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Genetic variation in Xanthomonas axonopodis pv. dieffenbachiae

University of Florida Institutional Repository

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GENETIC VARIATION IN Xanthomonas axonopodis pv. dieffenbachiae By RYAN SCOTT DONAHOO A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2003

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Copyright 2003 by Ryan S. Donahoo

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This document is dedicated to my grandparents, Doug and Valle Glover.

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ACKNOWLEDGMENTS I would like to first thank Dave Norman for providing me the opportunity to work on this project in an astonishing atmosphere as well as the assistance that he provided throughout the completion of this step in my academic career. Next, I would like to thank Jeff Jones for providing me with a lab in Gainesville, constantly taking time out of his schedule to address my questions, and giving me insight into bacteria, taxonomy, molecular genetics, and Gator sports. I am greatly appreciative to Jeanne Yuen, Rosa Resendiz, and Joe Boswell down at the Mid-Florida Research and Education Center for their help with handling of cultures, maintenance of plants, and so much of the work that was accomplished in Apopka. Next, I would like to thank Gerry Minsavage for all of his technical assistance, allowing a great deal of this research to come to completion. Last, I would like to thank my parents for believing in me and often providing me with much needed motivation and serenity. iv

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TABLE OF CONTENTS Page ACKNOWLEDGMENTS.................................................................................................iv LIST OF FIGURES..........................................................................................................vii ABSTRACT.....................................................................................................................viii CHAPTER 1 INTRODUCTION........................................................................................................1 2 LITERATURE REVIEW.............................................................................................3 History..........................................................................................................................3 Related Studies......................................................................................................5 Pathogenicity Tests................................................................................................5 PCR Based Analysis..............................................................................................7 3 MATERIALS AND METHODS...............................................................................10 Bacterial Strains Used.................................................................................................10 Genomic Comparisons................................................................................................10 Amplified Fragment Length Polymorphism (AFLP)..........................................11 Sequence Comparisons........................................................................................12 Pathogenicity and Host Range....................................................................................13 4 RESULTS...................................................................................................................20 Genomic Comparisons................................................................................................20 Rep-PCR..............................................................................................................20 Amplified Fragment Length Polymorphism.......................................................20 DNA Sequencing.................................................................................................21 Host Range Tests........................................................................................................22 Dieffenbachia Strains..........................................................................................22 Anthurium Strains...............................................................................................23 Syngonium Strains...............................................................................................23 v

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5 DISCUSSION.............................................................................................................31 LIST OF REFERENCES...................................................................................................34 BIOGRAPHICAL SKETCH.............................................................................................38 vi

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LIST OF FIGURES Figure page 1. Cluster analysis of 191 X.a.d strains isolated from ten host genera over a 20-year period based on rep-PCR using BOX, ERIC and REP primers..................25 2. Cluster analysis of 38X.a.d strains isolated from six host genera over a 20-year period based on AFLP using M02, and E00 primers combinations.........................26 3. Cluster analysis of 39X.a.d strains isolated from six host genera over a 20-year period based on AFLP using M02, and E02 primers combinations.........................27 4. Percent of X. a. dieffenbachiae strains isolated from Dieffenbachia spp. producing disease when inoculated into various aroid hosts...................................28 5. Percent of X. a. dieffenbachiae strains isolated from Anthurium spp. producing disease when inoculated into various aroid hosts.....................................................29 6. Percent of X. a. dieffenbachiae strains isolated from Syngonium spp. producing disease when inoculated into various aroid hosts.....................................................30 vii

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Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science GENETIC VARIATION WITHIN Xanthomonas axonopodis pv. dieffenbachiae By Ryan Scott Donahoo May 2003 Chair: David J. Norman Major Department: Plant Pathology Members of the Araceae are susceptible to the bacterial pathogen Xanthomonas axonopodis pv. dieffenbachiae, which is capable of causing crop losses up to 100 percent. When 187 strains isolated from nine aroid hosts were subjected to Rep-PCR, six genetic clusters were generated. One cluster was found to represent only strains isolated from Syngonium spp. Forty strains isolated from Anthurium, Dieffenbachia, and Syngonium were subjected to amplified fragment length polymorphism (AFLP) and tested for pathogenicity on five Aroid hosts. AFLP data correlated well with Rep-PCR data. Based on pathogenicity tests, the Syngonium strains were selectively pathogenic on Syngonium. None of the strains from other hosts caused significant disease on Syngonium. DNA from ten representative strains was amplified by PCR using primers to the ITS and hrpB. Phylogenetic analysis of sequenced PCR products reveals that the syngonium strains exist as a distinct group from other members of X.a.pv. dieffenbachiae and supports the use of pv. syngonii for strains isolated from Syngonium. viii

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CHAPTER 1 INTRODUCTION The United States tropical foliage plant industry has an annual wholesale value of more than 500 million dollars (Unites States Department of Agriculture, 2002), with approximately 61% of this production in Florida. Consistent growth has occurred in the tropical foliage industry due to the popularity of plants in the family Araceae. This family includes genera such as Aglaonema, Anthurium, Dieffenbachia, Philodendron, and Syngonium (www.aroid.org). Aroids are native to the tropics and vary greatly in growth habit. Some are aquatic and totally submerged, others are hemi-epiphytic, and yet others are totally epiphytic growing on rocks and trees. Aroids can be found in their native habitats from coastal locations to higher elevations of several thousand feet. However, most of the commercially cultivated species are found growing at low elevations under humid tropical rainforest conditions. Thus, most commercial production of these crops is done either in tropical areas of the world or under greenhouse conditions. Costa Rica, Guatemala, and Honduras are primary suppliers of propagative material while the Netherlands, California, Florida, and Hawaii are the primary producers of finished plants. Most aroids are known to be susceptible to bacterial blight caused by the bacterium Xanthomonas axonopodis pv. dieffenbachiae. Traditionally, it has been thought that the bacterium enters through the hydathodes. However, recent observations suggest that entry may also occur through the stomata. The hydathodes have been thought of as the main point of entry associated with this Xanthomonas due to the fact that these 1

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2 specialized cells localized to the leaf margin secrete glutamine and/or glutamate, both which can serve as chemotaxic stimuli as well as a sole carbon source for Xanthomonas dieffenbachiae. The guttation fluid from infected plants serves as a source of inoculum, and is easily spread by overhead irrigation and/or workers clothing. Since many of the aroids are propagated vegetatively, the disease is spread rapidly, on cutting utensils used in crop maintenance and propagation. Once the bacteria have entered a susceptible host, whether via the stomata, hydathodes or wounds, the bacteria multiply at the point of entry, spreading through the xylem vessels. If the pathogen entry is via the hydathodes, water soaking develops at the leaf margin within a few days. As the disease progresses, the water soaking becomes chlorotic then necrotic, forming classical V-shaped lesions of dead tissue. If plants are grown in close proximity to each other and under warm and humid conditions, spread within and between plants can be rapid with crop losses up to 100%.

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CHAPTER 2 LITERATURE REVIEW History Aroids, members of the plant family Araceae, constitute the majority of tropical foliage crops. McCulloch and Pirone published the first report of bacterial blight of Dieffenbachia (1939). They described an organism that caused leaf spots, blight, and in some cases death. They referred to the pathogen as Bacterium dieffenbachiae. During the 1920s and 1930s it was common practice to group all phytopathogenic bacteria in the genus Bacterium (Dowson 1943). Over time, the necessity for additional genera was realized. Through the research efforts of Migula, Dowson and others in the early 1900s, new genera such as Pseudomonas and Xanthomonas were gradually accepted. In 1939, Bergeys Manual renamed the pathogen as Phytomonas dieffenbachiae. Dowson reclassified Phytomonas dieffenbachiae as Xanthomonas dieffenbachiae, the pathogen causing bacterial blight of Dieffenbachia (Dowson 1943). Roughly 20 years after Dowsons contribution to the nomenclature Young et al. transferred the species dieffenbachiae to a pathovar of Xanthomonas campestris designating it X. campestris pv. dieffenbachiae (Xcd) (Young et al. 1978). In 1980 Dye et al. formally described the use of the pathovar system and included Xcd as a member. During the 1960s Xcd was described causing disease on other aroids including Aglaonema and Philodendron (McFadden 1962 and 1967). During the 1970s Xcd was also reported in Anthurium in Hawaii (Hayward 1972). In the 1980s, Xcd was described 3

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4 on numerous aroid hosts throughout the United States (Chase and Poole 1986, Cooksey 1985, Porhonenzy et al. 1985, Chase et al. 1992). In 1969 bacterial blight was described on Syngonium podophyllum (Wehlburg 1969). The pathogen was described as a Xanthomonas-like organism based on cultural and microscopic observations characterizing the pathogen as X. vitians. Only minor physiological differences between the two organisms were noted. In the 1980s when Dickey and Zumoff (1987) compared four strains from the Syngonium cultivar Cream (LX 103, LX105, LX106, LX114) and two strains from the cultivar White Butterfly (L 212, L215). These six strains were compared with two strains used in Wehlburgs study (XV29 = NCPPB2255 isolated from Syngonium ATCC19320) and type strains representing X. c. vitians (NCPPB 969), X. c. campestris (B-24), and strains of Xanthomonas campestris from aroid hosts with pathovar designations aracearum (XA-2), dieffenbachiae (XD-3), and zantedeschiae (XZ1). They noted that strains XA-2, XD-3, and ATCC 19320 produced disease symptoms on Syngonium when misted on plants at 10 8 CFU/ml. However it was also noted that the reactions produced by these strains were different from those produced by strains originally isolated from Syngonium. Dickey and Zumoff (1987) proposed designating the Syngonium organism as Xanthomonas campestris pv. syngonii. (Dickey and Zumoff 1987). This pathovar designation syngonii is available from culture collections (LMG 9055 and ICMP 9152, 9153, 9154, 9155 all representing strains collected by Dickey) but does not appear anywhere in-approved lists of bacterial names or taxonomic lists. The xanthomonad that infects Syngonium is currently referred to as X. campestris pv. dieffenbachiae (Chase et al. 1992).

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5 In 1995 the aroid pathogen was reclassified as X. axonopodis pv. dieffenbachiae based on DNA-DNA homology studies (Vauterin et al. 1995). In that study five X. campestris pv. dieffenbachiae strains were compared with 178 strains from 33 other pathovars of X. campestris. The five X. campestris pv. dieffenbachiae strains used, included three Anthurium strains from Brazil and two Dieffenbachia strains from the United States. The strains used in their study seem limited in regards to hosts of origin and excluded the incorporation of any strains from Aglaonema, Philodendron, Syngonium, or Xanthosoma. Related Studies The family Araceae is a large and diverse family of plants. Understanding this is important in recognizing the need to determine the diversity that exists in the many xanthomonads that infect and cause disease in these aroids. Traditionally, host range pathogenicity is a good starting point when describing bacterial plant pathogens. In recent years a number of PCR techniques have been developed and are used for investigating species, pathovars, strains, and races. The following is a brief review of the literature regarding host range, pathogenicity, and PCR-based phylogeny-type work. Pathogenicity Tests The history of Xanthomonas syngonii raises questions that as of yet have not been answered, especially in regards to host range and pathogenicity. Dickey and Zumoff (1987) showed that strains XA-2, XD-3, and ATCC 19320 produced symptoms on Syngonium when misted at 10 8 CFU/ml. However, it was also noted that the reactions produced by these strains were different from those produced by strains originally isolated from Syngonium. It was also observed that xanthomonads isolated from Syngonium were weakly pathogenic on the Dieffenbachia amoena and not at all

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6 pathogenic on D. maculata. Also, XD-3 (from Dieffenbachia) was capable of producing typical symptoms on Dieffenbachia yet only minor symptoms on Syngonium. The results from their study suggest Syngonium strains cause disease in Syngonium but appear to be less virulent when inoculated into Dieffenbachia. In addition, they concluded that there were typical and atypical syngonii strains based on pigmentation and growth on nutrient agar. It would appear that Dickey and Zumoff (1987) were suggesting that the syngonium strains are the only true pathogen of Syngonium. Similar research on the Syngonium strains was conducted by Chase et al. (1992). In that study 149 X. campestris dieffenbachiae and X. c. pv. syngonii strains isolated from a variety of aroid hosts were subjected to physiological, pathological, and fatty acid analyses. They showed that various strains were not necessarily host-specific though they suggested that groups of strains have overlapping hosts. It was observed when monitoring populations in leaves infiltrated with bacterial suspensions adjusted to 10 4 CFU/ml that all strains (with the exception of the Syngonium strains) were capable of multiplying one log unit regardless of host tested. The Syngonium strains only increased in populations in Syngonium and Aglaonema. Chase et al. (1992) concluded that differences in host specificity were not significant enough to be used as a means to differentiate strains of X. c. pv. dieffenbachiae from those of X. c. pv. syngonii. It appears that due to this ambiguity in host range among these various strains there has been no pathovar designation for all of these strains aside from the all-encompassing pathovar dieffenbachiae. Efforts by Lipp et al. (1992) to characterize strains isolated from Dieffenbachia and Syngonium employed a similar host range study. However, while the typical syngonium

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7 strains were virulent on Syngonium and weakly virulent to avirulent on other host genera, the atypical strains were indistinguishable from other Xcd based on bacteriological tests (Lipp et al, 1992). They found no significant correlation between hosts of origin and host range, and concluded that this group was extremely heterogeneous. PCR Based Analysis Recently PCR has been used to amplify specific regions of an organisms genome for comparison with other organisms as a means to determine evolutionary relationships. Several of the PCR based techniques include Repetitive Element PCR (rep-PCR), Amplified Fragment Length Polymorphism (AFLP), and sequence comparisons of the Hrp region and a region corresponding to the ribosomal DNA (Louws et al. 1994, Janssen et al. 1996, Leite Jr. et al. 1995, and Hauben et al. 1997). Rep-PCR is based on highly conserved regions of the genome. There are three regions that have been used: Repetitive Enterobacterial Palindrome (REP), (Gilson et al. 1984, Higgins et al. 1982) Enterobacterial Repetitive Intergenic Consensus (ERIC), (Hulton et al. 1991, Sharples and Lloyd 1990) and the BOX1A element from E. coli (BOX) (Martin et al. 1992). It has been shown that these elements are widely distributed in phytopathogenic bacteria including Pseudomonas and Xanthomonas and can be used for rapid molecular characterization especially at the pathovar level (Louws et al. 1994). Using rep-PCR Louws et al. (1994) were able to generate genomic fingerprints consisting of five to 20 bands ranging in size from 100 base pairs to 5 kbp. In comparing fingerprints from various Pseudomonas and Xanthomonas strains, they were able to distinguish P. syringae pv. syringae from P. syringae pv. tomato as well as X. oryzae pv. oryzae from X. oryzae pv. oryzicola. Using rep-PCR members from the same group they

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8 were able to place X. campestris pv. vesicatoria strains into two groups, groups A and B (Louws et al.1995). In 1995 the PCR based genomic fingerprinting method Amplified Fragment Length Polymorphism (AFLP) was developed. The basis of AFLP is to analyze the whole genome. First described by Vos et al. (1995), the process begins with fragmenting the whole genome with restriction endonucleases. Following the restriction or cutting of the genome, the second step employs the ligation of site-specific adapters to the restriction fragments. The adapted fragments are selectively amplified with primers designed to complement the adapters and allow specificity through 3 selective base(s) incorporated into the primers. This technique is useful with DNA of any origin and of any complexity (Vos et al. 1995). It has been suggested that this DNA fingerprinting technique is comparable in taxonomic value to that of DNA-DNA homology studies or fatty acid analyses (Janssen et al. 1996). That study demonstrated the usefulness of AFLP using Xanthomonas as a model. Various primer combinations were able to differentiate closely related bacterial strains. It was also observed that the placement of strains into groups deduced through this technique was in agreement with DNA homology studies as well as fatty acid data (Janssen et al. 1996). In a more recent study comparing AFLP and rep-PCR, Xanthomonas was used as the model and it was demonstrated that both fingerprinting methods were useful in determining taxonomic relationships (Rademaker et al. 2000). DNA sequence analysis of genes followed by analysis for phylogenetic relationships is useful in determining relatedness of organisms. One region that has been examined for determining relatedness is the hrp (hypersensitive response and

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9 pathogenicity). This region is commonly associated with the majority of plant pathogenic bacteria and is accountable for those organisms ability to cause disease or a hypersensitive response. It has been suggested that by subjecting this region to PCR and subsequent analysis it is possible to differentiate xanthomonads as well as detect the presence of X. c. vesicatoria on pepper and tomato seed (Leite Jr. et al. 1995). Similarly, this technique has been used for detecting X. fragariae in nurseries (Roberts et al. 1996). That study found that the 49 strains of X. fragariae shared identical restriction profiles. Another region used for taxonomic purposes is the ribosomal DNA. In the early 1990s, a study was conducted showing the high degree of variability that exists in ribosomal DNA restriction patterns of X. campestris pv. dieffenbachiae (Berthier et al.1993). This was supported by the data stating that 53 strains were characterized by five restriction patterns, and there were no patterns corresponding to geographic origin but clearly to host plant origin (Berthier et al.1993). In a later study Hauben et al. (1997) determined that the use of rDNA signatures to differentiate Xanthomonas species was not ideal due to the restricted variability. In that study only one strain of X. axonopodis pv. dieffenbachiae was used. However, a more recent study shows that rDNA analysis is sufficient in the identification of closely related xanthomonads such as X. axonopodis pv. citri, and X. axonopodis pv. aurantifolii, the causal agent of bacterial citrus canker types A and B, respectively (Cubero and Graham 2002).

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CHAPTER 3 MATERIALS AND METHODS This chapter includes all scientific and data collecting procedures. All experiments were conducted between May 2001 and May 2003. A majority of the experiments were conducted at the Mid Florida Research and Education Center-Apopka, including greenhouse studies, genomic comparisons, and storage of bacterial strains. All DNA sequencing work and growth chamber studies were conducted at the University of Florida in Gainesville. Bacterial Strains Used One hundred and eighty seven X. a. pv. dieffenbachiae strains were used in this study (Table 1). These strains were isolated over a 20-year period from ten aroid genera. Strains were revived from cryogenic storage when needed. Genomic Comparisons All strains in Table 1 were subjected to Repetitive Element PCR (rep-PCR) using BOX, ERIC, and REP primers. DNA from these strains was prepared using the GenomicPrep Kit, (Amersham Pharmacia Biotech, Pistcataway, NJ). Instead of using a master mix, PCR was done using RAPD Analysis Beads (Amersham). A modified program suggested by Amersham Pharmacia Biotech was used to simulate results obtained using a master mix. Template DNA was amplified using a PTC-100 thermocycler (M.J. Research Inc., Watertown, Mass). The thermocycler was programmed as follows. For BOX initial denaturation at 95 for five minutes, followed by 45 cycles denaturing at 95 for one minute and annealing at 53 for one minute. ERIC 10

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11 and REP had annealing temperatures of 52 and 40, respectively. Final extension was conducted at 72 for two minutes; upon completion the block was held at 4. PCR products were separated on 1.5% gels, stained with ethidium bromide and photographed under a UV transluminator with a Kodak Digital Documentation System 120 (Rochester, NY). All gels photographed were stored as Tiff files. Banding patterns were compared between strains using Pearson Correlation and Unweighted Pair Group Means Analysis (UPGMA) with the BioNumerics program ver. 2.1 (Applied Maths, Kortrijk, Belgium). For strains with unique banding patterns DNA was extracted again, PCR was redone, and the analysis was repeated. Using the BioNumerics program, a combined comparison was made of banding patterns from all three rep-PCR primers. Cluster cut-off values were also calculated using the BioNumerics software in each comparison. The ATCC X.a.d Type strain 23379 was used as a standard. Amplified Fragment Length Polymorphism (AFLP) Based on rep-PCR clusters, 40 strains were selected from seven distinct genetic clusters. Due to the large number of Anthurium strains an emphasis was placed on these as well as those representing strains from Syngonium and Dieffenbachia. These strains were compared using AFLP. DNA was prepared as above using the kit obtained from Amersham. Template was prepared using LI-COR (Lincoln, NE) template preparation kit according to the provided protocol. The protocol was modified only for the selective amplification step by following a procedure according to Janssen et al., (1996) LI-CORs template preparation kit uses the EcoRI and MseI restriction enzymes and a two-dye system for labeling rather than the traditional radiolabelling. An MseI primer containing 3cytosine was obtained from Genomechanics (Alachua, FL). Following the

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12 nomenclature provided by Janssen et al. (1996) this primer will be referred to as M02. Using the LI-COR system, the EcoRI primers were labeled with an IR-dye. Two dyes were obtained from LI-COR: one 700 EcoRI primer with no selective base (E00) and one 800 dye with cytosine as a selective base (E02). Another 700 dye was obtained for (E02) as the 800 dye gave less than ideal products. Acrylamide gels were prepared using KB plus gel matrix (LI-COR), temed and ammonium persulfate according to manufacturers specifications. Gels were run 4 hours at 1500 volts on a Global LI-COR IR 2 System. Images were recorded using the SAGA software (LI-COR). Tiff files were analyzed using the BioNumerics program. Sequence Comparisons Two regions were chosen for sequence comparison, the HrpB (for hypersensitivity response and pathogenicity) and the intergenic transcribed sequence (ITS). DNA for the 12 selected strains was prepared as previously mentioned. The hrp region was selected to determine whether or not this group of organisms contained hrp genes. PCR products were obtained using Hrp (RST65-RST69) primers used for amplification of a 420bp product in X. campestris pv. vesicatoria. Products obtained from strains isolated from various hosts were subjected to restriction digests using CfoI, and HaeIII. Different profiles were generated and observed on NuSieve 3:1 agarose gels. Based on the variation in restriction profiles, PCR was repeated, products were cleaned using a spin column (Qiagen, Germantown, MD) and submitted for sequencing at the University of Floridas Interdisciplinary Center for Biotechnology Research (ICBR) sequencing lab in Gainesville. The ITS region was not subjected to restriction digest, but was amplified using (J13-J14) primers and sequenced in the same manner. Sequences were assembled

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13 using the Sequencher software and alignments were performed using the European Bioinformatics Institutes CLUSTALW (www.ebi.ac.uk/clustalw/). Pathogenicity and Host Range Using the 40 pre-selected strains, a limited host range was conducted using both mist and leaf infiltration techniques. Strains used in the pathogenicity tests were grown on Nutrient Agar (Difco) amended with 5% sucrose at 28 C for 24-48 hours prior to use. Bacteria were harvested from the petri plates and suspended in 0.1M NaCl solution. Bacterial suspensions were adjusted with a spectrophotometer to 1 X 10 8 CFU/ml with an optical density 0.3 (A 600 ). This suspension was applied to leaf surfaces using hand-pump sprayers. Immediately after misting, plants were placed in plastic bags for 24 hours to maintain high relative humidity. The inoculated plants were observed for progression of disease weekly for two months. Plant species utilized were aglonema (Aglonema commutatum, Schott Maria), dieffenbachia (Dieffenbachia maculata (Lodd) G. Donn., compacta), philodendron, (Philodendron scandens oxycardium), syngonium (Syngonium podophyllum, White Butterfly), and three species of anthurium (Anthurium crystalinum Linda Andre, Crystal Hope; a complex interspecific hybrid of A. amnicola Dressler x A. andreaneum Linden ex Andr, Red Hot; and A. andraeanum Hearts Desire). Plants were kept under identical cultural conditions in a fiberglass shadehouse with maximum irradiance of 125 mol s -1 m -2 temperature range of 21 to 37 C with natural photoperiod, and high relative humidity. For leaf infiltrations, bacterial suspensions were serially diluted to 1 X 10 5 CFU/ml. The same host plant species mentioned above were utilized with the exception of only using one anthurium cultivar (Red Hot). Leaf tissue, approximately 4 cm 2 was infiltrated on two leaves on each of two plants of each host plant species using a 25gauge

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14 hypodermic needle. Younger leaves were preferentially used. The infiltrated areas were observed for symptom expression over a two-month period. For both methods of inoculation a saline buffer and a strain of X. campestris pv. campestris (ATCC 33913) were used as negative controls. All greenhouse tests were conducted between June and September 2002.

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15 Table1. Sources and hosts of Xanthomonas axonopodis pv. dieffenbachiae strains utilized in this study. Strain ID Original source ID Host Geographic origin 01-156 MREC Anthurium Florida 13 MREC Anthurium Florida 14 MREC Anthurium Florida 82 A990-5 Anthurium Hawaii 84 A844-1 Anthurium Hawaii 158 MREC Syngonium Florida 159 MREC Syngonium Florida 161 MREC Syngonium Florida 162 MREC Syngonium Florida 166 MREC Syngonium Florida 170 MREC Philodendron Florida 172 MREC Syngonium Unknown? 173 MREC Syngonium Unknown? 175 MREC Dieffenbachia Florida 176 MREC Epipremnum Florida 178 MREC Syngonium Florida 181 MREC Syngonium Florida 183 MREC Dieffenbachia Florida 187 MREC Syngonium Florida 191 MREC Syngonium Florida 192 MREC Syngonium Florida 195 GWS 2218-83 Dieffenbachia Florida 265 ICMP 9586 Philodendron Florida 268 D 150 Anthurium Hawaii 269 D 158 Anthurium Hawaii 271 D 36.12 Syngonium Hawaii 272 D 129.12 Syngonium Hawaii 326 PDD 1145-87 Anthurium Florida 328 DPI P87-2307 Anthurium Florida 376 PDD 1399-87 Aglaonema Florida 422 PDD 21-88 Anthurium Florida 423 PDD 20-88 Anthurium Florida 430 D129.1M Syngonium Hawaii 431 D129.1D Syngonium Hawaii 451 D61.11 Anthurium Hawaii 452 D68.00 Anthurium Hawaii 453 D184.00 Aglaonema Hawaii 454 D182.00 Anthurium Hawaii 455 D183.00 Anthurium Hawaii 456 D185.00 Aglaonema Hawaii 457 D206.00 Colocasia Hawaii 458 D191.00 Epipremnum Hawaii 460 D228.00 Colocasia Hawaii

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16 Table 1 (Continued). Strain ID Original source ID Host Geographic origin 462 D36.10 Syngonium Hawaii 463 D18.00 Anthurium Hawaii 464 D139.00 Anthurium Hawaii 465 D227.00 Colocasia Hawaii 466 D135.00 Anthurium Hawaii 467 D122.00 Colocasia Hawaii 468 D206.00 Colocasia Hawaii 469 D204.00 Colocasia Hawaii 471 D162.00 Anthurium Hawaii 472 D170.00 Anthurium Hawaii 473 D17.10 Anthurium Hawaii 474 D145.00 Anthurium Hawaii 475 D147.00 Anthurium Hawaii 476 D150.00 Anthurium Hawaii 477 D145.00 Anthurium Hawaii 478 D1.21 Anthurium Hawaii 479 D30.00 Spathiphyllum Hawaii 480 D55.1 Anthurium Hawaii 481 D110.00 Anthurium Hawaii 482 D120.00 Anthurium Hawaii 483 D131.00 Anthurium Hawaii 484 D93.00 Spathiphyllum Hawaii 485 D94.40 Anthurium Hawaii 487 D101.00 Anthurium Hawaii 488 D160.00 Anthurium Hawaii 489 D46.20 Anthurium Hawaii 490 D52.00 Anthurium Hawaii 491 D69.10 Anthurium Hawaii 492 D70.00 Anthurium Hawaii 493 D71.50 Anthurium Hawaii 494 D38.10 Anthurium Hawaii 495 135.00 Anthurium Hawaii 497 D40.10 Anthurium Hawaii 585 MREC Anthurium Florida 586 MREC Anthurium Florida 606 DPI 072-743 Anthurium Florida 628 DPI 076-953 Philodendron Florida 641 DPI 072-745 Anthurium Florida 642 MREC Philodendron Florida 661 PDD 1772-88 Aglaonema Florida 696 DPI P88-3370 Philodendron Florida 697 DPI P87-2081 Dieffenbachia Florida 746 MREC Anthurium Florida 747 MREC Anthurium Florida

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17 Table 1 (Continued). Strain ID Original source ID Host Geographic origin 757 PDD 15017-89 Anthurium Florida 758 PDD 1500-89 Anthurium Florida 764 PDD 2033-89 Aglaonema Florida 765 PDD 2025-89 Anthurium California 766 PDD 2050-89 Anthurium Florida 767 PDD 2050-89 Anthurium Florida 768 PDD 2050-89 Anthurium Florida 785 PDD 2129-89 Anthurium Florida 786 PDD 2129-89 Anthurium Florida 788 MREC Anthurium Florida 790 PDD 2444-89 Anthurium Florida 805 PDD 2507A-89 Dieffenbachia Florida 807 MREC Caladium Florida 811 MREC Aglaonema Florida 813 MREC Caladium Florida 818 DPI 89-3142-1 Colocasia Florida 830 PDD 2912-89 Anthurium Florida 831 PDD 2927-89 Anthurium Florida 834 MREC Anthurium Florida 840 MREC Syngonium Florida 841 MREC Anthurium Florida 851 PDD 3523-89 Philodendron Florida 868 DPI P89-4119-1 Dieffenbachia Florida 875 DPI P89-4526-10 Caladium Florida 979 ATCC33913 Cabbage United Kingdom 1176 MREC Anthurium Florida 1181 MREC Philodendron Florida 1185 MREC Anthurium Florida 1186 MREC Anthurium Florida 1188 MREC Caladium Florida 1268 MREC Aglaonema Florida 1272 Cooksey Anthurium California 1277 DPI P90-5399 Anthurium Florida 1278 DPI P90-3705 Philodendron Florida 1279 DPI P90-3919 Philodendron Florida 1283 DPI P90-3731-1 Syngonium Florida 1341 PDD 3466-91 Syngonium Florida 1343 DPI P91-2557 Anthurium Florida 1353 MREC Anthurium Florida 1354 MREC Anthurium Florida 1390 DPI P91-3902 Epipremnum Florida 1420 MREC Philodendron Florida 1474 MREC Philodendron Florida 1476 PDD 359A-92 Syngonium Florida

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18 Table 1 (Continued). Strain ID Original source ID Host Geographic origin 1477 PDD 359B-92 Syngonium Florida 1559 MREC Aglaonema Florida 1564 MREC Aglaonema Florida 1567 MREC Anthurium Florida 1568 MREC Anthurium Florida 1610 MREC Aglaonema Florida 1617 M 101 Xanthosoma Florida 1618 M 102 Xanthosoma Florida 1619 M 103 Xanthosoma Florida 1620 M 105 Xanthosoma Florida 1621 M 106 Xanthosoma Florida 1622 M 110 Xanthosoma Florida 1623 M 113 Xanthosoma Florida 1624 M 114 Xanthosoma Florida 1625 M 115 Xanthosoma Florida 1626 M 116 Xanthosoma Florida 1627 M 117 Xanthosoma Florida 1628 M 123 Xanthosoma Florida 1629 M 124 Xanthosoma Florida 1630 M 126 Xanthosoma Florida 1631 M 127 Xanthosoma Florida 1671 MREC Anthurium Florida 1672 MREC Anthurium Florida 1673 MREC Anthurium Florida 1674 MREC Syngonium Florida 1688 Z 27 Anthurium Puerto Rico 1689 Z 23 Anthurium Puerto Rico 1694 MREC Difffenbachia Florida 1697 MREC Syngonium Florida 1698 MREC Syngonium Florida 1699 MREC Epipremnum Guatemala 1701 MREC Syngonium Florida 1702 MREC Syngonium Florida 1703 MREC Dieffenbachia Florida 1704 MREC Syngonium Florida 1705 MREC Anthurium Florida 1706 MREC Caladium Florida 1707 MREC Syngonium Florida 1708 MREC Dieffenbachia Florida 1709 MREC Dieffenbachia Florida 1711 MREC Syngonium Florida 1713 MREC Aglaonema Florida 1718 MREC Dieffenbachia Florida 1750 LMG7399 Z Dieffenbachia USA

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19 Table 1 (Continued). Strain ID Original source ID Host Geographic origin 1751 LMG695 Anthurium Brazil 1752 LMG7400 Dieffenbachia USA 1753 LMG7484 Anthurium Brazil 1754 LMG8664 Anthurium Brazil y Strains were obtained from the following laboratories: (A or D) A. Alvarez, Department of Plant Pathology, University of Hawaii at Manoa, Honolulu, HI, 96822. (ATCC) American Type Culture Collection, Manassas, VA 20108. (BCCM/LMG) Belgian Coordinated Collections of Micro-organisms, Brussels, Belgium. (ICMP) International Collection of Micro-Organisms from Plants, Mt Albert, Auckland, New Zealand. (M) K. Pernezny, Everglades Research and Education Center, Belle Glade, FL 33430. (MREC) Mid-Florida Research and Education Center, University of Florida, Apopka, FL 32703. D. Cooksey, University of California Riverside. (DPI) J. Miller, Division of Plant Industry, Florida Department of Agriculture and Consumer Services, Gainesville, FL 32602. (GWS) G. Simone, Department of Plant Pathology, University of Florida, Gainesville, FL 32611. (NZTCC) New Zealand Type Culture Collection. (PDD) D. Brunk, Plant Disease Diagnostics, Inc., Apopka, FL 32703. (NCPPB) National Collection of Plant Pathogenic Bacteria, York, UK. (Z) M. Zapata, University of Puerto Rico, Mayasuez, PR. z Pathovar reference strain for X. a pv. dieffenbachiae.

PAGE 28

CHAPTER 4 RESULTS Genomic Comparisons Rep-PCR The profiles for BOX, REP, and ERIC were combined and used in computer analysis. The results of this analysis yielded six distinct clusters, shown in Figure 1. Cluster III contained 81 strains and was comprised primarily of strains originally isolated from Anthurium spp. This cluster consists of two sub-clusters of 37 and 42 strains. The two sub-clusters represent those strains isolated from Anthurium spp. with 86% and 93% of the strains originating from Anthurium spp., respectively. There were a couple of outliers from this cluster containing two strains isolated from Syngonium spp. Cluster II contained 33 strains and was very heterogeneous in regards to host of origin, with nine of the ten host genera being represented. Cluster I consisted of 15 strains sharing 75% similarity, which was isolated from Xanthosoma spp. Three outliers representing three strains isolated from Aglaonema, Dieffenbachia, and Epiprenum also were grouped between cluster II and cluster III. Strains originally isolated exclusively from Syngonium spp. comprised cluster IV containing 19 strains. The fifth cluster was relatively small and mixed in regards to host of origin. Cluster VI was composed of 15 strains in which ten or 67% of the strains were isolated from Philodendron spp.. Amplified Fragment Length Polymorphism Forty representative strains based on the results from the rep-PCR with an emphasis on strains originating from Anthurium, Dieffenbachia, and Syngonium spp. 20

PAGE 29

21 were used in AFLP analysis. Strains isolated from Aglaonema, Caladium, Colocasia, and Philodendron spp. were also represented in AFLP analysis. Results using the M02 and E00 primer combination generated clusters for 38 strains and are shown in Figure 2. The cluster analysis yielded similar results as that of the rep-PCR technique. There were five distinct clusters. Cluster I contained strains showing 53% similarity and contained both type strains (LMG 7399, 7400) originating from Dieffenbachia spp. as well as two of the three strains isolated from Aglaonema spp. There was also a strain isolated from Caladium spp. within this cluster. Cluster II consisted of nine strains showing 66% similarity, of which six were strains from Anthurium. Two representing the Anthurium type strains (LMG 7484, 8664), and three from Dieffenbachia spp. Cluster III contained six strains all of which were isolated from Anthurium spp. one of which represented the Anthurium type strain (LMG 695). Cluster IV contained six strains all of which were isolated from Syngonium spp. and shared 54% similarity. Between clusters IV and V, there was an outlier, X-457, isolated from Colocasia spp. Cluster V had slightly lower similarity of 44%, and contained six strains isolated from four different host genera. Three of these strains were isolated from Dieffenbachia and the other three representing strains isolated from Anthurium, Syngonium, and Philodendron spp. Another outlier, X-271, was below cluster V, isolated from Syngonium spp. Strain X-805 isolated from Dieffenbachia spp was most distantly related to all other strains. DNA Sequencing Sequence comparisons were done using the European Bioinformatics Internet CLUSTALW program. The results for the ribosomal DNA placed all strains tested within two groups with which one group was composed of strains originating from Syngonium. Two strains, X-430 and X-1674, grouped together but were clearly different

PAGE 30

22 from other strains. Most of the variation between strains was observed in the ITS region. The Syngonium strains varied primarily in positions in between the 16S and 23S known as the ITS region, and were two nucleotides shorter in overall length. The hrp sequences revealed similar results placing Syngonium strains in one group distinct from others. Host Range Tests The pathogenicity tests proved the heterogeneity of X. axonopodis pv. dieffenbachiae with strains varying immensely in their ability to produce symptoms on the hosts tested. Host range tests did not show distinct relationships between strains based on host of origin as was predicted by preliminary rep-PCR data. Although many of the strains isolated from their host of origin showed pathogenicity on other aroids, there were some important trends observed. Results from representative strains originally isolated from Dieffenbachia, Anthurium, and Syngonium are presented in Figures 3,4, and 5 respectively. Dieffenbachia Strains In pathogenicity tests with ten strains originally isolated from Dieffenbachia (Figure 3), disease on Dieffenbachia was 60% and 72% when misted at 10 8 cfu/ml and infiltrated at 10 5 cfu/ml, respectively. Four strains failed to produce symptoms on Dieffenbachia maculata after mist inoculations (X-183, X-697, X-805, and LMG 7399). Whereas they produced some symptoms on D. maculata in the infiltration inoculations, X-805 failed to produce symptoms on any of the hosts tested. In addition to causing disease in D. maculata, a number of these Dieffenbachia strains produced symptoms on the Anthurium spp. in both tests. Of the strains tested, X-1709 and X-1718 appeared the most aggressive towards Anthurium even resulting in systemic necrosis. Strains producing symptoms in Anthurium also produced symptoms on Aglaonema commutatum.

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23 Only two strains (X-1703 and LMG 7400) produced symptoms on Syngonium podophyllum and on all hosts tested with the exception of Philodendron scandens oxycardium. Only X-1718 consistently produced symptoms on the Philodendron spp. tested. Anthurium Strains Figure 4 illustrates the results obtained from 12 strains originally isolated from Anthurium. Anthurium strains produced symptoms in a similar manner to that of the Dieffenbachia strains. A majority of the strains (greater than 70%) produced symptoms on Dieffenbachia whether infiltrated or misted. Symptom production was greater in D. maculata than that of any of the Anthurium cultivars tested. Nine of the twelve strains reliably produced symptoms in misting tests. The exceptions (X-14, X-84, X-1705) failed to produce symptoms on Anthurium in either test and failed to produce consistent symptoms in any of the hosts tested. It was observed during the infiltration tests that leaf age plays a role in susceptibility particularly when working with the cultivar Red Hot. This may account for the low number of strains producing symptoms when using the infiltration technique. The results for the misting yielded 50%, 56%, and 62% disease on the cultivars Red Hot, Crystal Hope, Hearts Desire respectively. The strains producing symptoms in Anthurium spp. did not consistently produce symptoms in A. commutatum as was observed with the strains isolated from Dieffenbachia. None of the strains originating from Anthurium produced any symptoms on S. podophyllum nor the Philodendron spp. tested. This exclusiveness in pathogenicity is noteworthy. Syngonium Strains Results from strains isolated from Syngonium are shown in Figure 5. The Syngonium strain inoculations appeared considerably different from those with strains

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24 isolated from Anthurium or Dieffenbachia. Of these strains 25% produced symptoms when misted and 68% produced symptoms on D. maculata when infiltrated. Only one of these strains (X-191) produced symptoms on the Red Hot and Hearts Desire cultivars of Anthurium and failed to produce symptoms on Red Hot in the infiltration inoculations. Syngonium appears to be the most susceptible host for these strains. Three (X-159, X-187, X-271) of the ten strains failed to produce symptoms on S. podophyllum in the misting test. Strains X-159 and X-187 expressed partial symptoms when infiltrated. Strain X-271 failed to produce symptoms on any of the hosts tested. The Aglaonema tests were similar to those of the Anthurium isolates except, that fewer strains induced symptoms. In both the infiltration and mist inoculations no Syngonium strains produced symptoms on Anthurium spp.

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25 100 90 70 50 30 10 Similarity coefficient 15 Xanthosoma strains (100%) 35 strains (9 genera) 40 strains (35/40 A nthurium) 42 strains (39/42 A nthurium) 3 strains 2 strains 19 strains (100% Sy n g onium) 12 strains (7 genera) 15 strains (67% P hilodendron) I II III a III b IV V VI Figure 1. Cluster analysis of 183 X.a.d strains isolated from ten host genera over a 20-year period based on rep-PCR using BOX, ERIC and REP primers. Banding patterns were compared between strains using Pearson Correlation and Unweighted Pair Group Means Analysis (UPGMA) with the BioNumerics program ver. 2.1

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26 1009070503010Similarity coefficient 6 strains (3 genera) 9 strains (6 Anthurium 3 Dieffenbachia) 6 strains (100% Anthurium) 6 strains (100% Syngonium) 6 strains (4 genera) X 457 (Colocasia)2 strains (1 Dieffenbachia 1 Syngonium) X805 (Dieffenbachia)I X 271 (Syngonium)IIIIIIVV Figure 2. Cluster analysis of 38X.a.d strains isolated from six host genera over a 20-year period based on AFLP using M02, and E00 primers combinations. Banding patterns were compared between strains using Pearson Correlation and Unweighted Pair Group Means Analysis (UPGMA) with the BioNumerics program ver. 2.1

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27 100907050 30 10 100907050 30 10 Similarity coefficient 6 strains (3 genera) 9 strains (7 A nthurium 2 D ie ff enbachia) 5 strains (100% A nthurium ) 6 strains (100% Sy n g onium ) 8 strains (4 genera) 3 strains (1 D ie ff enbachia 1 Sy n g onium 1 P hilodendron ) X805I X 271 Sy n g oniumIIIIIIVV Figure 3. Cluster analysis of 39X.a.d strains isolated from six host genera over a 20-year period based on AFLP using M02, and E02 primers combinations. Banding patterns were compared between strains using Pearson Correlation and Unweighted Pair Group Means Analysis (UPGMA) with the BioNumerics program ver. 2.1

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28 0 10 20 30 40 50 60 70 80 90 100Anthurium 'Crystal Hope' Anthurium 'Heart's Desire' Anthurium 'Red Hot' Dieffenbachia Syngonium Aglaonema Philodendron Figure 4. Percent of X. a. dieffenbachiae strains isolated from Dieffenbachia spp. producing disease when inoculated into various aroid hosts. Blue bars represent mist inoculations of bact erial suspension adjusted to 1.0 X 108 CFU/ml. Red bars represent infilt ration inoculations using bacterial suspensions adjusted to 1.0 X 105 CFU/ml. Results based on two mistings and four infiltrations.

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29 0102030405060708090100Anthurium 'Crystal Hope'Anthurium 'Heart'sDesire'Anthurium 'Red Hot'Dieffenbachia SyngoniumAglaonemaPhilodendron Figure 5. Percent of X. a. dieffenbachiae strains isolated from Anthurium spp. producing disease when inoculated into various aroid hosts. Blue bars represent mist inoculations of bacterial suspension adjusted to 1.0 X 10 8 CFU/ml. Red bars represent infiltration inoculations using bacterial suspensions adjusted to 1.0 X 10 5 CFU/ml. Results based on two mistings and four infiltrations.

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30 0102030405060708090100Anthurium 'CrystalHope'Anthurium 'Heart'sDesire'Anthurium 'Red Hot'Dieffenbachia SyngoniumAglaonemaPhilodendron Figure 6. Percent of X. a. dieffenbachiae strains isolated from Syngonium spp. producing disease when inoculated into various aroid hosts. Blue bars represent mist inoculations of bacterial suspension adjusted to 1.0 X 10 8 CFU/ml. Red bars represent infiltration inoculations using bacterial suspensions adjusted to 1.0 X 10 5 CFU/ml Results based on two mistings and four infiltrations.

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CHAPTER 5 DISCUSSION In this study I addressed the variation that exists within Xanthomonas affecting aroid hosts. Using rep-PCR it was observed that six genetic clusters were generated by the 183 strains tested. Four of these clusters appeared to cluster based on host of origin. Clusters clearly represented strains originating Philodendron, Xanthosoma, Anthurium, and Syngonium. These results suggest that each of the four clusters may represent a different pathovars as previously described by Louws et al. (1994). When representative strains were subjected to AFLP, similar results were observed to that of the rep-PCR. Distinct clusters representing strains virulent on Anthurium and Syngonium were observed. Genetic differences between these two clusters and between the others provide considerable evidence for the variation within X. a. dieffenbachiae, and particularly for the Syngonium strains. These results when combined with the rep-PCR data may reveal that these are significant species since the techniques provide information close to the resolution of DNA-DNA hybridization (Rademaker et al. 2000). Upon phylogenetic analysis of rDNA, the same differentiation was observed between X. a. dieffenbachiae and those strains isolated from Syngonium. A great degree of variability had been observed in restriction patterns within pathovar dieffenbachiae (Berthier et al. 1993). These results also elicited some minor differences within strains from Anthurium and Dieffenbachia. However, quite significant variation was observed between these strains and those from Syngonium, particularly in the ITS region. The same variation was found upon investigating the hrpB region. Individually or in 31

PAGE 40

32 combination these two techniques were useful in differentiating X. a. dieffenbachia from pathovar syngonii. Many of the strains tested exhibited overlapping pathogenicity on host plants, which was observed in other studies (Lipp et al. 1992, Chase et al. 1992). This was especially the case when looking at the data from those strains isolated from Anthurium and Dieffenbachia. Interestingly, the Syngonium strains did not exhibit pathogenicity in a manner similar to the X. a. dieffenbachiae strains. Strains from Philodendron and Xanthosoma were found in distinct genetic clusters based on rep-PCR data and might also exhibit specialization in pathogenicity. The pathovar, syngonii, was described earlier based on NaCl tolerance, pH optima, gelatin hydrolysis, and growth on SX media (Dickey and Zumoff 1987). However, there were many reports on the inadequacy of the tests used. Hodge et al. (1990) concluded that there was not enough variation in fatty acid profiles to differentiate X. a. dieffenbachiae from X. c. syngonii, and strains from Syngonium and should be included in X. a. dieffenbachiae. In an attempt to differentiate the two with the use of monoclonal antibodies, Lipp et al. (1992) came to a similar conclusion stating that typical Syngonium strains are most similar to Anthurium strains while the atypical Syngonium strains are more similar to those strains isolated from Dieffenbachia. Even when using physiological, pathological and fatty acid analysis, Chase et al. (1992) concluded that none of these tests were useful in differentiating X. a. dieffenbachiae from X. c. syngonii. The Syngonium cluster (cluster V in figures 4 and 5) represents fastidious xanthomonads, which are specialized in pathogenicity in Syngonium. The pathovar, syngonii, previously

PAGE 41

33 described by Dickey and Zurmoff would be an appropriate designation for these strains (1987). This work has provided genetic evidence for the pathovar designation syngonii when referring to those strains specialized in pathogenicity on Syngonium spp. The genetic data is convincingly reinforced by host range tests. These results illustrate the usefulness of molecular tools in bacterial taxonomy. In order to be absolute in this matter it may still be necessary to conduct DNA-DNA hybridization experiments with additional strains representing pathovar syngonii. The strains comprising X.a.d. used by Vauterin et al. (1995) proved to be very similar upon rep-PCR and AFLP analysis though they varied in their pathogenicity. None of these strains were representative of those strains representing pathovar syngonii and further justifies the need for them to be included in a DNA-DNA homology study.

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LIST OF REFERENCES Bergey, D. H., Harrison, F. C., Breed, R. S., Hammer, B. H. & Huntoon, F. M. (1939). Manual of Determinative Bacteriology. Baltimore, MD: The Williams & Wilkins company. Berthier, Y., Verdier, A., Guesdon, J., Chevrier, D., Denis, J., Decoux, G., & LeMattre, M. (1993). Characterization of Xanthomonas campestris pathovars by rRNA gene restriction patterns. Appl & Envir Micro 59, 851-859 Bouzar, H., Jones, J. B., Stall, R. E., Hodge, N. C., Minsavage, G. V., Benedict, A. A., & Alvarez, A. M. (1994). Physiological, chemical, serological, and pathogenic analysis of a worldwide collection of Xanthomonas campestris pv. vesicatoria strains. Phytopathology 84, 663-671 Chase, A. R., & Poole, R.T. (1986). Effects of host nutrition on growth and susceptibility of Anthurium scherzeranum to Xanthomonas leaf spot. Nurserymens Digest 20, 58-59 Chase, A. R., Stall, R. E., Hodge, N. C., & Jones, J. B. (1992). Characterization of Xanthomonas campestris strains from Aroids using physiological, pathological, and fatty acid analyses. Phytopathology 82, 754-759 Cooksey, D. A. (1985). Xanthomonas blight of Anthurium andreanum in California. Plant Disease 69, 727 Cubero, J. & Graham, J.H. (2002). Genetic relationships among worldwide strains of Xanthomonas causing canker in Citrus species and design of new primers for their identification by PCR. Appl & Envir Micro 68, 1257-1264 Dickey, R. S. & Zumoff (1987). Bacterial blight of Syngonium caused by a pathovar of Xanthomonas campestris. Phytopathology 77, 1257-1262 Dowson, W. J. (1943). On the generic names Pseudomonas, Xanthomonas, and Bacterium for certain bacterial plant pathogens. Transactions Of The British Mycological Society 27, 3-14 Dye, D. W., Bradbury, M. G., Hayward, A. C., Lelliott, R. A., & Schroth, M. N. (1980). The inadequacy of the usual determinative tests for the identification of Xanthomonas spp. N Z J Sci 5, 393-416 34

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35 Gilson, E., Clement, J. M., Brutlag, D., & Hofnung, M. (1984). A family of dispersed repetitive extragenic palindromic DNA sequences in E. coli. EMBO J 3, 1417-1421 Gonclaves, E. R., & Rosato Y. B. (2002). Phylogenetic analysis of Xanthomonas species based upon 16S-23S rDNA intergenic spacer sequences. Int J Syst & Envir Micro 52, 355-361 Hauben, L., Vauterin, L., Swings, J., & Moore, E. R. B. (1997). Comparison of 16S ribosomal sequences of all Xanthomonas species. Int J Syst Bacteriol 47, 328-335 Hayward, A. C. (1972). A bacterial disease of Anthurium in Hawaii. Plant Disease Reporter 56, 904-908 Higgins, C. F., Ames, G. F.-L., Barnes, W. M., Clement, J. M., & Hofnung, M. (1982) A novel intercistronic regulatory element of prokaryotic operons. Nature 298, 760-762 Hodge, N. C., Chase, A. R., & Stall, R. E. (1990). Characterization of xanthomonads from Araceae by fatty acid analyses. Phytopathology 80, 158 Hulton, C. S. J., Higgins, C. F., & Sharp, P. M. (1991). ERIC sequences: A novel family of repetitive elements in the genomes of Escherichia coli, Salmonella typhimurium and other enterobacteria. Mol Microbiol 5, 825-834 Hyndman, S., Zhu, G., & Mottl, T. (2003). International Aroid Society. Jill Bell Designs http://www.aroid.org (April 2003) Janse, J. D., Rossi, M. P., Gorkink, R. F. J., Derks, J. H. J., Swings, J., Janssens, D., & Scortichini, M. (2001). Bacterial leaf blight of strawberry (Fragaria (x) ananassa) caused by a pathovar of Xanthomonas arboricola, not similar to Xanthomonas fragariae Kennedy & King. Description of the causal organism as Xanthomonas arboricola pv. fragariae (pv. nov., comb. nov.). Plant Pathology 50, 653-665 Janssen, P., Coopman, R., Huys G., Swings, J., Bleeker, M., Vos, P., Zabeau, M., & Kersters, K. (1996). Evaluation of the DNA fingerprinting method AFLP as a new tool in bacterial taxonomy. Microbiology 142, 1881-1893 Jones, J. B., Bouzar, H., Stall, R. E., Almira, E. C., Roberts, P. D., Bowen, B. W., Sudberry, J., Strickler, J., & Chun, J. (2000). Systematic analysis of xanthomonads (Xanthomonas spp.) associated with pepper and tomato lesions. Int J Syst & Envir Micro 50, 1211-1219 Leite, R. P. Jr., Jones, J. B., Somodi, G. C., Minsavage, G. V., & Stall, R. E. (1995). Detection of Xanthomonas campestris pv. vesicatoria associated with pepper and tomato seed by DNA amplification. Plant Disease 79, 917-922

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36 Lipp, R. L., Alvarez, A. M., Benedict, A. A., & Berestecky (1992). Use of monoclonal antibodies and pathogenicity tests to characterize strains of Xanthomonas campestris pv. dieffenbachiae from Aroids. Phytopathology 82, 677-682 Louws, F. J., Fulbright, D. W., Taylor Stephens, C., & DeBruijn, F. J. (1994). Specific genomic fingerprints of phytopathogenic Xanthomonas and Pseudomonas pathovars and strains generated with repetitive sequences and PCR. Appl & Envir Micro 60, 2286-2295 Louws, F. J., Fulbright, D. W., Taylor Stephens, C., & DeBruijn, F. J. (1995). Differentiation of genomic structure by rep-PCR fingerprinting to rapidly classify Xanthomonas campestris pv. vesicatoria. Phytopathology 85, 528-536 Martin, B., Humbert, O., Guenzi, E., Walker, J., Mitchell, T., Andrew, P., Prudhomme, M., Alloing, G., Hackenbeck, R., Morrison, D. A., Boulnois, G. J., & Clarveys, J.-P. (1992). A highly conserved repeated DNA element located in the chromosome of Streptococcus pneumoniae. Nucleic Acids Res 20, 3479-3483 McCulloch, L., & Pirone, P.P. (1939). Bacterial leaf spot of Dieffenbachia. Phytopathology 29, 956-962 McFadden, L. A. (1962). Two bacterial pathogens affecting the leaves of Aglaonema robelinii. Phytopathology 52, 20 McFadden, L. A. (1967). A Xanthomonas infection of Philodendron oxycardium leaves. Phytopathology 57, 343 Pohronenzy, K., Volin, R. B., & Dankers, W. (1985). Bacterial leaf spot of cocoyam (Xanthosoma caracu) by Xanthomonas campestris pv. dieffenbachiae, in Florida. Plant Disease 69, 170-173 Rademaker, J. L. W., Hoste, B., Louws, F. J., Kersters, K., Swings, J., Vauterin L., Vauterin, P., & DeBrujn, F. J. (2000). Comparison of AFLP and rep-PCR genomic fingerprinting with DNA-DNA homology studies: Xanthomonas as a model system. Int J Syst & Envir Micro 50, 665-677 Roberts, P. D., Jones, J. B., Chandler, C. K., Stall, R. E., & Berger, R. D. (1996). Survival of Xanthomonas fragariae on strawberry in summer nurseries in Florida detected by specific primers and nested polymerase chain reaction. Plant Disease 80, 1283-1288 Sharples, G. J., & Lloyd, R. G. (1990). A novel repeated DNA sequence located in the intergenic regions of bacterial chromosomes. Nucleic Acids Res. 18, 6503-6508 United States Deptartment of Agriculture (2002). Foliage, floriculture and cut greens. U.S. Department of Agriculture Statistices Board, U.S. Government Printing Office, Washington, D.C.

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37 Vauterin, L., Hoste, B., Kesters, K., & Swings, J. (1995). Reclassification of Xanthomonas. Int J Syst Bacteriol 45, 472-489 Vos, P., Hogers, R., Bleeker, M., Reijans, M., van de Lee, T., Hornes, M., Frijters, A., Pot, J., Peleman, J., Kuiper, M., & Zabeau, M. (1995). AFLP: a new technique for DNA fingerprinting. Nuc Acids Res 23, 4407-4414 Wehlburg, C. (1969). Bacterial leaf blight of Syngonium podophyllum. Phytopathology 59, 1056 Young, J. M., Dye, D. W. Bradbury, J. F., Panagopoulos, C. G., & Robbs, C. F. (1978). A proposed nomenclature and classification for plant pathogenic bacteria. N Z J of Agric Res 21, 153-177

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BIOGRAPHICAL SKETCH Ryan Donahoo is a Florida native with a vested interest in the tropical foliage industry. He received his B.S. in plant science-plant pathology with a minor in plant molecular and cellular biology from the University of Florida in 2001. While completing this undergraduate degree, Mr. Donahoo worked with various microorganisms in laboratory research studies. This experience fostered an interest in bacterial pathogens, which was expanded upon in this study. Mr. Donahoo plans to continue his work in plant pathology and microbiology in a professional research setting. 38


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Title: Genetic variation in Xanthomonas axonopodis pv. dieffenbachiae
Physical Description: Mixed Material
Creator: Donahoo, Ryan Scott ( Author, Primary )
Publication Date: 2003
Copyright Date: 2003

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GENETIC VARIATION IN Xanthomonas axonopodis pv. dieffenbachiae


By

RYAN SCOTT DONAHOO













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

UNIVERSITY OF FLORIDA


2003


































Copyright 2003

by

Ryan S. Donahoo

































This document is dedicated to my grandparents, Doug and Valle Glover.















ACKNOWLEDGMENTS

I would like to first thank Dave Norman for providing me the opportunity to work

on this project in an astonishing atmosphere as well as the assistance that he provided

throughout the completion of this step in my academic career. Next, I would like to

thank Jeff Jones for providing me with a lab in Gainesville, constantly taking time out of

his schedule to address my questions, and giving me insight into bacteria, taxonomy,

molecular genetics, and Gator sports. I am greatly appreciative to Jeanne Yuen, Rosa

Resendiz, and Joe Boswell down at the Mid-Florida Research and Education Center for

their help with handling of cultures, maintenance of plants, and so much of the work that

was accomplished in Apopka. Next, I would like to thank Gerry Minsavage for all of his

technical assistance, allowing a great deal of this research to come to completion. Last, I

would like to thank my parents for believing in me and often providing me with much

needed motivation and serenity.
















TABLE OF CONTENTS
Page

A C K N O W L E D G M E N T S ................................................................................................. iv

L IST O F F IG U R E S .... ...... ................................................ .. .. ..... .............. vii

A B S T R A C T .......................................... .................................................. v iii

CHAPTER

1 IN TRODU CTION ................................................. ...... .................

2 LITER A TU R E REV IEW ............................................................. ....................... 3

H history ........................................................ 3
Related Studies ......................................... ........................ ............
P athogenicity T ests.................................................. 5
PCR B ased A nalysis.................................................... 7

3 M ATERIALS AND M ETHOD S ....................................................... 10

B bacterial Strains U sed ......................................................................10
Genomic Comparisons.........................0..........................10
Amplified Fragment Length Polymorphism (AFLP) .............. ...............11
S equ en ce C om p arison s .................................................................................. 12
Pathogenicity and H ost R ange .............................................. .......... .... ............ .. 13

4 RE SU LTS ................................. .............. .... .... ............. ..... 20

G enom ic C om parisons..................................................................................20
R ep -P C R ............... ... ... ...... ..... .. .......... .. .. ... 2 0

D N A Sequ encing ..................................................................... ..................... 2 1
H ost R ange T ests ................................................... ...... 22
D ieffenbachia Strains ............................................. ................ ............... 22
A nthurium Strains .................................. ................................................23
Syngonium Strains................................................................. .........................23







v









5 D IS C U S S IO N ................................................................................................ 3 1

LIST OF REFERENCES ..................................................................... ...............34

B IO G R A PH IC A L SK E T C H ...................................................................... ..................38















LIST OF FIGURES


Figure page

1. Cluster analysis of 191 Xa.d strains isolated from ten host genera over a
20-year period based on rep-PCR using BOX, ERIC and REP primers.................25

2. Cluster analysis of 38Xa.d strains isolated from six host genera over a 20-year
period based on AFLP using M02, and E00 primers combinations.......................26

3. Cluster analysis of 39Xa.d strains isolated from six host genera over a 20-year
period based on AFLP using M02, and E02 primers combinations.......................27

4. Percent ofX. a. dieffenbachiae strains isolated from Dieffenbachia spp.
producing disease when inoculated into various aroid hosts. ................................28

5. Percent ofX. a. dieffenbachiae strains isolated from Anthurium spp. producing
disease when inoculated into various aroid hosts............................................29

6. Percent ofX. a. dieffenbachiae strains isolated from Syngonium spp. producing
disease when inoculated into various aroid hosts............................................30















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

GENETIC VARIATION WITHIN Xanthomonas axonopodis pv. dieffenbachiae

By

Ryan Scott Donahoo

May 2003

Chair: David J. Norman
Major Department: Plant Pathology

Members of the Araceae are susceptible to the bacterial pathogen Xanthomonas

axonopodis pv. dieffenbachiae, which is capable of causing crop losses up to 100 percent.

When 187 strains isolated from nine aroid hosts were subjected to Rep-PCR, six genetic

clusters were generated. One cluster was found to represent only strains isolated from

Syngonium spp. Forty strains isolated from Anthurium, Dieffenbachia, and Syngonium

were subjected to amplified fragment length polymorphism (AFLP) and tested for

pathogenicity on five Aroid hosts. AFLP data correlated well with Rep-PCR data. Based

on pathogenicity tests, the Syngonium strains were selectively pathogenic on Syngonium.

None of the strains from other hosts caused significant disease on Syngonium. DNA

from ten representative strains was amplified by PCR using primers to the ITS and hrpB.

Phylogenetic analysis of sequenced PCR products reveals that the syngonium strains exist

as a distinct group from other members ofX.a.pv. dieffenbachiae and supports the use of

pv. syngonii for strains isolated from Syngonium.














CHAPTER 1
INTRODUCTION

The United States tropical foliage plant industry has an annual wholesale value of

more than 500 million dollars (Unites States Department of Agriculture, 2002), with

approximately 61% of this production in Florida. Consistent growth has occurred in the

tropical foliage industry due to the popularity of plants in the family Araceae. This

family includes genera such as Aglaonema, Anthurium, Dieffenbachia, Philodendron, and

Syngonium (www.aroid.org).

Aroids are native to the tropics and vary greatly in growth habit. Some are aquatic

and totally submerged, others are hemi-epiphytic, and yet others are totally epiphytic

growing on rocks and trees. Aroids can be found in their native habitats from coastal

locations to higher elevations of several thousand feet. However, most of the

commercially cultivated species are found growing at low elevations under humid

tropical rainforest conditions. Thus, most commercial production of these crops is done

either in tropical areas of the world or under greenhouse conditions. Costa Rica,

Guatemala, and Honduras are primary suppliers of propagative material while the

Netherlands, California, Florida, and Hawaii are the primary producers of finished plants.

Most aroids are known to be susceptible to bacterial blight caused by the bacterium

Xanthomonas axonopodis pv. dieffenbachiae. Traditionally, it has been thought that the

bacterium enters through the hydathodes. However, recent observations suggest that

entry may also occur through the stomata. The hydathodes have been thought of as the

main point of entry associated with this Xanthomonas due to the fact that these









specialized cells localized to the leaf margin secrete glutamine and/or glutamate, both

which can serve as chemotaxic stimuli as well as a sole carbon source for Xanthomonas

dieffenbachiae. The guttation fluid from infected plants serves as a source of inoculum,

and is easily spread by overhead irrigation and/or worker's clothing. Since many of the

aroids are propagated vegetatively, the disease is spread rapidly, on cutting utensils used

in crop maintenance and propagation.

Once the bacteria have entered a susceptible host, whether via the stomata,

hydathodes or wounds, the bacteria multiply at the point of entry, spreading through the

xylem vessels. If the pathogen entry is via the hydathodes, water soaking develops at the

leaf margin within a few days. As the disease progresses, the water soaking becomes

chlorotic then necrotic, forming classical V-shaped lesions of dead tissue. If plants are

grown in close proximity to each other and under warm and humid conditions, spread

within and between plants can be rapid with crop losses up to 100%.














CHAPTER 2
LITERATURE REVIEW

History

Aroids, members of the plant family Araceae, constitute the majority of tropical

foliage crops. McCulloch and Pirone published the first report of bacterial blight of

Dieffenbachia (1939). They described an organism that caused leaf spots, blight, and in

some cases death. They referred to the pathogen as Bacterium dieffenbachiae. During

the 1920's and 1930's it was common practice to group all phytopathogenic bacteria in

the genus Bacterium (Dowson 1943).

Over time, the necessity for additional genera was realized. Through the research

efforts of Migula, Dowson and others in the early 1900's, new genera such as

Pseudomonas and Xanthomonas were gradually accepted. In 1939, Bergey's Manual

renamed the pathogen as Phytomonas dieffenbachiae. Dowson reclassified Phytomonas

dieffenbachiae as Xanthomonas dieffenbachiae, the pathogen causing bacterial blight of

Dieffenbachia (Dowson 1943). Roughly 20 years after Dowson's contribution to the

nomenclature Young et al. transferred the species dieffenbachiae to a pathovar of

Xanthomonas campestris designating it X campestris pv. dieffenbachiae (Xcd) (Young et

al. 1978). In 1980 Dye et al. formally described the use of the pathovar system and

included Xcd as a member.

During the 1960's Xcd was described causing disease on other aroids including

Aglaonema and Philodendron (McFadden 1962 and 1967). During the 1970's Xcd was

also reported in Anthurium in Hawaii (Hayward 1972). In the 1980's, Xcd was described









on numerous aroid hosts throughout the United States (Chase and Poole 1986, Cooksey

1985, Porhonenzy et al. 1985, Chase et al. 1992).

In 1969 bacterial blight was described on Syngonium podophyllum (Wehlburg

1969). The pathogen was described as a Xanthomonas-like organism based on cultural

and microscopic observations characterizing the pathogen as X vitians. Only minor

physiological differences between the two organisms were noted. In the 1980's when

Dickey and Zumoff (1987) compared four strains from the Syngonium cultivar Cream

(LX 103, LX105, LX106, LX114) and two strains from the cultivar White Butterfly (L

212, L215). These six strains were compared with two strains used in Wehlburg's study

(XV29 = NCPPB2255 isolated from Syngonium ATCC19320) and type strains

representing X c. vitians (NCPPB 969), X c. campestris (B-24), and strains of

Xanthomonas campestris from aroid hosts with pathovar designations aracearum (XA-2),

dieffenbachiae (XD-3), and zantedeschiae (XZ1). They noted that strains XA-2, XD-3,

and ATCC 19320 produced disease symptoms on Syngonium when misted on plants at

108 CFU/ml. However it was also noted that the reactions produced by these strains were

different from those produced by strains originally isolated from Syngonium. Dickey and

Zumoff (1987) proposed designating the Syngonium organism as Xanthomonas

campestris pv. syngonii. (Dickey and Zumoff 1987).

This pathovar designation syngonii is available from culture collections (LMG

9055 and ICMP 9152, 9153, 9154, 9155 all representing strains collected by Dickey) but

does not appear anywhere in-approved lists of bacterial names or taxonomic lists. The

xanthomonad that infects Syngonium is currently referred to as X campestris pv.

dieffenbachiae (Chase et al. 1992).









In 1995 the aroid pathogen was reclassified as X axonopodis pv. dieffenbachiae

based on DNA-DNA homology studies (Vauterin et al. 1995). In that study five X

campestris pv. dieffenbachiae strains were compared with 178 strains from 33 other

pathovars ofX. campestris. The five X campestris pv. dieffenbachiae strains used,

included three Anthurium strains from Brazil and two Dieffenbachia strains from the

United States. The strains used in their study seem limited in regards to hosts of origin

and excluded the incorporation of any strains from Aglaonema, Philodendron,

Syngonium, or L~ ulitl, /, ,imit

Related Studies

The family Araceae is a large and diverse family of plants. Understanding this is

important in recognizing the need to determine the diversity that exists in the many

xanthomonads that infect and cause disease in these aroids. Traditionally, host range

pathogenicity is a good starting point when describing bacterial plant pathogens. In

recent years a number of PCR techniques have been developed and are used for

investigating species, pathovars, strains, and races. The following is a brief review of the

literature regarding host range, pathogenicity, and PCR-based phylogeny-type work.

Pathogenicity Tests

The history of Xanthomonas syngonii raises questions that as of yet have not been

answered, especially in regards to host range and pathogenicity. Dickey and Zumoff

(1987) showed that strains XA-2, XD-3, and ATCC 19320 produced symptoms on

Syngonium when misted at 108 CFU/ml. However, it was also noted that the reactions

produced by these strains were different from those produced by strains originally

isolated from Syngonium. It was also observed that xanthomonads isolated from

Syngonium were weakly pathogenic on the Dieffenbachia amoena and not at all









pathogenic on D. maculata. Also, XD-3 (from Dieffenbachia) was capable of producing

typical symptoms on Dieffenbachia yet only minor symptoms on Syngonium. The

results from their study suggest Syngonium strains cause disease in Syngonium but appear

to be less virulent when inoculated into Dieffenbachia. In addition, they concluded that

there were typical and atypical syngonii strains based on pigmentation and growth on

nutrient agar. It would appear that Dickey and Zumoff (1987) were suggesting that the

syngonium strains are the only true pathogen of Syngonium.

Similar research on the Syngonium strains was conducted by Chase et al. (1992).

In that study 149 X campestris dieffenbachiae and X c. pv. syngonii strains isolated from

a variety of aroid hosts were subjected to physiological, pathological, and fatty acid

analyses. They showed that various strains were not necessarily host-specific though

they suggested that groups of strains have overlapping hosts. It was observed when

monitoring populations in leaves infiltrated with bacterial suspensions adjusted to 104

CFU/ml that all strains (with the exception of the Syngonium strains) were capable of

multiplying one log unit regardless of host tested. The Syngonium strains only increased

in populations in Syngonium and Aglaonema. Chase et al. (1992) concluded that

differences in host specificity were not significant enough to be used as a means to

differentiate strains ofX. c. pv. dieffenbachiae from those ofX. c. pv. syngonii. It

appears that due to this ambiguity in host range among these various strains there has

been no pathovar designation for all of these strains aside from the all-encompassing

pathovar dieffenbachiae.

Efforts by Lipp et al. (1992) to characterize strains isolated from Dieffenbachia and

Syngonium employed a similar host range study. However, while the typical syngonium









strains were virulent on Syngonium and weakly virulent to avirulent on other host genera,

the atypical strains were indistinguishable from other Xcd based on bacteriological tests

(Lipp et al, 1992). They found no significant correlation between hosts of origin and host

range, and concluded that this group was extremely heterogeneous.

PCR Based Analysis

Recently PCR has been used to amplify specific regions of an organism's genome

for comparison with other organisms as a means to determine evolutionary relationships.

Several of the PCR based techniques include Repetitive Element PCR (rep-PCR),

Amplified Fragment Length Polymorphism (AFLP), and sequence comparisons of the

Hrp region and a region corresponding to the ribosomal DNA (Louws et al. 1994,

Janssen etal. 1996, Leite Jr. et al. 1995, and Hauben et al. 1997).

Rep-PCR is based on highly conserved regions of the genome. There are three

regions that have been used: Repetitive Enterobacterial Palindrome (REP), (Gilson et al.

1984, Higgins et al. 1982) Enterobacterial Repetitive Intergenic Consensus (ERIC),

(Hulton et al. 1991, Sharples and Lloyd 1990) and the BOX1A element from E. coli

(BOX) (Martin et al. 1992). It has been shown that these elements are widely distributed

in phytopathogenic bacteria including Pseudomonas and Xanthomonas and can be used

for rapid molecular characterization especially at the pathovar level (Louws et al. 1994).

Using rep-PCR Louws et al. (1994) were able to generate genomic "fingerprints"

consisting of five to 20 bands ranging in size from 100 base pairs to 5 kbp. In comparing

fingerprints from various Pseudomonas and Xanthomonas strains, they were able to

distinguish P. syringae pv. syringae from P. syringae pv. tomato as well as X oryzae pv.

oryzae from X oryzae pv. oryzicola. Using rep-PCR members from the same group they









were able to place X campestris pv. vesicatoria strains into two groups, groups A and B

(Louws et a. 1995).

In 1995 the PCR based genomic "fingerprinting" method Amplified Fragment

Length Polymorphism (AFLP) was developed. The basis of AFLP is to analyze the

whole genome. First described by Vos et al. (1995), the process begins with fragmenting

the whole genome with restriction endonucleases. Following the restriction or cutting of

the genome, the second step employs the ligation of site-specific adapters to the

restriction fragments. The adapted fragments are selectively amplified with primers

designed to complement the adapters and allow specificity through 3' selective base(s)

incorporated into the primers. This technique is useful with DNA of any origin and of

any complexity (Vos et al. 1995). It has been suggested that this DNA fingerprinting

technique is comparable in taxonomic value to that of DNA-DNA homology studies or

fatty acid analyses (Janssen et al. 1996). That study demonstrated the usefulness of

AFLP using Xanthomonas as a model. Various primer combinations were able to

differentiate closely related bacterial strains. It was also observed that the placement of

strains into groups deduced through this technique was in agreement with DNA

homology studies as well as fatty acid data (Janssen et al. 1996). In a more recent study

comparing AFLP and rep-PCR, Xanthomonas was used as the model and it was

demonstrated that both fingerprinting methods were useful in determining taxonomic

relationships (Rademaker et al. 2000).

DNA sequence analysis of genes followed by analysis for phylogenetic

relationships is useful in determining relatedness of organisms. One region that has been

examined for determining relatedness is the hrp (hypersensitive response and









pathogenicity). This region is commonly associated with the majority of plant

pathogenic bacteria and is accountable for those organisms' ability to cause disease or a

hypersensitive response. It has been suggested that by subjecting this region to PCR and

subsequent analysis it is possible to differentiate xanthomonads as well as detect the

presence ofX. c. vesicatoria on pepper and tomato seed (Leite Jr. et al. 1995). Similarly,

this technique has been used for detecting X fragariae in nurseries (Roberts et al. 1996).

That study found that the 49 strains of X fragariae shared identical restriction profiles.

Another region used for taxonomic purposes is the ribosomal DNA. In the early

1990's, a study was conducted showing the high degree of variability that exists in

ribosomal DNA restriction patterns ofX. campestris pv. dieffenbachiae (Berthier et

al. 1993). This was supported by the data stating that 53 strains were characterized by

five restriction patterns, and there were no patterns corresponding to geographic origin

but clearly to host plant origin (Berthier et al. 1993). In a later study Hauben et al. (1997)

determined that the use of rDNA signatures to differentiate Xanthomonas species was not

ideal due to the restricted variability. In that study only one strain ofX. axonopodis pv.

dieffenbachiae was used. However, a more recent study shows that rDNA analysis is

sufficient in the identification of closely related xanthomonads such as X axonopodis pv.

citri, and X axonopodis pv. aurantifolii, the causal agent of bacterial citrus canker types

A and B, respectively (Cubero and Graham 2002).














CHAPTER 3
MATERIALS AND METHODS

This chapter includes all scientific and data collecting procedures. All experiments

were conducted between May 2001 and May 2003. A majority of the experiments were

conducted at the Mid Florida Research and Education Center-Apopka, including

greenhouse studies, genomic comparisons, and storage of bacterial strains. All DNA

sequencing work and growth chamber studies were conducted at the University of Florida

in Gainesville.

Bacterial Strains Used

One hundred and eighty seven X a. pv. dieffenbachiae strains were used in this

study (Table 1). These strains were isolated over a 20-year period from ten aroid genera.

Strains were revived from cryogenic storage when needed.

Genomic Comparisons

All strains in Table 1 were subjected to Repetitive Element PCR (rep-PCR) using

BOX, ERIC, and REP primers. DNA from these strains was prepared using the

GenomicPrep Kit, (Amersham Pharmacia Biotech, Pistcataway, NJ). Instead of using a

master mix, PCR was done using RAPD Analysis Beads (Amersham). A modified

program suggested by Amersham Pharmacia Biotech was used to simulate results

obtained using a master mix. Template DNA was amplified using a PTC-100

thermocycler (M.J. Research Inc., Watertown, Mass). The thermocycler was

programmed as follows. For BOX initial denaturation at 950 for five minutes, followed

by 45 cycles denaturing at 950 for one minute and annealing at 530 for one minute. ERIC









and REP had annealing temperatures of 52 and 400, respectively. Final extension was

conducted at 720 for two minutes; upon completion the block was held at 4. PCR

products were separated on 1.5% gels, stained with ethidium bromide and photographed

under a UV transluminator with a Kodak Digital Documentation System 120 (Rochester,

NY). All gels photographed were stored as Tiff files.

Banding patterns were compared between strains using Pearson Correlation and

Unweighted Pair Group Means Analysis (UPGMA) with the BioNumerics program ver.

2.1 (Applied Maths, Kortrijk, Belgium). For strains with unique banding patterns DNA

was extracted again, PCR was redone, and the analysis was repeated. Using the

BioNumerics program, a combined comparison was made of banding patterns from all

three rep-PCR primers. Cluster cut-off values were also calculated using the

BioNumerics software in each comparison. The ATCC Xa.d Type strain 23379 was

used as a standard.

Amplified Fragment Length Polymorphism (AFLP)

Based on rep-PCR clusters, 40 strains were selected from seven distinct genetic

clusters. Due to the large number of Anthurium strains an emphasis was placed on these

as well as those representing strains from Syngonium and Dieffenbachia. These strains

were compared using AFLP. DNA was prepared as above using the kit obtained from

Amersham. Template was prepared using LI-COR (Lincoln, NE) template preparation

kit according to the provided protocol. The protocol was modified only for the selective

amplification step by following a procedure according to Janssen et al., (1996) LI-COR's

template preparation kit uses the EcoRI and Msel restriction enzymes and a two-dye

system for labeling rather than the traditional radiolabelling. An Msel primer containing

3'cytosine was obtained from Genomechanics (Alachua, FL). Following the









nomenclature provided by Janssen et al. (1996) this primer will be referred to as M02.

Using the LI-COR system, the EcoRI primers were labeled with an IR-dye. Two dyes

were obtained from LI-COR: one 700 EcoRI primer with no selective base (E00) and one

800 dye with cytosine as a selective base (E02). Another 700 dye was obtained for (E02)

as the 800 dye gave less than ideal products. Acrylamide gels were prepared using KB

plus gel matrix (LI-COR), temed and ammonium persulfate according to manufacturer's

specifications. Gels were run 4 hours at 1500 volts on a Global LI-COR IR2 System.

Images were recorded using the SAGA software (LI-COR). Tiff files were analyzed

using the BioNumerics program.

Sequence Comparisons

Two regions were chosen for sequence comparison, the HrpB (for hypersensitivity

response and pathogenicity) and the intergenic transcribed sequence (ITS). DNA for the

12 selected strains was prepared as previously mentioned. The hrp region was selected to

determine whether or not this group of organisms contained hrp genes. PCR products

were obtained using "Hrp" (RST65-RST69) primers used for amplification of a 420bp

product in X campestris pv. vesicatoria. Products obtained from strains isolated from

various hosts were subjected to restriction digests using CfoI, and HaeIII. Different

profiles were generated and observed on NuSieve 3:1 agarose gels. Based on the

variation in restriction profiles, PCR was repeated, products were cleaned using a spin

column (Qiagen, Germantown, MD) and submitted for sequencing at the University of

Florida's Interdisciplinary Center for Biotechnology Research (ICBR) sequencing lab in

Gainesville. The ITS region was not subjected to restriction digest, but was amplified

using (J13-J14) primers and sequenced in the same manner. Sequences were assembled









using the Sequencher software and alignments were performed using the European

Bioinformatics Institute's CLUSTALW (www.ebi.ac.uk/clustalw/).

Pathogenicity and Host Range

Using the 40 pre-selected strains, a limited host range was conducted using both

mist and leaf infiltration techniques. Strains used in the pathogenicity tests were grown

on Nutrient Agar (Difco) amended with 5% sucrose at 280 C for 24-48 hours prior to use.

Bacteria were harvested from the petri plates and suspended in 0.1M NaCl solution.

Bacterial suspensions were adjusted with a spectrophotometer to 1 X 108 CFU/ml with an

optical density 0.3 (A600). This suspension was applied to leaf surfaces using hand-pump

sprayers. Immediately after misting, plants were placed in plastic bags for 24 hours to

maintain high relative humidity. The inoculated plants were observed for progression of

disease weekly for two months. Plant species utilized were aglonema (Aglonema

commutatum, Schott 'Maria'), dieffenbachia (Dieffenbachia maculata (Lodd) G. Donn.,

compactta', philodendron, (Philodendron scandens oxycardium), syngonium

(Syngonium podophyllum, 'White Butterfly'), and three species of anthurium (Anthurium

crystalinum Linda Andre, 'Crystal Hope'; a complex interspecific hybrid ofA. amnicola

Dressler x A. andreaneum Linden ex Andre, 'Red Hot'; and A. andraeanum 'Hearts

Desire'). Plants were kept under identical cultural conditions in a fiberglass shadehouse

with maximum irradiance of 125 .imol s-1 m-2, temperature range of 21 to 37 C with

natural photoperiod, and high relative humidity.

For leaf infiltrations, bacterial suspensions were serially diluted to 1 X 105 CFU/ml.

The same host plant species mentioned above were utilized with the exception of only

using one anthurium cultivar ('Red Hot'). Leaf tissue, approximately 4 cm2was

infiltrated on two leaves on each of two plants of each host plant species using a 25gauge






14


hypodermic needle. Younger leaves were preferentially used. The infiltrated areas were

observed for symptom expression over a two-month period. For both methods of

inoculation a saline buffer and a strain ofX. campestris pv. campestris (ATCC 33913)

were used as negative controls. All greenhouse tests were conducted between June and

September 2002.









Table. Sources and hosts ofXanthomonas axonopodis pv. dieffenbachiae strains
utilized in this study.
Strain ID Original source ID Host Geographic origin
01-156 MREC Anthurium Florida
13 MREC Anthurium Florida
14 MREC Anthurium Florida
82 A990-5 Anthurium Hawaii
84 A844-1 Anthurium Hawaii
158 MREC Syngonium Florida
159 MREC Syngonium Florida
161 MREC Syngonium Florida
162 MREC Syngonium Florida
166 MREC Syngonium Florida
170 MREC Philodendron Florida
172 MREC Syngonium Unknown?
173 MREC Syngonium Unknown?
175 MREC Dieffenbachia Florida
176 MREC Epipremnum Florida
178 MREC Syngonium Florida
181 MREC Syngonium Florida
183 MREC Dieffenbachia Florida
187 MREC Syngonium Florida
191 MREC Syngonium Florida
192 MREC Syngonium Florida
195 GWS 2218-83 Dieffenbachia Florida
265 ICMP 9586 Philodendron Florida
268 D 150 Anthurium Hawaii
269 D 158 Anthurium Hawaii
271 D 36.12 Syngonium Hawaii
272 D 129.12 Syngonium Hawaii
326 PDD 1145-87 Anthurium Florida
328 DPI P87-2307 Anthurium Florida
376 PDD- 1399-87 Aglaonema Florida
422 PDD 21-88 Anthurium Florida
423 PDD 20-88 Anthurium Florida
430 D129.1M Syngonium Hawaii
431 D129.1D Syngonium Hawaii
451 D61.11 Anthurium Hawaii
452 D68.00 Anthurium Hawaii
453 D184.00 Aglaonema Hawaii
454 D182.00 Anthurium Hawaii
455 D183.00 Anthurium Hawaii
456 D185.00 Aglaonema Hawaii
457 D206.00 Colocasia Hawaii
458 D191.00 Epipremnum Hawaii
460 D228.00 Colocasia Hawaii









Table 1 (Continued).
Strain ID Original source ID
462 D36.10
463 D18.00
464 D139.00
465 D227.00
466 D135.00
467 D122.00
468 D206.00
469 D204.00
471 D162.00
472 D170.00
473 D17.10
474 D145.00
475 D147.00
476 D150.00
477 D145.00
478 D1.21
479 D30.00
480 D55.1
481 D110.00
482 D120.00
483 D131.00
484 D93.00
485 D94.40
487 D101.00
488 D160.00
489 D46.20
490 D52.00
491 D69.10
492 D70.00
493 D71.50
494 D38.10
495 135.00
497 D40.10
585 MREC
586 MREC
606 DPI 072-743
628 DPI 076-953
641 DPI 072-745
642 MREC
661 PDD- 1772-88
696 DPI P88-3370
697 DPI P87-2081
746 MREC
747 MREC


Host
Syngonium
Anthurium
Anthurium
Colocasia
Anthurium
Colocasia
Colocasia
Colocasia
Anthurium
Anthurium
Anthurium
Anthurium
Anthurium
Anthurium
Anthurium
Anthurium
Spathiphyllum
Anthurium
Anthurium
Anthurium
Anthurium
Spathiphyllum
Anthurium
Anthurium
Anthurium
Anthurium
Anthurium
Anthurium
Anthurium
Anthurium
Anthurium
Anthurium
Anthurium
Anthurium
Anthurium
Anthurium
Philodendron
Anthurium
Philodendron
Aglaonema
Philodendron
Dieffenbachia
Anthurium
Anthurium


Geographic origin
Hawaii
Hawaii
Hawaii
Hawaii
Hawaii
Hawaii
Hawaii
Hawaii
Hawaii
Hawaii
Hawaii
Hawaii
Hawaii
Hawaii
Hawaii
Hawaii
Hawaii
Hawaii
Hawaii
Hawaii
Hawaii
Hawaii
Hawaii
Hawaii
Hawaii
Hawaii
Hawaii
Hawaii
Hawaii
Hawaii
Hawaii
Hawaii
Hawaii
Florida
Florida
Florida
Florida
Florida
Florida
Florida
Florida
Florida
Florida
Florida









Table 1 (Continued).
Strain ID Original source ID


PDD
PDD
PDD
PDD
PDD
PDD
PDD
PDD
PDD


15017-89
1500-89
2033-89
2025-89
2050-89
2050-89
2050-89
2129-89
2129-89


757
758
764
765
766
767
768
785
786
788
790
805
807
811
813
818
830
831
834
840
841
851
868
875
979
1176
1181
1185
1186
1188
1268
1272
1277
1278
1279
1283
1341
1343
1353
1354
1390
1420
1474
1476


Host
Anthurium
Anthurium
Aglaonema
Anthurium
Anthurium
Anthurium
Anthurium
Anthurium
Anthurium
Anthurium
Anthurium
Dieffenbachia
Caladium
Aglaonema
Caladium
Colocasia
Anthurium
Anthurium
Anthurium
Syngonium
Anthurium
Philodendron
Dieffenbachia
Caladium
Cabbage
Anthurium
Philodendron
Anthurium
Anthurium
Caladium
Aglaonema
Anthurium
Anthurium
Philodendron
Philodendron
Syngonium
Syngonium
Anthurium
Anthurium
Anthurium
Epipremnum
Philodendron
Philodendron
Svngonium


Geographic origin
Florida
Florida
Florida
California
Florida
Florida
Florida
Florida
Florida
Florida
Florida
Florida
Florida
Florida
Florida
Florida
Florida
Florida
Florida
Florida
Florida
Florida
Florida
Florida
United Kingdom
Florida
Florida
Florida
Florida
Florida
Florida
California
Florida
Florida
Florida
Florida
Florida
Florida
Florida
Florida
Florida
Florida
Florida
Florida


MREC
PDD 2444-89
PDD 2507A-89
MREC
MREC
MREC
DPI- 89-3142-1
PDD- 2912-89
PDD 2927-89
MREC
MREC
MREC
PDD- 3523-89
DPI- P89-4119-1
DPI- P89-4526-10
ATCC33913
MREC
MREC
MREC
MREC
MREC
MREC
Cooksey
DPI- P90-5399
DPI- P90-3705
DPI- P90-3919
DPI- P90-3731-1
PDD- 3466-91
DPI- P91-2557
MREC
MREC
DPI- P91-3902
MREC
MREC
PDD 359A-92









Table 1 (Continued).
Strain ID Original source ID
1477 PDD- 359B-92
1559 MREC
1564 MREC
1567 MREC
1568 MREC
1610 MREC
1617 M 101
1618 M102
1619 M 103
1620 M 105
1621 M 106
1622 M 110
1623 M 113
1624 M 114
1625 M115
1626 M 116
1627 M 117
1628 M 123
1629 M 124
1630 M 126
1631 M127
1671 MREC
1672 MREC
1673 MREC
1674 MREC
1688 Z 27
1689 Z 23
1694 MREC
1697 MREC
1698 MREC
1699 MREC
1701 MREC
1702 MREC
1703 MREC
1704 MREC
1705 MREC
1706 MREC
1707 MREC
1708 MREC
1709 MREC
1711 MREC
1713 MREC
1718 MREC
1750 LMG7399z


Host
Syngonium
Aglaonema
Aglaonema
Anthurium
Anthurium
Aglaonema
















Anthurium
Anthurium
Anthurium

Anthurium
Anthurium
Difffenbacthia











Epipremnum
Dieffenbacthia











Anthurium
Caladium
Syngonium
Dieffenbachia
Dieffenbachia






Syngonium
Aglaonema
Dieffenbachia
Dieffenbachia
Anthurium
Anthurium
Anthurium
Syngonium


Dieffenbachia







Syngonium



Dieffenbachia
Dieffenbachia



Dieffenbachia


Geographic origin
Florida
Florida
Florida
Florida
Florida
Florida
Florida
Florida
Florida
Florida
Florida
Florida
Florida
Florida
Florida
Florida
Florida
Florida
Florida
Florida
Florida
Florida
Florida
Florida
Florida
Puerto Rico
Puerto Rico
Florida
Florida
Florida
Guatemala
Florida
Florida
Florida
Florida
Florida
Florida
Florida
Florida
Florida
Florida
Florida
Florida
USA









Table 1 (Continued).
Strain ID Original source ID Host Geographic origin
1751 LMG695 Anthurium Brazil
1752 LMG7400 Dieffenbachia USA
1753 LMG7484 Anthurium Brazil
1754 LMG8664 Anthurium Brazil
Y Strains were obtained from the following laboratories: (A or D) A. Alvarez,
Department of Plant Pathology, University of Hawaii at Manoa, Honolulu, HI, 96822.
(ATCC) American Type Culture Collection, Manassas, VA 20108. (BCCM/LMG)
Belgian Coordinated Collections of Micro-organisms, Brussels, Belgium. (ICMP)
International Collection of Micro-Organisms from Plants, Mt Albert, Auckland, New
Zealand. (M) K. Pernezny, Everglades Research and Education Center, Belle Glade, FL
33430. (MREC) Mid-Florida Research and Education Center, University of Florida,
Apopka, FL 32703. D. Cooksey, University of California Riverside. (DPI) J. Miller,
Division of Plant Industry, Florida Department of Agriculture and Consumer Services,
Gainesville, FL 32602. (GWS) G. Simone, Department of Plant Pathology, University of
Florida, Gainesville, FL 32611. (NZTCC) New Zealand Type Culture Collection. (PDD)
D. Brunk, Plant Disease Diagnostics, Inc., Apopka, FL 32703. (NCPPB) National
Collection of Plant Pathogenic Bacteria, York, UK. (Z) M. Zapata, University of Puerto
Rico, Mayasuez, PR.
z Pathovar reference strain for X a pv. dieffenbachiae.














CHAPTER 4
RESULTS

Genomic Comparisons

Rep-PCR

The profiles for BOX, REP, and ERIC were combined and used in computer

analysis. The results of this analysis yielded six distinct clusters, shown in Figure 1.

Cluster III contained 81 strains and was comprised primarily of strains originally isolated

from Anthurium spp. This cluster consists of two sub-clusters of 37 and 42 strains. The

two sub-clusters represent those strains isolated from Anthurium spp. with 86% and 93%

of the strains originating from Anthurium spp., respectively. There were a couple of

outliers from this cluster containing two strains isolated from Syngonium spp. Cluster II

contained 33 strains and was very heterogeneous in regards to host of origin, with nine of

the ten host genera being represented. Cluster I consisted of 15 strains sharing 75%

similarity, which was isolated from X iIneil,,init spp. Three outliers representing three

strains isolated from Aglaonema, Dieffenbachia, and Epiprenum also were grouped

between cluster II and cluster III. Strains originally isolated exclusively from Syngonium

spp. comprised cluster IV containing 19 strains. The fifth cluster was relatively small and

mixed in regards to host of origin. Cluster VI was composed of 15 strains in which ten or

67% of the strains were isolated from Philodendron spp..

Amplified Fragment Length Polymorphism

Forty representative strains based on the results from the rep-PCR with an

emphasis on strains originating from Anthurium, Dieffenbachia, and Syngonium spp.









were used in AFLP analysis. Strains isolated from Aglaonema, Caladium, Colocasia,

and Philodendron spp. were also represented in AFLP analysis. Results using the M02

and EOO primer combination generated clusters for 38 strains and are shown in Figure 2.

The cluster analysis yielded similar results as that of the rep-PCR technique. There were

five distinct clusters. Cluster I contained strains showing 53% similarity and contained

both type strains (LMG 7399, 7400) originating from Dieffenbachia spp. as well as two

of the three strains isolated from Aglaonema spp. There was also a strain isolated from

Caladium spp. within this cluster. Cluster II consisted of nine strains showing 66%

similarity, of which six were strains from Anthurium. Two representing the Anthurium

type strains (LMG 7484, 8664), and three from Dieffenbachia spp. Cluster III contained

six strains all of which were isolated from Anthurium spp. one of which represented the

Anthurium type strain (LMG 695). Cluster IV contained six strains all of which were

isolated from Syngonium spp. and shared 54% similarity. Between clusters IV and V,

there was an outlier, X-457, isolated from Colocasia spp. Cluster V had slightly lower

similarity of 44%, and contained six strains isolated from four different host genera.

Three of these strains were isolated from Dieffenbachia and the other three representing

strains isolated from Anthurium, Syngonium, and Philodendron spp. Another outlier, X-

271, was below cluster V, isolated from Syngonium spp. Strain X-805 isolated from

Dieffenbachia spp was most distantly related to all other strains.

DNA Sequencing

Sequence comparisons were done using the European Bioinformatics Internet

CLUSTALW program. The results for the ribosomal DNA placed all strains tested

within two groups with which one group was composed of strains originating from

Syngonium. Two strains, X-430 and X-1674, grouped together but were clearly different









from other strains. Most of the variation between strains was observed in the ITS region.

The Syngonium strains varied primarily in positions in between the 16S and 23 S known

as the ITS region, and were two nucleotides shorter in overall length. The hrp sequences

revealed similar results placing Syngonium strains in one group distinct from others.

Host Range Tests

The pathogenicity tests proved the heterogeneity of X axonopodis pv.

dieffenbachiae with strains varying immensely in their ability to produce symptoms on

the hosts tested. Host range tests did not show distinct relationships between strains

based on host of origin as was predicted by preliminary rep-PCR data. Although many of

the strains isolated from their host of origin showed pathogenicity on other aroids, there

were some important trends observed. Results from representative strains originally

isolated from Dieffenbachia, Anthurium, and Syngonium are presented in Figures 3,4, and

5 respectively.

Dieffenbachia Strains

In pathogenicity tests with ten strains originally isolated from Dieffenbachia

(Figure 3), disease on Dieffenbachia was 60% and 72% when misted at 108 cfu/ml and

infiltrated at 105 cfu/ml, respectively. Four strains failed to produce symptoms on

Dieffenbachia maculata after mist inoculations (X-183, X-697, X-805, and LMG 7399).

Whereas they produced some symptoms on D. maculata in the infiltration inoculations,

X-805 failed to produce symptoms on any of the hosts tested. In addition to causing

disease in D. maculata, a number of these Dieffenbachia strains produced symptoms on

the Anthurium spp. in both tests. Of the strains tested, X-1709 and X-1718 appeared the

most aggressive towards Anthurium even resulting in systemic necrosis. Strains

producing symptoms in Anthurium also produced symptoms on Aglaonema commutatum.









Only two strains (X-1703 and LMG 7400) produced symptoms on Syngonium

podophyllum and on all hosts tested with the exception of Philodendron scandens

oxycardium. Only X-1718 consistently produced symptoms on the Philodendron spp.

tested.

Anthurium Strains

Figure 4 illustrates the results obtained from 12 strains originally isolated from

Anthurium. Anthurium strains produced symptoms in a similar manner to that of the

Dieffenbachia strains. A majority of the strains (greater than 70%) produced symptoms

on Dieffenbachia whether infiltrated or misted. Symptom production was greater in D.

maculata than that of any of the Anthurium cultivars tested. Nine of the twelve strains

reliably produced symptoms in misting tests. The exceptions (X-14, X-84, X-1705) failed

to produce symptoms on Anthurium in either test and failed to produce consistent

symptoms in any of the hosts tested. It was observed during the infiltration tests that leaf

age plays a role in susceptibility particularly when working with the cultivar Red Hot.

This may account for the low number of strains producing symptoms when using the

infiltration technique. The results for the misting yielded 50%, 56%, and 62% disease on

the cultivars 'Red Hot', 'Crystal Hope', 'Heart's Desire' respectively. The strains

producing symptoms in Anthurium spp. did not consistently produce symptoms in A.

commutatum as was observed with the strains isolated from Dieffenbachia. None of the

strains originating from Anthurium produced any symptoms on S. podophyllum nor the

Philodendron spp. tested. This exclusiveness in pathogenicity is noteworthy.

Syngonium Strains

Results from strains isolated from Syngonium are shown in Figure 5. The

Syngonium strain inoculations appeared considerably different from those with strains









isolated from Anthurium or Dieffenbachia. Of these strains 25% produced symptoms

when misted and 68% produced symptoms on D. maculata when infiltrated. Only one of

these strains (X-191) produced symptoms on the 'Red Hot' and 'Heart's Desire' cultivars

of Anthurium and failed to produce symptoms on 'Red Hot' in the infiltration

inoculations. Syngonium appears to be the most susceptible host for these strains. Three

(X-159, X-187, X-271) of the ten strains failed to produce symptoms on S. podophyllum

in the misting test. Strains X-159 and X-187 expressed partial symptoms when

infiltrated. Strain X-271 failed to produce symptoms on any of the hosts tested. The

Aglaonema tests were similar to those of the Anthurium isolates except, that fewer strains

induced symptoms. In both the infiltration and mist inoculations no Syngonium strains

produced symptoms on Anthurium spp.















15 Xanthosoma
strains (100%)



35 strains (9 genera)


3 strains


40 strains
(35/40 Anthurium)



42 strains
(39/42 Anthurium)


2 strains


19 strains
(100% Syngonium)

v 12 strains (7 genera)


15 strains
(67% Philodendron)


Figure 1. Cluster analysis of 183 Xa.d strains isolated from ten host genera over a 20-
year period based on rep-PCR using BOX, ERIC and REP primers. Banding
patterns were compared between strains using Pearson Correlation and
Unweighted Pair Group Means Analysis (UPGMA) with the BioNumerics
program ver. 2.1















10 30
I I


50 70 90 100
I I I


Sinilarity coefficient


6 strains (3 genera)


9 strains
(6Anthriwn 31ieffenbachia)





6 strains (1000/oAnthwimn)



6 strains (100/oSyngonimn)


X457 (Cdoaisia)


6 strains (4 genera)

X271 (Syngoniun)


2 strains (1 ieffaebachia 1 Syngonim)



X805 (ieffenbachia)


Figure 2. Cluster analysis of 38Xa.d strains isolated from six host genera over a 20-year
period based on AFLP using M02, and EOO primers combinations. Banding
patterns were compared between strains using Pearson Correlation and
Unweighted Pair Group Means Analysis (UPGMA) with the BioNumerics
program ver. 2.1











10 30 50 70 90 100

Similarity coefficient


6 strains (3 genera)


9 strains
(7 Anthurium 2 Dieffenbachia)





5 strains (100% Anthurium)



6 strains (100% Syngonium)





8 strains (4 genera)

X 271 Syngonium

3 strains (1 Dieffenbachia 1 Syngonium
1 Philodendron )



X805


Figure 3. Cluster analysis of 39Xa.d strains isolated from six host genera over a 20-year
period based on AFLP using M02, and E02 primers combinations. Banding
patterns were compared between strains using Pearson Correlation and
Unweighted Pair Group Means Analysis (UPGMA) with the BioNumerics
program ver. 2.1

























co E co a
SE 2
0 'E o
0 -
'S o' o
: a_


Figure 4. Percent ofX. a. dieffenbachiae strains isolated from Dieffenbachia spp.
producing disease when inoculated into various aroid hosts. Blue bars
represent mist inoculations of bacterial suspension adjusted to 1.0 X 108
CFU/ml. Red bars represent infiltration inoculations using bacterial
suspensions adjusted to 1.0 X 105 CFU/ml. Results based on two mistings
and four infiltrations.


- w
CI)
01










100
90
80
70
60
50
40 1
30
20



E E &
E 0 E E 0
Eo E" Eo o o
T- D L L D T
L M Ucj) 0





Figure 5. Percent of X a. dieffenbachiae strains isolated from Anthurium spp. producing
disease when inoculated into various aroid hosts. Blue bars represent mist
inoculations of bacterial suspension adjusted to 1.0 X 108 CFU/ml. Red bars
represent infiltration inoculations using bacterial suspensions adjusted to 1.0
X 105 CFU/ml. Results based on two mistings and four infiltrations.










100
90
80
70
60
50
40
30
20
10

o E o c
E E,- E = E
0E (D 0
T 0

( < <- < (D <(






Figure 6. Percent of X a. dieffenbachiae strains isolated from Syngonium spp.
producing disease when inoculated into various aroid hosts. Blue bars
represent mist inoculations of bacterial suspension adjusted to 1.0 X 108
CFU/ml. Red bars represent infiltration inoculations using bacterial
suspensions adjusted to 1.0 X 105 CFU/ml Results based on two mistings and
four infiltrations.














CHAPTER 5
DISCUSSION

In this study I addressed the variation that exists within Xanthomonas affecting

aroid hosts. Using rep-PCR it was observed that six genetic clusters were generated by

the 183 strains tested. Four of these clusters appeared to cluster based on host of origin.

Clusters clearly represented strains originating Philodendron, lnll/nhUl via, Anthurium,

and Syngonium. These results suggest that each of the four clusters may represent a

different pathovars as previously described by Louws et al. (1994).

When representative strains were subjected to AFLP, similar results were observed

to that of the rep-PCR. Distinct clusters representing strains virulent on Anthurium and

Syngonium were observed. Genetic differences between these two clusters and between

the others provide considerable evidence for the variation within X a. dieffenbachiae,

and particularly for the Syngonium strains. These results when combined with the rep-

PCR data may reveal that these are significant species since the techniques provide

information close to the resolution of DNA-DNA hybridization (Rademaker et al. 2000).

Upon phylogenetic analysis of rDNA, the same differentiation was observed

between X a. dieffenbachiae and those strains isolated from Syngonium. A great degree

of variability had been observed in restriction patterns within pathovar dieffenbachiae

(Berthier et al. 1993). These results also elicited some minor differences within strains

from Anthurium and Dieffenbachia. However, quite significant variation was observed

between these strains and those from Syngonium, particularly in the ITS region. The

same variation was found upon investigating the hrpB region. Individually or in









combination these two techniques were useful in differentiating X a. dieffenbachia from

pathovar syngonii.

Many of the strains tested exhibited overlapping pathogenicity on host plants,

which was observed in other studies (Lipp et al. 1992, Chase et al. 1992). This was

especially the case when looking at the data from those strains isolated from Anthurium

and Dieffenbachia. Interestingly, the Syngonium strains did not exhibit pathogenicity in a

manner similar to the X a. dieffenbachiae strains. Strains from Philodendron and

Atllhei\,n,,ia were found in distinct genetic clusters based on rep-PCR data and might

also exhibit specialization in pathogenicity.

The pathovar, syngonii, was described earlier based on NaCl tolerance, pH optima,

gelatin hydrolysis, and growth on SX media (Dickey and Zumoff 1987). However, there

were many reports on the inadequacy of the tests used. Hodge et al. (1990) concluded

that there was not enough variation in fatty acid profiles to differentiate X a.

dieffenbachiae from X c. syngonii, and strains from Syngonium and should be included in

X a. dieffenbachiae. In an attempt to differentiate the two with the use of monoclonal

antibodies, Lipp et al. (1992) came to a similar conclusion stating that typical Syngonium

strains are most similar to Anthurium strains while the atypical Syngonium strains are

more similar to those strains isolated from Dieffenbachia. Even when using

physiological, pathological and fatty acid analysis, Chase et al. (1992) concluded that

none of these tests were useful in differentiating X a. dieffenbachiae from X c. syngonii.

The Syngonium cluster (cluster V in figures 4 and 5) represents fastidious xanthomonads,

which are specialized in pathogenicity in Syngonium. The pathovar, syngonii, previously









described by Dickey and Zurmoff would be an appropriate designation for these strains

(1987).

This work has provided genetic evidence for the pathovar designation syngonii

when referring to those strains specialized in pathogenicity on Syngonium spp. The

genetic data is convincingly reinforced by host range tests. These results illustrate the

usefulness of molecular tools in bacterial taxonomy. In order to be absolute in this matter

it may still be necessary to conduct DNA-DNA hybridization experiments with additional

strains representing pathovar syngonii. The strains comprising Xa.d. used by Vauterin et

al. (1995) proved to be very similar upon rep-PCR and AFLP analysis though they varied

in their pathogenicity. None of these strains were representative of those strains

representing pathovar syngonii and further justifies the need for them to be included in a

DNA-DNA homology study.















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BIOGRAPHICAL SKETCH

Ryan Donahoo is a Florida native with a vested interest in the tropical foliage

industry. He received his B.S. in plant science-plant pathology with a minor in plant

molecular and cellular biology from the University of Florida in 2001. While completing

this undergraduate degree, Mr. Donahoo worked with various microorganisms in

laboratory research studies. This experience fostered an interest in bacterial pathogens,

which was expanded upon in this study. Mr. Donahoo plans to continue his work in plant

pathology and microbiology in a professional research setting.