ABSTRACT BECKER, LINDSEY ELIZABETH. Increased Flower

ABSTRACT

BECKER, LINDSEY ELIZABETH. Increased Flower Production and Plant Weight of Calibrachoa x hybrida by the Soil Fungus Mortierella elongata. (Under the direction of Dr. Marc A. Cubeta).

Calibrachoa (Calibrachoa x hybrida), is a flowering ornamental plant, commonly known as a mini-petunia or million bells and is rapidly gaining popularity as a flowering bedding and hanging plant. It has a range of desirable horticultural characteristics, including attractive flowers, range of flower colors, drought tolerance, and plant habit. The saprobic fungus

Mortierella elongata isolated as an endophyte from Populus deltoides (eastern cottonwood) roots has been shown to increase seedling growth of corn, red oak and tomato in pasteurized sand and untreated field soil. In this context, we conducted two greenhouse experiments in

2017 to evaluate the effect of potting media amendments of millet colonized with M. elongata at 1 and 2% (v/v) on plant growth of Calibrachoa cv. ‘Kabloom Deep Blue’ rooted cuttings. Plants were assessed weekly for flower production and at 86 days post inoculation for above/below ground dry weight. M. elongata isolate 624- significantly increased the number of flowers produced for inoculum volumes 1 and 2% over a period of 3- 5 weeks for both experiments and increased above and belowground plant dry weight during experiment

1. Our findings suggest that M. elongata 624- consistently promotes flower production of

Calibrachoa.

Calibrachoa, valued for its aesthetic qualities, requires constant surveillance for disease symptoms. Susceptibility of Calibrachoa to the soilborne fungal pathogen

Thielaviopsis basicola (causal agent of black root rot) is informally well-known but has not been the subject of comprehensive investigation. When Calibrachoa are infected with T. basicola, they exhibit stunting and chlorosis, which makes them unsuitable for market. In two greenhouse experiments conducted in 2017, we screened seven commercially available

Calibrachoa cultivars for resistance to a virulent isolate of T. basicola. In this study, we developed an experimental protocol and root rating scale for evaluating severity of black root rot disease on Calibrachoa plants. Calibrachoa cultivars exhibited varying susceptibility to the pathogen. Two cultivars, ‘Minifamous Compact Hot Pink’ and ‘Deep Blue Kabloom’ had significantly higher dry root weight and lower disease severity ratings compared to ‘Callie

Scarlet’. These results suggest that ‘Minifamous Compact Hot Pink’ and ‘Deep Blue

Kabloom’ may exhibit partial resistance to T. basicola. Our results highlight the need for continued evaluation of Calibrachoa cultivars to T. basicola, which may aid breeders in identifying sources of resistance to black root rot disease in Calibrachoa.

by Lindsey Elizabeth Becker

A thesis submitted to the Graduate Faculty of North Carolina State University in partial fulfillment of the requirements for the degree of Master of Science

Plant Pathology

Raleigh, North Carolina

2017

APPROVED BY:

______Dr. Shuijin Hu Dr. Howard D. Shew

______Dr. Marc A. Cubeta Committee Chair

DEDICATION

This manuscript is dedicated to my parents, Don and Polly Becker, my grandparents,

Marguerite and Fitzhugh Turner, and to my partner in love, life, and scientific pursuit of excellence, Camilo Humberto Parada Rojas.

BIOGRAPHY

Lindsey Elizabeth Becker was born on May 21st, 1988 in Durham, North Carolina.

She received her bachelor of art (B.A.) in biological sciences at The College of Wooster in

Wooster, Ohio. While pursuing her bachelors, Ms. Becker studied the spatial/temporal destruction of Ash trees (Fraxinus spp.) by the emerald ash borer in southeastern Michigan for her senior thesis. Post-graduation, Ms. Becker worked in the labs of Dr. Jim Clark and

Dr. Rytas Vilgalys at Duke University for four years, acting as lead technician on a project investigating the fungal endophytic community of tree seedlings commonly found in forests in the Southeast US. She was mentored by Dr. Soledad Benitez Ponce during this time, who introduced Ms. Becker to the field of plant pathology. This experience led Ms. Becker to pursue a Master’s of Science at NC State University in plant pathology under the guidance of

Dr. Marc Cubeta.

Ms. Becker’s research focuses on understanding the effects of the soil fungi

Mortierella elongata on the ornamental plant Calibrachoa, and also detailing the susceptibility of Calibrachoa cultivars to the common soilborne fungal pathogen

Thielaviopsis basicola, causual agent of black root rot. Ms. Becker has presented the results from this thesis in 2017 at the annual meeting of the Mycological Society of America in

Athens, GA. She is currently serving as president of the Plant Pathology Graduate Student

Association and has served as the representative of the PPGSA for the University Graduate

Student Association.

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ACKNOWLEDGMENTS

I would first like to thank Dr. Brigid Hogan, for offering me a job in her lab when I was still in high school. Thank you for being so supportive of me every step of the way, even when my interests shifted to fungi and ecology rather than epithelial stem cells. Your encouragement strengthened my resolve to pursue what I loved.

I would like to thank my advisor, Dr. Marc Cubeta, for encouraging me to work on a project with many unknowns. I highly value the wisdom you impart during casual chats in the lab and long car rides. Your unconventional thinking challenges me to approach everything with an open mind. I would also like to thank you for giving me the opportunity to work on this project and to be in your lab. Many thanks to my committee members, Drs.

Shuijin Hu and Dave Shew, for their advice and input throughout the course of this project.

I would also like to thank Dr. Soledad Benitez Ponce for encouraging me to combine my love of plants and fungi via the world of plant pathology. Dr. Rytas Vilgalys practically adopted me into his mycology lab when I was working on a fungal endophyte project, and kindly gave me opportunities to attend MASMC and connect with others in mycology. The

Vilgalys lab ethos of community, support, and love was always inviting and inclusive, and I continue to appreciate that to this day.

To my fellow graduate students in the plant pathology program at NCSU, thank you for being an inspiring and wonderful group of people to work with.

I would like to also thank my parents for their continuous love and support. They have always emphasized the importance of self-care in relation to enjoying work. I thank my

mother for long walks and for always being available to listen. I would also like to thank my father for all of the long bike rides that never failed to make me feel better.

Last, but certainly not least, a huge thank you to Camilo Parada for all of your love, encouragement, support, numerous ice cream cones at the library, and your thorough reviews of this thesis.

TABLE OF CONTENTS

LIST OF TABLES…………………………………………………………………………viii

LIST OF FIGURES………………………………………………………………………....ix

CHAPTER I. Literature Review…………………………………………………………....1

Calibrachoa…………………………………………………………………………..1

Calibrachoa and micronutrients…………………………………………………... 3

Common soilborne fungal pathogens of Calibrachoa………………...…….…...... 4

Thielaviopsis basicola……………………………...... ……..4

Black root rot symptoms on Calibrachoa…...... …5

Management of black root rot disease...... 6

Cultural management…...... 6

Chemical management…...... 6

Biological management…...... 7

Host resistance…...... …7

Fungal endophytes…...... 8

Plant growth promotion by fungal endophytes…...... 10

Mechanisms of plant growth promotion by fungal endophytes…...... 11

Mortierella elongata…...... 11

Ecology of M. elongata…...... 12

Research hypotheses and objectives…...... 13

LITERATURE CITED…...... 14

CHAPTER II. Increased flower production and plant weight of Calibrachoa x hybrida by the soil fungus Mortierella elongata…………………………………...... …...... 23

ABSTRACT………………………………………………………………...... ……23

INTRODUCTION……………………………………………………………...... 25

MATERIALS AND METHODS...... 28

RESULTS………………………………………………………………………...... 31

DISCUSSION…………………………………………………………………….....36

ACKNOWLEDGMENTS……………………………………………………….…41

LITERATURE CITED………………………………………………………….…42

CHAPTER III. Susceptibility of Calibrachoa cultivars to Thielaviopsis basicola, causal agent of black root rot disease………………………………………………………….….72

ABSTRACT…………………………………………………………………...... …72

INTRODUCTION……………………………………………………………...... 74

MATERIALS AND METHODS...... 77

RESULTS……………………………………………………………………...... ….80

DISCUSSION…………………………………………………………………….....82

ACKNOWLEDGMENTS………………………………………………………….85

LITERATURE CITED………………………………………………………….…86

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LIST OF TABLES

Chapter I: Literature Review

Table 1.1 Recommended fungicides for application if symptoms of black root rot are widespread in a greenhouse...... 21

Table 1.2 Recommended fungicides for application if symptoms of black root rot are sporadic in a greenhouse...... 22

Chapter II: Increased flower production and plant weight of Calibrachoa x hybrida by the soil fungus Mortierella elongata.

Table 2.1 ANOVA table for simple effects of Isolate x Volume interaction for leaf/stem, root, and total dry weight in experiment 1…...... …67

Table 2.2 ANOVA table for simple effects of Isolate x Volume interaction for flower count of experiment 1, including weeks 5, 6, and 7 post inoculation…...... …68

Table 2.3 ANOVA table for simple effects of Isolate x Volume interaction for leaf/stem, root, and total dry weight in experiment 2……...... 69

Table 2.4 ANOVA table for simple effects of Isolate x Volume interaction for flower count of experiment 2, including weeks 4, 6, and 7 post inoculation…...... …70

Table 2.5 Average minimum and maximum greenhouse temperatures (C) by month (every 30 days) for experiments 1 and 2……...... 71

Chapter III: Susceptibility of Calibrachoa cultivars to Thielaviopsis basicola, causal agent of black root rot disease.

Table 3.1 ANOVA table for main effect of black root rot root disease rating and dry weight (g) for combined experiments (1 and 2)……...... 90

Table 3.2 Mean black root rot disease severity and root dry weight of Calibrachoa cultivars screened for resistance to Thielaviopsis basicola for combined experiments……...... 91

Table 3.3 Dry root weight for inoculated (I) and non-inoculated control (C) for Calibrachoa cultivars screened for resistance to Thielaviopsis basicola for experiment 1 and 2……...... 92

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LIST OF FIGURES

Chapter I: Literature Review

Fig 1.1 Drawing of Nierembergia intermedia Graham, a synonym for Salpigloss linearis which was renamed as Calibrachoa linearis in 1990 (Wijsman) sourced from The British Flower Garden (Sweet, 1835)………...... 20

Chapter II: Increase of flower production and plant weight of Calibrachoa x hybrida by the soil fungus Mortierella elongata.

Figure 2.1 Dry leaf/stem weight of Calibrachoa cultivar ‘Kabloom Deep Blue’ grown in Fafard potting media amended with either 624+, 624-, 93+, or 93- isolates of M. elongata at 1% v/v harvested at 12 wk post inoculation in experiment 1. Data represents contrast of each isolate against the 95th percentile range of non-inoculated 1% v/v millet control using student’s t-statistic. The 95th percentile upper and lower limits for control are represented by the horizontal blue line, while the means of the isolates are represented by length of vertical lines...... 47

Figure 2.2 Dry root weight of Calibrachoa cultivar ‘Kabloom Deep Blue’ grown in Fafard potting media amended with either 624+, 624-, 93+, or 93- isolates of M. elongata at 1% v/v harvested at 12 wk post inoculation in experiment 1. Data represents contrast of each isolate against the 95th percentile range of non-inoculated 1% v/v millet control using student’s t- statistic. The 95th percentile upper and lower limits for control are represented by the horizontal blue line, while the means of the isolates are represented by length of vertical lines…...... 48

Figure 2.3 Dry total weight of Calibrachoa cultivar ‘Kabloom Deep Blue’ grown in Fafard potting media amended with either 624+, 624-, 93+, or 93- isolates of M. elongata at 1% v/v harvested at 12 wk post inoculation in experiment 1. Data represents contrast of each isolate against the 95th percentile range of non-inoculated 1% v/v millet control using student’s t- statistic. The 95th percentile upper and lower limits for control are represented by the horizontal blue line, while the means of the isolates are represented by length of vertical lines…...... 49

Figure 2.4 Dry leaf/stem weight of Calibrachoa cultivar ‘Kabloom Deep Blue’ grown in Fafard potting media amended with either 624+, 624-, 93+, or 93- isolates of M. elongata at 2% v/v harvested at 12 wk post inoculation in experiment 1. Data represents contrast of each isolate against the 95th percentile range of non-inoculated 2% v/v millet control using student’s t-statistic. The 95th percentile upper and lower limits for control are represented by the horizontal blue line, while the means of the isolates are represented by length of vertical lines……...... 50

Figure 2.5 Dry root weight of Calibrachoa cultivar ‘Kabloom Deep Blue’ grown in Fafard potting media amended with either 624+, 624-, 93+, or 93- isolates of M. elongata at 2% v/v harvested at 12 wk post inoculation in experiment 1. Data represents contrast of each isolate against the 95th percentile range of non-inoculated 2% v/v millet control using student’s t- statistic. The 95th percentile upper and lower limits for control are represented by the horizontal blue line, while the means of the isolates are represented by length of vertical lines……...... 51

Figure 2.6 Dry total weight of Calibrachoa cultivar ‘Kabloom Deep Blue’ grown in Fafard potting media amended with either 624+, 624-, 93+, or 93- isolates of M. elongata at 2% v/v harvested at 12 wk post inoculation in experiment 1. Data represents contrast of each isolate against the 95th percentile range of non-inoculated 2% v/v millet control using student’s t- statistic. The 95th percentile upper and lower limits for control are represented by the horizontal blue line, while the means of the isolates are represented by length of vertical lines……...... 52

Figure 2.7 Means of flower production of Calibrachoa cv. ‘Kabloom Deep Blue’ by M. elongata isolates 624+, 624-, 93+, 93- and non-inoculated millet controls (inoculum volumes 0, 1, and 2%) assessed weekly 2 to 11wk post inoculation in experiment 1………………...53

Figure 2.8 Means of flower production of Calibrachoa cv. ‘Kabloom Deep Blue’ by isolates of M. elongata 93+, 93-, 624+, and 624- and non-inoculated millet control at 1% inoculum volume for 5, 6, and 7 wk post inoculation for experiment 1………………...... 54

Figure 2.9 Means of flower production of Calibrachoa cv. ‘Kabloom Deep Blue’ by isolates of M. elongata 93+, 93-, 624+, and 624- and non-inoculated millet control at 2% inoculum volume for 5, 6, and 7 wk post inoculation for experiment 1……………...... 55

Figure 2.10 Dry leaf/stem weight of Calibrachoa cultivar ‘Kabloom Deep Blue’ grown in Fafard potting media amended with either 624+, 624-, 93+, or 93- isolates of M. elongata at 1% v/v harvested at 12 wk post inoculation in experiment 2. Data represents contrast of each isolate against the 95th percentile range of non-inoculated 1% v/v millet control using student’s t-statistic. The 95th percentile upper and lower limits for control are represented by the horizontal blue line, while the means of the isolates are represented by length of vertical lines…………...... 56

Figure 2.11 Dry root weight of Calibrachoa cultivar ‘Kabloom Deep Blue’ grown in Fafard potting media amended with either 624+, 624-, 93+, or 93- isolates of M. elongata at 1% v/v harvested at 12 wk post inoculation in experiment 2. Data represents contrast of each isolate against the 95th percentile range of non-inoculated 1% v/v millet control using student’s t- statistic. The 95th percentile upper and lower limits for control are represented by the horizontal blue line, while the means of the isolates are represented by length of vertical lines…………...... 57

Figure 2.12 Dry total weight of Calibrachoa cultivar ‘Kabloom Deep Blue’ grown in Fafard potting media amended with either 624+, 624-, 93+, or 93- isolates of M. elongata at 1% v/v harvested at 12 wk post inoculation in experiment 2. Data represents contrast of each isolate against the 95th percentile range of non-inoculated 1% v/v millet control using student’s t- statistic. The 95th percentile upper and lower limits for control are represented by the horizontal blue line, while the means of the isolates are represented by length of vertical lines…………...... 58

Figure 2.13 Dry leaf/stem weight of Calibrachoa cultivar ‘Kabloom Deep Blue’ grown in Fafard potting media amended with either 624+, 624-, 93+, or 93- isolates of M. elongata at 2% v/v harvested at 12 wk post inoculation in experiment 2. Data represents contrast of each isolate against the 95th percentile range of non-inoculated 2% v/v millet control using student’s t-statistic. The 95th percentile upper and lower limits for control are represented by the horizontal blue line, while the means of the isolates are represented by length of vertical lines………………...... 59

Figure 2.14 Dry root weight of Calibrachoa cultivar ‘Kabloom Deep Blue’ grown in Fafard potting media amended with either 624+, 624-, 93+, or 93- isolates of M. elongata at 2% v/v harvested at 12 wk post inoculation in experiment 2. Data represents contrast of each isolate against the 95th percentile range of non-inoculated 2% v/v millet control using student’s t- statistic. The 95th percentile upper and lower limits for control are represented by the horizontal blue line, while the means of the isolates are represented by length of vertical lines………………...... 60

Figure 2.15 Dry total weight of Calibrachoa cultivar ‘Kabloom Deep Blue’ grown in Fafard potting media amended with either 624+, 624-, 93+, or 93- isolates of M. elongata at 2% v/v harvested at 12 wk post inoculation in experiment 2. Data represents contrast of each isolate against the 95th percentile range of non-inoculated 2% v/v millet control using student’s t- statistic. The 95th percentile upper and lower limits for control are represented by the horizontal blue line, while the means of the isolates are represented by length of vertical lines……………...... 61

Figure 2.16 Means of flower production of Calibrachoa cv. ‘Kabloom Deep Blue’ by M. elongata isolates 624+, 624-, 93+, 93- and non-inoculated millet controls (inoculum volumes 0, 1, and 2%) assessed weekly 2 to 11wk post inoculation in experiment 2……………...... 62

Figure 2.17 Means of flower production of Calibrachoa cv. ‘Kabloom Deep Blue’ by isolates of M. elongata 93+, 93-, 624+, and 624- and non-inoculated millet control at 1% inoculum volume for 4, 6, and 7 wk post inoculation for experiment 2…………...... ……...63

Figure 2.18 Means of flower production of Calibrachoa cv. ‘Kabloom Deep Blue’ by isolates of M. elongata 93+, 93-, 624+, and 624- and non-inoculated millet control at 2% inoculum volume for 4, 6, and 7 wk post inoculation for experiment 2………………...... 64

Figure 2.19 Box and whisker plot of greenhouse minimum temperatures (C) between experiment 1 and experiment 2 by month (defined as a 30 day period of the experiment)….65

Figure 2.20 Box and whisker plot of greenhouse maximum temperatures (C) between experiment 1 and experiment 2 by month (defined as a 30 day period of the experiment)….66

Chapter III: Susceptibility of Calibrachoa cultivars to Thielaviopsis basicola, causal agent of black root rot disease.

Figure 3.1 Disease Rating Scale for black root rot of Calibrachoa roots. a) 0= healthy, no disease symptoms; b) 1= 1-10%; c) 2= 11-20%; d) 3= 21-50%; e) 4= 51-80%; f) 5= 81%- 100% disease severity……………………………………………………………………………………….89

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CHAPTER I

Literature Review

Calibrachoa. Calibrachoa (Calibrachoa x hybrida) is rapidly gaining popularity as a flowering bedding and hanging plant. Calibrachoa, valued for its aesthetic qualities, requires constant surveillance for disease symptoms. Common nursery environmental variables such as high humidity, temperature and soil moisture are conducive for pathogens that reside in potting media. Susceptibility of Calibrachoa to common soilborne fungal pathogens is informally well-known but has not been reported in depth for Thielaviopsis basicola (causal agent of black root rot). As Calibrachoa is a highly valued ornamental plant, numerous flowers are a highly desirable trait. Recently, fungal endophytes have been implicated in shortening time to flowering and promoting proliferation of flowers (Das et al., 2012,

Ghanem et al., 2014).

The flowering ornamental plant Calibrachoa Llave & Lex. is commonly known as a mini-petunia or million bells. Calibrachoa is commercialized due to its desirable horticultural characteristics, including attractive flowers, range of flower colors, drought tolerance, and plant habit. Calibrachoa belongs to the family Solanaceae and is a close relative of Petunia and likely originated in southern South America (Olmstead et al., 2008). The family

Solanaceae includes plants of importance, both for horticulture and floriculture, and includes vegetables such as eggplant, pepper, potato, and tomato, as well as, tobacco and flowering petunia. Wijsman and De Jong (1985) formerly separated the genus Petunia into Petunia and

Calibrachoa based on chromosome number (x=18 and 14, respectively (2N)), morphology of leaf shape, bud aestivation, and seed coat morphology. In 1990, 15 species of Petunia were

transferred to the genus Calibrachoa (Wijsman, 1990) and 10 additional species were added by Stehmann and Semir (1997). A dichotomous key of Calibrachoa based on leaf anatomy and cellular structure was developed by Reis et al. (2002). Separation of Petunia and

Calibrachoa was confirmed using Restriction Fragment Length Polymorphism (RFLP) genetic markers from chloroplast DNA. The analysis revealed separation of Petunia and

Calibrachoa with 100% bootstrap support (Ando et al., 2005).

The genus Calibrachoa Llave and Lex. is an annual or perennial shrub-like groundcover plant with woody stems, and numerous small trumpet-shaped flowers. In 1832, seeds of the plant were procured by British traders and grown in the UK, where it was described by D. Don “As the plant is found… to be readily increased by cuttings, we hope soon to see it a common ornament of the flower border, to which its graceful habit, and successive profusion of blossoms of the deepest purple, shaded partly with brown, and of a rich velvety hue, cannot fail to render it a most welcome addition” (Sweet, 1835). The etymology of the name Calibrachoa comes from a Brazilian botany professor, A. de la Cal e

Bracho (Wijsman, 1990). Calibrachoa x hybrida are bred from either Petunia and

Calibrachoa spp. or result as a hybrid of two Calibrachoa spp. The first filed patent for

Calibrachoa x hybrida was a new cultivar of Petunia ‘Suntory SP-R’ a hybrid between

Petunia and a wildtype Petunia (a species of Calibrachoa) found in Gramado, Rio Grande

Do Sul, Brazil (Kenichi Suzuki et al., 1995). Later in a patent for a Calibrachoa cultivar, the same grower references the ‘Suntory SP-R’ cultivar as a species of Calibrachoa (Kanaya,

2005).

Species of Calibrachoa found in southern Brazil and Uruguay exhibit a wide variety of flower color, including yellow, orange, red, and pink, with the majority of species exhibiting white and purple flower colors (Kanaya et al., 2010). This natural range of flower color has aided breeding of Calibrachoa hybrida over the last 20 years. Currently, there are

408 US patents for cultivars of Calibrachoa (USPTO, 2017). The production value of

Calibrachoa in the US was recently estimated to be approximately $45 million US dollars

(USDA, 2014). Calibrachoa are primarily produced as vegetative cuttings in the US with production located in South America and Africa. Commercial seed production is limited to a single breeding line ‘Kabloom’ produced by Ball Seed Company.

Calibrachoa and micronutrients. Most micronutrients are insoluble at high pH in potting media used to grow Calibrachoa. Calibrachoa is an iron-inefficient crop and a potting media pH above 6.2 reduces the ability of the plant to solubilize iron for growth and development

(Argo & Fisher, 2002). Iron deficiency can be detrimental for plants, as it can be a limiting factor for plant growth at high pH in potting media (Fisher et al., 2003). To ameliorate this issue, potting media is typically acidified with ammonium-based fertilizer, acidified irrigation water and supplemental iron chelate drenches (Fe-EDDHA and Fe-DTPA) (Argo

& Fisher, 2002). Amending plants with iron is known to improve flowering. Fisher et al.

(2003) discovered a significant increase in the number of flower buds compared to the control when Fe-EDDHA was applied to Calibrachoa as a foliar spray at 20, 40 and 80 mg L-

1 (ppm) Fe.

Symptomatically, iron deficiency initially appears as a yellowing of young

Calibrachoa leaves that turn light green. Over time chlorosis occurs in older leaves of the

plant. Eventually the youngest leaves turn brown and necrotic (Dole et al., 2002). Dickson et al. (2016) reported that Calibrachoa cultivars vary in iron uptake efficiency at high pH. They measured shoot dry weight and leaf chlorophyll index of 24 cultivars grown at both low pH

(5.4) and high pH (7.1) potting media. Their findings indicate that pH plays an important role in iron uptake of the majority of Calibrachoa cultivars.

Common soilborne fungal pathogens of Calibrachoa.

The fungal-like oomycete Phytophthora can be introduced into greenhouse production via untreated field soil. Plants that exhibit symptoms of Phytophthora root rot such as above ground wilting, stunted growth and crown lesions cannot be sold to consumers. Enzenbacher et al. (2015) found six Calibrachoa cultivars (‘Cabaret Yellow’, ‘Callie Gold with Red Eye’,

‘Can-Can Apricot’, ‘Celebration Purple Star’, ‘Million Bells Cherry Pink’, and ‘Superbells

White’) were susceptible to Phytophthora capsici Leonian and P. tropicalis Aragaki & J.Y.

Uchida. Becktell et al. (2006) reported varying susceptibility of 10 cultivars of Calibrachoa to two isolates of Phytophthora infestans (Montagne) de Bary obtained from potato and tomato. Nine of 10 cultivars were susceptible to at least one of the isolates. Omer et al.

(2011) reported susceptibility of one Calibrachoa cultivar, ‘Colorburst Violet’, to

Phytophthora cinnamoni Rands, P. citrophthora (R.E. Sm. & E.H. Sm.) Leonian and P. nicotianae Breda de Haan. Calibrachoa cultivar ‘Million Bells Trailing Blue’ has also been reported as being susceptible to P. drechsleri Tucker (Slinski et al., 2003).

Thielaviopsis basicola. Thielaviopsis basicola Berk. and Broome (synanomorph = Chalara elegans Nag Raj and Kendrick) is a soilborne plant pathogenic fungus and causal agent of black root rot disease. T. basicola is classified within the Phylum Ascomycota, Class

Sordariomycetes, sub-class Hypocreomycetidae, Order Microascales, and Family

Ceratocystidaceae (IndexFungorum, 2017). Aleuriospores (chlamydospores) are overwintering propagules, which can survive for at least 9 months in soil with low moisture due to their melanized, thick cell walls (Papavizas, 1970). Thin-walled endoconidia serve as the source of secondary inoculum and are produced after germination of aleuriospores and root infection (Meyer & Shew, 1994). The host range of Thielaviopsis basicola encompasses

137 different genera across 15 plant families (Yarwood, 1981). Several solanaceous genera of plants are susceptible, including Calibrachoa, Nicotiana, Petunia, Physalis, and Solanum.

T. basicola is found worldwide in temperate areas (CABI, 2015). Other ornamental flowering plants affected by T. basicola include begonia, impatiens, pansy, phlox, poinsettia, and snapdragon (Hausbeck & Dudek, 2008).

Black root rot symptoms on Calibrachoa. T. basicola may be introduced into a greenhouse environment via untreated potting media or peat moss, diseased rooted cuttings, and re-used plug trays and flats. Moist conditions (Moisture Holding Capacity (MHC) above 30%) and temperatures > 10 C accelerate germination of T. basicola and are ideal for colonization and reproduction of T. basicola in a greenhouse environment (Enzenbacher et al., 2015,

Papavizas, 1970). To date, there is no peer-reviewed published disease report of T. basicola infection of Calibrachoa, but the susceptibility of Calibrachoa is well known among extension agents, breeders, and greenhouse growers (Daughtrey, 2006, Schoellhorn, 2013,

Thomas & Williams-Woodward, 2016, Dudek, 2016, Winners, 2017). Disease management of root rot of ornamental plants such as Calibrachoa is intensive due to the plant’s purely

aesthetic value for marketing to consumers. Plants with stunting symptoms will not be sold by growers.

Above ground symptoms of black root rot are challenging to distinguish from common nutrient deficiencies, such as nitrogen. Diseased plants are often stunted and exhibit wilting in older leaves. Chlorosis is also a common symptom and yellowing of mature leaves with green veins can occur (Hausbeck & Harlan, 2017). Below ground symptoms include dark discoloration of roots, associated with the formation of abundant aleuriospores and pseudoparenchymatous stromatic tissues on the root surface. In some cases the lesions are brown when spores are less abundant or located within the root cortex (Daughtrey, 1995).

Management of black root rot disease.

Cultural management. Producers should acquire only non-infected plants and avoid propagating cuttings from plants that exhibit symptoms of T. basicola infection. The use of new or sterilized pots and flats can reduce the probability of introducing T. basicola to the greenhouse. It is also recommended that growers acidify irrigation water to pH 5.2 to decrease disease, as a pH of 5.6 or higher will enhance black root rot severity (Harrison &

Shew, 2001). Ammonium (NH4) or nitrate (NO3) based nitrogen fertilizer is also recommended to reduce soil pH and populations of T. basicola (Harrison & Shew, 2001).

Fungus gnats can act as a vector for T. basicola, spreading the fungus to healthy plants within the same greenhouse (Harris, 1995).

Chemical Management. The number of effective fungicides registered for black root rot is limited (Chase, 2014, Chase, 2016). When growing Calibrachoa in a greenhouse with a known history of black root rot, or if symptoms of black root rot are widespread, the

following fungicides are recommended for ornamentals at the high application rate: Cleary’s

336 WP/ OHP 6672 FL, Emblem, Medallion WG, and Terraguard (Table 1.1) (Hausbeck &

Harlan, 2017, Dudek, 2016, Wollaeger, 2016, Daughtrey, 2006). When growing Calibrachoa in a greenhouse with very few plants exhibiting symptoms and no history of black root rot disease, the following fungicides are recommended for ornamentals: Affirm WDG, Orkestra,

Empress Intrinsic, and Trinity (Table 1.2) (Hausbeck & Harlan, 2017, Daughtrey, 2006).

Biological management. Paenibacillus alvei strain K-165 applied to cotton seed (Schoina et al., 2011), significantly reduced black root rot disease symptoms and increased plant height and fresh weight of seedlings compared to non-treated control seedlings. Co-culturing of P. alvei K-165 and T. basicola on V8 juice medium significantly reduced mycelial growth of the fungus. Reddy and Patrick (1992) examined the potential utility of Pseudomonas fluorescens strain RD1 as a biological control agent against T. basicola. These authors found that the bacterium reduced mycelial growth of T. basicola on potato dextrose agar (PDA).

The authors also reported significantly reduced black root rot disease severity of cotton seedlings dipped in a RD1 bacterial suspension and grown in T. basicola infested soil compared to non-treated seedlings.

Host resistance. The company Proven Winners claims increased tolerance to T. basicola in the breeding line Superbells. However, this claim comes with guidelines to utilize cultural control and recommended fungicides. Previous research by Margery Daughtrey at Cornell suggests Calibrachoa cultivars can vary in susceptibility to T. basicola. Daughtery reported that ‘Cabaret Red’, ‘Cabaret Cherry Rose’, ‘Cabaret Apricot’, ‘Cabaret White’, ‘Cabaret

Scarlet’, ‘Cabaret Purple’, ‘Superbells White’, ‘Superbells Pink Kiss’, ‘Superbells Trailing

Rose’, ‘Superbells Blue’, ‘Million Bells Cherry Pink’ and ‘Million Bells Crackling Fire’ were less susceptible to T. basicola than ‘Million Bells Terracotta’ (Daughtrey, 2006).

Fungal endophytes. Fungal endophytes are characterized as asymptomatic fungal infections, where the fungus resides subcuticularly or within plant flowers, leaves, seeds, and stems, and in roots without forming mycorrhizal structures (Rodriguez et al., 2009, Carroll, 1988) or causing apparent harm (Petrini, 1991). Fungal endophytes often colonize host plant roots inter and intra-cellularly (Schulz & Boyle, 2006). Endophytes can be obligate heterotrophs and rely on host plants for nutrition (Arnold & Lewis, 2005). Some fungal endophytes are host specific, such as Rhabdocline parkeri Sherwood, J.K. Stone & G.C. Carroll on Douglas

Fir (Pseudotsuga menziesii (Mirb) Franco), while others such as Hypoxylon serpens (Pers.) J

Kickx f. is a generalist (Carroll, 1988). An endophytic interaction represents a continuum of

‘phenotypes’ between the fungus and a plant host, (e.g. saprobic to mutualistic, depending on developmental stage, genetics, nutrition, and environmental factors (Schulz & Boyle, 2006).

Fungal endophytes have historically been divided into two groups, Clavicipitaceous

(C) endophytes sampled from graminaceous plants and Nonclavicipitaceous (NC) endophytes sampled from angiosperms, gymnosperms and ferns (Rodriguez et al., 2009). NC endophytes are classified into three groups based on transmission of the endophyte from one host generation to the next, method of host colonization, ecological function, and in planta level of biodiversity (Rodriguez et al., 2009). Class 2 NC endophytes are predominantly fungi in the phyla Ascomycota and Basidiomycota, and increase host root and shoot weight, alter flowering time, produce beneficial secondary metabolites for the plant host (Das et al.,

2012), and protect the plant against fungal pathogens (Narisawa et al., 1998). There is less

species diversity among NC class 2 endophytes (Rodriguez et al., 2008), but this may be due to thorough colonization of the fungus in above and below ground plant tissue (Rodriguez et al., 2009).

In woody plants, fungal endophytes are commonly horizontally transmitted to hosts, meaning that plants are colonized by fungi from the surrounding environment (Rodriguez et al., 2009, Saikkonen et al., 1998). In similar fashion, fungal endophytes can be transmitted maternally by colonizing seeds of host plants, also known as vertical transmission

(Saikkonen et al., 1998). Saikkonen et al. (1998) proposed a model to predict frequency of the transmission of fungal endophytes (vertical vs. horizontal) in grasses or woody plants and likelihood of the benefit conferred to the plant host (antagonistic, neutral and mutualistic).

They suggested that vertically transmitted fungal endophytes of grasses were most likely to occur and interact in a mutualistic fashion with the host plant. Horizontally transmitted, non- systemic fungal endophytes of woody plants are proposed to occur least frequently and to rarely have an antagonistic or mutualistic interaction with the host plant. This theory is interesting, yet the dearth of studies examining fungal endophytes of woody plants suggests that the overall nature of the association between horizontally transmitted fungal endophytes and woody plants is yet to be determined.

Rodriguez et al. (2008) suggested that Class 2 fungal endophytes confer increased tolerance to drought, desiccation, heat and salinity stress in a wide range of plants. Narisawa et al. (1998) screened fungal endophytes from host plants in search of novel biological control agents for managing Plasmodiophora brassicae (causal agent of clubroot disease) on

Chinese cabbage (Brassica campestris L.) cv. Muso, which lacks mycorrhizal associations

The authors identified an endophyte, Heteroconium chaetospira, with biological control potential for P. brassicae.

Zygomycetous fungi are not known as root endophytes, but there are several studies that suggest they can colonize the surface and internal cells of roots. Huang et al. (2015) isolated two Zygomycetous fungi, Umbeleopsis dimorpha Mahoney & W. Gams and

Syzygites sp. from the roots of Kadsura angustifolia A.C.Sm. (2015). Species of Mortierella have been isolated as endophytes from roots of field mustard (Brassica campestris)

(Narisawa et al., 1998) and Changnienia amoena (Jiang, 2011, Narisawa et al., 1998).

Mortierella alpina Peyronel has been isolated as an endophyte from tall fescue (Festuca arundinacea L.) (Gan et al., 2017) and Scots pine (Pinus sylvestris L.) (Peršoh, 2013). More recently, Bonito et al. (2016) identified species of Mortierella from Quercus alba L., Populus deltoides W. Bartram ex Marshall, and Pinus taeda L. roots and associated soil. While these isolates of Mortierella spp. have been isolated from internal plant tissues within roots, it is important to note that their ecology within the rhizosphere is unknown (Bonito et al., 2016).

Plant growth promotion by fungal endophytes. Benefits of fungal endophytes for host plants include improved growth via access to nutrients, minerals and microbial synthesis of phytohormones (Schulz & Boyle, 2006). Das et al. (2012) demonstrated plant growth promotion of Coleus forskohlii Andrews by the endophyte Piriformospora indica (Sav.

Verma, Aj. Varma, Rexer, G. Kost & P. Franken), which increased shoot and root weight, shortened time to flowering and increased abundance of flowers. Mandyam et al. (2013) found that dark septate endophyte (DSE) isolates of Periconia macrospinosa Lefebvre &

Aar.G. Johnson and Microdochium sp. resulted in differential growth responses in shoot

weight on different accessions of Arabidopsis thaliana. A diverse genera of endophytes, including Heteroconium, Chaetomium, Fusarium, and Stagonospora have been implicated in growth promotion (Schulz, 2006).

Mechanisms of plant growth promotion by fungal endophytes. A majority of fungal root endophytes surveyed by Schulz et al. (2002) produced secondary metabolites exhibiting antimicrobial (bacterial and fungal) and herbicidal properties that may inhibit other microbes or plants competing for the same resources or space. One mechanism proposed for endophyte-enabled plant growth promotion is that fungi may be able to access nitrogen, phosphorous and carbon and make it available to the host plant (Schulz, 2006).

Fungal endophytes also produce phytohormones (such as gibberellins and indole-3- acetic-acid (IAA), which have been implicated in altering host plant structure (Hardoim et al., 2015). Dark septate endophytes produce hydrolytic enzymes capable of releasing C, N and P from detritus and increase P concentration within plant tissue (Mandyam &

Jumpponen, 2005). Recently, a meta-analysis examined plant growth promotion activity of

DSEs, and concluded that DSEs increased plant weight, and shoot nitrogen and potassium content (Newsham, 2011).

Mortierella elongata. Mortierella elongata Linnem. is a member of the phylum Zygomycota in the Domain Eukarya, Kingdom Fungi, Subphylum Mortierellomycotina, Order

Mortierellales, and Family Mortierellaceae (Linneman, 1941). The genus Mortierella was described in honor of M. Du Mortier, president of the Societe de Botanique de Belgique

(Wagner et al., 2013). Estimates for the number of species within the genus Mortierella range from 90 to 100 according to the literature, and as many as 126 species based on a

model of the ratio between described species and molecular operational taxonomic units

(MOTUs) for genera obtained from GenBank (Nagy et al., 2011).

Most genera within the Order Mortierellales exhibit rosette shaped colonies in pure culture that are divided into even zones, with whitish cottony tufts of mycelium at the edge of each zone (Wagner et al., 2013). Production of sexual zygospores by M. elongata was confirmed by Gams et al. (1972). M. elongata produces arachidonic acid, a Polyunsaturated

Fatty Acid (PUFA) thought to trigger plant defense signaling networks and increase jasmonic acid (JA) levels within Arabidopsis and tomato (Yamada et al., 1987, Savchenko et al.,

2010). The fungus also produces another PUFA, eicosapentaenoic acid (EPA). Recently, researchers have identified endosymbiotic endohyphal bacteria associated with M. elongata

(Sato et al., 2010, Ohshima et al., 2016, Uehling et al., 2017).

Ecology of M. elongata. Fungi in the Order Mortierellales are saprobic soil inhabiting microorganisms, subsisting on decaying organic matter (Wagner et al., 2013). M. elongata is commonly found as a soil saprobe throughout temperate areas of the globe (Tedersoo et al.,

2014) and has been isolated from soil in the Netherlands, Germany, Austria (Wagner et al.,

2013), US (Gams et al., 1972), China (Yao et al., 2012), and Japan (Sato et al., 2010).

However, M. elongata has also been isolated from roots of Populus deltoides, Brassica campestris and Phragmites australis (Cav.) Trin. ex Seud. suggesting that it may also behave as an endophyte (Bonito et al., 2014, Narisawa et al., 1998, Khalmuratova et al., 2015). The ecology of Mortierella elongata is not very well understood, and function as an endophyte has yet to be confirmed. The isolation of previously classified saprobic fungi as endophytes is not without precedent. Chaverri and Gazis (2011) examined the fungal community of

Hevea brasiliensis (rubber tree) endophytes and surrounding soil, and identified

Perisporiopsis lateritia isolated from a decaying leaf, as well as an endophyte of leaves and sapwood. Additionally, Oidioendron maius can grow as a saprobe in peat, but is also known to develop ericoid mycorrhizae within host plant roots (Rice & Currah, 2006). Isolates of

Mortierella elongata sampled from Populus deltoides internal root tissue (endosphere) by

Bonito et al. (2016) were utilized as inoculum added to soil for tomato (Solanum lycopersicum L.), red oak (Quercus rubra L.) and corn (Zea mays L.). Plants grown in M. elongata amended soil exhibited significantly greater plant height than non-inoculated control plants (Vilgalys, unpublished).

Research hypotheses and objectives. The overall goals of this research project were to investigate the promotion of plant growth and flowering of Calibrachoa with the soil fungus

Mortierella elongata, and the susceptibility of Calibrachoa cultivars to the common soilborne pathogen Thielaviopsis basicola. We tested the hypotheses that 1) M. elongata would promote growth of the solanaceous plant Calibrachoa x hybrida and 2) C. hybrida cultivars will be susceptible to the black root rot pathogen T. basicola. To address these hypotheses, we 1) evaluated plant growth promotion and flowering of C. hybrida plants inoculated with isolates of M. elongata and 2) evaluated host resistance of C. hybrida cultivars to the fungal pathogen T. basicola, causal agent of black root rot.

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Figure 1.1 Drawing of Nierembergia intermedia Graham, a synonym for Salpigloss linearis, which was renamed as Calibrachoa linearis in 1990 (Wijsman) sourced from The British

Flower Garden (Sweet, 1835).

Table 1.1 Recommended fungicides for application if symptoms of black root rot are widespread in a greenhouse.

Product Name Active Ingredient FRAC Code

Cleary’s 336 WP/ OHP 6672 FL Thiophanate-methyl 1

Emblem, Medallion WG Fludioxonil 12

Terraguard Triflumizole 3

Table 1.2 Recommended fungicides for application if symptoms of black root rot are sporadic in a greenhouse.

Product Name Active Ingredient FRAC Code

Affirm WDG Polyoxin D zinc salt 19

Orkestra Fluxapyroxad/pyroclostrobin 7/11

Empress Intrinsic Pyraclostrobin 11

Trinity Triticonazole 3

CHAPTER II

Increased flower production and plant weight of Calibrachoa x hybrida by the soil

fungus Mortierella elongata

L. E. Becker and M. A. Cubeta

Department of Entomology and Plant Pathology, North Carolina State University, Raleigh,

NC 2795, USA.

ABSTRACT

The saprobic fungus Mortierella elongata isolated as an endophyte from Populus deltoides

(eastern cottonwood) roots increases seedling growth of corn, red oak and tomato in pasteurized sand and untreated field soil. Two greenhouse experiments were conducted to examine the potential utility of M. elongata for promoting growth of the solanaceous floriculture plant Calibrachoa. Rooted cuttings of Calibrachoa cv. ‘Kabloom Deep Blue’ were transplanted into potting media amended with 1 or 2% (v/v) of millet colonized with one of four isolates of M. elongata. Two isolates with endosymbiotic bacteria, 93+ and 624+, and two without the bacteria, 93- and 624-, were used. Plants were assessed weekly for flower production and after 86 days for above/below ground dry weight. M. elongata isolate

624- significantly increased the number of flowers produced at 1 and 2% v/v compared to the

1 and 2% v/v non-inoculated millet controls 5, 6 and 7 weeks after amendment. A significant increase in above and belowground plant dry weight for plants amended with M. elongata isolate 624- at 1% v/v compared to non-inoculated 1% v/v controls was observed during experiment 1 (P< 0.005), but not in experiment 2. Our results suggest that M. elongata 624-

promotes the production of flowers consistently, but that an increase in plant dry weight may be variable.

INTRODUCTION

Fungal endophytes are characterized as asymptomatic fungal infections, where the fungus resides within plant tissue without causing apparent harm (Petrini, 1991) and are thought to be present in all plants (Strobel & Daisy, 2003, Carroll, 1988). Beneficial fungal endophytes can increase plant weight (Khalmuratova et al., 2015, Newsham, 2011), assist in mineral nutrition of the plant host (Usuki & Narisawa, 2017, Haselwandter & Read, 1982,

Newsham, 2011), provide protection against plant pathogens (Kari Dolatabadi et al., 2011,

Schulz, 2006), and ameliorate abiotic stress (Rodriguez et al., 2008). These fungi are highly adapted to the rhizosphere and exhibit ecological plasticity (Šišić et al., 2016). Fungal endophytes are emerging as a potential alternative to chemical amendments, due to their reduced environmental impact and dual activity as promoters of plant growth.

Fungi in the Ascomycota represent the majority of known fungal endophytes (Das et al., 2012, Ghanem et al., 2014, Lingfei et al., 2005, Newsham, 2011, Saikkonen et al., 1998,

Hoff et al., 2004). Members of the phylum Zygomycota account for less than 10% of recovered isolates from plants (Huang et al., 2015, Chowdhary & Kaushik, 2015, Gan et al.,

2017, Russo et al., 2016, Khalmuratova et al., 2015, Hoff et al., 2004). The zygomycetous fungus Mortierella elongata Linnem. is a plant and soil associated saprobe found throughout temperate regions of the world (Wagner et al., 2013, Parkinson & Clarke, 1960, Buée et al.,

2009, Tedersoo et al., 2014). M. elongata has recently been isolated as an endophyte from

Phragmites australis (Cav.) Trin. ex Seud. (common reed), Brassica campestris L. (field mustard), and Populus deltoides W. Bartram ex Marshall (eastern cottonwood)

(Khalmuratova et al., 2015, Narisawa et al., 1998, Bonito et al., 2016) and investigated as a

host to bacterial endosymbionts (Sato et al., 2010, Ohshima et al., 2016, Uehling et al.,

2017). Other fungal saprobes isolated as endophytes include Perisporiopsis lateritia P.

Chaverri & Gazis (2011) isolated from Hevea brasiliensis Müll. Arg. (rubber tree) sapwood and decaying leaves. The fungus Oidiodendron maius Barron isolated as an ericoid mycorrhizal endophyte from Rhodendron sp. roots was also identified as a saprobe in peat land bogs and fens (Rice & Currah, 2006). Compared to ascomycetous fungi such as

Trichoderma spp., zygomycetous fungi remain an overlooked taxon of plant growth promoting fungi. A few research groups have evaluated species of Mortierella for plant growth promotion activity and reported neutral (Gil et al., 2013, Khalmuratova et al., 2015) or positive effects (Wani et al., 2017, Zhang et al., 2011).

Calibrachoa (Calibrachoa x hybrida), a solanaceous ornamental and close relative of petunia, is rapidly gaining popularity as a flowering bedding and hanging plant since its introduction two decades ago. Calibrachoa is valued for its aesthetic qualities of small, numerous and colorful trumpet-shaped flowers. As of 2014, annual sales of Calibrachoa plants in the United States were valued at 45 million US dollars (USDA, 2014). Beyond a healthy plant, growers prize Calibrachoa plants for their abundant flower production and long duration of production. Fungal endophytes have recently been implicated in shortening time to flowering and promoting proliferation of flowers in Coleus forskohlii Briq. [syn. P. barbatus Andr.] (Indian coleus) and Cyclamen persicum Mill. (cyclamen) (Das et al., 2012,

Ghanem et al., 2014).

The benefit of a fungal endophyte such as M. elongata for promotion of flowering and plant weight in Calibrachoa plants has not been examined. In this study, we investigated

the interaction between the endophytic fungus M. elongata and solanaceous floriculture plant

Calibrachoa. The following objectives were addressed (i) to determine the effect of M. elongata isolates and inoculum volume on plant weight production, and (ii) to investigate the influence of M. elongata on flower production of Calibrachoa. We hypothesized that M. elongata has beneficial effects on plant dry weight and flowering when associated with

Calibrachoa roots.

MATERIALS AND METHODS

Isolates. Two isolates of Mortierella elongata (93+ and 624+) sampled from Populus deltoides W. Bartram ex Marshall (eastern cottonwood) roots along the Yadkin River in

North Carolina and obtained from Rytas Vilgalys (Duke University, Durham NC) (Bonito et al., 2014) were used in this study. These isolates were derived from internal root tissue and confirmed to have endosymbiotic bacteria (Bonito et al., 2016, Uehling et al., 2017).

Subsequently, two additional strains (93- and 624-) were generated by curing the original isolates of their endosymbiotic bacteria with antibiotics (Uehling et al., 2017). Pure cultures of each isolate of M. elongata were grown on Malt Extract Agar (Becton, Dickinson and

Company, Sparks, MD) for approximately 2 wk at 24 C temperature under photoperiod with a 12 h light/dark cycle (48 µE m-2 s-1). Isolates were maintained in long term storage in sterilized distilled water at room temperature and at -80 C in sterile glycerol solution.

Inoculum production. Millet seed (300 g) was mixed with equal volume of distilled H2O

(300 ml) in a 3.79 L plastic jug, plugged with a foam stopper capped with aluminum foil and autoclaved for 45 min at 121 C (124 kPa) for three consecutive days. After autoclaving, half of a 9-cm diam Petri dish of each isolate was cut into small plugs and aseptically added to each jug. Inoculated millet jugs were incubated at room temperature (21 C ±2 C) under fluorescent light for 4 wk. Non-inoculated millet control was amended with non-infected agar plugs.

Plant Growth Promotion Experiments. Tip cuttings (4-6 cm in length and less than one week old) of Calibrachoa (cv. ‘Kabloom Deep Blue’) obtained from Ball Seed Co., Chicago,

IL were dipped in rooting hormone powder Root Boost with 0.1% active ingredient indole-3

butyric acid (Tech Pac, LLC. Lexington KY). Cuttings were placed in soilless peat-based potting media (Farfard 2P with wetted pH of 5.5 to 6.5) in 128 cell flats for 3 wk. Plants were misted intermittently to promote root formation in a greenhouse at North Carolina State

University, Raleigh. Rooted cuttings of Calibrachoa were transplanted into 10-cm square pots (one plant per pot) with a 2:1:1 mix of Farfard 2P potting media: pasteurized soil: and pasteurized sand. Prior to transplanting, soil mix was infested with millet seed colonized by an isolate 93+, 93-, 624+, or 624- of Mortierella elongata Linnem. Infested millet seed was thoroughly incorporated into the soil mix by agitation at either 1% or 2% v/v. Controls consisted of uninfested millet seed added at 0, 1, or 2% v/v. There were five replicates of each treatment. Treatments were arranged in a randomized complete block design with five replications. The experiment was conducted twice. Experiment 1 was started on 18 March and harvested 11 June of 2017. Experiment 2 was conducted from 9 July to 2 October of

2017. Minimum and maximum greenhouse temperatures were recorded daily throughout the duration of the experiments.

Assessment of Plant Growth. The number of fully emerged flowers per plant was counted weekly following flower emergence (wk 2 to 11). Twelve weeks after transplanting, above ground leaves, stems and flowers were harvested by clipping each plant stem at the surface of the potting media and placing the plant tissue into a paper bag. Roots from each plant were rinsed under running tap water to remove excess media. Leaves/stems and roots were dried separately in paper bags for 8 h at 50-60 C in a Thelco drying oven (Precision Scientific

Company, Chicago IL). Dried leaves/stems and roots were weighed separately using a Metler

Toledo ME4002E scale (Columbus, OH).

Statistical Analysis. All statistical analyses were performed using SAS (version 9.4; SAS

Institute; Cary NC). Mean plant dry weight and flower values from experiment 1 and 2 were subject to analysis of variance (ANOVA) using the PROC GLIMMIX procedure of SAS. A generalized linear mixed model was utilized for the continuous and count data for leaves/stems and root weight and flowering assuming a normal distribution for continuous data and poisson distribution for count data (Stroup, 2015). When ANOVA was significant for isolate and volume interaction, simple effects were examined to compare isolates by individual inoculum volumes. Post hoc analysis of the data was performed with Tukey’s honestly significant difference test to examine significant differences among the means

(alpha= 0.05). Additionally, Dunnett’s multiple comparison against controls was utilized to discern significant differences between individual isolates and control for dry weight analysis.

RESULTS

Experiment 1.

Leaf/stem, root, and total dry weight differed significantly with isolate and there was a significant inoculum volume x isolate interaction for each plant growth parameter accessed

(P < 0.05) (see Table 2.1 for Analysis of Variance). Therefore, treatments were analyzed for each inoculum volume separately to examine main effects. Rooted cuttings grown in potting media amended with M. elongata isolate 624- had significantly higher (P< 0.0001, 0.0005 and <0.0001) leaf/stem, root, and total dry weight compared to the non-amended control at

1% v/v. In contrast, cuttings amended with M. elongata isolate 624+ exhibited significantly lower dry weight (P< 0.0001) for all three dependent variables (leaf/stem, root, and total dry weight) at 1% v/v (Figs. 2.1, 2.2, and 2.3). Rooted cuttings grown in potting media amended with M. elongata isolate 93+ at 1% inoculum volume exhibited significantly lower leaf/stem and total dry weight (P≤ 0.0001) compared to the non-inoculated control at 1% v/v (Figs. 2.1 and 2.3). Rooted cuttings amended with M. elongata isolate 93+ at a 2% inoculum volume exhibited significantly lower leaf/stem, root, and total dry weight (P< 0.0001, = 0.0096, and

=0.0001) compared to the non-amended 2% v/v controls (Figs. 2.4, 2.5 and 2.6).

Significant differences among isolates were observed for flower production for wk 5,

6, and 7 (Fig. 2.7). The inoculum volume x isolate interaction was significant for flower production in wk 5, 6, and 7 (Table 2.2) and treatments were examined separately for each inoculum volume to determine main effects. Rooted cuttings grown in potting media amended with M. elongata isolate 624- at 1% v/v produced significantly more (P< 0.0001,

=0.0003, <0.0001) flowers than the 1% v/v non-inoculated millet control at wk 5, 6 and 7

post inoculation (Fig. 2.8). Rooted cuttings inoculated with M. elongata isolate 624+ and 93+ at 1% v/v produced significantly less (624+: P= 0.0001, 0.0036 and 93+: P= 0.0036, 0.0002) flowers than the 1% v/v non-inoculated millet control at wk 6 and 7 (Fig 2.8). Calibrachoa cuttings amended with M. elongata isolate 624- at 2% v/v during wk 5 and 6 produced significantly more (P= 0.0049, 0.0001) flowers than the 2% v/v non-inoculated millet control at wk 5 and 6 (Fig. 2.9).

Significant differences for plant dry weight and flower production were observed between M. elongata isolates 624 and 93. However, the greatest differences in plant dry weight and flower promotion occurred within the same isolate, depending on the presence or absence of endosymbiotic bacteria. Cuttings exhibited greater flowering and total dry weight when amended with M. elongata isolate 624- at 1% v/v compared to the control, whereas cuttings exhibited significantly less flowering and total dry weight when amended with M. elongata isolate 624+ at 1% v/v (Figs. 2.3 and 2.8). Rooted cuttings amended with M. elongata isolate 93, regardless of presence or absence of endosymbiotic bacteria, produced either similar or significantly less flowers compared to the control at 1% inoculum volume

(Fig. 2.8). Isolate 93+ exhibited significantly less leaf/stem and total dry weight at 1% and

2% inoculum volume.

As indicated by the significant interaction between volume and treatment, significant differences (P< 0.0001) were observed among cuttings amended at volumes 0, 1, and 2% for leaf/stem, root, and total dry weight. Rooted cuttings amended with 2% inoculum volume exhibited significantly higher dry weight for all three metrics compared to rooted cuttings amended with 1% inoculum volume. Cuttings amended with 1% inoculum volume exhibited

significantly higher dry weight for all three metrics compared to cuttings amended with 0%

(control) inoculum volume. Although rooted cuttings amended with 2% inoculum volume exhibited higher dry weight overall, we observed fewer differences between isolates and the control. A similar pattern for flower production during wk 6 and 7, where cuttings inoculated with 2% inoculum volume exhibited significantly greater flower count than cuttings inoculated with 1% inoculum volume was observed. Cuttings inoculated with M. elongata

624- isolate at 2% volume during wk 6 exhibited significantly higher flower production compared to the control. Non-inoculated millet controls and non-amended controls exhibited higher leaf/stem, root, and total dry weight for 2% volume compared to 1% volume, and higher dry weight for 1% volume compared to 0% volume (P <0.0001).

Experiment 2.

Leaf/stem, root, and total dry weight differed significantly among isolates. We detected a significant inoculum x isolate interaction for each metric (P< 0.05) (see Table 2.3 for Analysis of Variance). Therefore, isolates were analyzed by inoculum volume to examine main effects. Rooted cuttings grown in potting media amended with M. elongata isolate 93- had significantly lower (P= 0.0103, 0.0484, and 0.0143) leaf/stem, root and total dry weight compared to the non-amended control at 1% v/v (Figs. 2.10, 2.11, and 2.12). At 2% inoculum volume, rooted cuttings grown in potting media amended with all M. elongata isolates did not exhibit significant differences in leaf/stem, root, and total dry weight compared to the non-inoculated control (Figs. 2.13, 2.14 and 2.15).

Flower production across all volumes of inoculum and isolates varied across weeks 2 through 12 of the experiment (Figure 2.16). Rooted cuttings amended with M. elongata

isolate 624- exhibited higher flower number compared to non-inoculated/non-amended controls for weeks 5,6, 7, 8, and 10 (P= 0.0184, <0.0001, and <0.0001, for wk 5, 8, and 10 respectively). We observed a significant interaction between inoculum volume and isolate for flower production during wk 4, 6, and 7 (see Table 2.4 for Analysis of Variance) and isolates were examined separately for each inoculum volume to determine simple effects. Rooted cuttings grown in potting media amended with M. elongata isolate 624- at 2% v/v produced significantly more flowers than the non-inoculated 2% v/v control at weeks 6 and 7 post inoculation (P< 0.0001 and < 0.0001) (Fig. 2.18). Rooted cuttings inoculated with M. elongata isolate 93- produced significantly more flowers than the non-inoculated/non- amended control at wk 5 (P= 0.0035) (Fig. 2.16). Plants inoculated with M. elongata isolate

93+ produced significantly more flowers than the non-inoculated/non-amended control at wk

8 (P= 0.0009). During weeks 6, and 7, when Calibrachoa plants were blooming, rooted cuttings amended with M. elongata isolate 624+ and 93+ exhibited significantly less flowers compared to non-inoculated millet control (Figs. 2.16, 2.17, and 2.18).

We observed significant differences in flower production between isolates harboring endosymbiotic bacteria compared to cured isolates without detectable endosymbiotic bacteria. As indicated by the significant interaction between volume and isolate for dry weight and flower production, we detected significant differences among amendment volumes 0, 1, and 2%. For foliage and total dry weight, plants amended with 2% volume exhibited significantly higher dry weight than plants amended with 1% volume, and plants inoculated with 1% volume exhibited significantly higher dry weight than plants inoculated with 0% volume (P< 0.01 for all comparisons). For dry root weight, plants amended with 2%

volume exhibited significantly greater dry weight compared to 0% volume (P= 0.0461). We observed a similar trend for flower production during weeks of blooming 6, 7, 8, and 10. We detected significant differences among 2, 1, and 0% volumes for flower production (P< 0.003 for all comparisons). Among the controls, inoculum volume 1% and 2% exhibited significantly higher leaf/stem, root, and total dry weight when compared to control volume

0% (P <0.0001 for foliage, root, and total weight). We recorded differences in greenhouse temperatures throughout the length of experiment 2. Table 2.5 presents the average minimum and maximum greenhouse temperatures for experiment 1 and 2 by month (analyzed as 30 day units). The mean minimum and maximum greenhouse temperatures for experiment 2 during month 1 were 22.8 and 36.5 C respectively. These temperature means differ from the average min. and max. greenhouse temperatures for experiment 1 during month 1, which were 20.7 and 34.2 C respectively (Figs. 2.19 and 2.20).

DISCUSSION

M. elongata isolate 624- increased flower number of Calibrachoa plants. However, leaf/stem and root dry weight of Calibrachoa plants amended with M. elongata isolate 624- only significantly increased in experiment 1. Our results represent the first report of a zygomycetous fungal endophyte increasing flower production over the period of several weeks in an ornamental plant species. The identification and application of novel fungal endophytes with plant growth potential is a growing area of interest for ornamental and agricultural researchers with the aim of increasing production and providing protection against abiotic stress, herbivores and plant pathogens (Le Cocq et al., 2016, Lugtenberg et al.,

2016).

An increase in plant dry weight and flowering during weeks 5, 6 and 7 for plants amended with isolate 624- was observed during experiment 1. In addition, the flowering promotion effect in experiment 2 expanded beyond weeks 5-7 to weeks 8 and 10. In contrast, plants amended with isolate 93+ exhibited lower flower counts and plant dry weight when compared against non-inoculated 1 and 2% control. We assessed flower production on a temporal scale instead of a single time point to reflect timespan of flower production corresponding to growers demands for marketable plants. The association between inoculation of fungal endophytes and increase in flower production has been previously reported in Indian coleus and cyclamen (Das et al., 2012, Ghanem et al., 2014), and promotion of plant weight by fungal endophytes is well documented (Rodriguez et al., 2009,

Mandyam et al., 2013, Schulz et al., 2002, Ansari et al., 2013, Newsham, 2011). The observed variation in plant growth promotion among isolates of by M. elongata and

experiments suggests that environmental factors and inoculum volume may affect root and leaf/stem dry weight. Schmid et al. (2017) suggested that fungal endophytes must confer benefits to their plant host to survive. However, these benefits may not be observable based on environmental conditions or fungus/plant genotypes.

The association of a diverse suite of endosymbiotic bacteria within endophytic fungi has been previously documented (Hoffman & Arnold, 2010). Variation of plant growth response to individual isolates of M. elongata depended largely on presence or absence of endosymbiotic bacteria. Our research suggests that the presence of endosymbiotic bacteria within M. elongata did not enhance plant growth promotion properties of the fungus. Radial hyphal growth of M. elongata isolate AG77 significantly decreases (P <0.05) when associated with the bacterial endosymbiont Mycoavidus cysteinexigens compared to an antibiotic cured strain of M. elongata AG77, suggesting a metabolic cost of harboring the bacterial endosymbiont (Uehling et al., 2017). In contrast, the presence of Rhizobium radiobacter within the plant growth promoting endophytic fungus Piriformospora indica

(Sav. Verma, Aj. Varma, Rexer, G. Kost & P. Franken) suggests that the endosymbiotic bacteria play a role in plant growth promotion activity of the fungus (Bonfante & Anca,

2009). In addition to the bacterial endosymbiont M. cysteinexigens, M. elongata isolates have been confirmed to contain Burkholderia sp., Stenotrophomonas sp. and Brevundimonas sp.

(Uehling, 2017). It is not known specifically how the endosymbiotic bacterial community is composed within M. elongata isolates 624+ and 93+, or if the community composition changes over time and/or responds to environmental factors. A direct link between the

presence and function of endosymbiotic bacteria sourced from endophytic fungi and plant growth has yet to be firmly established.

Among isolates of M. elongata, significant differences were observed by inoculum volume for flower production and plant dry weight. M. elongata strain 624- isolated as an endophyte exhibited plant growth promotion activity at 1% inoculum volume. However, isolates and controls exhibited a similar increase in plant dry weight when amended at 2% inoculum volume. We hypothesize that the higher plant dry weight observed at 2% inoculum volume across all treatments may result from increased nutrient availability to the host plant due to the addition of millet to the potting media. In contrast, while investigating optimal inoculum volumes of Trichoderma saturnisporum, Marín-Guirao et al. (2016) reported higher substrate inoculum volumes as beneficial for cucumber, pepper and tomato weight.

The authors concluded that without a high rice substrate volume, T. saturnisporum exhibits detrimental activities on its plant host. As a free-living soil fungus, T. saturnisporum requires increased nutrition to avoid competition with its host plant.

Plants in Experiment 1 exhibited higher leaf/stem, root, and total plant dry weight (P<

0.05, data not shown) compared to plants in experiment 2, which suggests that greenhouse environmental conditions may have been more conducive for plant and fungal growth during experiment 1. Greenhouse temperatures differed between the two experiments, with the greatest difference in minimum and maximum temperatures observed during days 1-30.

Mean maximum temperature was 34.2 and 36.5 C for experiment 1 and 2, respectively, while mean minimum temperature was 20.7 and 22.8 C for experiment 1 and 2 (Table 2.5). We hypothesize that the higher minimum and maximum temperature at the beginning of the

second experiment may possibly explain differences in dry weight between the two experiments. In addition, the delayed flowering observed in experiment 2 compared to experiment 1 might be explained by the higher temperature observed during month 1 of experiment 2. Growth of Calibrachoa and M. elongata are sensitive to higher temperatures.

In ornamental plants, higher mean daily temperatures are inversely related to plant weight

(Vaid & Runkle, 2014). Cultures of M. elongata exhibit increased radial growth at 24 C and the closely related Mortierella alpina exhibits increased fungal weight when incubated at 25

C (Yamada et al., 1987, Dedyukhina et al., 2015). Gams et al. (1972) demonstrated that growth and survival of M. elongata are inhibited by temperatures at or above (≥) 35 C. The optimal temperatures described above support our hypothesis that the temperatures observed during the second experiment may have decreased plant and fungal growth and delayed flowering.

We evaluated flower production of the Calibrachoa plants on a weekly basis as a means to obtain more detailed information of how M. elongata affects flower production over time. Despite observing higher flower production for plants grown in potting media amended with M. elongata isolate 624- in both experiments, we observed variation in the response. Further experiments could pinpoint the optimal inoculum substrate and volume to control for presence of a nutrient source in the form of inoculum substrate, to better elucidate the mechanism(s) of flowering promotion by M. elongata. A better understanding of environmental conditions in which flowering promotion occurs will also clarify the requirements for plant growth promotion by a fungal endophyte, such as M. elongata. In our study, M. elongata isolate 624- promoted an increase in plant dry weight in experiment 1, but

environmental conditions greatly influenced the response of plants grown with the fungus. In addition, future experiments would benefit by expanding upon the plant hosts evaluated for growth promotion by M. elongata. Future experiments that examine the sensitivity of M. elongata to commonly applied chemical products under floriculture production conditions are warranted. Fungicide sensitivity trials would mirror conditions in which M. elongata could be exposed to in a greenhouse setting.

Fungal endophytes represent novel biological-based sources of plant growth promotion with broad applications across agriculture and horticulture. Zygomycetous endophytes are potentially an un-tapped source of mutualistic fungal endophytes. Our results suggest that amendment of potting media with M. elongata increased flower production. The floriculture industry relies on consistent production of aesthetically valuable flowering plants. Recently, biological-based amendments are increasing in use in greenhouse production systems and therefore increasing in market value (Bailey et al., 2010). Our studies suggest that M. elongata isolate 624- has the potential to increase flowering during marketable periods of Calibrachoa and become a valuable component of biological-based amendments.

ACKNOWLEDGEMENTS

We would like to thank Mike Klopmeyer at Ball seed for providing plants. We would also like to thank Dr. Helen Kraus for providing needed greenhouse space for rooting cuttings and

Dr. Barbara Shew for statistical assistance

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Figure 2.19 Box and whisker plot of greenhouse minimum temperatures (C) between experiment 1 and experiment 2 by month (defined as a 30 day period of the experiment).

Figure 2.20 Box and whisker plot of greenhouse maximum temperatures (C) between experiment 1 and experiment 2 by month (defined as a 30 day period of the experiment).

Table 2.1 ANOVA table for simple effects of Isolate x Volume interaction for leaf/stem, root, and total weight in experiment 1.

Plant Source DF Mean Square F Pr>F Tissue Leaf/Stem Model 10 13.9218 41.51 <0.0001 Isolate 4 8.7217 26.00 <0.0001 Volume 2 49.5828 147.83 <0.0001 Isolate*Volume 4 1.4925 4.45 0.0017 Error 264 0.3354 R2 0.6112 Coefficient of 29.97 Variance (%) Root Model 10 1.7337 15.81 <0.0001 Isolate 4 1.3938 12.71 <0.0001 Volume 2 4.9757 45.38 <0.0001 Isolate*Volume 4 0.5061 4.62 0.0013 Error 264 0.1096 R2 0.3746 Coefficient of 30.59 Variance (%) Total Model 10 25.0555 36.12 <0.0001 Isolate 4 16.9850 24.49 <0.0001 Volume 2 84.8381 122.31 <0.0001 Isolate*Volume 4 3.7044 5.34 0.0004 Error 264 0.6936 R2 0.5778 Coefficient of 27.62 Variance (%)

Table 2.2 ANOVA table for simple effects of Isolate x Volume interaction for flower count of experiment 1, including weeks 5, 6, and 7 post inoculation.

Week Source DF Mean Square F Pr>F 5 Model 10 84.2658 8.09 <0.0001 Isolate 4 98.0164 9.41 <0.0001 Volume 2 6.9842 0.67 0.5122 Isolate*Volume 4 80.0192 7.68 <0.0001 Error 264 10.4128 R2 0.2346 Coefficient of 72.7372 Variance (%) 6 Model 10 224.9948 22.93 <0.0001 Isolate 4 260.0484 26.50 <0.0001 Volume 2 469.2982 47.83 <0.0001 Isolate*Volume 4 29.9786 3.06 0.0174 Error 264 9.8127 R2 0.4648 Coefficient of 69.1366 Variance (%) 7 Model 10 537.0979 28.10 <0.0001 Isolate 4 399.2922 20.89 <0.0001 Volume 2 1800.3753 94.18 <0.0001 Isolate*Volume 4 62.4156 3.27 0.0123 Error 264 19.1161 R2 0.5156 Coefficient of 60.18 Variance (%)

Table 2.3 ANOVA table for simple effects of Isolate x Volume interaction for leaf/stem, root, and total weight in experiment 2.

Plant Source DF Mean Square F Pr>F Tissue Leaf/stem Model 10 5.0376 9.89 <0.0001 Isolate 4 3.7327 7.33 <0.0001 Volume 2 13.1038 25.72 <0.0001 Isolate*Volume 4 2.2837 4.48 0.0016 Error 255 0.5094 R2 0.2794 Coefficient of 52.95 Variance (%) Root Model 10 0.6981 3.46 0.0003 Isolate 4 0.7530 3.73 0.0057 Volume 2 0.7602 3.77 0.0243 Isolate*Volume 4 0.6725 3.34 0.0110 Error 255 0.2016 R2 0.1195 Coefficient of 50.38 Variance (%) Total Model 10 8.9136 7.16 <0.0001 Isolate 4 7.8085 6.27 <0.0001 Volume 2 18.8468 15.14 <0.0001 Isolate*Volume 4 5.2294 4.20 0.0026 Error 255 1.2447 R2 0.2193 Coefficient of 49.82 Variance (%)

Table 2.4 ANOVA table for simple effects of Isolate x Volume interaction for flower count of experiment 2, including weeks 4, 6, and 7 post inoculation.

Week Source DF Mean Square F Pr>F 4 Model 10 14.2377 5.64 <0.0001 Isolate 4 7.2714 2.88 0.0232 Volume 2 40.0433 15.87 <0.0001 Isolate*Volume 4 7.1641 2.84 0.0249 Error 255 2.5236 R2 0.1812 Coefficient of 116.09 Variance (%) 6 Model 10 303.8807 10.11 <0.0001 Isolate 4 287.2771 9.55 <0.0001 Volume 2 581.0865 19.32 <0.0001 Isolate*Volume 4 143.7266 4.78 0.0010 Error 255 30.0710 R2 0.2838 Coefficient of 91.68 Variance (%) 7 Model 10 360.5224 10.57 <0.0001 Isolate 4 249.9075 7.33 <0.0001 Volume 2 931.7754 27.33 <0.0001 Isolate*Volume 4 111.1888 3.26 0.0124 Error 255 34.0957 R2 0.2931 Coefficient of 86.48 Variance (%)

Table 2.5 Average minimum and maximum greenhouse temperatures (C) by month (every 30 days) for experiments 1 and 2.

Month (day) Experiment Average Minimum Average Maximum Temperature (C) Temperature (C)

1 (d 1-30) Exp.1 20.7 34.2 Exp.2 22.8 36.5 2 (d 31-60) Exp.1 21.6 33.5 Exp.2 22.0 34.6 3 (d 61-86) Exp.1 22.2 35.5 Exp.2 20.6 33.7

CHAPTER III

Susceptibility of Calibrachoa cultivars to Thielaviopsis basicola, causal agent of black root rot disease.

L. E. Becker and M. A. Cubeta

Department of Entomology and Plant Pathology, North Carolina State University, Raleigh,

NC 2795, USA.

ABSTRACT

Thielaviopsis basicola, causal agent of black root rot, is an important soilborne fungal pathogen of ornamental plants grown throughout the world. When ornamental plants, valued for their aesthetic qualities, are infected with T. basicola, they exhibit symptoms that make them undesirable for market. In this study, two experiments were conducted to evaluate seven commercially available cultivars of Calibrachoa, also known as mini-petunia, for resistance to T. basicola. Roots of Calibrachoa cultivars exhibited a range of discoloration and necrosis that appeared near the top of the roots. Black root rot disease severity ranged from 0 (healthy) to 5 (81-100% root area diseased) for cultivars. Two cultivars, ‘Minifamous

Compact Hot Pink’ and ‘Deep Blue Kabloom’ had significantly higher dry root weight and lower disease severity ratings when compared to ‘Callie Scarlet’, after inoculation with T. basicola isolate. The Calibrachoa cultivars screened in the study exhibited varying susceptibility to the pathogen and suggested cultivars ‘Minifamous Compact Hot Pink’ and

‘Deep Blue Kabloom’ may exhibit partial resistance to T. basicola. Our results highlight the

need to evaluate susceptibility of Calibrachoa cultivars to T. basicola, which may aid breeders in identifying sources of resistance to black root rot in Calibrachoa.

INTRODUCTION

Thielaviopsis basicola (Berk. and Broome) Ferraris (syn. Chalara elegans Nag Raj &

Kendrick) is a pathogenic fungus that causes black root rot disease of plants in cultivated and non-cultivated soils throughout temperate regions of the world (Noshad, 2007). T. basicola has a wide host range across 15 families of plants (Yarwood, 1981), and within the floriculture industry, the fungus can infect african daisy, begonia, cyclamen, geranium, impatiens, pansy, petunia, phlox, poinsettia, snapdragon, verbena, and calibrachoa

(Calibrachoa Llave and Lex) (Hausbeck & Dudek, 2008, Daughtrey, 1995, Wollaeger, 2016,

Johnson, 1916). The genus Calibrachoa Llave and Lex. is closely related to Petunia and contains approximately 27 species (Fregonezi, 2012) valued for their numerous colorful trumpet-like flowers. Calibrachoa hybrids (Calibrachoa x hybrida) have been marketed widely since their introduction by breeders approximately 20 years ago, and in 2014 the production value in the United States (US) was estimated to be $45 million (USDA, 2014).

In 2014, North Carolina (NC) was ranked 3rd in the US for total flowering and foliar annuals sold. Sales of Calibrachoa were valued at $980,000 (USDA, 2014).

Calibrachoa, valued for its ornamental qualities, requires constant surveillance for disease symptoms. Calibrachoa are often grown in the late fall and winter for market in the spring, and may be exposed to low light levels, cold greenhouses, moist soil, and reduced air circulation (Schoellhorn, 2013). T. basicola infects plants at cooler temperatures, due to its wide range for optimal growth (12-32 C) that extends beyond the optimal growing temperatures of 20-25 C for Calibrachoa (Lucas, 1955). This combination of factors leads to an increased risk of black root rot disease outbreak if proper precautions have not been taken.

Disease symptoms caused by T. basicola result in economic losses to growers and can be challenging to identify in early stages of plant propagation and production. Above ground symptoms of black root rot on Calibrachoa are often challenging to distinguish from nitrogen deficiency. Diseased plants are often stunted and exhibit wilting and chlorosis in older leaves

(Dole et al., 2002, Hausbeck & Harlan, 2014). Below ground symptoms include dark discoloration of roots, due to necrosis of root tissue and presence of melanized chlamydospores of the fungus (Daughtrey, 1995).

Approaches for managing black root rot disease include preventive measures such as sanitation that involve utilization of soilless potting media (Daughtrey, 1995), sterile pots and flats, non-diseased plugs, and acidification of potting media and irrigation water (Harrison &

Shew, 2001). Fungicide applications can also be applied by growers (Hausbeck & Harlan,

2014), however the number of fungicides registered for black root rot is limited (Chase,

2014, Chase, 2016). Host resistance is an important and cost-effective management strategy for black root rot disease but mechanism(s) of resistance to T. basicola in ornamental crops are poorly understood. Single and multiple gene resistance of other hosts to T. basicola, such as tobacco (Nicotiana tabacum) have been sourced from close relatives (Clayton, 1969,

Hood, 1996, Trojak-Goluch & Berbeć, 2011, Gasser et al., 1988, Trojak-Goluch & Berbeć,

2005). Resistance to T. basicola has also been identified in cultivars and closely related species of cotton (Wheeler et al., 1999, Niu et al., 2008). Identifying resistant cultivars of floriculture crops can improve management of black root rot as part of an integrated disease management program in a greenhouse setting.

Evidence of varying susceptibility to T. basicola among Calibrachoa cultivars has been reported (Daughtrey, 2006), but has not been the subject of comprehensive experimental investigation. Little is known about the susceptibility of Calibrachoa to black root rot beyond anecdotal observations from extension agents and commercial grower practices (Wollaeger, 2016, Willmott, 2006, Thomas & Williams-Woodward, 2016,

Hausbeck & Harlan, 2017, Schoellhorn, 2013, Chase & Daughtrey, 2013). Calibrachoa cultivars in the breeding line Superbells are partially resistant according to the company

Proven Winners (Winners, 2017). To our knowledge, there are no peer-reviewed publications that have critically investigated cultivars of Calibrachoa for resistance to T. basicola.

Determining the susceptibility of commercially available Calibrachoa cultivars and describing the symptoms of black root rot infected plants may aid breeders in identifying sources of resistance to black root rot of Calibrachoa in a greenhouse setting. In this study, we developed an experimental protocol and root rating scale for evaluating severity of black root rot disease on Calibrachoa plants to examine the susceptibility of commercially available

Calibrachoa cultivars to T. basicola.

MATERIALS AND METHODS

Plant Material. Seven cultivars of Calibrachoa were evaluated in two separate experiments conducted in 2017 (Table 3.2). Tip cuttings (Ball Horticultural Co., Chicago IL) were rooted by dipping each cutting in rooting hormone powder (Root Boost; Tech Pac, LLC. Lexington

KY) which contains active ingredient 0.10% Indole-3 Butyric Acid (IBA) and placing them in soilless potting media (Farfard 2P Mix Sun Gro Horticulture Agawam MA) in 128 cell flats. Cuttings were grown for 4 wk under an intermittent misting program to encourage rooting (4-8 s of misting every 6-12 min) in a greenhouse at North Carolina State University,

Raleigh. Cuttings of each cultivar of Calibrachoa were transplanted into 10 cm2 plastic pots

(one plant per pot) with a 2:1:1 mix (v/v) of Farfard 2P potting media: pasteurized sand: and pasteurized soil immediately prior to inoculation with T. basicola for the susceptibility assessment experiments.

Chlamydospore production. One isolate of Thielaviopsis basicola (MD1) cultured from an infected Calibrachoa cv. ‘Million Bells Terra Cotta’ was obtained from Margery Daughtrey

(Cornell University, Riverhead, NY) and used to produce chlamydospores (aleuriospores) and endoconidia for Calibrachoa resistance screening (Daughtrey, 2006). Chlamydospores of

T. basicola were produced by growing the isolate in 15% V8 juice medium (150 ml filtered

V8 juice, 850 ml distilled water, and 15 g Difco agar) for approximately 3 wk at room temperature (21-23 C). After incubation, plates were flooded with sterile distilled H2O with

0.01% Tween 20 (Sigma, Saint Louis, MO) and mycelium on the surface of each plate was gently scraped with a rubber policeman to detach chlamydospores and endoconidia. This process was repeated 3 times. The spore suspension solution was passed through a No. 400

sieve (38 µ, Fisher Scientific Company, Hampton, NH) to retain chlamydospores on the sieve surface and remove smaller endoconidia. Chlamydospores were rinsed from the sieve surface, diluted with distilled H2O and placed in a Waring blender for 30 s to separate large chains of chlamydospores and separate chlamydospores from mycelium. The suspension was diluted to a concentration of 1250 chlamydospores/ml with sterilized distilled H2O using a hemacytometer. Isolates were stored in sterilized distilled water at room temperature and in glycerol at -80 C.

Susceptibility of Calibrachoa to black root rot disease. Immediately following transplanting, Calibrachoa plants from each cultivar were inoculated by adding 40 ml of a

1250 chlamydospores/ml suspension to obtain an inoculum density of 100 chlamydospores per gram of soil (Harrison & Shew, 2001) in each pot. For each cultivar, five Calibrachoa cuttings were inoculated. Five additional plants of the same cultivar were not inoculated and served as the control. Treatments were arranged in a randomized complete block design with two replications. The experiment was conducted twice. Experiment 1 was conducted from 8

May to 30 June, 2017 and experiment 2 was conducted from 4 August to 26 September, 2017 in the greenhouse at North Carolina State University in Raleigh. Disease severity of black root rot was assessed using the following scale; 0 = healthy, no disease, 1 = (1-10% root area diseased), 2 = (11-20% root area diseased), 3 = (21-50% root area diseased), 4 = (51%-80% root area diseased) and 5 = (81%-100% root area diseased) (Figure 3.1). Minimum and maximum air temperatures were recorded daily during each experiment and used to calculate mean minimum and maximum temperatures for each experiment.

After 54 days post inoculation (dpi) cuttings were harvested, roots were rinsed with tap water to remove potting media, and photographed with a Nikon D5100 Digital Single

Lens Reflex (DSLR) camera. Roots from each experimental unit were placed into a paper bag and dried for 8 h at 50-60 C in a Thelco drying oven (Precision Scientific Company,

Chicago IL). Dried root weight was determined using a Metler Toledo ME4002E scale

(Columbus OH).

Statistical Analysis. Mean black root rot severity rating and dry root weight were determined for each cultivar and subjected to analysis of variance (ANOVA) using PROC

GLM in SAS (version 9.4, SAS Institute Inc., Cary, NC). Post-hoc analysis was performed using PROC GLIMMIX in SAS to perform Tukey’s Honestly Significant Difference (HSD) test to determine significant differences (alpha= 0.05) among cultivars. The GLIMMIX model was chosen based on unequal treatment sizes and to incorporate gamma distribution for root weight due to non-normal distribution of the dependent variable.

RESULTS

Root Disease Rating. Fifty-four days after inoculation with T. basicola, Calibrachoa roots exhibited a range of discoloration and necrosis that appeared near the top of the roots and black root rot disease severity ranged from 0 (healthy) to 5 (81-100% root area diseased) for cultivars. No disease or significantly less disease was observed in the non-inoculated control.

ANOVA of black root rot disease severity data were not significant for run source of variation (P≥ 0.06, Table 3.1) and data were combined for comparison of means. We observed significant differences in root ratings among Calibrachoa cultivars (P= 0.0012).

Calibrachoa cultivar ‘Minifamous Compact Hot Pink’ which had the lowest mean black root rot disease rating (2.4), and cultivar ‘Callie Scarlett’ had the highest mean black root rot disease rating (3.7) (Table 3.2). Significant differences were observed among inoculated

Calibrachoa cultivars with ‘Minifamous Compact Hot Pink’, ‘Minifamous Double Lemon’, and ‘Kabloom Deep Blue’ exhibiting significantly lower black root rot severity ratings compared to ‘Callie Scarlet’ (P = 0.0003, 0.0286 and 0.0488, respectively) (Table 3.2).

Cultivars ‘Starshine Apricot’, ‘Cabaret Mango Tango’, and ‘Noa Papaya’ exhibited intermediate susceptibility and values for black root rot disease ratings.

Dry Root Weight. Dry root weights (mg) of inoculated treatments for all cultivars from experiment 1 and 2 were combined after examining for significant differences between the experiments (see Table 3.1 for Analysis of Variance). We observed significant differences in dry root weights among the Calibrachoa cultivars evaluated (P= 0.0109). Calibrachoa cultivar ‘Kabloom Deep Blue’ had the highest mean root dry weight 151.5 mg while ‘Callie

Scarlett’ had the lowest mean root dry weight 76.0 mg. The cultivars ‘Kabloom Deep Blue’,

‘Minifamous Compact Hot Pink’, and ‘Starshine Apricot’ had significantly higher dry root weights compared to ‘Callie Scarlet’ (P= 0.0026, 0.0366, and 0.0449, respectively). Root dry weight means for Calibrachoa cultivars ‘Minifamous Double Lemon’, ‘Cabaret Mango

Tango’, and ‘Noa Papaya’ were not significantly different than ‘Callie Scarlet’.

Mean root dry weight (mg) varied between experiments for non-inoculated controls and cultivars inoculated with T. basicola (data not shown), so experiments were analyzed separately. Significant differences between inoculated and control treatments were observed for all cultivars, with the exception of ‘Deep Blue Kabloom’, in experiment 1 (Table 3.3).

Mean root dry weight of ‘Callie Scarlet’, ‘Minifamous Compact Hot Pink’, ‘Mango Tango’,

‘Minifamous Double Lemon’, ‘Noa Papaya’ and ‘Starshine Apricot’ inoculated with T. basicola differed (P ≤ 0.01) from the non-inoculated control in experiment 1. In experiment

2, mean root dry weight of cultivars ‘Mango Tango’, ‘Kabloom Deep Blue’, ‘Noa Papaya’,

‘Starshine Apricot’, ‘Callie Scarlet’, and ‘Minifamous Double Lemon’ inoculated with T. basicola differed (P ≤ 0.004) from the non-inoculated control, with the exception of

‘Minifamous Compact Hot Pink’ (Table 3.3). Mean minimum greenhouse temperature was

22.0 C for experiment 1 and 21.5 C for experiment 2. Mean maximum greenhouse temperature was 35.0 C for experiment 1 and 34.1 C for experiment 2.

DISCUSSION

Resistance to T. basicola in Calibrachoa cultivars is a fundamental management strategy of interest to breeders and growers. In our study, we evaluated seven commercially available Calibrachoa cultivars for susceptibility to T. basicola under greenhouse conditions.

The Calibrachoa cultivars investigated exhibited varying degrees of susceptibility to T. basicola. Daughtrey (2006) reported similar variation in susceptibility among Calibrachoa cultivars in a monthly extension report for the US Northeast ornamental industry. In the current study, we identified two commercially available cultivars of Calibrachoa that exhibited decreased black root rot disease severity and high root dry weight when artificially inoculated with T. basicola isolate MD1.

Calibrachoa cultivar ‘Kabloom Deep Blue’, which produces purple flowers, and cultivar ‘Minifamous Compact Hot Pink’, with pink flowers, consistently exhibited the lowest black root rot disease severity rating and highest root dry weight compared to other cultivars of Calibrachoa examined in our study. Additionally, root dry weight for cultivars

‘Kabloom Deep Blue’ and ‘Minifamous Compact Hot Pink’ artificially inoculated with T. basicola were not significantly different compared to their respective non-inoculated controls in experiments 1 and 2, respectively. These results suggest that ‘Kabloom Deep Blue’ and

‘Minifamous Compact Hot Pink’ may possibly exhibit partial resistance to black root rot.

Additionally, cultivar ‘Callie Scarlet’ consistently exhibited the highest black root rot disease rating and lowest root dry weight, suggesting that this cultivar is highly susceptible to black root rot disease. The differential responses of Calibrachoa cultivars ‘Kabloom Deep Blue’,

‘Minifamous Compact Hot Pink’ and ‘Callie Scarlet’ can provide plant breeders with

valuable information to aid integrated pest management (IPM) strategies to combat black root rot disease.

Calibrachoa is not currently a target of breeding programs with the goal of identifying black root rot disease resistance. Therefore, there is a need for resistant Calibrachoa cultivars and information about the relative susceptibility of commercially available cultivars.

Breeding programs rely on this information to develop hybrids of resistant cultivars by crossing germplasm. This approach has been employed to manage T. basicola in other crops, such as tobacco, cotton, and garland flower (Daphne cneurom L.) (Hood, 1996, Trojak-

Goluch & Berbeć, 2011, Kumar et al., 2012, Wheeler et al., 1999, Noshad, 2007). The development of resistant cultivars of tobacco and cotton rely on a fundamental understanding of levels of resistance across commercial cultivars. Calibrachoa cultivars tested in our study did not exhibit complete resistance, and therefore could not be utilized as potential sources of resistance in breeding programs. However, an integrated management approach involving fungicides, cultural practices and cultivars identified in this study that exhibited reduced or intermediate susceptibility to T. basicola could be potentially deployed by growers.

We identified two cultivars that exhibited partial resistance to T. basicola. Noshad

(2007), used one isolate of T. basicola to assess resistance of 32 Daphne spp. and observed varying susceptibility of species to T. basicola. Further research must expand the number of genetically diverse isolates of T. basicola that exhibit varying levels of aggressiveness in order to access a wider range of cultivars for resistance to T. basicola. Limited resources and information are available to researchers to identify genetic sources of black root rot resistance in commercially available cultivars. Identification of cultivars with less

susceptibility to black root rot disease will aid growers in selecting cultivars of Calibrachoa.

Our results represent the first report of the response of varying susceptibility among

Calibrachoa cultivars to T. basicola.

ACKNOWLEDGEMENTS

We would like to thank Dr. Margery Daughtrey for providing guidance and an isolate for this study. Thanks to Dr. David Shew for providing guidance on the inoculation procedures. We would also like to thank Dr. Helen Kraus for providing us with greenhouse space for rooting cuttings and Camilo Parada for criticial review of the manuscript.

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Figure 3.1 Disease Rating Scale for black root rot of Calibrachoa roots. a) 0= healthy, no disease symptoms; b) 1= 1-10%; c) 2= 11-20%; d) 3= 21-50%; e) 4= 51-80%; f) 5= 81%- 100% disease severity.

Table 3.1 ANOVA table for main effect of black root rot root disease rating and dry weight (g) for combined experiments (1 and 2).

Source DF Mean Square F Pr>F Disease Model 1 3.2004 3.61 0.0596 rating Error 135 0.8869 R2 0.0260 Coefficient of 31.1646 Variance (%) Dry root Model 1 0.0040 0.75 0.3874 weight Error 137 0.0053 R2 0.0055 Coefficient of 63.3940 Variance (%)

Table 3.2 Mean black root rot disease severity and root dry weight of Calibrachoa cultivars screened for susceptibility to Thielaviopsis basicola for combined experiments.

Cultivar Root dry Root rating† weight (mg)* Kabloom Deep Bluea 151.5 ±(18.8) a 2.8421 ±(0.2045) b Minifamous Compact Hot Pinkc 132.1 ±(16.8) a 2.4211 ±(0.2045) b Starshine Apricotc 129.5 ±(16.1) a 3.0000 ±(0.1993) ab Minifamous Double Lemonc 126.0 ±(15.7) ab 2.8000 ±(0.1993) b Cabaret Mango Tangoa 98.5 ±(12.2) ab 3.1000 ±(0.1993) ab Noa Papayad 92.0 ±(11.4) ab 3.2500 ±(0.1993) ab Callie Scarletb 76.0 ±(9.4) b 3.7000 ±(0.1993) a a- Ball Flora Plant b- Syngenta Flower c- Selecta d- Danziger *Means within a column followed by the same letter are not significantly different (P =0.05). † Disease Rating Scale for black root rot of Calibrachoa roots. 0= healthy, no disease symptoms; 1= 1-10%; 2= 11-20%; 3= 21-50%; 4= 51-80%; 5= 81%-100% disease severity.

Table 3.3 Dry root weight for inoculated (I) and non-inoculated control (C) for Calibrachoa cultivars screened for susceptibility to Thielaviopsis basicola for experiment 1 and 2.

Treat- Exp. 1 dry root weight Exp. 2 dry root weight Cultivar ment (mg)* (mg) Starshine Apricotc C 335.0 ±(41.3) a 310.0 ±(48.6) ab Callie Scarletb C 321.0 ±(39.6) ab 282.0 ±(44.2) ab Kabloom Deep Bluea C 298.0 ±(36.7) abc 466.0 ±(73.0) a Cabaret Mango Tangoa C 292.0 ±(36.0) abc 476.0 ±(74.6) a Noa Papayad C 256.0 ±(31.6) abc 351.4±(65.8) ab Minifamous Compact Hot C 241.0 ±(29.7) abc 291.0 ±(45.6) ab Minifamous Double Lemonc C 204.0 ±(25.2) abcd 419.0 ±(65.6) a Pinkc Kabloom Deep Bluea I 183.0 ±(22.6) bcd 120.0 ±(18.8) cd Starshine Apricotc I 170.0 ±(21.0) cde 89.0 ±(13.9) cde Noa Papayad I 129.0 ±(15.9) def 55.0 ±(8.6) e Cabaret Mango Tangoa I 120.0 ±(14.8) def 77.0 ±(12.1) de Minifamous Compact Hot I 95.0 ±(11.7) efg 173.3 ±(28.6) bc Minifamous Double Lemonc I 87.0 ±(10.7) fg 165.0 ±(25.9) bc Pinkc Callie Scarletb I 58.0 ±(7.2) g 94.0 ±(14.7) cde a- Ball Flora Plant b- Syngenta Flower c- Selecta d- Danziger *LS means within a column for each experiment followed by the same letter are not significantly different (P =0.05).