EVALUATION OF CHEMICAL AND PHYSICAL CONTROL STRATEGIES ACROSS LIFE HISTORY STAGES OF THE INVASIVE Kalanchoe xhoughtonii

By

JESSICA L. SOLOMON

A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE

UNIVERSITY OF FLORIDA

2019

© 2019 Jessica L. Solomon

To my mom and brother

ACKNOWLEDGMENTS

I want to express the utmost gratitude to my advisor Dr. Stephen Enloe, for his compassion, support, knowledge, and passion in this research. Dr. Enloe has been a mentor in the field of research, education, and outreach. His energy helped inspire me in times I lacked the motivation and his kindness and support helped me through times of difficulty. I am also thankful for my committee members, Dr. Jay Ferrell and Dr. Carrie Reinhardt Adams for their support, time, and guidance. I would also like to extend immense gratitude towards my lab mates

Jonathan Glueckert and Kaitlyn Quincy for all of their encouragement, friendship, and tutoring through our coursework and research. I am especially thankful for my lab mate Mackenzie Bell, who has been an incredibly supportive friend, science partner, and plant enthusiast with me for the past three years. I would also like to thank Lara Colley, Dr. James Leary, Dr. Benjamin

Sperry, and the rest of the faculty at The Center of Aquatic and Invasive Center (CAIP) for their insight and passion for research and education. A special thank you is needed for Sara

Humphrey, Conrad Oberweger, Ethan Church, and Matt Shinego who supports CAIP on a day- to-day basis. I am extremely grateful to my friends Lauren Natwick and Dominick Holden for always dedicating their time, encouragement, and endearment when I needed it the most. I would also like to thank my mom and brother for their continual support in my long-time passion for education and science. This research was made possible through collaborations with Kelly Ussia and the rest of the staff at St. Johns County Parks and Recreation and Beaches Departments.

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TABLE OF CONTENTS

page

ACKNOWLEDGMENTS ...... 4

LIST OF TABLES ...... 7

LIST OF FIGURES ...... 8

ABSTRACT ...... 10

CHAPTER

1 LITERATURE REVIEW ...... 12

Crassulaceae ...... 12 Genus of Kalanchoe ...... 13 Bufadienolides in Kalanchoe ...... 15 Kalanchoe species of concern ...... 16 Plantlets and Reproduction ...... 21 Invasiveness of Kalanchoe ...... 23 Site of Concern: Beach dunes ...... 26 Control and Management ...... 28

2 Kalanchoe CONTROL AND RESPONSE TO HERBICIDE ...... 40

Materials and Methods ...... 44 Plant material ...... 44 Herbicide Treatments and Data Collection ...... 45 Statistical Analyses ...... 48 Results and Discussion ...... 49 Experiment 1: Auxin Herbicides ...... 49 Experiment 2: Amino Acid Inhibiting Herbicides ...... 51 Experiment 3: Miscellaneous Herbicides ...... 53 Conclusion ...... 54

3 OVERCOMING THE NURSE PLANT EFFECT TO CONTROL Kalanchoe WITH HERBICIDE VIA CANOPY PENETRATION ...... 69

Materials and Methods ...... 73 Field Site ...... 73 Treatments and Data Collection ...... 74 Statistical Analyses ...... 76 Results and Discussion ...... 76 Conclusion ...... 78

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4 CUT ONE, GET TEN: Kalanchoe ASEXUAL REGENERATION IN RESPONSE TO MOWING ...... 87

Materials and Methods ...... 91 Plant material ...... 92 Simulated Mowing Study and Data Collection ...... 92 Regeneration Study and Data Collection ...... 93 Statistical Analyses ...... 94 Results and Discussion ...... 94 Simulated Mowing Study ...... 94 Regeneration Study ...... 96 Conclusion ...... 97

5 CONCLUSIONS AND FUTURE RESEARCH ...... 109

LIST OF REFERENCES ...... 114

BIOGRAPHICAL SKETCH ...... 123

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LIST OF TABLES

Table page

1-1 Herbicides tested on Kalanchoe species in available publications ...... 38

2-1 Kalanchoe xhoughtonii herbicide trials list of active ingredients, mode of action, and the application rates, sorted by experiment (Exp)...... 57

2-2 The average shoot and root dried biomass for Experiment 1, auxin mimicking herbicides...... 59

2-3 Plantlet development on treated Kalanchoe xhoughtonii adult plants...... 62

2-4 The average shoot and root dried biomass for Experiment 2, amino acid inhibiting herbicides...... 64

2-5 The influence of herbicide on average shoot and root dry weight in grams for Experiment 3...... 67

3-1 Range of canopy heights, K. xhoughtonii canopy (over 10cm and under 10cm), and total canopy for each block...... 81

3-2 Spray Card treatment application and surfactant concentrations...... 81

3-3 Average spray coverage of Organosilicone surfactant treatments for the upper canopy cards...... 85

3-4 Average spray coverage of OS treatment for the lower canopy cards...... 86

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LIST OF FIGURES

Figure page

2-1 Kalanchoe xhoughtonii adult plant live leaf count response to auxin herbicide treatments (Experiment 1) at 45 days after treatment...... 58

2-2 Kalanchoe xhoughtonii plantlet response to auxin herbicide treatments (Experiment 1) at 45 days after treatment...... 60

2-3 Kalanchoe xhoughtonii plantlet response to auxin herbicide treatments (Experiment 1) at 125 days after treatment for trial 2 only...... 61

2-4 Kalanchoe xhoughtonii adult live leaf response to amino acid inhibitor herbicide treatments (Experiment 2) at 80 days after treatment...... 63

2-5 Kalanchoe xhoughtonii plantlet response to amino acid inhibitor herbicide treatments (Experiment 2) at 125 days after treatment...... 65

2-6 Kalanchoe xhoughtonii adult live leaf response to miscellaneous herbicide treatments (Experiment 3) at 45 days after treatment...... 66

2-7 Kalanchoe xhoughtonii plantlet response to miscellaneous herbicide treatments (Experiment 3) at 125 days after treatment...... 68

3-1 Vegetation of field site on 29 August 2019 at Butler Beach in St. Augustine, Florida .....80

3-2 Plot set-up, arrow indicates the direction the sprayer moved across plot...... 82

3-3 Average percent spray coverage for the upper spray cards to indicate spray retention for the upper canopy ...... 83

3-4 Average percent spray coverage for the lower spray cards positioned approximately 5cm above the soil surface to indicate spray retention on the lower canopy...... 84

4-1 Kalanchoe xhoughtonii average change in plant height from 0-30 DATa (30 DAT) and 30-60 DAT (60 DAT) for established (left) and small (right) plants...... 100

4-2 Kalanchoe xhoughtonii average leaf count for established (left) and small (right) plants...... 101

4-3 Kalanchoe xhoughtonii average change in leaf number from 0-30 DATa (30 DAT) and 30-60 DAT (60 DAT) for established (left) and small (right) plants...... 102

4-4 Kalanchoe xhoughtonii average plantlet development for established (left) and small (right) plants...... 103

4-5 Kalanchoe xhoughtonii treatment 1 (apical shoot placed on sand) at 1 days after treatment (left) and 7 days after treatment (right) ...... 104

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4-6 Kalanchoe xhoughtonii average plant height at 0 DATa and 60 DAT for treatment 1 (apical shoot on sand, left) and treatment 2 (apical shoot in sand, right)...... 105

4-7 Kalanchoe xhoughtonii average leaf count at 0 DATa, 30 DAT, and 60 DAT for treatment 1 (apical shoot on sand - left) and treatment 2 (apical shoot in sand...... 106

4-8 Kalanchoe xhoughtonii average plantlet development at 0 DATa, 30 DAT, and 60 DAT for treatment 1 (apical shoot on sand, top left), treatment 2 (apical shoot in ...... 107

4-9 Kalanchoe xhoughtonii average fresh weight at 0 DATa and 60 DAT for treatment 1 (apical shoot on sand, top left), treatment 2 (apical shoot in sand, top right)...... 108

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Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science

EVALUATION OF CHEMICAL AND PHYSICAL CONTROL STRATEGIES ACROSS LIFE HISTORY STAGES OF THE INVASIVE Kalanchoe xhoughtonii

By

Jessica L. Solomon

December 2019

Chair: Stephen Enloe Major: Agronomy

Kalanchoe is a genus of succulent species native to the warm, dry regions of Eastern

Africa, Southeast Asia, and Madagascar. Many species are now common around the world due to the ornamental plant trade. Individual species have since become invasive. The Kalanchoe species mentioned here have the ability to produce vegetative clones (plantlets) on the margin of their leaves. Plantlets are the main contributing factor to the high fecundity of these species and are considered the driving force of establishment during the initial phase of invasion. In Florida,

Kalanchoe has been observed as invading beach dunes around the coast. This research investigated methods of control of K. xhoughtonii through physical and chemical methods as well as exploring different application techniques for the use of chemical control in beach dunes.

In greenhouse studies triclopyr, fluroxypyr, aminopyralid and the commercial standard glyphosate all provided excellent control of adult plants and plantlets. Several other active ingredients, including penoxsulam and flumioxazin, showed limited to no control of adult plants and variable control of plantlets.

Field study observations showed herbicide application in beach dunes would be difficult due to Kalanchoe species use of ‘nurse’ plants, living under the canopy of native vegetation to avoid harsh environmental conditions. A field study was conducted on the penetration of spray

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through the upper canopy of native vegetation to the lower canopy of Kalanchoe plantlets using varying rates of an organosilicone surfactant (OS) and spray application volumes. Results showed that an increase in spray application volume has the potential to increase spray coverage to the lower canopy.

Mowing as a control method was also quantified as well as the ability of the clipped plant parts to regenerate and/or produce plantlets in greenhouse studies. Results showed that mowing to ground level (0 cm) controlled K. xhoughtonii. However, plant parts have shown to have the ability to regenerate quickly via plantlets and adventitious roots. The results of these studies provide land managers with information needed to manage invasive populations of Kalanchoe as well as groundwork for future research on Kalanchoe management.

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CHAPTER 1 LITERATURE REVIEW

Crassulaceae

Mother of Thousands, Devil’s backbone, Mother of Millions, and Chandelier Plant are all exchangeable common names for Kalanchoe delagoensis and Kalanchoe xhoughtonii, two very common succulent plants known in the ornamental plant industry since the 1800’s. These species have escaped cultivation and have since become invasive in many ecosystems in several countries (Houghton 1935, Ward 2006). The genus Kalanchoe belongs to the family

Crassulaceae, a relatively diverse family of dicotyledons that is very broadly described as succulent herbs from warm dry regions. There are approximately 1500 species in this family.

Controversy around the infrafamilial classification of the Crassulaceae genera are still subject to ongoing debate. Two major viewpoints prevail, (1) there should be three separate genera of

Bryophyllum, Kalanchoe, and Kitchingia or that (2) all species should be in the single genus of

Kalanchoe (Chernetskyy 2011, Hart and Eggli 1995). The majority of the members of

Crassulaceae are notable for their xeromorphic structure, specifically their occurrence of water- storage tissues in the leaf and stem (Metcalfe, C.R. and Chalk 1950). Generally, most species in the Crassulaceae family are herbaceous; however, the family does include some shrub-like, tree- like, and aquatic species.

As the name implies, Crassulacean Acid Metabolism (CAM) was discovered in this family, specifically in the plant Bryophyllum calycinum, now called Kalanchoe pinnata. In 1885, a German agricultural chemist, Adolf Mayer observed a higher proportion of titratable free acid in the early mornings and that the acidity diminishes after the leaves are exposed to light for a few hours (Pucher and Vickery 1942, Ranson, S.L. and Thomas 1960). Crassulacean Acid

Metabolism’s name refers to the acid that is metabolized, mainly malic acid, and not the

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metabolism of "crassulacean acid." CAM photosynthesis is the ability of a plant to temporally differentiate carbon sequestration and fixation. This involves plants keeping their stomates closed during the day and open at night to capture carbon dioxide. During the nocturnal phase of

CAM photosynthesis, CO2 is taken up through stomatal openings, phosphoenolpyruvate carboxylase (PEPC) fixes carbon dioxide in the cytoplasm into an organic acid, usually malic acid, which is transported into large central vacuoles for storage (Lüttge 2004, Niechayev et al.

2019). In the daytime while the stomata are closed, these organic acids are remobilized, decarboxylated, and then converted back into carbon dioxide before being shunted into the

Calvin Cycle. This adaptation allows the plants to preserve more water in arid conditions by reducing evapotranspiration and, consequently, improves water-use efficiency (WUE). Plants with CAM abilities display plasticity in the extent to which species engage in net nocturnal CO2 uptake, ranging from 0 to 100% (Niechayev et al. 2019). Though CAM was discovered and more notably examined in species within the family Crassulaceae, it has evolved across the plant kingdom, in more than 28 families (Edwards and Ogburn 2012). The improved WUE as well as environmental plasticity are two of the physiological advantages that contribute to population growth of CAM species in their native ranges and are also the traits that make CAM species well known in the ornamental plant industry (Schafer and Luttge 1987). The low water requirements, wide range of light tolerance, and general low maintenance are what have made CAM species such as Aloe vera and Snake Plant (Sansevieria trifasciata) common houseplants.

Genus of Kalanchoe

The first member of the presently named genus of Kalanchoe was identified by botanist

Michel Adason in China in 1763, Cotyledon laciniata, now known as Kalanchoe laciniata. The genus Bryophyllum was introduced into the family Crassulaceae by the British botanist Richard

Anthony Salisbury in 1805, describing the plant Bryophyllum calycinum, now Kalanchoe

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pinnata, where adventive buds on the margin of the leaves were observed (Baldwin 1938,

Chernetskyy 2011). Bryophyllum is derived from the Greek “bryon” + “phyllon,” meaning

“sprout leaf;” this most likely refers to the ability of the plants in this genus to form epiphyllous buds along the leaf margins. Due to the very publicized work of German botanist Hermann

Jacobsen in 1954 on Handbuch der Sukkulenten Pflanzen, translated to “Handbook of succulent plants,’ the recognition of a single genus of Kalanchoe was generally accepted (Chernetskyy

2011, 2012). The relatively large genus is taxonomically difficult and its classification is in a state of flux with some botanists still treating Bryophyllum as a separate genus, leaving some species names unresolved (Chernetskyy 2011, Descoings 2003, Ward 2008). The ability for some species to hybridize also contributes to the confusion (Baldwin 1938, Shaw 2008). Due to this uncertainty, the number of species within this genus ranges from 125, based on French botanist Raymond Hamet’s concept of the group from 1907, and up to over 150 species from

Bernard Descoings’ 2006 publication in French “Le genre Kalanchoe (Crassulaceae): structure et definition” (Abdel-Raouf 2012, Baldwin 1938, Chernetskyy 2012, Descoings 2003, 2006).

Kalanchoe, is thought to be derived from the Chinese term ‘Kalan Chauhuy’ roughly meaning “that which falls and grows,” assumingly referring to the plantlets, though no bulbilferous taxa are native to China. It is also thought that the genus name may be derived from the Indian ‘kalanka’ + ‘chaya’ which translates to ‘spot/rust’ + ‘gloss,’ perhaps referring to the glossy and often reddish leaves of the taxa found in India (Descoings 2003). Species in the genus have a wide distribution of their native habitat ranging from Eastern Africa to Southeast Asia with Madagascar being the center of the highest diversity. Kalanchoe species tend to be monocarpic, perennial, succulent shrubs, rarely but on occasion individuals may be biennial and annual as well as small trees to epiphytes. Inflorescences in this genus all generally have a

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tetramerous arrangement with four calyx lobes, four corolla lobes, eight anthers, and four pistils.

(Allorge-Boiteau 1996, Baldwin 1938, Descoings 2006, Karper and Doorenbos 1983). Flowering is somewhat erratic throughout the genus for cultivated plants. Some species do not flower, some need relatively low temperatures, long days, or short days preceded by long days, with majority of species needing short days to induce flowering (Karper and Doorenbos 1983). Common features for all taxa are the succulent, bifacial leaf structure, enlarged central storage vacuoles of the chlorenchyma cells storing organic acid with reduced intercellular air space. Roots of

Kalanchoe species tend to be very shallow and fine. Shallow root systems can optimize the use of limited (seasonal or intermittent) rainfall and extends survival during the dry season

(Niechayev et al. 2019). For much of the year, water is the main soil resource limiting the plant growth in arid environments. Under environmental stress, some species will change biomass production towards provisions for life preservation by increasing ratio of plantlets to dry leaf weight of leaves. Plantlets offer the chance to survive harsh living conditions (Widmann et al.

1990). It has also been found that the proportion of the biomass assigned to belowground roots will increase with an increase in sunlight received (Guerra-García et al. 2018). However, species within the genus differ in terms of appearance and considerable variation in the structure and presence of trichomes, collenchyma cells, and stomata, in addition to epidermal thickness, embryology, viviparous plant formation, and flower morphology. (Chernetskyy 2012,

Kuligowska et al. 2015, Ting 1989).

Bufadienolides in Kalanchoe

The plant tissues, mainly leaves, of many species of Kalanchoe contain alkaloids, triterpenes, glycosides, flavonoids, and steroids. Of particular interest to chemical, phytochemical, and pharmacological scientists, are a type of cardiac glycoside called bufadienolides. The biological capacities of the bufadienolides include insecticidal, cytotoxic,

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antitumor, anticancer, anti-viral, and cardiotonic activities (Kolodziejczyk-Czepas and Stochmal

2017, Maharani, R., Fajriah, S., Hardiawan, R., & Supratman 2008, Majaz et al. 2011).

Poisoning of livestock as well as pets from the bufadienolides of K. lanceolata, K. pinnata, K. daigremontiana and other species of Kalanchoe have been reported relatively frequent in areas of South Africa, Australia, and Indonesia (Smith 2004). In calves, the lethal dose is estimated to be 7g of flowers kg-1 body weight or 40g of leaves kg-1 body weight (McKenzie et al. 1987).

Toxicity to animals from bufadienolides is caused by producing an inhibition of the sodium- potassium pump in the myocardial cellular membrane, leading to frequent and irregular depolarization of the cell, which will result in disorganized cardiac electrical activity. This causes a variety of arrhythmias that eventually lead to cardiac arrest (Anderson et al. 1984,

Majaz et al. 2011, McKenzie et al. 1987, Smith 2004). The insecticidal activity of bufadienolide compounds have been investigated in some species of Kalanchoe, in search of novel insecticidal compounds as well as to better understand the natural enemies of these species (Kolodziejczyk-

Czepas and Stochmal 2017, Maharani, R., Fajriah, S., Hardiawan, R., & Supratman 2008, Majaz et al. 2011, Supratman et al. 2014).

Kalanchoe species of concern

It is not well documented when Kalanchoe first arrived in the United States in the ornamental plant trade, but in a 1938 publication by biologist Dr. John Baldwin, Jr., there were multiple Kalanchoe species cultivated in the United States at the time (Baldwin 1938). In

Florida, there are approximately six species of Kalanchoe documented in natural areas with an additional five species that have been reported but are believed to not persist outside of cultivation (Ward 2008, Wunderlin and Hansen 2011). K. pinnata, K. daigremontiana, K. delagoensis, and K. xhoughtonii are the species currently observed as invading natural areas in the United States. (Ward 2006, 2008).

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K. pinnata has been one of the most popular of Kalanchoe species in the ornamental plant industry (Ward 2008). One of the common names for this species is “miracle leaf,” possibly derived from its worldwide ethnomedicinal uses on a variety of medical conditions, such as anti- diabetic, anti-neoplastic, antioxidant, immunomodulation, anti-lipidaemic, and anti-allergenic.

Another common name is “air plant,” born from the common practice of pinning a leaf on a window curtain that would then induce foliar plantlet production as a way to propagate more plants (Hershey 2009, Ward 2008). K. pinnata leaves are simple to pinnately compound with 3-

5and occasionally 7 leaflets. Leaflets are 6-12cm in length and broadly elliptic with crenate margins. Leaf coloration is light green with reddish margins. Excluding the inflorescences, individual plants grow up to 1.5meters tall (Majaz et al. 2011, Wunderlin and Hansen 2011). The inflorescence is a dense grouping of terminal, slender, pendulous flowers in the formation known as a compound cyme. The 25-40mm calyx is papery and inflated in appearance and attached to a slender 1-2.5cm pedicel. The papery green to purplish or reddish corolla inserted in the calyx can be up to 7cm long, making individual flowers up to 10cm in length with dozens of flowers in each terminal inflorescence. Besides the ease of care, another selling point of this species in the horticulture industry is the large, vibrant flowers that will stay in bloom for multiple months throughout the year, as is true for the rest of the Kalanchoe species described here. Flowering can start as early as October, usually December and last to late spring, when there is often a lack of flowering species in gardens and landscapes (García-Sogo et al. 2010). Native to Asia, it is now found in the warmer and more temperate parts of China, Australia, New Zealand, the West

Indies, the Philippines, the Galapagos Islands, Melanesia, Polynesia, the Virgin Islands, and the

United States. In many of these countries it is regarded as an invasive species (Boiteau and

Allorge-Boiteau 1995, CABI 2019, Gué Zou et al. 2010, Hannan-Jones and Playford 2002, Ting

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1989, Wang et al. 2016). In the U.S., K. pinnata has been reported as invading native ecosystems in Hawaii and throughout the peninsular and east coast of Florida (Benitez et al. 2012,

EDDMapS 2019a). Synonyms for this species in literature include: Bryophyllum pinnatum,

Cotyledon pinnata, Crassuvia floripendia, Vereia pinnata, and Bryophyllum calycinum.

K. daigremontiana, also known as ‘Devil’s backbone,’ ‘Mother of thousands,’ and

‘Mexican hat plant,’ is native to Madagascar and was first described in 1914 (Baldwin 1938).

Currently, this species is invasive in Africa, Arabia, Southeast Asia, and the United States

(Hannan-Jones and Playford 2002, Herrera and Nassar 2009, Ting 1989, Wang et al. 2016).

There are dozens of varieties of cultivars of this species (Shaw 2008). The cultivars, as well as the wild types, range in leaf and flower size, shape, and coloration (Shaw 2008). In 1934, botanist Charles Swingle wrote an article entitled “The easiest plant in the world to propagate,” about K. daigremontiana and it’s highly prolific plantlet production (Swingle 1934). The species can be described in general terms as a perennial, succulent, glaucous herb growing up to 1.8 meters tall. Leaves are opposite, simple, triangular to lanceolate in shape, serrated margins, green with purple blotching on the bottom side and range in size from 5cm to 25cm. The inflorescence, much like a smaller version of the K. pinnata inflorescence, are a compound cyme of inflated, papery, pendulous flowers that are greenish to pink or lavender in coloration with individual flowers only up to approximately 2.5cm in length (Baldwin 1938, Batygina et al. 1996). This species is often confused with K. xhoughtonii. K. daigremontiana has been identified in almost every county along the southern and eastern coasts of Florida as well as in a few counties in

Texas (Jasper, San Patricio, and Nueces Counties) (EDDMapS 2019b). Allelopathic influence was investigated in this species in 1988, showing that ferulic acid isolated from the plants’ growing medium contribute to inhibitory effects on growth of nearby plants and seedlings (Nair

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et al. 1988). In multiple investigations of K. daigremontiana in the semiarid zones of Cerro

Saroche National Park in Venezuela, it was found that this species can alter the mineralization of carbon and nitrogen in the soil (Chacón et al. 2009, Herrera et al. 2018). Synonyms for this species in literature include: Bryophyllum daigremontianum and Kalanchoe laetevirens.

K. delagoensis, also known as the ‘Chandelier plant’ and ‘Mother of thousands,’ was introduced into the United States as the result of a single introduction from its native range in

Madagascar, collected by botanists Charles Swingle and Henri Humbert during a USDA sponsored expedition in 1928 (Trager 2005). This species is considered one of the most invasive plants in Australia. It is also regarded as hazardous to livestock with many recorded cases of poisoning due to the bufadienolides (Hannan-Jones and Playford 2002, McKenzie et al. 1987).

K. delagoensis is also well known as an invasive plant in South Africa, Cuba, Mexico, the Virgin

Islands, China, and parts of the United States (Batianoff and Butler 2002, Hannan-Jones and

Playford 2002, Henderson 2007, LR et al. 2012, SEMARNAT 2016, Ting 1989, Wang et al.

2016). In Florida, this species has scattered reports throughout the peninsular part of the state

(EDDMapS 2019c). K. delagoensis grows over 1 meter tall, often falling horizontally from being too top heavy, turns upward towards the sun, and continues to grow vertically, in what is called phototropism. Phototropic growth pattern occurs in the other Kalanchoe species described here and is very often seen in K. delagoensis in the field (Ward 2006). Leaves are simple, mostly opposite or whorled in sets of three, the petiole and blade are indistinguishable, very narrowly oblong and sub-cylindric in shape with a longitudinal groove along the adaxial surface, 3-15cm in length with 3-9 conic teeth at the apex. Coloration varies, depending on the amount of light, from a light green base with dark green blotching around cylindrical leaf to a brown leaf with brownish-purple blotching. Inflorescences are similar to those of K. daigremontiana with a

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compound cyme in a dense terminal of inflated, papery, pendulous flowers that are greenish to orange or reddish in coloration with individual flowers only up to approximately 2.5cm in length.

Synonyms for this species in literature include: Kalanchoe tubiflora, Kalanchoe verticillata,

Bryophyllum tubiflorum, Bryophyllum verticillatum, Bryophyllum delagoense, Geaya purpurea

K. xhoughtonii is a hybrid of K. daigremontiana (female) and K. delagoensis (male ) (i.e.

Bryophyllum tubiflora or Kalanchoe verticillata). It was synthesized by medical Doctor and botanist Dr. Author Houghton in 1935 in his gardens in San Fernando, California and named it

Bryophyllum tubimontanum (Baldwin 1949, Houghton 1935, Shaw 2008, Ward 2006). It was not published by Dr. Houghton and was not officially named and described as the current usage of K. xhoughtonii until very recently (Ward 2006). In that article, Ward described a plant that has become established in multiple points around the state of Florida due to escape from cultivation.

Ward said that even though this plant has been known for over 65 years in the horticulture industry, it had yet received a scientific binomial. The plants had usually been misidentified as the maternal plant from the hybrid as K. daigremontiana. Ward formally described the holotype from a specimen he collected in February of 2000 from a patch in a roadside ditch in Merritt

Island, Florida that is currently held at the University of Florida Herbarium (Baldwin 1949, Ward

2006). K. xhoughtonii is a great example of hybrid intermediacy in its morphology between its parental plants. The leaves are lanceolate to oblong with coarsely serrated margin, much like K. daigremontiana but smaller and have a boat-like shape, as if an expanded version of the longitudinal grove along the tubular K. delagoensis. Adaxial leaf surface is light to dark green, often but not always, with some darker blotching while the adaxial surface is green with dark brown to purplish blotching. The petiole is 2-2.5cm long and varies in coloration from bright green to dark purple, seen with and without darker blotching. When plants are in less than ideal

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conditions (i.e. low light intensity), it has been noted that the leaves resemble more that of the K. delagoensis, with a narrower oblong and less-lanceolate shape with less teeth near the base of the leaf, having serration near the apex. Leaves can be arranged opposite, like K. daigremontiana, and whorled in sets of three, like that of K. delagoensis, with branching possible on the upper leaves. The pendulous compound cyme inflorescence is another example of intermediacy in the hybrid between the orange to reddish flowers of the K. delagoensis and the pink to lavender of the K. daigremontiana mixing to create flowers with a deeper pink to purplish coloration.

Plantlets and Reproduction

All of the Kalanchoe species described here have the ability to reproduce asexually through the vegetative propagation of epiphyllial buds, known here as plantlets. Synonyms of the term ‘plantlet’ in literature includes: ‘buds,’ ‘foliar meristems,’ ‘embryos,’ ‘foliar embryos,’

‘foliar buds,’ ‘adventitious buds,’ ‘epiphyllous buds,’ ‘leaf-embryos,’ and ‘pseudobulbils’

(Batygina et al. 1996, Warden 1972). Of the species in the genus Kalanchoe that produce plantlets, some produce plantlets only when placed under stress (induced), some spontaneously

(constitutive), and others produce plantlets under stress as well as constitutively (semi- constitutive). All plantlet producing Kalanchoe demonstrate the totipotency of differentiated cells to regenerate into whole plants. This was found to be linked to specific gene expression along the leaf margin (Garcês and Sinha 2009, Garces et al. 2007). Plantlets develop on the flange-like projections created by the serrations on leaf margin. The projections are a unique organ on the plant that are developed from the bottom of the leaf’s marginal notches or serrations. Plantlet development is gradual and basipetally symmetrical along the leaf margin; the first plantlets begin development at the tip and the following plantlets begin to develop equally down along each side of the leaf. Morphologically, the first stage of plantlet development is a small, green to brownish, globular growth on the mother plant’s leaf tooth. The dome-like

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growth will then split into two small cotyledon-like leaves. More leaves grow on the plantlet along with two hypocotyl-like roots. The plantlet’s vascular system is connected to the mother plant’s through the flange-like tooth the plantlet sits on during development until it is detached.

Plantlets can continue to grow and develop on the mother plant until a disturbance (i.e. animal, strong wind or rain, etc.) knocks the plantlet off of its pedestal. At the pedestal there is a swollen base which serves as a storage organ for water and food as well as a fulcrum point at the abscission point, allowing the plantlet to easily disconnect and fall to the ground. (Garcês and

Sinha 2009, Garces et al. 2007, Johnson 1934). Plantlet development is generally restricted to the long days of the year (i.e. summer), opposite to flowering that is during the shorter days of the year (i.e. winter). Plantlet development can become dormant after the early stages of differentiation, preventing the continuation of development of plantlets prior to leaf and root development. During the winter months, or when conditions are not ideal, the embryonic tissue remains dormant on the mother plant’s leaf notches. Dormancy can be broken by longer days, in some species leaf severing (i.e. extreme stress), or in more ideal growing condition, allowing for vigorous plantlet production in a very short time (Johnson 1934). The ability of Kalanchoe species to form somatic embryos outside of the seed environment has attracted many researchers to investigate the molecular and genetic morphogenic processes in these species (Garcês and

Sinha 2009). Plantlets can develop and spread within the same growing season as the parental plant they fell from generating multiple asexual cycles per year that contributions to the high fecundity of these species. In other words, the plantlets that were dropped to the ground in the beginning of the growing season can grow and develop plantlets that will be mature enough to drop and grow that same year (Herrera et al. 2012).

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Studies of infestations of K. delagoensis, K. daigremontiana, and K. xhoughtonii in

Mexico have shown that each are composed of a single clone and possibly the product of a single introduction. The main cause of invasion was determined to be due to the clonal spread of the plantlets (Guerra-García et al. 2015). Herrera et. al. (2012) investigated the invasion dynamics of

K. daigremontiana in Mexico, concluding that the ability to rapidly increase the population through asexual reproduction of plantlets during the initial phase of invasion is the driving force of establishment. They also determined that the contribution of sexual reproduction of this species had little to no significance in the success of establishment, possibly due to the longer timeframe of approximately three years for mature plants to produce seeds. In Venezuela, K. daigremontiana has been found invading protected arid ecosystems mainly through the extremely high propagule pressure of plantlets (Herrera and Nassar 2009).

Sexual reproduction is not a driving force of invasion for most species of Kalanchoe.

Though they can produce extremely large quantity of seeds, >20,000 seeds per plant, the viability and germination rates are low, and even lower for K. xhoughtonii (Hannan-Jones and

Playford 2002). In the novel ecosystems Kalanchoe species are seen invading, there are little to no observed pollinators accessing the high nectar content of the flowers, therefore they are not being pollinated or pollinated at a very low frequency (Herrera and Nassar 2009). K. daigremontiana, which is self-compatible, is capable of autonomous self-pollination and can produce over 16,000 seeds per year but with only a 17.8% viability and an exponentially decreasing viability rate after each year (Hannan-Jones and Playford 2002, Herrera and Nassar

2009).

Invasiveness of Kalanchoe

There is limited research on the impacts on ecosystems from the invasion of Kalanchoe.

Allelopathy, poisonings, alterations in soil nutrients, and changes in pollinator community have

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been observed and investigated. Groner (1974, 1975) investigated the possible role of allelopathy in K. daigremontiana and concluded that extracts from this plant had an inhibitory effect on the germination and survival of various plants, including its own plantlets. Nair et al. (1988) attributed the allelopathic influence shown by K. daigremontiana to free ferulic acid. Multiple events of cattle and other livestock deaths have been reported due to the ingestion of Kalanchoe species where invaded habitats border livestock enclosures. In Queensland, Australia, between

1960 to 1984 there were over 370 cattle poisoned by ingestion of the Kalanchoe species mentioned here, plus K. prolifera. In New South Wales, Australia, 122 cattle died due to a single poisoning event from ingesting K. delagoensis in the adjoining enclosure (Hannan-Jones and

Playford 2002, McKenzie et al. 1987, McKenzie and Dunster 1986). Kalanchoe species have been shown to increase nutrient availability in nitrogen and carbon poor environments in arid and semiarid zones. This was identified as being due to the large amount of organic matter created by the above ground biomass that is uncommon for the native species (Guerra-García et al. 2018,

Herrera et al. 2018, Widmann et al. 1990). It has also been found that Kalanchoe species can enhance the carbon mineralization rate, ammonification, and enzymatic activities in the soil, leading to changes in the chemical and biological properties of the soil by increasing nutrient bioavailability (Chacón et al. 2009, Herrera et al. 2018). A study into pollinator dynamics of beach dunes, which are ecologically important native bee sanctuaries, investigated the differences in dunes near urban development and dunes near protected/undeveloped sites.

Researchers found that urban locations had significantly lower pollinator species richness and abundance, threatening the integrity of coastal ecosystems. K. delagoensis was listed as one of the non-native plants that are populating the urban sites and whose invaded range has been expanding throughout Florida (Abbate et al. 2019). Herrera and Nassar (2009), found that,

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though K. daigremontiana produces numerous flowers that are rich in nectar, they have a very low frequency of flower visits in invaded ecosystems.

Multiple studies in Australia, Mexico, and Africa have documented the growing invasion and invasive qualities of Kalanchoe species. In a 2002 Assessment of invasive plants in southeast Queensland, K. delagoensis (i.e. Bryophyllum delagoense) was in the top ten most invasive species, with K. xhoughtonii (i.e. Bryophyllum daigremontianum x B. delagoense) and

K. pinnata (i.e. Bryophyllum pinnatum) in the top 50 (Batianoff and Butler 2002). Guerra-García et. al. (2018) examined multiple traits of invasibility of K. delagoensis plantlets in greenhouse settings. They discovered that plantlet survival in the most extreme case of full sun and no watering for 60 days had a survival rate of almost 30% and an almost 100% survival rate in watered and shaded conditions. They also found that stress conditions can trigger the onset of plantlet production from the margin of the adult plant leaves, increasing propagule pressure in times of stress for the rest of the species in the ecosystem. Guerra-García et. al. concluded that plantlets are the main reason for population persistence in K. delagoensis invasions in Mexico, where this species is listed as invasive alien species (SEMARNAT 2016), as well as due to the plant’s environmental tolerance and plasticity. It appears that the northern extent of these species is confined by these succulents’ frost-sensitivity and inability to tolerate cold temperature long- term. In Madagascar, a Kalanchoe native hotspot, temperatures range from only 16°C to 29°C

(Moran 2009, Shaw 2008, Ward 2008). K. pinnata and K. xhoughtonii have only recently been recognized as invasive in Florida, placed on Florida Exotic Pest Plant Council’s list of invasive plant species, since 2013 and 2017, respectively (FLEPPC 2019).

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Site of Concern: Beach dunes

For the purposes of the research in this thesis, our main focus is on the invasion of

Kalanchoe species, mainly K. delagoensis and K. xhoughtonii, into beach dune habitats. The field staff of St. Johns County Parks and Recreation (anecdotal1,2) have observed an increase in abundance of these species in the swales between the primary and secondary beach dunes in St.

Augustine, Florida over the last five years. There is great concern of invasive species in these habitats due to the highly sensitive nature of beach dunes, as well as the ecological and financial importance of functioning dune systems. Introduced species is one of the primary threats to beach ecosystems, severely impacting the integrity of the coastal sites by displacing native plants, degrading wildlife habitat, and disrupting the ecosystem processes of the community

(Batianoff and Franks 1997, Gordon 1998, Marshall et al. 2014, Williams 2007, Zarnetske et al.

2010).

Coastal environments are characterized as low, moderate, or high energy depending on the wave and wind forces acting on the shoreline. The intensity of the energy will then depict the type of vegetation and wildlife that would be present. High to moderate energy coastlines usually result in beach or dune habitat, while marshes or mangrove habitats occur on low energy coastlines (Johnson and Barbour 1990). Of the more than 1,300 miles of coastline in Florida, approximately 800 miles are sandy beaches. These coastlines contribute more than $15 billion annually, indicating that ecologically functioning beaches are an extremely important economic asset to the state (Shivlani et al. 2003). In a 2010 study into the stakeholder perceptions on beach management in Florida, it was found that most stakeholders identify prevention of dune

1 Observations of staff at St. Johns County Park and recreation Department, 2018

2 Jessica Solomon, personal observations

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degradation as one of the most important goals (Ariza et al. 2014). A major ecological function of dunes systems is erosion control from the daily battering of wind, rain, and surf as well as protection from hurricanes and tropical storms that frequent Florida’s coastline. Many of the native plants in dune communities have intense, expansive root systems that maintain the integrity of the dune, stabilizing the sand and soil. Beach dunes are predominantly herbaceous communities and usually built by sea oats (Uniola paniculata), a federally protected perennial rhizomatous grass. Other native plants common in the harsh conditions of Florida beach dunes include: Dune Greenbrier (Smilax auriculata), multiple Prickly Pear Cactus species (Opuntia spp.), Seacoast marshelder (Iva imbricata), railroad vine (Ipomoea pes-caprae), beach elder (Iva imbricata), and Coastal Sandbur (Cenchrus spinifex) (FNAI 2010, Johnson and Barbour 1990,

Williams 2007). Invasive plants, such as Kalanchoe, with a very minor underground root system, contribute to destabilization of beach dune sands as well as potentially decline in native species abundance and diversity (Castillo and Moreno-Casasola 1996, Gordon 1998, Zarnetske et al.

2010). In the swales of the beach dunes of St. Augustine, Florida, dense patches of adult

Kalanchoe are observed reaching for the sunlight through the shorter, slower growing native vegetation. When investigating the coverage of vegetation more closely, carpets of Kalanchoe plantlets are seen at the base of the sea oats, greenbrier, and other shading vegetation. It is thought that this creates a nurse plant effect, allowing the Kalanchoe plantlets to avoid the harsh sun and salt spray in the shadow of the native vegetation. Frequent invasive plant removal and treatment in sandy dune habitats has shown to prevent re-establishment of the native community

(Zarnetske et al., 2010). Re-establishment for native plants is a slow process in most plant communities, let alone in the low nutrient and salty conditions of beach dunes (Mason and

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French, 2007). This leads to the notion that the initial treatment of attack on Kalanchoe in

Florida beach dunes will be of crucial importance and need to be of the highest efficacy.

Control and Management

There is relatively limited research into the management of Kalanchoe species, with no published literature found on the management of these species in the sensitive and unique ecosystems of beach dunes. In an updated 2018 publication from the University of Florida’s

Center for Aquatic and Invasive Plants (CAIP), “Integrated Management of Nonnative Plants in

Natural Areas of Florida,” K. pinnata and K. xhoughtonii are the only Crassulaceae members in the species-specific list of control methods for nonnative plants in use by land managers in

Florida. CAIP’s publication indicates that the current commercial standard in Florida for treating

K. pinnata and/or K. xhoughtonii is a foliar herbicide treatment of 5% (volume/volume) of

Roundup (i.e. glyphosate). Also noted, Roundup is an effective treatment for this species because it kills individual leaves on the plants that would otherwise produce plantlets, but also noted that a follow-up hand removal of individual leaves is necessary to prevent those leaves from producing a new plant (Enloe et al. 2018).

The active ingredient of glyphosate has a very low toxicity in fish and wildlife, binds quickly and tightly to soil organic matter. and is broken down by bacteria in the soil. Glyphosate was first registered for use in the United States in 1974 and is one of the most widely used herbicides used in agriculture, forestry, industrial areas, as well as use by the general public on their lawns and gardens. The main issue with the use of glyphosate is the lack of selectivity of this herbicide. Glyphosate is a broad-spectrum herbicide that works by blocking a key point in a plant’s shikimic acid pathway. All plants and some microorganisms have a shikimic acid pathway for the production of folate and aromatic amino acids, which are essential to plants and animals’ biological function and growth. Humans and other animals acquire these folates and

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amino acids through the consumption of the plants and microorganisms that produce them on their own (Henderson et al. 2010, Maeda and Dudareva 2012).

Many publications since the early 1990’s indicated a great need for control and management of the invasive ornamental Kalanchoe species but reported little on the methods and efficacy of control efforts. Batianoff and Franks(1997), documented the increase in the proportion of invasive ornamental species in the dunes along the east coast of Queensland, which included multiple Kalanchoe (i.e. Bryophyllum) species as the most abundant, noting that the main contributing factor to the ornamental weed invasions were through human assisted introductions from the dumping of garden plants into native vegetation. They concluded that immediate and urgent action is required to prevent further degradation of the dunes, suggesting removal of invasive species and revegetating with native species as well as establish outreach to educate the public, new policies, and fines for polluters/violators.

Field studies on the chemical control of K. delagoensis (i.e. B. tubiflorum) found that fluroxypyr (Starane 200™ [350 mL 100L-1 water]), metsulfuron (Brush off™ [5 g 100L-1 water]), 2,4-D (Tornado™ [2 kg ha-1], Baton™ [2 kg ha-1], and AF300™ [700 mL 100L-1 water]), all with a nonionic surfactant (1 L 1000L-1 water), had great control of K. delagoensis by nine months after treatment, with 2,4-D products being the most cost effective control strategy

(Armstrong and Prior 1999). Sparkes et al. (2002) investigated herbicide activity on K. pinnata

(i.e. B. pinnatum) in the field. They found that out of 2,4-D, fluroxypyr, metsulfuron, and triclopyr + picloram, (with a nonionic surfactant spreading agent added to all treatments), the most cost efficient and effective treatment was 2,4-D with >90% kill rate after ten weeks. The authors reported a site by site interaction that caused statistically different efficacy ratings for treatments at the three different sites that accounted for almost 24% of the variance in the data.

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The authors also noted that complete efficacy of all herbicides tested may not of been visible during the study due to the assessment period of ten weeks which is not long enough for all the herbicides to show complete efficacy (such as the triclopyr + picloram and metsulfuron treatments) as well as possibly not using enough wetter-spreader adjuvant to penetrate the thick cuticle of K. pinnata. In a recent study into plantlet survival, herbicidal activity was investigated on plantlets of K. delagoensis in greenhouse settings. Trials showed that 2,4-D and glyphosate +

2,4-D were the only effective treatments, though no surfactant was added and could have had more efficacy with the other herbicides tested. They concluded that 2,4-D was the most economical herbicide to use in chemical control of plantlets, mostly due to the concern about using the non-selective herbicide glyphosate (Guerra-García et al. 2018).

2,4-D is a systemic herbicide, meaning it has movement in the plant’s xylem and phloem, that has selectivity on dicotyledons (ex: oaks, daisies, roses, cacti, legumes, etc.) but not on monocotyledons (ex: grasses, grains, palms, gingers, etc.). There are multiple formulations including salt, ester, and acid. Toxicity and environmental fate of the herbicide depends on the formulation. 2,4-D, in all its formulations, is an auxin-type herbicide, meaning it mimics natural auxins in the plant. Auxin is a plant hormone that is responsible for the elongation of cells in the meristems (i.e. the growing portions of the plant) and regulation of plant growth. When 2,4-D is applied, it is absorbed through the leaves, stems, and/or roots, translocated to the meristems. It causes uncontrolled and unsustainable growth, and eventually causes death in the plant (Song

2014). 2,4-D was first registered in the 1940’s, was the first selective herbicide on the market, and is one of the oldest herbicides still registered for use today (Jervais et al. 2008). All herbicides found in publications tested on Kalanchoe species can be seen in Table 1-1 with the

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author, location of the research, specific species tested, herbicides and rates used, with the reported efficacy.

Hand removal of Kalanchoe species has been explored on a limited basis. Sparkes et. al.

(2002), added hand pulling as a component of their herbicide activity study on K. pinnata, concluding that hand pulling was the most expensive of methods at approximately $2,095 per hectare with the most economical method of 2,4-D being only $148.25 per hectare. The final efficacy or success rate of the hand pull method was not mentioned in the publication but in the graphical representation of the treatment responses, the graphs show a steady increase in plantlet numbers through the 10-week assessment timeline (Sparkes et al. 2002). In a 2002 in-depth publication on the biology of Kalanchoe (i.e. Bryophyllum) weeds in Australia, unpublished work on hand weeding by “P. Whyte” on K. delagoensis (i.e. B. delagoense) was reported. All obvious plants, plantlets, and leaves were removed and over 100 plantlets m2 were observed nine weeks later. It was unclear whether the returning plantlets were from seed banks and/or hidden/unseen plantlets. Also noted, the removed K. delagoensis were mounded to assess decomposition and after ten weeks most horizontal stems were rooting, tips were vertical (i.e. phototropism) with new growth, and there were no signs of decomposition of plants inside the mound (Hannan-Jones and Playford 2002). Hand removal of K. daigremontiana was investigated through computer modeling with population viability analysis under different management scenarios. It was found that hand pulling may be an efficient control method for this species only if applied at relatively high intensities (65-80%) promptly after the introduction to a new foci.

The extinction probability decreases rapidly with time since establishment, regardless of harvesting intensity. Also noted, a strict control of plantlets and a continuous monitoring of new

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invasions should be first priority, reducing the number of propagules introduced to almost zero to achieve a significant reduction in the probability of establishment (Herrera et al. 2012).

An issue not yet discussed in literature is the difficulty of herbicide application to

Kalanchoe species in habitats where the plants use other native vegetation as nurse plants. The

“nurse plant” effect is when a plant species uses other plants to protect itself from undesired environmental conditions, such as intense sunlight, cold front, and/or salt spray. Nurse plants have been shown to enhance plant survival and growth of the plants in their shadow (Niering et al. 1963, Padilla and Pugnaire 2006). In semi-arid plant communities, the shrub canopies act as a nurse plant and has been shown to improve the microclimatic conditions as well as the soil physical and chemical properties. On average, maximum temperature were 5 °C and 10 °C higher in open canopies as compared to under the nurse plant shrub canopies. This decrease in temperate also creates a decrease in soil water evaporation resulting in an increase in WUE. Soil nutrient, nitrogen and phosphorus, was also increased under the canopy of nurse plants (Armas and Pugnaire 2005). With limited research on herbicide intercept from nurse plants, the decrease in sunlight under the canopy can give an estimate of the reduction of an herbicidal spray to under a canopy of nurse plants. Percent light interception was measured under shrub canopies and found that a significant amount of light was reduced from upper nurse plant canopy to the lower plant canopy (Yelenik et al. 2015).

The lack of literature on this may be due to the monocultures of Kalanchoe species in many of the countries where these species are being studied (Guerra-García et al. 2018, Herrera et al. 2018). K. delagoensis plantlets have been found to have almost 100% survival in shaded conditions as compared to full sun conditions (Guerra-García et al. 2018). Kalanchoe plants and plantlets have been observed in high densities growing under native vegetation, eventually

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growing through the canopy of the other plants (anecdotal3,4 and Ileana Herrera, Ferrer-Paris,

Hernández-Rosas, & Nassar, 2016). The vegetation that the Kalanchoe grows through can intercept herbicide applications, resulting in reduced efficacy on the target species with increased non-target damage. The increased non-target damage from herbicide interception could potentially result in a localized proliferation of Kalanchoe due to the reduced competition. Two methods of promoting herbicide penetration through a canopy are to increase application carrier volume and the use of high concentrations of organosilicone (OS) adjuvant.

Increasing application carrier volume has been studied in cropping systems to increase spray coverage, increase penetration of herbicide through cover crops, and to reduce crop injury.

In a study on the control of Palmer amaranth weeds (Amaranthus palmeri) with postemergence contact herbicides, there was a correlation between increased spray volumes and increased control. When sprayed at 94 L/ha, to smaller weeds had approximately 90% control at 21 DAT with larger weeds having 80% control. When the carrier volume was increased to 187 L/ha, the smaller weed control slightly increased to approximately 93% while the larger weed control was significantly higher at 89% control. The improved control of palmer amaranth from the increased carrier volume was due to the increased spray coverage. At 94 L/ha carrier volume, there was approximately 15-22% spray coverage, depending on nozzle type, while the higher carrier volume of 187 L/ha increased spray coverage to approximately 30-48% (Berger et al. 2014). A study on the control of common cocklebur (Xanthium strumarium) investigated the efficacy of contact herbicides among carrier volumes and droplet sizes. They found that 3 weeks after treatment the application rate of 112L/ha and 250-µm droplet size had the greatest control as

3Observations of staff at St. Johns County Park and recreation Department, 2018

4 Jessica Solomon, personal observations

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compared to application rates ranging from 56L/ha to 169 L/ha and droplet size ranging from

245µm to 450µm. The improved control was due to the increase in deposition of herbicide

(Shaw et al. 2000). In another investigation on the impact of carrier volume and droplet size on herbicide, it was found that the herbicide had significantly greater control of rigid ryegrass

(Lolium rigidum) in wheat crops (Triticum aestivum) with an increase in carrier volume (Borger et al. 2013). Ferguson et. al. (2016) found that canopy penetration and spray coverage was increased by an increase in application volume rate rather than droplet size for the control of agronomic pests in oats (Avena sativa), concluding that further research was needed to understand if the minimum level of biologically effective coverage can be determined to further aid in technology selection for spraying of pest control. Sperry (2019) found a positive relationship between carrier volume and efficacy on herbicides tested in the control of barnyard grass (Echinochloa crus-galli) and waterhemp (Amaranthus tuberculatus), though the quantity of herbicide that reached the soil from high carrier volume applications was unclear. Further research is needed in the field of post-herbicide application and increased carrier volumes on canopy interception in non-cropping systems.

A more novel method of increasing penetration through a plant canopy is the use of exceptionally high concentrations of OS adjuvants. At label rates, OS adjuvants greatly reduce surface tension and increased stomatal infiltration and subsequently can increase the efficacy of the agrochemicals mixed within. The extreme spreading of OS adjuvants, especially in high concentrations and high volume combinations may lead to consolidation of spray droplets resulting in reduced spray retention and reduced weed control (Stevens 1993). The increased run- off could be beneficial in the application of herbicides to lower canopy targeted plants. OS adjuvants have been shown to reduce herbicide spray retention and consequent reduce foliar

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injury on crop species while maintain weed control (Nelson and Penner 2006a, 2006b).

Greenhouse experiments evaluated an OS adjuvant in aiding herbicide penetration through wheat crop residue to the soil surface. They found that weed control could be maximized with the inclusion of an OS adjuvant at 0.7 to 1.7% v/v, depending on the herbicide active ingredient used, assumingly from the increased spray penetration through the canopy of wheat crop residue

(Sperry 2019). Further research on increased rates of OS adjuvants on weed control through a non-target plant canopy on non-cropping systems is needed.

There have been a few biological control agents studied for the control of Kalanchoe species. In Madagascar, the native range of many Kalanchoe species, a weevil species (Osphilia tenuipes) was observed to attack K. delagoensis (i.e. B. delagoense), where the larvae bore into the main stems of plants, weakening them considerably. The adult weevils feed externally on the stem and occasionally the plantlets and leaves. In an initial screening of this weevil in Australia, where K. delagoensis is extremely invasive, it was found that O. tenuipes has a narrow field host range but will feed on seven non-target species in the Crassulaceae family. Due to the few native

Crassulaceae in Australia and need for biocontrol for K. delagoensis, O. tenuipes is considered to have potential for release (Palmer 2011, Witt 2004). Another weevil, Alcidodes sedi, was discovered in South Africa feeding on the invaded K. delagoensis (i.e. B. delagoense) and studied in laboratory screenings as a potential biocontrol in Australia as well as South Africa.

Adult and larval feeding of A. sedi caused significant reduction in stem length and number of leaves, but much like that of O. tenuipes, it feeds on many other ornamental Crassulaceae species and more host-range trials are needed (Witt et al. 2004). In greenhouse and garden settings, scale insects, leaf blotch, mealybugs, and powdery mildew have been observed as attacking Kalanchoe species, but not observed in population controlling numbers in the wild (Hannan-Jones and

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Playford 2002). Kalanchoe species has also been observed to be susceptible to infection by several viruses, affecting the growth and CAM activities but not causing death across populations (Izaguirre‐Mayoral et al. 1990). In the analysis by Herrera et al., (2012), it was found that biological agents targeting plantlets were capable of reducing the survivorship and growth of that stage and could be effective in a small/local eradication of new locations. In a separate study, simulations of K. pinnata populations in Mexico suggest that an intense (over

80%) and sustained weeding campaign, will reduce populations within a 10 year timeframe

(González de León et al. 2016).

Other methods that have been explored but not well studied in control settings for the control of Kalanchoe species include fire, pasture suppression, and engineered male-sterile plants. Fire could potentially assist in suppressing the spread of Kalanchoe species where adequate fuel load can be burned and encourage grass competition. Though herbicide application is still needed in unburnt Kalanchoe patches. Where Kalanchoe infestations are in areas to be turned into pasture or crop production, strip blade ploughing and sowing of fast growing and highly competitive native grasses and plants have been used to suppress the growth and spread of

Kalanchoe (Hannan-Jones and Playford 2002). A reliable and efficient Agrobacterium-mediated protocol was developed that engineered male-sterile K. blossfeldiana, a very common ornamental species found around the world, making the pollen of this plant not viable. The authors suspect this male-sterile approach could be combined with a female-sterility system to reduce the invasive potential of new ornamentals (García-Sogo et al. 2010). Though this method only targets the reduction of sexual reproduction and does not address the greater issue of asexual reproduction in Kalanchoe species.

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Seed germination and seedling survival rates for all mentioned Kalanchoe species are very low. Sexual reproduction is extremely low or unknown in population growth and establishment in invaded ecosystems due to limited research (González de León et al. 2016,

Herrera and Nassar 2009, Ward 2006). In all of the publications on Kalanchoe invasion and management, the concurring conclusion is that the spread and survivorship of plantlets is the key component of the life cycle contributing to the population growth and establishment (González de León et al. 2016, Guerra-García et al. 2018, Herrera et al. 2012, Herrera and Nassar 2009).

The studies in this thesis aim to find effective management techniques for Kalanchoe species. Chemical control through herbicide application is investigated at depth. We are also aiming to find techniques specifically for the use in the sensitive and ecologically important habitats of beach dunes.

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Table 1-1: Herbicides tested on Kalanchoe species in available publications Standardized Rate(s) Author(s) Location Species Herbicide(s) Product(s) Reported Rate(s) (g a.i. L-1) Reported Efficacy Armstrong and Queensland, K. fluroxypyr* Starane 200 350 mL 100 L-1 water 0.70 Effective control up to 9 months Prior, 1999 Australia delagoensis after treatment metsulfuron* Brushoff 5 g 100 L-1 water 0.03 Effective control up to 9 months after treatment 2,4-D* Tornado 2 kg 1000 L-1 water 1.40 Best method (1) 2,4-D* Baton 2 kg 1000 L-1 water 1.60 Best method (2) 2,4-D* AF300 700 mL 100 L-1 water 2.10 Effective control up to 9 months after treatment Sparkes et. al., Queensland, K. pinnata 2,4-D* Amicide 500 mL 100 L-1 water 2.50 >90% mortality after 9 weeks 2002 Australia 2,4-D* Amicide 1000 mL 100 L-1 water 5.00 ~90% mortality after 9 weeks

fluroxypyr* Starane 500 mL 100 L-1 water 1.80 Efficacy varied across sites fluroxypyr* Starane 1000 mL 100 L-1 water 3.60 ~90% mortality after 9 weeks

metsulfuron* Brushoff 5 g 100 L-1 water 0.03 Efficacy varied across sites metsulfuron* Brushoff 10 g 100 L-1 water 0.06 Efficacy varied across sites triclopyr + Grazon 350 mL 100 L-1 water 1.05 (tri.) + Efficacy varied across sites picloram* 0.30 (pic.) Guerra-Garcia Mexico K. glyphosate Takle360 14.6 g L-1 water 5.26 93% mortality after 4 weeks et. al., 2018 delagoensis (plantlets) glyphosate + Takle360 + 25 mL L-1 water (gly.) + 9.00 (gly.) + ~85% mortality after 4 weeks atrazine Coach 14.6 mL L-1 (atra.) 5.26 (atra.) metsulfuron + Anti-Yuyo + 0.1 g L-1 water (met.) + 0.06 (met.) + ~95% mortality after 4 weeks glyphosate Takle360 11.25 mL L-1 water 4.05 (gly.) (gly.) glyphosate + Takle360 + 14.6 g L-1 water (gly.)+ 5.26 (gly.) + ~90% mortality after 4 weeks diuron/paraquat Fogonazo 11.25 mL L-1 water 2.25 (diur.) + (diur./para.) 1.15 (para.) picloram/2,4-D Tordon 472 30.5 mL L-1 water 0.68 (pic.) >93% mortality after 4 weeks M 10.98 (2,4-D)

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Table 1-1: (Continued) Standardized Rate(s) Author(s) Location Species Herbicide(s) Product(s) Reported Rate(s) (g a.i. L-1) Reported Efficacy Guerra-Garcia Mexico K. et. al., 2018 delagoensis glyphosate/2,4- Desmonte 8.21 (gly) (plantlets) D plus 37.5 mL L-1 water 6.00 (2,4-D) 99% mortality after 2 weeks 2,4-D Esteron 47- 5 mL L-1 water 2.00 99% mortality after 2 weeks M *Addition of nonionic surfactant

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CHAPTER 2 Kalanchoe CONTROL AND RESPONSE TO HERBICIDE

Kalanchoe species are native to the subtropical, arid habitats of Eastern Africa to Southeast

Asia and Madagascar, with Madagascar having the highest number of species (Allorge-Boiteau

1996, Baldwin 1938). Common features for all species in the genus Kalanchoe are the succulent, bifacial leaf structure and the enlarged central storage vacuoles of the chlorenchyma cells storing organic acid with reduced intercellular air space. Kalanchoe species also tend to have a thick waxy cuticle (Chernetskyy 2012, Ting 1989). Multiple species of Kalanchoe are listed as invasive and/or noxious weeds in Australia, Mexico, South Africa, Cuba, China, Virgin Islands,

South America, and the United States (Batianoff and Butler 2002, FLEPPC 2019, Hannan-Jones and Playford 2002, Henderson 2007, LR et al. 2012, SEMARNAT 2016, Wang et al. 2016). The majority of research on the negative impacts and management of Kalanchoe species is from

Australia and Mexico, where they are high priority species (Batianoff and Butler 2002,

SEMARNAT 2016).

Four species which have been observed as invading natural areas in the United States are K. pinnata, K. daigremontiana, K. delagoensis, and K. xhoughtonii (FLEPPC 2019, Ward 2006,

2008). These four species, along with many other Kalanchoe species, have the ability to reproduce rapidly through the asexual reproduction of plantlets (Garcês and Sinha 2009, Garces et al. 2007, Johnson 1934). Plantlets are produced along the margin of the leaf during the longer days of the year (i.e. summer) and suppressed in December through February (Johnson 1934).

Each leaf of K. xhoughtonii, for example, can produce over 20 plantlets, with mature plants being able to produce hundreds of plantlets. As the plantlets mature, the slightest disturbance causes them to fall to the ground where they can rapidly root and establish. Plantlets can develop

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and spread within the same growing season as the parental plant they fell from, generating multiple asexual cycles per year that contributes to the high fecundity of these species (Herrera et al. 2012).

The consensus among researchers appears to be that the driving force of establishment is via the ability to very rapidly increase the population through asexual reproduction of plantlets during the initial phase of invasion (Chacón et al. 2009, Guerra-García et al. 2015, Herrera et al.

2012). Formation of plantlet banks (similar to seed banks) allow for Kalanchoe species to create dense monospecific patches, an effective strategy for the exclusion of interspecific competitors

(Herrera et al. 2012). Kalanchoe species do flower and produce seed, but seed germination and survival rates are very low and the amount of population growth contributed to sexual reproduction in Florida is unknown (González de León et al. 2016, Herrera and Nassar 2009,

Ward 2006). Research into the management of Kalanchoe species has consisted of a limited examination of a few herbicides, two prospective species for biocontrol, and hand removal.

There have been two weevils under investigation as possible biological control agents for

Kalanchoe species in Australia but are still in need of further research in the extent of all possible host plants (Witt 2004, Witt et al. 2004). There are no current publications on research into a biological control agent of Kalanchoe species in the United States. Other forms of control that are readily available are needed.

Hand pulling is the most discriminative form of invasive plant control, with humans selectively removing only the individual plants that need to be removed. In sensitive habitats, however, like that of dune communities, hand removal is not necessarily a beneficial form of control. This approach may involve localized disturbance with soil compaction, root disturbance,

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and trampling of native vegetation. Management regimes of hand weeding in dune systems must be considered as a form of disturbance by land managers (Mason and French 2007).

The literature on herbicidal activity across studies and Kalanchoe species is variable.

Herbicidal activity on the plantlets of K. delagoensis was investigated in controlled greenhouse experiments and included glyphosate, glyphosate + atrazine, metsulfuron + glyphosate, glyphosate + diuron + paraquat, and picloram + 2,4-D. The experiment concluded that 2,4-D and glyphosate + 2,4-D were the only effective treatments. The experiment did not utilize an adjuvant and the authors indicated this could have significantly altered the efficacy of all herbicides on penetrating the thick and waxy cuticle of K. delagoensis plantlets (Guerra-García et al. 2018).

The active ingredients atrazine, diuron, and picloram are not options for management in natural areas Florida as they are not registered to be used outside of agricultural uses and are not to be used near or around water (Baur et al. 1972, Diana et al. 2000, Pesce et al. 2006, Pratt et al.

1997). Further research in other herbicide active ingredients that are safe for use in natural areas as well as aquatic safe formulations is needed.

In an Australian field study, the herbicidal activity on K. pinnata was investigated finding that a low rate of 2,4-D was the most effective and cost-efficient treatment with over 90% kill at ten weeks after treatment. The other herbicides tested were a high rate 2,4-D, a low and high rate of fluroxypyr, low and high rate metsulfuron, and triclopyr + picloram, where efficacy varied across the multiple experimental sites (Sparkes et al. 2002). In a field herbicidal efficacy study on K. delagoensis in Australia, 2,4-D products were the most cost efficient treatments and provided control nine months after treatment with similar efficacy as the more expensive fluroxypyr and metsulfuron treatments (Armstrong and Prior 1999).

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The current commercial standard for treating K. pinnata and K. xhoughtonii in Florida is 5%

(v/v) glyphosate foliar application (Enloe et al. 2018). Glyphosate is a non-selective, broad- spectrum herbicide that prevents the production of folate and aromatic amino acids. This prevents protein synthesis, which results in a cessation of growth and subsequent death

(Henderson et al. 2010, Maeda and Dudareva 2012).

Many herbicide active ingredients and modes of action have yet to be investigated on their efficacy on Kalanchoe species. Other active ingredients registered for use in Florida natural areas should be explored. Auxin mimicking herbicides such as aminocyclopyrachlor, triclopyr amine, acid, and choline formulations, aminopyralid, and florpyrauxifen-benzyl have yet to be investigated on their potential use on control of Kalanchoe. All are approved for use in natural areas in Florida and triclopyr choline, triclopyr amine, and florpyrauxifen-benzyl are also approved for use in aquatic areas. Amino acid inhibitors imazamox, imazapyr, penoxsulam, and bispyribac-sodium are all aquatic herbicides, safe to apply in aquatic habitats, are other potential herbicide active ingredients that merit investigation. PPO inhibitors flumioxazin and carfentrazone, HPPD inhibitor topramezone, and the PSI electron divertor are all contact-type herbicides that have aquatic labels, are safe to use in natural areas, and warrant an investigation into their potential activity on Kalanchoe species as well.

There is a lack of investigations in many of the other currently available herbicide active ingredients. The available literature on herbicide efficacy are across varying Kalanchoe species and varying life stages. Kalanchoe in Florida lack natural enemies and hand removal has shown to be an ineffective method of control (Mason and French 2007). Therefore, a comprehensive herbicide efficacy study on Kalanchoe is needed. Our objective was to assess efficacy of a broad

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range of herbicide modes of action and active ingredients registered for use in aquatic areas in

Florida on K. xhoughtonii adult plants as well as on the plantlets at standard label rates.

Materials and Methods

Greenhouse studies on K. xhoughtonii were carried out in 2018 and early 2019 at the

UF/IFAS Center for Aquatic and Invasive Plants (CAIP) in Gainesville, Florida. Three studies were performed to test the efficacy of a variety of herbicide active ingredients and modes of actions on K. xhoughtonii adult plants and plantlets.

Plant material

K. xhoughtonii was collected on 7 February 2018 from a small infestation near Vilano Beach

Nature Trail in St. Augustine, Florida (29°55'06.8"N 81°17'38.2"W). Plants were removed with permission of St. Johns County Parks and Recreation. Plants were dug out with a hand shovel, placed in trays, then transported back to CAIP. The following day, each individual plant was transplanted into 6.4cm by 6.4cm by 8.9cm square plastic pots. Substrate used was 50% sand1 and 50% soil2. Plants were fertilized3 on 19 February 2018 and were then fertilized every 6- months thereafter.

Plantlet development on the leaf margins of the newly potted plants was observed four days after the initial fertilization on 23 February 2018. After plantlets matured on the leaf margins for almost three weeks and had developed at least four leaves and two roots, they were individually removed for additional study. Plantlets were gently detached from parent plants by running a finger across the leaf margin, allowing plantlets to fall into a common receptacle.

1 DOT sand, Argos USA, 924 S Main Street, Gainesville, FL 32601

2 Fafard 4P Professional Growing mix, Sun Gro Horticulture, 770 Silver Street, Agawam, MA, 01001-2907

3 4-10-3 Root Stimulator, ferti-lome, 230 FM 87, Bonham, TX 75418

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Using tweezers, plantlets were randomly selected and transplanted into 100% sand potting medium into 8.9cm by 8.9cm by 8.9cm square plastic pots. Plantlets were placed into three rows of four, totaling 12 plantlets per pot. Plantlets were allowed to grow in transplanted pots for at least 3 weeks prior to first experiment. Plantlets were replaced with live ones if any died after transplant. We had over 99% success rate with only three of 1,152 plantlets dying and needing replacement due to the transplant process.

All plants and plantlets were maintained in a greenhouse covered with a 50% shade cloth and temperature set at 31°C, though air temperature fluctuated between 18°C and 35°C throughout the duration of the experiments. Plants were watered two to three times a week, allowing potting medium to dry completely between watering, as this is common in the dune habitats where these plants proliferate. Plants exhibited no visual symptoms of water stress at any time during each experiment.

All adult K. xhoughtonii plants and plantlet pots were randomly selected for each trial of each experiment and numbered. Ten adult plants and six plantlet pots (72 plantlets total) were randomly selected for each treatment within each trial of each experiment. All plantlets were under 2cm in height at time of treatments.

Herbicide Treatments and Data Collection

Experiment 1 herbicide treatments occurred on 24 April 2018 and 19 June 2018 for trial 1 and 2, respectively. Experiment 1 treatments consisted of seven auxin mimicking herbicide treatments and one untreated control treatment. Herbicide treatments included the active

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ingredients: aminocyclopyrachlor4, fluroxypyr5, triclopyr amine6, triclopyr acid7, triclopyr choline8, aminopyralid9, and florpyrauxifen-benzyl10. Experiment 2 herbicide treatments occurred on 14 July 2018 and 26 September 2018 for trials 1 and 2, respectively. Experiment 2 treatments consisted of four ALS inhibitor herbicides, one EPSP synthase inhibitor, and an untreated control. Herbicide treatments included the active ingredients: imazamox11, imazapyr12, penoxsulam13, bispyribac-sodium14, and glyphosate15. Experiment 3 herbicide treatments occurred on 24 October 2018 and 14 January 2019 for trials 1 and 2, respectively. Experiment 3 treatments consisted of two PPO inhibitors, one HPPD inhibitor, one PSI electron divertor, and an untreated control. Herbicide treatments included the active ingredients: flumioxazin16, carfentrazone17, topramezone18, and diquat19 (Table 2-1).

4 Method® 240SL, Bayer Environmental Science, 2 T. W. Alexander Drive, Research Triangle Park, NC 27709

5 Vista® XRT, Dow AgroSciences, 9330 Zionsville Rd, Indianapolis, IN 46268

6 Garlon® 3A, Dow AgroSciences, 9330 Zionsville Rd, Indianapolis, IN 46268

7 Trycera®, Helena Agri-Enterprises, LLC, 25 Schilling Blvd., Collierville, TN 38017

8 Vastlan®, Dow AgroSciences, 9330 Zionsville Rd, Indianapolis, IN 46268

9 MilestoneTM, Dow AgroSciences, 9330 Zionsville Rd, Indianapolis, IN 46268

10 ProcellaCOR™ SC, SePRO Corporation, 11550 North Meridian Street, Suite 600, Carmel, IN 46032

11 Clearcast®, SePRO Corporation, 11550 North Meridian Street, Suite 600, Carmel, IN 46032

12 Habitat®, SePRO Corporation, 11550 North Meridian Street, Suite 600, Carmel, IN 46032

13 Galleon® SC, SePRO Corporation, 11550 North Meridian Street, Suite 600, Carmel, IN 46032

14 TradewindTM, Valent U.S.A. Corporation, P.O. Box 8025, Walnut Creek, CA 94596-8025

15 Roundup CustomTM, Monsanto Company, 800 N. Lindbergh Blvd., St. Louis, MO 63167

16 Clipper TM, Valent U.S.A. Corporation, P.O. Box 8025, Walnut Creek, CA 94596-8025

17 Stingray®, SePRO Corporation, 11550 North Meridian Street, Suite 600, Carmel, IN 46032

18 Oasis®, SePRO Corporation, 11550 North Meridian Street, Suite 600, Carmel, IN 46032

19 Reward®, Syngenta Crop Protection, LLC, P.O. Box 18300, Greensboro, NC 24719-8300

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On the day of treatment, all plants and plantlets were watered prior to application. All plantlets on adult plants were gently removed just prior to herbicide application in order to observe post-treatment plantlet development. Baseline measurements included plant height and number of leaves with height being measured from the soil surface to the maximum leaf height.

Every 30 days after treatment, number of live leaves and number of live plantlets per pot were counted. A ‘live’ adult leaf was defined as a leaf where the petiole of the leaf had no succulence but could still show succulence in the anterior portion of the leaf. Plantlet development and flowering on treated adult plants was also noted throughout the experiments. A ‘live’ plantlet was defined as having any leaf succulence and green coloration; any plantlet that was completely brown and desiccated was recorded as dead.

For herbicide treatments, established plants and plantlets were removed from the greenhouse and placed in rows on the ground, ensuring no plants were in another plants’ spray shadow. Foliar applications were made with a three-nozzle boom using TeeJet® 8003 nozzles20

- spaced 45.7cm apart. We used a CO2 pressurized backpack sprayer at the equivalent of 374 L ha

1 and an operating pressure of 40 psi (275.8 kPa). Each treatment consisted of one pass across the row of plants and plantlets with a constant boom height of approximately 1m above the ground.

All treatments, including the untreated control, included a nonionic surfactant21 (0.25% v/v) or a methylated seed oil22 at 2.34 L ha-1. After treatment, plants and plantlets were allowed to dry in open air until leaf surfaces were dry. Plants were then moved back into greenhouse and randomized across the bench.

20 TeeJet® nozzle, TeeJet Technologies Illinois, LLC, 1801 Business Park Drive, Springfield, IL 62703

21 Induce NIS, Helena Agri-Enterprises, LLC, 25 Schilling Blvd., Collierville, TN 38017

22 MSO Concentrate, Loveland Products, Inc., 14520 Co Rd 64, Greeley, CO 80631

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Each experimental trial was carried out to different end dates, depending on the herbicide mode of action being tested and the length of time needed to observe complete herbicide activity.

Each trial of experiments 1 (Auxin mimics) and 3 (Misc. Herbicides) were carried out to 45 days after treatment, while experiment 2 (Amino acid inhibiting herbicides) was carried out to 80 days after treatment. Experiments involving plantlets were carried out to 125 DAT to ensure the herbicidal efficacy was visually observable as ‘live’ or ‘dead’ on the small leaf surfaces. With the exception of trial 1 of experiment 1, the plantlet data was only carried out to 45 DAT, which was the original protocol for plantlets to match the end date of the adult plants. The protocol for plantlets was extended after this experimental trial to 125 DAT. A second trial of the auxin study on plantlet efficacy is still needed to verify the data presented is accurate. Data represented on plantlets for experiment 1 is first represented to 45 DAT for both trials and then 125 DAT for the second trial.

At the end of each experimental run, either 45 or 80 days after treatment, live leaf count and plantlet development was noted. Additionally, live plants were separated into above and below ground plant material, dried at 65°C, and weighed for comparison across treatments. To test the viability of the plantlets that developed on the treated adult plants, all plantlets from individual adult plants were placed in 100% sand potting medium in 6.35cm by 6.35cm by 8.9cm tall square plastic pots and maintained for 30 days in greenhouse. A ‘live’ plantlet was defined as previously stated.

Statistical Analyses

Experiments were conducted using a randomized complete block design with two experimental trials for each of the three experiments. Data was analyzed in Rstudio® (RStudio

Team 2015) as a single factorial ANOVA with herbicide as a fixed variable. Tukey’s honest significant difference test (Tukey’s HSD) was performed for post-hoc analysis with a 95%

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confidence interval (P<0.05) using the ‘emmeans’ package in Rstudio (Lenth 2019, RStudio

Team 2015). If there was a missing value, for example a missing shoot biomass number, the average shoot biomass for that experimental trial was used in place. All data, including live leaf count, above ground biomass, below ground biomass, and live plantlet count, was checked for and achieved normality and homogeneity of variance. Plantlet data were subjected to arcsine and square root transformation for nonparametric analysis of percentage live (Ahrens et al. 1990).

Results and Discussion

Experiment 1: Auxin Herbicides

There was a significant difference between trial 1 and trial 2 for plant height and live leaf number for adult K. xhoughtonii for baseline (0 DAT) measurements (P<0.0001). This was likely due to the experimental trials being separated by multiple weeks, and the plants continued to grow. Therefore, there was a significant difference in plant size between trials. At the end of both experimental trials (45 DAT), there was a significant difference between treatments and trials

(P<0.0001), but no significant interaction between treatment and trial for live leaf number

(P=0.0827). Therefore, data was pooled across trials for live leaf count for this experiment. The live leaf count 45 DAT measurements showed the florpyrauxifen-benzyl treatment reduced leaf number slightly compared to the untreated control. The rest of the treatments significantly reduced the number of live leaves on adult K. xhoughtonii plants to very low numbers compared to the untreated control (Figure 2-1). The shoot and root biomass data indicated that the florpyrauxifen-benzyl treatment performed similar to or only slightly better than the untreated control with the rest of the herbicides resulting in significant shoot and root biomass reduction

(Table 2-2).

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Live plantlet count data at 45 DAT indicated a significant effect of treatment (P<0.0001), trial (P<0.0001), and the interaction between treatment and trial (P<0.0001). At 45 DAT, the triclopyr acid treatment provided the greatest control of plantlets across trials.

Aminocyclopyrachlor, fluroxypyr and triclopyr amine provided variable control between trials with significant plantlet mortality in the first trial and significantly less plantlet mortality in the second trial (Figure 2-2). For trial 2 that was carried out to 125 DAT, the plantlet control improved for most treatments and resulted in similar results as the adult treated plants, with the florpyrauxifen-benzyl treatment performing only slightly better than the untreated control and the rest of the treatments significantly reducing plantlets to 0 or 1 live plantlets per pot (Figure 2-

3). However, this study needs to be repeated on plantlets to 125 DAT in order to verify these findings. Trial 3 for the auxin herbicide experiment on plantlets was initiated on 5 November

2019 to repeat trial 2 out to 125 DAT.

For trial 1, plantlets developed on adult K. xhoughtonii plants for all treatments except for the aminocyclopyrachlor and aminopyralid. For the treatments that plantlets developed, all but the triclopyr acid treatment produced viable plantlets during the 4-week assessment period. For trial 2, only triclopyr choline, aminopyralid, and florpyrauxifen-benzyl treatments produced plantlets. Of the plantlets produced on the treated plants in trial 2, all succeeded in the 4-week assessment period except for the aminopyralid treatment. Plantlets were able to successfully develop and mature on plants treated with triclopyr choline and florpyrauxifen-benzyl in both trials (Table 2-3).

Overall, all auxin-type herbicides tested provided adult and plantlet K. xhoughtonii mortality, with the exception of the florpyrauxifen-benzyl treatment. These results are similar to the previous herbicide efficacy studies where 2,4-D and fluroxypyr have demonstrated K.

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delagoensis or K. pinnata mortality (Armstrong and Prior 1999, Guerra-García et al. 2018,

Sparkes et al. 2002). It is possible that the reduced activity observed with florpyrauxifen-benzyl was due to the use rate utilized for these experiments. The maximum allowed rate according to the EPA’s approved label in the state of Florida is 59.9 g ae ha-1, the rate used in these experiments. However, due to the significant control observed with the other auxin mimicking herbicides, an increased application rate or tank mixes with florpyrauxifen-benzyl could be effective and could be investigated in future Kalanchoe management research.

Aminocyclopyrachlor, triclopyr acid, and aminopyralid treatments did not produce viable plantlets on treated plants in both trials, indicating that these are likely to be the most consistent and beneficial of the auxin mimic herbicides. Aminocyclopyrachlor and aminopyralid are effective broadleaf herbicides that may be used in the presence of perennial grasses without causing damage. Neither herbicide should not be applied directly to water and aminocyclopyrachlor should also not be used in natural areas where conifers, deciduous trees, and some ornamental shrubs and trees (ex: crape myrtle, hydrangea, and magnolia) are present

(Anonymous 2015). Triclopyr acid, amine, and choline formulations are permitted for use in aquatic areas, have low soil mobility and are permitted to use in natural areas. However, triclopyr ester is toxic to fish and should not be applied directly to water. Conifer species can also be sensitive to triclopyr acid and care should be taken when treating with this active ingredient

(Anonymous 2011a, 2016, Enloe et al. 2018).

Experiment 2: Amino Acid Inhibiting Herbicides

There was a significant difference between trial 1 and trial 2 for plant height and live leaf number for adult K. xhoughtonii for baseline (0 DAT) measurements (P<0.0001). This was likely due to the experimental trials being separated by multiple weeks, and the plants continued to grow. Therefore, there was a significant difference in plant size between trials. At the end of both

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experimental trials (80 DAT), there was a significant difference between treatments and trials

(P<0.0001) as well as a significant interaction with trial for live leaf number (P<0.0001), shoot biomass (P=0.0001), and root biomass (P=0.0001). For live leaf count (Figure 2-4) and shoot and root biomass (Table 2-4), only the glyphosate, imazamox, and imazapyr treatments provided significant control in both trials.

For plantlet control in this experiment, there was a significant effect for treatment

(P<0.0001), trial (P=0.0002), and an interaction between the two (P=0.0002) (Figure 2-5).; therefore data was separated by trials. Glyphosate had the most reliable plantlet control between both trials. Trial 1 showed significant control by glyphosate and imazapyr. Trial 2 showed limited control with glyphosate and bispyribac-sodium. These differences of efficacy across these treatments between trials was driving the interaction. This interaction showed substantial variability and consequently, difficulty in controlling plantlets in regulated greenhouse settings.

This efficacy variability would likely lead to greater variability of Kalanchoe control in the field.

Glyphosate and imazapyr treatments were the only herbicides in this experiment that did not produce viable plantlets on the treated adult plants in both trials after the 4-week assessment period. Imazamox only had a limited amount of adult plants produce viable plantlets after treatment in the second trial with no adult plants producing viable plantlets in the first trial

(Table 2-3).

Previously tested amino acid inhibiting herbicides were metsulfuron and glyphosate.

Significant control of K. delagoensis adult plants and plantlets and K. pinnata was seen with glyphosate products and varied control with metsulfuron (Armstrong and Prior 1999, Guerra-

García et al. 2018, Sparkes et al. 2002). The previously examined amino acid inhibiting herbicide

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results are similar to results found here, with glyphosate having significant control. However, we saw varied control across the other amino acid inhibiting herbicides.

Glyphosate and imazapyr were the most effective and consistent of the auxin mimicking herbicides in respect to adult plant death, plantlet death, as well as not producing viable plantlets post-treatment. Glyphosate is safe to use in natural areas and has many aquatic safe formulations.

It has little potential for causing nontarget damage from to root absorption, due to negligible mobility in the soil, although care must be taken to avoid contact with non-target vegetation

(Enloe et al. 2018, Maeda and Dudareva 2012). Imazapyr can be applied directly to water and controls most grasses, and broadleaf weeds. Precaution is needed when applying imazapyr near non-target species, especially oaks and other hardwoods, due to its persistence in the soil and high potential for subsequent root absorption (Anonymous 2011b, Enloe et al. 2018).

Experiment 3: Miscellaneous Herbicides

Though there was a significant difference between treatments and trials for adult plant live leaf number (P<0.0001) and shoot and root biomass (P<0.0001), there was no adult plant mortality for any of the herbicides tested in this experiment. All herbicide treatments resulted in a rapid burndown of all foliage within the first 15-days. However, plants flushed out new shoot growth by 45 DAT (Figure 2-6). By 45 DAT, shoot biomass was significantly reduced compared to the untreated control for all herbicides in trial 1 and only flumioxazin and carfentrazone in the second trial (Table 2-5). For root biomass, the first trial showed significant reduction for diquat, flumioxazin, and carfentrazone as compared to the untreated control. In the second trial for root biomass, flumioxazin was the only herbicide that had significant reduction as compared to the untreated control.

For plantlet control, there was a significant difference between treatments (P<0.0001), trials (P=0.0002), and an interaction between the two (P=0.0002). Flumioxazin in both trials

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provided significant plantlet control and trial 1 showed marginal plantlet control with diquat. All other herbicides did not have a significant reduction in live plantlets as compared to the untreated control (Figure 2-7).

There were no herbicides in this experiment that did not prevent viable plantlet production on the treated adult plants. Flumioxazin and carfentrazone did not produce viable plantlets in the first trial but viable plantlets were produced on the majority of the treated adult plants in the second trial after the 4-week assessment period (Table 2-3).

All herbicides tested in this experiment did not provide adequate reduction in adult plant live leaves, biomass, or subsequent death in adult plants. Viable plantlets were produced on all treated adult plants. Flumioxazin was the only herbicide that provided any control of plantlets in both trials. Flumioxazin is a broad spectrum contact herbicide that has aquatic safe formulations and is commonly used on agriculture and food crop weeds (Wisconsin Department of Natural

Resources 2012). This herbicide would be beneficial in treating areas that have had adult plants removed or in a tank mix with another herbicide active ingredient that has proven efficacy on adult plants.

Conclusion

Land managers should select the herbicide that is adequate for the habitat for which

Kalanchoe has invaded. Between all three experiments, the herbicide active ingredients aminocyclopyrachlor, triclopyr acid, aminopyralid, glyphosate, and imazapyr all showed significant control of adult plants, had the most consistent control of plantlets, and did not produce viable plantlets post-treatment on adult K. xhoughtonii plants. Flumioxazin did not control adult plants but showed significant control on plantlets. Where possible, tank mixes of different modes of action could be used to increase control of all life stages of Kalanchoe in a single foliar application.

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There was variability in mortality for adult plants across experiments and between each trial. This is possibly due to the difference in plant size, and therefore the available above and below ground biomass, between trial one and trial two. It is possible that the larger plants were able to recover from the herbicide stress due to the greater biomass available. This hypothesis needs to be directly tested by conducting herbicide treatments on similar sized-large plants and similar sized-small plants and comparing defoliation, plantlet productions, and mortality between sizes. The variability in the controlled greenhouse settings suggests that herbicide performance in the field will produce even more variability. There was also variability observed in plantlet control between trials across all experiments as well as compared to the herbicide efficacy of adult K. xhoughtonii. There was less control observed in plantlets as compared to adult plants, especially when looking at similar timeframes of 45 DAT. This difference in efficacy may be due to the limited leaf space for a foliar application. It is also possible that there is a dormancy factor in effect, that has greatly reduced or halted the photosynthetic processes of plantlets, and subsequently greatly reducing the movement of water and chemical uptake. Dormancy of plantlets while still attached to the mother leaf has been determined to occur after the early stages of differentiation, preventing the continuation of development of plantlets prior to leaf and root development. During the winter months or nonideal conditions, the embryonic tissue remains dormant on the mother plant’s leaf notches. Dormancy can be broken by longer days, in some species leaf severing, or when more ideal growing condition warrant it (Johnson 1934, Stoudt

1938). There is little known about the potential for plantlets to return to a dormant state once detached from the parental plant.

Further research on herbicide efficacy of Kalanchoe adult plants and plantlets could include the use of herbicide tracer experiments. The movement of the different active ingredients

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and modes of action could be beneficial in understanding the differences of efficacy across the different size classes. Plantlet control in the Auxin herbicide experiment (Experiment 1) will be repeated in order to validate findings out to 125 DAT. More research is also needed in the efficacy of the herbicide active ingredients tested here in the control of other Kalanchoe species as well as varying rates of the herbicides. Florpyrauxifen-benzyl is an aquatic safe auxin mimic that has proven effective against a multitude of broadleaf agronomic weeds as well as herbicide- resistant barnyardgrass and warrant more testing on its efficacy on Kalanchoe species (Epp et al.

2016, Ryan Miller et al. 2017). K. xhoughtonii adult plants and plantlets were examined in these studies, but the efficacy of the same herbicides at varying rates on the other Kalanchoe species of concern, such as K. pinnata, K. daigremontiana, and K. delagoensis, is needed. Integrated management plans (IMP) with multiple control methods should also be investigated. Further research on IMP for Kalanchoe species could include hand removal of larger plants and chemical control of the smaller plants and plantlets as well as the combination of mowing and chemical control. Application timing, such as during winter or summer, and its effect on plantlet development and efficacy on adult plants and plantlets in the field should also be investigated.

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Table 2-1: Kalanchoe xhoughtonii herbicide trials list of active ingredients, mode of action, and the application rates, sorted by experiment (Exp). Each experiment also included the use of a non-ionic surfactant, including the untreated controls.

Standardized rate Exp Mode of Action Active Ingredient (g ae ha-1) 1 Auxin mimic aminocyclopyrachlor 314 Auxin mimic fluroxypyr 565 Auxin mimic triclopyr amine 3,362 Auxin mimic triclopyr acid 3,366 Auxin mimic triclopyr choline 3,362 Auxin mimic aminopyralid 123 Auxin mimic florpyrauxifen-benzyl 60 -- untreated control 1 -- 2 EPSP Synthase Inhibitor glyphosate 6,051 ALS inhibitor imazamox 898 ALS inhibitor imazapyr 898 ALS inhibitor penoxsulam 98 ALS inhibitor bispyribac-sodium 112 -- untreated control 2 -- 3 PPO inhibitor flumioxazin 284 PPO inhibitor carfentrazone 224 HPPD inhibitor (type of carotenoid topramezone 393 biosynthesis inhibitor) PSI Electron diverter diquat 281 -- untreated control 3 --

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Figure 2-1: Kalanchoe xhoughtonii adult plant live leaf count response to auxin herbicide treatments (Experiment 1) at 45 days after treatment. Data is pooled across both trials due to no significant difference (P=0.0827) Means with the same letter are not different (P<0.05) and error bars represent ± 1 standard error.

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Table 2-2: The average shoot and root dried biomass for Experiment 1, auxin mimicking herbicides. Data and letters of significance are separated by trial and biomass type (shoot and root). Means within the same column with the same letter are not significantly different (P<0.05). Average Shoot Average Root Biomass (g) Biomass (g) Trial 1 Trial 2 Trial 1 Trial 2 Treatment (SEa = 0.446) (SE = 0.582) (SE = 0.070) (SE = 0.048) aminocyclopyrachlor 0.70 (b) 0.00 (b) 0.05 (b) 0.00 (c) fluroxypyr 0.58 (b) 0.00 (b) 0.10 (b) 0.00 (c) triclopyr amine 1.50 (b) 1.75 (b) 0.15 (b) 0.15 (c) triclopyr acid 1.71 (b) 0.00 (b) 0.24 (b) 0.00 (c) triclopyr choline 1.24 (b) 0.70 (b) 0.14 (b) 0.07 (c) aminopyralid 1.72 (b) 1.23 (b) 0.15 (b) 0.09 (c) florpyrauxifen-benzyl 6.40 (a) 8.77 (a) 0.92 (a) 0.97 (b) untreated control 6.68 (a) 10.22 (a) 1.10 (a) 1.30 (a) a SE = Standard Error

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Figure 2-2: Kalanchoe xhoughtonii plantlet response to auxin herbicide treatments (Experiment 1) at 45 days after treatment. Data and letters of significance are separated by trial due to significant difference (P<0.0001). Means with the same letter within trials are not different (P<0.05) and error bars represent ± 1 standard error. Lowercase letters refer to trial 1 and uppercase refer to trial 2.

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Figure 2-3: Kalanchoe xhoughtonii plantlet response to auxin herbicide treatments (Experiment 1) at 125 days after treatment for trial 2 only. Means with the same letter are not different (P<0.05) and error bars represent ± 1 standard error.

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Table 2-3: Plantlet development on treated Kalanchoe xhoughtonii adult plants. Numbers indicates the number of adult plants that plantlets developed on out of the total amount of adult plants in the trial. Yes/no indicates plantlet survival at the end of a 4-week assessment period after transplant to another pot (i.e. Plantlets survival). Trial 1 Trial 2 Plantlets Plantlet Plantlets Plantlet Experiment Treatment developed survival developed survival 1: Auxins aminocyclopyrachlor 0/10 - 0/10 - fluroxypyr 10/10 yes 0/10 - triclopyr amine 3/10 yes 0/10 - triclopyr acid 2/10 no 0/10 - triclopyr choline 7/10 yes 2/10 yes aminopyralid 0/10 - 3/10 no florpyrauxifen-benzyl 10/10 yes 6/10 yes untreated control 1 10/10 yes 10/10 yes 2: Amino Acid glyphosate 0/10 - 0/10 - Inhibitors imazamox 0/10 - 1/10 yes imazapyr 0/10 - 2/10 no penoxsulam 10/10 yes 10/10 yes bispyribac-sodium 4/10 yes 1/10 yes untreated control 2 10/10 yes 10/10 yes 3: Misc. flumioxazin 0/10 - 6/10 yes carfentrazone 0/10 - 8/10 yes topramezone 9/10 yes 9/10 yes diquat 5/10 yes 10/10 yes untreated control 3 6/10 yes 10/10 yes

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Figure 2-4: Kalanchoe xhoughtonii adult live leaf response to amino acid inhibitor herbicide treatments (Experiment 2) at 80 days after treatment. Data are separated by trial (P<0.0001). Means with the same letter are not different (P<0.05) and error bars represent ± 1 standard error. Lowercase letters refer to trial 1 and uppercase refer to trial 2.

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Table 2-4: The average shoot and root dried biomass for Experiment 2, amino acid inhibiting herbicides. Data are separated by trial and biomass type (shoot and root). Means within the same column with the same letter are not significantly different (P<0.05). Average Shoot Average Root Biomass (g) Biomass (g) Trial 1 Trial 2 Trial 1 Trial 2 Treatment (SEa = 0.275) (SE = 0.216) (SE = 0.078) (SE = 0.097) glyphosate 0.00 (d) 0.00 (d) 0.00 (c) 0.00 (c) imazamox 0.41 (d) 6.87 (c) 0.11 (c) 1.06 (b) imazapyr 0.00 (d) 4.32 (cd) 0.00 (c) 0.58 (bc) penoxsulam 6.57 (b) 14.14 (ab) 1.40 (ab) 2.49 (a) bispyribac-sodium 4.26 (c) 8.99 (bc) 1.04 (b) 2.11 (a) untreated control 9.24 (a) 16.32 (a) 1.74 (a) 2.92 (a) a SE = Standard Error

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Figure 2-5: Kalanchoe xhoughtonii plantlet response to amino acid inhibitor herbicide treatments (Experiment 2) at 125 days after treatment. Data and letters of significance are separated by trial due to significance difference (P=0.0002). Means with the same letter are not different (P<0.05) and error bars represent ± 1 standard error. Lowercase letters refer to trial 1 and uppercase refer to trial 2.

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Figure 2-6: Kalanchoe xhoughtonii adult live leaf response to miscellaneous herbicide treatments (Experiment 3) at 45 days after treatment. Data and letters of significance are separated by trial due to significance difference (P=0.003). Means with the same letter are not different (P<0.05) and error bars represent ± 1 standard error. Lowercase letters refer to trial 1 and uppercase refer to trial 2.

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Table 2-5: The influence of herbicide on average shoot and root dry weight in grams for Experiment 3. Treatment means sharing the same letter, within a column are not significantly different according to Tukey’s honestly significant difference test with 95% confidence intervals at P ≤ 0.05 in parentheses. Average Shoot Average Root Biomass (g) Biomass (g) Trial 1 Trial 2 Trial 1 Trial 2 Treatment (SEa = 1.43) (SE = 0.822) (SE = 0.141) (SE = 0.168) flumioxazin 5.04 (b) 8.64 (c) 1.43 (bc) 2.00 (b) carfentrazone 7.15 (b) 11.90 (bc) 1.38 (bc) 2.27 (ab) topramezone 10.70 (b) 16.84 (a) 1.92 (ab) 2.93 (a) diquat 5.12 (b) 14.58 (ab) 1.06 (c) 2.92 (a) untreated control 17.43 (a) 15.36 (a) 2.45 (a) 2.92 (a) a SE = Standard Error

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Figure 2-7: Kalanchoe xhoughtonii plantlet response to miscellaneous herbicide treatments (Experiment 3) at 125 days after treatment. Data and letters of significance are separated by trial due to significance difference (P<0.0001). Means with the same letter are not different (P<0.05) and error bars represent ± 1 standard error. Lowercase letters refer to trial 1 and uppercase refer to trial 2.

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CHAPTER 3 OVERCOMING THE NURSE PLANT EFFECT TO CONTROL Kalanchoe WITH HERBICIDE VIA CANOPY PENETRATION

Herbicides within differing modes of action have been identified that control K. xhoughtonii adult plants and plantlets (Chapter 2). The active ingredients aminocyclopyrachlor, triclopyr acid, aminopyralid, glyphosate, and imazapyr all showed significant control in greenhouse trials and warrant field testing to examine efficacy in real world settings. K. delagoensis and K. xhoughtonii have been observed as invading beach dunes in St. Augustine,

Florida and are a primary concern of this research (anecdotal1,2 and EDDMapS 2019).

The Kalanchoe species of concern are often observed growing under and through other dune swale vegetation, using other plants to shade from full sun, frosts, salt spray, and other undesirable environmental conditions. This is referred to as the ‘nurse plant’ effect, where a species grows under the canopy of another that serves as protection. This is especially important during the more vulnerable immature phase of a plant’s life cycle (Niering et al. 1963, Padilla and Pugnaire 2006). Nurse plant effects are often critical to many species’ success in hot, dry environments. Under shrub canopies in semi-arid plant communities, the microclimatic conditions as well as the soil physical and chemical properties are more conducive to successful establishment of some species. Armas and Pugnaire (2005), found that maximum temperatures were 5°C to 10°C higher in open canopies compared to beneath nurse plant shrub canopies. This decrease in temperature also creates a decrease in soil water evaporation, resulting in an increase in the water use efficiency (Armas and Pugnaire 2005).

1Observations of staff at St. Johns County Park and recreation Department, 2018

2 Jessica Solomon, personal observations

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Kalanchoe plantlets have a wide tolerance to light and water conditions, making them more likely to survive in diverse habitats, though they have been observed as more successful in shaded conditions (Guerra-García et al. 2018). In a study on K. delagoensis survivorship in varying light and water conditions, it was found that in the most extreme conditions (100% sunlight and no water for 60 days) there was approximately 12% plantlet survival. In less severe conditions, similar to the conditions under the canopy of other vegetation (70-100% water and

70% shade), survival was almost 100% (Guerra-García et al. 2018). In an investigation of recruitment of Stapelia gigantea and K. daigremontiana in arid environments of South America, the species were found at a significantly higher abundance under native trees compared to open spaces (Herrera et al. 2016).

Due to Kalanchoe species’ observed propensity to colonize habitat underneath other vegetation as nurse plants, targeting Kalanchoe with a foliar herbicide application in the composition of species of the beach dunes of St. Augustine may be difficult. The nurse plants may not only prevent herbicide spray from reaching the majority of the target species in the lower canopy but may also be injured or killed. No published literature was found on the difficulty of herbicide application to Kalanchoe species in habitats where it uses the canopy of other vegetation to serve as nurse plants. The lack of literature on this may be due to the monotypic stands of Kalanchoe species in many of the countries where these species are being studied (Guerra-García et al. 2018, Herrera et al. 2018). There was also no available data found on spray intercept from nurse plants that did not include the use of pre-emergent herbicides. This is possibly due to the majority of available research on spray quality and retention being conducted in agricultural cropping systems.

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The native vegetation that the species of Kalanchoe grow through can intercept herbicide applications, potentially resulting in reduced efficacy on the target species with increased non- target damage. One method of promoting herbicide penetration through a canopy is to increase application carrier volume. An alternative method to canopy penetration is the use of exceptionally high concentrations of organosilicone (OS) adjuvant (Berger et al. 2014, Ferguson et al. 2016, Sperry 2019).

A method used in cropping systems to increase spray coverage, increase penetration of herbicide through cover crops, and to reduce crop injury, is to increase the application carrier volume. In multiple studies on higher carrier volume for herbicide application on the control of crop weeds, an increase in carrier volume will increase the spray coverage. An increase in spray coverage, especially in combination with contact-type herbicides, has shown to improve weed control in cropping systems such as wheat (Triticum aestivum), peanut (Arachis hypogaea), and corn (Zea mays) on weeds such as Palmer amaranth (Amaranthus palmeri), common cocklebur

(Xanthium strumarium), and rigid ryegrass (Lolium rigidum) (Berger et al. 2014, Borger et al.

2013, Shaw et al. 2000). In a study on the control of agronomic pests in oat (Avena sativa) cropping systems, it was found that by increasing application volume there was an increase in canopy penetration and spray coverage. They concluded that canopy penetration could be improved with proper nozzle selection and operation parameters (Ferguson et al. 2016). Sperry

(2019) found a positive relationship between carrier volume and efficacy to assist herbicide penetration through wheat straw mulch for the control of barnyard grass (Echinochloa crus- galli) and waterhemp (Amaranthus tuberculatus). Though, the authors indicated that the quantity of herbicide that reached the soil from the high carrier volume applications was unclear and further research is needed.

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Another method of increasing spray penetration through a plant canopy is through the use of high concentrations of OS adjuvants. OS adjuvants greatly reduce surface tension, increase stomatal infiltration and subsequently can increase the efficacy of the pesticides when applied at label rates (Stevens 1993). The extreme spreading of OS adjuvants, especially in high concentrations and high volume combinations may lead to consolidation of spray droplets resulting in reduced spray retention and reduced weed control (Stevens 1993). This increased run-off is potentially beneficial when targeting lower canopy plants. OS adjuvants have been shown to reduce herbicide spray retention and foliar injury on crop species while maintaining weed control (Nelson and Penner 2006a, 2006b). Greenhouse experiments evaluated an OS adjuvant in aiding herbicide penetration through wheat crop mulch to the soil surface. It was found that weed control could be maximized with the use of an OS adjuvant at concentrations of

0.7 to 1.7% (v/v), approximately twice label rates (Sperry 2019).

The Kalanchoe species observed in the beach dunes of St. Augustine, FL are typically observed growing through the canopy of native shrubs and forbs with dense stands of juvenile plants in the understory. The open spaces of the dune swales are generally scarcely populated with Kalanchoe. Invasive plant species in dune systems can reduce or prevent natural dune formation, encourage erosion, alter vegetation structure, alter nutrient availability, reduce native plant and wildlife densities, and disrupt ecological function (Castillo and Moreno-Casasola 1996,

Gordon 1998, Marshall et al. 2014, Williams 2007). Invasive plant management in beach dune communities can be difficult due to destabilization of dunes from plant removal, negative public perception of chemical application, low nutrient and droughty conditions of soil resulting in slow recovery of native vegetation, and the need for long-term monitoring following removal ((FNAI)

2010, FNAI 2010, Williams 2007). Hand weeding is considered impractical other than on a

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small scale for non-rhizomatous species, requiring constant vigilance and potentially causing a disturbance that could, consequently, encourage more nonnative species (Bergin 2011, Gordon

1998).

An adapted application technique is needed for the use of herbicides in dune communities that will result in little-to-no non-target damage, especially for the treatment of invasive species that reside in the understory of the native vegetation. An increase in canopy penetration to the lower vegetation as well as a decrease in upper canopy retention is a desirable outcome. Our objective was to examine the canopy interaction of spray on Kalanchoe in beach dune settings.

Specifically, we were looking to explore the difference in penetration of spray through different application rates and OS concentrations. We hypothesized that the higher application volume will result in greater penetration of spray to the lower vegetation. We also hypothesize that the higher OS concentration will result in greater penetration of spray into the lower vegetation through the upper canopy. The hypothesized increased spray penetration is thought to be due to greater run-off from the upper canopy with a higher OS concentration and, therefore, should result in a reduced spray retention on the upper canopy as compared to the lower concentration of OS.

Materials and Methods

Field Site

A field study was carried out on 29 August 2019 at Butler Beach in St. Augustine,

Florida (29°47'16.3"N 81°15'29.3"W) (Figure 3-1) to evaluate the effect of different concentrations of OS surfactant and application volumes on penetration of spray to the lower level canopy. K. xhoughtonii populates the swale between the primary and secondary beach dunes at densities of approximately 150 plants and plantlets per meter on average in patches, under the native vegetation. This density was calculated by counting individual plants in one

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quarter of a plot for four plots. The dominant native plant species included Dune greenbrier

(Smilax auriculata), Partridge pea (Chamaecrista fasciculata), Indian blanket flower (Gaillardia pulchella), and Beach sunflower (Helianthus debilis). On the day of spraying, the wind ranged from 0 to 9.8 km hr-1. Test plots 1m2 in size were set up in a swale invaded with K. xhoughtonii with a minimum of 1m2 spacing between each plot. Percent canopy of individual species was estimated by two observers for each plot and added together for a total canopy coverage for each plot that could exceed 100% due to the multiple layering of the canopy structure. In order to separate the species into two size classes, K. xhoughtonii cover was separated between less than10cm and greater than10cm in height. It is worth mentioning that the majority of K. xhoughtonii observed over 10cm in height in the plots were considerably taller than 10cm, mostly growing up an into the upper canopy with a relatively small amount of intermediate sizes.

Plots were separated into three blocks by total canopy, without the inclusion of the lower canopy cover of Kalanchoe (<10cm): (1) 45-95%, (2) 96-120%, and (3) 121-136% total canopy. The lower canopy of Kalanchoe was not included as the main outcome of this study was to examine the difference of spray coverage between treatments reaching the lower canopy of <10cm K. xhoughtonii. Canopy height was also measured in each plot. Within each block, K. xhoughtonii canopy, total canopy, and canopy height varied (Table 3-1). The first run took place between

8:00am and 10:00am and the second run took place, after ample time for the first spray to dry, between 11:00am and 12:30pm on the same plots.

Treatments and Data Collection

Each plot within each block received one of four possible spray treatments (Table 3-2), with treatment 1 (40 GPA, low OS) considered the standard application rate and the label concentration rate of OS (Anonymous 2014). The higher concentration of OS used was obtained from the previous research on the use of OS for increasing canopy penetration (Sperry 2019).

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Each treatment had the addition of a blue dye3 at a concentration of 0.125% v v-1. Treatments were applied across the center of each plot with a CO2-pressurized backpack sprayer equipped with a TeeJet® 8002E nozzle4, held approximately 9 inches (22.86cm) above the top of the plot’s canopy, adjusted to 40 psi (275.8 kPa). To quantify the amount of spray penetrating to the lower level K. xhoughtonii (<10cm), 15 cm by 10cm spray card collectors with chrome-coated photo-paper5 were placed directly on top of the plantlets, approximately 5cm from the soil surface, hereafter referred to as the “lower” cards and at the top of the plot’s canopy, hereafter referred to as the “upper” cards. The upper cards were held in place with double sided tape attached to ring stand clamps that were clamped to a metal rebar (approximately 1m in height).

The rebar was positioned in the middle of each plot, pushed into the sand until the bottom of the card was touching the top of the vegetation. The card was intentionally faced in the direction of the sprayer to prevent any spray shadow or spray reduction due to the rebar interference (Figure

3-2). Two spray mixes were used with the two concentration of OS. In order to apply at the two different application rates, the output per minute was calculated and the speed was adjusted. The pressure and nozzle remained the same for both application volumes.

After application, the photo-paper collectors were left to dry for approximately 5 min before they were removed, stored in an acid-free notebook, protected from sunlight, and transported to the University of Florida’s Center for Aquatic and Invasive Plants (CAIP) to be analyzed. All evaluations were made on the center 12cm by 7cm of each 15 cm by 10cm spray card to eliminate spray build-up on edges. Spray cards were scanned, converted to 8-bit format,

3FarmWorks™ Spray Indicator Blue, Tangi-Pac, LLC, 101 Ponchatoula Pkwy, Ponchatoula, LA 70454-8313

4 TeeJet® nozzle, TeeJet Technologies Illinois, LLC, 1801 Business Park Drive, Springfield, IL 62703

5 14PT Carolina® Coated Cover C2S, WestRock Company, 501 South 5th Street Richmond, VA 23219

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the black/white threshold adjusted to ensure the 8-bit image accurately represented the colored spray card, and analyzed for percent coverage using ImageJ software (C.A. et al. 2012).

Statistical Analyses

Experiments were a randomized complete block design (RCBD) with a factorial arrangement of treatments, three replications within each treatment, two trials, and three blocks.

The factors were two application volumes, 374 L ha-1 (40 GPA) and 748 L ha-1 (80 GPA), two surfactant concentrations, 0.6% v/v (0.6 g L-1) and 1.5% v/v (1.5g L-1) and three blocks, 45-95%,

96-120%, and 121-136% total canopy (with the exclusion of the <10cm K. xhoughtonii canopy cover). Data analysis was conducted in Rstudio® (RStudio Team 2015) under the ‘emmeans’ package. Data was tested for and achieved normality and homogeneity of variance. Data was subjected to ANOVA to test the significance of main effects and interactions. Post hoc multiple comparisons were performed using Tukey’s honest significant difference test (Tukey’s HSD) with a 95% confidence interval (P<0.05).

Results and Discussion

The upper cards showed a significant difference between treatments (P<0.0001). Neither the trial (P=0.913) or interaction between trial and treatments were significant (P=0.456). There was also no statistical significance between blocks for the upper cards (P=0.606) or interactions involving block (P=0.908). Data were pooled across trials but kept separated by block for comparison to lower cards, that did show a significance between blocks. The upper cards showed the percent of spray coverage for treatment 1 (40 GPA, low OS) in block 1 (45-95% total canopy) was the only statistically different grouping from the rest of the treatments (Figure 3-3).

Treatments 1 (40 GPA, low OS) and 2 (40 GPA, high OS) had on average 82-93% spray coverage while treatment 3 (80 GPA, low OS) and 4 (80 GPA, high OS) had on average 93-

100% spray coverage on the upper cards across all three blocks.

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The lower cards had a significant difference between treatments (P=0.0002), with no trial significant (P=0.535) or interaction between the treatment and trial (P=0.519). Therefore, data was pooled across trials for the lower cards as well. There was a significant difference between blocks for the lower cards (P=0.024) and a block interaction with the treatments (P=0.029).

Treatment 3 and 4 (80 GPA) for block 2 (96-120% total canopy) showed the highest percent spray coverage on the lower cards (Figure 3-4). Treatment 4 (80 GPA, high OS) had the highest average percent spray coverage as compared to the standard rate of treatment 1 (40 GPA, low

OS), with the exception of block 1 (45-95% total canopy) in treatment 1 being statistically similar to all treatments in all blocks. This was possibly due to the open and bare spots in the plots with lower total canopy that the plots with higher total canopy did not have. Treatment 4

(80 GPA, high OS) had the most consistent higher average percent coverage as compared to the other three treatments for block 1 (45-95% total canopy) and 2 (96-120% total canopy).

Treatment 1 and 2 were combined (40 GPA) and evaluated against the combination of treatment 3 and 4 (80 GPA) in order to examine the differences of spray coverage between application rates across the three blocks as well as their interactions. For the upper canopy, there was a significant difference between the 40 GPA and the 80 GPA treatments (P<0.0001) with no block effect (P=0.351) or interaction (P=0.582). The 80 GPA treatments had an average of 12% more spray coverage than the 40 GPA treatments (Table 3-3). For the lower cards, there was also a significant difference between the 40 GPA treatments and the 80 GPA treatment (P=0.0026), as well as a block effect (P=0.0335) and an interaction between GPA and block (P=0.0354). There was no statistical difference between 40 GPA and 80 GPA across all three blocks except for 80

GPA in block 2 (96-120% total canopy), with 80 GPA in block 1 (45-95% total canopy) having

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statistically similar spray coverage averages as all treatments (Table 3-4). The 80 GPA treatment for block 2 had an average of 29.2% more spray coverage than all other treatments.

Treatments 1 and 3 were combined (low OS) and evaluated against the combination of treatment 2 and 4 (high OS) in order to examine the differences of spray coverage between OS concentrations across the three blocks as well as their interactions. There was no statistical difference of spray coverage between OS treatments for upper cards (P=0.468) or lower cards

(P=0.537). Average spray coverage for the upper spray cards across all OS treatments was 93.6% and for the lower cards the average spray coverage was 28.0%.

Conclusion

These results show that increasing the application rate can potentially increase the spray coverage of the lower canopy, but the increased OS did not result in an increase of spray coverage to the upper or lower canopy. Though, at the higher application rate (80 GPA), the higher concentration of OS did have an increase of spray coverage on the lower cards, especially in the plots with lower average percent total canopy, though not consistently enough to be in a statistically separate group. The desired result was to increase spray penetration into the lower canopy where the plantlets of Kalanchoe are found, however the increased spray coverage on the lower canopy resulted in increased spray coverage on the upper canopy as well. This would result in a significantly greater percent of Kalanchoe targeted in the lower canopy; however, there would likely be a significant amount of non-target damage to the upper canopy plants if herbicide was applied in this manner.

The spray retention on the upper canopy needs to be reduced in order to obtain the desired outcome of reducing non-target damage of the upper canopy, consisting of native plants, while effectively targeting the understory of Kalanchoe. Other adjuvants need to be examined in field spray card studies on penetrating through the upper canopy; adjuvants with the possible

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properties of increased run-off to the soil and increased droplet size. The herbicide needs to be able to penetrate through the upper canopy with little to no retention, land on the ground (on and around Kalanchoe plantlets) and prevent absorption into the soil to prevent loss of herbicide and non-target plants’ roots taking-up the herbicide, possibly through larger droplet size (Shaw et al.

2000). Another form of application of herbicide that should be explored is the use of granular formulations of herbicides. This would possibly allow the applicator to apply the solid form of herbicide in a manner that would prevent all retention on the upper canopy. Greenhouse studies are needed to investigate the potential of Kalanchoe species’ shallow and fine root system capacity to uptake soil applied granular herbicides while avoiding uptake from native plants’ deeper and larger root systems.

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Figure 3-1: Vegetation of field site on 29 August 2019 at Butler Beach in St. Augustine, Florida (29°47'16.3"N 81°15'29.3"W), facing Northwest.

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Table 3-1: Range of canopy heights, K. xhoughtonii canopy (over 10cm and under 10cm), and total canopy for each block. The canopy cover for Block was determined by the total canopy cover minus the <10cm K. xhoughtonii cover to isolate the upper canopy that intercepts the spray to the lower K. xhoughtonii. Range of Canopy Cover (%) Range of Canopy K. xhoughtonii K. xhoughtonii Total Canopy Block height (cm) >10cm <10cm Cover 1 (45 - 95%) 30 - 72 2 - 15 12 - 55 82 -118 2 (96 - 120%) 36 - 73 3 - 25 20 - 70 125 - 147 3 (121 - 136%) 30 - 85 12 - 35 15 - 75 151 - 185

Table 3-2: Spray Card treatment application and surfactant concentrations. Organosilicone surfactanta Treatment Name Application Rate concentration -1 -1 -1 -1 (Gal acre ) (L ha ) (oz gal ) (g L ) 1: 40 GPA, low OS 40 374 0.8 6.0 2: 40 GPA, high OS 40 374 1.92 14.4 3: 80 GPA, low OS 80 748 0.8 6.0 4: 80 GPA, high OS 80 748 1.92 14.4

a Dyne-Amic®, Helena Chemical Company, 225 Schilling Blvd., Suite 300, Collierville, TN 38017

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Figure 3-2: Plot set-up, arrow indicates the direction the sprayer moved across plot. Lower card (1) positioned directly on top of K. xhoughtonii plantlets, approximately 5cm above the soil surface and just left of the middle of the plot to ensure it was not in the spray shadow of the upper card. Upper card (2) attached to rebar with a ring stand clamp and positioned in the middle of the plot with the card facing the direction of the incoming sprayer.

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Figure 3-3: Average percent spray coverage for the upper spray cards to indicate spray retention for the upper canopy, data pooled across blocks (P=0.606) and trials (P=0.913). Means with the same letter are not different (P<0.05) and error bars represent ± 1 standard error.

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Figure 3-4: Average percent spray coverage for the lower spray cards positioned approximately 5cm above the soil surface to indicate spray retention on the lower canopy. Data is pooled across trials (P=0.535) and separated by block (P=0.0004), letters of significance are separated by block. Blocks are defined as (1) 45-95%, (2) 96-120%, and (3) 121-136% total canopy cover (with the reduction of <10cm K. xhoughtonii in the targeted lower canopy). Means with the same letter are not different (P<0.05) and error bars represent ± 1 standard error. *ns=no significance

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Table 3-3: Average spray coverage of Organosilicone surfactant treatments for the upper canopy cards. Treatment Average spray coverage (%) Standard Error 40 GPA 87.5 (b) ± 1.91 80 GPA 99.5 (a) ± 1.88

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Table 3-4: Average spray coverage of OS treatment for the lower canopy cards. Blocks are defined as (1) 45-95%, (2) 96-120%, and (3) 121-136% total canopy cover (with the reduction of <10cm K. xhoughtonii in the targeted lower canopy) Treatment Block Average spray coverage (%) Standard Error 40 GPA 1 27.9 (b) ± 5.40 40 GPA 2 18.5 (b) ± 5.97 40 GPA 3 13.0 (b) ± 5.97 80 GPA 1 31.4 (ab) ± 5.40 80 GPA 2 52.6 (a) ± 6.34 80 GPA 3 26.2 (b) ± 5.40

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CHAPTER 4 CUT ONE, GET TEN: Kalanchoe ASEXUAL REGENERATION IN RESPONSE TO MOWING

Kalanchoe is a genus of plants in the family Crassulaceae. Species of Kalanchoe are generally described as succulent herbaceous plants with bifacial leaf structure with a thick and waxy cuticle, a fine and shallow root system, and enlarged central storage vacuoles that store organic acids (Chernetskyy 2012, Ting 1989). Many of the species in this genus are found in the ornamental plant industry around the world and considered easy to care for due to their very low maintenance requirements. The Crassulacean Acid Metabolism (CAM) photosynthetic process used by members of this genus produce plants with low water requirements, a wide range of light tolerance, and a wide range of environmental plasticity (Schafer and Luttge 1987, 1988).

Unfortunately, the characteristics that have made Kalanchoe a successful horticultural species have also resulted in it being invasive in certain areas. A few Kalanchoe species are now regarded as noxious and invasive in certain parts of the world. K. pinnata, K. daigremontiana, K. delagoensis, and K. xhoughtonii are listed as invasive in parts of Australia, Mexico, South

Africa, China, and the United States (Batianoff and Butler 2002, FLEPPC 2019, Hannan-Jones and Playford 2002, Henderson 2007, LR et al. 2012, SEMARNAT 2016, Wang et al. 2016).

Negative impacts on invaded ecosystems include livestock poisonings, alterations in soil nutrients, and changes in the structure of the native community (Abbate et al. 2019, Chacón et al.

2009, Guerra-García et al. 2018, Herrera et al. 2018, Widmann et al. 1990). The driving force of establishment of the invasive Kalanchoe species is attributed to the ability to very rapidly increase the population through asexual reproduction of plantlets during the initial phase of invasion (Chacón et al. 2009, Guerra-García et al. 2015, Herrera et al. 2012). Plantlets are vegetative clones of the parent plant that, in the case of the previously listed Kalanchoe species,

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are produced along the margin of the leaves (Garcês and Sinha 2009, Garces et al. 2007, Johnson

1934). Plantlets develop cotyledon-like leaves and hypocotyl-like roots while attached to the parent plant. Plantlets are produced during the longer days of the year (i.e. summer) and suppressed in December through February, during the flowering stage (Johnson 1934).

Individual leaves of a Kalanchoe plant can produce dozens of plantlets, with mature plants being able to produce hundreds of plantlets. In a study on the survivorship of plantlets in varying light and water conditions, it was found that plantlets watered daily and in shaded conditions had almost 100% survival rate (Guerra-García et al. 2018). Plantlets are able to develop and spread within the same growing season resulting in multiple asexual cycles per year (Herrera et al.

2012). Plantlet production on the margin of the leaves of some species of Kalanchoe can be induced or increased from stress, such as the detachment from the parent plant or environmental stress. This is believed to be a form of life preservation by increasing ratio of plantlets to dry leaf weight, with the idea that plantlets offer the chance to survive harsh living conditions (Garcês and Sinha 2009, Garces et al. 2007, González de León et al. 2016, Widmann et al. 1990). It is thought that environmental stress triggers a release from dormancy, allowing plantlets to complete their development on the margins of leaves. Plantlets become dormant after the very early stages of differentiation, prior to leaf and root development, on the mother leaf. Dormancy can be broken by longer days, leaf severing, and extreme water stress, allowing for vigorous plantlet production in a very short time (Johnson 1934). The plantlets of K. rotundifolia have been found to remain dormant on the parent leaf until removed, even where root development has been initiated (Stoudt 1938). Further research is needed in understanding the state of dormancy a plantlet incurs once released from the mother plant or leaf (Batygina et al. 1996,

Johnson 1934).

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Hand pulling is a discriminative form of invasive plant control, with humans selectively removing only the individual plants that need to be removed and able to remove all visible plants. In sensitive habitats, like that of dune communities, hand removal is not necessarily a beneficial form of control. On 28 November 2018, a Kalanchoe hand-pull event occurred in a small section of the sand dunes in the swale between the primary and secondary dunes at Butler

Beach in St. Augustine, Florida. Intentions were to observe the efficacy of the invasive plant control method as well as the recovering properties of the dunes. The dunes at this beach are populated sporadically with K. xhoughtonii, at densities of approximately 150 plants and plantlets per meter (when present). The K. xhoughtonii was commonly found under the native vegetation composed of Dune greenbrier (Smilax auriculata), Partridge pea (Chamaecrista fasciculata), Indian blanket flower (Gaillardia pulchella), and Beach sunflower (Helianthus debilis). Nine volunteers weeded a total area of approximately 4m X 3m in four hours and removed approximately 150-200lbs of plant material. Volunteers expressed the difficulty of removing all plants and plantlets from under the native vegetation. Once some of the plants with intact roots were removed from the sandy soil, the other smaller plants and plantlets were buried under sand and difficult to see to remove. Also observed by the end of the hand pull event was considerable trampling damage to native plants in area. The hand pull area at Butler Beach was surveyed 60-days later and found that bare spots and damaged native vegetation was still visible, though no large K. xhoughotnii plants were observed. On closer inspection, however, the swale was heavily infested with small K. xhoughotnii plants and plantlets under the vegetation. In some portions of the previously hand weeded plot, the repopulated patches had approximately 300 plants and plantlets per meter. Though the larger Kalanchoe plants were successfully removed,

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the disturbance caused by the hand weeding in the sensitive and slow growing dune system had potentially allowed for the proliferation of K. xhoughtonii plantlets and smaller plants.

Mowing is a management tool often used to control annuals, occasionally used to reduce seed production and sometimes can provide suppression of biennials and perennials when used repeatedly (Ditomaso et al. 2010). However, mowing plants that reproduce vegetatively will stimulate production of new stems from the below ground biomass (Ditomaso et al. 2010, Nice and Johnson 2008). Mowing of dune vegetation as a management tool has been examined in the control of the grass species Arrhenatherum elatius in sand dunes in Wales, UK. There was a significant decline in the abundance of the invasive grass from the mowing technique, specifically in the plots located in the low dunes. Native vegetation showed either no significant difference or an increase in abundance post mowing treatment. Though the authors mention there was a high likelihood of the grass to resume its invasive role should mowing cease (Hewett

1985). The study showed that mowing in dune systems may be beneficial to the native plant community.

Kalanchoe species have the majority of biomass allocated to aboveground tissues with their root systems tending to be very shallow and fine (Guerra-García et al. 2018). The shallow root systems optimize the use of limited rainfall and extend survival during dry seasons, a time when plant growth is typically limited by low soil water content in arid environments (Niechayev et al. 2019). The limited biomass allocated to the root structure of Kalanchoe species could potentially be a weak point of the genus’s morphology. Theoretically, if there is not enough stored energy reserves or available meristems to generate new shoots, photosynthesis will cease, and the plant roots will perish. Conversely, plantlet production is the strongest component of

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Kalanchoe reproduction. Plantlet production on the margin of fallen leaves is an exemplary form of life preservation; when one leaf falls, many plants are created.

The development of multiple plantlets has been observed1 when leaves fall from the mother plant, but there is a lack of available quantified information. There is also a lack of data documenting the effect of mowing on Kalanchoe control or regeneration. Therefore, our objectives were to (1) quantify the ability of K. xhoughtonii to root and grow from cut material,

(2) quantify plantlet production from cut plant material, and (2) document the effect of mowing at different heights on K. xhoughtonii regeneration.

Materials and Methods

Two greenhouse studies were conducted in July through September of 2019 at the

University of Florida’s Center for Aquatic and Invasive Plants (CAIP) in Gainesville, Florida.

The studies were performed on small and established K. xhoughtonii plants to quantify the growth after mowing and the growth of cut material. The studies were separated into a simulated mowing study and a regeneration study. The simulated mowing study consisted of two types of plant material, established and small, with three mowing treatments of 0cm from soil surface,

10cm from soil surface, and no mowing (untreated control). The regeneration study consisted of

4 treatments, 15cm of the apical shoot placed on the sand surface, 15cm of the apical shoot planted 2 cm into the sand, a single leaf placed on the sand, and a single leaf planted 2 cm into the sand. We used small plants as well as established plants to compare the regenerative abilities with differing biomasses. The established plants have more above ground and below ground biomass available to potentially allow for greater regenerative effects as compared to smaller plants.

1 Jessica Solomon, personal observation at Center for Aquatic and Invasive Plants

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Plant material

For the ‘established’ plants of K. xhoughtonii, plants were harvested from Vilano Beach

Nature Trail in St Augustine, FL (29°55'06.8"N 81°17'38.2"W) on 7 February of 2018 with permission of St. Johns County Parks and Recreation. Plants were dug out with a hand shovel, placed in trays, then transported back to CAIP. The following day, plants were transplanted into a medium of 50% sand2, 50% soil3 into 6cm X 6cm X 9cm square pots and maintained in a greenhouse. Plants were fertilized4 every 6 months. All plants were maintained in a greenhouse covered with a 50% shade cloth and temperature set at 31°C, though air temperature fluctuated between 23°C and 35°C throughout the duration of the experiment. Plants were watered two to three times a week, allowing potting medium to dry-out completely between watering as this is common in their native habitats. Plants exhibited no visual symptoms of water stress at any time during each experiment. Plants were transplanted into larger, 6.8 L round plastic pots (19cm diameter X 13cm) on 10 June 2019 in the same potting medium of 50% sand and 50% soil. For the ‘small’ plants, K. xhoughtonii was harvested from the same location as before on 6 June

2019, plants were transported, transplanted into 6cm X 6cm X 9cm square plastic pots, fertilized, and maintained as the previous plants were.

Simulated Mowing Study and Data Collection

Six established plants and six small plants were randomly assigned to one of three treatments: (1) 10cm mow, (2) 0cm mow, and (3) no mow. Mowing height was measured up from soil surface and was performed using sharp, sterilized garden shears. Baseline data taken on

2 DOT sand, Argos USA, 924 S Main Street, Gainesville, FL 32601

3 Fafard 4P Professional Growing mix, Sun Gro Horticulture, 770 Silver Street, Agawam, MA, 01001-2907

4 4-10-3 Root Stimulator, ferti-lome, 230 FM 87, Bonham, TX 75418

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the day of treatment included: root collar diameter (RCD), plant height, and leaf count before and after mowing. These same data were collected again at 30 days after mowing, with the addition of plantlet count. The slightest disturbance caused plantlets to fall off of the established plants when manipulated for data collection; therefore, an estimate of the number of plantlets was taken instead in order to reduce the number of plantlets lost. Plantlet count for the established plants was estimated by counting plantlets on 5 random and separate leaves in the top

20 cm of the plant and averaged, then multiplied by one-half of the total amount of leaves. The experiment ran for 60 days. Final measurements included: RCD, plant height, leaf count, plantlet count, and above and below ground biomass. The above and below ground plant material was dried at 65°C for comparison across treatments. The first trial was initiated on 15 July 2019 and the second trial was initiated on 22 July 2019.

Regeneration Study and Data Collection

Experiments began with established plants as described previously. Plant material was clipped using sharp and sterilized garden shears and all plantlets on the leaves were removed prior to clipping. Potting medium for the study was 100% sand. Plant material was clipped in one of four ways: (1) 15cm of the apical shoot placed onto the sand surface in 6.8 liter pots (19cm diameter x 13cm), (2) 15cm of the apical shoot planted 2cm into the sand, (3) 1 leaf with its petiole placed on the top of the sand in 18.6oz pots (10cm diameter x 7cm tall), and (4) 1 leaf with its petiole planted 2 cm into the sand. Each treatment had 6 replicates. Plants and plant parts were maintained in the greenhouse with watering 2-3 times a week. Baseline data taken on the day of treatment included: number of leaves, stem diameter at cutting, and fresh biomass prior to planting. At 30 days after treatment, measurements taken were a visual estimate of root development, number of plantlets, number of leaves, and RCD at the cut or at sand level. The experiment ran for 60 days and final measurements included: stem height, number of plantlets, 93

and fresh biomass. The plant material was dried at 65°C and weighed for a constant biomass for comparison across treatments. Each trial of this experiment was initiated on the same day as the simulated mowing experiments, 15 July 2019 and 22 July 2019 for the first and second trial, respectively.

Statistical Analyses

Experiments were conducted using a randomized complete block design (RCBD) with two experimental trials for each of the two experiments. For the simulated mowing experiment, the

RCBD had a two factorial arrangement that included plant size and mowing height. For the regeneration experiment, the RCBD also had a two factorial arrangement, placement in or on sand and plant part (15cm of apical shoot or singular leaf). Data was analyzed in Rstudio®

(RStudio Team 2015). All data was checked for and achieved normality and homogeneity of variance. Data was subjected to ANOVA to test significance of main effects and interactions.

Tukey’s honest significant difference test (Tukey’s HSD) was performed for post-hoc analysis with a 95% confidence interval (P<0.05) using the ‘emmeans’ package in Rstudio (Lenth 2019,

RStudio Team 2015).

Results and Discussion

Simulated Mowing Study

Overall, the un-mowed (tx3) treatment had a 100% survival rate, the 10cm (tx2) mowing treatment had approximately 92% survival and the 0cm (tx1) mow height had a 0% survival rate between both trials and across both the small and established plants. There was a significant difference between initial plant size in all measurements (P<0.0001) so data was analyzed for established and small plants separately. The baseline measurements showed that there was a significant difference between RCD for the two different sized plants (P<0.0001), small plants

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averaged 5.9mm RCD while established plants averaged 10.0mm. There was no significant change in RCD over time for all plants (P=0.092).

For both, established and small plants, the heights were significantly different across time

(P<0.0001) and treatments (P<0.0001). This is to be expected since plants were mowed to different heights. Therefore, the change in height between 0-30 DAT and 30-60 DAT was compared instead. For established plants, height change between 0-30 DAT and 30-60 DAT showed that the un-mowed treatment (tx3) plants’ height grew significantly faster over time as compared to the 0cm (tx1) and 10cm (tx2) mowing treatments. For the small plants, 10cm mow height (tx3) grew faster than the un-mowed (tx3) treatment between 0 and 30 DAT. However, by

60 DAT the un-mowed (tx3) plants grew faster than the 10cm (tx2) and 0cm (tx1) mow heights.

The 0cm (tx1) mow height had no growth over the 60-day experiment (Figure 4-1).

For leaf count, the established un-mowed (tx3) plants always had significantly more leaves than the two mow treatments (tx1 and tx2) (Figure 4-2). Leaf count change over time showed that the 10cm (tx2) mow height developed leaves quicker than the 0cm (tx1) and un-mowed

(tx3) treatments for both small and established plants. For established plants, the un-mowed (tx3) treatments had a reduction of leaves between 30 and 60 DAT. For small plants, the 10cm (tx2) mow height and un-mowed (tx3) treatments had a reduction of leaves between 30 and 60 DAT with the 0cm (tx1) mow height treatment having no leaves developed at all (Figure 4-3). The reduction in leaves is possibly due to natural plant leaf shedding.

The number of plantlets produced varied across treatments and size classes. For established plants, plantlets only developed on the un-mowed plants. No plantlets developed within 60 days on either the 0cm mow height or the 10cm mow height. For small plants, plantlets developed on the 10cm (tx2) and un-mowed (tx3) treatments. There were significantly more plantlets on the

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un-mowed plants compared to the plantlet development on the 10cm (tx2) mow height (Figure 4-

4). This difference is likely due to the greater leaf area with the un-mowed plants having significantly greater reproductive opportunity.

Regeneration Study

All apical shoot treatments in this study survived and developed adventitious roots regardless if they were placed on the sand surface or planted in the sand. Within 24-hours of experiment initiation, the top portion of the stem and leaves of Treatment 1 (apical shoot placed on sand) were observed to orient upward and by 7 DAT the bottom portion of the stem was also angled upwards (Figure 4-5). In Treatment 3 (leaf on sand surface), the leaves did not develop roots or become established in either experimental run. While all Treatment 3 leaves withered and eventually died, many of these successfully developed viable plantlets prior to complete desiccation. While 50% of Treatment 4 (leaf planted in sand) desiccated, the rest established and developed adventitious roots. All Treatment 4 leaves developed viable plantlets. Although plantlet development in Treatment 3 and 4 were not high (approximately 3 per leaf, on average) it is noteworthy that reproduction occurred simultaneously with desiccation.

Treatment 1 (apical shoot placed on sand) showed a significant increase in height between 0 and 60 DAT (Figure 4-6). Treatment 2 (apical shoot planted in sand) had a greater average height in 60 DAT as compared to 0 DAT but due to the large variance between measurements in the 60 DAT data, there was not a statistical significance between heights

(Figure 4-6). Leaf count increased between 0 and 30 DAT for both Treatment 1 and 2 but decreased by 60 DAT (Figure 4-7). This is possibly due to the apical shoot partitioning materials from the leaves towards root development. A significant number of plantlets developed on all four treatments between 0 and 30 DAT. Treatment 1 (apical shoot placed on sand) and Treatment

2 (apical shoot planted in sand) continued to show an increase in plantlet production between 30 96

to 60 DAT while Treatment 3 (leaf on sand) and 4 (leaf push in sand) had relatively no change in plantlet development (Figure 4-8). This was most likely due to space availability on treatments 1 and 2 having significantly more leaves than that of treatments 3 and 4 which consisted of a single leaf. Fresh weight for treatment 1 (apical shoot placed on sand) and treatment 2 (apical shoot planted in sand) showed a decrease between 0 DAT and 60 DAT (Figure 4-9). The leaf count data showed that 60 DAT had either significantly fewer leaves or similar number of leaves as 0

DAT. Though plant height increased, the majority of the biomass is in the leaves and a reduction of leaf number will consequently reduce the total fresh biomass. Fresh biomass for treatment 3

(leaf on sand) and treatment 4 (leaf planted in sand) showed a similar significant decrease in fresh weight between 0 DAT and 60 DAT, similar to the pattern observed in the stem treatments

(Figure 4-9).

Conclusion

Plantlet development for both studies presented results that agree with previous research concluding that plantlet production is a major biological role in the life cycle of Kalanchoe species through the ability to increase rapidly from stress stimuli as well as during the initial phase of invasion when extinction risks are generally higher (Chacón et al. 2009, Guerra-García et al. 2015, Herrera et al. 2012). Kalanchoe demonstrated the ability to generate numerous plantlets in response to either mowing or plant disturbance. By 30 DAT, all four treatments in the regeneration study had developed a significant number of plantlets. In the simulated mowing study, no plantlets developed on the 10cm mow height by 60 DAT, though we hypothesize that plantlets could develop if the experiment had run for longer than 60-days. For small plants in the simulated mowing study, an average of 40 plantlets developed by 30 DAT and an average of 74 plantlets by 60 DAT. It was possible the small plants had the appropriate root:shoot ratio to allocate nutrient and material to plantlet production post-mowing treatment. 97

The simulated mowing study showed that K. xhoughtonii plants could potentially be mowed at ground level (0cm) and may lack available meristems or energy reserves to redevelop a plant successfully with both established and small plants having had no leaves or plantlets developed post treatment. For both small and established plants, mowing to the average mower height of 10cm will most likely not cause plant death and in small plants the plantlet development will continue immediately post mowing. Small plants and established plants were used to compare the regenerative abilities with differing biomasses.

This study needs to be expanded to a field experiment. Guerra-Garcia et al. (2018), found that K. delagoensis has significantly larger root biomass in the field as compared to greenhouse plants. They hypothesized that it was due to the readily available and systematically reoccurring water source in a greenhouse as compared to the natural environment. Plants in natural settings outside must allocate more resources to belowground structures to capture water (Guerra-García et al. 2018). It is possible the larger root system in the field may allow for growth of leaves and plantlet development from the 0cm mowing height. The regenerative abilities of the native beach vegetation post-mowing treatment also need to be investigated.

The regeneration study showed that plant parts of K. xhoughtonii have the potential to develop adventitious roots and plantlets, especially if the cut portion is buried in the ground where it can more easily access water in the soil. Apical shoots, whether placed on the sand or planted into the sand, will very likely develop roots and plantlets. The horizontal apical shoot had remarkably fast response to sunlight, turning upwards within the first 24-hours of being placed on the sand surface. Plant leaves that did not produce roots and died, still produced an average of three plantlets. This experiment showed that 100% of plant material will need to be

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removed from a site if not using a chemical control that effectively kills the plants before producing plantlets.

Further research should include field experiments of the mowing and regeneration greenhouse studies conducted here. The water availability and, in turn, root structure of

Kalanchoe species in the field have been shown to be significantly different and may react differently to the mowing and regeneration treatments. Further research could also include smaller segments of plant portions to investigate their capabilities to produce roots, leaves, and plantlets. The timing of mowing plants and regeneration abilities of plant parts also needs to be investigated. It may be possible that mowing during the winter may have significantly more control of Kalanchoe species when they have been shown to allocate less production to plantlets and more to reproductive structures (i.e. inflorescence). This may also be true for the regenerative capabilities of plant parts, possibly producing less plantlets and less likely to produce adventitious roots.

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Figure 4-1: Kalanchoe xhoughtonii average change in plant height from 0-30 DATa (30 DAT) and 30-60 DAT (60 DAT) for established (left) and small (right) plants. Tx1 is 0cm mow height, tx2 is a 10cm mow height, and tx3 was un-mowed. Data was pooled across trials (P=0.613) and separated by treatment (P<0.0001). Means with the same letter are not different (P<0.05) and error bars represent ± 1 standard error. Lowercase letters refer to treatment 1, uppercase refer to treatment 2, and numbers refer to treatment 3. aDAT= Days After Treatment

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Figure 4-2: Kalanchoe xhoughtonii average leaf count for established (left) and small (right) plants. Tx1 is 0cm mow height, tx2 is a 10cm mow height, and tx3 was un-mowed. Data was pooled across trials (P=0.562) and separated by size class (P<0.0001) and treatment (P<0.0001). Means with the same letter or number are not different (P<0.05) and error bars represent ± 1 standard error. Lowercase letters refer to treatment 1, uppercase refer to treatment 2, and numbers refer to treatment 3. aDAT= Days After Treatment

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Figure 4-3: Kalanchoe xhoughtonii average change in leaf number from 0-30 DATa (30 DAT) and 30-60 DAT (60 DAT) for established (left) and small (right) plants. Tx1 is 0cm mow height, tx2 is a 10cm mow height, and tx3 was un-mowed. Data and letters of significance are separated by size class (P<0.0001) and treatment (P<0.0001). Means with the same letter are not different (P<0.05) and error bars represent ± 1 standard error. Lowercase letters refer to treatment 1, uppercase refer to treatment 2, and numbers refer to treatment 3. aDAT= Days After Treatment

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Figure 4-4: Kalanchoe xhoughtonii average plantlet development for established (left) and small (right) plants. Tx1 is 0cm mow height, tx2 is a 10cm mow height, and tx3 was un-mowed. Data was pooled across trials (P=0.968). Data and letters of significance are separated by size class (P<0.0001) and treatment (P<0.0001). Means with the same letter are not different (P<0.05) and error bars represent ± 1 standard error. Lowercase letters refer to treatment 1, uppercase refer to treatment 2, and numbers refer to treatment 3. aDAT= Days After Treatment

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Figure 4-5: Kalanchoe xhoughtonii treatment 1 (apical shoot placed on sand) at 1 days after treatment (left) and 7 days after treatment (right) showing the phototropism of the top and bottom of the stem.

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Figure 4-6: Kalanchoe xhoughtonii average plant height at 0 DATa and 60 DAT for treatment 1 (apical shoot on sand, left) and treatment 2 (apical shoot in sand, right). Data was pooled across trials due to no statistical difference. Letters of significance are separated across treatments. Means with the same letter are not different (P<0.05) and error bars represent ± 1 standard error. aDAT= Days After Treatment

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Figure 4-7: Kalanchoe xhoughtonii average leaf count at 0 DATa, 30 DAT, and 60 DAT for treatment 1 (apical shoot on sand - left) and treatment 2 (apical shoot in sand - right). Data was pooled across trials due to no statistical difference. Letters of significance are separated across treatments. Means with the same letter are not different (P<0.05) and error bars represent ± 1 standard error. aDAT= Days After Treatment

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Figure 4-8: Kalanchoe xhoughtonii average plantlet development at 0 DATa, 30 DAT, and 60 DAT for treatment 1 (apical shoot on sand, top left), treatment 2 (apical shoot in sand, top right), treatment 3 (leaf on sand, bottom left), and treatment 4 (leaf in sand, bottom right). Letters of significance are separated across treatments. Means with the same letter are not different (P<0.05) and error bars represent ± 1 standard error. aDAT= Days After Treatment

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Figure 4-9: Kalanchoe xhoughtonii average fresh weight at 0 DATa and 60 DAT for treatment 1 (apical shoot on sand, top left), treatment 2 (apical shoot in sand, top right), treatment 3 (leaf on sand, bottom left), and treatment 4 (leaf in sand, bottom right). Letters of significance are separated across treatments. Means with the same letter are not different (P<0.05) and error bars represent ± 1 standard error. aDAT= Days After Treatment

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CHAPTER 5 CONCLUSIONS AND FUTURE RESEARCH

Kalanchoe is a genus of succulent plants in the family Crassulaceae and is native to the subtropical warm, dry regions of Eastern Africa, Southeast Asia, and Madagascar (Allorge-

Boiteau 1996, Descoings 2006). Since the 1800’s, many individual species have been spread around the world in the ornamental plant industry due to the variation of unique appearances as well as the ease of care (Baldwin 1938). Today, a few individual species of the 150 in the genus have become invasive in parts of China, Australia, New Zealand, the West Indies, the

Philippines, the Galapagos Islands, Melanesia, Polynesia, the Virgin Islands, and the United

States (Batianoff and Butler 2002, FLEPPC 2019, Hannan-Jones and Playford 2002, Henderson

2007, LR et al. 2012, SEMARNAT 2016, Wang et al. 2016).

The Kalanchoe species that have been observed as invading ecosystems around the world have the ability to reproduce rapidly through the asexual reproduction of plantlets (Garcês and

Sinha 2009, Garces et al. 2007, Johnson 1934). The consensus among researchers appears to be that the driving force of establishment is via the ability to very rapidly increase the population through asexual reproduction of plantlets during the initial phase of invasion (Chacón et al. 2009,

Guerra-García et al. 2015, Herrera et al. 2012). The plantlet production of some species of

Kalanchoe can be induced or increased from stress, such as leaf detachment from the parent plant or environmental stress (i.e. drought). This is believed to be a form of life preservation by allocating more biomass to plantlet production, with the idea that plantlets offer the chance to survive harsh living conditions (Garcês and Sinha 2009, Garces et al. 2007, González de León et al. 2016, Widmann et al. 1990).

The species of concern, for the purpose of this thesis, are K. pinnata, K. daigremontiana, K. delagoensis, and K. xhoughtonii which have been observed as invading natural areas in the

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United States (EDDMapS 2019c, FLEPPC 2019, Ward 2006, 2008, Wunderlin and Hansen

2011). In Florida, K. delagoensis and K. xhoughtonii have been observed as invading the sensitive and integral ecosystems of beach dunes around the coast (anecdotal1,2 and EDDMapS

2019). Chemical control methods have been investigated for K. delagoensis and K. pinnata in previous studies using mostly 2,4-D, glyphosate, metsulfuron, and fluroxypyr products as well as a study that encompasses mostly glyphosate in combination with other active ingredients on plantlets of K. delagoensis. Results showed great control with 2,4-D and glyphosate products, though the assessment periods for most of these experiments did not extend past 9 weeks of monitoring and did not include the development of plantlets on treated plants. 2,4-D is commonly used in agriculture but is not preferred for use in natural areas in Florida due to many formulations having moderate soil mobility, leaching into groundwater, toxicity to fish, and aquatic animals (Enloe et al. 2018, Jervais et al. 2008). Glyphosate is one of the most commonly used herbicide active ingredients in the United States due to its lack of soil mobility, low toxicity profile, and aquatic safe formulations (Henderson et al. 2010, Maeda and Dudareva 2012).

Though the lack of selectivity in glyphosate, killing both broadleaf plants and grasses, researchers and land managers prefer more selective and diverse active ingredients in their herbicide tool belt. A more thorough study of additional herbicide active ingredients and modes of action for the use in natural areas and aquatic areas for the control of Kalanchoe species was needed.

The research in this thesis investigates the biology of K. xhoughtonii and methods of control through herbicide screenings as well as exploring different techniques of application for the

1 Observations of staff at St. Johns County Park and recreation Department, 2018

2 Jessica Solomon, personal observations

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specific use of chemical control in beach dunes. Greenhouse herbicide trials were conducted at the University of Florida’s Center for Aquatic and Invasive Plants (CAIP) on K. xhoughtonii adult plants and plantlets looking at the efficacy of various herbicide active ingredients and modes of action. Using a foliar application, the active ingredients aminocyclopyrachlor, triclopyr acid, aminopyralid, glyphosate, and imazapyr all showed significant control of adult plants, plantlets, and did not produce viable plantlets post-treatment on adult Kalanchoe plants. These results expand upon the previous studies on herbicide efficacy, where auxin mimicking herbicides (2,4-D and fluroxypyr) and glyphosate products showed significant control on the tested Kalanchoe species (Armstrong and Prior 1999, Guerra-García et al. 2018, Sparkes et al.

2002). These studies have provided a land managers options for the control of Kalanchoe species in natural areas, where they should select the herbicide(s) that is adequate for the habitat for which Kalanchoe has invaded and chemical control is an option. Where possible, tank mixes of different modes of action could be used to increase control of all life stages of Kalanchoe in a single foliar application.

A more novel application technique for spraying herbicide in beach dunes arose as a need once the vegetative habit of the native plants and Kalanchoe was observed. Kalanchoe species in the arid and sunny dunes tend to use other native plant species as “nurse” plants. Nurse plants provide a positive influence on another species, mainly seedlings, by providing improved microclimatic conditions in its shadow. Nurse plants interactions are very common in arid and dry habitats (Niering et al. 1963, Padilla and Pugnaire 2006). These results of the spray card study on field plots in St. Augustine Beach, Florida showed that an increase in the application rate can potentially increase spray coverage to the lower canopy. These results are conclusive with previous work in agricultural field studies of increasing application rate through mulch (i.e.

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crop residue) and upper canopy weeds (Berger et al. 2014, Borger et al. 2013, Shaw et al. 2000,

Sperry 2019). However, the increased spray coverage on the lower canopy resulted in increased spray coverage on the upper canopy as well. This would result in a significantly greater percent of Kalanchoe targeted in the lower canopy while there would likely be a significant amount of non-target damage to the upper canopy plants if herbicide was applied in this manner. Further research is needed on reducing spray retention on the upper canopy in order to obtain the desired outcome of reducing non-target damage of the upper canopy, consisting of native plants, while effectively targeting the understory of Kalanchoe. Possibly other adjuvants with properties of increased run-off to the soil and increased droplet size could be examined in future field studies on targeting Kalanchoe. The use of granular formulations of herbicides, applied in their solid form, could be investigated. Greenhouse studies are needed to examine the control of root active herbicides on Kalanchoe species’ shallow and fine root systems.

Mowing as a control method for Kalanchoe species had yet to be quantified in prior studies as well as the ability of the clipped plant parts to re-grow, re-root, and/or produce plantlets.

Greenhouse experiments were conducted in order to examine regenerative abilities of

Kalanchoe from its limited root biomass as well as plant tissues regenerative abilities. We concluded that mowing to ground level (0 cm) has the possibility to significantly control

Kalanchoe. When mowed to this height, neither small nor large plants regenerated above ground biomass. The limited root biomass may be a weak point of this genus that could be exploited for control tactics. Field studies on mowing to ground level are needed as Kalanchoe species in the field tend to have a larger root biomass as compared to greenhouse grown plants (Guerra-García et al. 2018). The larger root:shoot ratio could allow regeneration of above ground biomass in field settings.

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The greenhouse regeneration study showed that plant parts, as small as a single leaf, of K. xhoughtonii have the potential to develop adventitious roots and then continue to grow successfully. Only the single leaf treatments (one leaf placed in or planted in sand) had death of the plant part in the regeneration studies. However, an average of 3 plantlets developed on the leaves, whether the leaf had died or re-rooted.

Plantlet development for all studies agree with earlier studies concluding that plantlets are the driving force of invasion for Kalanchoe species (Chacón et al. 2009, Guerra-García et al. 2015,

Herrera et al. 2012). The limitation of Kalanchoe species to regenerate from below ground biomass appears to be outweighed by the ability to produce numerous new plants from the stimulus of stress, in the form of plantlets, that have shown to have a very high survival rate

(Guerra-García et al. 2018).

The studies in this thesis builds on the previous herbicide and biological studies on

Kalanchoe species from Mexico, Australia and Africa (Armstrong and Prior 1999, Batianoff and

Butler 2002, González de León et al. 2016, Guerra-García et al. 2015, 2018, Herrera et al. 2012,

Sparkes et al. 2002). These studies also provide framework for future research into the biology and control of Kalanchoe species Florida, especially in beach dune habitats.

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LIST OF REFERENCES

(FNAI) FNAI (2010) Guide to the Natural Communities of Florida: Beach Dune. 1–5 p

Abbate A, Campbell J, Kimmel C, Kern WH (2019) Urban development decreases bee abundance and diversity within coastal dune systems. Glob Ecol Conserv 20

Abdel-Raouf HS (2012) Anatomical traits of some species of Kalanchoe (Crassulaceae) and their taxonomic value. Ann Agric Sci 57:73–79

Ahrens W, Cox D, Budhwar G (1990) Use of the arcsine and square root transformations for subjectively determined percentage data. Weed Sci 38:452–458

Allorge-Boiteau L (1996) Madagascar centre de speciation et d’origine du genre Kalanchoe (Crassulaceae). Biogeogr Madagascar 8:137–145

Anderson LAP, Steyn PS, Van Heerden FR (1984) The Characterization of Two Novel Bufadienolides, Lanceotoxins A and B from Kalanchoe lanceohts [ Forssk.] Pers. J Chem Soc Perkin Trans I:1573–1575

Anonymous (2011a) Triclopyr 4 herbicide product label. Indianapolis, IN: Dow AgroSciences LLC

Anonymous (2011b) Habitat® herbicide product label. Carmel, IN: SePRO Corporation

Anonymous (2014) Dyne-Amic® Product Label. Collierville, TN: Helena Chemical Company

Anonymous (2015) Method ® 240SL Herbicide herbicide product label. Research Triangle Park, NC: Bayer Environmental Science

Anonymous (2016) Trycera herbicide product label. Collierville, TN: Helena Chemical Company

Ariza E, Lindeman KC, Mozumder P, Suman DO (2014) Beach management in Florida: Assessing stakeholder perceptions on governance. Ocean Coast Manag 96:82–93

Armas C, Pugnaire FI (2005) Plant interactions govern population dynamics in a semi-arid plant community. J Ecol 93:978–989

Armstrong T, Prior S (1999) Control of mother of millions, Bryophyllum tubiflorum (Harvey) using herbicides. Page 627 in Twelfth Australian Weed Conferenc. Tasmania, Hobart: Weed Society of Tasmania, Hobart

Baldwin JT (1938) Kalanchoe : The Genus and its Chromosomes. Am J Bot 25:572–579

Baldwin JT (1949) Hybrid of Kalanchoe Daigremontiana and K . verticillata. Bull Torrey Bot Club 76:343–345

114

Batianoff G, Franks A (1997) Invasion of sand beachfronts by ornamental plant species in Queensland. Plant Prot Q 12:180–186

Batianoff GN, Butler DW (2002) Assessment of invasive naturalised plants in south-east Queensland. Plant Prot Q 17:27–34

Batygina TB, Bragina EA, Titova GE (1996) Morphogenesis of Propagules in Viviparous Species Bryophyllum Daigremontianum annd B. Calycinum

Baur J, Bovey R, Merkle M (1972) Concentration of Picloram in Runoff Water. Weed Sci 20:309–313

Benitez D, Loh R, Tunison T, Zimmer N, Makaike J, Mattos R, Casali M (2012) Distribution of Invasive Plant Species of Concern in the Kīlauea and Mauna Loa Strip Areas of Hawai’i Volcanoes National Park, 2000–2010. Honolulu, Hawai’i. 120 p

Berger ST, Dobrow MH, Ferrell JA, Webster TM (2014) Influence of Carrier Volume and Nozzle Selection on Palmer Amaranth Control. Peanut Sci 41:120–123

Bergin D (2011) Weed Control Options for Coastal Sand Dunes – a Review

Boiteau P, Allorge-Boiteau L (1995) Kalanchoe (Crassulacées) de Madagascar. Systématique, écophysiologie et phytochimie. Paris: Karthala

Borger CPD, Riethmuller GP, Ashworth M, Minkey D, Hashem A, Powles SB (2013) Increased Carrier Volume Improves Preemergence Control of Rigid Ryegrass ( Lolium rigidum ) in Zero-Tillage Seeding Systems . Weed Technol 27:649–655

C.A. S, W.S. R, K.W. E (2012) NIH image to ImageJ: 25 years of image analysis. Nat Methods 9:671–675

CABI (2019) Kalanchoe pinnata [original text by Rojas-Sandoval, J. and Acevedo-Rodríguez, P.]. Wallingford, UK: Invasive Species Conmpendium. CAB International

Castillo SA, Moreno-Casasola P (1996) Coastal sand dune vegetation: an extreme case of species invasion. J Coast Conserv 2:13–22

Chacón N, Herrera I, Flores S, González JA, Nassar JM (2009) Chemical, physical, and biochemical soil properties and plant roots as affected by native and exotic plants in Neotropical arid zones. Biol Fertil Soils 45:321–328

Chernetskyy M (2011) Problems in nomenclature and systematics in Kalanchoideae (Crassulaceae) over the years. ACTA Agrobot 64:67–74

Chernetskyy MA (2012) The Role of Morpho-Anatomical Traits of the Leaves in the Taxonomy of Kalanchoideae Berg. Subfamily (Crassulaceae DC.). Mod Phytomorphology 1:15–18

115

Descoings B (2003) Illustrated handbook of succulent plants: Crassulaceae. Pages 143–180 in U. Eggli. Berlin: Springer-Verlag

Descoings B (2006) Le genre Kalanchoe (Crassulaceae): structure et definition. 2–3 p

Diana SG, Resetarits WJ, Schaeffer DJ, Beckmen KB, Beasley VR (2000) Effects of atrazine on amphibian growth and survival in artificial aquatic communities. Environ Toxicol Chem 19:2961–2967

Ditomaso JM, Masters RA, Peterson VF (2010) Rangeland Invasive Plant Management. Rangelands 32:43–47

EDDMapS (2019a) Cathedral Bells Kalanchoe pinnata (Lam.) Pers. The University of Georgia - Center for Invasive Species and Ecosystem Health.

EDDMapS (2019b) Devil’s backbone Kalanchoe daigremontiana Hamet & Perrier. The University of Georgia - Center for Invasive Species and Ecosystem Health.

EDDMapS (2019c) Chandelier plant Kalanchoe delagoensis Ecklon & Zeyh. The University of Georgia - Center for Invasive Species and Ecosystem Health.

Edwards EJ, Ogburn RM (2012) Angiosperm Responses to a Low-CO 2 World: CAM and C 4 Photosynthesis as Parallel Evolutionary Trajectories. Int J Plant Sci

Enloe SF, Langeland K a, Ferrell J a, Sellers B, Macdonald GE (2018) Integrated Management of Nonnative Plants in Natural Areas of Florida

Epp JB, Alexander AL, Balko TW, Buysse AM, Brewster WK, Bryan K, Daeuble JF, Fields SC, Gast RE, Green RA, Irvine NM, Lo WC, Lowe CT, Renga JM, Richburg JS, Ruiz JM, Satchivi NM, Schmitzer PR, Siddall TL, Webster JD, Weimer MR, Whiteker GT, Yerkes CN (2016) The discovery of ArylexTM active and RinskorTM active: Two novel auxin herbicides. Bioorganic Med Chem 24:362–371

Ferguson JC, Chechetto RG, Hewitt AJ, Chauhan BS, Adkins SW, Kruger GR, O’Donnell CC (2016) Assessing the deposition and canopy penetration of nozzles with different spray qualities in an oat (Avena sativa L.) canopy. Crop Prot 81:14–19

FLEPPC (2019) List of Invasive Plant Species. https://www.fleppc.org/list/list.htm

FNAI (2010) Guide to the Natural Communities of Florida: Coastal Uplands. Florida Narual Areas Invent:77–79

Garcês H, Sinha N (2009) The “Mother of Thousands” (Kalanchö daigremontiana): A plant model for asexual reproduction and CAM studies. Cold Spring Harb Protoc 4:1–10

Garces HMP, Champagne CEM, Townsley BT, Park S, Malho R, Pedroso MC, Harada JJ, Sinha NR (2007) Evolution of asexual reproduction in leaves of the genus Kalanchoe. Proc Natl Acad Sci 104:15578–15583

116

García-Sogo B, Pineda B, Castelblanque L, Antón T, Medina M, Roque E, Torresi C, Pío Beltrán J, Moreno V, Antonio Cañ as L, Torresi C (2010) Efficient transformation of Kalanchoe blossfeldiana and production of male-sterile plants by engineered anther ablation. Plant Cell Rep 29:61–77

González de León S, Herrera I, Guevara R (2016) Mating system, population growth, and management scenario for Kalanchoe pinnata in an invaded seasonally dry tropical forest. Ecol Evol 6:4541–4550

Gordon DR (1998) Effects of Invasive, Non-Indigenous Plant Species on Ecosystem Processes: Lessons from Florida. Ecol Appl 8:975–989

Gué Zou A, Trueman M, Buddenhagen CE, Chamorro S, Guerrero AM, Pozo P, Atkinson R (2010) An Extensive Alien Plant Inventory from the Inhabited Areas of Galapagos. PLoS One 5

Guerra-García A, Barrales-Alcalá D, Argueta-Guzmán M, Cruz A, Mandujano MC, Arévalo- Ramírez JA, Milligan BG, Golubov J (2018) Biomass Allocation, Plantlet Survival, and Chemical Control of the Invasive Chandelier Plant (Kalanchoe delagoensis) (Crassulaceae). Invasive Plant Sci Manag 11:33–39

Guerra-García A, Golubov J, C. Mandujano M (2015) Invasion of Kalanchoe by clonal spread. Biol Invasions 17:1615–1622

Hannan-Jones MA, Playford J (2002) The biology of Australian weeds 40. Bryophyllum Salisb. species. Plant Prot Q 17:42–57

Hart H t, Eggli U, eds. (1995) Evolution and Systematics of the Crassulaceae. Leiden: Backhuys

Henderson AM, Gervais JA, Luukinen B, Buhl K, Stone D, Cross A, Jenkins J (2010) Glyphosate General Fact Sheet. Oregon State University Extension Services. http://npic.orst.edu/factsheets/glyphogen.html. Accessed June 8, 2019

Henderson L (2007) Invasive, naturalized and casual alien plants in southern Africa: a summary based on the Southern African Plant Invaders Atlas (SAPIA). Bothalia 37:215–248

Herrera I, Ferrer-Paris JR, Benzo D, Flores S, Garcia B, Nassar JM (2018) An Invasive Succulent Plant (Kalanchoe daigremontiana) Influences Soil Carbon and Nitrogen Mineralization in a Neotropical Semiarid Zone. Pedosphere 28:632–643

Herrera I, Ferrer-Paris JR, Hernández-Rosas JI, Nassar JM (2016) Impact of two invasive succulents on native-seedling recruitment in Neotropical arid environments. J Arid Environ 132:15–25

Herrera I, Hernandez MJ, Lampo M, Nassar JM (2012) Plantlet recruitment is the key demographic transition in invasion by Kalanchoe daigremontiana. Popul Ecol 54:225–237

117

Herrera I, Nassar JM (2009) Reproductive and recruitment traits as indicators of the invasive potential of Kalanchoe daigremontiana (Crassulaceae) and Stapelia gigantea (Apocynaceae) in a Neotropical arid zone. J Arid Environ 73:978–986

Hershey DR (2009) Using the Kalanchoe daigremontiana Plant to Show the Effects of Photoperiodism on Plantlet Formation. Sci Act Classr Proj Curric Ideas 39:30–34

Hewett DG (1985) Grazing and Mowing as Management Tools on Dunes. Vegetatio 62:441–447

Houghton AD (1935) An intersting hybrid. Cactus Succul J 7:44

Izaguirre‐Mayoral ML, Carballo O, Gil F (1990) Purification and Partial Characterization of Isometric Virus‐like Particles in Kalanchoe Species. J Phytopathol 130:303–311

Jervais G, Luukinen B, Buhl K, Stone D (2008) 2,4-D General Fact Sheet. Oregon State University Extension Services

Johnson AF, Barbour MG (1990) Dune and maritime forests. Pages 429–473 in RL Myers, JJ Ewel, eds. Ecosystems of Florida. Gainesville, Florida: University Press of Florida

Johnson M (1934) The Origin of the Foliar Pseudo-Bulbils in Kalanchoe daigremontiana. Bull Torrey Bot Club 61:355–366

Karper JJ, Doorenbos J (1983) Kalanchoe at Wageningen. Gard 108:237–240

Kolodziejczyk-Czepas J, Stochmal A (2017) Bufadienolides of Kalanchoe species: an overview of chemical structure, biological activity and prospects for pharmacological use. Phytochem Rev 16:1155–1171

Kuligowska K, Lütken H, Christensen B, Skovgaard I, Linde M, Winkelmann T, Müller R (2015) Evaluation of reproductive barriers contributes to the development of novel interspecific hybrids in the Kalanchoë genus. BMC Plant Biol 15

Lenth R (2019) emmeans: Estimated Marginal Means, aka Least-Squares Means

LR G-T, R R, A P, eds. (2012) Plantas Invasoras en Cuba [Invasive plants in Cuba]. Pages 1–140 in Bissea: Boletín sobre Conservación de Plantas del Jardin Botánico Nacional

Lüttge U (2004) Ecophysiology of Crassulacean Acid Metabolism (CAM). Ann Bot 93:629–652

Maeda H, Dudareva N (2012) The Shikimate Pathway and Aromatic Amino Acid Biosynthesis in Plants. Annu Rev Plant Biol 63:73–105

Maharani, R., Fajriah, S., Hardiawan, R., & Supratman U (2008) Insecticidal bufadienolides from the leaves of Kalanchoe daigremontiana (Crassulaceae). Proceeding Int Semin Chem 2008:236–239

118

Majaz Q, Tatiya AU, Khurshid M, Nazim S, Siraj S (2011) The miracle plant ( Kalanchoe pinnata ): A phytochemical and pharmacological review ISSN 2229-3566. Int J Res Ayurveda Pharm 2:1478–1482

Marshall FE, Banks K, Cook GS (2014) Ecosystem indicators for Southeast Florida beaches. Ecol Indic 44:81–91

Mason TJ, French K (2007) Management regimes for a plant invader differentially impact resident communities. Biol Conserv 136:246–259

McKenzie RA, Dunster PJ (1986) Hearts and flowers: Bryophyllum poisoning of cattle. Aust Vet J 63:222–227

McKenzie RA, Franke FP, Dunster PJ (1987) The toxicity to cattle and bufadienolide content of six Bryophyllum species. Aust Vet J 64:298–301

Metcalfe, C.R. and Chalk L (1950) Crassulaceae. Pages 578–581 in Anatomy of dicotyledons : leaves, stem, and wood in relation to taxonomy, with notes on economic uses

Moran RV (2009) Crassulaceae. Pages 147–230 in Flora of North America North of MexicoVol. 8.

Nair MG, Epp MD, Burke BA (1988) Ferulate esters of higher fatty alcohols and allelopathy in Kalanchöe daigremontiana. J Chem Ecol

Nelson EA, Penner D (2006a) Enhancing Herbicide Selectivity with Water-Repellent Adjuvants. Weed Technol 20:677–681

Nelson EA, Penner D (2006b) Reduction of Isoxaflutole Injury to Corn (Zea mays) with Herbicide Safeners and Water-Repellent Adjuvants. Weed Technol 20:999–1003

Nice G, Johnson B (2008) A Prickly Situation that Pricklypear

Niechayev NA, Pereira PN, Cushman JC (2019) Understanding trait diversity associated with crassulacean acid metabolism (CAM). Curr Opin Plant Biol 49:74–85

Niering WA, Whittaker RH, Lowe CH (1963) The Saguaro: A Population in Relation to Environment. Science (80- ) 142:15–23

Padilla FM, Pugnaire FI (2006) The role of nurse plants in the restoration of degraded environments. Front Ecol Env

Palmer WA (2011) Australia ’ s Current Approval Procedures for Biological Control with Particular Reference to its Biological Control Act. Pages 84–88 in XIII International Symposium on Biological Control of Weeds

119

Pesce S, Fajon C, Bardot C, Bonnemoy F, Portelli C, Bohatier J (2006) Effects of the phenylurea herbicide diuron on natural riverinemicrobial communities in an experimental study. Aquat Toxicol 78:303–314

Pratt JR, Melendez AE, Barreiro R, Bowers NJ (1997) Predicting the ecological effects of herbicides. Ecol Appl 7:1117–1124

Pucher GW, Vickery HB (1942) On the Identity of the so called Crassulacean Malic Acid with Isocitric Acid. J Biol Chem 145:525–532

Ranson, S.L. and Thomas M (1960) Crassulacean Acid Metabolism. Annu Rev Plant Physiol 11:81–110

RStudio Team (2015) RStudio: Integrated development for R. Boston, MA: RStudio, Inc.

Ryan Miller M, Norsworthy JK, Scott RC (2017) Evaluation of florpyrauxifen-benzyl on herbicide-resistant and herbicide-susceptible barnyardgrass accessions. Weed Technol 32:126–134

Schafer C, Luttge U (1987) Water translocation in Kalanchoe daigremontiana during periods of drought. Plant; Cell Environ 10:761–766

Schafer C, Luttge U (1988) Effects of High Irradiances on Photosynthesis , Growth and Crassulacean Acid Metabolism in the Epiphyte Kalanchoë uniflora. Oecologia 75:567–574

SEMARNAT (2016) Acuerdo por el que se determina la lista de especies exóticas invasoras para México. Mexico

Shaw DR, Morris WH, Webster EP, Smith DB (2000) Effects of Spray Volume and Droplet Size on Herbicide Deposition and Common Cocklebur ( Xanthium strumarium ) Control. Weed Technol 14:321–326

Shaw JMH (2008) An investigation of the cultivated Kalanchoe daigremontiana group , with a checklist of Kalanchoe cultivars. Hanburyana 3:17–79

Shivlani MP, Letson D, Theis M (2003) Visitor Preferences for Public Beach Amenities and Beach Restoration in South Florida. Coast Manag 31:367–385

Smith G (2004) Kalanchoe species poisoning in pets. Vet Med:933–936

Song Y (2014) Insight into the mode of action of 2,4-dichlorophenoxyacetic acid (2,4-D) as an herbicide. J Integr Plant Biol 56:106–113

Sparkes EC, Grace S, Panetta FD (2002) The effects of various herbicides on Bryophyllum pinnatum (Lam.) Pers in Nudgee Wetlands Reserve, Queensland. Plant Prot Q 17:77–80

Sperry B (2019) Optimizing residual herbicides in mid-south cropping systems

120

Stevens PJG (1993) Organosilicone Surfactants as Adjuvants for Agrochemicals*. Page Pestic. Sci. 103–122 p

Stoudt H (1938) Gemmipary in Kalanchoe rotundifolia and Other Crassulaceae. Am J Bot 25:106–110

Supratman U, Fujita T, Akiyama K, Hayashi H (2014) New Insecticidal Bufadienolide, Bryophyllin C, from Kalanchoe pinnata Unang. Biosci Biotechnol Biochem 64:1310–1312

Swingle CF (1934) The easiest plant in the world to propagate. J Hered 25:73–74

Ting I (1989) Photosynthesis of Arid and Subtropical Succulent Plants. Aliso 12:387–406

Trager JN (2005) The Huntington Botanical Gardens Presents the 2019 Offering of the International Succulent Introductions. Cactus Succul J 77:101

Wang ZQ, Guillot D, Ren MX, López-Pujol J (2016) Kalanchoe (Crassulaceae) as invasive aliens in China - new records, and actual and potential distribution. Nord J Bot 34:349–354

Ward DB (2006) A name for a hybrid Kalanchoe now naturalized in Florida. Cactus Succul J 78:92–95

Ward DB (2008) Keys to the Flora of Florida:18, Kalanchoe (Crassulaceae). Phytologia 90:41– 46

Warden J (1972) Leaf-plantlet meristems (="leaf-embryos") of Bryophyllum. Revision of terminology. Port Acta Biol Ser A 12:97–100

Widmann K, Gebauer G, Rehder H, Ziegler H (1990) Biomass production and nitrogen contents of the CAM plants Kalanchoe damigremontiana and K. tubitlora in cultures with different nitrogen and water supply. Page Oecologia. Springer-Verlag. 478–483 p

Williams MJ (2007) Native Plants for Coastal Restoration: What, When, and How for Florida. USDA, NRCS, Brooksv Plant Mater Cent:51

Wisconsin Department of Natural Resources (2012) Flumioxazin Chemical Fact Sheet. Madison, WI

Witt ABR (2004) Initial screening of the stem-boring weevil Osphilia tenuipes, a candidate agent for the biological control of Bryophyllum delagoense in Australia. Page BioControl. 197– 209 p

Witt ABR, McConnachie AJ, Stals R (2004) Alcidodes sedi (Col.: Curculionidae), a natural enemy of Bryophyllum delagoense (Crassulaceae) in South Africa and a possible candidate agent for the biological control of this weed in Australia. Biol Control 31:380–387

Wunderlin RP, Hansen BF (2011) Guide to the vascular plants of Florida. 3rd ed. Gainesville: University Press of Florida. 396 p

121

Yelenik SG, Dimanno N, D’Antonio CM (2015) Evaluating nurse plants for restoring native woody species to degraded subtropical woodlands. Ecol Evol 5:300–313

Zarnetske PL, Seabloom EW, Hacker SD (2010) Non-target effects of invasive species management: Beachgrass, birds, and bulldozers in coastal dunes. Ecosphere 1

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BIOGRAPHICAL SKETCH

Jessica Solomon was born and raised in Florida. She received her Associates in Zoo

Animal Technology at Santa Fe College Teaching Zoo before moving on to receive her Bachelor of Science at The University of Florida in Wildlife Ecology and Conservation in 2013. Jessica moved to California where she worked at an ecological consulting firm, responsible for plant and wildlife surveys. She spent most weekends working at International Bird Rescue doing aquatic bird medical rehabilitation. She moved back to Florida in 2015 where her passion for the outdoors and ecology lead her to work at the Florida Park Service working with the AmeriCorps program Florida Conservation Corps (FLCC), learning firsthand about herbicide application and large scale infestations. Through a collaboration of AmeriCorps with The University of Florida’s

Center for Aquatic and Invasive Plants (CAIP), Jessica meet professor Dr. Stephen Enloe who gave her the opportunity to pursue a Master of Science degree in Agronomy. Her studies at UF and CAIP has focused on invasive plant management. She plans on graduating in December of

2019. Jessica also continues to collaborate with FLCC on herbicide safety and application.

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