Laurel Wilt

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Title:
Laurel Wilt Assessing the Risk of Pruning Tool Transmission of Raffaelea Lauricola
Physical Description:
1 online resource (56 p.)
Language:
english
Creator:
Beckman, Fredrick C
Publisher:
University of Florida
Place of Publication:
Gainesville, Fla.
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Thesis/Dissertation Information

Degree:
Master's ( M.S.)
Degree Grantor:
University of Florida
Degree Disciplines:
Forest Resources and Conservation
Committee Chair:
Smith, Jason A.
Committee Members:
Minogue, Patrick J.
Ploetz, Randy C

Subjects

Subjects / Keywords:
avocado -- clean -- disinfect -- disinfest -- laurel -- lauricola -- persea -- prune -- pruning -- raffaelea -- redbay -- sanitize -- swampbay -- tools -- wilt
Forest Resources and Conservation -- Dissertations, Academic -- UF
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Forest Resources and Conservation thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract:
Laurel wilt, an exotic disease caused by the fungus Raffaelea lauricola and transmitted to members of the Lauraceae family by the redbay ambrosia beetle (Xyleborus glabratus), is rapidly killing native hosts and is now distributed in commercial avocado groves. In addition to dispersal and transmission by the vector, alternative modes of pathogen transmission may be important for disease management; but have yet to be explored. Due to the frequent topping and pruning in avocado groves and removal of affected trees by arborists, the risk of transmission of R. lauricola via pruning tools should be researched. An investigation was conducted to assess potential transmission of the laurel wilt pathogen on commercial pruning tools to healthy avocado (Persea americana) and swampbay (Persea palustris) trees. Plants were top pruned with inoculum-contaminated tools, simulating the pruning of an infested tree prior to a healthy tree. Symptoms were only observed in positive control plants injected with artificial inoculum. Supplementary studies investigated basipetal systemic movement of R. lauricola through vascular tissues. Plants were wounded by drilling with a small diameter bit (simulating beetle vector attack), or top pruning. Once wounded the artificial inoculum, in the form of a spore suspension or fungal plug, was applied directly to the wound. Symptoms were observed on the majority of treated plants, indicating the pathogen is capable of basipetal movement.Finally, disinfestation of pruning tools contaminated with R. lauricola was demonstrated with varying degrees of success using commercial disinfesting agents. These results suggest that although pruning tool transmission is not likely, their disinfestation is possible with commercially available agents.
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 Fredrick C Beckman.
Thesis:
Thesis (M.S.)--University of Florida, 2012.
Local:
Adviser: Smith, Jason A.
Electronic Access:
RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2013-12-31

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UFRGP
Rights Management:
Applicable rights reserved.
Classification:
lcc - LD1780 2012
System ID:
UFE0045096:00001


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1 LAUREL WILT: ASSESSING THE RISK OF PRUNING TOOL TRANSMISSION OF Raffaelea lauricola By FREDRICK CHARLES BECKMAN A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIRE MENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2012

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2 2012 Fredrick Charles Beckman

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3 To my wife Kristi, for without her love and support, this would not have been possible and t o my parent s Michael and Melinda for their lov e and support from my beginning

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4 ACKNOWLEDGMENTS I would like to express my gratitude for the patience and support of my Committee Chair, Jason Smith, who first allowed me to join his lab in August 2009. I would also like to thank my committee members, Patrick Minogue and Randy Ploetz for their time and support of this project. Additionally, I want to acknowledge the USDA Critical Issues Grant FLA FOR 004924 which provided funding for the research. I would also like to expr Gainesville, FL for her charitable donations and advice, John Alvarez of Fiberlock niversity of Florida Forest Pathology lab for all of their assistance. Finally, I wish to thank my wife and all of my family for their never ending support.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF TABLES ................................ ................................ ................................ ............ 6 LIST O F FIGURES ................................ ................................ ................................ .......... 7 ABSTRACT ................................ ................................ ................................ ..................... 8 CHAPTER 1 INTRODUCTION ................................ ................................ ................................ .... 10 2 MATERIALS & M ETHODS ................................ ................................ ..................... 13 Study Site ................................ ................................ ................................ ............... 13 Plant Material ................................ ................................ ................................ .......... 13 Inoculum ................................ ................................ ................................ ................. 14 Fungal Strain Used For Positive Control ................................ .......................... 14 Infested Host Material ................................ ................................ ...................... 14 Recov ery of Raffaelea lauricola ................................ ................................ .............. 14 Mechanical Transmission Assessment ................................ ................................ ... 15 Basipetal Vascular Movement of R. lauricola in Susceptible H osts ........................ 22 Comparison of Disinfestants ................................ ................................ ................... 26 3 RESULTS ................................ ................................ ................................ ............... 36 Mechanical T ransmission Assessment ................................ ................................ ... 36 Basipetal Vascular Movement of R. lauricola in Susceptible Hosts ........................ 36 Comparison of Disinfestants ................................ ................................ ................... 37 4 DISCUSSION ................................ ................................ ................................ ......... 50 Mechanical Transmission Assessment ................................ ................................ ... 50 Basipetal Vas cular Movement of Raffaelea lauricola ................................ .............. 50 Comparison of Disinfestants ................................ ................................ ................... 52 LIST OF REFERENCES ................................ ................................ ............................... 54 BIOGRAPHICAL SKETCH ................................ ................................ ............................ 56

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6 LIST OF TABLES Table page 3 1 Effect of ten disinfestants at four concentrations on Raffaelea lauricola survival ................................ ................................ ................................ ............... 44 3 2 Comparison of Bleach, Lysol Pine Sol disinfestation of Raffaelea lauricola on contaminated tools ................................ 47

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7 LIST OF FIGURES Figure page 1 1 Laurel wilt distribution map ................................ ................................ ................. 12 2 1 C ontamination types for preliminary mechanical transmission assessment ....... 31 2 2 Series of wound locations for preliminary mechanical transmission assessment ................................ ................................ ................................ ........ 32 2 3 Raffaelea lauricola infested field logs with cut passes; field inoculum ................ 33 2 4 Representative 200 L pipette tip funnel, used in the basipetal vascular movement study ................................ ................................ ................................ 34 2 5 Representative 3 0 cm section subsequently sampled at intervals of 5, 15, 25 cm for basipetal vascular movement study ................................ .................... 35 3 1 Early symptoms of laurel wilt caused by Raffaelea lauricola .............................. 39 3 2 Mean external symptom severity rating over time for the first mechanical t ransmission experiment ................................ ................................ ..................... 40 3 3 Mean external symptom severity rating over time for the second mechanical transmission experiment ................................ ................................ ..................... 40 3 4 Series of laurel wilt symptoms for basip etal vascular movement study .............. 41 3 5 Representative 200 L pipette tip funnel, plugged with plant exudates from basipetal vascular movement study ................................ ................................ .... 42 3 6 Inhibition of Raffaelea lauricola as a result of b leach in three plate columns for tool disinfestant assay ................................ ................................ ................... 43 3 7 Inhibition of Raffaelea lauricola on plates treated with represented in three plate columns for tool disinfestant assay ........................... 45 3 8 Three plate columns providing the contrast between the controls for the in vitro growth inhibition test ................................ ................................ ................... 46 3 9 Representative p lates providing contrast of controls for the t ool disinfestant assay ................................ ................................ ................................ .................. 48 3 10 Series of Raffaelea lauricola contaminated links for tool disinfestant assay ....... 49

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8 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 LAUREL WILT: ASSESSING THE RISK OF PRUNING TOOL TRANSMISSION OF Raffaelea lauricola By Fredrick Charles Beckman December 2012 Chair: Jason Smith Major: Forest Resources and Conservation Laurel wilt, an exotic disease caused by the fungus Raffaelea laur icola and transmitted to members of the Lauraceae family by the redbay ambrosia beetle ( Xyleborus glabratus ) is rapidly killing native hosts and is now distributed in commercial avocado groves. In addition to dispersal and transmission by the vector, alte rnative modes of pathogen transmission may be important for disease management ; but have yet to be explored. Due to the frequent topping and pruning in avocado groves and removal of affected trees by arborists, the risk of transmission of R. lauricola via pruning tools should be research ed An investigation was conducted to assess potential transmission of the laurel wilt patho gen on commercial pruning tools to healthy avocado ( Persea americana ) and swamp bay ( Persea palustris ) trees P lants were top pruned with inoculum contaminated tools simulating the pruning of an infest ed tree prior to a healthy tree. Symptoms were only observed in positive control plants injected with artificial inoculum. Supplementary studies investigated basipetal systemic movement o f R. lauricola through vascular tissues. P lants were wounded by drill ing with a small diameter bit (s imulating beetle vector attack ) or top pruning. Once wounded the artificial inoculum in the form of a spore suspension or fungal plug was applied direct ly to the

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9 wound. Symptoms were observed on the majority of treated plants, indicating the pathogen is capable of basipetal movement. Finally, disinfestation of pruning tools contaminated with R. lauricola was demonstrated with varying degrees of success us ing commercial disinfesting agents These results suggest that although pruning tool transmission is not likely, their disinfestation is possible with commercially available agents.

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10 CHAPTER 1 INTRODUCTION Laurel wilt, caused by the fungus Raffaelea lauri cola T.C. Harr., Fraedrich & Aghayeva, is a vascular wilt disease that kills American members of the Lauraceae plant family and cultivated non native members, which include avocado, Persea americana (Harrington et al ., 2008; Ploetz et al 2011a). The path ogen was first detected in 2003, one year following the initial detection of the non native ambrosia beetle vector, Xyleborus glabratus Eichhoff ( Coleoptera: Curculionidae : Scolytinae ) X. glabratus was originally detected in May 2002 (and likely introduce d on solid wood packing material ), at a seaport near Savannah, Georgia ( Fraedrich et al ., 2008; Harrington et al ., 2008; Harrington et al ., 2010 a ; Rabaglia et al ., 2006 ) The initial detection of this pathogen was not recognized until after mortality of re dbay, Persea borbonia near Savannah (Cameron et al., 2008; Fraedrich et al ., 2008, Rabaglia et al ., 2006) R. lauricola was first described in 2008 by Harrington et al 2008. As of May 2012 laurel wilt ha d been reported in at least 105 counties in southe ast United States (US) (Figure 1 1) (USDA Forest Service. 2012) ; one of which contains a major commercial US avocado production region in Florida (Evans et al ., 2010; Ploetz et al ., 2011a). The rapid movement of laurel wilt poses a serious threat to the c ommercial avocado production in Florida, the National Germplasm Repository for avocado in Miami (USDA ARS) and major (California) and minor avocado commerce (Texas, Hawaii, and Puerto Rico) elsewhere in the US (Evans et al ., 2010; Ploetz et al ., 2011a). W ithin the last 3 years laurel wilt has been reported in areas of Mississippi (Riggins et al ., 2010) and Alabama ( USDA Forest Service. 2012 )

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11 The first avocado trees were killed by laurel wilt in 2006 in Duval County, FL (Mayfield et al ., 2008 ) ; and the d isease has since been documented on this host as far south as Miami Dade County, FL in which practically all commercial avocado production occurs in Florida (Ploetz et al ., 2011 a, b). Transmission of the pathogen by the beetle vector from tree to tree is the critical element for dissemination of the pathogen from unhealthy host to healthy host. However, due to the common practice of topping and pruning of trees within avocado groves alternative modes of pathogen transmission, in addition to dispersal and t ransmission by the vector, need ed to be assessed as they may be important for disease management. Diverse disease management strategies have been examined for avocado, including host resistance and the use of fungicides and insecticides; however, to date, no highly effective and cost efficient management strategy has been identified (Ploetz et al ., 2011a; 2011c). The objectives of this study were to : 1) assess the risk of transmission of R. lauricola via pruning tools; 2) investigate basipetal systemic move ment of R. lauricola through host vascular tissues; and 3) assess sanitization agents against R. lauricola on pruning tools.

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12 Figure 1 1 Laurel w ilt d istribution m ap (USDA Forest Service 2012)

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13 CHAPTER 2 MATERIALS & METH ODS Study Site All experiments were conducted at greenhouse, growth chamber or nursery facilities maintained by the School of Forest Resources and Conservation (SFRC) located on University of Florida (UFL) property; Gainesville, Alachua County, Florida. Al achua County has been a positive county in Florida for laurel wilt and the redbay ambrosia beetle, Xyleborus glabratus (Eichhoff) since 2007 (USDA Forest Service 2012). Therefore, R. lauricola was obtainable from infested host material located at Austin C ary Forest (ACF), a 2,080 acre teaching and research forest northeast of Gainesville. Plant Material All plants used for these assessments were either swampbay ( Persea palustris (Raf.) Sarg ) or avocado ( Persea americana Mill et al ., 2011 d ) obtained from commercial nurseries. Avocado plants were clonal scions grafted on seedling rootstocks, which had a single, dominant central stem. Plants were an average of 1.5 m in height and about 3 cm in diameter above the graft union (abou t 10 cm from the soil line). Swampbay plants were either young saplings or seedlings, which had a single, dominant central stem. Saplings were an average of 3.5 m in height and were about 6 cm in diameter about 10 cm from the soil line. Seedlings were an a verage of 1 m in height and were about 2 cm in diameter about 10 cm from the soil line. All plants were maintained with standard fertilizer and irrigation practices to maintain health and vigor prior to use in these studies.

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14 Inoculum Fungal S train U sed F o r P ositive C ontrol The AvoB isolate of R. lauricola (Mayfield et al ., 2008) was used to induce laurel wilt disease in greenhouse (2010), field (2010 and 2011), growth chamber (2011 and 2012), and in in vitro experiments ( 2012 ) AvoB was originally recovere d from a laurel wilt affected avocado tree in Florida, and a SSU rDNA sequence for AvoB is deposited in GenBank under accession no. EU257806 (Mayfield et al ., 2008 ). Infest ed H ost M aterial Laurel wilt affected redbay ( Persea borbonia (L.) Spreng ) materia l was obtained from ACF and utilized for pruning transmission experiments in 2010 and 2011. Prior to obtaining infested redbay material, small samples of discolored sapwood discoloration were surface sterilized by submersion in a 3% sodium hypochlorite sol ution for 30 sec rinsed with deionized water and allowed to air dry on a sterile surface. Wood chips were then pla ced on a semi selective medium developed by Harrington (1981), CSMA ( 1% malt extract, 1.5% agar, and 200 ppm of cycloheximide and 100 ppm of streptomycin sulfate added after autoclaving) I solation plates were typically evaluated after 7 to 14 days. Suspect R. lauricola colonies were sub cultured, and then confirmed via Sanger DNA sequencing of the SSU rDNA (Dreaden et al ., 2008). Recovery of Raffaelea lauricola CSMA was used to recover R. lauricola in all experiments. When isolating from avocado plant material, a modified version of CSMA was also used to isolate the pathogen, which contained 0.6 g cycloheximide, 0.25 g ampicillin and 0.005 ri famycin L 1 ; referred to as CSMA + (Ploetz et al ., 2011d). Raffaelea lauricola is readily recovered on both CSMA and CSMA + from naturally or artificially affected plants showing

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15 symptoms. The increased cycloheximide concentration and additional antibiotics in CSMA + resulted in cleaner isolations (reduced recovery of other fungal and bacterial contaminants) from redbay, swampbay, and avocado (Ploetz et al ., 2011 d ). Mechanical Transmission Assessment A preliminary assessment was conducted in May 2010 to deter mine whether Raffaelea lauricola could be transferred to healthy containerized avocado and swampbay via contaminated pruning equipment The avocado experiment was carried out within a greenhouse ; and the swampbay experiment in an outdoor nursery plot resp ectively Eighteen 2 3 years old, 1.5 m in ht, in 26.5 liter (7 gallon) containers ) and 18 swampbay saplings ( 5 6 years old, 4 m in ht, in 132.5 liter (35 gallon) containers ) were used Due to the use of greenhouse facilities to conduct one of the assessments, a 3 6 randomized complete block design (RCBD) was utilized to remove the effect of confounding factors caused by environmental gradients within the greenhouse facility. Each of the three blocks contained one of each in dividual treatment for each tool, in addition to one positive control, and one mock inoculation (negative control) The mechanical pruning treatments were: (1) handsaw contaminated by spraying with artificial inoculum (Figure 2 1 A ); (2) handsaw contamina ted with infested field log debris (Figure 2 1 C ) ; (3) chainsaw contaminated by spraying artificial inoculum (Figure 2 1 B ); (4) chainsaw with infested field log debris ( Figure 2 1 D ) Each treatment was replicated once with one positive control and one mock inoculation per block with a total of three blocks in each experiment For the positive control and spray inoculum, the AvoB isolate was grown on CSMA for 14 days. Conidia were collected by flooding plates with approximately 2 mL of

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16 sterile deionized water, and conidia were then loosened with a polypropylene cell spreader. Spore concentrations were determined with a haemacytometer and ranged from 1.5 10 6 to 2.6 10 6 conidia/mL. For field inoculum, laurel wilt affected redbay material was collected from the field The presence of R. lauricola in affected logs was confirm ed on CSMA (Fraedrich et al ., 2008). Mechanical wound treatments were created either by using arborist pruning handsaws (Silky ZUBAT 330), or a small arborist pruning chainsaw (Stihl MS 192 TC) Prior to the ir use, they were thoroughly cleaned and disinfested with 70% ethyl alcohol. Three m echanical pruning wounds were applied to each experimental unit, including controls The wounds were applied : (1) on the primary stem; just above the graft union for avocado, or 30 cm above the soil line for swampbay (Figure 2 2, A); (2) on a lower canopy branch collar (Figure 2 2, B); (3) in the middle of an upper canopy branch (Figure 2 2, C) W ounding sites were similar to locations for standard tree pruning practices (Bedker et al ., 1995). The wound site in the middle of an upper canopy branch represented accidental wounding during pruning, to investigate whether transmission could occur via this occurrence. Avocado plants were wounded just above the grafting union point on the primary stem (avg. dia. 2.1 cm), at a midlevel height branch collar (avg. dia. 1.06 cm), and in the middle of an upper crown branch (avg. dia. 1.06 cm). Wounds were made similar with swampbay hosts except the lowest wounds were made approximately 30 cm from the soil line on the primary stem, rather than just above the graft union point, as with the avocado plants. P rimary stem average wound depth was 0.9 cm with an average width of 1.75 cm; br anch collar average wound depth

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17 was 0.9 cm with an average width of 0.9 cm on the branch co llar and mid branch wound depth w as 0.4 cm with an average width of 1.0 cm on the branch collar. For the experimental treatment, p runing devices were contaminated with one of the two sources of R. lauricola : 1) spore suspension of isolate AvoB or 2) naturally infest ed redbay logs prior to applying the three mechanical wounds to each experimental unit The spray contamination method used 1000 mL Dynalon Quick ware, Rochester, NY) (Figure 2 1, A C) One bottle contain ed sterile deionized water (mock control) and the other the spore suspension of the fungal isolate. Each device was sprayed twice, once on each side of the pruning tool for every individual treate d and then wound treatments were administered. The field log contamination method involved making ten cut passes into the infest ed redbay wood for every individual treated and then wound treatments were administered. The number of passes was determined fo llowing observation of the relative amount of residual wood debris on the pruning device prior to treatment application (Figure 2 3 ) Treatments were applied to a total of six avocado scions by chainsaw and another six by handsaw, cutting to a depth which was approximately one third the diameter of the selected area on each individual experimental unit Out of the six scions for each pruning treatment, three scions were inoculated with spore suspension spray contaminated tools and the other three were inoc ulated with tools contaminated with naturally infest ed redbay material Treatments were also applied to six swampbay saplings by chainsaw and another six by handsaw, cutting to a depth which was approximately one third the diameter of the selected area on each individual. Out of the

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18 six seedlings for each pruning treatment, three seedlings were inoculated with spore suspension spray contaminated tools and the other three were inoculated with tools contaminated with naturally infest ed redbay material. There fore, each block consisted of one of each of the four treatments, which was replicated three times. The remaining 12 plants of each host type (6 avocado; 6 swampbay), out of the total 36 plants, were used as control for this study; each group with three po sitive inoculation plants and 3 mock inoculation plants. Positive control avocado scions were wounded 10 cm above graft union by drilling three holes (2.75 mm in diameter and 3 to 7 mm deep) at a 45 downward angle into the primary stem and 100 L of a spo re suspension (10 6 spores/mL) of the fungal isolate was pipetted into the drill wounds and holes were wrapped with Parafilm M (Pechiney Plastic Packaging, Menasha, WI ). Positive control swampbay seedlings were wounded 30 cm abov e the soil line by drilling five holes (2.75 mm in diameter and 3 to 7 mm deep) at a 45 downward angle into the primary stem and 100 L of a spore suspension of the fungal isolate was pipetted into the drill wounds and holes were wrapped with Parafilm M (Pechiney Plastic Packaging Menasha, WI ) The increased inoculum volume utilized for the swampbay seedlings was due to the relative difference in host size as compared to the avocado scions ; this was merely to assure disease caused by R. lauricola Mock inoculations were carried ou t with the same technique described for the positive controls, except with sterile deionized water in place of the spore suspension of the fungal isolate. All control plants, both positive and mock, were wounded with sterilized pruning tools, to replicate the pruning treatment technique described previously. These pruning wounds allowed for any stress response resulting from the wound to be observed in addition to the wilt

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19 symptoms which resulted due to disease caused by R. lauricola Two of the three plant s of each host type were wounded with a chainsaw and one with a pruning handsaw. Two plants were selected for wounding by chainsaw due to the difficulty of working with small diameter subjects and the wider wound created by the chainsaw bar and chain. Afte r nine weeks, disease symptoms were rated for crown condition (i.e., foliage primarily green, wilting, or brown). Plants with mostly brown or wilting foliage were considered dead (Fraedrich et al ., 2008). At this time, bark was removed from plant stems wit h a knife to assess the presence of sapwood discoloration (internal symptom development). Pieces of stem sapwood tissue 10 to 20 cm above the primary stem wound points were excised and surface sterilized for 30 s in 3% bleach, rinsed in sterile water, blot ted dry on sterile paper towels and plated on CSMA and a variant of CSMA referred to a CSMA + (avocado only) which contained 0.6 g cycloheximide, 0.25 g ampicillin and 0.005 rifamycin L 1 to confirm the presence of the inoculated fungal isolate (Ploetz et al ., 2011 d ). A second assessment was conducted from December 2010 until February 2011 at the University of Florida SFRC facilities, to re examine laurel wilt mechanical transmission potential based on the results of the preliminary study. The second asse ssment, a modified version of the first assessment, consisted of a CRD with o ne mixed test group of avocado within a temperature controlled greenhouse using only naturally infest ed redbay material as treatment inoc ulum, as described above, and partial crown removal by a hydraulic pole pruning chainsaw (Husqvarna 327P4). Treatment consisted of contamination of the hydraulic pole pruning saw with infested redbay wood and then removing a portion of

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20 the treated plants healthy crown. This tr eatment was replicated 42 times (32 avocado; 14 swampbay) for this assessment, with four positive controls (2 avocado; 2 swampbay) and four mock inoculations (2 avocado; 2 swampbay) for a total of 54 containerized plants. The alterat ion in the pruning treatment method was designed to simulate pruning practices in Florida avocado groves and door yard avocado trees (Randy Ploetz, Department of Plant Pathology, University of Florida, TREC, Homestead, Florida, personal communication). The hydraulic pole pruning chainsaw was selected to provide a better representation of tools utilized to prune avocado trees (Randy Ploetz, personal communication). Avocado scions were 2 3 years old, with an average height of 1.5 m, and grown in 38 liter (10 gallon) containers. Swampbay saplings were 5 6 years old, with an average height of 4 m, and grown in 132.5 liter (35 gallon) containers. Following this initial assessment, the RCBD was not used due to a lack of any apparent confounding effect caused by th e greenhouse conditions versus the outdoor field plot. All subsequent experiments were conducted using a completely randomized design (CRD ) Randomization assignment was carried out using a computerized random number generator in Microsoft Excel Plants were maintained prior to inoculation treatment, as previously described. A spore suspension of the AvoB isolate (Mayfield et al ., 2008 ) was used for positive inoculation control plants. AvoB inoculum was produced as previously described, and inoculum conce ntrations for the isolate ranged from 1.5 10 6 to 2.6 10 6 conidia/mL. Forty six plants (32 avocado; 14 swampbay) were mechanically wounded using a hydraulic pole pruning chainsaw (Husqvarna 327P4 ) by complete removal of the upper third of the crown (a maintenance practice often used to control

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21 the size of trees) involving the indiscriminate cutting of branches and stems at right angles leaving lo ng stubs, known as top pruning or topping (Bedker et al ., 1995). The hydraulic pole pruning chainsaw was clea ned and sterilized prior to inoculation, and contaminated as previously described by making ten cut passes into naturally infested redbay material. An additional eight plants (4 of each host type ) were then used for positive inoculation and mock inoculatio n controls). Positive control avocado scions were wounded 10 cm above graft union by drilling 3 holes (2.75 mm in diameter and 3 to 7 mm deep) at a 45 downward angle into the primary stem and 100 L of a spore suspension (10 6 spores/mL) of the fungal isol ate was pipetted into the drill wounds and holes were wrapped with Parafilm M (Pechiney Plastic Packaging, Menasha, WI ) Positive control swampbay seedlings were wounded 30 cm above the soil line by drilling 5 holes (2.75 mm in diameter and 3 to 7 mm deep ) at a 45 downward angle into the primary stem and 100 L of a spore suspension of the fungal isolate was pipetted into the drill wounds and holes were wrapped with Parafilm M (Pechiney Plastic Packaging, Menasha, WI). The increased inoculum volume utili zed for the swampbay seedlings was due to the relative difference in host size as compared to the avocado scions ; this was merely to assure disease caused by R. lauricola Mock inoculations were carried out with the same technique described for the positiv e controls, except with sterile deionized water in place of the spore suspension of the fungal isolate. All control plants, both positive and mock, were wounded with the sterilized hydraulic pole pruning chainsaw, as to replicate the pruning treatment tech nique described previously. These pruning wounds allowed for any stress response resulting from the wound to be observed, in addition to the wilt symptoms which resulted due to disease caused by R.

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22 lauricola Plants were rated weekly for laurel wilt extern al symptom severity, following onset of symptoms using a subjective 1 to 10 scale, where 1 = no symptoms; 2 = 1 11% of the canopy showing symptoms; 3 = 12 99%; and 10 = dead (Ploetz et al ., 2011 d ). After nine weeks, symptom evaluation was conc luded and pieces of stem sapwood tissue 10 to 20 cm above the primary stem wound points were surface sterilized and plated on CSMA + to confirm the presence of the inoculated fungal isolate. The above experiment was repeated in September 2011 at the Univers ity of Florida SFRC facilities, with the exception of using an outdoor field plot instead of greenhouse facilities. The repeated experiment also consisted of 70 containerized plants (35 avocado; 35 swampbay). To evaluate whether there were any significant differences among trea tment and control plant mean rating s, SAS software was utilized to perform a two sample pooled t for each of the mechanical transmission assessments. Basipetal Vascular Movement of R. lauricola in Susceptible Hosts Two studies were conducted to evaluate basipetal movement of R. lauricola in suscep tible hosts. The first assessment was carried out in October 2011 at the University of Florida SFRC facilities. The assessment, a modified inoculation protocol of Banfield et al (1941), consisted of a CRD with 20 containerized swampbay seedlings approxima tely 1 2 years old, with an average height of 1 m, grown in 11.5 liter (3 gallon) containers with commercial nursery mix. Four plants were reserved for use as experimental control plants; two were mock inoculations and the other two inoculated at the base with the prepared R. lauricola The remaining plants were assigned to funnel inoculation treatments, with 16 replicates. Seedlings were placed within a growth

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23 chamber with a 16 h photoperiod constant 25C, and 99% r.h Before inoculation, plants were mai ntained with standard fertilizer and irrigation practices for a minimum of 1 month to ensure plant health and limit the presence of latent biotic and abiotic stress factors. AvoB inoculum was produced as previously described, and inoculum concentrations f or the isolate ranged from 1.5 10 6 to 2.6 10 6 conidia/mL. A 100 L volume of R. lauricola was introduced through artificial wounds in the tops of 16 swampbay seedling plants using passive uptake through vascular tissues. These wounds were applied appro ximately 30 cm from apex of the dominant central stem, by drill. Mock inoculations were carried out with sterile deionized water. Positive control plant inoculum was introduced 30 cm from the soil line, rather than the 30 cm from the stem apex. The inocul um was introduced through sterile 200 L pipette tips, with approximately 5 mm removed from the tapered tip end, which acted as a funnel into which 100 L of the spore suspension was pipetted. The 200 L pipette tips were placed and secured with Parafilm within a single drill hole (2.4 mm in diameter and 3 to 5 mm deep) at a 45 downward angle into the dominant central stem after the addition of inoculum the pipette tips were wrapped with Parafilm M (Pechiney Plastic Packaging, Menasha, WI) (Figure 2 4 ) Plants were rated weekly for laurel wilt external symptom severity, following onset of symptoms post inoculation on a subjective 1 to 10 scale, as previously described. After seven weeks, symptom evaluation was concluded and samples were taken from represe ntative points throughout the plant.

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24 The entire primary stem of all experimental units were sampled from the apex tip t o just above the soil line, at 10 cm intervals from +25 cm, +15 cm, +5 cm, 5 cm 15 cm 25 cm 35 cm 45 cm of the point of inoculation (Figure 2 5) Samples were removed the entire length of the plant whether or not sapwood discoloration was observed. The bark was completely removed from sample material, exposing the sapwood tissues. Plants typically averaged 4 sections, with a total of 12 sample discs, approximately 5 mm thick Sample discs were divided vertically providing 2 samples per interval; 1 half leaf trace side and 1 half non trace side. Samples were surface sterilized and plated on CSMA to confirm the presence of the inoculated fungus, as previously described Two weeks post plating, the distribution of the fungus was then qualitatively evaluated based on the recovery of R. lauricola from stem samples. The second study was carried out in December 2011 until February 2 012 at the University of Florida SFRC facilities, as a replication of the first assessment. The assessment consisted of a CRD with 20 containerized swampbay seedlings approximately 1 2 years old, with an average height of 1 m, grown in 11.5 liter (3 gallon ) containers with commercial nursery mix. Four plants out of the total number of plants were reserved for use as experimental control plants; two were mock inoculations and the other two inoculated at the base with the prepared R. lauricola The remaining plants were assigned to top pruned fungal plug inoculation treatments, with 16 replicates. Plants were maintained prior to inoculation treatment, as previously described. Plants were rated weekly for laurel wilt external symptom severity, following onset o f symptoms post inoculation on a subjective 1 to 10 scale, as previously described.

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25 The methodology to induce laurel wilt was modified, by changing inoculation technique, based on observations of the initial assessment. As opposed to using a spore suspens ion of AvoB for inoculum, plants were inoculated using mycelia and spores collected by scraping an actively growing AvoB fungal culture on CSMA, similar to Ploetz et al. (2011 d ), where an actively growing fungal plug on an agar based medium is applied to a wound to inoculate. The plug of fungal tissue was applied directly to the xylem with a sterilized scalpel, after removing approximately 10 cm from the shoot tip of the dominant central stem with sterilized pruning shears. Positive controls were inoculated 30 cm from the soil line, as opposed to the shoot tip wound site, by applying a plug of fungal tissue directly to a small downward flap cut into the bark which was sealed with Parafilm similar to Ploetz et al (2011 d ). Mock inoculations received CSMA me dium only applied to wounds. Negative controls and treated plants were not sealed with Parafilm Plants were rated weekly for laurel wilt external symptom severity, following onset of symptoms post inoculation on a subjective 1 to 10 scale, as previously described. After seven weeks, symptom evaluation was concluded and samples were taken from representative points throughout the plant and processed, as previously described in the initial basipetal vascular movement assessment. Two weeks post plating, the distribution of the fungus was qualitatively evaluated based on the recovery of R. lauricola from stem samples. To evaluate whether there were any significant differences among treatment and control plant mean rating s on the final date of each basipetal v ascular movement study, SAS software was utilized to perform a two sample pooled t test for difference of population means assessments.

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26 Comparison of Dis infest ants C hemical dis infest ants were evaluated in two studies in March and May 2012 at the University of Florida SFRC facilities. The methodology for each of these studies was modifi ed from Tevotdale et al (1991), for application against R. lauricola The comparison was conducted in two phases; the in vitro growth inhibition test (March 2012) examined the effect of 10 chemical dis infest ants on survival of R. lauricola in vitro and t he tool dis infest ant assays (May 2012) which assessed the effectiveness of the best disinfest ants in vitro when applied to metallic tool surfaces contaminated with pathogen infested wood debris. Seeded plates in these comparison tests were carried out in 2 4 h darkness at approximately 25C within an incubation chamber. The disinfest ants (active ingredients by volume in parentheses) evaluated were: Lysol Brand Concentrate, Original Scent (O Benzyl p Po Pine Sol Rubbing Alcohol (Isopropyl Alcohol, 70, 91, and 99%) Ethyl Alcohol, 1.5%), CuPro 2005 T/N/O (Cupric Hydroxide, 53.8%), and AvoB inoculum was produced as previously described. Spore concentrations were determined with a haemacytometer and ranged from 1.5 10 4 to 2.6 10 4

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27 conidia/mL, a 100 fold decrease in concentration as compared to previously described experiments Inoculum c oncentration reduction was a result of trials to generate sufficient visibility of individual developing fungal colonies once spread onto media plates. Inoculum conc entrations ranging from 1.5 10 6 to 2.6 10 6 conidia/mL, as in the previous experiments, resulted in undefined clusters of colony forming units once spread onto media plates The in vitro growth inhibition test consisted of a CRD of 40 treatments plate d onto CSMA plates Each treatment was replicated three times at each of the four dilution ratios for the chemical disinfestant s examined. The controls for this assessment consisted of three positive control plates (spore suspension) and three water contro l plates. For the in vitro growth inhibition test, 1 mL of the prepared AvoB suspension was pipetted into 9 mL of each of the 10 disinfest ant solutions at four different dilution ratios of the highest labeled rate; undiluted, 1:2, 1:5, and 1:10. All disinf est ants, undiluted and diluted 1:2, 1:5, and 1:10, were obtained from unopened containers and once opened were immediately tested. The solution mixtures were combined by inversion 3 4 times and then 30 L was spread over each of 3 CSMA culture plates with a sterilized glass rod. Mock disinfest ant application was carried out with sterile deionized water in the place of the disinfest ants. Undiluted and diluted disinfest ants were also cultured to confirm lack of contaminants prior to application. Seeded cultur e plates were i ncubated for approximately 84 h to guarantee any delayed spore germination post plate seed. Following the incubation period, the number of colony forming units (CFU) for each plate was evaluated within a predefined 937 mm circular area.

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28 The number of colony forming units were calculated as an arcsine transformation to normalize variance of the means of three plates (n=3), calculations performed in Microsoft Excel The t ransformed means were compared with Fisher's least significant differen ce (LSD) procedure using SAS software to determine whether there wa s a significant difference among disin festants Surviving CFUs were expressed as a mean of the three plates of the specific dilution ratio of disinfestant, and the mean was considered a si ngle replicate (Table 3 1). The range of CFUs per plate was determined for each dilution ratio in each disinfectant (treatment). The mean and the standard error of the mean (SE, standard deviation divided by the square root of the number of plates per trea tment) of the proportion of plates at dilution ratio 1:10 were compared by Fisher's least significant difference (LSD) means comparison test. Following evaluation of disinfest ants at their respective dilutions from the in vitro growth inhibition test, thre e methods were utilized for cleansing in the tool disinfest ant assays, using the lowest concentration (1:10) of those disinfest ants which remained effective in complete R. lauricola growth reduction. The disinfest ants which were successful at a dilution of 1:10 were Pine Sol Lysol This assessment consisted of a CRD of 4 treatments. Each treatment was replicated 10 times at the 1:10 dilution ratio per chemical disinfestant examined. The controls for this assessment consisted of 1 0 positive control plates (slurry mixture) and 10 water control plates where chain saw chain links were placed directly onto the culture media. Individual chainsaw chain links were surface contaminated by immersion into an inoculum slurry mixture ( a combin ation of sterilized Lauraceous wood debris and a volume of AvoB spore suspension ) Each disinfest ant solution was applied to the links,

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29 following a brief shake off of access debris, by: 1) one dip, 2) one spray on each side of the link using a hand held pu mp sprayer, or 3) one 2 minute soak. Immediately after disinfest ant application the link was placed on a CSMA plate. Ten m ock disinfest ant application was carried out with sterile deionized water. Due to concern of a fungitoxic effect caused by oil applie d to chain links by the manufacturer, the inoculum slurry mixture was sampled following contamination of 20, 50, 80, 110, 140 links, to positively confirm inoculum viability throughout the entire study. New links were also plated without any surface applic ation to verify deficiency of surface contaminants prior to the study. Seeded plates were incubated for nine days to guarantee any delayed fungal development post plate seed. During this time, the plates were qualitatively evaluated for any laurel wilt dev elopment. Evaluation ceased on the 9th day; however, seeded plates were kept for two additional weeks with no further development. To create the inoculum slurry mixture, two swampbay saplings, 1 healthy and 1 dead from laurel wilt, were ground into sawdust using a Wiley Mill to pass a 3 mm stainless steel sieve. Sawdust was autoclaved to sterilize and provide the necessary basis for contaminant debris. The sterile sawdust was then combined with an equal volume of AvoB spore suspension within a sterile cont ainer, in a ratio which successfully adhered to individual chainsaw chain links. Prepared inoculum concentrations for the isolate ranged from 1.5 10 4 to 2.6 10 4 conidia/mL, as previously described. The inoculum slurry mixture modeled infested wood debr is found on a pruning device post pruning. New OREGON D series (Vanguard 72V) chainsaw chain was completely disassembled into individual links for use in this comparison test.

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3 0 The data analysis for this paper was generated using SAS software, Version 9.3 of the SAS System for Microsoft Windows Copyright 2012 SAS Institute Inc. SAS and all other SAS Institute Inc. product or service names are registered trademarks or trademarks of SAS Institute Inc., Cary, NC, USA.

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31 A B C D Figure 2 1 C ontamination types for preliminary mechanical transmission assessment A) H andsaw s pray i noculum B) Chainsaw spray i noculum C) Handsaw f ield i noculum and D) Chainsaw f ield i noculum (Photos courtesy of Keumchul Shin )

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32 A B C Figure 2 2 Series of wound locations for preliminary mechanical transmission assessment. A) Primary st em B) Branch c ollar and D) Mid branch (Photos courtesy of Keumchul Shin)

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33 Figure 2 3. Raffaelea lauricola infest ed field logs with cut passes; field inoculum (Photo courtesy of Keumchul Shin)

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34 Figure 2 4 Representative 200 L pipette tip funnel, used in the basipetal vascular movement study Funnel is sealed and attached to plant with Parafilm M (Pechiney Plastic Packa ging, Menasha, WI) (Photo courtesy of Fredrick Beckman)

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35 Figure 2 5 Representative 30 cm section subsequently sampled at intervals of 5, 15, 25 cm for basipetal vascular movement study. Representative samples are relative to inoculation point which is 5 cm above the 5 cm sample point (Photo courtesy of Fredrick Beckman)

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36 CHAPTER 3 RESULTS Mechanical Transmission Assessment No symptoms were observed on any treated plants; however, complete wilt occurred as early as six weeks and as late as nine we eks post inoculation for positive control plants. There was no significant difference between species for initial time for onset of wilt symptoms (p > 0.05 ) (Figure 3 1 ) Recovery rate of R. lauricola was 100% from controls. There was no significant differ ence between plant species and recovery success of R. lauricola from sampled sapwood tissues (p > 0.05). N o significant difference was observed for wilt symptom development of control plants for the first assessment ( p=0.95 ) (Figure 3 2) and for the second experiment ( p=0.92 ) (Figure 3 3 ) However, for both experiments there was an highly significant difference between treated experimental units and positive controls (p<0.0001) Basipetal Vascular Movement of R. lauricola in Susceptible Hosts For the first v ascular movement assessment 75% of inoculated trees exhibited external wilt symptoms with a 69% recovery rate of the pathogen. None of the negative controls displayed symptoms (Figure 3 4 A) and 100% of positive controls wilted (Figure 3 4 B) Complete w ilt (Figure 3 4 C) occurred as early as five weeks post inoculation and there were treated plants that did not completely wilt in the time frame of the study. Recovery rate of R. lauricola was 100% from controls. There were 12% of plants which displayed w ilt symptoms and the pathogen was not recovered successfully due to the presence of additional fungi; however, there were also 6% of plants that did not exhibit wilt symptoms with positive recovery of R. lauricola (Figure 3 4 D) This is similar to result s obtained by Ploetz et al (2011 d ) where susceptible plants were

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37 positive for R. lauricola yet the onset of external wilt symptoms were minimal to nonexistent Additionally, 12% of plants produced exudates which plugged the wound site and pipette tip dra stically reducing the potential for passive uptake of the source inoculum (Figure 3 5 ) For the second vascular movement assessment 88% of inoculated trees exhibited external wilt symptoms with a 94% recovery rate of the pathogen. None of the negative cont rols displayed symptoms and 100% of positive controls wilted. Recovery rate of R. lauricola was 100% from controls. There were two plants that were asymptomatic; however, the pathogen was recovered successfully from the sapwood of these plants. Complete wi lt occurred as early as five weeks post inoculation and there were plants that did not completely wilt in the time frame of the study. There was no significant difference between initial onset of wilt symptoms from positive control plants as compared to th e treatment plants ( p=0.32 ). Comparison of Disinfest ants The most consistent growth reduction of R. lauricola on CSMA medium, for the in vitro growth inhibition test, occurred following exposure to Pine Sol Lysol bleach (NaClO) (Figure 3 6 ) or ShockW 3 1 ). This was supported by Fisher's least significant difference (LSD) means comparison test which indicated that Pine Sol Lysol were significant compared to the other disinfestin g agents (p = 0.0006). Raffaelea lauricola survived after (Figure 3 7 ) ; after treatment with 1:5 and 1:10 dilutions of 70, 91, and 99% isopropyl alcohol, and after treatment with all concentrations of CuPR O 2005 T/N/O and Trisodium Phosphate (TSP). A total of 449

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38 c olony forming units of R. lauricola were present within the 937mm circular area on the control culture plates used for this study (Table 3 1) (Figure 3 8 ) The lowest dilution ratio with the most consistent R. lauricola growth reduction, for the tool disinfest ant assays (Figure 3 9 ) was obtained by either 2 second dipping or 2 minute soaking the contaminated chain link in 1:10 dilution of b leach, or 2 minute soaking in 1:10 dilution of Pine Sol (T able 3 2). Failure to decontaminate the chainsaw chain link occurred most often with the spray cleansing method. Bleach (NaClO) (Figure 3 10 B D) was most effective in R. lauricola growth reduction, however; not one of the disinfest ants provided 100% cont rol at a 1:10 dilution ( Table 3 2) This suggests that even though the 1:10 dilution was successful in vitro that contamination may still occur when tool s are contaminated with plant debris.

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39 Figure 3 1. Early symptoms of laurel wilt caused by Raffa elea lauricola A vocado ( P ersea a mericana ) left; S wampbay ( P ersea palustris ) right (Photos courtesy of Fredrick Beckman)

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40 Figure 3 2 Mean external symptom severity rating over time for the first mechanical transmission experiment Legend symbols next to test plant species indicate (+) for positive control plants, ( ) for negative control plants, and (T) for experimental treatment plants Figure 3 3 Mean external symptom severity rating over time for the second mechanical transmission experiment Legend symbols next to test plant species indicate (+) for positive control plants, ( ) for negative control plants, and (T) for experimental treatment plants

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41 A B C D Figure 3 4 Series of laurel wilt symptoms for basipet al vascular movement study. A) Negative c ontrol B) Positive c ontrol C) Treatment and D) Asymptomatic t reatment w/ positive recovery of pathogen (Photos courtesy of Fredrick Beckman)

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42 Figure 3 5 Representative 200 L pipette tip funnel, plugged wi th plant exudates from basipetal vascular movement study (Photo courtesy of Fredrick Beckman)

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43 Figure 3 6 Inhibition of Raffaelea lauricola as a result of b leach in three plate columns for tool disinfest ant assay, each column represents dilution rat ios left to right, u ndiluted; 1:2; 1:5 and 1:10 (Photos courtesy of Fredrick Beckman)

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44 Table 3 1 E ffect of ten disinfestants at four concentrations on Raffaelea lauricola survival* Arcsine transformed me an CFUs and standard e rror (n=3) Disinfestants Undiluted 1:2 1:5 1:10 Pine Sol 0 b 0 b 0 b 0 b Lysol 0 b 0 b 0 b 0 b Sodium Hypochlorite 0 b 0 b 0 b 0 b 0 b 0 b 0 b 0 b Isopropyl Alcohol, 70% 0 b 0 b 1.20 0. 21 a 1.21 0. 19 a Isopropyl Alcohol, 91% 0 b 0 b 1.42 0 15 a 0.97 0. 31 a Isopropyl Alcohol, 99.8+% 0 b 0 b 1.18 0. 19 a 1.15 0. 22 a 0 b 0 b 0.52 0. 52 bc 0.52 0. 52 ab CuPRO 2005 T/N/O 1.20 0. 19 a 1.35 0. 12 a 1.34 0. 12 a 1.10 0. 27 a Trisodium Phosphate 1.29 0 1 4 a 1.24 0. 19 a 1.06 0. 26 ba 1.21 0. 20 a Water Control 1.38 0.1 1 Values are mean s standard error of triplicate contaminated links c ultured on cycloheximide streptomycin malt agar (CSMA) for 84 hours at 24C. (n=3). ** ** Spore su spension spread over plates; c olony forming units (CFUs) counted within 937mm circular area. = CFUs outside of specified circular area on CSMA media plate, excluded from data. Means with two letters (ab ac, or bc ) represent subgroups that are not statis tically significant (p > 0.05).

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45 Figure 3 7 Inhibition of Raffaelea lauricola on plates treated with represented in three plate columns for tool disinfest ant assay, each column represents dilution ratios left to right u ndiluted; 1:2; 1:5 and 1:10 (Photos courtesy of Fredrick Beckman)

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46 Figure 3 8 Three plate columns providing the contrast between the controls for the i n vitro growth inhibition test; p ositive control (spore suspension) left column, and the n egative control (water) right column (Photo courtesy of Fredrick Beckman)

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47 Table 3 2 Comparison of Bleach, Lysol Pine Sol disinfestation of R affaelea lauricola on contaminated tools Method applied Bleach (1:10) Lysol (1:10) Pine Sol (1:10) Water control Spray 10% 80% 60% 100% 100% Dip, 2 sec 0 % 60% 50% 80% 100% Soak, 2 min 0 % 10% 0 % 10% 100% Values represent percentage of Raffaelea lauricola positive chainsaw chain links following treatment (n=10) ** Links contaminated with R affaelea lauricola by immersion into inoculum slurry mixture, then cleansed by each disinfestant before plating onto a semi selective medium = Cycloheximide streptomycin malt agar (CSMA)

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48 Figure 3 9 Representative plates providing contrast of controls for the t ool disinfest ant assay i noculum slurry mixture, top ; c hain link only on medium, bottom The dark pigmentation shown in the lower photograph is a result of diffusion and stai ning of oil from the chain link (Photos courtesy of Fredrick Beckman)

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49 A B C D Figure 3 10 Series of Raffaelea lauricola contaminated links for to ol disinfest ant assay A) Positive c ontrol B) Bleach 1:10 s pray treatment C) Bleach 1:10 2 sec dip treatment and D) Bleach 1:10 2 min treatment Raffaelea lauricola growth present only in positive control (A) and the 1:10 spray treatment (B) (Ph otos courtesy of Fredrick Beckman)

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50 CHAPTER 4 DISCUSSION Mechanical Transmission Assessment Although transmission of R. lauricola through pruning has yet to be documented, the potential for transmission needed to be considered. The hypothesis tested was th at R. lauricola could be transmitted to new trees via pruning tool contamination as has been shown with two similar diseases, Dutch elm disease ( caused by Ophiostoma novo ulmi ) (Dreistadt 2004) Fusarium wilt of palm ( Fusarium oxysporum f. sp. palmarum ) (Elliot, 2010) R. lauricola appears to be unique in that transmission of the pathogen from host to host is facilitated only by means of a beetle vector (Harrington et al ., 2010b ) with probable root graft transmission ( Ploetz et al ., 2011d) Our attempts to successfully transmit R. lauricola through pruning of infested material to healthy host tissues were unsuccessful. Given the large number of pruned trees and variable factors, including condition of trees, types of pruning tools and local conditions, pr uning transmission may still occur infrequently. However, these results indicate that it is not likely to play a significant role in laurel wilt disease epidemiology. Basipetal Vascular Movement of Raffaelea lauricola To determine if an absence of basipet al colonization by the pathogen was responsible for lack of pruning tool transmission, apical portions of plants were inoculated The hypothesis tested was that R. lauricola could be transmitted in top worked (pruned) trees via basipetal movement of the pa thogen The observations from this study demonstrate that introduction of R. lauricola to susceptible host vascular tissues including tissue near the primary shoot apex allowed basipetal vascular movement through the xylem This was supported by successf ul recover y of R. lauricola

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51 throughout the entire length of inoculated plants above and below the point of inoculation similar to results by Inch et al (2011) These results conflict with the results of the mechanical pruning transmission experiments, wh ich demonstrated transmission via pruning tools was not likely. The reason transmission did not occur in the mechanical transmission experiments, more than likely was a result of R. lauricola rapid rate of desiccation outside of the host tissues which i s supported by experiments evaluating R. lauricola survival in infested wood that has been chipped (D Spence, unpublished data) The residual debris on saws, created when cutting into infested material, is so fine and due to exposure to the elements, the f ungus is no longer in an ideal environment in which it can survive. Additionally, basipetal vascular movement can be explained by the cohesion tension theory of xylem movement, where negative pressure can occur within the xylem during night time cycles wh en transpiration is reduced or nonexistent (Tatt a r et al ., 1999) This negative pressure can cause reduction in vascular transport within the xylem, which may have provided the pathogen with the opportunity for bimodal movement from the point of inoculati on (Banfield 1941; Tatt a r et al ., 1999). During the first vascular movement study two plants developed wilt symptoms one week earlier than the other plants treated. P lant stress factors were minimal throughout this study due to the optimal environmental g rowing conditions in the growth chamber A symptomatic plants were a rare occurrence; however, they did occur E xudates plugging the pipette tip funnel during the first vascular movement study may have also played a role in reducing the interaction of R. la uricola inoculum with the healthy host

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52 vascular tissues ; these responses may be a consequence of artificially wounding plants ( Figure 3 5 ) Comparison of Disinfest ants The results of this study can be used to determine how pruning tool s should be disinfest ed in laurel wilt affected avocado orchards The methodology was modified from Teviotdale et a l (1991), where the focus was reduction in transmission of the fire blight pathogen ( E rwinia amylovora ) on apple and A sian pear trees. Since E. amylovora is a Gr am negative bacterium and R. lauricola is an asexual ascomycete, some of the chemical agents and methods had to be altered to fit a fungal pathogen Chemical agents were selected specifically focusing on fungitoxicity, and the semi selective medium cyclohe ximide streptomycin malt agar (CSMA) was used for R. lauricola propagation The in vitro growth inhibition test s indicated that all 10 chemical agents inhibited growth of R. lauricola to some degree compared to the water control. These results were support ed by similar results from Teviotdale et al (1991) The results of the tool disinfest ant assays also paralleled Teviotdale et al (1991) and reflect those disinfestants typically used by commercial arborists ; however, the spray method which they described as quite affective was the least effective method in the present work The cost effectiveness and corrosion of these materials need to be considered Although the corrosion potential of each disinfesting agent was not within the scope of this study each of the disinfest ants has the potential for corrode metal. P otential economic losses of laurel wilt could cause in hav e been estimated between $27 and $54 million (Evans et al ., 2010 ) Although t he

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53 results of this investigation suggest that pruning tool transmission is not likely, their disinfestation is possibl e with several commercially available agents It is critical that additional efforts be made to implement preventative management policies and sanitation regimes to decelerate the spread of the disease and to invest in research on effective treatments.

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54 LIST OF REFERENCES Banfield WM, 1941. Distribution by the sap stream of spores of three fungi that induce vascular wilt diseases of elm. Journal of Agricultural Research 62, 637 81. Bedker P, O'Brien JG, Mielke ME, 1995. How to prune trees USDA For. Serv State and Private Forestry NA FR 01 95. Cameron RS, Bates C, Johnson J, 2008. Distribution and spread of laurel wilt disease in Georgia: 2006 08 survey and field observations. Georgia Forestry Commission.US Forest Service. [ http://fhm.fs.fed.us/em/funded/ 09/so em 08 02 report.pdf ] Accessed 25 May 2012. Dreaden T, Smith J, Mayfield A, 2008. Development of a real time PCR assay for detection of the Raffaelea species causing laurel wilt disease. Phytopathology 98, S48. Dreistadt SH, 2004. Pests of landscape t rees and shrubs 2nd ed. Oakland: Univ. Calif. Agric. Nat. Res. Publ. 3359. Elliott ML, 2010. Fusarium Wilt of Queen Palm and Mexican Fan Palm (PP 278). Gainesville: University of Florida Institute of Food and Agricultural Sciences. Evans EA, Crane J, Hod ges A, Osborne JL, 2010. Potential economic impact of laurel wilt disease on the Florida avocado industry. HortTechnology 20, 234 8. Fraedrich S, Harrington T, Rabaglia R, Ulyshen M, Mayfield III A, Hanula J, Eickwort J, Miller D, 2008. A fungal symbiont o f the redbay ambrosia beetle causes a lethal wilt in redbay and other Lauraceae in the southeastern United States. Plant Disease 92, 215 24. Harrington T, 1981. Cycloheximide sensitivity as a taxonomic character in Ceratocystis Mycologia 73: 1123 29. Harr ington T, Aghayeva D, Fraedrich S, 2010 a New combinations in Raffaelea, Ambrosiella, and Hyalorhinocladiella, and four new species from the redbay ambrosia beetle, Xyleborus glabratus Mycotaxon 111, 337 61. Harrington T, Fraedrich S, 2010 b Quantificatio n of propagules of the laurel wilt fungus and other mycangial fungi from the redbay ambrosia beetle, Xyleborus glabratus Phytopathology 100, 1118 23. Harrington T, Fraedrich S, Aghayeva D, 2008. Raffaelea lauricola a new ambrosia beetle symbiont and path ogen on the Lauraceae. Mycotaxon 104, 399 404. Inch S, Ploetz R, 2011. Impact of laurel wilt, caused by Raffaelea lauricola on xylem function in avocado, Persea americana Forest Pathology

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55 Mayfield III A, Smith J, Hughes M, Dreaden T, 2008. First report of laurel wilt disease caused by a Raffaelea sp. on avocado in Florida. Plant Disease 92, 976 Ploetz R C Harrington T, Hulcr J, Bostock R, Eskalen A, Faber B, Crane J, Harmon C, Inch S, Palmateer A, 2011a. Recovery Plan for Laurel Wilt of Avocado (caused by Raffaelea lauricola ). National Plant Disease Recovery System USDA ARS: HSPD 9. [http://www.ars.usda.gov/SP2UserFiles/Place/00000000/opmp/Avocado%20LW %20110829.pdf] Ploetz R C Pea JE, Smith JA, Dreaden TJ, Crane JH, Schubert T, Dixon W, 2011b. Laurel Wi lt, Caused by Raffaelea lauricola is Confirmed in Miami Dade County, Center of Florida's Commercial Avocado Production. Plant Disease 95, 1589. Ploetz RC, Prez Martnez JM, Evans EA, Inch SA, 2011c. Toward fungicidal management of laurel wilt of avocado. Plant Disease 95, 977 82. Ploetz R C Prez Martnez J, Smith J, Hughes M, Dreaden T, Inch S, Fu Y, 2011d. Responses of avocado to laurel wilt, caused by Raffaelea lauricola Plant Pathology Rabaglia RJ, Dole SA, Cognato AI, 2006. Review of American Xyleb orina (Coleoptera: Curculionidae: Scolytinae) Occurring North of Mexico, with an Illustrated Key. Annals of the Entomological Society of America 99, 1034 56. Riggins J, Hughes M, Smith J, Mayfield III A, Layton B, Balbalian C, Campbell R, 2010. First occur rence of laurel wilt disease caused by Raffaelea lauricola on redbay trees in Mississippi. Plant Disease 94, 634. SAS Institute Inc., 2011. SAS/STAT Cary, NC: SAS Institute Inc. Tattar TA, Tattar SJ, 1999. Evidence for the downward movem ent of materials injected into trees. Journal of Arboriculture 25, 325 32. Teviotdale B, Wiley M, Harper D, 1991. How disinfectants compare in preventing transmission of fire blight. California Agriculture 45, 21 3. USDA Forest S ervice. 2012. Laurel Wilt D istribution Map USDA Forest Service. [ http://www.fs.fed.us/r8/foresthealth/laurelwilt/dist_map.shtm ]. Accessed 25 May 2 012

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56 BIOGRAPHICAL SKETCH Fredrick Charles Beckman was born in Valparaiso, Indiana a town found in the northwestern part of the state It was in this rural setting where Fredrick truly developed his desire for knowledge in the biological workings of plants and his passion for science. Following graduation from high school, Fredrick attended Purdue University where in May 2008, he obtain ed a Bachelor of Science in plant biology, an Associate of Agriculture, and a minor in plant pathology. He immediately went to work for the Walt Disney World Company as a plant science intern in the Epcot Science Professional Internship Program where he further expanded his knowledge in applied plant sciences. Fredrick went on to purse graduate level education and joined the School of Forest August 2009. It was Fredrick support from his loving wife which allowed him to obtain a Master of Science in forest pathology from the University of Florida.