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Uptake, Distribution and Effects of Phosphite on Fusarium Oxysporum in Palms

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Title:
Uptake, Distribution and Effects of Phosphite on Fusarium Oxysporum in Palms
Physical Description:
1 online resource (41 p.)
Language:
english
Creator:
Yu, Jia Ming
Publisher:
University of Florida
Place of Publication:
Gainesville, Fla.
Publication Date:

Thesis/Dissertation Information

Degree:
Master's ( M.S.)
Degree Grantor:
University of Florida
Degree Disciplines:
Horticultural Sciences, Environmental Horticulture
Committee Chair:
Broschat, Timothy K
Committee Members:
Elliott, Monica Lynn
Moore, Kimberly Anne

Subjects

Subjects / Keywords:
fusarium-wilt -- ion-chromatography -- palm -- phosphite
Environmental Horticulture -- Dissertations, Academic -- UF
Genre:
Horticultural Sciences thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract:
Phosphite compounds have been shown to reduce disease, however the action mode of phosphite is complex and not fully understood. Knowledge of phosphite distribution in plants is essential to determine the optimum timing for fungicide treatment. Phosphite has control on avocado Fusarium wilt disease. Its effects on Fusarium wilt in palms are unknown. Our objectives for this study include: 1) to determine the uptake and distribution of phosphite in coconut palms over time that have been trunk injected. 2) to determine if Fusarium oxysporum f. sp. palmarum is sensitive to phosphite in vitro, and compare the results with greenhouse trials on Washingtonia robusta(W.r.). From this study, it shows that phosphite has a long persistence and not evenly distributed in coconut palm. It is concentrated in spear and young leaf rachis. Two Fusarium oxysporum isolates show sensitively to phosphite in vitro, and with a concentration of about 350 µg/g inoculated W.r. seedlings had a lower mortality rate.
General Note:
In the series University of Florida Digital Collections.
General Note:
Includes vita.
Bibliography:
Includes bibliographical references.
Source of Description:
Description based on online resource; title from PDF title page.
Source of Description:
This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility:
by Jia Ming Yu.
Thesis:
Thesis (M.S.)--University of Florida, 2013.
Local:
Adviser: Broschat, Timothy K.

Record Information

Source Institution:
UFRGP
Rights Management:
Applicable rights reserved.
Classification:
lcc - LD1780 2013
System ID:
UFE0045969:00001

MISSING IMAGE

Material Information

Title:
Uptake, Distribution and Effects of Phosphite on Fusarium Oxysporum in Palms
Physical Description:
1 online resource (41 p.)
Language:
english
Creator:
Yu, Jia Ming
Publisher:
University of Florida
Place of Publication:
Gainesville, Fla.
Publication Date:

Thesis/Dissertation Information

Degree:
Master's ( M.S.)
Degree Grantor:
University of Florida
Degree Disciplines:
Horticultural Sciences, Environmental Horticulture
Committee Chair:
Broschat, Timothy K
Committee Members:
Elliott, Monica Lynn
Moore, Kimberly Anne

Subjects

Subjects / Keywords:
fusarium-wilt -- ion-chromatography -- palm -- phosphite
Environmental Horticulture -- Dissertations, Academic -- UF
Genre:
Horticultural Sciences thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract:
Phosphite compounds have been shown to reduce disease, however the action mode of phosphite is complex and not fully understood. Knowledge of phosphite distribution in plants is essential to determine the optimum timing for fungicide treatment. Phosphite has control on avocado Fusarium wilt disease. Its effects on Fusarium wilt in palms are unknown. Our objectives for this study include: 1) to determine the uptake and distribution of phosphite in coconut palms over time that have been trunk injected. 2) to determine if Fusarium oxysporum f. sp. palmarum is sensitive to phosphite in vitro, and compare the results with greenhouse trials on Washingtonia robusta(W.r.). From this study, it shows that phosphite has a long persistence and not evenly distributed in coconut palm. It is concentrated in spear and young leaf rachis. Two Fusarium oxysporum isolates show sensitively to phosphite in vitro, and with a concentration of about 350 µg/g inoculated W.r. seedlings had a lower mortality rate.
General Note:
In the series University of Florida Digital Collections.
General Note:
Includes vita.
Bibliography:
Includes bibliographical references.
Source of Description:
Description based on online resource; title from PDF title page.
Source of Description:
This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility:
by Jia Ming Yu.
Thesis:
Thesis (M.S.)--University of Florida, 2013.
Local:
Adviser: Broschat, Timothy K.

Record Information

Source Institution:
UFRGP
Rights Management:
Applicable rights reserved.
Classification:
lcc - LD1780 2013
System ID:
UFE0045969:00001


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1 UPTAKE, DISTRIBUTION AND EFFECTS OF PHOSPHITE ON FUSARIUM OXYSPORUM IN PALMS By JIAMING YU A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE O F MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2013

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2 2013 Jiaming Yu

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3 To my family and friends

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4 ACKNOWLEDGMENTS I would like to thank my advisors Drs. Timothy Broschat and Monica Elliott for their support, encouragement, patience, motivation, and immense knowledge. I extend my gratitude to Dr. Kimberly Moore for instruction, assistance, and serving on my committee. Additionally, I would like to thank William Latham for helping develop the ion chromatography experimental method. Many thanks to Elizabeth Des Jardin, Susan Thor, and Sebastian Ortiz for helping with experi ment work and data collection.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF TABLES ................................ ................................ ................................ ............ 7 LIST OF FIGURES ................................ ................................ ................................ .......... 8 LIST OF ABBREVIATIONS ................................ ................................ ............................. 9 ABSTRACT ................................ ................................ ................................ ................... 10 CHAPTER 1 INTRODUCTION ................................ ................................ ................................ ..... 11 2 LITERATURE REVIEW ................................ ................................ ........................... 13 Phosphite ................................ ................................ ................................ ................ 13 Fusarium Wilt of Palms ................................ ................................ ........................... 14 3 MATERIALS AND METHODS ................................ ................................ ................. 17 Dynamics and Distribution of Injected HPO 3 2 ................................ ......................... 17 Plant Material ................................ ................................ ................................ ... 17 Injection System ................................ ................................ ............................... 17 Sample Collection ................................ ................................ ............................ 18 Plant Analysis ................................ ................................ ................................ ... 18 Ion Chromatography ................................ ................................ ......................... 19 Effects of Phosphite on Fusarium O xysporum In Vitro ................................ ........... 19 Isolates ................................ ................................ ................................ ............. 19 Base Media ................................ ................................ ................................ ...... 20 Growth of Myceliu m and Radial Measurements ................................ ............... 20 Effects of Phosphite on Fusarium Oxysporum In Vivo ................................ ............ 20 Growing Substrate ................................ ................................ ............................ 20 Phosphite Treatment ................................ ................................ ........................ 21 Horticulture Control ................................ ................................ .......................... 21 Plant Inoculation ................................ ................................ ............................... 21 Plant Analysis ................................ ................................ ................................ ... 21 4 RESULTS AND DISCUSSION ................................ ................................ ................ 23 Dynamics of Injected HPO 3 2 ................................ ................................ .................. 23 Distribution of Injected HPO 3 2 ................................ ................................ ................ 25 Effects of P hosphite on Fusarium Oxysporum In Vitro ................................ ........... 25 Effects of P hosphite on Fusarium Oxysporum In Vivo ................................ ............ 26

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6 5 CONCLUSION ................................ ................................ ................................ ........ 37 LIST OF REFERENCES ................................ ................................ ............................... 38 BIOGRAPHICAL SKETCH ................................ ................................ ............................ 41

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7 LIST OF TABLES Table page 4 1 Phosphite contents in different parts of coconut p alm at 40 weeks after injection with potassium phosphite. ................................ ................................ .... 32 4 2 Mortality of Washingtonia robusta seedlings at 14 weeks after first treatment in trial 1. ................................ ................................ ................................ .............. 35 4 3 Mortality of Washingtonia robusta seedlings at 14 weeks after first treatment in trial 2 ................................ ................................ ................................ .............. 36

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8 LIST OF FIGURES Figure page 4 1 Phosphite concentration in old rachis of coconut through 30 weeks following trunk injection with potassium phosphite. ................................ ........................... 28 4 2 Phosphite c oncentration i n s pear of coconut through 30 weeks following trunk injection with potassi um phosphite ................................ ................................ .... 29 4 3 Phosphite concentration in young leaflets of coconut through 30 weeks following trunk injecti on with potassium phosphite ................................ ............ 30 4 4 Phosphite concentration in old leaflets of coconut through 30 weeks following trunk injection with potassium phosphite. ................................ ........................... 31 4 5 Colony diameters of two isolates of F. o. palmarum in1/5 PDA medium containing potassium phosphite. ................................ ................................ ........ 33 4 6 Growth of PLM 249A and PLM 140B myceli um on 1/5 PDA with seven phosphite concentrations. ................................ ................................ ................... 34

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9 LIST OF ABBREVIATIONS DI Deionized Water KOH Potassium Hydroxide PD A Potato Dextrose Agar

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10 Abstract of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science UPTAKE, DISTRIBUTION AND EFFECTS OF PHOSPHITE ON FUSARIUM OXYSPORUM IN PALMS By Jiaming Yu August 2013 Chair: Timothy Broschat Major: Horticultural Sciences Phosphite compounds have been shown to reduce plant disease h owever the mode of action for phosphite is complex and not fully understood. Knowledge of phosphite distribution and dynamics in plants is essential to determine which diseases it may control and to determine the optimum timing for fungicide treatment P hosphite has controlled Phytophthora bud rot in palms. Its effects on Fusarium wilt in palms are unknown. Our objectives for this study were: 1) to determine the uptake and distribution of phosphite in trunk injected coconut palms over time, 2) to determine if Fusarium oxysporum f. sp palmarum is sensitive to phosphite in vitro and 3) compare the results with greenhouse trials on Washingtonia robusta This study shows that phosphite has a long persistence and is not evenly distributed in coconut palm, but it is concentrated in the spear leaf and young and old leaf rachises. Two Fusarium oxysporum isolates showed sensitivity to phosphite in vitro Inoculated W. robusta seedlings with a fo liar phosphite concentration of about 350 1 had a lower mortality rate than those with lower foliar concentrations of phosphite.

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11 CHAPTER 1 INTRODUCTION Phosphites (syn. phosphonates) (H 2 PO 3 ; HPO 3 2 ) are alkali metal salts of phosphorous acid (H 3 PO 3 ) a reduced form of phosphate (PO 4 3 ). Phosp hite has a higher degree of solubility and mobility, as it has one less oxygen molecule than phosphate. This characteristic gives phosphite the ability to rapidly cross the membranes of leaves or roots. It is also considered an ambimobile nutrient, which m eans it has both xylem and phloem mobility (Guest and Grant, 1991). Other benefits of phosphite include: persistence in plant tissue, controlled release of phosphorus during crop growth, enhanced plant and root development, improved plant health, multiple action sites as a fungicide, and low environmental toxicity. Among the advantages of phosphite compounds, the most widely recognized property is its extremely effective control of plant diseases caused by Phytophthora spp. (Fenn and Coffey, 1984; Forster e t al., 1998; Grant et al., 1992; Guest and Grant, 1991; Guest et al., 1995; Jackson et al., 2000; Jee et al., 2002; Smillie et al., 1989). Phosphite compounds have been shown to reduce disease ( Phytophthora infestans Fusarium solani and Rhizoctonia solani ) severity in potato seed tubers and foliage(Lobato et al ., 2008; Lobato et al., 2010), and also have a positive effect on hot and sweet pepper against Phytophthora capsici (Fernando Cesar Sala, 2004). However, the action mode of phosphite is complex and n ot fully understood. Knowledge of phosphite distribution and dynamics in plant tissues is essential to determine the optimum timing for fungicide treatment (Bezuidenhout et al., 1987) A study on the dynamics and distribution of phosphite in avocado trees treated with phosetyl Al indicates that the phosphite concentrations were higher in branches than in

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12 the roots or leaves (Bezuidenhout et al., 1987). Its concentration in the branches and root samples taken from the Fuerte avocado trees reached its peak a month after trunk injection of phosetyl Al. However, in mature leaves, the phosphite peak was broader. As a result, old branches stored about 50% of the phosphite, the roots around 30%, and the last 20% was in young braches, leaves and leaf stems. The con clusion from this study was that phosphite was not evenly distributed among different plant organs. Nothing is known about the dynamics and distribution of phosphite in palms. The objectives for this study include: 1) determine the uptake and distribution of phosphite in trunk injected coconut palms over time, 2) determine if Fusarium oxysporum f. sp palmarum is sensitive to phosphite in vitro and 3) compare the in vitro results with greenhouse trials on W ashingtonia r obusta inoculated with Fusarium oxysp orum f. sp palmarum to determine if phosphite had direct or indirect effects on this pathogen.

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13 CHAPTER 2 LITERATURE REVIEW Phosphite Palms are beautiful ornamental plants and are economically important. As monocots, single stemmed palms are relatively ea sy to grow but are also easily killed because they have a single apical meristem. Once the meristem is damaged, the palm usually dies. This damage could be caused by infectious diseases or physiological disorders. For example, palm bud rot, is a common dis ease caused by P hytophthora palmivora (Oomycetes ) The first symptom of a bud rot caused by this pathogen is the discoloration and wilting of the spear leaf, the youngest and unopened leaf. The next youngest leaves may also appear discolored and wilted. Ev entually, all leaves become desiccated, turn brown and collapse. The leaf bases often have distinct brown or necrotic areas. Fosetyl Al, which has phosphite as its active ingredient, is the recommended fungicide for juvenile palms for control of bud rot ca used by Phytophthora (Elliott, 2009). It has been shown that phosphite is effective in controlling Phytophthora in coconut palms, and that stem injection is more effective than other metho ds (de Franqueville and Renard, 1989). In another study, optimum lon g term control of Phytophthora palmivora was achieved in cocoa by using stem injected potassium phosphite (Guest et al., 1994). Trunk injection of potassium phosphite showed efficient and durable control of Phytophthora diseases in avocado (Pegg et al., 1985). A study on the dynamics and distribution of phosphite in avocado trees treated with phosetyl Al indicated that the phosphite concentrations were higher in branches than in the roots or leaves (Bezuidenhout et al., 1987). Its concentration in the br anches and root samples taken

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14 from the Fuerte avocado trees reached its peak a month after the injection of phosetyl Al. However, in mature leaves, the phosphite peak was broader. As a result, old branches stored about 50% of the phosphite, the roots aroun d 30%, and the last 20% was in young braches, leaves and leaf stems. The conclusion from this study was that phosphite was not evenly distributed among the different plant organs. Knowledge of phosphite distribution and dynamics in plants is essential to d etermine optimum time for fungicide treatment (Bezuidenhout et al., 1987). However, nothing is known about the dynamics and distribution of phosphite in palms. Foliar sprays and soil drenches are two common methods of applying phosphite. Studies have shown that foliar treatment with phosphite can control Phytophthora cinnamoni in infected avocado trees (Pegg et al. 1985). Although there are differences among phosphite salts applied, all of them enhance efficacy in controlling Phytophthora. In addition, agr onomic performance of infected trees, including number of leaves, photosynthetic activity, etc. improved significantly in phosphite treated trees (Cervera et al. 2007). In plant tissues, phosetyl Al degrades to ethanol and phosphite, the latter is the to xophore by either activating defense mechanisms in the plant (Bompeix and Saindrenan, 1984) or by acting directly on the fungus (Fenn and Coffey, 1984). Plants cannot utilize phosphite as a phosphorus source (Maclntyre et al 1950 ). However, some bacteria are capable of converting phosphite to phosphate (Malacinski and Konetzka, 1966). Fusarium Wilt of Palms Fusarium wilt is a common vascular disease in some species of palm. It is caused by specific formae speciales of the fungal pathogen Fusarium oxysporu m

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15 When the fungus grows in the xylem of palms, xylem tissues become obstructed by the fungus causing a failure to conduct water and other nutrients throughout the trunk, stem s and leaves of palms. Currently, there are four known formae speciales of F. oxysporum affecting palms throughout the world: Fusarium oxysporum f. sp. albedinis ( F. o. albedinis ), Fusarium oxysporum f. sp. canariensis ( F. o. canariensis ), Fusarium oxyspor um f. sp. elaeidis ( F. o. elaeidis ), and Fusarium oxysporum f. sp. palmarum ( F. o. palmarum ). No matter which Fusarium wilt is affecting palms, they die within several weeks to months after the initial infection (Elliott et al., 2004; Elliott et al., 2010) Fusarium oxysporum f. sp. palmarum attacks Syagrus romanzoffiana (queen palm) and Washingtonia robusta (Mexican fan palm) and it has also been reported to attack Phoenix canariensis (Canary Island date palm) in Florida (Elliott, 2011). No teleomorph of Fusarium oxysporum (Ascomycota) has been observed in culture. On potato dextrose agar (PDA), F. o. palmarum colonies have pale pinkish or purple color on the upper surface. Macroconidia, formed in orange sporodochia, are three to four septate with a curved apical cell and a foot shaped basal cell. Microconidia are unicellular and oval, elliptical or reniform in shape. Within 4 weeks after isolation, chlamydospores are produced. On PDA, some isolates produce sclerotia that are blue in color (Elliott et al., 2010) Fusarium oxysporum is a soilborne fungus. Chlamydospores produced by this fungus can survive in plant debris and soil for years (Elliott et al., 2004) Transmission of disease caused by Fusarium oxysporum depends on the movement of infected trees or infested soil and is also possible through the use of contaminated pruning tools.

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16 Once palms are affected by Fusarium wilt there is no known effective control. Preventive chemical controls have also not been shown to be efficacious or cost effective. C urrently, the control of this disease of palms is focused on prevention. An important aspect of prevention involves proper sterilization of tools between pruning different palms. Selection of non host palm species is important, especially if there is a his tory of previous disease incidences. This is important because even if diseased palms have already been removed, infective spores are likely to still be contaminating the soil. Once a palm is known to be affected by Fusarium, it should be cut down and burn ed or otherwise removed because as long as it remains in place it provides a potential source of infection for the surrounding landscape (Elliott et al., 2004)

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17 CHAPTER 3 MATERIALS AND METHODS Dynamics and Distribution of Injected HPO 3 2 Plant Material Twenty trees of Cocos nucifera University of Florida Fort Lauderdale Research and Education Center, Davie, FL., were used in this study. The Coconut palms were approximately 30 years old, 8 10 m in overall h eight, and were fruiting at the time of this study. Twenty replicates were arranged in a completely randomized design. Coconut palm s were growing in a Margate Fine Sand soil, with individual palms spaced about 6 m apart. Palms were fertilized every 3 month s by broadcasting an 8N 0.9P 10K 4Mg plus micronutrients controlled 2 to the entire plot. Injection System The Arborjet @ tree I.V. Micro infusion system (Arborjet, Inc., Woburn, MA) was used to inject the following four treatments: deionized water (DI) only (control), 30 mL potassium phosphite in 90 mL DI, 60 mL potassium phosphite in 180 mL DI, and 90 mL potassium phosphite in 270 mL DI. The commercial source of the potassium ph osphate was StarPhite @ (Loveland Products, Inc., Greeley, CO). Holes 9.5 mm in diameter and 10 cm deep were drilled approximately one meter above the ground. All of the holes were on the south side of trunk. Treatments were replicated 5 times and were arra nged in a completely randomized design. Injections were performed May 17, 2012.

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18 Sample Collection Plant tissues were harvested at 1, 5, 10, 15, 20, and 30 weeks after application. The following plant tissues were harvested to determine phosphite dynamics i n coconut: spear leaf tip, old leaf rachis base, central leaflets from an old leaf, and central leaflets from the youngest fully expanded leaf. On February 11, 2013, forty weeks after injection of potassium phosphite, ten different plant tissues were harv ested from 90 mL potassium phosphite treatment palms and numbered as: 1. spear leaf ( newest unopened leaf ) tip 2. rachis base from an old leaf, 3. rachis base from the youngest fully expanded leaf, 4. distal portion of the rachis on an old leaf, 5. distal portion of the rachis of the youngest fully expanded leaf, 6. basal leaflets from an old leaf, 7. basal leaflets from the youngest fully expanded leaf, 8. distal leaflets from an old leaf, 9. basal half of the distal leaflets from an old leaf, and 10. dis tal half of the distal leaflets from an old leaf. These plant tissues were analyzed for determination of phosphite distribution in coconut. Plant Analysis Plant samples were dried at 60C for 48 hrs and ground in a Wiley Mill to pass through a 40 mesh scre en. Homogeneous powder samples (0.5g) were extracted with deionized water, shaken by Labquake shaker (Labindustries, Inc, Berkeley, CA) for 10 minutes and centrifuged for 10 minutes at 2400 rpm The liquid superna tant was then filtered through a 0.22 m ny lon membrane filter using a vacuum pump. The filtrate was collected and refiltered through C18 SEP PAK Cartridges This filtrate was then brought to a final sample volume of 5 mL. Samples were kept frozen at 11C until they could be analyzed.

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19 Ion Chromat ography Ion chromatography is an ideal method for analyzing anions in plant materials. There is a published a method using ion chromatography to determine HPO 3 2 in tomato leaflets (Fenn and Coffey, 1989). Smilie and Grant (1988) described a rapid and feas ible method of using ion chromatography to determine phosphite and phosphate concentrations in plant materials. Samples were injected by hand using a 5 ml syringe into a Dionex 100 Ion Chromatograph (Model DX1 03) (Dionex Corp., Sunnyvale, CA) with integra ted Anion Self regenerating Suppressor Ultra, an AS 17 column, eluent gradient system and were eluted with 100 mM potassium hydroxide at a flow rate of 0.56 mL min 1 during the retention time of 0 0.45 min and 7.45 8.15 min at 18% of 100 mM KOH, and 5. 45 7.45 min at 100% of 100mM KOH.. Anions were detected by a conductivity detector and the data processed using Peak Net Data acquisition system (Dionex Corp., Sunnyvale, CA). Standards containing chloride, nitrite, phosphite, bromide, and nitrate were pr epared daily at three dilutions. Phosphite concentration data from coconut leaves at 40 weeks were analyzed (SAS Institute, Cary, N.C.) Effects of Phosphite on Fusarium oxysporum In V itro Isolates Two isolates of Fusarium oxysporum f. sp. p almarum from W ashington ia robusta (Mexican fan palm) (PLM 249A) and from Syagrus romanzoffiana ( Queen Palm ) (PLM 140B) were used in this experiment.

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20 Base Media The base medium was 1/5 strength Difco potato dextrose agar (1/5 PDA). Various amounts of potassium phosphite (StarPhit e, Loveland Products, Inc., Greeley, CO) were added to 1/5 PDA to obtain 0 (control), 1, 5, 10, 20, 40, or 60 gmL 1 of phosphite StarPhite contains 28% phoshorous acid (H 3 PO 3 ) that has been neutralized with potassium hydroxide. Growth of Mycelium and R adial Measurements Twenty eight plates of PLM 249A (four replicates per level of phosphite) and 28 plates (four replicates per level of phosphite) of PLM 140B were inoculated and then placed in 28 C incubator in darkness. Mycelial growth in each plate was determined by measuring the diameter of the colony (in two directions) per plate every 24 hours from 2 to 6 days following inoculation (Davis, et al., 1994). The experiment was conducted twice, and the data combined for analysis. The mycelia diameter on s ix dates at all rates were analyzed using regression analysis and the diameters on the sixth day at all rates were analyzed using ANOVA (SAS, SAS Institute, Cary, N.C.). Effects of Phosphite on Fusarium Oxysporum In Vivo Growing Substrate Washingtonia robu sta seedlings were grown in a greenhouse at the Fort Lauderdale Research and Education Center in 1 liter containers filled with 50% pine bark, 40% sedge peat and 10% sand substrate amended with 15N 9P 2 O 5 12K 2 O Osmocote (Scotts Co., Marysville, OH) controll ed release fertilizer incorporated at a rate of 10.86 kg/m 3 No herbicides were used to control weeds An overhead irrigation system provided ca. 2 cm of water daily.

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21 Phosphite Treatment Eight replicate plants per treatment were used for inoculation with t he pathogen and 8 additional replicates were used as horticulture controls and for leaf phosphite analysis Potassium phosphite ( Starphite ) was used in this treatment with foliar sprays or soil drenches applied The control group received only water. This experiment was conducted twice. The first trial was conducted from February to June in 2012 with 5 treatments including: control, foliar sprays (spray to run off) or soil drenches (50 mL) every 3 weeks at 0.74 gmL 1 (2.64 mL L 1 ) or 1.48 gmL 1 (5.28 m L L 1 ). The second trial was performed from November 2012 to March 2013 with 9 treatments including: control, foliar spray or soil drench applied only once at the 2 rates, or foliar sprays and soil drenches applied every 3 weeks at the 2 rates. Horticultur e Control The horticulture control seedlings were harvested on May 16, 2012 for trial one and on February 22, 2013 for trial two. Recently matured leaves of W. robusta palms were collected for leaf analysis of phosphite concentrations 14 weeks after first treatment. Plant Inoculation One month after the first phosphite treatment, the small palms (5 or 6 leaves) were inoculated with a spore suspension of PLM 249A (10 6 spores ml 1 ; 25 ml per palm) by using soil drench inoculation on February 29, 2012 for tria l one and on November 23, 2012 for trial 2. Plant Analysis Leaf samples were dried at 60C at 48 hrs ground in a Wiley Mill to pass through a 40 mesh screen. The powdered 0.5 g samples were extracted with deionized

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22 water; shaken by Labquake shaker (Labin dustries, Inc., Berkeley, CA) for 10 minutes and centrifuged for 10 minutes at 2400 rpm The liquid superna tant was then filtered through a 0.22 m nylon membrane filter using a vacuum pump. The filtrate was collected and refiltered through C18 SEP PAK Ca rtridges This filtrate was then brought to a final sample volume of 5 mL. Samples were kept frozen at 11C until they could be analyzed. Plant dry weight and phosphite concentration data were analyzed using ANOVA (SAS, SAS Institute, Cary, N.C.).

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23 CHAPTER 4 RESULTS AND DISCUSSION Dynamics of Injected HPO 3 2 The phosphite concentrations from the first sampling of the control palms which received no phosphite showed that there very little (<25 gg 1 ) naturally occuring phosphite in mature coc onut palms In old leaf rachis tissue, phosphite was rapidly taken up within the first week after injection, with the phosphite content reaching a peak at 242.7 gg 1 for the 30 mL phosphite treatment, 324.58 gg 1 for the 60 mL phosphite treatment, and 677.51 gg 1 for the 90 mL phosphite treatment (Fig. 4 1) After the first week, phosphite concentrations in all three treatments decreased until around five weeks. At that time, the concentration of phosphite in the 90 mL phosphite treatment increased an d reached a peak of 1 and then decreased, instead of decreasing for the first ten weeks like the other two treatments. However at the tenth week, the 30 mL treatment concentration increased from 80 gg 1 to 180 gg 1 for 5 weeks and then, as t he other treatments, phosphite concentrations in all three rates stabilized at 151 gg 1 for the 30 mL, 174 gg 1 for the 60 mL, and about 340 gg 1 for the 90mL treatments for another 20 weeks (Fig. 4 1). The increased concentration of phosphite occurr ed at 90 mL treatment between 5 10 weeks and 30 mL treatment between 10 15 weeks with an approximate increased value of 100 gg 1 (450 550 gg 1 and 80 180gg 1 for treatment 90 mL and 30 mL, respectively. ) After application, phosphite is rapidly absorbed in xylem, but is also translocated within the phloem (Guest and Grant, 1991). The decreasing and increasing concentration during the first twenty weeks could possibly be caused by phosphite equilibration and mobilization. Since the rachis serves a s the connection of vascular part of the palm and

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24 the leaf blade, the mobilization of phosphite is expected. These numbers indicate that phosphite concentrations are highest at one week after injection, and after 15 weeks, phosphite concentrations in old l eaf rachis tissue remains fairly stable for at least another 6 months (Fig. 4 1). In the spear leaf, it took ten weeks to reach the phosphite concentration peak of 313 gg 1 for the 30 mL treatment, 297.03 gg 1 for the 60 mL treatment, and 831.3 gg 1 f or the 90 mL treatment. After 15 weeks, phosphite concentration declined slowly over time for the 90 mL treatment. This indicates that the maximum accumulation of phosphite occurred about 10 weeks after application for the spear leaf. The phosphite concent rations of these three rates were around 350 gg 1 for 90 mL treatment and around 150 250 gg 1 for the other two treatments (Fig. 4 2) Unlike the spear leaf, young leaflets take up phosphite immediately after application. Phosphite concentration reac hed their peaks within one week after application at 374.26 gg 1 for the 30 mL treatment, 395.732 gg 1 for the 60 mL treatment, and 860.26 gg 1 for the 90 mL treatment (Fig. 4 3). During the next four weeks, phosphite concentrations decreased signifi cantly. In young leaf leaflets, the 30 mL and 60 mL treatments were similar for all sampling dates. The 90 mL treatment had twice the phosphite concentrations compared to the other two treatments. However, those three treatments had similar concentrations after the fifth week. A similar trend was observed in the old leaflets, although the inflection point appeared at the tenth week (Fig. 4 4). In old leaf leaflets, phosphite was also absorbed rapidly after application with concentrations at one week of 127 .73 gg 1 and 279 gg 1 for the 30 mL/90 mL and 60 mL/180 mL treatments, respectively, and 504.74 gg 1 for

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25 the 90 mL/270 mL treatment. The phosphite concentration in highest treatment (90 mL/270 mL) decrease from 500 gg 1 to less th a n 100 gg 1 in t he first ten weeks, and then phosphite concentration from all rates stay every similar, except for the 30 mL/90 mL and 60 mL/180 mL treatments, after 5 weeks of phosphite application, the phosphite concentration in old leaflets start to be stable around 70 90 gg 1 Which indicate that for a n effective disease control concentration, after 10 weeks these three rates provide similar efficacy. Distribution of Injected HPO 3 2 At 40 weeks after injection with 90 mL potassium phosphite, 10 different parts of t he coconut palm were sampled and phosphite content was determined. The spear leaf tip contained an average of 249.45 gg 1 phosphite For the same plant position, tissue from young leaves consistently had higher phosphite content than older leaves. For ex ample, young leaf rachis bases had 420.22 gg 1 of phosphite compared to 321.63 gg 1 for old leaf rachis bases. The same was true for distal rachis, basal leaflet an d distal leaflet tissues (Tab. 4 1). The high concentrations in leaf rachis indicate tha t phosphite accumulated at the position where the pathogen Fusarium oxysporum would infect the palm. Phosphite content decreased gradually from basal to distal portions in both young and old leaves (Tab. 4 1). This was expected since the palms were treate d via trunk injection and phosphite should be translocated through xylem to the canopy Spear leaves maintained a fairly high concentration for at least 10 months. The higher phosphite concentration in spear leaves come from phosphite remobilization from o ld tissue. This also explains why phosphite showed efficacy on P hytophthora bud rot on

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26 palms, since the spear leaf is where the phosphite accumulation occurred and where the pathogen first infects the palm. Effects of P hosphite on Fusarium Oxysporum In Vi tro In vitro phosphite directly inhibited PLM 249A and PLM 140B growth. As phosphite concentration increased, inhibition of phosphite on these two isolates of F. o. palmarum increased. PLM 140B growth was similar for 1/5 PDA with no phosphite and 1 gmL 1 phosphite. However, growth rates decreased from 10.86 mm at 5 gmL 1 10.36 mm at10 gmL 1 7.21 mm at 20 gmL 1 to 3.58 mm at 40 gmL 1 (Fig. 4 5). For PLM 249A, when phosphite concentration was increased from 0 gmL 1 to 60 gmL 1 the growth of isolates decreased significantly. Compared with PLM 140B, phosphite caused greater growth suppression in PLM 249A (Fig. 4 5). conclusion, both isolates of F. o. palmarum app eared to be sensitive to phosphite in vitro Effects of P hosphite on Fusarium Oxysporum In Vivo In the first trial, the lowest mortality of W. robusta was achieved by application of 0.74 gmL 1 soil drench, which led to a phosphite concentration of 209.6 g g 1 However, there is no significant difference with control, which contained a phosphite concentration of 187.92 g g 1 (Tab. 4 2). The relatively high concentration of phosphite in untreated W. robusta is unexpected as there is no natural phosphite in plant tissue documented. This requires a further determination of phosphite in W. robusta leaf tissue In the second trial, phosphite concentrations in the youngest fully expanded leaves of W. robusta seedlings treated every 3 weeks with 0.74 or 1.48 gm L 1

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27 averaged 330.49 gg 1 and 377.7 gg 1 respectively. The mortality rate of these seedlings averaged 6.25% which is significantly lower than the other treatments (Tab. 4 3). The average mortality was calculated based on similar foliar phosphite concen trations because this was a better way to show the relationship between phosphite sensitivity and concentration (Tab. 4 3). This suggested the possibility that a lower concentration of a foliar spray applied every 3 weeks was a feasible application of pota ssium phosphite from this experiment. More treatments and repeats need be added, and inoculation of F. o. palmarum for consistency in mortality needs to be improved. The dry weights from the seedlings surviving after inoculation were analyzed, but there wa s no significant relationship between seedling dry weight and phosphite content or phosphite treatment This suggests that phosphite d oes not quantitati vely reduce growth inhibition caused by Fusarium wilt.

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28 Figure 4 1. Phosphite concentration ( g g 1 ) in old rachis of coconut through 30 weeks following trunk injection with 3 rates of potassium phosphite: 30mL potassium phosphite in 90mL DI ( ), 60mL potassium phosphite in 180mL DI( ), and 90mL potassium phosphite in 270mL DI ( ).

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29 Figu re 4 2. Phosphite concentration ( g g 1 ) in s pear of coconut through 30 weeks following trunk injection with 3 rates of potassium phosphite: 30mL potassium phosphite in 90mL DI ( ), 60mL potassium phosphite in 180mL DI( ), and 90mL potassium phosphite in 270mL DI ( ).

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30 Figure 4 3. Phosphite concentration ( g g 1 ) in young leaflets of coconut through 30 weeks following trunk injection with 3 rates of potassium phosphite: 30mL potassium phosphite in 90mL DI ( ), 60mL potassium pho sphite in 180mL DI( ), and 90mL potassium phosphite in 270mL DI ( ).

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31 Figure 4 4 Phosphite concentration ( g g 1 ) in old leaflets of coconut through 30 weeks following trunk injection with 3 rates of potassium phosphite: 30mL potassi um phosphite in 90mL DI ( ), 60mL potassium phosphite in 180mL DI( ), and 90mL potassium phosphite in 270mL DI ( ).

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32 Table 4 1. Phosphite contents (average of 5 replicates) in different parts of coconut palm a t 40 weeks after injection with 9 0 mL pot assium phosphite in 270 mL DI. Position Mean ( g g 1 SE) Spear 249.45 17.86 Young basal rachis 420.22 23.05 Young distal rachis 234.98 19.96 Young basal leaflets 146.04 17.86 Young distal leaflet basal half 25.04 19.96 Young dista l leaflet distal half 13.11 17.86 Old basal rachis 321.63 23.05 Old distal rachis 64.73 17.86 Old basal leaflets 18.49 17.86 Old distal leaflets 16.10 17.86

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33 Fig ure 4 5. Colony diameters (mean of 8 replicates) of two isolates of F. o. palmarum: PLM 249A ( ) and PLM 140B ( ) grown in1/5 PDA medium containing potassium phosphite.

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34 Fig ure 4 6. Growth of PLM 249A (A) and PLM 140B (B) mycelium on 1/5 PDA with seven phosphite concentrations (0, 1, 5, 10, 20, 40, or 60 gmL 1 ).

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35 Table 4 2. Mortality (%) of Washingtonia r obusta seedlings at 14 weeks after first treatment in trial 1. Treatment Application frequency Mean of phosphite concentration in the first new leaf ( g g 1 ) z Mortality (%) 0.74 g mL 1 foliar s pray e very 3 weeks 265.65 60 1.48 g mL 1 foliar spray every 3 weeks 314 .00 75 0.74 g mL 1 soil drench every 3 weeks 209.6 0 25 1.48 g mL 1 soil drench every 3 weeks 242 .00 75 control 197.92 60 z Values are not significantly different based on mean sep

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36 Table 4 3. Mortality (%) of Washingtonia r obusta seedlings at 14 weeks after first treatment in trial 2 Treatment Application frequency Mean of phosphi te concentration in the first new leaf ( g g 1 ) y Mortality (%) Average mortality (%) z 0.74 gmL 1 foliar spray every 3 weeks 377.70 a 12.5 6.25 1.48 gmL 1 foliar spray every 3 weeks 330.49 a 0 ------------------------------------------------------------------------------------------------------------1.48 gmL 1 soil drench every 3 weeks 131.63 bc 12.5 20.83 0.74 gmL 1 foliar spray One time 146.96 bc 12.5 1.48 gmL 1 foliar spray One time 187.54 b 37.5 ------------------------------------------------------------------------------------------------------------0.74 gmL 1 soil drench One time 69.36 c 37.5 31.25 1.48 gmL 1 soil drench One time 78.62 c 62.5 0.74 gmL 1 soil drench every 3 weeks 77.27 c 12.5 control 76.5 9 c 12.5 y Values followed by the same letter are not significantly different based on mean z Average mortality is based on the 3 groups shown.

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37 CHAPTER 5 CONCLUSION Trunk injected potassium phosphite can be translocated by coconut palm s immediatel y after application. Phosphite concentrations in old lea f rachis and all leaflet tissue reached their peaks within a week after application. However, phosphite concentrations in the spear leaf required 10 weeks to reach their highest concentrations. Spear leaves maintained a fairly high concentration for at least 10 months. Regardless of phosphite application rate, phosphite concentrations became stable ten weeks after application. Phosphite is not evenly distributed in coconut palms, but was concentrated i n the spear leaf, young leaf rachis, old leaf rachis base, and young leaf basal leaflet tissues. In vitro, PLM 249A and PLM 140B showed sensitivity to phosphite. As the concentration of phosphite was increased, growth of both isolates decreased. In vivo, W robusta seedling mortality was reduced at an average foliar phosphite concentration of 3 5 0.49 g g 1 when applied as a foliar spray every 3 weeks

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38 LIST OF REFERENCES Bezuidenhout, J.J. J.M. Darvas and J.M. Kotze. 1987. The dynamics and distribution of phosphite in avocado trees treated with phosetyl Al. South Africa Avocado 103. Bompeix, G. and P. Saindrenan. 1984. In vitro antifungal activity of fosetyl Al and phosphorous acid on Phytophthora species. Fruits 39:77 7 786. Cervera, M., R.Cautin and G.Jeria. 2007. Evaluation of calcium phosphite; magnesium phosphite and potassium phosphite in the control of Phytophthora cinnamon i in H ass avocado trees ( Persea a mericana Mill) grown in container. Proc VI World Avocado C ongress. Via Del Mar, Chile. 12 16 Nov. 2007. Davis, A.J., M. Say, A.J. Snow and B.R. Grant. 1994. Sensitivity of Fusarium oxysporum f. sp. cubense to phosphonate. Plant Pathol. 43:200 205. de F ranqueville, H. and J.L. Renard. 1989. Effectiveness of Foset yl Al in coconut Phytophthora control. Application methods. Olagineux. 44(7):351 358. Elliott, M.L. 2011. First Report of Fusarium Wilt Caused by Fusarium oxysporum f. sp. palmarum on Canary Islan d Date Palm in Florida. Amer. P hytopath Soc PP 356 Ellio tt, M.L. 2010. Fusarium wilt of queen palm and Mexican fan palm. U niv. Fla., Inst. Food Agr. Sci. Fact Sheet PP 278. Elliott, M.L. 2009. Bud rots of p alm. Univ. Fla., Inst. Food Agr. Sci. Fact Sheet PP 220. Elliott, M.L. T.K. Broschat, J.Y. Uchida, and G. W. Simone 2004. Compendium of Ornamental Palm Disease and Dis orders. Amer. Phytopath. Soc., St. Paul. MN Elliott, M.L., E.A. Des Jardin, K. O'Donnell, D.M. Geiser, N.A. Harrion and T.K. Broschat. 2010,. Fusarium oxysporum f.sp. palmarum a novel forma sp ecialis causing a lethal disease of Syagrus romanzoffiana and Washingtonia subusta in Florida. Plant Dis 94:31 38. Fenn, M.E. and M.D. Coffey. 1984. Antifungal activity of fosetyl Al and phosphorous acid. Phytopathology 74: 606 611. Fenn, M.E. and M D. Co ffey. 1989. Quantification of phosphonate and ethyl phosphonate in tobacco and tomato tissues and significance for the mode of action of two phosphonate fungicides. Phytopathology 79:76 82.

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39 Fernando C.S., S.P. da Costa; M. de Moraes Echer, M.C. Martins, an d S.F. Blat. 2004. Phosphite effect on hot and sweet pepper reaction to Phytophthora capsici Sci. Agric. Piracicaba, Braz. PP 492 495 Forster J.E., Adaskaveg, D.H. Kim, and M.E. Stanghellin. 1998. Effect of phosphite on tomato and pepper plants and on s usceptibility of peppers to Phytophthora root and crown rot in h ydroponic culture. Plant Dis. 82:1165 1170. Grant, B.R., J. Grant, and J. Harris. 1992. Inhibition of growth of Phytophthora infestans by phosphate and phosphonate in defined media. Exp. Mycol 16: 240 244. Guest, D.I. and B.R. Grant. 1991. The complex action of phosphonates as antifungal agents. Biol. Rev. 66:159 187. Guest, D.I., R.D. Anderson, H.J.Foard, D.Phillips, S.Worboys, and R.M.Middleton. 1994. Long term control of P hytophthora diseas es of cocoa using trunk injected phosphonate. Plant Pathol. 43:479 492. Guest D.I., K.G. Pegg, and A.W. Whiley. 1995. Control of phyphthora diseases of tree crops using trunk inj ected phosphonates. Hort. Rev. 17: 299 330. Jackson, T.J., T. Burgess, I.Colq uhoun, and G.E.S. Hardy. 2000. Action of fungicide phosphite on Eucalyptus marginata inoculated with Phytophthora cinnamomi Plant Path. 49:147 154. Jee, H.J., W.D. Cho, and C.H. Kim. 2002. Effect of potassium phosphanate on the control of Phytophthora roo t rot of lettuce in hydroponics. Plant Pathol. J. 18(3): 142 146. Lobato, M.C., F.P. Olivieri, G.R. Daleo and A.B. Andreu. 2010. Antimicrobial activity of phosphites against different potato pathogens. Plant Dis and Prot 117(3):102 109. Lobato, M.C., F.P Olivieri, E.A. Gonzalez Altamiranda, E.A. Wolski, G.R. Daleo, D.O. Caldiz and A.B. Andreu. 2008. Phosphite compounds reduce disease severity in potato seed tubers and foliage. Eur. J. Plant Pathol. 122: 349 358. Malacinski G. a nd W.A. Konetzka. Bacterial oxidation of orthophosphate. J. Bacteriol. 91(2): 578 82. Maclntire, W.H., S.H. Winterberg, L.J. Hardin, and A.J. Sterges. 1950. Fertilizer evaluation of certain phosphorus, phosphorous and phosphoric materials by means of pot cultures J. Amer. Soc. Agrono. 42: 543 549.

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40 Pegg, K.G., A.W. Whiley, J.B. Saranh, and R.J. Glass. 1985. Control of Phytophthora root rot of avocado with pohsphorous acid. Austral Plant Patho l. 14:25 29. Smillie, R.H. and B. Grant. 1988. Determination of phosphate and phosphite i n plant material by gas chromatography mass spectrometry and i on c hromatography. J of Chromatog. 455:253 261. Smillie, R., B.R. Grant, and D. Guest. 1989. The mode of action of phosphite: Evidence for both direct and indirect modes of action on three Phyt ophthora spp. in plants. Phytopathology 79: 921 926.

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41 BIOGRAPHICAL SKETCH Jiaming Yu was born in Lingyuan, Liaoning, China, in 1986. She received a Heilongjiang, China an State University, East Lansi ng, Michigan, U.S. A. in 2010. After her graduation, Jiaming worked in a flower bu lbs breeding center for a year. In 2011, Jiaming was admitted as a graduate student to the Department of Environmental Horticulture, under the superv ision of Dr. Timothy Broschat. with a concentration in Environmental Horticulture in the Summer of 2013 and was admitted as a Ph.D student by University of Florida in the Department of Plant Pathology. Her career goal is to be a plant pathologist.