OARDC/OSU

1680 Madison Ave.

Wooster, OH 44691

Phone: (330) 263-3935

Fax: (330) 263-3686

E-mail: [email protected]

POINSETTIA: Euphorbia pulcherrima Willd ex Klotzsch

SCREENING FOR WHITEFLY RESISTANCE IN POINSETTIAS, SUMMER

2006

Cultivars: 1) ‗Early Freedom Red‘, 2) ‗Early Prestige Red‘, 3) ‗Freedom Red‘, 4) ‗Monet Twilight‘, 5) ‗Prestige Red‘, and 6) ‗Snowcap White‘ were provided by:

Paul Ecke Ranch

800 Ecke Ranch Road

Encinitas, CA 92024

Phone: (760) 753-1134

Fax: (760) 944-4002

E-mail: [email protected]

122

Cultivar ‗Zapoteca‘ wild type was provided by: John Sanderson, Associate Professor,

Cornell University, [email protected] , 2134 Comstock Hall, Ithaca, NY 14853.

123

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