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Effect of Oxygen on the Growth of Xylella fastidiosa

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PAGE 1

1 EFFECT OF OXYGEN ON THE GROWTH OF Xylella fastidiosa By ANTHONY D. SHRINER 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 2007

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2 Copyright 2007 by Anthony D. Shriner

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3 To every stray soul who made a wrong turn in life and struggled to make things right.

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4 ACKNOWLEDGMENTS I wish to give special thanks to Trisha Sp ears, Fred Spears, Robert Reeves and Jane Reeves, for having faith in me when I doubted. I also thank my friends Dean Paini, Michelle Stuckey, Tobin Northfield, Enoch Osekre, Francis Tsigbey, Susan Bambo, and Towanga Katsvairo for putting up with the whole manic process that is graduate school I also thank some anonymous people for allowing me to have this chance to develop my professional skills.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS...............................................................................................................4 LIST OF TABLES................................................................................................................. ..........7 LIST OF FIGURES................................................................................................................ .........8 ABSTRACT....................................................................................................................... ..............9 CHAPTER 1 LITERATURE REVIEW.......................................................................................................11 The Host Environment........................................................................................................... .13 The Host Environment with Respect to Xylella fastidiosa .....................................................14 Oxygen Levels in Xylem Fluid...............................................................................................15 General Overview of Electron Transport...............................................................................18 Xf s Electron Transport System..............................................................................................19 Xf Use of Oxygen as Terminal Oxidase.................................................................................19 Fermentation................................................................................................................... ........20 The Added Influence of the Disulfide Bond Forming System...............................................21 Physiological Modifications Due to Oxygen Availability......................................................22 Bacterial Attachment and Appendages...................................................................................24 2 EFFECT OF OXYGEN ON THE GROWTH AND BIOFILM FORMATION OF Xylella fasdtidiosa ..................................................................................................................28 Introduction................................................................................................................... ..........28 Materials and Methods.......................................................................................................... .30 Bacteria Used.................................................................................................................. .30 Cultivation Protocol.........................................................................................................30 PW+ Media, Broth and Solid...........................................................................................31 CHARDS medium...........................................................................................................32 XDM2-PR meduim.........................................................................................................32 The BCYE Agar Plates....................................................................................................32 Grams Staining...............................................................................................................33 PCR Protocol...................................................................................................................33 Deeps.......................................................................................................................... .....34 Stabs.......................................................................................................................... ......34 Oxygen Levels.................................................................................................................35 Growth Conditions..........................................................................................................35 Optical Density (OD)......................................................................................................36 Biofilm Qualification.......................................................................................................36 Media Analysis................................................................................................................36 Statistical Analysis..........................................................................................................37

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6 Results........................................................................................................................ .............38 Agar Deeps..................................................................................................................... .38 Experimental Conditions.................................................................................................38 Xylella vs. an Obligate Aerobe and a Facultative Anaerobe...........................................39 Xylella vs. Xanthomonas .................................................................................................39 Defined Media Growth....................................................................................................40 Organic Acid Analysis....................................................................................................40 Discussion..................................................................................................................... ..........40 3 MEDIA COMPONENT EFFECTS ON GROW TH AND BIOFILM FORMATION OF Xylella fastidiosa ....................................................................................................................57 Introduction................................................................................................................... ..........57 Materials and Methods.......................................................................................................... .59 Bacteria Used.................................................................................................................. .59 Cultivation Protocol.........................................................................................................59 PW+ Media, Broth and Solid...........................................................................................60 The BCYE (Buffered Charcoal Yeast Extract) Agar Plates............................................60 CHARDS Medium..........................................................................................................61 XDM2 and XDM2-PR Media.........................................................................................61 Grams Staining...............................................................................................................62 PCR Protocol...................................................................................................................62 Growth Conditions..........................................................................................................63 Optical Density (OD)......................................................................................................64 Biofilm Qualification.......................................................................................................64 Media Analysis................................................................................................................64 Statistical Analysis..........................................................................................................66 Results........................................................................................................................ .............66 Discussion..................................................................................................................... ..........67 LIST OF REFERENCES............................................................................................................. ..80 BIOGRAPHICAL SKETCH.........................................................................................................86

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7 LIST OF TABLES Table page 1-1 Oxygen concentrations f ound in various xylem fluids...........................................................27 2-1. Concentrations of oxygen found in various xylem fluids......................................................44 2-2. Organic acid profiles in the CHARDS medi a after 15 days..................................................54 2-3. Organic acid profiles in th e XDM2-PR media after 15 days...............................................55 3-1. A Phenol red series experiment done in the XDM2 media using Temecula......................71 3-2. Effect of phenol red on the grow th and behavior on three strains of Xylella fastidiosa .......72 3-3. Amino acid analysis CHARDS af ter 15 days of bacterial growth........................................75 3-4. Amino acid analysis CDM2-PR af ter 15 days of bacterial growth.......................................76 3-5. Amino acids used in the XDM2-PR and CHARDS..............................................................77 3-6. CHARDS constituents in mM amounts.................................................................................78 3-7. XDM2-PR constituents in mM amounts...............................................................................79

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8 LIST OF FIGURES Figure page 2-1. Growth of Xylella fastidiosa in agar deeps to determine oxygen limitations........................45 2-2. Dissolved oxygen levels detected in dH2O and CHARDS media as a function of treatment levels and over time...........................................................................................47 2-3. Comparison of Xylella fastidiosa cultivated under reduced oxygen levels...........................48 2-4. Xylella fastidiosa growth in PW+ for 15 days unde r aerobic and hypoxic conditions. Each point is the average of three replicates......................................................................50 2-5. Xanthomonas campestris pv. campestris growth in PW+ for 5 days under aerobic and hypoxic conditions. Each point is th e average of three replicates....................................51 2-6. Growth and performance of Pierces disease strain Te mecula in the CHARDS media under two gas treatments...................................................................................................52 2-7. Growth and performance of Pierces disease strain Te mecula in the XDM2-PR media under two gas treatments...................................................................................................53 3-1. Growth of Xylella fastidiosa Temecula using variations of CHARDS based on XDM2-PR media composition..........................................................................................73

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9 Abstract of Thesis Presen ted to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science EFFECT OF OXYGEN ON THE GROWTH OF Xylella fastidiosa By Anthony D. Shriner May 2007 Chair: Peter C. Andersen Major Department: Horticultural Sciences Xylella fastidiosa, a bacterial plant pathogen, is the causative agent of many water-stress type diseases, such as almond leaf scorch, oak le af scorch, and Pierces disease of grapes. The disease symptoms are putatively due to blocking of the transpiration st ream by forming biofilms (bacteria and exopolysaccharide th ree-dimensional structures) and/ or inducing the formation of tylosis, a plant-generated plug that protects th e plant from cavitation events. The dissolved oxygen levels in the xylem environment duri ng the growing season can reach hypoxic levels (range, 20 to 60 mol L-1). In 1987 Wells et al. reported that Xylella fastidiosa was an obligate aerobe and did not grow under an anoxic enviro nment, which is similar to the growth Xanthomonas campestris Xylella s closest genetic relative. In a growth comparison of three Xylella strains, an obligate aerobe, and a facultative an aerobe using various levels of oxygen for 21 to 0%, the pattern of growth for Xylella closely resembled that of the facultative anaerobe and not the obligate aerobe We showed that Xylella fastidiosa is capable of growing in an anoxic environment in nutrient broth as well as in two defined media, C HARDS and XDM2-PR, in which Xylella planktonic population and al so biofilm formation incr eased more in the anoxic treatment in the XDM2-PR medium In addition to the growth of Xylella under hypoxic

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10 conditions, the bacterium produces a different organic acid prof ile (most likely fermentation) which indicates that they are capable of alternative means of energy production.

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11 CHAPTER 1 LITERATURE REVIEW Xylella fastidiosa ( Xf ) is a gram-negative, rod shaped bacterial plant pathogen, which belongs to the -proteobacteria, such as Escherichia coli and whose closest genetic relation is another plant pathogen, the xanthomonads (Simpson et al. 2000). Xf resides within the xylem vessels of many plant species and in the foregut of leafhoppers. Xf is considered to be xylemlimited, and transmission from host to host is accomplished by sharpshooters (Hemiptera: Cicadillidae), which feed on xylem fluid. Pierce s disease of grapevine, phony peach disease, plum leaf scorch, and citrus variegated ch lorosis are a few of the diseases caused by Xf Plant symptoms are thought to be mainly caused by the occlusion of xylem vessels. Xylem dysfunction is thought to be cause d by bacterial aggregation and biofilm formation (Purcell & Hopkins, 1996), an adhered, organized community of bacteria and their polymeric matrix which encapsulates them (Costerton et al. 1999). The bacteria may also cau se an increase formation of tyloses, a plug in the xylem element create d by the xylem parenchyma cells (Esau, 1948). Xf is predominantly found in the continental Amer icas, as far north as Canada and south to Argentina. The only location outside of the cont inent is Taiwan, which has a Pear Leaf Scorch pathovar (anonymous, 2004). The host range of Xf is extremely broad and diverse, which includes many monocots and angiosperms. Gymnos perms are the largest gr oup of plants without any report of disease or Xf presence (Purcell, A. H., The Xylella fastidiosa website, http://www.cnr.berk eley.edu/xylella/ December, 2005). Although many endemic host plants contain Xf in most cases it resides in a benign presence, in that not all plants that serve as a host to the bacteria will develop the disease. Within Vitis some species are highly susceptible and others are tolerant (Mollenhaue r & Hopkins, 1976). Other bacteria l host-plants may not develop the disease or severe symptoms until they suffer a stress situation, such as Virginia creeper

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12 (McElrone et al. 1999; McElrone et al. 2003). In such cases it is be lieved that the synergistic effect of bacterial inf ection and the negative effects of wa ter stress conditions lead to the development or progression of the disease. The underlying basis of host plant resistance is unknown and may vary with plant species. Many plant species known to harbor Xf (Purcell, A. H., The Xylella fastidiosa website, http://www.cnr.berk eley.edu/xylella/ December, 2005) and many endemic, highly-resistant species that act as natural reser voirs for the bacterium. This is a problem for the management for these diseases because there is no reasonable way to remove the pathogen from the environment. There are no acceptable treatments that can cure pl ants of the disease, however some antibiotics will cause a reduction in disease symptoms (Hop kins, 1989). The only feasible action is to remove and destroy the infected host. An exampl e of this control techni que is found in Brazil, where citrus variegated chlorosis caused by a strain of Xf is controlled by is pruning the infected tissue or removing the infected trees. This is al so a method used to control Pierces disease and other Xf diseases. The proactive method for disease prevention, in California, is the application of pesticides to kill the vector thereby inhibiting the spread of the bacteria from an infected (resistant) host to a more susceptible host. Th is is not practical in the southeast because too many sprays are required. Several Xf pathovars can induce Pierces diseas e (PD) (Purcell & Hopkins, 1996). Symptoms for PD include the formation of chloro tic and necrotic leaf margins that develop over the whole leaf, water stress, stunting and root di e back, and vine death. Other symptoms include the formation of green islands (Krivanek et al. 2005), which may be attributed to the lack of cork development (Esau, 1948), and the developmen t of match sticks, a petiole which remains

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13 attached to the cane af ter the leaf has been sheared off (Krivanek et al. 2005). To date there are no associated bacterial toxins associ ated with disease symptoms (Goodwin et al. 1988). The Host Environment The xylem vessels are specialized cells which ha ve been killed in an apoptotic fashion, leaving behind a system connected hollow cells wh ich allows for the transpiration of water and nutrients from the root zone to the photosyntheti c and growing tissues of the plant. The xylem cells have been specially modified in several wa ys. One modification is strengthening them to withstand the negative tension of the transpiration stream by lignification, a process in which phenylpropanoids, called monolignols, are modified by peroxidases to form a support for the cell walls (Christensen et al. 1998). Another modification of xylem tissue is a coating of cellulose, an insoluble car bohydrate, (cellulose is a glucose polymer bonded via a 1-4 epoxy bond and starch is glucose polymer with an 1-4 epoxy bond), which is intertwined with the lignin to create a hydrop hobic barrier on the inside of the xylem elements. The formation of pits and perforated ends, which are other modifications of xylem cells, allow for the lateral and longitudinal movement, respectively, of solute through the plant. The pits are specialized holes in the vessel which have a torus, gate, in the middle attached to tethers that allows solute to travel la terally through the plant. Pit memb ranes are not permeable to large (>25 nm) molecule, including bacteria. In the case of a cavitation event, the sudden formation of an air pocket within the transpiration stream is contained and does not a ffect the rest of the transpiration stream. The transpiration stream (xylem fluid) runs from the roots to the apical meristem transporting nutrients collected from the root zone to active growing and photosynthesis portions of the plant. Xylem fluid contai ns amino acids, and organic acids, such as citrate, succinate and lactate (Andersen & Brodbeck, 1989; Andersen & Brodbeck, 1991; Andersen et al. 1992;

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14 Andersen et al. 2005), glutathione (Herschbach & Rennenberg, 2001), a tripeptide made of glycine, cysteine and glutamine as well as i norganic ions (Andersen et al 2005). The total osmolarity of xylem fluid, combining the enti re amount of compounds and molecules, is ~20 mM with water making up the balance of th e volume, 95% (Andersen & Brodbeck, 1991). Glutamine is the compound of highest concentration in Vitis xylem fluid (Andersen & Brodbeck, 1989; Andersen & Brodbeck, 1991; Andersen et al. 1992; Andersen et al. 2005), and is thought to be a transport molecule to deliver nitrogen to the photosynthetic tissue. Sulfur compounds are the most limited within xylem fluid w ith cysteine comprising 8-27 M, for Vitis (Andersen et al. 2005), and 0.005-2.6 M for many trees (Herschbach & Rennenberg, 2001), methionine at 5-12 M for Vitis (Andersen et al. 2005), and 0-39 M glutathione at 0.002-31.2 M and SO4 at 25 4697 M in deciduous trees (Herschbach & Rennenberg, 2001). The Host Environment with Respect to Xylella fastidiosa The low nutrient concentrati ons found in xylem fluid probably caused some unusual adaptations in Xf in order for it to survive. Under lower nutrient conditions Xf has been shown to produce more biofilm. The conve rse to this occurs when Xf is grown in enrichment media; e. g. PW+ and then there is a dramatic reduction in the amount of biofilm produced compared to Xf grown in a defined media with re stricted nutrient levels (Leite et al. 2004). The formation of a biofilm could also allow protec tion of the bacteria population from defensive responses of the plant such as oxidative bursts (Wojtaszek, 1997) and the production of antibiotics (Andersen et al. 2004; Ishida et al. 2004). The nutrient requirements for the bacterium appe ar to be minimal, (i.e., a few amino acids and ferric pyrophosphate) (Cha ng & Donaldson, 2000; Leite et al. 2004; Lemos et al. 2003). A constituent for many of the media used to grow Xf is glutamine (Campanharo et al. 2003; Chang & Donaldson, 2000; Leite et al. 2004; Lemos et al. 2003). It has the highest concentration of

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15 any of the organic molecules in xylem fl uid (Andersen & Brodbeck, 1989; Andersen & Brodbeck, 1991; Andersen et al. 1992; Andersen et al. 2005) and is thought to be the primary carbon and nitrogen source for Xf Sulfur is an essential element for all organism s and is critical for st ructure and function of many proteins of the plant and bacteria. The re quirement for sulfur in the bacterias gene products begins with methionine, disulfide bond s, a structural bond formation which requires two cysteines, as well as many protei ns which contain an iron sulfur cluster as part of their active sites. Another sulfur compound is glutathione a tripeptide consisting of glycine-cysteineglutamine, which is thought of as an antioxidant compound by functioning as a co-factor for some enzymes and acting as a buffer to maintain key sulfur residues. Glutathione has many functions in cellular activity. In red blood cells the free sulfhydr yl group in reduced glutathione serves as a buffer to maintain an unbound cystei ne, which is used for binding oxygen. The class I type ribonuclotide reductases, an o xygen-dependent enzyme which is found in Xf also utilize glutathione in the reduction of ribose to deoxyribose elements for the synthesis of DNA (Jordan & Reichard, 1998). Glutathione is also utilized for disulfide bond maintena nce in the cytoplasm, either for the formation of cytoplasmic prot eins or breakage for transporting across the membrane (Ritz & Beckwith, 2001). All of these situations ar e possible and many are probably true for Xf according to the published genomes fo r the CVC and PD pathovar (Kanehisa Laboratories, Kyoto Encyclope dia of Genes and Genomics, www.kegg.com December, 2005). Oxygen Levels in Xylem Fluid The levels of oxygen found in xylem fluid ha ve been shown to range from atmospheric levels to almost anoxic, 230 mol O2 L-1 (Gansert et al. 2001) to less than 2.88 mol O2 L-1 (Eklund, 2000). The maximum air-satur ation in water is 288 mol O2 L-1 at 20 C, (also denoted as 100% air-saturated water, 21% oxygen and 9.2 ppm (g/ml) dissolved oxygen in water).

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16 Across a radial section of a stem there is a fluc tuation with a rapid drop from the outside, 21% to the cambium, about 7%, and a slight rise, 15%, in the pith (Dongen et al. 2003). Inside the xylem system three physical factors influence the ca pacity of a liquid, or xylem fluid, to take up and retain dissolved oxygen; they are temperature, osmolarity and pressure. As a liquid rises in temperature the capacity to retain oxygen lowers. The higher the solute concentration of a solution also inhibits th e capacity to retain oxygen, for xylem fluid of Vitis this is around 20 mM (Andersen & Brodbeck, 1991) and could be considered similar to fresh water. A decrease in pressure will also decrea se the amount of dissolved oxygen in the system, and the tension in the transpiration stream of Vitis can be as low as .6 MPa (Andersen et al. 1992), approximately psi. Most of the oxygen in xylem fluid travels by means of the transpiration stream (Dongen et al. 2004; Gansert et al. 2001; Gansert, 2003; Mancuso & Ma rras, 2003). Entry into the transpiration stream is a difficult arrangement wh ere there are some obstacles to overcome such as an adequate supply of oxygenate d water, at the root zone, whic h needs to be accessible to the newly dividing and elongating sect ion of the root tip. Once the root matures and suberization occurs around the stele the influx of oxygen dr ops drastically (Mancuso & Boselli, 2002). During two extreme events, flooding and drought (because the plant inhibits the osmosis of water out of the plant into th e arid environment), the dissolv ed oxygen levels around the rootzone become anoxic which can inhibit the transpor t of nutrients across the root tissue. However some plants have survival techniques to combat the flooding event, such as the formation of aerenchyma, a system to transport air from th e air to the root zone and the creation of adventitious roots which form a bove the ground in an attempt to a void the deeper anaerobic root zone (Hook et al. 1972). During the night when the tran spiration stream is not appreciably

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17 flowing, there is an influx, 13 to 40% (Ganse rt, 2003; Mancuso & Marras, 2003) of the oxygen can enter through the lenticels a nd moves through the tissue apoplas ticly, through the gas spaces. The density of the wood influences this abilit y of lateral oxygen tran sport into the xylem vessels(Mancuso & Marras, 2003), also the discon tinuous nature of xylem rays, which are begun and ended by the cambium each year, hinder direct adsorption into the xylem vessels. During the winter months, when the transpiration st ream has stopped and the high demands for oxygen are low, lateral diffusion is the means which allo ws the levels of dissolved oxygen in the xylem fluid to approach ambient levels (Eklund, 2000). Oxygen to serve as the favored electr on acceptor for energy production (oxidative phosphorylation) for dividing cambial regions (Eklund et al. 1998), and in the roots is primarily transported via the transpirati on stream (Mancuso & Boselli, 2002). However the amount of oxygen supplied to the growing tissue can fall below adequate levels and the plant must therefore rely on other pathways for energy production such as fermentation, the use of the substrate as an electron donor and then acceptor for energy production as a means to regenerate NADH. The anaerobic fermentation (substrate phosphoryla tion) by-products such as lactate (Andersen et al. 2005), ethanol and acetaldehyde (Kimmerer & St ringer, 1988; MacDonald & Kimmerer, 1991; MacDonald & Kimmerer, 1993) have been reported in the xylem fluid of Vitis Populus Acer Quercus and other trees. Ethanol has been shown to be incorporated along the vascular tissue of Populus where radiolabeled ethanol wa s injected into the transpiration stream of excised leaves and stems, with less then 1% being released as CO2 (MacDonald & Kimmerer, 1993). There are several repor ts that describe the amount of oxygen found in the xylem fluid of plants. The researchers from these reports have come to similar conc lusions using different techniques varying from GC-MS (Eklund, 2000), a homemade clark-type sensor (Mancuso &

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18 Marras, 2003), or a novel technique using fi ber optics and fluorescent compound which is quenched in the present of oxygen (Dongen et al. 2003; Gansert et al. 2001; Hierro et al. 2002). Each group reports their data either as a percen tage of oxygen that satura tes a liquid (100 to 0% air-saturated), with 100% saturate d being equal to 21% air (21 to 0%), or a direct molar amount 288 mol O2 L-1 (at saturation) in Table 1, all three, as well as g L-1 (ppm) are shown together. The lowest levels of oxygen were found in Picea albies (Norway Spruce) (Eklund, 2000), which ranged between <1 5% oxygen, the high est oxygen levels were detected during the winter months, 17 19% oxygen (Hierro et al. 2002) which is almost at air-saturation levels. The levels during the growing season for a few a ngiosperms can get as low as 14% air-saturation (Eklund, 2000). For an overall summary see Table 1. Since the major influx of oxygen is through the transpiration stream it is possible that Xf can monitor the concentration as a function of fl ow. The oxygen in the xylem is exhausted as it ascends the plant (Eklund et al. 1998; Eklund, 2000), therefore concentrations in the transpiration stream would be highest near th e roots and lowest near the apical meristem. General Overview of Electron Transport The electron transport chain utilizes a gra dual reduction of redox potential to move electrons along a pathway thereby generating a pr oton gradient, high on the outside of the inner membrane and low on the inside, which is used to produce ATP, and power flagella. The pathway begins with complex I, NADH dehydrogenase in which two protons are shuttled to the outside of the cell and two elec trons which are transferred to quinone (which becomes quinol), a compound found amidst the lipid bilayer of the i nner membrane. Then complex II, succinate dehydrogenase and fumarate reductase (fumarase), which converts succinate to fumarate in order to shuttle two protons outside a nd transfers two electrons to qu inone pool, the collective amount of quinone found in the inner membrane. Next, is complex III, cytochrome bc, which transfers

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19 the electrons from quinol to an enzyme called cytochrome c reductase, where the change in energy facilitates the transport of two more pr otons across the membrane. Finally complex IV, the terminal oxidase, also transfers two protons to the outside of the cell but also reduces the number of protons inside by binding them to molecular oxygen, O or O2. Therefore the end result of this is that 10 protons removed, 8 are expelled from the cytoplasm and 2 protons removed by the synthesis of water, from the cy toplasm thereby increasing the difference in proton gradient that is used to drive ATP synthesis. Xf s Electron Transport System Xf is missing a few of the components associat ed with the electron tr ansport chain. The complex II system, of which there is only fumera te reductase and comple x three, is absent altogether. The components that are present are complex I, a partia l complex II (succinate dehydrogenase) and complex IV. It is with complex IV that th e ability to use oxygen becomes significant. The enzyme kinetics for cytochrome bo (for E. coli ) are an affinity of Km = 0.2 M, and rate of Vmax = 1.1 1.5 mol O2 nmol protein-1 min-1 (Rice & Hempfling, 1978), so that it is known as the high through-put, low affin ity terminal oxidase (as compare to E. coli s cytochrome bd, which has a Km of 0.02 M (great er affinity) and a reaction speed of 0.7 mol O2 nmol protein-1 min-1 (half the reaction rate of cyt. bo) which makes it known as the low through-put, high affinity terminal oxidase). Th e result of these missing components is that it requires more of the complexs substrates, NADH succinate and oxygen, to generate the proton motive force which operates the ATP synt hesis machinery. The one up side for Xf is that there are no flagella which would require th e use of the proton motive force. Xf Use of Oxygen as Terminal Oxidase Aerobic bacteria use as the final electron ac ceptor in producing a proton gradient which is then used as the driving force for ATP synthesis. The terminal oxidase for Xf is cytochrome

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20 (cyt.) bo, which has four subunits, with subun it B possessing the metallic prosthetic groups, heme b, heme o and copper (Kita et al. 1984). Cyt. bo is found in many other bacteria, the most studied is in E. coli a facultative anaerobe (capable of aer obic and anaerobic growth) which also possesses another terminal oxidase cytochrome bd. The interesting thing about these two oxidases is that cyt. bd, a two subunit prot ein, is used during low oxygen concentration environments and cyt. bo, a four subunit pr otein, is used in high oxygen concentration environments. One of the uses for oxygen in Xf biochemistry is that it is re quired at the terminal electron acceptor by the cyt. bo. A BLAST an alysis of the sub-units of Xf s cyt. bo against E. coli s showed a 40 60% similarity to each other. S ubunit B (also known as subunit I), which contains the metallic cofactors, have th e highest similarity, identical and analogous chemical properties amino acids, with a BLAST score of ~68%. The amino acids which are considered essential for the binding of the metallic cofactors are present. Fermentation The production of energy is a method used to classify bacteria, depending on the terminal electron acceptor. Aerobic or ganisms use oxygen as the term inal electron acceptor, i.e. oxidative-phosphorylation. Anaerobic organisms ar e a more complicated situation. When one speaks of anaerobic metabolism, ones first though maybe of sulfate or nitrate reduction where the terminal electron acceptor is an inorganic molecule. However, another form of anaerobic metabolism is fermentation or substrate based phosphorylation. Xylella fastidiosa breaks down glucose using the EM P (Embden-Meyer-Parnas) pathway (Facincani et al. 2003), which utilizes glucose-6-phosphate rather then 6-phosphogluconate, the pentose phosphate and Entner-Doudoroff pathways, to initiate the conversion of glucose into pyruvate. The fate of pyruvate is to enter th e citric acid cycle. A by-product of these

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21 cycles/pathways is the reduction of NAD and FAD to NADH and FADH2, respectively. These reduced compounds must then be oxid ized back to there reductive form s, this is carried out either through respiration, us ing oxygen or other inorganic molecule, or by fermentation. Respiration can be aerobic or anaerobic (wit h varying degrees of oxygen tolerance), both utilize membrane bound enzymatic reactions. Ae robic respiration is a membrane bound process which utilizes the electron transport system and a terminal oxidase to create a proton potential to operate the ATP synthase, by reducing NADH co mpounds. Anaerobic respiration is also a membrane bound process but can use sulfate or nitr ate, to name a few, inorganic compounds as the electron sink which drives the reduction of NADH. Another alternative is fermentation which occurs with in the cytosol and uses orga nic molecules, food, as the electron donors and as the electron acceptors during the reduction of NADH. The fermentation processes are broken into six main classes based by the end product homologies they each produce such as ethanol, lactic, butyric, propionic, homo acetic and mixed acid. These electron acceptors are then deposited into the media. The Added Influence of the Disulfide Bond Forming System The disulfide bond (dsb) protein system is predominantly concerned with the correct formation of disulfide bonds that are excreted from the cytoplas m to the periplasm or through the periplasm to the outer membrane. The machinery for this family of enzymes is located in the periplasm and associated with the inner me mbrane oriented to the periplasm (Bader et al. 1999; Collet & Bardwell, 2002). The part that is german e for the correct formation of disulfide binds is the periplasm dsbA, which is reduced by the me mbrane bound disulfide protein B (dsbB), which is recharged by contributing two el ectrons to the quinone pool. The affinity for quinone to dsbB is Km = 0.31 M (Fabianek et al. 2000). The appendage structures which are associated with the outer membrane all have the ability to have a disulfide bond per prot ein subunit, as seen on

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22 the amino acid sequences (Kanehisa Laboratories, Kyoto Encyclopedia of Genes and Genomics, www.kegg.com December, 2005), (pilE, a subunit for type-IV pili formation is known to have one and a completed pilus as many copies of the subunit), thereby the creati on of these structures rely on the presence of the quinone and terminal ox idase in order to function. A point of interest is that Xf has two dsbA genes and these are locate d adjacent to each other, the only other organism that could be found that has a similar gene arrangement is Xanthomonas campestris and X. axonipodis (Kanehisa Laboratories, Kyoto En cyclopedia of Genes and Genomics, www.kegg.com December, 2005). In addition of disulfide bond formation, another f unction of this gene family is that it is the chaperone component for type-I pilus (fimbr ia) production (Fernandez & Berenguer, 2000). The type I pilus system utilizes helpers all the way. From the cytoplasm P-pili (a type-I pilus from E. coli ) uses a sec-dependent pathway to enter the cy toplasm. Once in the periplasm, the pilus subunits are transported to the outer memb rane using DsbD (Soto & Hultgren, 1999). Physiological Modifications Due to Oxygen Availability During the 1970s and 1980s studies of the causati ve agent of Pierces disease, then known as a rickettsia-like bacterium, were usually in the form of scanning electron microscopy of dissected Vitis sp. canes and petioles. Two differe nt morphological characteristics were described for the rickettsia-like bacterium of infected grapevines, the rippled and the smooth outer membranes. In V. rotundifolia Mitchx. (Carlos), the small tracheary elements had mostly rippled bacteria and the larger tracheary elements the smooth bacteria were prevalent. In the bunch grapes ( V. vinifera L.), the bacterial populations ha d higher counts with smooth outer membranes and a reduced number with rippled membranes (Huang et al. 1986). This led to an idea that different cell morphologies are an indication of the pathogens virulence.

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23 There are other changes associated with the appearance of the bacterium as a function of the environment in which it was grown. If th e bacterium was recently inoculated into a grapevine the bacteria had a less dense morphology th en if it was grown longer in the plant or for four days in enrichment media (Mollenha uer & Hopkins, 1976). These denser morphology images are very similar to the effects of aerobic verses an anaerobic environment upon the morphology of mitochondria, an ance stral proteobacteria, found in Oryza sp. A mutational analysis of cyt. bo in Pseudomonas putida which is lethal but also creates plasmolysis and polar pitting (Duque et al. 2004), which appears to be present in images of Xf (French, unpublished). The oxygen levels may also be attributed to th e changes in the expression appendage, pilus and fimbriae, as well as the metabolic activity. The Schembri model, based on E. coli attachment mechanisms, describes a change in the expression of these outer membrane structures due to changes in the OxyRS system, a two component system for determining the oxygen levels. These changes can lead to a cascade of other gene changes such as the formation of biofilms (pili mediated changes) or the activation of motility complexes (Bespalov et al. 1996). The bacteria may utilize the cha nges in oxygen as an indicator to its environment. Bacterial hemoglobin, also known as soluble cytochrome o has also attributed to the production of degradation enzymes as a f unction of oxygen levels. In Vitreoscilla sp., an -proteobacter, when the hemoglobin detects a hypoxic environment it s timulates the cell to produces an amylase, a carbohydrate degradation enzyme, and when this hemoglobin is expressed in E. coli similar results occurred (Kallio et al. 1994). This type of sens or system may modify Xf genome to produce different enzymes in order to survive on other carbon source, i.e., glutamine or cellulose, either form the xylem stream or as attached biofilms, respectively. Aerotaxis and

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24 redoxtaxis are sensory responses which direct movement towards a favorable oxygen levels and a redox environment, respectively. There are othe r responses to oxygen levels such as biofilm formation, such as Pseudomonas aerigionsa in the lungs of multiple sclorosis patients produce more biofilm when und er hypoxic stress (Boes et al. 2006). Bacterial Attachment and Appendages Bacterial infections occur af ter the bacteria have been introduced to a sterile environment and are then able to attach, coloni ze, and form defenses, biofilm formation, within the host (Schembri et al. 2002). These initial steps of in fection are poorly understood for Xf due to the inaccessibility of the host environment and fastidious nature of the pathogen. However the proposed method for attachment of gram-negative bacteria to the surface s of its environment (host) is obtained through the pili appendages (Schembri et al. 2002), for Xf these include type I pilus which uses the chaperone-usher pathway me diated with the disulf ide bond protein family and the type IV pilus which uses general secr etion pathway, which constructs the pilus on the inner membrane and then protrudes through the outer membrane (Kanehisa Laboratories, Kyoto Encyclopedia of Genes and Genomics, www.kegg.com December, 2005). It is thought that the type IV pili occur on the polar ends of the bacteria and are longer then the type-I pili which cover the entire bacteria. The type I pili have been asso ciated with the ability of other bacteria such as Escherichia coli Klebsiella pneumonia and Bordetella pertussis to attach and colonize their hosts (Soto & Hultgren, 1999). In E. coli the inter action with the pili and the bacterias surroundings lead to changes in protein composition of the outer membrane (Otto et al. 2001). The utilization of type-I and type-IV pili of Xf have been documented for biofilm production and movement (Feil et al. 2003; Meng et al. 2005), respectively. The loss of pili/fimbriae expression has been associated with the e ffects of sub-culturing, which maybe associated with the reduction

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25 of virulence of the bacteria, after 18 months of subculturing (Hopkins, 1984), but not with a triply cloned still virulent (Li et al. 1999). There maybe differences in the attachment to various substrates, namely the foregut of the vector and the xylem tissue. Some of the EM shots of the bacteria in the media and in the foregut of vector indicate that there maybe two different attachme nt mechanisms. In the vector, the bacteria appear to be standing up (Brlansky et al. 1983; Newman et al. 2004), which would indicate a polar expression of attachment mechanisms, to which type-IV pili have been associated to the polar ends of the bacteria. Images from the plant and from media show the bacteria laying about in no discernable pattern (Leite et al. 2002; Lopes et al. 2000; Mollenhauer & Hopkins, 1974; Newman et al. 2003; Newman et al. 2004) which may indica te that the type I pili (fimbria) attachment which are expressed all over the bacteria. The attachment to both of these substrat es is probably mediated through adhesin expression differences. Another piece of evidence for this maybe that when comparing the movement down the vine the production only of the type-IV pili moved even farther then the wild-type, which produces both ty pe-I pili and type-IV pi li, which may indicate that the lack of fimbria may inhibit the colonization of the xylem as the bacteria pass through (Meng et al. 2005). The type-I pili have also been implicated in the attachment of ot her bacterial pathogens to there respective hosts, an example is the, well described, PAP system in E. coli which attaches to the urethra, a conduit that passes urine from the bladder to outside the body (Fernandez & Berenguer, 2000; Soto & Hultgren, 1999). The previously mentioned adhesion techniques for Xf are based on the model for other proteobacteria (mainly E. coli ) proposed by Schembri, et al, 2 002. This model indicates that there is a phase variance in the expression of certain appendages in E. coli which is triggered by

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26 the oxygen sensor system of OxyRS, which is a two component system consisting of a sensor and a transcription factor which in turn activ ates an adaptation response to the change in environment (Storz & Imlay, 1999). Another part of this model is that there is a sequence of events which the bacterium goes through in order to form a biofilm, first is attachment, followed by the formation of a micro-colony and then the expression of biofilm. Bacterial attachment comes in two stages, one is a temporary form which lasts few seconds to minutes and the second is a permanent adhesion to the surface. During the temporary adhesion stage the bacteria sens es its environment around it, eith er using adhesin molecules at the tips of the pili, used to rest on the surf ace, or sensors embedded in the membranes of the bacteria. If during the temporar y adhesion the bacteria determines it is a suitable habitat the bacteria will begin to change the attachment mechanism to form a permanent link, however if the environment is not to the bacteria s liking the bacteria will not stay attached to the surface (Agladze et al. 2003; Agladze et al. 2005). Based on Schembris model biofilm is not created until a microcolony is formed on a surface, and there has been a change in the produc tion of appendages. The type I pili have been implicated in the formation of biofilm and by knockout mutations the lack of production (Feil et al. 2003; Meng et al. 2005). Type I pili in E. coli has also been implicated in the reduction of virulence based on a changes of gene expression due to surface interactio n with the appendages (Otto et al. 2001) Cell-to-cell aggregation, which contributes to the stability in the formation of colonies within the host tissue (Schembri et al. 2002), has been attributed to two hemagglutinin proteins, binding proteins not associated with pilus/fimbria formations, are involved in cell-to-cell aggregation (Guilhabert & Kirkpatrick, 2005).

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27Table 1-1 Oxygen concentrations found in various xylem fluids Species (Conditions) DO (mol L-1) DO (% air saturated solution)DO (% Air) ppm (g L-1) Reference Ricinus communis 95.8 33.3 7 3.1 (Dongen, et al. 2003) Betula pendula 42 to 230 14.6 to 80 3.1 to 16.8 1.3 to 7.4 (Gansert, et al. 2001) Olea europea (day) 80 to 90 27.9 to 31.3 5.8 to 6.6 2.6 to 2.9 (Mancuso & Marras 2003) O. europea (night) 20 to 30 9.96 to 10.4 1.46 to 2.19 0.64 to 0.96 (Mancuso & Marras 2003) Picea abies (irrigation) 68.5 23.8 5 2.2 (Eklund 2000) P. abies (no irrigation) 13.7 to 41.1 4.76 to 14.3 1 to 3 0.43 to 1.3 (Eklund 2000) P. abies (drought) <13.7 <4.76 <1 <0.43 (Eklund 2000) P. abies (winter) 273.8 95.2 20 8.8 (Eklund 2000) Fagus orientalis (day) 58.36 20.3 4.26 1.87 (Hierro, et al. 2002) F. orientalis (night) 50.31 17.5 3.68 1.61 (Hierro, et al. 2002) Laurus nobilis 51.75 18* 3.78 1.66 (Hierro, et al. 2002) L. nobilis (flooding) 37.38 13* 2.73 1.20 (Hierro, et al. 2002) = The data as it was presented in th e primary literature. The conversions were made assuming a temperature of 20 C, and that 288 mol L-1 = 100% air-saturated solu tion = 21% air = 9.2 ppm.

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28 CHAPTER 2 EFFECT OF OXYGEN ON THE GROWTH AND BIOFILM FORMATION OF Xylella fasdtidiosa Introduction Xylella fastidiosa ( Xf ) is a Gram negative bacillus bacterium which resides in the xylem vessels of many plants. The bacterium is vector ed between host plants by xylophagus insects, such as the glassy-winged sharpshooter [ Homalodisca coagulata (Say)] (Pur cell & Hopkins, 1996). Xf is believed to cause diseases in its host by restricting the transpiration stream by clogging the xylem elements and/or stimulating the plant to form tyloses, protrusions from the parenchyma cells, which plug the xylem elements (Esau, 1948). Xf was described as being an obligate aerobe (Wells et al. 1987). However recent publications describe the dissolved oxygen cont ent at hypoxic and anaero bic levels in xylem vessels (Table 1) during the growing season when the demand for oxygen in the metabolically active tissue is at its highest. The amount of dissolved oxygen found in the xylem fluid can range from ~20-30 mol L-1 (~9% of air-saturated water) (M ancuso & Marras, 2003), and up to 280 mol L-1 (~95% of air-saturated wate r), at 20C (Gansert, 2003). The xylem elements are constructed predominan tly of lignin and cellulose, for strength and water proofing respectively. Xy lem fluid consists of 95 99% water, with the remaining constituents mainly consisting of amino acids, small sugars and ions (Andersen & Brodbeck, 1989; Andersen & Brodbeck, 1991; Andersen et al. 1992; Andersen et al. 2005). Another constituent of xylem fluid, which is impor tant to the survival capabilities of Xf is the concentration of dissolved oxygen. The dissolved oxygen predominantly enters though the root zone (Mancuso & Boselli, 2002), with a small pe rcentage enters through radial diffusion through the trunk and stems (Gansert et al. 2001; Mancuso & Marras, 2003) and the concentration decreases as the transpiration stream moves away from the r oots (Eklund, 2000). The levels of

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29 O2 get low enough that the plants may begin a fe rmentation (anaerobic respiration) process in order to meet the required energy needs, as seen with the generation of fermentation by-products such as ethanol and aldehydes (Kimmerer & Stringer, 1988; MacDonald & Kimmerer, 1991). This is especially true for the xylem environment at the center of large trees. It is also interesting that the tissue at the terminal en ds of the xylem system i.e. le aves, are capable of metabolizing these fermentation products (MacDonald & Kimmerer, 1993). There are three physical components which regulate the ability for oxygen to become dissolved in water; one is the temperature, in that the higher the temperature the less the solubility of oxygen in water, a nother is osmolarity, the more constituents a solution has the less oxygen can be retained, and the third regulator is pressure where a decr ease in pressure will decrease the amount of oxygen in water. In plants these components regulate the amount of dissolved oxygen that can enter in to the inner plant tissues. The contributions of these conditions for Vitis are such that during the grow ing season it creates what could be an environment with low levels of dissolved oxygen. The temperature rises into to the mid-90s (degrees Fahrenheit) in north Florida, the tension of the transpiration stream can get as low as -1.6 MPa (or -230 psi) and the osmolarity of the xylem fluid is approximately 20 mM (Andersen & Brodbeck, 1989; Andersen & Brodbeck, 1991; Andersen et al. 1992), which is higher than in winter but probably has little overall effect. Other factors whic h effect the dissolved oxygen concentration is flooding, in which the water becomes anoxic aro und the root zone, and during drought, where the roots isolate themselves off to the environment to prevent further water loss. The genomes for two strains (pathovars) of Xf have been sequenced. The first was citrus variegated chlorosis (Simpson et al. 2000) which causes a disease in citrus that result in unmarketable fruit. The second strain to be se quenced was the Pierces disease strain Temecula

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30 (M. A. Van Sluys et al. 2003), which infects grapevines and redu ces the fruit quality and kills the most susceptible Vitis species such as Vitis vinifera There are also two other genomes which have been partially sequenced, one Dixon, almo nd leaf scorch pathovar, and the other Ann, oleander leaf scorch (M. A. Van Sluys et al. 2003). These sequenced ge nomes indicate that the pathovars contain the similar oxidative phos phorylation pathways a nd the capability for anaerobic energy producti on by sulfate reduction. Materials and Methods Bacteria Used The Xylella fastidiosa strains were obtained Dr. Purcell and Christina Wistrom (UC Berkeley). The Xf strains used were the Pierces dis ease strains: Temecula, UCLA, STL, Conn Creek, and Santa Cruz; and the al mond leaf scorch strains used were: Tulare and Dixon. The other bacterial cultures used were: Erwinia sp, Pseudomonas sp, Xanthomonas sp, Xanthomonas campestris pv vitians (from Dr. Momols lab group) and Xanthomonas campestris pv. campestris (from Dr. Jones) Cultivation Protocol Bacterial suspensions in PW+ and 10% glycerol were stored in a -80C freezer (Baxtor Scientific Products, Cryo-Fridge). Initial cultures were starte d by scrapping a sterile wooden applicator stick over the frozen stock culture and streaked out onto a Buffered Charcoal Yeast Extract (BCYE) plate (Wells et al. 1987). After 10 days bacterial growth was scraped off with an inoculation loop and placed into 5 mL of PW+ broth (Davis et al 1981) and incubated between 10 to 14 days, (until a visible turbid so lution) before being used to make working cultures. These 5 mL cultures we re transferred to 50 mL of PW+ media in 250 mL Erlenmeyer flasks with cotton plugs or sili cone rubber plugs with a filter to allow gas exchange. All

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31 incubations were done in New Br unswick incubator (model# G25-R) set at 28C and all liquid cultures were placed on an orbital shaker set to 150 rpm. Experiments were carried out in 15 mL polypr opylene Falcon tubes with red-rubber septa or screw caps and 16 X 150 mm glass test tubes with slip cap closures. The media used included an enrichment media, PW+, the defined media, CHARD2 (Le ite et al, 2004), which resembles the xylem chemistry of Vitis vinifera Chardonnay and the varian t CHARDS, and the defined media XDM2 (Lemos et al, 2003), which was cr eated by studying the genomic requirements of the citrus variegated chlorosis strain and the variant XDM2-PR, XDM2 which lacks phenol red. PW+ Media, Broth and Solid PW+ media (Davis et al. 1981) was made in two solutions which were then combined. Solution A consisted of (for 1L) 900mL dH2O to which these components were added, 1.0 g soluble starch, 4.0 g soytone, 1.0 g tryptone, 1.2 g K2HPO4, 1.0 g KH2PO4, 0.4 g MgSO4-7H2O, 0.85 g (NH4)2HPO4, 1.0 g histidine, 25.0 mg cyclohexami de, 10.0 mL (0.1%) hemin-Cl, 10.0 mL (0.2%) phenol red. Media were sterilized by au toclaving. Solution B was made by dissolving 4.0 g of glutamine into 50 mL dH2O at C, and 6.0 g bovine seru m albumin (fraction V) into 30 mL of dH2O, which were then combined. Next media were sterilized through a 0.22 m filter. The two solutions were then combin ed to form the completed broth medium. PW+ agar plates were made by adding 12.0 g agar to soluti on A before sterilizati on in the autoclave. Solution B was then combined when solution A c ooled down to 55C in a hot water bath. Then PW+ medium was aseptically ali quoted into Petri dishes. PW+ soft agar deeps were made using 2.0 g of agar in solution A before sterilization in the autoclave. Solution B was then combined when solution A cooled down to 55C in a hot wa ter bath. The soft agar was then dispensed aseptically into the appropriate test tube s, either the polypropylene or glass.

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32 CHARDS medium CHARDS was a modified version of CHARD2 which was a defined media based on the xylem fluid chemistry of Vitis vinifera Chardonnay (Leite et al. 2004). Solution A consisted of (for 1 L) 819 mL of dH2O to which these components were added, 1.0 g KH2PO4, 1.5 g K2HPO4, 0.2 g MgSO4-7H2O, 0.17 g alanine, 0.58 g aspartic aci d, 1.8 g glutamine, 1.05 g arginine, 0.01g cysteine-HCl, 1 mL (1 mg/ L) biotin, and 1.0 g of soluble starch. The pH was then adjusted to 6.6 using concentrated KOH and then sterilized by autoclaving. Solution B consisted of 0.25 g of ferric pyrophosphate dissolved in 60 mL dH2O and 4.0 g glutamine dissolved in 50 mL dH2O at C which was then combined and was st erilized through a 0.22 m filter. The two solutions were then combined, asepti cally, to form the completed media. XDM2-PR meduim XDM2-PR was a modified version of XDM2 (Lemos et al. 2003) which was a defined media based on the genome of X. fastidiosa pv. citrus variegated chloro sis. For 1 Liter, into 980 mL of dH2O these components were added 10.0 g glucose, 2.1 g K2HPO4, 0.8 g KH2PO4, 0.4 g MgSO4-7H2O, 0.125 g ferric pyrophosphate, 4.0 g glutam ine, 1.0 g asparagine, 0.4 g serine, 0.4 g methionine, and 10 mL of vitamin solution. The pH was then adjusted to 6.6 using concentrated KOH and then sterilized through a 0.22 m filter. The vitamin solution consisted of (for 100mL) 350.0 mg myo-inositol, 10.0 mg pyridoxine-HCl, 10.0 g thiamine, 5.0 mg nicotinic acid, and 0.2 mg of D-biotin. The vitamin solution was sterilized through a 0.22 m filter and stored at 4C in a light-protected bottle. The BCYE Agar Plates This was the media used by Wells et al 1987, for the initial growth of the Xylella strains (ATCC medium 1099 CYE; buffered (Charcoal yeas t extract) medium). Solution A was made by mixing 0.4 g L-cysteine-HCl into 10 mL of dH2O and sterilized through a 0.22 m filter.

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33 Solution B was made by mixing 0.25 g fe rric pyrophosphate into 10 mL of dH2O and sterilized through a 0.22 m filter. Solution C was made by mixing 10.0 g of ACES buffer (n-2acetamido-2-aminoethane sulfoni c acid) into 250 mL of dH2O. The pH was adjusted to 6.9 +/0.05; by this means the chemical will go into so lution. Solution D consisted of 10 g of yeast extract was added into 730 mL of dH2O, 2.0 g of charcoal, 17.0 g of agar. pH was adjusted to 6.9 +/0.05 using KOH. The media was autoclaved to sterilize and then tempered to 50C in a water bath. Solutions A, B, C, and D were comb ined aseptically. The media was dispensed into Petri dishes and gently swirled to keep the charcoal in suspension thereby ensuring even distribution. Grams Staining Grams staining protocol was conducted on a sample of cells either a single CFU applied to a glass slide or 1 mL of liquid cultur e spun down and re-suspended in sterile dH2O and 20 l placed on a glass slide and allowed to air dry. Then the bacterium on the glass slide was heat fixed by passing the slide over a Bunsen burner three ti mes, fixing the bacteria to the slide. Once cool, crystal violet was applied on to the fixed bacteria for one minute, and then the slide was rinsed with dH2O. Next, iodine was placed onto the cu lture for one minute, and then the slide was rinsed with dH2O. The slide was then de-stained in an acetone: ethanol mixture, for thirty seconds, and then the slide was rinsed with dH2O. Finally, safranin was applied to the bacteria for one minute, and then the slide was rinsed with dH2O. Gram positive bacteria stain purple due to the crystal violet, iodine compound, where th e Gram negative bacteria stain pink with the lighter stain basic safranin. PCR Protocol Once purity had been established by Grams stain, PCR (polymerase chain reaction) was used to confirm the identity of the cultures. DNA extractions were c onducted using the Quiagen

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34 DNeasy mini prep. The PCR protocol used a ne sted primer technique (Pooler & Hartung, 1995). The first reaction used the primers 2721 (5-AGCGGGCCAATATTCAATTGC-3) and 272-2 (5-AGCGGGCCAAAACGATGCGTG -3). The final concentration of reagents for a 20 l reaction was 1X PCR Buffer, 2.0 mM MgCl2, 0.2 mM dNTPs, 0.5 M primer 272-1, 0.5 M primer 272-2, and 1 U Taq. The second reactio n used the primers 272-2int (5-GCCGCTTCGGAGAGCATTCCT-3) and CVC-1 (5-AGATGAAA ACAATCATGCAAA-3). The final concentration of reagents for a 20 l reaction was 1X PCR Buffer, 2.0 mM MgCl2, 0.2 mM dNTPs, 0.3 M primer CVC-1, 0.3 M primer 272-2int, and 1 U Taq. The thermocycler protocol (same protocol for both reactions) was 94 C for 2min, then 35 cycles of 94 C for 1 min, 62 C for 1 min, and 72 C for 1.5 min, followed by a final extension at 72 C for 5 min. The reactions were stored at 4 C until they were assayed by elec trophoresis through 1% agarose gel slab and stained with ethidium bromide, with positive results indicated by a band at ~500 bp. Deeps Soft agar was tempered to 50C in a wa ter bath. This was inoculated with Xylella fastidiosa to a final optical density at 600 nm of 0.05 and then di spensed into 16 X 150 mm glass test tubes with slip-cap closur es or 15 mL polypropylene falcon tubes with rubber septa sealed with parafilm and treated with air or nitrogen. These tubes were then placed in a 28C incubator and the bacterial development was then tr acked over time. Results were photographed. Stabs Soft agar deeps, consisting of PW+ media and 0.2% agar, was inoculated and dispensed into 16 X 150 mm glass test tube with slip cap cl osures, to ensure adequate gas exchange. An inoculation needle was dipped into a PW+ culture at an optical density at 600 nm of 0.2 (or greater) and stabbed into the cen ter of the tube. Other experiments used 15 mL polypropylene falcon tubes with rubber septa was sealed with pa rafilm and treated with air or nitrogen. The

PAGE 35

35 tubes were then incubated at 28C until initial grow th was visible. At that point a photograph of the tube/growth was taken. The tube was then pl aced back into the incubator. The bacterial development was then tracked over time. Oxygen Levels The LaMottes Dissolved Oxygen Test K it (Model EDO-Code 7414), was used to determine the treatment effects over tim e in the aqueous environments of dH2O and CHARDS. The assays were conducted with in red-rubber septa closed tu be by injecting 100 l of the manganous sulfate mixed well, then 100 l of th e alkaline potassium iodide azide mixed well and when the precipitant starte d to settle a 100 l of 50% H2SO4 mixed well until all the precipitant dissolved. The tubes were then opene d and the solution was transferred to a supplied beaker with a lid designed to fit around the tip of the grad uated titration syringe. Then thiosulfate was titrated into the sample and when the color became clear, against a white background, the volume used represented the amount of dissolved oxygen in the sample, measured in ppm. Growth Conditions Mature bacterial cultures, in 50 mL batches, were removed from the incubator and pooled together into a sterilized media flask. The op tical density of the culture was obtained, using a spectrophotometer set at a wavelength of 600 nm. Aliquots were placed into sterile polypropylene/glass tubes and centrifuged at 2380 x g for 15 minutes. The supernatant was decanted and the pellet was resuspended into 3 mL of PBS (phosphate buffered saline, 0.1 M phosphate, 0.8% NaCl and a pH of 6.8). Th e suspension was then centrifuged again. The supernatant was discarded and the pellet resuspended into the appr opriate media. This was the centrifuged as before and the supernatant discarde d. The pellet was resuspended into 5 mL of the appropriate media. Then the tubes were capped with red-rubber se pta and secured with

PAGE 36

36 parafilm. Gas treatments were done each day. The tops of the septa were sterilized by rinsing with 70% isopropyl alcohol and dabbed dry with a kimwipe. The gas was passed through a 0.22 m syringe filter then injected through a one in ch, sterilized 22 gauge needle, and then another sterilized 22 gauge needle was placed into the se pta to act as an exhaust port to ensure gas exchange. The tubes were placed in the New Br unswick incubator set at 28C, on an orbital shaker set at 150 rpm. Optical Density (OD) Optical density was a quantitative measurement of the concentration of planktonic bacteria in solution based on the absorption of light thro ugh a liquid culture. OD was measured using a Genosys 8 spectrophotometer at a wavelength of 600nm. The machine was zeroed using an aliquot of sterile media or suspension buffer. Then an aliquot of the bacterial suspension was placed into the cuvette and measured. Biofilm Qualification The biofilm assay was a qualitative measurement of the concentration of bacteria that have become sessile on the tube or flask in which they were grown (Espinosa-Urgel et al. 2000). The bacteria were decanted and the vessel was rinsed with dH2O and air dried. The vessels were then filled with 0.1% crystal violet, and placed on a shaker for 1 hour. The crystal violet was decanted and the tubes were rinsed until all the excess dye has been removed and air dried. The vessels were then de-stained using 70% isopropan ol. An aliquot will then be placed into a cuvette and the optical density was measured at a wavelength of 600nm. The spectrophotometer was zeroed using a cuvette with 70% isopropanol used as a blank. Media Analysis Xf strain Temecula was inoculated into C HARDS and XDM2-PR media by dispensing 1 mL of mature culture (~0.35 OD at 600nm) into 15 mL polypr opylene tube and centrifuged

PAGE 37

37 (Jouan C3-12) at 2,380 g for 15 minutes. The su pernatant was discarded and the pellet was resuspended in 3 mL of PBS. This was centrif uged at 2,380 g for 15 minut es and the supernatant was discarded. The pellet was resuspended in 2 mL of the appropriate media and then centrifuged at 2,380 g for 15 minutes. The supe rnatant was discarded and the pellet was resuspended in 5 mL of the appropriate media and sealed with red-rubber septa and sealed with parafilm. The tubes were then subjected to gas treatment which consisted of various concentrations of oxygen, 21% (breathing air) and 0% (nitrogen) for 5 mi nutes every day for the duration of the experiment. Each treatment consis ted of inserting a syringe needle connected to a compressed gas tank with a positive pressure of ~5 psi through the rubber-septa and an exhaust needle was inserted to allow for gas exchange. Three tubes from each media-gas interaction we re removed every five days and assayed. The optical density was obtained at 600 nm. A drop was placed on PW+ agar media and incubated at 28C for 10 days to determine vi ability. A 2 mL sample from each tube was centrifuged at 5,000 g at 4C for 15 minutes (Sorva l Biofuge, Model: Stratos) in a temperature regulated centrifuge, then th e supernatant was stored at -80C. Then the tubes were then emptied and assayed for biofilm formation. The frozen supernatant samples were filtered through a 10,000 molecular weight cutoff filter and then prepared for HPLC analysis, us ing a Water automated system operating with the Millennium operating program. Organic acid analysis was conducted on a polymeric cation exchange column using 0.01 N H2SO4 elution at 0.4 mL per minute, at 40C. Product was detected by UV absorbance at 210 nm, which corre sponds to the presence of carboxyl groups. Statistical Analysis All experiments were designed so that each data collection would consist of three independent replicates. The bact eria were divided and washed in order to insure that each

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38 response observed was dependent on each treatme nt (and not the three measurements of the initial bacteria treatment interaction). The experiments were done at least twice, with fresh cultures and media, to confirm the results obtaine d were reproducible. The data was analyzed with ANOVA, t-tests, means and standard er ror as well as regression using the SAS V9.0 software package. The error bars represent standard error. Results Agar Deeps The initial growth (Figure 2-1a), for all Xf strains tested, began just below the surface. This area was still considered to be aerobic; however, it indicates that optimum growth of the bacterium occurred in a reduced oxygen environm ent. The growth of the bacterium proceeded down into the tube away from th e initial growth development. After 2 months of incubation in either the air or nitrogen treatments bacterial gr owth development continued from just below the surface down to the bottom of the tube for deeps and along the path of the inoculating needle for stabs as well as radiating out from the path of the inoculating needle (Figure 2-1b and 2-1c). During this growth there was no evidence of gas production, which woul d be indicated by the formation of bubbles in the agar. Experimental Conditions The oxygen levels were determined using a La Mottes Dissolved Oxygen Test Kit (model EDOcode 7414). Using deionized water and C HARDS media the experimental conditions were assayed to determine the dissolved oxygen levels as a function of treatment and effects over time. Figure 2-2 shows that the initial treat ment was enough to establish the dissolved oxygen level. Once the level had been set it was stable after daily treatments for the length of the experiment. The LaMottes test kit has the lim itation in which there may be dissolved oxygen in

PAGE 39

39 the liquid reagents and therefore may overestim ate the oxygen concentr ations as well as erroneously detecting oxygen fo r the nitrogen treatment. Xylella vs. an Obligate Aerobe and a Facultative Anaerobe The growths of Xf stains UCLA, STL and Tulare-ALS (Figure 2-3a) was compared to Pseudomonas sp, an obligate aerobe and Erwinia sp, a facultative anaerobe (Figure 2-3b) in PW+ at various oxygen levels. The patte rns of growth for all three of the Xf strains under the four gas treatments were similar in that the optical density and biofilm droppe d as a function of the reduction of the oxygen levels. The growth of the obligate aerobe ( Pseudomonas sp) only occurred under the 21% oxyge n levels. The optical density of the facultative anaerobe decreased as a function of the oxygen levels. It should be noted that the growth from an OD of 0.1 to 0.2 for the Pseudomonas could be attributed to a delay prior to the gas treatments. However it would appear that there was no growth after the subseque nt gas treatments, as indicated by the lack of growth of another obligate aerobe, Xanthomonas campestris (Figure 2-5), under nitrogen treatments. Xylella vs. Xanthomonas The comparison of growth rates, in nutrient broth, of two putative obligate aerobes, X. fastidiosa and the closely related X. campestris pv. campestris under aerobic and hypoxic conditions show that there was a difference in the response of these two bact eria. The growth of X. fastidiosa Pierces disease strain Temecula (Figure 2-4) (as well as UCLA and the ALS strain Tulare, data not shown) showed a c ontinued growth under th e aerobic (air) treatment; however, the anoxic (nitrogen) tr eatment also showed steady growth in the nutrient broth. X. campestris pv. campestris (Figure 2-5) was able to grow under aerobic treatment as evidenced by the rise in the optical density, whereas it wa s unable to grow under th e anoxic conditions as

PAGE 40

40 indicated by the negligible slop e of the line (this was also seen in other experiments conducted with X. campestris pv. vitians data not shown). Defined Media Growth Xf Temecula when grown under anoxic and aerobic conditions in defined media had continual growth over thirty days of gas treatments in both media. The changes in the optical density for Xf in CHARDS media (Figure 2-6a) under the gas treatm ents were not significant as a function of gas treatment. The biofilm pr oduction in the CHARDS (Figure 2-6b) was not significant. In the XDM2-PR media growth and biofilm production (F igure 2-7a and 2-7b) increased significantly under the nitrogen gas treatment. Organic Acid Analysis Organic acid profiles of CHARDS and XDM 2-PR media varied between the air and nitrogen treatments. Comparisons of the peak areas at each retention time (which correspond to a compound eluting from the HPLC column) are shown in Tables 2-2 and 2-3. The rows of zeros are compounds that vanished either as a f unction of bacterial growth or as a function of time when the 15 day old media was compared to da y 0, which were used to determine the initial optical densities as well as the negative contro l which had no bacteria in it.. Both media had significant differences associate with presence of bacteria and the gas treatment as well as the interaction of both. Discussion Based on the results of the agar deeps Xylella fastidiosa appears to prefer a reduced oxygen atmosphere based on the initial growth (arro ws in Figure 2-1a) below the surface. When the tubes were allowed to incubate the growth c ontinued down to the bottom of the tube, and did not occur on the surface. The growth at th e area below the surface would increase thereby creating a biologically active barrier, thereby util izing the oxygen as it diffused into the agar

PAGE 41

41 deep, and preventing the transfer of oxygen fr om the surface to ar eas under the growth formation. In the agar deep experiment, when gas exchange allowed oxygen to remain at the 21% levels, using the slip-cap tubes, growth never developed on the surface of the agar, but always just below. However, on occasion colonies would form on the surface for sealed tubes of the agar stabs but usually after many weeks. Thes e growth patterns indicate a negative effect of either a gaseous environment or high (21%) oxygen levels. The growth of the three Xf strains in various oxygen concen trations (Figur e 2-3a) shows a gradual step down growth pattern were the gr eatest optical density was under the 21% oxygen treatment and dropped with the subsequent 2.1% oxygen and the 0.21% and 0% treatments, as indicated by the ANOVA analysis and Duncan sepa rations. This growth pattern was not similar to the growth pattern of the obligate aerobe, Pseudomonas sp (Figure 2-3b) where growth only occurred in the 21% oxygen treatment. The grow th pattern was very similar to the facultative anaerobe, Erwinia sp (Figure 2-3b), which also has an incremental growth pattern down the oxygen gradient except for the 0% oxygen treatmen t were the anaerobic metabolism allowed for an increased growth compared to the 0.21%, which may indicate th e oxygen labile nature of the anaerobic machinery. The comparison of these gr owth patterns raised the question as to the respiration classification of Xf as an obligate aerobe. The comparison of growth between Xylella (figure 2-4) and its close relative, Xanthomonas (Figure 2-5) indicated that there was ev en more of a reason to doubt the obligate aerobic status of Xylella as stated by Wells et al. 1987. The growth pattern for Xylella under the gas treatments of air was as expected with c ontinuous growth; however the presence of steady growth after 15 days in the hypoxic treatments (n itrogen) was quite remarkable (these results were similar for two other Xylella strains, UCLA-PD and Tulare-ALS, data not shown). There

PAGE 42

42 was more growth for the aerobi c (air) treatment verses the hypoxic (nitrogen) treatments. Aerobic respiration produces a bout 32 to 34 ATP (per glucose molecule) whereas fermentation tends to produce 4 ATP (per glucose molecule). Xylella has the genetic capability for sulfate reduction (M. A. Van Sluys et al. 2003; Simpson et al. 2000); however, end products such as hydrogen sulfide or elemental sulfur (o r iron sulfide) were not detected. The growth of Xf in CHARDS was not significantly different as measured by optical density and biofilm formation; however it should be noted that growth was steady and continuous. However the growth in the XDM2-PR media was significantly different, in that there was an increase in optical density as well as biofilm form ation under the anoxic (nitrogen) treatment verses the aerobic (air) treatment. Analysis of the tw o defined media after the growth experiment yielded more evidence for the possib ility of anaerobic capabilities (Tables 2-2 and 23) as indicated by the differing organic acid profil es, due to the interaction of the bacteria and gas treatments. The bacteria and gas treatment interactions indicate that there is a change in metabolic activities as a function of oxygen availability. This switch in metabolism is could be a change from oxidative phosphorylation which us es oxygen as a terminal electron acceptor to a fermentation pathway, which uses the substrate as the electron donor as well as the electron acceptor. During the growing season the dissolved oxyge n in the xylem fluid becomes low enough to induce fermentation within the plants tissues (Kimmerer & Stringer, 1988), with the results presented here it was likely that Xylella fastidiosa was capable of the same. The ability of Xf to modify its metabolism and/or respiration pathwa ys implies that during the growing season when the oxygen levels drop and yet the demand becomes greater that the bact eria are capable of continuing their growth and development. Not on ly are they apparently capable of sustained

PAGE 43

43 growth, but biofilm production incr eases as well, which probably leads to the onset of disease symptoms in late summer and early fall (Purcell & Hopkins, 1996). Goodwin et al, 1988, investigated the presen ce of possible toxic compounds generated by Xf ; however the results were based on the washes of aerobically gr own cultures on solid media. The results of our experiment s dispute or raise doubts rega rding the production of toxic compounds (Goodwin et al. 1988). Since Xf has always been considered to be an obligate aerobe (Wells et al. 1987) studying the different metabolic by-pr oducts, as a function of fermentation, which could either bring about tylosis formati on, for which no known trigge r exists, by the plant or simulating the symptoms of the disease, such as the abscission of the l eaf from the petiole and green islands or the lack of cork production (Esau, 1948; Krivanek et al. 2005). These results, the continued growth as well as changes in the orga nic acid profiles under the anoxic treatments raise serious questions regarding Xylellas oxygen requirements for growth. The strains UCLA and Tulare show ed growth under the a noxic conditions in the nutrient broth (data not shown), si milarly to the Temecula strain. Other evidence includes three other strains of Peirces disease, Conn Creek, Santa Cruz, STL, and another almond leaf scorch Dixon as soft agar stabs (data not shown) which indicates that th is anaerobic ability may include many more (or all the ) strains. The differences in the metabolic by-products may also indicate that there is a compound produced by the bacteria which may in turn be a toxin or a stimulator for disease symptoms in the plant environment. We are in the process of naming as well as quantifying the organic acid data with HPLC-MS (D r. Jodie Johnson, University of Florida) and will hopefully be able to identify the fermentation substrates as well as end products.

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44 Table 2-1. Concentrations of oxyge n found in various xylem fluids Species (Conditions) DO (mol L-1) DO (% air-saturated solution) DO (% Air) ppm (g L-1) Reference Ricinus communis 95.8 33.3 7 3.1 (Dongen, et al. 2003) Betula pendula 42 to 230 14.6 to 80 3.1 to 16.8 1.3 to 7.4 (Gansert, et al. 2001) Olea europea (day) 80 to 90 27.9 to 31.3 5.8 to 6.6 2.6 to 2.9 (Mancuso, Marras 2003) O. europea (night) 20 to 30 9.96 to 10.4 1.46 to 2.19 0.64 to 0.96 (Mancuso, Marras 2003) Picea abies (irrigation) 68.5 23.8 5 2.2 (Eklund 2000) P. abies (no irrigation) 13.7 to 41.1 4.76 to 14.3 1 to 3 0.43 to 1.3 (Eklund 2000) P. abies (drought) <13.7 <4.76 <1 <0.43 (Eklund 2000) P. abies (winter) 273.8 95.2 20 8.8 (Eklund 2000) Fagus orientalis (day) 58.36 20.3 4.26 1.87 (Hierro, et al. 2002) F. orientalis (night) 50.31 17.5 3.68 1.61 (Hierro, et al. 2002) Laurus nobilis 51.75 18* 3.78 1.66 (Hierro, et al. 2002) L. nobilis (flooding) 37.38 13* 2.73 1.20 (Hierro, et al. 2002) = The data as it was presented in th e primary literature. The conversions were made assuming a temperature of 20 C, and that 288 mol L-1 = 100% air-saturated solu tion = 21% air = 9.2 ppm.

PAGE 45

45 Figure 2-1. Growth of Xylella fastidiosa in agar deeps to determine oxygen limitations A) Initial growth of Xylella fastidiosa (center three tubes with arrows, left to right UCLA, STL, and Tulare (almond leaf scor ch)) after 5 days of growth, when compared to the growth of Pseudomonas sp. (far left), an obligate aerobe and Erwinia B C A

PAGE 46

46 sp. (far right), a facultative anaerobe. B) Growth of Tula re (almond leaf scorch) after two weeks of growth with the left tube grown under nitrogen gas, the middle tube under air and the right tube is an un-inoc ulated deep. C) Growth of Temecula (Pierces disease) after two weeks with the left tube grown under nitrogen gas, the middle tube under air and the right t ube is an un-inoculated deep.

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47 0 1 2 3 4 5 6 7 21%10%2.10%0% Oxygen Levels ppm dH20 CHARDS Day 1 CHARDS Day 3 CHARDS Day 6 Figure 2-2. Dissolved oxyge n levels detected in dH2O and CHARDS media as a function of treatment levels and over time. The dissolved oxygen levels in dH2O were determined after one treatment. Each poi nt is the average of three replicates.

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48 0 0.1 0.2 0.3 0.4 0.5 0.6 21.00%2.10%0.21%0.00%21.00%2.10%0.21%0.00%21.00%2.10%0.21%0.00% UCLASTLALS OD OD Biofilm -0.2 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 21.00%2.10%0.21%0.00%21.00%2.10%0.21%0.00% PseudomonasErwinia OD OD Biofilm Figure 2-3. Comparison of Xylella fastidiosa cultivated under reduced oxygen levels. A) The comparison of growth of three Xylella fastidiosa strains (two Pierces disease, STL and UCLA, and Tulare, almond leaf scorch ) under different oxygen levels, each with an initial optical density of 0.05 were inc ubated for 15 days. B) The comparison of A B a b c c a a b b c c c c c c b b b b a a

PAGE 49

49 growth of an obligate aerobe ( Pseudomonas sp) and a facultative anaerobe ( Erwinia sp), under different oxygen levels, each with an initial optical density of 0.1 were incubated for 15 days. Error bars represen t standard error and the optical densities were separated using Duncans.

PAGE 50

50 y = 0.026x + 0.0069 y = 0.0068x + 0.046 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45 0246810121416 Days OD Air Nitrogen Linear (Air) Linear (Nitrogen) Figure 2-4. Xylella fastidiosa growth in PW+ for 15 days un der aerobic and hypoxic conditions. Each point is the average of three replicates.

PAGE 51

51 y = 0.1979x 0.0325 y = -0.0007x + 0.0789 0.0 0.2 0.4 0.6 0.8 1.0 1.2 0123456 Days OD Air Nitrogen Linear (Air ) Linear (Nitrogen) Figure 2-5. Xanthomonas campestris pv. campestris growth in PW+ for 5 days under aerobic and hypoxic conditions. Each point is the average of three replicates. .

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52 0 0.05 0.1 0.15 0.2 0.25 05101530 Days OD CHARDS Air CHARDS Nitrogen Figure 2-6. Growth and performance of Pierces disease strain Temecula in the CHARDS media under two gas treatments. A) is optical de nsity and B) is biofilm formation under air (open squares) and nitrogen (closed square s) gas treatments. Each point is the average of three replicates. 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 5 10 1530 Days Biofilm CHARDS Air CHARDS NitrogenB A

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53 0 0.05 0.1 0.15 0.2 0.25 05101530 Days OD XDM2-PR Air XDM2-PR Nitrogen 0 0.02 0.04 0.06 0.08 0.1 0.12 0.14 0.16 5101530 Days Biofilm XDM2-PR Air XDM2-PR Nitrogen Figure 2-7. Growth and performance of Pierce s disease strain Temecula in the XDM2-PR media under two gas treatments. A) is optic al density and B) is biofilm formation under air (open squares) and nitrogen (closed squares) gas treatments. Each point is the average of three replicates. A B

PAGE 54

54 Table 2-2. Organic acid profiles in the CHARDS media after 15 days with Xylella fastidiosa without X. fastidiosa Significance Retention times a Air Nitrogen Air Nitrogen Bacteria Gas B*G 6.646 151.67 360.67 1074.67 354.67 ** 6.906 1796.00 1198.00 1403.00 1265.33 ** 7.586 165606.00 152676.33 117838.00 146047.67 8.019 11530.67 18154.33 0.00 0.00 **** ** ** 8.457 8551.33 8666.00 0.00 0.00 **** 9.322 10307.33 8147.33 6731.67 9277.67 10.643 b 0.00 0.00 0.00 0.00 11.027 2736.33 3296.33 1706.33 4685.33 11.405 14469.67 0.00 15258.67 21957.33 ** ** 11.54 0.00 14432.33 0.00 0.00 * 11.834 0.00 0.00 6483.67 9962.67 ** 12.429 2093.00 0.00 153436.67 208947.67 **** 12.588 0.00 852.67 0.00 0.00 12.633 15805.33 9539.33 0.00 0.00 ** 13.226 0.00 0.00 1973.00 0.00 13.494 35029.33 31094.00 24227.33 35293.67 14.462 1020.33 0.00 0.00 0.00 15.33 36966.67 9077.33 2563.33 13060.67 *** ** **** 15.791 10655.33 10573.67 10363.67 11561.67 16.515 21332.67 22276.00 25589.33 32490.67 ** 17.242 34096.33 33374.67 25217.00 33577.00 17.53 22030.00 21202.00 18546.67 24383.67 18.429 11610.00 11330.33 10987.67 14889.00 19.208 10286.67 10284.00 9753.00 128259.33 19.743 27483.67 24209.67 16215.33 21433.67 20.363 0.00 0.00 0.00 0.00 21.062 0.00 2363.00 0.00 0.00 *** *** *** 21.797 19638.33 22025.00 17241.67 22095.67 24.309 900.67 1381.00 0.00 0.00 25.000 2395.33 806.33 467526.00 602806.67 **** 25.937 1710.33 0.00 0.00 0.00 27.383 0.00 2473.67 0.00 0.00 ** ** ** 27.993 10129.33 17086.33 17819.00 20589.67 * 36.736 24926.67 39429.33 1203003.67 35877.67 a = Retention time on HPLC column. b = Presence of a peak found at day 0, the initial samples (data not shown). P-values are re presented = 0.05, ** = 0.01, *** = 0.001, **** = 0.00001.

PAGE 55

55 Table 2-3. Organic acid profiles in the XDM2-PR media after 15 days With Xylella fastidiosa Without X. fastidiosa Significance Retention times a Air Nitrogen Air Nitrogen Bacteria Gas B*G 6.597 1426.33 548.00 1051.33 548.50 6.844 1575.33 1182.67 1347.33 1286.50 ** 7.58 177952.00 231268.67 153846.00 132244.00 8.02 6547.33 20048.33 0.00 0.00 *** ** ** 8.457 7740.67 18153.33 0.00 0.00 **** ** ** 9.338 6482.67 9041.67 4630.33 4085..5 ** 10.297 b 0.00 0.00 0.00 0.00 10.636 0.00 0.00 0.00 0.00 11.046 445.33 5638.00 760.00 1436.00 11.375 4620.67 0.00 2436.00 2066.00 **** **** **** 11.78 0.00 6830.67 0.00 0.00 12.395 60088.33 0.00 72173.00 62718.50 * 12.655 0.00 12197.33 0.00 0.00 * 13.162 7901.67 8628.00 4443.00 3516.00 13.722 22658.33 26896.67 11057.33 9664.00 ** 14.434 3056.67 4751.33 0.00 0.00 **** * 15.345 0.00 0.00 0.00 0.00 15.437 599108.33 0.00 270378.67 0.00 ** 15.546 0.00 489081.67 0.00 241595.00 **** **** **** 16.359 0.00 0.00 0.00 0.00 16.511 18813.33 39624.00 3791.00 8849.50 *** *** 17.265 78816.00 683194.67 11843.67 13438.00 **** **** **** 17.544 0.00 0.00 11792.00 12036.50 ** 18.002 0.00 7660.00 4107.33 5883.00 ** 18.319 16433.67 25531.67 0.00 0.00 ** 18.433 0.00 0.00 5697.67 6892.00 ** 19.218 7353.33 12031.00 5430.67 5851.50 19.726 21426.00 54879.67 9372.33 882.00 *** ** 21.333 3739.00 0.00 0.00 0.00 * 21.561 0.00 179943.33 0.00 0.00 **** **** **** 21.749 12142.33 0.00 3212.33 1382.00 ** 22.157 8151.33 19473.00 2221.00 0.00 **** ** 24.291 4701.67 0.00 2038.00 1662.50 ** ** 24.742 0.00 22175.67 0.00 0.00 **** **** **** 25.018 0.00 0.00 6744.00 5403.50 *** a = Retention time on HPLC column. b = Presence of a peak at initi ation of the experiment, time point 0. P-values are represented = 0.05, ** = 0.01, *** = 0.001, **** = 0.00001.

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56 Table 2-3. Continued With Xylella fastidiosa Without X. fastidiosa Significance Retention times a Air Nitrogen Air Nitrogen Bacteria Gas B*G 26.442 0.00 1601.00 0.00 0.00 ** ** ** 27.099 0.00 2754.33 0.00 0.00 27.985 12253.33 15074.33 14736.33 10256.00 28.555 1656.33 15492.00 875.33 0.00 *** *** *** 36.73 23171.00 30152.00 12734.67 10931.50 ** a = Retention time on HPLC column. b = Presence of a peak at initi ation of the experiment, time point 0. P-values are represented = 0.05, ** = 0.01, *** = 0.001, **** = 0.00001.

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57 CHAPTER 3 MEDIA COMPONENT EFFECTS ON GROW TH AND BIOFILM FORMATION OF Xylella fastidiosa Introduction Xylella fastidiosa ( Xf ) is a Gram negative bacillus bacterium which resides in the xylem vessels of many plants. The bacterium is vector ed between host plants by xylophagus insects, such as the glassy-winged sharpshooter [ Homalodisca coagulate (Say)] (Purcell & Hopkins, 1996). It is believed to cause diseases in it s host by restricting the transpiration stream by clogging the xylem elements and/or stimulating the plant to form tyloses, protrusions from the parenchyma cells which plug the xylem elements (Esau, 1948). Xf consists of many strains which reside in many different hosts and in various environmental conditions throughout the Americas. One of the greatest difficulties in working with Xf is the variance among the strains such as doubling times (Wells et al. 1987), biofilm production, and nutritional requirements (Leite et al. 2004; Lemos et al. 2003). There is also variance in pathogenicity ( in vivo ) that develops as the bacteria is sub-cultured (Hopkins, 1984)), which could be due to a reducti on in fimbriae production which w ould be a reduction in adhesion and subsequent biofilm production in vitro Several defined media have been created for the cultivation of Xf In recent years two media had been created which we re based on the genomic (Lemos et al. 2003) and xylem chemistry (Leite et al. 2004). Lemos et al., 2003 created a defined media series (XDM) based on the genomic capabilities of the citrus variegated chlorosis pathovar of Xf (and confirmed its ability to cultivate other strain s such as Pierces disease (PD) which contained 4 amino acids, glucose, magnesium sulfate, and iron pyrophos phate as well as vitamins suspended in a phosphate buffer. Leite et al 2005, produced a defined media (CHARD2) based on the chemical

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58 composition of Vitis vinifera Chardonnay which consist of 5 amino acids, magnesium sulfate and iron pyrophosphate suspended in a phosphate buffer. A concern about these media is that they contain phenol red (phe nolsulfonephthalein) which is commonly used as a pH indicator in bact erial media (also as a color indicator used to test kidney function) but has no (known) biological function. Phenol red is normally a colorimetric pH indicator which changes from br ight yellow in acidic conditions, to dark red in alkaline, and it does indicate that Xf does produce an alkaline product as a function of growth in PW+ media. The structure of phenol red consists of two phenols and a benzene motif attached to a central carbon atom along with a sulfite group, any part of which could interact with the receptors and sensors associated with the bacter ia. The question arises as to the function of phenol red as a necessary additive to these define d media, as well as the nutrient media such as PW+ (Davis et al. 1981) PWG and PD2 (American Type Culture Collection, www.atcc.org). The two media XDM2-PR (a variant of XDM2 which lacks phenol) and CHARDS (a variant of CHARD2 which has the addition of soluble starch as well as lacks phenol red) have many similarities such as a phosphate buffer, magnesium sulfate, iron pyrophosphate and an abundance of glutamine, as well as a similar pH, 6.8 to 6.6, respectively. However there are a couple of differences which should be noted such as the presence a carbon source, glucose, vitamins, and the amino acids. Can the differences in optical density and biofilm formation (as seen in chapter 2) between the tw o media be explained as a functi on of these differences? So we will examine the effects of glucose and vitamins on the growth of Xf in the CHARDS background. By culturing the bacteria in the defined media it is possible to analyze the utilization of the constituent amino acids. When the bacteria was grown in xylem fluid from grapevines

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59 (unpublished data) many of the amino acids correl ated with growth were ones which were found at low concentrations (Andersen et al. 2005). Materials and Methods Bacteria Used The Xylella fastidiosa strains were obtained from Dr. Purcell and Christina Wistrom (UC Berkeley). The Xf strains used were: Temecula-PD, UCLA-PD, STL-PD, and Tulare, an almond leaf scorch strain. Cultivation Protocol Bacterial suspensions of PW+ and 10% glycerol were stored in a -80C freezer (Baxtor Scientific Products, Cryo-Fridge). Initial cultures were starte d by scrapping a sterile wooden applicator stick over the frozen stock culture and streaked out onto a BCYE (Buffered Charcoal Yeast Extract) plate. After 10 days bacterial growth was scraped off with an inoculation loop and placed into 5 mL of PW+ and incubated between 10 to 14 da ys, (until an optical density of 0.2 at 600nm, which corresponds to the mid-log phase) before being used to make working cultures. These 5 mL cultures we re transferred to 50 mL of PW+ media in 250 mL Erlenmeyer flasks with cotton plugs or sili cone rubber plugs with a filter to allow gas exchange. All incubations were done in New Br unswick incubator (model# G25-R) set at 28C and all liquid cultures were placed on an orbital shaker set to 150 rpm. Experiments were carried out in 15 mL polypr opylene Falcon tubes with re-rubber septa or screw caps and 16 X 150 mm glass test tubes with slip cap closures. The media included an enrichment media, PW+, the defined media CHARD2 (Leite et al. 2004) which is based on xylem fluid chemistry of Vitis vinifera Chardonnay, and the variant CHARDS, and the defined media XDM2 (Lemos et al. 2003)), which is based on the genomic needs of the citrus variegated chlorosis strain.

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60 PW+ Media, Broth and Solid PW+ media (Davis et al. 1981) was made in two solutions which were then combined. Solution A consisted of (for 1L) 900 mL dH2O to which these components were added, 1.0 g soluble starch, 4.0 g soytone, 1.0 g tryptone, 1.2 g K2HPO4, 1.0 g KH2PO4, 0.4 g MgSO4-7H2O, 0.85g (NH4)2HPO4, 1.0 g histidine, 25.0 mg cyclohexami de, 10.0 mL (0.1%) hemin-Cl, 10.0 mL (0.2%) phenol red. Solution A was sterilized by autoclaving. Solution B was made by dissolving 4.0 g of glutamine into 50 mL dH2O at C, and 6.0 g bovine serum albumin (fraction V) into 30 mL of dH2O, which were then combined. Next, solution B was sterilized through a 0.22 m filter. The two solutions we re then added to form the completed broth medium. PW+ agar plates were made by adding 12.0 g agar to solution A before sterilization in the autoclave. Solution B was then added when solution A cooled down to 55C in a hot water bath. Then PW+ medium was aseptically ali quoted into Petri dishes. PW+ soft agar deeps were made using 2.0 g of agar in solution A before ster ilization in the autoclav e. Solution B was then added when solution A cooled down to 55C in a hot water bath. The soft agar was then dispensed aseptically into the appropriate te st tubes, either the polypropylene or glass. The BCYE (Buffered Charcoal Yeast Extract) Agar Plates This is the media used by Wells et al 1987, for the initial growth of the Xylella strains (ATCC medium 1099 CYE; buffered (Cha rcoal yeast extract) medium). Solution A is made by mixing 0.4 g Lcysteine-HCl into 10 mL of dH2O and sterilized through a 0.22 m filter. Solution B is made by mixing 0.25 g ferric pyrophosphate into 10 mL of dH2O and sterilized through a 0.22 m filter. Solution C is made by mixing 10.0 g of ACES buffer (n-2-acetamido-2-aminoethane sulfonic acid into 250 mL of dH2O. The pH is adjusted to 6.9 +/0.05; by this means the chemical will go in to solution. Solution D consisted of 10 g of yeast extract was added into 730 mL of dH2O, 2.0 g of charcoal, 17.0 g of agar. pH was adjusted

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61 to 6.9 +/0.05 using KOH. The media was autoclav ed to sterilize and then tempered to 50C in a water bath. Solutions A, B, C, and D were added aseptically. The media was dispensed into Petri dishes and gently swirled to keep the charcoal in suspension thereby ensuring even distribution. CHARDS Medium CHARDS was a modified version of CHARD2 which was a defined media based on the xylem fluid chemistry of Vitis vinifera Chardonnay (Leite et al. 2004). Solution A consisted of (for 1 L) 819 mL of dH2O to which these components were added, 1.0 g KH2PO4, 1.5 g K2HPO4, 0.2 g MgSO4-7H2O, 0.17 g alanine, 0.58 g aspart ic acid, 1.8 g glutamine, 1.05 g arginine, 0.01g cysteine-H Cl, 1 mL (1 mg/ L) biotin, and 1.0 g of soluble starch. The pH was then adjusted to 6.6 using concentrated KOH an d then sterilized by autoclaving. Solution B consisted of 0.25 g of ferric pyrophosphate dissolved in 60 mL dH2O and 4.0 g glutamine dissolved in 50 mL dH2O at C which was then combined and was sterilized through a 0.22 m filter. The two solutions were then combined, aseptically, to form the completed media. CHARDS-F is CHARDS but autoclavi ng the starch only in 750 mL of dH2O and the rest of the ingredients were combined into 250 mL of dH2O, which was filtered through a 0.22 m filter and added aseptically to the starch solution. CHARDS-G is CHARDS without the starch but using 10 g of glucose instead. The glucose was dissolved in 500 mL of dH2O and filtered through a 0.22 m filter and the other constituents were brought in 500 mL a nd autoclaved, then added aseptically. CHARDS-V is CHARDS with the addition of 10 mL of the XDM2 vitamin solution. XDM2 and XDM2-PR Media XDM2 is a defined media based on the genome of X. fastidiosa pv. citrus variegated chlorosis (Lemos et al. 2003)). For 1 Liter, into 980 mL of dH2O these components were added

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62 10.0 g glucose, 2.1 g K2HPO4, 0.8 g KH2PO4, 0.4 g MgSO4-7H2O, 0.125 g ferric pyrophosphate, 4.0 g glutamine, 1.0 g asparagine, 0.4 g serine, 0.4 g methionine, 10 mL (1%) phenol red and 10 mL of vitamin solution. Then the pH was adju sted to 6.6 and then f iltered through a 0.22 m membrane. For the creation of XDM2-PR, phenol red was omitted from the recipe. The vitamin solution consisted of (for 100mL) 350.0 mg myo-inositol, 10.0 mg pyridoxine-HCl, 10.0 g thiamine, 5.0 mg nicotinic acid, and 0.2 mg of D-biotin and was sterilized through a 0.22 m filter and stored at 4C in a light-protected bottle. Grams Staining Grams staining protocol was conducted on a sample of cells, either a single colony applied to a glass slide or 1 mL of liquid cult ure spun down and re-suspended in 100 l sterile dH2O, because of interference caused by some of the media constituents, and 20 l placed on a glass slide and allowed to air dry. Then, the bacterium on the glass slide was heat fixed by passing the slide over a B unsen burner three times. Once cool, crystal violet was applied on to the fixed bacteria for one minute, a nd then the slide was rinsed with dH2O. Next, iodine was placed onto the culture for one minute, and then the slide was rinsed with dH2O. The slide was then de-stained in a 4:1 ethanol: acetone mixture, for thirty seconds and then the slide was rinsed with dH2O. Finally, safranin was applied to the bact eria for one minute, and then the slide was rinsed off with dH2O. Gram positive bacteria stain purple due to the crystal violet, iodine compound, where the Gram negative bacteria stain pink with the lighter stain basic safranin. PCR Protocol Once purity had been established by Grams stain, PCR (polymerase chain reaction) was used to confirm the identity of the cultures. DNA extractions were c onducted using the Quiagen DNeasy mini prep. The PCR protocol used a nested primer technique (Pooler & Hartung, 1995; Pooler et al. 1997)). The first reaction used the primers 272-1 (5-AGCGGGCCAATATTCAA-

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63 TTGC-3) and 272-2 (5-AGCGGGCCAAAACGATGCGT G -3). The final concentration of reagents for a 20 l reaction was 1X PCR Buffer, 2.0 mM MgCl2, 0.2 mM dNTPs, 0.5 M primer 272-1, 0.5 M primer 272-2, and 1 U Taq. Th e second reaction used the primers 272-2int (5-GCCGCTTCGGAGAGCATTCCT-3) and CVC-1 (5-AGATGAAAACAATCATGCAAA3). The final concentration of reagents fo r a 20 l reaction was 1X PCR Buffer, 2.0 mM MgCl2, 0.2 mM dNTPs, 0.3 M primer CVC-1, 0.3 M primer 272-2int, and 1 U Taq. The thermocycler protocol, same pr otocol for both reactions was 94 C for 2min, then 35 cycles of 94 C for 1 min, 62 C for 1 min, and 72 C for 1.5 min, followed by a final extension at 72 C for 5 min. The reactions were stored at 4 C until they were assayed by electrophoresis through 1% agarose gel slab and stained with ethidium brom ide. A positive reaction is indicated by the appearance of a band at ~500bp. Growth Conditions Mature bacterial cultures, between two week s and four weeks, were removed from the incubator. The optical dens ity of the culture was obtaine d, using a wavelength of 600 nm. Aliquots were placed into st erile polypropylene/glass tubes and centrifuged at 2380 x g for 15 minutes. The supernatant was decanted and th e pellet was resuspended into 3 mL of PBS (phosphate buffered saline, 0.1 M phosphate and 0.8% NaCl and a pH of 6.8). The suspension was then centrifuged again. The supernatant was discarded and th e pellet resuspended into the appropriate media. This was the centrifuged as before and the supernatan t discarded. The pellet was resuspended into 5 mL of th e appropriate media. Then the tubes were capped appropriately, either slip caps for the glass tubes, or plastic screw caps or red-rubber se pta and parafilm for the 15 mL polypropylene Falcon tubes. The red-rubber septa capped tubes were then subjugated to gas treatments.

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64 Gas treatments were done each day. The tops of the septa were sterilized by rinsing with 70% isopropyl alcohol and dabbed dry with a k imwipe. The gas was passed through a 0.22 m syringe filter then injected through a one inch, sterilized 22 gauge needle, and then another sterilized 22 gauge needle was placed into the se pta to act as an exhaust port to ensure gas exchange. Gas was passed through at ~5 psi for 5 minutes. The tubes were placed in the New Brunswick incubator set at 28 C, on an orbital shaker set at 150 rpm. Optical Density (OD) Optical density is a quantitative measurement of the concentration of planktonic bacteria in solution based on the absorption of light through a liquid culture. OD was measured using a Genosys 8 spectrophotometer at a wavelength of 600nm. The machine was calibrated using an aliquot of sterile media or suspension buffer. Then an aliquot of the suspension was placed into the cuvette and measured. Biofilm Qualification Biofilm is a qualitative measurement of the c oncentration of bacteria that have become sessile on the tube or flask in whic h they were grown (Espinosa-Urgel et al. 2000)). The bacteria were decanted and the vessel was rinsed with dH2O and air dried. The vessels were then filled with 0.1% crystal violet, and placed on a shaker for 1 hour. The crystal violet was decanted and the tubes were rinsed until all the excess dye has been removed and air dried. The vessels were then de-stained using 70% isopropan ol. An aliquot will then be placed into a cuvette and the optical density was measured at a wavelength of 600nm. The spectrophotometer was calibrated using a cuvette filled with 70% isopropanol. Media Analysis Xf strain Temecula was inoculated into C HARDS and XDM2-PR media by dispensing 1 mL of mature culture (~0.35 OD at 600nm) into 15 mL polypropyl ene tube and centrifuged at

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65 2,380 g for 15 minutes. The supernatant was discar ded and the pellet was resuspended in 3 mL of PBS. This was centrifuged at 2,380 g for 15 minutes and the supernatant was discarded. The pellet was resuspended in 2 mL of the appropriate media and th en centrifuged at 2,380 g for 15 minutes. The supernatant was discarded and the pellet was resuspended in 5 mL of the appropriate media and sealed with red-rubber sept a and sealed with parafilm. The tubes were then subjected to gas treatment which consis ted of various concentrations of oxygen, 21% (breathing air) and 0% (nitrogen) for 5 minutes every day for the duration of the experiment. Each treatment consisted of inserting a syringe needle connected to a compressed gas tank with a positive pressure of ~5 psi through the rubber-septa and an exhaust needle was inserted to allow for gas exchange. Three tubes from each media-gas interaction we re removed every five days and assayed. The optical density was obtained by placing 1 mL into a cuvette and absorbance was measured at 600 nm. A drop was placed on PW+ agar media and incubated at 28C for 10 days to determine the viability of the culture. A two m illiliter sample from each tube was centrifuged at 5,000 g at 4C for 15 minutes (Sorval Biofuge Model: Stratos), temperature regulated centrifuge), then the supe rnatant was stored at -80C. Then the tubes were emptied and assayed for biofilm. Amino acid analysis required a 1:50 dilution of the samples prior to pico N tagging. Tagging consisted of drying a 30 l sample of eac h media-gas treatment in a speed-vac. Then the pellet was resuspended in 100 l of 2:1:1 (100% ethanol: triethylamine: dH2O), in order to adjust the pH, then speed-vac dried. The pelle t was resuspended in 30 l of 7:1:1:1 (100% ethanol: triethylamine: dH2O: phenol isothiocyanate) and in cubated under nitrogen gas for 20 minutes at room temperature. After this incuba tion the sample is then speed-vac dried for over

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66 two hours (it is very important that this be very dry). The pellet is resuspended in the suspension buffer (5 mM sodium phosphate (monobasic) and 6% acetonitrile) and th en 20 l was loaded onto a Pico tag column (Waters) eluted with a proprietary gradient of Buffer A (14 mM sodium acetate-trihydrate and 0.05% triethylamine at a pH of 6.4) and Buffer B ( 60% acetonitrile) at a flow rate of 1.0 mL per minute at 38C. Produc t was detected at 253 nm which corresponds to the pico N tag. Statistical Analysis Data was analyzed with ANOVA, t-tests, mean s and standard error as well as regression using the SAS V9.0 software package. The error bars represent standard error. The letters represent separation by Duncans. Results Phenol red has a severe effect upon the form ation of biofilm within the XDM2 media. Concentrations (Table 3-1) as low as 0.02% inhi bited the formation of biofilm, and the increase to 0.1% had no increased effect on inhibiting biofilm formation. The effect was seen across the three strains used (Table 3-2). However the effect on the optical density was not consistent, in Table 3-1, the lack of phenol red increased the optical de nsity of Temecula, but in Table 3-2, it had a negative effect on UCLA and Temecula, but was not significantly different for STL. The comparison of the CHARDS variants (Fi gure 3-1 a and b) indicates that neither vitamins, nor filtering had any significant effect u pon the growth of the bacteria. However there was a significant negative effect of glucose in the air treatment wh ich retarded the optical density as well as biofilm production, wher eas the nitrogen treatment produced more biofilm then the air treatment, but still had a signifi cant reduction in optical density. The CHARDS media (Table3-4) was unaffected by the gas treatment so the gas treatment data was pooled. The bacteria produced significant subtractive differences in aspartic acid,

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67 glutamine, and cysteine, which were some of th e added constituents of the CHRADS media, and the amino acid that was significant due to an increase were glutamic acid. There was no significant change in the alanine or arginine, two other constituen ts of the CHARDS media. The XDM2-PR media (Table 3-5) was affected by th e bacteria, gas and the interaction of the treatments. The bacteria significant modifica tions were glutamic acid, serine, glutamine, threonine, alanine, and valine. The modificatio ns based on gas were glutamic acid, glutamine, threonine and leucene. The signi ficances due to interaction were glutamic acid and threonine. The amino acid data (Tables 3-4 and 3-5) show that the amino acids liste d in the media are not exclusively the amino acids found in the media. Table 3-6 was a comparison of the individual amino acids used in the media, at the concentrations used in each media, with th e glutamine concentration used at the CHARDS amount. The amino acids were sterilized through a 0.22 m nylon filter, except for one of the glutamines which was autoclaved. Each ami no acid was labeled as being 98 to 99% pure. Asparagine, aspartic acid and methionine had va lues which were considerably different than those listed on their bottle labels. The concentra tion of cysteine used appears to be below the detection level of the HPLC detector. There wa s a difference in the glutamine concentrations due to the destructive nature of autoclaving. Discussion The use of phenol red in the defined media i nhibited the production of biofilm or sessile populations which may in turn increase to optical density or planktonic populations (Table 3-1). This effect was documented for Xf strains Temecula and UCLA, although there was no increase in optical density of the STL strain (Table 3-2) The production of more biofilm in the absence of phenol red could be answered in two ways either as an inhibitor for the bacteria to adhere to

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68 the surface and initiate biofilm production or inhibiting the production of exopolysaccharides or proteins that contribut e to biofilm formation. Biofilm production is considered as a protective barrier to a harsh environment, in that under stress inducing conditions a ba cterium will encapsulate itself in order to survive (Costern et al. 1999). Phenol red may promote a behavior th at causes the bacteria to remain in planktonic state. The phenol red may also act as an inhi bitor to biofilm production by interfering with the machinery which stimulates biofilm production, such as inhibiting the ability of the bacteria to adhere to a surface, either using the rings, wh ich may share a binding affinity for the lignin building blocks which are phenylalanine homologues, an by inhibiting the adhesion to a surface thereby inhibits biofilm production. Another possibility is that of sulfones which are used as to treat certain bacteria, such as Mycobacterium leperae (used with an antibiotic) and the urinary tract infectious strains of Escherichia coli through an ability to prevent the adhesion, maybe through inhibition of pili production (Vaisanen et al. 1982). In Figure 3-1 the augmentation of the CHAR DS media of filtering or the addition of vitamins had no significant effect upon the optic al density or biofilm production for the air and nitrogen treatments. However the switching of glucose for starch, as a proposed carbon source, had a negative impact upon both optical densit y and biofilm production, for both the air and nitrogen treatment. The negativ e effect of glucose was greater in the air treatment with the optical density actually decreasing from the init ial optical density of 0.05. Biofilm production was greater in the nitrogen treatments for the CHARDS variants; however this was not always the case with CHARDS with only about 70% of the experiments results ending that way.. The XDM2-PR media should have had a greater imp act on optical density and biofilm under the nitrogen treatments, as seen in Chapter 2, this lack of response could be due to the age of the

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69 media used in the experiment, which was seen in other experiments when the age of the media exceeded 1 month (data not shown). The amino acid utilization followed the growth experiment results as described in Chapter 2. The CHARDS media had no significant change s due to the gas treatments (Table3-4). The significance in the difference between glutamine a nd glutamic acid could be explained by the deanimation of glutamine by the bacteria to produce glutamic acid, which was the only amino acid which increased significantly. Other amino acids which were significantly reduced were aspartic acid and cysteine. The XDM2-PR media (Table 3-5) had significance due to presence of the bacteria as well as on the gas tr eatment, which could be associated with the growth and biofilm production (Chapter 2). The ami no acids which were significantly different as a function of the presence of the bacteria were glutamic acid, se rine, glutamine, threonine, alanine and valine. The gas treatment was significant for glutamic acid, glutamine, and threonine. The interactions which were significant were glut amic acid and threonine. The glutamine and glutamic acid response was similar to the CHARDS except th at the air (aerobic) treatment used more glutamine and the nitrogen treatment produced mo re glutamic acid, significantly. Threonine was only detected in the bacter ia and nitrogen treatments. The interesting point about the media experi ment was the detection of the other amino acids which were not added to the media but we re a component of the individual amino acid (Table 3-6). In CHARDS this had no effect exce pt for the glutamic acid, which is probably just the deaminated glutamine. However, in the XDM2-PR media alanine and valine were significant. The comparison of the amino acid concentrations (as percentages) between the recipes and uninnoculated media in the experiment al analysis in Table 3-6 and 3-3 and Table 3-

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70 7 and 3-4, respectively, were different which wa s caused by the impurities found the amino acids which were added to the media (Table 3-5). These result, the phenol red effect and the am ino acid constituents, show that even the supposed insignificant or minute compound can alter or contribute to the grow th and behavior of Xylella fastidiosa Since Xylella resides in, what can be considered, a sparse environment each media component becomes suspect and shoul d not be taken casually. Therefore Xylella fastidiosa is truly a fastidious ba cterium and a better understandi ng of its responses to the environment or media components will help in the study of the pathogenic qualities.

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71 Table 3-1. A Phenol red series experime nt done in the XDM2 media using Temecula % PR OD Biofilm 0% 0.120 .001 0.286 .204 20% 0.060 .002 0.030 .005 100% ** 0.064 .003 0.033 .006 The amount of phenol red used in CHAR DS. ** The amount of phenol red used by Lemos, et al. 2003 for the XDM series medi a. Each point is the average of three replicates

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72 Table 3-2. Effect of phe nol red on the growth and be havior on three strains of Xylella fastidiosa Strain Media OD Biofilm STL XDM2 0.046 .003 0.043 .003 XDM2-PR 0.041 .005 0.267 .007 Statistics P=0.3885 P=0.0001 Temecula XDM2 0.141 .012 0.042 .001 XDM2-PR 0.052 .003 0.207 .008 Statistics P=0.0021 P=0.0001 UCLA XDM2 0.092 .017 0.035 .002 XDM2-PR 0.034 .002 0.159 .005 Statistics P=0.0295 P=0.0001 XDM2-PR is XDM2 without phenol red. Each point is the average of three replicates.

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73 0 0.02 0.04 0.06 0.08 0.1 0.12 0.14 0.16 CHARDSXDM2-PRCHARDS-FCHARDS-VCHARDS-GOD OD Biofilmb a a a b a ab ab b c 0 0.05 0.1 0.15 0.2 0.25 CHARDSXDM2-PRCHARDS-FCHARDS-VCHARDS-GOD OD Biofilma a aa b a a a b c Figure 3-1. Growth of Xylella fastidiosa Temecula using variati ons of CHARDS based on XDM2-PR media composition. A) The growth under air treatments. B) The growth under nitrogen gas treatments. Growth unde r the various gas treatments was 15 days A B

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74 and each treatment had started with an optical density of 0.05. Each point is the average of three replicates.

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75 Table 3-3. Amino acid analysis CHARDS after 15 days of bacterial growth With Xylella Without Xylella Significance Asp a 14.603 15.845 **** Glu 2.907 0.901 **** Asn 0.119 0.130 Ser 0.108 0.157 Gln 47.471 50.404 **** Gly 2.210 1.409 His 0.000 0.000 Arg 20.776 20.612 Thr 0.000 0.000 Ala 6.804 6.860 Pro 1.721 1.488 Tyr 0.261 0.393 Val 0.103 0.148 Met 0.000 0.000 Cys 0.037 0.054 Iso 2.186 1.015 Leu 0.086 0.094 Phe 0.517 0.415 Lys 0.092 0.090 a = The values listed are the means of pe rcent total of each amino acid. Bold amino acids are constituents added to the defined media. P-values are represented by: = 0.05, ** = 0.01, *** = 0.001, **** = 0.0001. Where N = 6.

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76 Table 3-4. Amino acid analysis CDM2-P R after 15 days of bacterial growth With Xylella fastidiosa Without Xylella fastidiosa Significance Air Nitrogen Air Nitrogen Bacteria Gas Bacteria*Gas Asp a 0 0 0 0 Glu 2.89 3.2 0.77 0.74 **** * Asn 11.8 12.06 12.16 12.02 Ser 13.9 12.79 15.16 15.41 *** Gln 55.06 58.1 57.89 58.63 * Gly 2.27 1.7 1.45 1.05 His 0 0 0 0 Arg 2.59 3.1 2.84 3.09 Thr 0 0.171 0 0 **** **** **** Ala 0.16 0.22 0.134 0.04 Pro 0.11 0.08 0.257 0.07 Tyr 1.84 0.42 0.448 1.01 Val 0.38 0.71 0.219 0.18 Met 5.65 5.38 5.94 5.56 Cys 0.01 0.02 0.032 0.03 Iso 2.08 1.07 1.3 0.86 Leu 0.18 0.12 0.36 0.22 Phe 0.68 0.46 0.76 0.68 Lys 0.41 0.38 0.28 0.42 a= The values listed are the means of percent total of each amino acid. Bold amino acids are constituents added to the defined media. P-values are represented by: = 0.05, ** = 0.01, *** = 0.001, **** = 0.0001. Where N=3.

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77 Table 3-5. Amino acids used in the XDM2-PR and CHARDS Ala Arg Asn Asp Cys Met Ser Gln (autoclaved) Gln (filtered) Asp 0.00% 0.00% 1.22% 81.80% 0.00% 0.00% 0.00% 0.00% 0.00% Glu 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 1.41% 0.17% Asn 0.00% 0.00% 88.61% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Ser 0.33% 0.16% 0.00% 0.81% 4.92% 0.31% 93.09% 0.48% 0.00% Gln 0.34% 0.18% 0.00% 0.46% 3.38% 0.29% 0.91% 85.25% 97.76% Gly 0.00% 0.15% 0.33% 0.69% 3.92% 0.20% 0.63% 1.33% 1.55% His 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Arg 0.00% 96.55% 0.00% 0.00% 0.00% 3.16% 0.00% 0.00% 0.02% Thr 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% Ala 92.76% 0.03% 0.29% 0.57% 2.48% 0.15% 0.24% 1.08% 0.02% Pro 0.00% 0.00% 0.61% 5.30% 0.00% 0.00% 0.31% 0.46% 0.00% Tyr 0.00% 0.00% 0.00% 0.00% 6.70% 10.38% 0.00% 3.46% 0.09% Val 0.14% 0.12% 0.38% 0.22% 30.07% 0.89% 0.30% 0.52% 0.01% Met 0.00% 0.00% 0.00% 0.00% 0.00% 79.17% 0.00% 0.00% 0.00% Cys 0.47% 0.14% 0.70% 0.78% 9.55% 0.60% 0.37% 0.30% 0.03% Iso 5.11% 1.59% 5.13% 6.56% 29.53% 3.51% 2.49% 3.49% 0.24% Leu 0.84% 0.48% 0.97% 1.20% 3.21% 0.50% 0.67% 0.43% 0.06% Phe 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.37% 0.00% Lys 0.01% 0.59% 1.77% 1.63% 6.23% 0.84% 0.98% 1.42% 0.06% The concentrations used were the same as used in the media; the CHARDS glutamine concentration was used. All the amino acids were filtered sterilized except for one of glutamine (as noted).

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78 Table 3-6. CHARDS cons tituents in mM amounts Ingredient mM % Total K2HPO4 8.612 KH2PO4 7.348 MgSO4-7H2O 0.811 Starch 11.101 Ferric pyrophosphate 0.335 Glutamine 39.685 76.22 Alanine 1.930 3.71 Aspartic acid 4.358 8.37 Arginine 6.028 11.58 Cysteine-HCl 0.063 0.12

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79 Table 3-7. XDM2-PR cons tituents in mM amounts Ingredient mM % Total K2HPO4 12.056 KH2PO4 5.878 MgSO4-7H2O 1.623 Glucose 55.506 Ferric pyrophosphate 0.168 Glutamine 27.369 64.81 Asparagine 9.516 22.53 Serine 2.664 6.31 Methionine 2.681 6.35 Myo-inositol 0.194 Pyridoxine-HCl 0.005 Thiamine 0.003 Nicotinic acid 0.004 D-Biotin 0.001

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80 LIST OF REFERENCES Agladze, K., Jackson, D. & Romeo, T. (2003). Periodicity of cell attachment patterns during Escherichia coli biofilm development. Journal of Bacteriology 185 5632-5638. Agladze, K., Wang, X. & Romeo, T. (2005). Spatial periodicity of Escherichia coli K-12 biofilm microstructure initiates during a reversible, polar attachment phase of development and requires the polysaccharide adhesin PGA. Journal of Bacteriology 187 8237-8246. Andersen, P. C. & Brodbeck, B. V. (1989). Temerature and temp erature preconditioning on flux and chemical composition of xylem exudate from muscadine grapevines. Journal of American Society for Horticultural Science 114 440-444. Andersen, P. C. & Brodbeck, B. V. (1991). Influence of Fertilizatoin on xylem fluid chemistry of Vitis rotundifolia Noble and Vitis hybrid Suwannee. American Journal of Enology and Viticulture 42 245-251. Andersen, P. C., Brodbeck, B. V. & Russell F. Mizell, I. (1992). Feeding by the leafhopper, Homalodisca coagulata, in relation to xylem fluid chemistry and tension. Journal of Insect Physiology 38 611-622. Andersen, P. C., Ishida, M. L., Momol, E. A., Brodbeck, B. V., Leite, B. & Momol, M. T. (2004). Influence of Vitis xylem fluid and xylem fluid plus cecropin on growth of Xylella fastidiosa Vitis 43 19-25. Andersen, P. C., Brodbeck, B. V., Russell F. Mizell, I. & Oden, S. (2005). Abundance and Feeding of Homolodisca coagulata (Hemiptera: Auchenorrhyncha: Cicadellidae) on the Vitis Genotypes in North Florida. Environmental Entomolgy 34 466-478. anonymous (2004). Xylella fastidiosa Bulletin OEPP/EPPO Bulletin 34 187-192. Bader, M., Muse, W., Ballou, D. P., Gassner, C. & Bardwell, J. C. A. (1999). Oxidative protein folding is driven by the electron transport system. Cell 98 217-227. Bespalov, V. A., Zhulin, I. B. & Taylor, B. L. (1996). Behavioral responses of Escherichia coli to changes in redox potential. Proceedings of the Nati onal Academy of Science 93 10084-10089. Boes, N., Schreiber, K., Hartig, E., Jaensch, L. & Schobert, M. (2006). The Pseudomonas aeruginosa universal stress protein PA4352 is esse ntial for surviving anaerobic energy stress. Journal of Bacteriology 188 6529-6538. Brlansky, R. H., Timmer, L. W., French, W. J. & McCoy, R. E. (1983). Colonization of the sharpshooter vectors, Oncometopia nigricans and Homalodisca coagulata by xylem-limited bacteria. Phytopathology 73 530-535. Campanharo, J. C., Lemos, M. V. F. & Lemos, E. G. d. M. (2003). Growth optimization procedures for the phytopathogen Xylella fastidiosa Current Microbiology 46 99-102.

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82 Facincani, A. P., Ferro, J. A., Jr., J. M. P., Jr., H. A. P., Lemos, E. G. d. M., Prado, A. L. d. & Ferro, M. I. T. (2003). Carbohydrate metabolism of Xylella fastidiosa : Detection of glycolytic and pentose phosphate pathway enzy mes and cloning and expression of the enolase gene. Genetics and Molecular Biology 26 203-211. Feil, H., Feil, W. S., Detter, J. C., Purcell, A. H. & Lindow, S. E. (2003). Site-Directed Disruption of fimA and fimF fimbrial genes of Xylella fastidiosa Phytopathology 93 2003. Fernandez, L. A. & Berenguer, J. (2000). Secretion and assembly of regular surface structures in Gram-negative bacteria. FEMS Microbiology Reviews 24 21-44. Gansert, D., Burgdorf, M. & Losch, R. (2001). A novel approach to the in situ measurement of oxygen concentrations in th e sapwood of woody plants. Plant, Cell, and Environment 24 10551064. Gansert, D. (2003). Xylem sap flow as a major pathway for oxygen supply to the sapwood of birch ( Betula pubescens Ehr.). Plant, Cell, and Environment 26 1803-1814. Goodwin, P. H., DeVay, J. E. & Meredith, C. P. (1988). Roles of water stress and phytotoxins in the development of Pierce's disease of the grapevine. Physiological and Molecular Pathology 32 1-15. Guilhabert, M. R. & Kirk patrick, B. C. (2005). Identification of Xylella fastidiosa antivirulence genes: Hemagglu tinin adhesins contribute to X. fastidiosa biofilm maturation and colonization and attenuate virulence. Molecular Plant-Microbe Interaction 18 856-868. Herschbach, C. & Rennenberg, H. (2001). Sulfur nutrition of deciduous trees. Naturwissenchaften 88 25-36. Hierro, A. M. d., Kronberger, W., Hietz, P., Offenthaler, I. & Richter, H. (2002). A new method to determine the oxygen concentr ation inside the sapwood of trees. Journal of Experimental Botany 53 559-563. Hook, D. D., Brown, C. L. & Wetmore, R. H. (1972). Aeration in Trees. Botanical Gazette 133 443-454. Hopkins, D. L. (1984). Variability of virulence in the grap evine among isolates of the Pierce's disease bacterium. Phytopathology 74 1395-1398. Hopkins, D. L. (1989). Xylella fastidiosa : xylem-limited bacter ial pathogen of plants. Annual Review in Phytopathology 27 271-291. Huang, P.-Y., Milholland, R. D. & Daykin, M. E. (1986). Structural and Morphological changes associated with the Pierce's Disease bacterium in bunch and muscadine grape tissues. Phytopathology 76 1232-1238.

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83 Ishida, M. L., Andersen, P. C. & Leite, B. (2004). Effect of Vitis vinifera L. cv. Chardonnay xylem fluid on cecropin B activity against Xylella fastidiosa Physiological and Molecular Plant Pathology 64 73-81. Jordan, A. & Reichard, P. (1998). Ribonucleotide reductases. Annual Reviews in Biochmistry 67 71-98. Kallio, P. T., Kim, D. J., Tsai, P. S. & Bailey, J. E. (1994). Intracellular expression of Vitreoscilla hemoglobin alters Escherichia coli energy metabolism under oxygen-limited conditions. European Journal of Biochemistry 219 201-208. Kimmerer, T. W. & Stringer, M. A. (1988). Alcohol dehydrogenase an d ethanol in the stems of trees: evidence for the anaerobic metabolism in the vascular cambium. Plant Physiology 87 693-697. Kita, K., Konishi, K. & Anaraku, Y. (1984). Terminal oxidases of Escherichia coli aerobic respiratory chain. I. Purificati on and properties of cytochrome b562-o complex in the early exponential phase of aerobic growth. The Journal of Biological Chemistry 259 3368-3374. Krivanek, A. F., Stevenson, J. F. & Walker, M. A. (2005). Development and comparison of symptom indices for quantifying grapev ine resistance to Pierce's disease. Phytopathology 95 3643. Leite, B., Ishida, M. L., Alves, E., Carrer, H ., Pascholati, S. F. & Kitajima, E. W. (2002). Genomics and x-ray microanalysis indicate that Ca2+ and thiols mediatew the aggregation and adhesion of Xylella fastidiosa Brazilian Journal of Medica l and Biological Research 35 1-6. Leite, B., Andersen, P. C. & Ishida, M. L. (2004). Colony aggregation and biofilm formation in xylem chemistry-based media for Xylella fastidiosa FEMS Microbiology Letters 230 283290. Lemos, E. G. d. M., Alves, L. M. C. & Campanharo, J. C. (2003). Genomics-based design of defined growth media for the plant pathogen Xylella fastidiosa FEMS Microbiology Letters 219 39-45. Li, W. B., Fernandes, N. G., Mirnanda, V. S., Teixeira, D. C., Ayres, A. J., Garnier, M. & Bove, J. M. (1999). A triply cloned strain of Xylella fastidiosa multiples and induces symptoms of citrus variegated ch lorosis in sweet orange. Current Microbiology 39 106-108. Lopes, S. A., Ribeiro, D. M., Roberto, P. G., Franca, S. C. & Santos, J. M. (2000). Nicotiana tabacum as an experimenta; hos t for the study of plantXylella fastidiosa interactions. Plant Disease 84 827-830. MacDonald, R. C. & Kimmerer, T. W. (1991). Ethanol in the stems of trees. Physiologia Plantarium 82 582-588.

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84 MacDonald, R. C. & Kimmerer, T. W. (1993). Metabolism of transpir ed ethanol by eastern cottonwood ( Populus deltoides Bartr.). Plant Physiology 102 173-179. Mancuso, S. & Boselli, M. (2002). Characterisation of the o xygen fluxes in the division, elongation, and mature zones of Vitis roots: onfluence of oxygen availability. Planta 214 767774. Mancuso, S. & Marras, A. M. (2003). Different pathways of th e oxygen supply in the sapwood of young Olea europaea trees. Planta 216 1028-1033. McElrone, A. J., Sherald, J. L. & Pooler, M. R. (1999). Identification of alternative hosts of Xylella fastidiosa in the Washington, D.C., area using nested polymerase chain reactiopn (PCR). Journal of Arbiculture 25 258-263. McElrone, A. J., Sherald, J. L. & Forseth, I. N. (2003). Interactive effects of water stress and xylem-limited bacterial infection on th e water relations of a host vine. Journal of Experimental Botany 54 419-430. Meng, Y., Li, Y., Galvani, C. D., Hao, G., Turner, J. N., Burr, T. J. & Hoch, H. C. (2005). Upstream migration of Xylella fastidiosa via pilus-driven twitching motility. Journal of Bacteriology 187 5560-5567. Mollenhauer, H. H. & Hopkins, D. L. (1974). Ultrastructural study of Pierce's disease bacterium in grape xylem tissue. Journal of Bacteriology 119 612-618. Mollenhauer, H. H. & Hopkins, D. L. (1976). Xylem morphology of Pierce's disease-infected grapevines with different levels of tolerance. Physiological Plant Pathology 9 95-100. Newman, K. L., Almeida, R. P. P., Purce ll, A. H. & Lindow, S. E. (2003). Use of a green flourescent strain for analysis of Xylella fastidiosa colonization of Vitis vinifera Applied and Environmental Microbiology 69 7319-7327. Newman, K. L., Almeida, R. P. P., Purce ll, A. H. & Lindow, S. E. (2004). Cell-cell signaling controls Xylella fastidiosa interactions with both insects and plants. Proceedings of the Natioinal Academy of Science 101 1737-1742. Otto, K., Norbeck, J., Larsson, T., Karl sson, L.-a. & Hermansson, M. (2001). Adhesion of type 1-fimbriated Escherichia coli to abiotic surfaces leads to altered composition of outer membrane proteins. Journal of Bacteriology 183 2445-2453. Pooler, M. R. & Hartung, J. S. (1995). Specific Pcr Detection and Identification of XylellaFastidiosa Strains Causing Citrus Variegated Chlorosis. Current Microbiology 31 377-381. Purcell, A. H. & Hopkins, D. L. (1996). Fastidious xylem-limited bacterial plant pathogens. Annual Review in Phytopathology 34 131-151.

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85 Rice, C. W. & Hempfling, W. P. (1978). Oxygen-Limited Continuous Culture and Respiratory Energy-Conservation in Escherichia-Coli. Journal of Bacteriology 134 115-124. Ritz, D. & Beckwith, J. (2001). Roles of thiol-redox pathways in bacteria. Annual Review of Microbiology 55 21-48. Schembri, M. A., Givskov, M. & Klemm, P. (2002). An attractive surface: Gram-negative bacterial biofilms. Science's STKE http://stke.sciencemag.org/cgi/ content/full/OC_sigtrans;2002/132/re6 Simpson, A. J. G., Reinach, F. C ., Arruda, P. & other authors (2000). The genome sequence of the plant pathogen Xylella fastidiosa Nature 406 151-159. Soto, G. E. & Hultgren, S. J. (1999). Bacterial Adhesins: Common themes and variations in architecture and assembly. Journal of Bacteriology 181 1059-1071. Storz, G. & Imlay, J. A. (1999). Oxidative Stress. Current Opinion in Microbiology 2 188-194. Vaisanen, V., Lounatmaa, K. & Korhonen, T. K. (1982). Effects of sublethal concentrations of antimicrobial agents on the hemagglu tination, adhesion, and ultrastructure of pyelonephritogenic Escherichia coli strains. Antimicrobial Agents and Chemotherapy 22 120127. van Sluys, Oliveira, M. C. d., Monteiro-Vitorello, C. B. & other authors (2003). Comparative analysis of the complete genome seque nces of Pierce's disease and citru variegated chlorosis strains of Xylella fastidiosa Journal of Bacteriology 185 1018-1026. Wells, J. M., Raju, B. C., Hung, H.-Y., Weisbur g, W. G., Mandelco-Paul, L. & Brenner, D. J. (1987). Xylella fastidiosa gen. nov., sp. nov: gram-negative, xylem-limited, fastidious plant bacteria related to Xanthomonas spp. International Journal of Sysematic Bacteriology 37 136143. Wojtaszek, P. (1997). Oxidative burst: an ear ly plant response to pathogen infectioin. Biochemical Journal 322 681-692.

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86 BIOGRAPHICAL SKETCH Anthony Shriner was born and raised in nor th Florida. He received his high school diploma in 1991 from Florida High School. He then went on to receive his Bachelor of Science degree in biology from Florida St ate University in 1998. He has received his Master of Science from the University of Florid a in horticultural science in 2006 He is planning on pursuing a PhD in environmental microbiology.


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EFFECT OF OXYGEN ON THE GROWTH OF Xylella fastidiosa


By

ANTHONY D. SHRINER

















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

2007
































Copyright 2007

by

Anthony D. Shriner


































To every stray soul who made a wrong turn in life and struggled to make things right.










ACKNOWLEDGMENTS

I wish to give special thanks to Trisha Spears, Fred Spears, Robert Reeves and Jane

Reeves, for having faith in me when I doubted. I also thank my friends Dean Paini, Michelle

Stuckey, Tobin Northfield, Enoch Osekre, Francis Tsigbey, Susan Bambo, and Towanga

Katsvairo for putting up with the whole manic process that is graduate school. I also thank some

anonymous people for allowing me to have this chance to develop my professional skills.












TABLE OF CONTENTS


page

ACKNOWLEDGMENT S .............. ...............4.....


LI ST OF T ABLE S ................. ...............7......._.....


LI ST OF FIGURE S .............. ...............8.....


AB S TRAC T ......_ ................. ............_........9


CHAPTER


1 LITERATURE REVIEW ................. ...............11...............


The Host Environment ..........._...._ .. ........._..... ................ ............1
The Host Environment with Respect to Xylella fa;stidiosa. ..........._..._ .........................14
Oxygen Levels in Xylem Fluid............... ...............15.
General Overview of Electron Transport ................ ....................... ................18
Xf s Electron Transport System ..........._.._ ....... ...............19....
XfUse of Oxygen as Terminal Oxidase .............. .....................19
Ferm entation ..........._.... ...... .... ......... .. ....... ......__.........20
The Added Influence of the Disulfide Bond Forming System ....._____ ...... ....__..........21
Physiological Modifications Due to Oxygen Availability............... ..............2
Bacterial Attachment and Appendages............... ...............2


2 EFFECT OF OXYGEN ON THE GROWTH AND BIOFILM FORMATION OF

Xylella f a;sdtidiosa .............. ...............28....

Introducti on .................. ...............28......__ .....
M materials and M ethods .............. ...............30....
Bacteria Used............... ...............30..
Cultivation Protocol ..........._...__........ ...............30.....
PW' Media, Broth and Solid ..........._...__........ ...............3_ 1..
CHARD S medium ..........._...__........ ...............32.....
XDM2-PR meduim .............. ...............32....
The BCYE Agar Plates ............ ......__ ...............32..
Gram' s Staining ............ ......_ ............... 3....
PCR Protocol .............. ...............33....

D eeps .............. ...............3 4....
Stab s .............. ...............3 4....

Oxygen Level s............... ...............3 5
Growth Conditions .............. ...............35....

Optical Density (OD) .............. ...............36....
Biofilm Qualification............... .............3
M edia Analy si s............ ..... .._ ...............36..
Statistical Analysis .............. ...............37....












Re sults ................ ...............38.................

Agar Deeps ............... ...............38....
Experimental Conditions ................. .. ........... ... ......... .............3
Xylella vs. an Obligate Aerobe and a Facultative Anaerobe ................ ........._._. .....39
Xylella vs. Xanthomona~s .............. ...............39....
Defined Media Growth ............. ..... ._ ...............40.....

Organic Acid Analysis .............. ...............40....
Discussion ..........._...__........ ...............40.....


3 MEDIA COMPONENT EFFECTS ON GROWTH AND BIOFILM FORMATION OF

Xylella fastidiosa .............. ...............57....


Introducti on ................. ...............57.................
Materials and Methods .............. ...............59....
Bacteria Used............... ...............59..
Cultivation Protocol ................. ...............59.................

PW' Media, Broth and Solid .................... .. .......... ....... ...............60.
The BCYE (Buffered Charcoal Yeast Extract) Agar Plates............... .................6
CHARDS Medium .............. ...............61....
XDM2 and XDM2-PR Media .............. ...............61....

Gram s Staining ................. ...............62................
PCR Protocol .............. ...............62....
Growth Conditions .............. ...............63....

Optical Density (OD) .............. ...............64....
BioHilm Qualif cation............... .............6
M edia Analy si s............. ...... ._ ...............64...
S tati sti cal Analy si s .............. ...............66....
R e sults............. ...... ...............66....
Discussion ............. ...... ._ ...............67....


LIST OF REFERENCES ............. ...... ._ ...............80....


BIOGRAPHICAL SKETCH .............. ...............86....










LIST OF TABLES


Table page

1-1 Oxygen concentrations found in various xylem fluids ....._._.__ .......___.. ........._........27

2-1. Concentrations of oxygen found in various xylem fluids............... ...............44.

2-2. Organic acid profiles in the CHARDS media after 15 days............... ...............54..

2-3. Organic acid profiles in the XDM2-PR media after 15 days .............. .....................5

3-1. A Phenol red series experiment done in the XDM2 media using 'Temecula' ......................71

3-2. Effect of phenol red on the growth and behavior on three strains of Xylella fa;stidiosa .......72

3-3. Amino acid analysis CHARDS after 15 days of bacterial growth ................... ...............7

3-4. Amino acid analysis CDM2-PR after 15 days of bacterial growth .............. ...............76

3-5. Amino acids used in the XDM2-PR and CHARDS .............. ...............77....

3-6. CHARDS constituents in mM amounts............... ...............78

3-7. XDM2-PR constituents in mM amounts .............. ...............79....










LIST OF FIGURES


Figure page

2-1i. Growth of Xylella fastidiosa in agar deep s to determine oxygen limitati ons ................... .....4 5

2-2. Dissolved oxygen levels detected in dH20 and CHARDS media as a function of
treatment levels and over time. ............. ...............47.....

2-3. Compari son of Xylella fa;stidiosa cultivated under reduced oxygen levels. ................... .......48

2-4. Xylella fa;stidiosa growth in PW+ for 15 days under aerobic and hypoxic conditions.
Each point is the average of three replicates. ................. ....._.._........... .......5

2-5. Xanthomona~s campestris py. campestris growth in PW+ for 5 days under aerobic and
hypoxic conditions. Each point is the average of three replicates ................. ...............51

2-6. Growth and performance of Pierce's disease strain Temecula in the CHARDS media
under two gas treatments. ............. ...............52.....

2-7. Growth and performance of Pierce's disease strain Temecula in the XDM2-PR media
under two gas treatments. ............. ...............53.....

3-1. Growth ofXylella fa;stidiosa 'Temecula' using variations of CHARDS based on
XDM2-PR media composition .............. ...............73....









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

EFFECT OF OXYGEN ON THE GROWTH OF Xylella fastidiosa

By

Anthony D. Shriner

May 2007

Chair: Peter C. Andersen
Major Department: Horticultural Sciences

Xylella fastidiosa, a bacterial plant pathogen, is the causative agent of many water-stress

type diseases, such as almond leaf scorch, oak leaf scorch, and Pierce' s disease of grapes. The

disease symptoms are putatively due to blocking of the transpiration stream by forming biofilms

(bacteria and exopolysaccharide three-dimensional structures) and/or inducing the formation of

tylosis, a plant-generated plug that protects the plant from cavitation events. The dissolved

oxygen levels in the xylem environment during the growing season can reach hypoxic levels

(range, 20 to 60 Clmol L )~. In 1987 Wells et al. reported that Xylella fastidiosa was an obligate

aerobe and did not grow under an anoxic environment, which is similar to the growth

Xanthomona~s campestris, Xylella' s closest genetic relative. In a growth comparison of three

Xylella strains, an obligate aerobe, and a facultative anaerobe using various levels of oxygen for

21 to 0%, the pattern of growth for Xylella closely resembled that of the facultative anaerobe and

not the obligate aerobe. We showed that Xylella fastidiosa is capable of growing in an anoxic

environment in nutrient broth as well as in two defined media, CHARDS and XDM2-PR, in

which Xylella planktonic population and also biofilm formation increased more in the anoxic

treatment in the XDM2-PR medium. In addition to the growth of Xylella under hypoxic









conditions, the bacterium produces a different organic acid profile (most likely fermentation)

which indicates that they are capable of alternative means of energy production.









CHAPTER 1
LITERATURE REVIEW

Xylella fastidiosa (X~f) is a gram-negative, rod shaped bacterial plant pathogen, which

belongs to the y-proteobacteria, such as Escherichia coli, and whose closest genetic relation is

another plant pathogen, the xanthomonads (Simpson et al. 2000). Xf resides within the xylem

vessels of many plant species and in the foregut of leafhoppers. Xf is considered to be xylem-

limited, and transmission from host to host is accomplished by sharpshooters (Hemiptera:

Cicadillidae), which feed on xylem fluid. Pierce' s disease of grapevine, phony peach disease,

plum leaf scorch, and citrus variegated chlorosis are a few of the diseases caused by Xf: Plant

symptoms are thought to be mainly caused by the occlusion of xylem vessels. Xylem

dysfunction is thought to be caused by bacterial aggregation and biofilm formation (Purcell &

Hopkins, 1996), an adhered, organized community of bacteria and their polymeric matrix which

encapsulates them (Costerton et al. 1999). The bacteria may also cause an increase formation of

tyloses, a plug in the xylem element created by the xylem parenchyma cells (Esau, 1948).

Xfis predominantly found in the continental Americas, as far north as Canada and south to

Argentina. The only location outside of the continent is Taiwan, which has a Pear Leaf Scorch

pathovar (anonymous, 2004). The host range of Xfis extremely broad and diverse, which

includes many monocots and angiosperms. Gymnosperms are the largest group of plants without

any report of disease or Xfpresence (Purcell, A. H., The Xylella fa;stidiosa website,

http://www. cnr.berkeley .edu/xylella/, December, 2005). Although many endemic host plants

contain XJ; in most cases it resides in a benign presence, in that not all plants that serve as a host

to the bacteria will develop the disease. Within Vitis, some species are highly susceptible and

others are tolerant (Mollenhauer & Hopkins, 1976). Other bacterial host-plants may not develop

the disease or severe symptoms until they suffer a stress situation, such as Virginia creeper










(McElrone et al. 1999; McElrone et al. 2003). In such cases it is believed that the synergistic

effect of bacterial infection and the negative effects of water stress conditions lead to the

development or progression of the disease. The underlying basis of host plant resistance is

unknown and may vary with plant species.

Many plant species known to harbor Xf (Purcell, A. H., The Xylella fa;stidiosa website,

http://www. cnr.berkeley .edu/xylella/, December, 2005) and many endemic, highly-resistant

species that act as natural reservoirs for the bacterium. This is a problem for the management for

these diseases because there is no reasonable way to remove the pathogen from the environment.

There are no acceptable treatments that can cure plants of the disease, however some antibiotics

will cause a reduction in disease symptoms (Hopkins, 1989). The only feasible action is to

remove and destroy the infected host. An example of this control technique is found in Brazil,

where citrus variegated chlorosis caused by a strain of Xfis controlled by is pruning the infected

tissue or removing the infected trees. This is also a method used to control Pierce's disease and

other Xfdiseases. The proactive method for disease prevention, in California, is the application

of pesticides to kill the vector, thereby inhibiting the spread of the bacteria from an infected

(resistant) host to a more susceptible host. This is not practical in the southeast because too

many sprays are required.

Several Xfpathovars can induce Pierce' s disease (PD) (Purcell & Hopkins, 1996).

Symptoms for PD include the formation of chlorotic and necrotic leaf margins that develop over

the whole leaf, water stress, stunting and root die back, and vine death. Other symptoms include

the formation of "green islands" (Krivanek et al. 2005), which may be attributed to the lack of

cork development (Esau, 1948), and the development of "match sticks," a petiole which remains









attached to the cane after the leaf has been sheared off (Krivanek et at. 2005). To date there are

no associated bacterial toxins associated with disease symptoms (Goodwin et at. 1988).

The Host Environment

The xylem vessels are specialized cells which have been "killed" in an apoptotic fashion,

leaving behind a system connected hollow cells which allows for the transpiration of water and

nutrients from the root zone to the photosynthetic and growing tissues of the plant. The xylem

cells have been specially modified in several ways. One modification is strengthening them to

withstand the negative tension of the transpiration stream by lignification, a process in which

phenylpropanoids, called monolignols, are modified by peroxidases to form a support for the

cell walls (Christensen et at. 1998). Another modification of xylem tissue is a coating of

cellulose, an insoluble carbohydrate, (cellulose is a glucose polymer bonded via a P 1-4 epoxy

bond and starch is glucose polymer with an a 1-4 epoxy bond), which is intertwined with the

lignin to create a hydrophobic barrier on the inside of the xylem elements.

The formation of pits and perforated ends, which are other modifications of xylem cells,

allow for the lateral and longitudinal movement, respectively, of solute through the plant. The

pits are specialized holes in the vessel which have a torus, gate, in the middle attached to tethers

that allows solute to travel laterally through the plant. Pit membranes are not permeable to large

(>25 nm) molecule, including bacteria. In the case of a cavitation event, the sudden formation of

an air pocket within the transpiration stream is contained and does not affect the rest of the

transpiration stream.

The transpiration stream (xylem fluid) runs from the roots to the apical meristem

transporting nutrients collected from the root zone to active growing and photosynthesis portions

of the plant. Xylem fluid contains amino acids, and organic acids, such as citrate, succinate and

lactate (Andersen & Brodbeck, 1989; Andersen & Brodbeck, 1991; Andersen et at. 1992;









Andersen et al. 2005), glutathione (Herschbach & Rennenberg, 2001), a tripeptide made of

glycine, cysteine and glutamine as well as inorganic ions (Andersen et al 2005). The total

osmolarity of xylem fluid, combining the entire amount of compounds and molecules, is ~20

mM with water making up the balance of the volume, 95-99% (Andersen & Brodbeck, 1991).

Glutamine is the compound of highest concentration in Vitis xylem fluid (Andersen & Brodbeck,

1989; Andersen & Brodbeck, 1991; Andersen et al. 1992; Andersen et al. 2005), and is thought

to be a transport molecule to deliver nitrogen to the photosynthetic tissue. Sulfur compounds are

the most limited within xylem fluid with cysteine comprising 8-27 CIM, for Vitis (Andersen et al.

2005), and 0.005-2.6 CIM for many trees (Herschbach & Rennenberg, 2001), methionine at 5-12

CIM for Vitis (Andersen et al. 2005), and 0-39 CIM glutathione at 0.002-31.2 CIM and SO4 at 25-

4697 CIM in deciduous trees (Herschbach & Rennenberg, 2001).

The Host Environment with Respect to Xylella fastidiosa

The low nutrient concentrations found in xylem fluid probably caused some unusual

adaptations in Xf in order for it to survive. Under lower nutrient conditions Xf has been shown to

produce more biofilm. The converse to this occurs when Xfis grown in enrichment media; e. g.

PW' and then there is a dramatic reduction in the amount of biofilm produced compared to Xf

grown in a defined media with restricted nutrient levels (Leite et al. 2004). The formation of a

biofilm could also allow protection of the bacteria population from defensive responses of the

plant such as oxidative bursts (Wojtaszek, 1997) and the production of antibiotics (Andersen et

al. 2004; Ishida et al. 2004).

The nutrient requirements for the bacterium appear to be minimal, (i.e., a few amino acids

and ferric pyrophosphate) (Chang & Donaldson, 2000; Leite et al. 2004; Lemos et al. 2003). A

constituent for many of the media used to grow Xf is glutamine (Campanharo et al. 2003; Chang

& Donaldson, 2000; Leite et al. 2004; Lemos et al. 2003). It has the highest concentration of









any of the organic molecules in xylem fluid (Andersen & Brodbeck, 1989; Andersen &

Brodbeck, 1991; Andersen et al. 1992; Andersen et al. 2005) and is thought to be the primary

carbon and nitrogen source for Xf:

Sulfur is an essential element for all organisms and is critical for structure and function of

many proteins of the plant and bacteria. The requirement for sulfur in the bacteria' s gene

products begins with methionine, disulfide bonds, a structural bond formation which requires

two cysteines, as well as many proteins which contain an iron sulfur cluster as part of their active

sites. Another sulfur compound is glutathione, a tripeptide consisting of glycine-cysteine-

glutamine, which is thought of as an anti-oxidant compound by functioning as a co-factor for

some enzymes and acting as a buffer to maintain key sulfur residues. Glutathione has many

functions in cellular activity. In red blood cells the free sulfhydryl group in reduced glutathione

serves as a buffer to maintain an unbound cysteine, which is used for binding oxygen. The class

I type ribonuclotide reductases, an oxygen-dependent enzyme which is found in XJ; also utilize

glutathione in the reduction of ribose to deoxyribose elements for the synthesis of DNA (Jordan

& Reichard, 1998). Glutathione is also utilized for disulfide bond maintenance in the cytoplasm,

either for the formation of cytoplasmic proteins or breakage for transporting across the

membrane (Ritz & Beckwith, 2001). All of these situations are possible and many are probably

true for XJ; according to the published genomes for the CVC and PD pathovar (Kanehisa

Laboratories, Kyoto Encyclopedia of Genes and Genomics, www.kegg.com, December, 2005).

Oxygen Levels in Xylem Fluid

The levels of oxygen found in xylem fluid have been shown to range from atmospheric

levels to almost anoxic, 230 Clmol 02 L (Gansert et al. 2001) to less than 2.88 Clmol 02 L^

(Eklund, 2000). The maximum air-saturation in water is 288 Clmol 02 L^ at 20oC, (also denoted

as 100% air-saturated water, 21% oxygen and 9.2 ppm (Clg/ml) dissolved oxygen in water).









Across a radial section of a stem there is a fluctuation with a rapid drop from the outside, 21% to

the cambium, about 7%, and a slight rise, 15%, in the pith (Dongen et at. 2003).

Inside the xylem system three physical factors influence the capacity of a liquid, or xylem

fluid, to take up and retain dissolved oxygen; they are temperature, osmolarity and pressure. As

a liquid rises in temperature the capacity to retain oxygen lowers. The higher the solute

concentration of a solution also inhibits the capacity to retain oxygen, for xylem fluid of Vitis

this is around 20 mM (Andersen & Brodbeck, 1991) and could be considered similar to fresh

water. A decrease in pressure will also decrease the amount of dissolved oxygen in the system,

and the tension in the transpiration stream of Vitis can be as low as -1.6 MPa (Andersen et at.

1992), approximately -230 psi.

Most of the oxygen in xylem fluid travels by means of the transpiration stream (Dongen et

at. 2004; Gansert et at. 2001; Gansert, 2003; Mancuso & Marras, 2003). Entry into the

transpiration stream is a difficult arrangement where there are some obstacles to overcome such

as an adequate supply of oxygenated water, at the root zone, which needs to be accessible to the

newly dividing and elongating section of the root tip. Once the root matures and suberization

occurs around the stele the influx of oxygen drops drastically (Mancuso & Boselli, 2002).

During two extreme events, flooding and drought (because the plant inhibits the osmosis of

water out of the plant into the arid environment), the dissolved oxygen levels around the root-

zone become anoxic which can inhibit the transport of nutrients across the root tissue. However

some plants have survival techniques to combat the flooding event, such as the formation of

aerenchyma, a system to transport air from the air to the root zone, and the creation of

adventitious roots which form above the ground in an attempt to avoid the deeper anaerobic root

zone (Hook et at. 1972). During the night when the transpiration stream is not appreciably









flowing, there is an influx, 13 to 40% (Gansert, 2003; Mancuso & Marras, 2003) of the oxygen

can enter through the lenticels and moves through the tissue apoplasticly, through the gas spaces.

The density of the wood influences this ability of lateral oxygen transport into the xylem

vessels(Mancuso & Marras, 2003), also the discontinuous nature of xylem rays, which are begun

and ended by the cambium each year, hinder direct adsorption into the xylem vessels. During

the winter months, when the transpiration stream has stopped and the high demands for oxygen

are low, lateral diffusion is the means which allows the levels of dissolved oxygen in the xylem

fluid to approach ambient levels (Eklund, 2000).

Oxygen to serve as the favored electron acceptor for energy production oxidativee

phosphorylation) for dividing cambial regions (Eklund et al. 1998), and in the roots is primarily

transported via the transpiration stream (Mancuso & Boselli, 2002). However the amount of

oxygen supplied to the growing tissue can fall below adequate levels and the plant must therefore

rely on other pathways for energy production such as fermentation, the use of the substrate as an

electron donor and then acceptor for energy production as a means to regenerate NADH. The

anaerobic fermentation (substrate phosphorylation) by-products such as lactate (Andersen et al.

2005), ethanol and acetaldehyde (Kimmerer & Stringer, 1988; MacDonald & Kimmerer, 1991;

MacDonald & Kimmerer, 1993) have been reported in the xylem fluid of Vitis, Populus, Acer,

Quercus, and other trees. Ethanol has been shown to be incorporated along the vascular tissue of

Populus, where radiolabeled ethanol was inj ected into the transpiration stream of excised leaves

and stems, with less then 1% being released as CO2 (MacDonald & Kimmerer, 1993).

There are several reports that describe the amount of oxygen found in the xylem fluid of

plants. The researchers from these reports have come to similar conclusions using different

techniques varying from GC-MS (Eklund, 2000), a homemade clark-type sensor (Mancuso &









Marras, 2003), or a novel technique using fiber optics and fluorescent compound which is

quenched in the present of oxygen (Dongen et at. 2003; Gansert et at. 2001; Hierro et at. 2002).

Each group reports their data either as a percentage of oxygen that saturates a liquid (100 to 0%

air-saturated), with 100% saturated being equal to 21% air (21 to 0%), or a direct molar amount

288 Clmol 02 L (at saturation) in Table 1, all three, as well as Clg L^1 (ppm) are shown together.

The lowest levels of oxygen were found in Picea albies (Norway Spruce) (Eklund, 2000),

which ranged between <1 5% oxygen, the highest oxygen levels were detected during the

winter months, 17 19% oxygen (Hierro et at. 2002) which is almost at air-saturation levels.

The levels during the growing season for a few angiosperms can get as low as 14% air-saturation

(Eklund, 2000). For an overall summary see Table 1.

Since the maj or influx of oxygen is through the transpiration stream it is possible that Xf

can monitor the concentration as a function of flow. The oxygen in the xylem is exhausted as it

ascends the plant (Eklund et at. 1998; Eklund, 2000), therefore concentrations in the

transpiration stream would be highest near the roots and lowest near the apical meristem.

General Overview of Electron Transport

The electron transport chain utilizes a gradual reduction of redox potential to move

electrons along a pathway thereby generating a proton gradient, high on the outside of the inner

membrane and low on the inside, which is used to produce ATP, and power flagella. The

pathway begins with complex I, NADH dehydrogenase, in which two protons are shuttled to the

outside of the cell and two electrons which are transferred to quinone (which becomes quinol), a

compound found amidst the lipid bilayer of the inner membrane. Then complex II, succinate

dehydrogenase and fumarate reductase (fumarase), which converts succinate to fumarate in order

to shuttle two protons outside and transfers two electrons to quinone pool, the collective amount

of quinone found in the inner membrane. Next, is complex III, cytochrome bc, which transfers









the electrons from quinol to an enzyme called cytochrome c reductase, where the change in

energy facilitates the transport of two more protons across the membrane. Finally complex IV,

the terminal oxidase, also transfers two protons to the outside of the cell but also reduces the

number of protons inside by binding them to molecular oxygen, O or V/2 02. Therefore the end

result of this is that 10 protons removed, 8 are expelled from the cytoplasm and 2 protons

removed by the synthesis of water, from the cytoplasm thereby increasing the difference in

proton gradient that is used to drive ATP synthesis.

X~fs Electron Transport System

Xfis missing a few of the components associated with the electron transport chain. The

complex II system, of which there is only fumerate reductase and complex three, is absent

altogether. The components that are present are complex I, a partial complex II (succinate

dehydrogenase) and complex IV. It is with complex IV that the ability to use oxygen becomes

significant. The enzyme kinetics for cytochrome bo (for E. coli) are an affinity of Km = 0.2 CIM,

and rate of Vmax = 1.1 1.5 Clmol 02 nmO1 protein' min' (Rice & Hempfling, 1978), so that it

is known as the high through-put, low affinity terminal oxidase (as compare to E. coli's

cytochrome bd, which has a Km of 0.02 CIM (greater affinity) and a reaction speed of 0.7 Clmol

02 nmO1 protein' min' (half the reaction rate of cyt. bo) which makes it known as the 'low

through-put, high affinity' terminal oxidase). The result of these missing components is that it

requires more of the complex' s substrates, NADH, succinate and oxygen, to generate the proton

motive force which operates the ATP synthesis machinery. The one up side for Xfis that there

are no flagella which would require the use of the proton motive force.

XfUse of Oxygen as Terminal Oxidase

Aerobic bacteria use as the final electron acceptor in producing a proton gradient which is

then used as the driving force for ATP synthesis. The terminal oxidase for Xfis cytochrome










(cyt.) bo, which has four subunits, with subunit B possessing the metallic prosthetic groups,

heme b, heme o and copper (Kita et al. 1984). Cyt. bo is found in many other bacteria, the most

studied is in E. coli, a facultative anaerobe (capable of aerobic and anaerobic growth) which also

possesses another terminal oxidase cytochrome bd. The interesting thing about these two

oxidases is that cyt. bd, a two subunit protein, is used during low oxygen concentration

environments and cyt. bo, a four subunit protein, is used in high oxygen concentration

environments.

One of the uses for oxygen in Xfbiochemistry is that it is required at the terminal electron

acceptor by the cyt. bo. A BLAST analysis of the sub-units of Xf s cyt. bo against E. coli's

showed a 40 60% similarity to each other. Subunit B (also known as subunit I), which contains

the metallic cofactors, have the highest similarity, identical and analogous chemical properties

amino acids, with a BLAST score of ~68%. The amino acids which are considered essential for

the binding of the metallic cofactors are present.

Fermentation

The production of energy is a method used to classify bacteria, depending on the terminal

electron acceptor. Aerobic organisms use oxygen as the terminal electron acceptor, i.e.

oxidative-phosphorylation. Anaerobic organisms are a more complicated situation. When one

speaks of anaerobic metabolism, ones first though maybe of sulfate or nitrate reduction where

the terminal electron acceptor is an inorganic molecule. However, another form of anaerobic

metabolism is fermentation or substrate based phosphorylation.

Xylella fastidiosa breaks down glucose using the EMP (Embden-Meyer-Parnas) pathway

(Facincani et al. 2003), which utilizes glucose-6-phosphate rather then 6-phosphogluconate, the

pentose phosphate and Entner-Doudoroff pathways, to initiate the conversion of glucose into

pyruvate. The fate of pyruvate is to enter the citric acid cycle. A by-product of these










cycles/pathways is the reduction of NAD and FAD to NADH and FADH2, TOSpectively. These

reduced compounds must then be oxidized back to there reductive forms, this is carried out either

through respiration, using oxygen or other inorganic molecule, or by fermentation.

Respiration can be aerobic or anaerobic (with varying degrees of oxygen tolerance), both

utilize membrane bound enzymatic reactions. Aerobic respiration is a membrane bound process

which utilizes the electron transport system and a terminal oxidase to create a proton potential to

operate the ATP synthase, by reducing NADH compounds. Anaerobic respiration is also a

membrane bound process but can use sulfate or nitrate, to name a few, inorganic compounds as

the electron sink which drives the reduction ofNADH. Another alternative is fermentation

which occurs with in the cytosol and uses organic molecules, "food", as the electron donors and

as the electron acceptors during the reduction ofNADH. The fermentation processes are broken

into six main classes based by the end product homologies they each produce such as ethanol,

lactic, butyric, propionic, homoacetic and mixed acid. These electron acceptors are then

deposited into the media.

The Added Influence of the Disulfide Bond Forming System

The disulfide bond (dsb) protein system is predominantly concerned with the correct

formation of disulfide bonds that are excreted from the cytoplasm to the periplasm or through the

periplasm to the outer membrane. The machinery for this family of enzymes is located in the

periplasm and associated with the inner membrane oriented to the periplasm (Bader et al. 1999;

Collet & Bardwell, 2002). The part that is germane for the correct formation of disulfide binds is

the periplasm dsbA, which is reduced by the membrane bound disulfide protein B (dsbB), which

is recharged by contributing two electrons to the quinone pool. The affinity for quinone to dsbB

is Km = 0.31 CIM (Fabianek et al. 2000). The appendage structures, which are associated with

the outer membrane all have the ability to have a disulfide bond per protein subunit, as seen on









the amino acid sequences (Kanehisa Laboratories, Kyoto Encyclopedia of Genes and Genomics,

www.kegg.com, December, 2005), (pilE, a subunit for type-IV pili formation is known to have

one and a completed pilus as many copies of the subunit), thereby the creation of these structures

rely on the presence of the quinone and terminal oxidase in order to function. A point of interest

is that Xf has two dsbA genes and these are located adj acent to each other, the only other

organism that could be found that has a similar gene arrangement is Xanthomona~s campestris

and I. axonipodis (Kanehisa Laboratories, Kyoto Encyclopedia of Genes and Genomics,

www.kegg.com, December, 2005).

In addition of disulfide bond formation, another function of this gene family is that it is the

chaperone component for type-I pilus (fimbria) production (Fernandez & Berenguer, 2000). The

type I pilus system utilizes helpers all the way. From the cytoplasm P-pili (a type-I pilus from E.

coli) uses a sec-dependent pathway to enter the cytoplasm. Once in the periplasm, the pilus

subunits are transported to the outer membrane using DsbD (Soto & Hultgren, 1999).

Physiological Modifications Due to Oxygen Availability

During the 1970s and 1980s studies of the causative agent of Pierce's disease, then known

as a rickettsia-like bacterium, were usually in the form of scanning electron microscopy of

dissected Vitis sp. canes and petioles. Two different morphological characteristics were

described for the rickettsia-like bacterium of infected grapevines, the rippled and the smooth

outer membranes. In Y. rotundifolia Mitchx. (Carlos), the small tracheary elements had mostly

rippled bacteria and the larger tracheary elements the smooth bacteria were prevalent. In the

bunch grapes (y. vinifera L.), the bacterial populations had higher counts with smooth outer

membranes and a reduced number with rippled membranes (Huang et al. 1986). This led to an

idea that different cell morphologies are an indication of the pathogens virulence.









There are other changes associated with the appearance of the bacterium as a function of

the environment in which it was grown. If the bacterium was recently inoculated into a

grapevine the bacteria had a less dense morphology then if it was grown longer in the plant or for

four days in enrichment media (Mollenhauer & Hopkins, 1976). These denser morphology

images are very similar to the effects of aerobic verses an anaerobic environment upon the

morphology of mitochondria, an ancestral proteobacteria, found in Oryza sp. A mutational

analysis of cyt. bo in Pseudomona~s putida, which is lethal but also creates plasmolysis and

polar pitting (Duque et al. 2004), which appears to be present in images ofXf(French,

unpublished).

The oxygen levels may also be attributed to the changes in the expression appendage, pilus

and fimbriae, as well as the metabolic activity. The Schembri model, based on E. coli

attachment mechanisms, describes a change in the expression of these outer membrane structures

due to changes in the OxyRS system, a two component system for determining the oxygen

levels. These changes can lead to a cascade of other gene changes such as the formation of

biofilms pilii mediated changes) or the activation of motility complexes (Bespalov et al. 1996).

The bacteria may utilize the changes in oxygen as an indicator to its environment. Bacterial

hemoglobin, also known as "soluble cytochrome o", has also attributed to the production of

degradation enzymes as a function of oxygen levels. In Vitreoscilla sp., an a-proteobacter, when

the hemoglobin detects a hypoxic environment it stimulates the cell to produces an amylase, a

carbohydrate degradation enzyme, and when this hemoglobin is expressed in E. coli similar

results occurred (Kallio et al. 1994). This type of sensor system may modify Xf genome to

produce different enzymes in order to survive on other carbon source, i.e., glutamine or

cellulose, either form the xylem stream or as attached biofilms, respectively. Aerotaxis and









redoxtaxis are sensory responses which direct movement towards a favorable oxygen levels and

a redox environment, respectively. There are other responses to oxygen levels such as biofilm

formation, such as Pseudomona~s aerigionsa in the lungs of multiple sclorosis patients produce

more biofilm when under hypoxic stress (Boes et al. 2006).

Bacterial Attachment and Appendages

Bacterial infections occur after the bacteria have been introduced to a "sterile"

environment and are then able to attach, colonize, and form defenses, biofilm formation, within

the host (Schembri et al. 2002). These initial steps of infection are poorly understood for Xfdue

to the inaccessibility of the host environment and fastidious nature of the pathogen. However the

proposed method for attachment of gram-negative bacteria to the surfaces of its environment

(host) is obtained through the pili appendages (Schembri et al. 2002), for Xfthese include type I

pilus which uses the chaperone-usher pathway mediated with the disulfide bond protein family

and the type IV pilus which uses general secretion pathway, which constructs the pilus on the

inner membrane and then protrudes through the outer membrane (Kanehisa Laboratories, Kyoto

Encyclopedia of Genes and Genomics, www.kegg.com, December, 2005). It is thought that the

type IV pili occur on the polar ends of the bacteria and are longer then the type-I pili which cover

the entire bacteria. The type I pili have been associated with the ability of other bacteria such as

Escherichia coli, Klebsiella pneumonia, and Bordetella pertussis, to attach and colonize their

hosts (Soto & Hultgren, 1999). In E. coli the interaction with the pili and the bacteria' s

surroundings lead to changes in protein composition of the outer membrane (Otto et al. 2001).

The utilization of type-I and type-IV pili of Xf have been documented for biofilm production and

movement (Feil et al. 2003; Meng et al. 2005), respectively. The loss of pili/fimbriae expression

has been associated with the effects of sub-culturing, which maybe associated with the reduction









of virulence of the bacteria, after 18 months of subculturing (Hopkins, 1984), but not with a

triply cloned still virulent (Li et al. 1999).

There maybe differences in the attachment to various substrates, namely the foregut of the

vector and the xylem tissue. Some of the EM shots of the bacteria in the media and in the

foregut of vector indicate that there maybe two different attachment mechanisms. In the vector,

the bacteria appear to be standing up (Brlansky et al. 1983; Newman et al. 2004), which would

indicate a polar expression of attachment mechanisms, to which type-IV pili have been

associated to the polar ends of the bacteria. Images from the plant and from media show the

bacteria laying about in no discernable pattern (Leite et al. 2002; Lopes et al. 2000; Mollenhauer

& Hopkins, 1974; Newman et al. 2003; Newman et al. 2004) which may indicate that the type I

pili (fimbria) attachment which are expressed all over the bacteria.

The attachment to both of these substrates is probably mediated through adhesin

expression differences. Another piece of evidence for this maybe that when comparing the

movement down the vine the production only of the type-IV pili moved even farther then the

wild-type, which produces both type-I pili and type-IV pili, which may indicate that the lack of

fimbria may inhibit the colonization of the xylem as the bacteria pass through (Meng et al.

2005). The type-I pili have also been implicated in the attachment of other bacterial pathogens

to there respective hosts, an example is the, well described, PAP system in E. coli which attaches

to the urethra, a conduit that passes urine from the bladder to outside the body (Fernandez &

Berenguer, 2000; Soto & Hultgren, 1999).

The previously mentioned adhesion techniques for Xfare based on the model for other y-

proteobacteria (mainly E. coli) proposed by Schembri, et al, 2002. This model indicates that

there is a phase variance in the expression of certain appendages in E. coli, which is triggered by









the oxygen sensor system of OxyRS, which is a two component system consisting of a sensor

and a transcription factor which in turn activates an adaptation response to the change in

environment (Storz & Imlay, 1999). Another part of this model is that there is a sequence of

events which the bacterium goes through in order to form a bioHilm, first is attachment, followed

by the formation of a micro-colony and then the expression of "biofilm".

Bacterial attachment comes in two stages, one is a temporary form which lasts few seconds

to minutes and the second is a permanent adhesion to the surface. During the temporary

adhesion stage the bacteria senses its environment around it, either using adhesin molecules at

the tips of the pili, used to rest on the surface, or sensors embedded in the membranes of the

bacteria. If during the temporary adhesion the bacteria determines it is a suitable habitat the

bacteria will begin to change the attachment mechanism to form a permanent link, however if the

environment is not to the bacteria's "liking" the bacteria will not stay attached to the surface

(Agladze et al. 2003; Agladze et al. 2005).

Based on Schembri's model bionilm is not created until a microcolony is formed on a

surface, and there has been a change in the production of appendages. The type I pili have been

implicated in the formation of biofilm and by knockout mutations the lack of production (Feil et

al. 2003; Meng et al. 2005). Type I pili in E. coli has also been implicated in the reduction of

virulence based on a changes of gene expression due to surface interaction with the appendages

(Otto et al. 2001)

Cell-to-cell aggregation, which contributes to the stability in the formation of colonies

within the host tissue (Schembri et al. 2002), has been attributed to two hemagglutinin proteins,

binding proteins not associated with pilus/fimbria formations, are involved in cell-to-cell

aggregation (Guilhabert & Kirkpatrick, 2005).












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CHAPTER 2
EFFECT OF OXYGEN ON THE GROWTH AND BIOFILM FORMATION OF Xylella
fasdtidiosa

Introduction

Xylella fastidiosa (X~f) is a Gram negative bacillus bacterium which resides in the xylem

vessels of many plants. The bacterium is vectored between host plants by xylophagus insects,

such as the glassy-winged sharpshooter [Homalodisca coagulata (Say)] (Purcell & Hopkins,

1996). Xfis believed to cause diseases in its host by restricting the transpiration stream by

clogging the xylem elements and/or stimulating the plant to form tyloses, protrusions from the

parenchyma cells, which plug the xylem elements (Esau, 1948).

Xfwas described as being an obligate aerobe (Wells et al. 1987). However recent

publications describe the dissolved oxygen content at hypoxic and anaerobic levels in xylem

vessels (Table 1) during the growing season when the demand for oxygen in the metabolically

active tissue is at its highest. The amount of dissolved oxygen found in the xylem fluid can

range from ~20-30 Clmol L^1 (~9% of air-saturated water) (Mancuso & Marras, 2003), and up to

280 Clmol L^1 (~95% of air-saturated water), at 200C (Gansert, 2003).

The xylem elements are constructed predominantly of lignin and cellulose, for strength and

water proofing respectively. Xylem fluid consists of 95 99% water, with the remaining

constituents mainly consisting of amino acids, small sugars and ions (Andersen & Brodbeck,

1989; Andersen & Brodbeck, 1991; Andersen et al. 1992; Andersen et al. 2005). Another

constituent of xylem fluid, which is important to the survival capabilities of XJ; is the

concentration of dissolved oxygen. The dissolved oxygen predominantly enters though the root

zone (Mancuso & Boselli, 2002), with a small percentage enters through radial diffusion through

the trunk and stems (Gansert et al. 2001; Mancuso & Marras, 2003) and the concentration

decreases as the transpiration stream moves away from the roots (Eklund, 2000). The levels of










02 get low enough that the plants may begin a fermentation (anaerobic respiration) process in

order to meet the required energy needs, as seen with the generation of fermentation by-products

such as ethanol and aldehydes (Kimmerer & Stringer, 1988; MacDonald & Kimmerer, 1991).

This is especially true for the xylem environment at the center of large trees. It is also interesting

that the tissue at the terminal ends of the xylem system i.e. leaves, are capable of metabolizing

these fermentation products (MacDonald & Kimmerer, 1993).

There are three physical components which regulate the ability for oxygen to become

dissolved in water; one is the temperature, in that the higher the temperature the less the

solubility of oxygen in water, another is osmolarity, the more constituents a solution has the less

oxygen can be retained, and the third regulator is pressure where a decrease in pressure will

decrease the amount of oxygen in water. In plants these components regulate the amount of

dissolved oxygen that can enter into the inner plant tissues. The contributions of these conditions

for Vitis are such that during the growing season it creates what could be an environment with

low levels of dissolved oxygen. The temperature rises into to the mid-90s (degrees Fahrenheit)

in north Florida, the tension of the transpiration stream can get as low as -1.6 MPa (or -230 psi)

and the osmolarity of the xylem fluid is approximately 20 mM (Andersen & Brodbeck, 1989;

Andersen & Brodbeck, 1991; Andersen et al. 1992), which is higher than in winter but probably

has little overall effect. Other factors which effect the dissolved oxygen concentration is

flooding, in which the water becomes anoxic around the root zone, and during drought, where

the roots isolate themselves off to the environment to prevent further water loss.

The genomes for two strains (pathovars) ofXfhave been sequenced. The first was citrus

variegated chlorosis (Simpson et al. 2000) which causes a disease in citrus that result in

unmarketable fruit. The second strain to be sequenced was the Pierce's disease strain Temecula










(M. A. Van Sluys et al. 2003), which infects grapevines and reduces the fruit quality and kills the

most susceptible Yitis species such as Yitis vinifera. There are also two other genomes which

have been partially sequenced, one Dixon, almond leaf scorch pathovar, and the other Ann,

oleander leaf scorch (M. A. Van Sluys et al. 2003). These sequenced genomes indicate that the

pathovars contain the similar oxidative phosphorylation pathways and the capability for

anaerobic energy production by sulfate reduction.

Materials and Methods

Bacteria Used

The Xylella fa;stidiosa strains were obtained Dr. Purcell and Christina Wistrom (UC

Berkeley). The Xf strains used were the Pierce's disease strains: Temecula, UCLA, STL, Conn

Creek, and Santa Cruz; and the almond leaf scorch strains used were: Tulare and Dixon. The

other bacterial cultures used were: Erwinia sp, Pseudonzona~s sp, Xanthonzona~s sp, Xanthonzona~s

canspestris pv vitians, (from Dr. Momol's lab group) and Xanthonzona~s canspestris py.

canspestris (from Dr. Jones)

Cultivation Protocol

Bacterial suspensions in PW+ and 10% glycerol were stored in a ~800C freezer (Baxtor

Scientific Products, Cryo-Fridge). Initial cultures were started by scrapping a sterile wooden

applicator stick over the frozen stock culture and streaked out onto a Buffered Charcoal Yeast

Extract (BCYE) plate (Wells et al. 1987). After 10 days bacterial growth was scraped off with

an inoculation loop and placed into 5 mL of PW+ broth (Davis et al, 1981) and incubated

between 10 to 14 days, (until a visible turbid solution) before being used to make working

cultures. These 5 mL cultures were transferred to 50 mL of PW+ media in 250 mL Erlenmeyer

flasks with cotton plugs or silicone rubber plugs with a filter, to allow gas exchange. All









incubations were done in New Brunswick incubator (model# G25-R) set at 280C and all liquid

cultures were placed on an orbital shaker set to 150 rpm.

Experiments were carried out in 15 mL polypropylene Falcon tubes with red-rubber septa

or screw caps and 16 X 150 mm glass test tubes with slip cap closures. The media used included

an enrichment media, PW the defined media, CHARD2 (Leite et al, 2004), which resembles

the xylem chemistry of Vitis vinifera 'Chardonnay' and the variant CHARDS, and the defined

media XDM2 (Lemos et al, 2003), which was created by studying the genomic requirements of

the citrus variegated chlorosis strain and the variant XDM2-PR, XDM2 which lacks phenol red.

PW+ Media, Broth and Solid

PW' media (Davis et al. 1981) was made in two solutions which were then combined.

Solution A consisted of (for 1L) 900mL dH20 to which these components were added, 1.0 g

soluble starch, 4.0 g soytone, 1.0 g tryptone, 1.2 g K2HPO4, 1.0 g KH2PO4, 0.4 g MgSO4-7H20,

0.85 g (NH4)2HPO4, 1.0 g histidine, 25.0 mg cyclohexamide, 10.0 mL (0.1%) hemin-C1, 10.0 mL

(0.2%) phenol red. Media were sterilized by autoclaving. Solution B was made by dissolving

4.0 g of glutamine into 50 mL dH20 at ~600C, and 6.0 g bovine serum albumin (fraction V) into

30 mL of dH20, which were then combined. Next, media were sterilized through a 0.22 Clm

filter. The two solutions were then combined to form the completed broth medium. PW+ agar

plates were made by adding 12.0 g agar to solution A before sterilization in the autoclave.

Solution B was then combined when solution A cooled down to 550C in a hot water bath. Then

PW' medium was aseptically aliquoted into Petri dishes. PW' soft agar deeps were made using

2.0 g of agar in solution A before sterilization in the autoclave. Solution B was then combined

when solution A cooled down to 550C in a hot water bath. The soft agar was then dispensed

aseptically into the appropriate test tubes, either the polypropylene or glass.









CHARDS medium

CHARDS was a modified version of CHARD2 which was a defined media based on the

xylem fluid chemistry of Vitis vinifera 'Chardonnay' (Leite et al. 2004). Solution A consisted of

(for 1 L) 819 mL of dH20 to which these components were added, 1.0 g KH2PO4, 1.5 g K2HPO4,

0.2 g MgSO4-7H20, 0.17 g alanine, 0.58 g aspartic acid, 1.8 g glutamine, 1.05 g arginine, 0.01g

cysteine-HC1, 1 mL (1 mg/ L) biotin, and 1.0 g of soluble starch. The pH was then adjusted to

6.6 using concentrated KOH and then sterilized by autoclaving. Solution B consisted of 0.25 g

of ferric pyrophosphate dissolved in 60 mL dH20 and 4.0 g glutamine dissolved in 50 mL dH20

at ~600C which was then combined and was sterilized through a 0.22 Clm filter. The two

solutions were then combined, aseptically, to form the completed media.

XDM2-PR meduim

XDM2-PR was a modified version of XDM2 (Lemos et al. 2003) which was a defined

media based on the genome of X fastidiosa py. citrus variegated chlorosis. For 1 Liter, into 980

mL of dH20 these components were added 10.0 g glucose, 2. 1 g K2HPO4, 0.8 g KH2PO4, 0.4 g

MgSO4-7H20, 0.125 g ferric pyrophosphate, 4.0 g glutamine, 1.0 g asparagine, 0.4 g serine, 0.4

g methionine, and 10 mL of vitamin solution. The pH was then adjusted to 6.6 using

concentrated KOH and then sterilized through a 0.22 Clm filter. The vitamin solution consisted

of (for 100mL) 350.0 mg myo-inositol, 10.0 mg pyridoxine-HC1, 10.0 g thiamine, 5.0 mg

nicotinic acid, and 0.2 mg of D-biotin. The vitamin solution was sterilized through a 0.22 Clm

filter and stored at 40C in a light-protected bottle.

The BCYE Agar Plates

This was the media used by Wells et al, 1987, for the initial growth of the "Xylella" strains

(ATCC medium 1099 CYE; buffered (Charcoal yeast extract) medium). Solution A was made

by mixing 0.4 g L-cysteine-HCI into 10 mL of dH20 and sterilized through a 0.22 Clm filter.









Solution B was made by mixing 0.25 g ferric pyrophosphate into 10 mL of dH20 and sterilized

through a 0.22 Clm filter. Solution C was made by mixing 10.0 g of ACES buffer (n-2-

acetamido-2-aminoethane sulfonic acid) into 250 mL of dH20. The pH was adjusted to 6.9 +/-

0.05; by this means the chemical will go into solution. Solution D consisted of 10 g of yeast

extract was added into 730 mL of dH20, 2.0 g of charcoal, 17.0 g of agar. pH was adjusted to

6.9 +/- 0.05 using KOH. The media was autoclaved to sterilize and then tempered to 500C in a

water bath. Solutions A, B, C, and D were combined aseptically. The media was dispensed into

Petri dishes and gently swirled to keep the charcoal in suspension thereby ensuring even

di stributi on.

Gram's Staining

Gram's staining protocol was conducted on a sample of cells, either a single CFU applied

to a glass slide or 1 mL of liquid culture spun down and re-suspended in sterile dH20 and 20 Cll

placed on a glass slide and allowed to air dry. Then the bacterium on the glass slide was heat

fixed by passing the slide over a Bunsen burner three times, fixing the bacteria to the slide. Once

cool, crystal violet was applied on to the fixed bacteria for one minute, and then the slide was

rinsed with dH20. Next, iodine was placed onto the culture for one minute, and then the slide

was rinsed with dH20. The slide was then de-stained in an acetone: ethanol mixture, for thirty

seconds, and then the slide was rinsed with dH20. Finally, safranin was applied to the bacteria

for one minute, and then the slide was rinsed with dH20. Gram positive bacteria stain purple due

to the crystal violet, iodine compound, where the Gram negative bacteria stain pink with the

lighter stain basic safranin.

PCR Protocol

Once purity had been established by Gram's stain, PCR (polymerase chain reaction) was

used to confirm the identity of the cultures. DNA extractions were conducted using the Quiagen









DNeasy mini prep. The PCR protocol used a nested primer technique (Pooler & Hartung, 1995).

The first reaction used the primers 272-1 (5 '-AGCGGGCCAATATTCAATTGC-3 ') and 272-2

(5' -AGCGGGCCAAAACGATGCGTG -3'). The final concentration of reagents for a 20 Cll

reaction was lX PCR Buffer, 2.0 mM MgCl2, 0.2 mM dNTPs, 0.5 CIM primer 272-1, 0.5 CIM

primer 272-2, and 1 U Taq. The second reaction used the primers 272-2int (5'-GCCGCTTCGG-

AGAGCATTCCT-3') and CVC-1 (5' -AGATGAAAACAATCATGCAAA-3 '). The final

concentration of reagents for a 20 Cll reaction was lX PCR Buffer, 2.0 mM MgCl2, 0.2 mM

dNTPs, 0.3 CIM primer CVC-1, 0.3 CIM primer 272-2int, and 1 U Taq. The thermocycler

protocol (same protocol for both reactions) was 94oC for 2min, then 35 cycles of 94oC for 1 min,

62oC for 1 min, and 72oC for 1.5 min, followed by a final extension at 72oC for 5 min. The

reactions were stored at 4oC until they were assayed by electrophoresis through 1% agarose gel

slab and stained with ethidium bromide, with positive results indicated by a band at ~500 bp.

Deeps

Soft agar was tempered to 500C in a water bath. This was inoculated with Xylella

fastidiosa to a final optical density at 600 nm of 0.05 and then dispensed into 16 X 150 mm glass

test tubes with slip-cap closures or 15 mL polypropylene falcon tubes with rubber septa sealed

with parafilm and treated with air or nitrogen. These tubes were then placed in a 280C incubator

and the bacterial development was then tracked over time. Results were photographed.

Stabs

Soft agar deeps, consisting of PW+ media and 0.2% agar, was inoculated and dispensed

into 16 X 150 mm glass test tube with slip cap closures, to ensure adequate gas exchange. An

inoculation needle was dipped into a PW+ culture at an optical density at 600 nm of 0.2 (or

greater) and stabbed into the center of the tube. Other experiments used 15 mL polypropylene

falcon tubes with rubber septa was sealed with parafilm and treated with air or nitrogen. The









tubes were then incubated at 280C until initial growth was visible. At that point a photograph of

the tube/growth was taken. The tube was then placed back into the incubator. The bacterial

development was then tracked over time.

Oxygen Levels

The LaMotte's Dissolved Oxygen Test Kit (Model EDO-Code 7414), was used to

determine the treatment effects over time in the aqueous environments of dH20 and CHARDS.

The assays were conducted within red-rubber septa closed tube by inj ecting 100 Cll of the

manganous sulfate mixed well, then 100 Cll of the alkaline potassium iodide azide mixed well

and when the precipitant started to settle a 100 Cll of 50% H2SO4 mixed well until all the

precipitant dissolved. The tubes were then opened and the solution was transferred to a supplied

beaker with a lid designed to fit around the tip of the graduated titration syringe. Then

thiosulfate was titrated into the sample and when the color became clear, against a white

background, the volume used represented the amount of dissolved oxygen in the sample,

measured in ppm.

Growth Conditions

Mature bacterial cultures, in 50 mL batches, were removed from the incubator and pooled

together into a sterilized media flask. The optical density of the culture was obtained, using a

spectrophotometer set at a wavelength of 600 nm. Aliquots were placed into sterile

polypropylene/glass tubes and centrifuged at 2380 x g for 15 minutes. The supernatant was

decanted and the pellet was resuspended into 3 mL of PBS (phosphate buffered saline, 0. 1 M

phosphate, 0.8% NaCl and a pH of 6.8). The suspension was then centrifuged again. The

supernatant was discarded and the pellet resuspended into the appropriate media. This was the

centrifuged as before and the supernatant discarded. The pellet was resuspended into 5 mL of

the appropriate media. Then the tubes were capped with red-rubber septa and secured with









parafilm. Gas treatments were done each day. The tops of the septa were sterilized by rinsing

with 70% isopropyl alcohol and dabbed dry with a kimwipe. The gas was passed through a 0.22

Clm syringe filter then inj ected through a one inch, sterilized 22 gauge needle, and then another

sterilized 22 gauge needle was placed into the septa to act as an exhaust port to ensure gas

exchange. The tubes were placed in the New Brunswick incubator set at 280C, on an orbital

shaker set at 150 rpm.

Optical Density (OD)

Optical density was a quantitative measurement of the concentration of planktonic bacteria

in solution based on the absorption of light through a liquid culture. OD was measured using a

Genosys 8 spectrophotometer at a wavelength of 600nm. The machine was zeroed using an

aliquot of sterile media or suspension buffer. Then an aliquot of the bacterial suspension was

placed into the cuvette and measured.

Biofilm Qualification

The biofilm assay was a qualitative measurement of the concentration of bacteria that have

become sessile on the tube or flask in which they were grown (Espinosa-Urgel et al. 2000). The

bacteria were decanted and the vessel was rinsed with dH20 and air dried. The vessels were then

filled with 0. 1% crystal violet, and placed on a shaker for 1 hour. The crystal violet was

decanted and the tubes were rinsed until all the excess dye has been removed and air dried. The

vessels were then de-stained using 70% isopropanol. An aliquot will then be placed into a

cuvette and the optical density was measured at a wavelength of 600nm. The spectrophotometer

was zeroed using a cuvette with 70% isopropanol used as a blank.

Media Analysis

Xf strain 'Temecula' was inoculated into CHARDS and XDM2-PR media by dispensing 1

mL of mature culture (~0.35 OD at 600nm) into 15 mL polypropylene tube and centrifuged









(Jouan C3-12) at 2,380 g for 15 minutes. The supernatant was discarded and the pellet was

resuspended in 3 mL of PBS. This was centrifuged at 2,380 g for 15 minutes and the supernatant

was discarded. The pellet was resuspended in 2 mL of the appropriate media and then

centrifuged at 2,380 g for 15 minutes. The supernatant was discarded and the pellet was

resuspended in 5 mL of the appropriate media and sealed with red-rubber septa and sealed with

parafilm. The tubes were then subjected to gas treatment which consisted of various

concentrations of oxygen, 21% (breathing air) and 0% (nitrogen) for 5 minutes every day for the

duration of the experiment. Each treatment consisted of inserting a syringe needle connected to a

compressed gas tank with a positive pressure of ~5 psi through the rubber-septa and an exhaust

needle was inserted to allow for gas exchange.

Three tubes from each media-gas interaction were removed every five days and assayed.

The optical density was obtained at 600 nm. A 'drop' was placed on PW' agar media and

incubated at 280C for 10 days to determine viability. A 2 mL sample from each tube was

centrifuged at 5,000 g at 40C for 15 minutes (Sorval Biofuge, Model: Stratos) in a temperature

regulated centrifuge, then the supernatant was stored at -800C. Then the tubes were then emptied

and assayed for biofilm formation.

The frozen supernatant samples were filtered through a 10,000 molecular weight cutoff

filter and then prepared for HPLC analysis, using a Water automated system operating with the

Millennium operating program. Organic acid analysis was conducted on a polymeric cation

exchange column using 0.01 N H2SO4 elution at 0.4 mL per minute, at 400C. Product was

detected by UV absorbance at 210 nm, which corresponds to the presence of carboxyl groups.

Statistical Analysis

All experiments were designed so that each data collection would consist of three

independent replicates. The bacteria were divided and washed in order to insure that each










response observed was dependent on each treatment (and not the three measurements of the

initial bacteria treatment interaction). The experiments were done at least twice, with fresh

cultures and media, to confirm the results obtained were reproducible. The data was analyzed

with ANOVA, t-tests, means and standard error as well as regression using the SAS V9.0

software package. The error bars represent standard error.

Results

Agar Deeps

The initial growth (Figure 2-la), for all Xf strains tested, began just below the surface.

This area was still considered to be aerobic; however, it indicates that optimum growth of the

bacterium occurred in a reduced oxygen environment. The growth of the bacterium proceeded

down into the tube away from the initial growth development. After 2 months of incubation in

either the air or nitrogen treatments bacterial growth development continued from just below the

surface down to the bottom of the tube for deeps and along the path of the inoculating needle for

stabs as well as radiating out from the path of the inoculating needle (Figure 2-1Ib and 2-1c).

During this growth there was no evidence of gas production, which would be indicated by the

formation of bubbles in the agar.

Experimental Conditions

The oxygen levels were determined using a LaMotte's Dissolved Oxygen Test Kit (model

EDO~code 7414). Using deionized water and CHARDS media the experimental conditions were

assayed to determine the dissolved oxygen levels as a function of treatment and effects over

time. Figure 2-2 shows that the initial treatment was enough to establish the dissolved oxygen

level. Once the level had been set it was stable after daily treatments for the length of the

experiment. The LaMotte's test kit has the limitation in which there may be dissolved oxygen in










the liquid reagents and therefore may overestimate the oxygen concentrations as well as

erroneously detecting oxygen for the nitrogen treatment.

Xylella vs. an Obligate Aerobe and a Facultative Anaerobe

The growths of Xf stains UCLA, STL and Tulare-ALS (Figure 2-3a) was compared to

Pseudonzona;s sp, an obligate aerobe and Erwinia sp, a facultative anaerobe (Figure 2-3b) in PW+

at various oxygen levels. The patterns of growth for all three of the Xf strains under the four gas

treatments were similar in that the optical density and biofilm dropped as a function of the

reduction of the oxygen levels. The growth of the obligate aerobe (Pseudonzona~s sp) only

occurred under the 21% oxygen levels. The optical density of the facultative anaerobe decreased

as a function of the oxygen levels. It should be noted that the growth from an OD of 0. 1 to 0.2

for the Pseudonzona~s could be attributed to a delay prior to the gas treatments. However it would

appear that there was no growth after the subsequent gas treatments, as indicated by the lack of

growth of another obligate aerobe, Xanthonzona~s canspestris (Figure 2-5), under nitrogen

treatments.

Xylella vs. Xanthomonas

The comparison of growth rates, in nutrient broth, of two putative obligate aerobes, X.

fastidiosa and the closely related X. canspestris py. canspestris, under aerobic and hypoxic

conditions show that there was a difference in the response of these two bacteria. The growth of

X. fastidiosa Pierce's disease strain 'Temecula' (Figure 2-4) (as well as 'UCLA and the ALS

strain 'Tulare', data not shown) showed a continued growth under the aerobic (air) treatment;

however, the anoxic (nitrogen) treatment also showed steady growth in the nutrient broth. I.

canspestris py. canspestris (Figure 2-5) was able to grow under aerobic treatment as evidenced by

the rise in the optical density, whereas it was unable to grow under the anoxic conditions as









indicated by the negligible slope of the line (this was also seen in other experiments conducted

with X campestris py. vitians, data not shown).

Defined Media Growth

Xf 'Temecula' when grown under anoxic and aerobic conditions in defined media had

continual growth over thirty days of gas treatments in both media. The changes in the optical

density for Xf in CHARDS media (Figure 2-6a) under the gas treatments were not significant as

a function of gas treatment. The bioHilm production in the CHARDS (Figure 2-6b) was not

significant. In the XDM2-PR media growth and bioHilm production (Figure 2-7a and 2-7b)

increased significantly under the nitrogen gas treatment.

Organic Acid Analysis

Organic acid profiles of CHARDS and XDM2-PR media varied between the air and

nitrogen treatments. Comparisons of the peak areas at each retention time (which correspond to

a compound eluting from the HPLC column) are shown in Tables 2-2 and 2-3. The rows of

zeros are compounds that vanished either as a function of bacterial growth or as a function of

time when the 15 day old media was compared to day 0, which were used to determine the initial

optical densities as well as the negative control which had no bacteria in it.. Both media had

significant differences associate with presence of bacteria and the gas treatment as well as the

interaction of both.

Discussion

Based on the results of the agar deeps Xylella fastidiosa appears to prefer a reduced

oxygen atmosphere based on the initial growth (arrows in Figure 2-la) below the surface. When

the tubes were allowed to incubate the growth continued down to the bottom of the tube, and did

not occur on the surface. The growth at the area below the surface would increase thereby

creating a biologically active barrier, thereby utilizing the oxygen as it diffused into the agar










deep, and preventing the transfer of oxygen from the surface to areas under the growth

formation. In the agar deep experiment, when gas exchange allowed oxygen to remain at the

21% levels, using the slip-cap tubes, growth never developed on the surface of the agar, but

always just below. However, on occasion colonies would form on the surface for sealed tubes of

the agar stabs but usually after many weeks. These growth patterns indicate a negative effect of

either a gaseous environment or high (21%) oxygen levels.

The growth of the three Xf strains in various oxygen concentrations (Figure 2-3a) shows a

gradual step down growth pattern were the greatest optical density was under the 21% oxygen

treatment and dropped with the subsequent 2. 1% oxygen and the 0.21% and 0% treatments, as

indicated by the ANOVA analysis and Duncan separations. This growth pattern was not similar

to the growth pattern of the obligate aerobe, Pseudomona~s sp (Figure 2-3b) where growth only

occurred in the 21% oxygen treatment. The growth pattern was very similar to the facultative

anaerobe, Erwinia sp (Figure 2-3b), which also has an incremental growth pattern down the

oxygen gradient except for the 0% oxygen treatment were the anaerobic metabolism allowed for

an increased growth compared to the 0.21%, which may indicate the oxygen labile nature of the

anaerobic machinery. The comparison of these growth patterns raised the question as to the

respiration classification of Xfas an obligate aerobe.

The comparison of growth between Xylella (figure 2-4) and its close relative,

XanthomonasX~~XX~~~XX~~XX (Figure 2-5) indicated that there was even more of a reason to doubt the obligate

aerobic status ofXylella as stated by Wells et al. 1987. The growth pattern for Xylella under the

gas treatments of air was as expected with continuous growth; however the presence of steady

growth after 15 days in the hypoxic treatments (nitrogen) was quite remarkable (these results

were similar for two other Xylella strains, UCLA-PD and Tulare-ALS, data not shown). There









was more growth for the aerobic (air) treatment verses the hypoxic (nitrogen) treatments.

Aerobic respiration produces about 32 to 34 ATP (per glucose molecule) whereas fermentation

tends to produce 4 ATP (per glucose molecule). Xylella has the genetic capability for sulfate

reduction (M. A. Van Sluys et al. 2003; Simpson et al. 2000); however, end products such as

hydrogen sulfide or elemental sulfur (or iron sulfide) were not detected.

The growth ofXfin CHARDS was not significantly different as measured by optical

density and biofilm formation; however it should be noted that growth was steady and

continuous. However the growth in the XDM2-PR media was significantly different, in that

there was an increase in optical density as well as biofilm formation under the anoxic (nitrogen)

treatment verses the aerobic (air) treatment. Analysis of the two defined media after the growth

experiment yielded more evidence for the possibility of anaerobic capabilities (Tables 2-2 and 2-

3) as indicated by the differing organic acid profiles, due to the interaction of the bacteria and gas

treatments. The bacteria and gas treatment interactions indicate that there is a change in

metabolic activities as a function of oxygen availability. This switch in metabolism is could be a

change from oxidative phosphorylation which uses oxygen as a terminal electron acceptor to a

fermentation pathway, which uses the substrate as the electron donor as well as the electron

acceptor.

During the growing season the dissolved oxygen in the xylem fluid becomes low enough

to induce fermentation within the plants tissues (Kimmerer & Stringer, 1988), with the results

presented here it was likely that Xylella fa;stidiosa was capable of the same. The ability of Xfto

modify its metabolism and/or respiration pathways implies that during the growing season when

the oxygen levels drop and yet the demand becomes greater that the bacteria are capable of

continuing their growth and development. Not only are they apparently capable of sustained










growth, but biofilm production increases as well, which probably leads to the onset of disease

symptoms in late summer and early fall (Purcell & Hopkins, 1996).

Goodwin et al, 1988, investigated the presence of possible toxic compounds generated by

Xf; however the results were based on the washes of aerobically grown cultures on solid media.

The results of our experiments dispute or raise doubts regarding the production of toxic

compounds (Goodwin et al. 1988). Since Xfhas always been considered to be an obligate aerobe

(Wells et al. 1987) studying the different metabolic by-products, as a function of fermentation,

which could either bring about tylosis formation, for which no known trigger exists, by the plant

or simulating the symptoms of the disease, such as the abscission of the leaf from the petiole and

"green islands" or the lack of cork production (Esau, 1948; Krivanek et al. 2005).

These results, the continued growth as well as changes in the organic acid profiles under

the anoxic treatments raise serious questions regarding Xylella 's oxygen requirements for

growth. The strains UCLA and Tulare showed growth under the anoxic conditions in the

nutrient broth (data not shown), similarly to the Temecula strain. Other evidence includes three

other strains of Peirce's disease, Conn Creek, Santa Cruz, STL, and another almond leaf scorch

Dixon as soft agar stabs (data not shown) which indicates that this anaerobic ability may include

many more (or all the ) strains. The differences in the metabolic by-products may also indicate

that there is a compound produced by the bacteria which may in turn be a toxin or a stimulator

for disease symptoms in the plant environment. We are in the process of naming as well as

quantifying the organic acid data with HPLC-MS (Dr. Jodie Johnson, University of Florida) and

will hopefully be able to identify the fermentation substrates as well as end products.













OhO 0000
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e atttt



en 9 90000 p
ONONOmoo

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o, OO
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0 0
O ~ '~ O o00
a d ccr 000
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1


Figre -1.Groth f Xlela fstiios inaga deps o dterineoye iiain.A
Intil rothofXyelafatdoa(etrtretbswharwlftori t
UCLA,~~~~~~~~~~~~~ STadTlr amn efsoch)atr5dy fgotwe
copae t hegrwh fPsedmnss.(a eta biaearb n rii









sp. (far right), a facultative anaerobe. B) Growth of Tulare (almond leaf scorch) after
two weeks of growth with the left tube grown under nitrogen gas, the middle tube
under air and the right tube is an un-inoculated deep. C) Growth of Temecula
(Pierce's disease) after two weeks with the left tube grown under nitrogen gas, the
middle tube under air and the right tube is an un-inoculated deep.













-z- dH20
x -A- CHARDS Day 1
6 HRS a
-m CHARDS Day 3






















21% 10% 2.10% 0%
Oxygen Levels

Figure 2-2. Dissolved oxygen levels detected in dH20 and CHARDS media as a function of
treatment levels and over time. The dissolved oxygen levels in dH20 were
determined after one treatment. Each point is the average of three replicates.























































a aB
















i~i: ic Ic












21.00% 2.10% 0.21% 0.00% 21.00% 2.10% 0.21% 0.00%


-.

0.4




OD 0.3




0.2




0.1 I











1.8


1.6


1.4


1.2


1


OD 0.8









0.2






-0.2


Figure 2-3. Comparison of Xylella fa;stidiosa cultivated under reduced oxygen levels. A) The

comparison of growth of three Xylella fastidiosa strains (two Pierce' s disease, STL
and UCLA, and Tulare, almond leaf scorch) under different oxygen levels, each with

an initial optical density of 0.05 were incubated for 15 days. B) The comparison of










growth of an obligate aerobe (Pseudomona~s sp) and a facultative anaerobe (Erwinia
sp), under different oxygen levels, each with an initial optical density of 0. 1 were
incubated for 15 days. Error bars represent standard error and the optical densities
were separated using Duncan's.











0 45


4 6 8 10 12 14


Figure 2-4. Xylella fa;stidiosa growth in PW+ for 15 days under aerobic and hypoxic conditions.
Each point is the average of three replicates.














o Air
aNitrogen
-Linear (Air )
1.0 -I' **Linear (Nitrogen)
y=0.1979x -0.0325
y=0179r .03 O


0.8


OD
0.6 n




0.4




0.2

y=-0.0007x +0.0789


0.0
0 1 2 3 4 5 6

Days

Figure 2-5. Xanthomonas campestris py. campestris growth in PW+ for 5 days under aerobic
and hypoxic conditions. Each point is the average of three replicates.












































0 5 10 15 30
Days


0.4

B
0.5 --CHARDS Air
-a-CHARDS Nitrogen




0.


Biofilm


00 2








0.05




5 10 15 30

Days

Figure 2-6. Growth and performance of Pierce's disease strain Temecula in the CHARDS media
under two gas treatments. A) is optical density and B) is biofilm formation under air

(open squares) and nitrogen (closed squares) gas treatments. Each point is the
average of three replicates.


0.25


-0- CHARDS Air
| m-CHARDS Nitrogen|


0.2






OD0.15



0.1





0.05


P















A
-1-- XDM2-PR Air
-5-XDM2-PR Nitrogen IT


0.25


0.2




0.15


OD


0.1




0.05






0 5 10 15 30

Days


0.16
-i-XDM2-PR Air B
-mXDM2-PR Nitrogen
0.14


0.2




Biofilm 00




0.06


0.04


0.02



5 10 15 30

Days

Figure 2-7. Growth and performance of Pierce's disease strain Temecula in the XDM2-PR
media under two gas treatments. A) is optical density and B) is biofilm formation
under air (open squares) and nitrogen (closed squares) gas treatments. Each point is
the average of three replicates.

























































day 0, the initial samples
0.001, **** = 0.00001.


1


Table 2-2. Organic acid profiles in the CHARDS media after 15 days


Retention with Xylella fa;stidiosa


without I. fastidiosa


Significance
Bacteria Gas
**
* **


times a
6.646
6.906
7.586
8.019
8.457
9.322
10.643 b
11.027
11.405
11.54
11.834
12.429
12.588
12.633
13.226
13.494
14.462
15.33
15.791
16.515
17.242
17.53
18.429
19.208
19.743
20.363
21.062
21.797
24.309
25.000
25.937
27.383
27.993
36.736


Air
151.67
1796.00
165606.00
11530.67
8551.33
10307.33
0.00
2736.33
14469.67
0.00
0.00
2093.00
0.00
15805.33
0.00
35029.33
1020.33
36966.67
10655.33
21332.67
34096.33
22030.00
11610.00
10286.67
27483.67
0.00
0.00
19638.33
900.67
2395.33
1710.33
0.00
10129.33
24926.67


Nitrogen
360.67
1198.00
152676.33
18154.33
8666.00
8147.33
0.00
3296.33
0.00
14432.33
0.00
0.00
852.67
9539.33
0.00
31094.00
0.00
9077.33
10573.67
22276.00
33374.67
21202.00
11330.33
10284.00
24209.67
0.00
2363.00
22025.00
1381.00
806.33
0.00
2473.67
17086.33
39429.33


Air
1074.67
1403.00
117838.00
0.00
0.00
6731.67
0.00
1706.33
15258.67
0.00
6483.67
153436.67
0.00
0.00
1973.00
24227.33
0.00
2563.33
10363.67
25589.33
25217.00
18546.67
10987.67
9753.00
16215.33
0.00
0.00
17241.67
0.00
467526.00
0.00
0.00
17819.00
1203003.67


Nitrogen
354.67
1265.33
146047.67
0.00
0.00
9277.67
0.00
4685.33
21957.33
0.00
9962.67
208947.67
0.00
0.00
0.00
35293.67
0.00
13060.67
11561.67
32490.67
33577.00
24383.67
14889.00
128259.33
21433.67
0.00
0.00
22095.67
0.00
602806.67
0.00
0.00
20589.67
35877.67

peak found at
= 0.01, *** =


B*G


** **


**

**
****


**
* *


** ****


****


a = Retention time on HPLC column.


b = Presence of a


(data not shown). P-values are represented = 0.05, *"










Table 2-3.
Retention
times a
6.597
6.844
7.58
8.02
8.457
9.338
10.297 b
10.636
11.046
11.375
11.78
12.395
12.655
13.162
13.722
14.434
15.345
15.437
15.546
16.359
16.511
17.265
17.544
18.002
18.319
18.433
19.218
19.726
21.333
21.561
21.749
22.157
24.291
24.742
25.018


Organic acid profiles in the
With Xylella ~fa;stidiosa


XDM2-PR media after 15 days


Without I ~fastidiosa


Significance
Bacteria Gas

**


Air
1426.33
1575.33
177952.00
6547.33
7740.67
6482.67
0.00
0.00
445.33
4620.67
0.00
60088.33
0.00
7901.67
22658.33
3056.67
0.00
599108.33
0.00
0.00
18813.33
78816.00
0.00
0.00
16433.67
0.00
7353.33
21426.00
3739.00
0.00
12142.33
8151.33
4701.67
0.00
0.00


Nitrogen
548.00
1182.67
231268.67
20048.33
18153.33
9041.67
0.00
0.00
5638.00
0.00
6830.67
0.00
12197.33
8628.00
26896.67
4751.33
0.00
0.00
489081.67
0.00
39624.00
683194.67
0.00
7660.00
25531.67
0.00
12031.00
54879.67
0.00
179943.33
0.00
19473.00
0.00
22175.67
0.00


Air
1051.33
1347.33
153846.00
0.00
0.00
4630.33
0.00
0.00
760.00
2436.00
0.00
72173.00
0.00
4443.00
11057.33
0.00
0.00
270378.67
0.00
0.00
3791.00
11843.67
11792.00
4107.33
0.00
5697.67
5430.67
9372.33
0.00
0.00
3212.33
2221.00
2038.00
0.00
6744.00


Nitrogen
548.50
1286.50
132244.00
0.00
0.00
4085..5
0.00
0.00
1436.00
2066.00
0.00
62718.50
0.00
3516.00
9664.00
0.00
0.00
0.00
241595.00
0.00
8849.50
13438.00
12036.50
5883.00
0.00
6892.00
5851.50
882.00
0.00
0.00
1382.00
0.00
1662.50
0.00
5403.50


B*G



**

**


***
****
**


**** ****


**


****


* *


**
****


***
****
**


***


****


****
**


**
****


**


****

**
**

****


a = Retention time on HPLC column.


Presence of a peak at initiation of the experiment, time


point 0. P-values are represented = 0.05, ** = 0.01, *** = 0.001, **** = 0.00001.










Table 2-3. Continued
Retention With Xylella fastidiosa Without X. fastidiosa Significance
times a Air Nitrogen Air Nitrogen Bacteria Gas B*G
26.442 0.00 1601.00 0.00 0.00 **** *
27.099 0.00 2754.33 0.00 0.00
27.985 12253.33 15074.33 14736.33 10256.00*
28.555 1656.33 15492.00 875.33 0.00 *** *** ***
36.73 23171.00 30152.00 12734.67 10931.50 **
a= Retention time on HPLC column. b = Presence of a peak at initiation of the experiment, time
point 0. P-values are represented = 0.05, ** = 0.01, *** = 0.001, **** = 0.00001.









CHAPTER 3
MEDIA COMPONENT EFFECTS ON GROWTH AND BIOFILM FORMATION OF Xylella
fastidiosa

Introduction

Xylella fastidiosa (X~f) is a Gram negative bacillus bacterium which resides in the xylem

vessels of many plants. The bacterium is vectored between host plants by xylophagus insects,

such as the glassy-winged sharpshooter [Homalodisca coagulate (Say)] (Purcell & Hopkins,

1996). It is believed to cause diseases in its host by restricting the transpiration stream by

clogging the xylem elements and/or stimulating the plant to form tyloses, protrusions from the

parenchyma cells which plug the xylem elements (Esau, 1948).

Xf consists of many strains which reside in many different hosts and in various

environmental conditions throughout the Americas. One of the greatest difficulties in working

with Xfis the variance among the strains such as doubling times (Wells et al., 1987), biofilm

production, and nutritional requirements (Leite et al., 2004; Lemos et al., 2003). There is also

variance in pathogenicity (in vivo) that develops as the bacteria is sub-cultured (Hopkins, 1984)),

which could be due to a reduction in fimbriae production which would be a reduction in adhesion

and subsequent biofilm production in vitro.

Several defined media have been created for the cultivation ofXf: In recent years two

media had been created which were based on the genomic (Lemos et al., 2003) and xylem

chemistry (Leite et al., 2004). Lemos et al., 2003 created a defined media series (XDM) based

on the genomic capabilities of the citrus variegated chlorosis pathovar of Xf(and confirmed its

ability to cultivate other strains such as Pierce's disease (PD) which contained 4 amino acids,

glucose, magnesium sulfate, and iron pyrophosphate as well as vitamins suspended in a

phosphate buffer. Leite et al 2005, produced a defined media (CHARD2) based on the chemical










composition of Vitis vinifera 'Chardonnay' which consist of 5 amino acids, magnesium sulfate

and iron pyrophosphate suspended in a phosphate buffer.

A concern about these media is that they contain phenol red (phenol sulfonephthalein)

which is commonly used as a pH indicator in bacterial media (also as a color indicator used to

test kidney function) but has no (known) biological function. Phenol red is normally a

colorimetric pH indicator which changes from bright yellow in acidic conditions, to dark red in

alkaline, and it does indicate that Xf does produce an alkaline product as a function of growth in

PW' media. The structure of phenol red consists of two phenols and a benzene motif attached to

a central carbon atom along with a sulfite group, any part of which could interact with the

receptors and sensors associated with the bacteria. The question arises as to the function of

phenol red as a necessary additive to these defined media, as well as the nutrient media such as

PW+ (Davis et al., 1981) PWG and PD2 (American Type Culture Collection, www.atcc.org).

The two media XDM2-PR (a variant of XDM2 which lacks phenol) and CHARD S (a

variant of CHARD2 which has the addition of soluble starch as well as lacks phenol red) have

many similarities such as a phosphate buffer, magnesium sulfate, iron pyrophosphate and an

abundance of glutamine, as well as a similar pH, 6.8 to 6.6, respectively. However there are a

couple of differences which should be noted such as the presence a carbon source, glucose,

vitamins, and the amino acids. Can the differences in optical density and biofilm formation (as

seen in chapter 2) between the two media be explained as a function of these differences? So we

will examine the effects of glucose and vitamins on the growth ofXfin the CHARDS

background.

By culturing the bacteria in the defined media it is possible to analyze the utilization of the

constituent amino acids. When the bacteria was grown in xylem fluid from grapevines










(unpublished data) many of the amino acids correlated with growth were ones which were found

at low concentrations (Andersen et al., 2005).

Materials and Methods

Bacteria Used

The Xylella fa;stidiosa strains were obtained from Dr. Purcell and Christina Wistrom (UC

Berkeley). The Xf strains used were: Temecula-PD, UCLA-PD, STL-PD, and Tulare, an almond

leaf scorch strain.

Cultivation Protocol

Bacterial suspensions of PW+ and 10% glycerol were stored in a ~800C freezer (Baxtor

Scientific Products, Cryo-Fridge). Initial cultures were started by scrapping a sterile wooden

applicator stick over the frozen stock culture and streaked out onto a BCYE (Buffered Charcoal

Yeast Extract) plate. After 10 days bacterial growth was scraped off with an inoculation loop

and placed into 5 mL of PW' and incubated between 10 to 14 days, (until an optical density of

0.2 at 600nm, which corresponds to the mid-log phase) before being used to make working

cultures. These 5 mL cultures were transferred to 50 mL of PW' media in 250 mL Erlenmeyer

flasks with cotton plugs or silicone rubber plugs with a filter, to allow gas exchange. All

incubations were done in New Brunswick incubator (model# G25-R) set at 280C and all liquid

cultures were placed on an orbital shaker set to 150 rpm.

Experiments were carried out in 15 mL polypropylene Falcon tubes with re-rubber septa or

screw caps and 16 X 150 mm glass test tubes with slip cap closures. The media included an

enrichment media, PW the defined media CHARD2 (Leite et al., 2004) which is based on

xylem fluid chemistry of Vitis vinifera 'Chardonnay, and the variant CHARDS, and the defined

media XDM2 (Lemos et al., 2003)), which is based on the genomic needs of the citrus

variegated chlorosis strain.









PW+ Media, Broth and Solid

PW' media (Davis et al., 1981) was made in two solutions which were then combined.

Solution A consisted of (for 1L) 900 mL dH20 to which these components were added, 1.0 g

soluble starch, 4.0 g soytone, 1.0 g tryptone, 1.2 g K2HPO4, 1.0 g KH2PO4, 0.4 g MgSO4-7H20,

0.85g (NH4)2HPO4, 1.0 g histidine, 25.0 mg cyclohexamide, 10.0 mL (0.1%) hemin-C1, 10.0 mL

(0.2%) phenol red. Solution A was sterilized by autoclaving. Solution B was made by

dissolving 4.0 g of glutamine into 50 mL dH20 at ~600C, and 6.0 g bovine serum albumin

(fraction V) into 30 mL of dH20, which were then combined. Next, solution B was sterilized

through a 0.22 Clm filter. The two solutions were then added to form the completed broth

medium. PW' agar plates were made by adding 12.0 g agar to solution A before sterilization in

the autoclave. Solution B was then added when solution A cooled down to 550C in a hot water

bath. Then PW' medium was aseptically aliquoted into Petri dishes. PW' soft agar deeps were

made using 2.0 g of agar in solution A before sterilization in the autoclave. Solution B was then

added when solution A cooled down to 550C in a hot water bath. The soft agar was then

dispensed aseptically into the appropriate test tubes, either the polypropylene or glass.

The BCYE (Buffered Charcoal Yeast Extract) Agar Plates

This is the media used by Wells et al, 1987, for the initial growth of the "Xylelkl`' strains

(ATCC medium 1099 CYE; buffered (Charcoal yeast extract) medium).

Solution A is made by mixing 0.4 g L-cysteine-HCI into 10 mL of dH20 and sterilized

through a 0.22 Clm filter. Solution B is made by mixing 0.25 g ferric pyrophosphate into 10 mL

of dH20 and sterilized through a 0.22 Clm filter. Solution C is made by mixing 10.0 g of ACES

buffer (n-2-acetamido-2-aminoethane sulfonic acid into 250 mL of dH20. The pH is adjusted to

6.9 +/- 0.05; by this means the chemical will go into solution. Solution D consisted of 10 g of

yeast extract was added into 730 mL of dH20, 2.0 g of charcoal, 17.0 g of agar. pH was adjusted









to 6.9 +/- 0.05 using KOH. The media was autoclaved to sterilize and then tempered to 500C in

a water bath. Solutions A, B, C, and D were added aseptically. The media was dispensed into

Petri dishes and gently swirled to keep the charcoal in suspension thereby ensuring even

di stributi on.

CHARDS Medium

CHARDS was a modified version of CHARD2 which was a defined media based on the

xylem fluid chemistry of Vitis vinifera 'Chardonnay' (Leite et al., 2004). Solution A consisted

of (for 1 L) 819 mL of dH20 to which these components were added, 1.0 g KH2PO4, 1.5 g

K2HPO4, 0.2 g MgSO4-7H20, 0.17 g alanine, 0.58 g aspartic acid, 1.8 g glutamine, 1.05 g

arginine, 0.01g cysteine-HC1, 1 mL (1 mg/ L) biotin, and 1.0 g of soluble starch. The pH was

then adjusted to 6.6 using concentrated KOH and then sterilized by autoclaving. Solution B

consisted of 0.25 g of ferric pyrophosphate dissolved in 60 mL dH20 and 4.0 g glutamine

dissolved in 50 mL dH20 at ~600C which was then combined and was sterilized through a 0.22

Clm filter. The two solutions were then combined, aseptically, to form the completed media.

CHARDS-F is CHARDS but autoclaving the starch only in 750 mL of dH20 and the rest

of the ingredients were combined into 250 mL of dH20, which was filtered through a 0.22 Clm

filter and added aseptically to the starch solution.

CHARDS-G is CHARDS without the starch but using 10 g of glucose instead. The

glucose was dissolved in 500 mL of dH20 and filtered through a 0.22 Clm filter and the other

constituents. were brought in 500 mL and autoclaved, then added aseptically.

CHARDS-V is CHARDS with the addition of 10 mL of the XDM2 vitamin solution.

XDM2 and XDM2-PR Media

XDM2 is a defined media based on the genome ofX. fastidiosa py. citrus variegated

chlorosis (Lemos et al., 2003)). For 1 Liter, into 980 mL of dH20 these components were added









10.0 g glucose, 2.1 g K2HPO4, 0.8 g KH2PO4, 0.4 g MgSO4-7H20, 0.125 g ferric pyrophosphate,

4.0 g glutamine, 1.0 g asparagine, 0.4 g serine, 0.4 g methionine, 10 mL (1%) phenol red and 10

mL of vitamin solution. Then the pH was adjusted to 6.6 and then filtered through a 0.22 Clm

membrane. For the creation of XDM2-PR, phenol red was omitted from the recipe.

The vitamin solution consisted of (for 100mL) 350.0 mg myo-inositol, 10.0 mg

pyridoxine-HC1, 10.0 g thiamine, 5.0 mg nicotinic acid, and 0.2 mg of D-biotin and was

sterilized through a 0.22 Clm filter and stored at 40C in a light-protected bottle.

Gram's Staining

Gram's staining protocol was conducted on a sample of cells, either a single colony

applied to a glass slide or 1 mL of liquid culture spun down and re-suspended in 100 Cll sterile

dH20, because of interference caused by some of the media constituents, and 20 Cll placed on a

glass slide and allowed to air dry. Then, the bacterium on the glass slide was heat fixed by

passing the slide over a Bunsen burner three times. Once cool, crystal violet was applied on to

the fixed bacteria for one minute, and then the slide was rinsed with dH20. Next, iodine was

placed onto the culture for one minute, and then the slide was rinsed with dH20. The slide was

then de-stained in a 4: 1 ethanol: acetone mixture, for thirty seconds and then the slide was rinsed

with dH20. Finally, safranin was applied to the bacteria for one minute, and then the slide was

rinsed off with dH20. Gram positive bacteria stain purple due to the crystal violet, iodine

compound, where the Gram negative bacteria stain pink with the lighter stain basic safranin.

PCR Protocol

Once purity had been established by Gram's stain, PCR (polymerase chain reaction) was

used to confirm the identity of the cultures. DNA extractions were conducted using the Quiagen

DNeasy mini prep. The PCR protocol used a nested primer technique (Pooler & Hartung, 1995;

Pooler et al., 1997)). The first reaction used the primers 272-1 (5' -AGCGGGCCAATATTCAA-









TTGC-3') and 272-2 (5' -AGCGGGCCAAAACGATGCGTG -3'). The final concentration of

reagents for a 20 Cll reaction was lX PCR Buffer, 2.0 mM MgCl2, 0.2 mM dNTPs, 0.5 CIM

primer 272-1, 0.5 CIM primer 272-2, and 1 U Taq. The second reaction used the primers 272-2int

(5 '-GCCGCTTCGGAGAGCATTCCT-3 ') and CVC-1 (5' -AGATGAAAACAATCATGCAAA-

3'). The final concentration of reagents for a 20 Cll reaction was lX PCR Buffer, 2.0 mM MgCl2,

0.2 mM dNTPs, 0.3 CIM primer CVC-1, 0.3 CIM primer 272-2int, and 1 U Taq. The

thermocycler protocol, same protocol for both reactions was 94oC for 2min, then 35 cycles of

94oC for 1 min, 62oC for 1 min, and 72oC for 1.5 min, followed by a final extension at 72oC for 5

min. The reactions were stored at 4oC until they were assayed by electrophoresis through 1%

agarose gel slab and stained with ethidium bromide. A positive reaction is indicated by the

appearance of a band at ~500bp.

Growth Conditions

Mature bacterial cultures, between two weeks and four weeks, were removed from the

incubator. The optical density of the culture was obtained, using a wavelength of 600 nm.

Aliquots were placed into sterile polypropylene/glass tubes and centrifuged at 2380 x g for 15

minutes. The supernatant was decanted and the pellet was resuspended into 3 mL of PBS

(phosphate buffered saline, 0. 1 M phosphate and 0.8% NaCl and a pH of 6.8). The suspension

was then centrifuged again. The supernatant was discarded and the pellet resuspended into the

appropriate media. This was the centrifuged as before and the supernatant discarded. The pellet

was resuspended into 5 mL of the appropriate media. Then the tubes were capped appropriately,

either slip caps for the glass tubes, or plastic screw caps or red-rubber septa and parafilm for the

15 mL polypropylene Falcon tubes. The red-rubber septa capped tubes were then subjugated to

gas treatments.









Gas treatments were done each day. The tops of the septa were sterilized by rinsing with

70% isopropyl alcohol and dabbed dry with a kimwipe. The gas was passed through a 0.22 Clm

syringe filter then inj ected through a one inch, sterilized 22 gauge needle, and then another

sterilized 22 gauge needle was placed into the septa to act as an exhaust port to ensure gas

exchange. Gas was passed through at ~5 psi for 5 minutes. The tubes were placed in the New

Brunswick incubator set at 28 C, on an orbital shaker set at 150 rpm.

Optical Density (OD)

Optical density is a quantitative measurement of the concentration of planktonic bacteria in

solution based on the absorption of light through a liquid culture. OD was measured using a

Genosys 8 spectrophotometer at a wavelength of 600nm. The machine was calibrated using an

aliquot of sterile media or suspension buffer. Then an aliquot of the suspension was placed into

the cuvette and measured.

Biofilm Qualification

Biofilm is a qualitative measurement of the concentration of bacteria that have become

sessile on the tube or flask in which they were grown (Espinosa-Urgel et al., 2000)). The

bacteria were decanted and the vessel was rinsed with dH20 and air dried. The vessels were then

filled with 0. 1% crystal violet, and placed on a shaker for 1 hour. The crystal violet was

decanted and the tubes were rinsed until all the excess dye has been removed and air dried. The

vessels were then de-stained using 70% isopropanol. An aliquot will then be placed into a

cuvette and the optical density was measured at a wavelength of 600nm. The spectrophotometer

was calibrated using a cuvette filled with 70% isopropanol.

Media Analysis

Xf strain 'Temecula' was inoculated into CHARDS and XDM2-PR media by dispensing 1

mL of mature culture (~0.35 OD at 600nm) into 15 mL polypropylene tube and centrifuged at









2,380 g for 15 minutes. The supernatant was discarded and the pellet was resuspended in 3 mL

of PBS. This was centrifuged at 2,380 g for 15 minutes and the supernatant was discarded. The

pellet was resuspended in 2 mL of the appropriate media and then centrifuged at 2,3 80 g for 15

minutes. The supernatant was discarded and the pellet was resuspended in 5 mL of the

appropriate media and sealed with red-rubber septa and sealed with parafilm. The tubes were

then subj ected to gas treatment which consisted of various concentrations of oxygen, 21%

(breathing air) and 0% (nitrogen) for 5 minutes every day for the duration of the experiment.

Each treatment consisted of inserting a syringe needle connected to a compressed gas tank with a

positive pressure of ~5 psi through the rubber-septa and an exhaust needle was inserted to allow

for gas exchange.

Three tubes from each media-gas interaction were removed every five days and assayed.

The optical density was obtained by placing 1 mL into a cuvette and absorbance was measured at

600 nm. A 'drop' was placed on PW+ agar media and incubated at 280C for 10 days to

determine the viability of the culture. A two milliliter sample from each tube was centrifuged at

5,000 g at 40C for 15 minutes (Sorval Biofuge, Model: Stratos), temperature regulated

centrifuge), then the supernatant was stored at -800C. Then the tubes were emptied and assayed

for biofilm.

Amino acid analysis required a 1:50 dilution of the samples prior to pico N tagging.

Tagging consisted of drying a 30 Cll sample of each media-gas treatment in a speed-vac. Then

the pellet was resuspended in 100 Cll of 2:1:1 (100% ethanol: triethylamine: dH20), in order to

adjust the pH, then speed-vac dried. The pellet was resuspended in 30 Cll of 7: 1:1:1 (100%

ethanol: triethylamine: dH20: phenol isothiocyanate) and incubated under nitrogen gas for 20

minutes at room temperature. After this incubation the sample is then speed-vac dried for over









two hours (it is very important that this be very dry). The pellet is resuspended in the suspension

buffer (5 mM sodium phosphate (monobasic) and 6% acetonitrile) and then 20 Cll was loaded

onto a Pico tag column (Waters) eluted with a proprietary gradient of Buffer A (14 mM sodium

acetate-trihydrate and 0.05% triethylamine at a pH of 6.4) and Buffer B (60% acetonitrile) at a

flow rate of 1.0 mL per minute at 380C. Product was detected at 253 nm which corresponds to

the pico N tag.

Statistical Analysis

Data was analyzed with ANOVA, t-tests, means and standard error as well as regression

using the SAS V9.0 software package. The error bars represent standard error. The letters

represent separation by Duncans.

Results

Phenol red has a severe effect upon the formation of bioHilm within the XDM2 media.

Concentrations (Table 3-1) as low as 0.02% inhibited the formation of bioailm, and the increase

to 0.1% had no increased effect on inhibiting bioailm formation. The effect was seen across the

three strains used (Table 3-2). However the effect on the optical density was not consistent, in

Table 3-1, the lack of phenol red increased the optical density of 'Temecula', but in Table 3-2, it

had a negative effect on 'UCLA' and 'Temecula', but was not significantly different for 'STL'.

The comparison of the CHARDS variants (Figure 3-1 a and b) indicates that neither

vitamins, nor filtering had any significant effect upon the growth of the bacteria. However there

was a significant negative effect of glucose in the air treatment which retarded the optical density

as well as bioHilm production, whereas the nitrogen treatment produced more bioHilm then the air

treatment, but still had a significant reduction in optical density.

The CHARDS media (Table3-4) was unaffected by the gas treatment so the gas treatment

data was pooled. The bacteria produced significant subtractive differences in aspartic acid,










glutamine, and cysteine, which were some of the added constituents of the CHRADS media, and

the amino acid that was significant due to an increase were glutamic acid. There was no

significant change in the alanine or arginine, two other constituents of the CHARDS media. The

XDM2-PR media (Table 3-5) was affected by the bacteria, gas and the interaction of the

treatments. The bacteria significant modifications were glutamic acid, serine, glutamine,

threonine, alanine, and valine. The modifications based on gas were glutamic acid, glutamine,

threonine and leucene. The significances due to interaction were glutamic acid and threonine.

The amino acid data (Tables 3-4 and 3-5) show that the amino acids listed in the media are not

exclusively the amino acids found in the media.

Table 3 -6 was a comparison of the individual amino acids used in the media, at the

concentrations used in each media, with the glutamine concentration used at the CHARDS

amount. The amino acids were sterilized through a 0.22 Clm nylon fi1ter, except for one of the

glutamines which was autoclaved. Each amino acid was labeled as being 98 to 99% pure.

Asparagine, aspartic acid and methionine had values which were considerably different than

those listed on their bottle labels. The concentration of cysteine used appears to be below the

detection level of the HPLC detector. There was a difference in the glutamine concentrations

due to the destructive nature of autoclaving.

Discussion

The use of phenol red in the defined media inhibited the production of bioailm or sessile

populations which may in turn increase to optical density or planktonic populations (Table 3-1).

This effect was documented for Xfstrains Temecula and UCLA, although there was no increase

in optical density of the STL strain (Table 3-2). The production of more bioHilm in the absence

of phenol red could be answered in two ways either as an inhibitor for the bacteria to adhere to









the surface and initiate biofilm production or inhibiting the production of exopolysaccharides or

proteins that contribute to biofilm formation.

Biofilm production is considered as a protective barrier to a harsh environment, in that

under stress inducing conditions a bacterium will encapsulate itself in order to survive (Costern

et al. 1999). Phenol red may promote a behavior that causes the bacteria to remain in planktonic

state. The phenol red may also act as an inhibitor to biofilm production by interfering with the

machinery which stimulates biofilm production, such as inhibiting the ability of the bacteria to

adhere to a surface, either using the rings, which may share a binding affinity for the lignin

building blocks which are phenylalanine homologues, an by inhibiting the adhesion to a surface

thereby inhibits biofilm production. Another possibility is that of sulfones which are used as to

treat certain bacteria, such as M~ycobacterium leperae (used with an antibiotic) and the urinary

tract infectious strains ofEscherichia coli through an ability to prevent the adhesion, maybe

through inhibition of pili production (Vaisanen et al. 1982).

In Figure 3-1 the augmentation of the CHARDS media of filtering or the addition of

vitamins had no significant effect upon the optical density or biofilm production for the air and

nitrogen treatments. However the switching of glucose for starch, as a proposed carbon source,

had a negative impact upon both optical density and biofilm production, for both the air and

nitrogen treatment. The negative effect of glucose was greater in the air treatment with the

optical density actually decreasing from the initial optical density of 0.05. Biofilm production

was greater in the nitrogen treatments for the CHARDS variants; however this was not always

the case with CHARDS with only about 70% of the experiments results ending that way.. The

XDM2-PR media should have had a greater impact on optical density and biofilm under the

nitrogen treatments, as seen in Chapter 2, this lack of response could be due to the age of the









media used in the experiment, which was seen in other experiments when the age of the media

exceeded 1 month (data not shown).

The amino acid utilization followed the growth experiment results as described in Chapter

2. The CHARDS media had no significant changes due to the gas treatments (Table3-4). The

significance in the difference between glutamine and glutamic acid could be explained by the de-

animation of glutamine by the bacteria to produce glutamic acid, which was the only amino acid

which increased significantly. Other amino acids which were significantly reduced were aspartic

acid and cysteine. The XDM2-PR media (Table 3-5) had significance due to presence of the

bacteria as well as on the gas treatment, which could be associated with the growth and biofilm

production (Chapter 2). The amino acids which were significantly different as a function of the

presence of the bacteria were glutamic acid, serine, glutamine, threonine, alanine and valine.

The gas treatment was significant for glutamic acid, glutamine, and threonine. The interactions

which were significant were glutamic acid and threonine. The glutamine and glutamic acid

response was similar to the CHARDS except that the air (aerobic) treatment used more

glutamine and the nitrogen treatment produced more glutamic acid, significantly. Threonine was

only detected in the bacteria and nitrogen treatments.

The interesting point about the media experiment was the detection of the other amino

acids which were not added to the media but were a "component" of the individual amino acid

(Table 3-6). In CHARDS this had no effect except for the glutamic acid, which is probably just

the deaminated glutamine. However, in the XDM2-PR media alanine and valine were

significant. The comparison of the amino acid concentrations (as percentages) between the

recipes and uninnoculated media in the experimental analysis in Table 3-6 and 3-3 and Table 3-









7 and 3-4, respectively, were different which was caused by the impurities found the amino acids

which were added to the media (Table 3-5).

These result, the phenol red effect and the amino acid constituents, show that even the

supposed insignificant or minute compound can alter or contribute to the growth and behavior of

Xylella fa;stidiosa. Since Xylella resides in, what can be considered, a sparse environment each

media component becomes suspect and should not be taken casually. Therefore Xylella

fastidiosa is truly a fastidious bacterium and a better understanding of its responses to the

environment or media components will help in the study of the pathogenic qualities.










Table 3-1. A Phenol red series experiment done in the XDM2 media using 'Temecula'
% PR OD Biofilm
0% 0.120 10.001 0.286 10.204
20% 0.060 10.002 0.030 10.005
100% ** 0.064 10.003 0.033 10.006
* The amount of phenol red used in CHARDS. ** The amount of phenol red used
by Lemos, et al. 2003 for the XDM series media. Each point is the average of three
replicates












Table 3-2. Effect of phenol red on the growth and behavior on three strains of Xylella fa;stidiosa
Strain Media OD Biofilm
STL


XDM2
XDM2-PR


0.046 10.003
0.041 10.005

P=0.3885



0.141 10.012
0.052 10.003

P=0.0021



0.092 10.017
0.034 10.002


0.043 10.003
0.267 10.007

P=0.0001



0.042 10.001
0.207 10.008

P=0.0001



0.035 10.002
0.159 10.005


Stati stics

Temecula


XDM2
XDM2-PR


Stati stics


UCLA


XDM2
XDM2-PR


Stati stics P=0.0295 P=0.000 1
XDM2-PR is XDM2 without phenol red. Each point is the
average of three replicates.






























O

0.08 -



0.06 -



0.04 -



0.02 -








0.25 -






0.2 -






0.15 -



O


0.1 -






0.05 -











Figure 3-1.


CHARDS XDM2-PR CHARDS-F CHARDS-V CHARDS-G


CHARDS XDM2-PR CHARDS-F CHARDS-V CHARDS-G


Growth ofXylella fa;stidiosa 'Temecula' using variations of CHARDS based on

XDM2-PR media composition. A) The growth under air treatments. B) The growth

under nitrogen gas treatments. Growth under the various gas treatments was 15 days









and each treatment had started with an optical density of 0.05. Each point is the
average of three replicates.










Table 3-3. Amino acid analysis CHARDS after 15 days of bacterial growth
With Xylella Without Xylella Significance
Asp a 14.603 15.845 ****
Glu 2.907 0.901 ****
Asn 0.119 0.130
Ser 0.108 0.157
Gln 47.471 50.404 ****
Gly 2.210 1.409
His 0.000 0.000
Arg 20.776 20.612
Thr 0.000 0.000
Ala 6.804 6.860
Pro 1.721 1.488
Tyr 0.261 0.393
Val 0.103 0.148
Met 0.000 0.000
Cys 0.037 0.054*
Iso 2.186 1.015
Leu 0.086 0.094
Phe 0.517 0.415
Lys 0.092 0.090
a= The values listed are the means of percent total of each amino acid. Bold
amino acids are constituents added to the defined media. P-values are
represented by: = 0.05, ** = 0.01, *** = 0.001, **** = 0.0001. Where N = 6.









































a= The values listed are the means of percent total of each amino acid. Bold amino acids are
constituents added to the defined media. P-values are represented by: = 0.05, ** = 0.01, *** =
0.001, **** = 0.0001. Where N=3.


Table 3-4. Amino acid analysis CDM2-PR after 15 days of bacterial growth


With Xylella fastidiosa


Without Xylella fastidiosa
Air Nitrogen
0 0
0.77 0.74
12.16 12.02
15.16 15.41
57.89 58.63
1.45 1.05
0 0
2.84 3.09
0 0


Significance
Bacteria Gas


Air
0
2.89
11.8
13.9
55.06
2.27
0
2.59
0
0.16
0.11
1.84
0.38
5.65
0.01
2.08
0.18
0.68
0.41


Nitrogen
0
3.2
12.06
12.79
58.1
1.7
0
3.1
0.171
0.22
0.08
0.42
0.71
5.38
0.02
1.07
0.12
0.46
0.38


B acteri a*Gas


Asp a
Glu
Asn
Ser
Gln
Gly
His
Arg
Thr
Ala
Pro
Tyr
Val
Met
Cys
Iso
Leu
Phe
Lys


* *


**** ****


0.134
0.257
0.448
0.219
5.94
0.032
1.3
0.36
0.76
0.28


0.04
0.07
1.01
0.18
5.56
0.03
0.86
0.22
0.68
0.42






















00 omonom-onvece0
00 30000000000000


0
00


-d
m
5
8

e












a

O


co






ha

9
X

Co
a~


1 a
ode a







04\000



030000






00000









00000









000000


03
Ob



00
co


00


OO
00


OO






coo



@0


NO
O 0





co








00


0000000 0000





OM oo *C
OOOMOBO 000

O3 000 ivv0 MO








000300000 O




0~000 a 0 m







00 000030 co


h~3 --~ ~ ~ ~ C h
~ 5~m
ag d d ~m ~a~ c~ ~ ~
~~~~~~X~~~a~~Cu~~a~










Table 3-6. CHARDS constituents in mM amounts
Ingredient mM % Total
K2HPO4 8.612
KH2PO4 7.348
MgSO4-7H20 0.811
Starch 11.101
Ferric
pyrophosphate 0.335

Glutamine 39.685 76.22
Alanine 1.930 3.71
Aspartic acid 4.358 8.37
Arginine 6.028 11.58
Cysteine-HCI 0.063 0.12










Table 3-7. XDM2-PR constituents in mM amounts
Ingredient mM % Total
K2HPO4 12.056
KH2PO4 5.878
MgSO4-7H20 1.623
Glucose 55.506
Ferric
pyrophosphate 0.168

Glutamine 27.369 64.81
Asparagine 9.516 22.53
Serine 2.664 6.31
Methionine 2.681 6.35


Myo-inositol 0.194
Pyridoxine-HCI 0.005
Thiamine 0.003
Nicotinic acid 0.004
D-Biotin 0.001










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BIOGRAPHICAL SKETCH

Anthony Shriner was born and raised in north Florida. He received his high school

diploma in 1991 from Florida High School. He then went on to receive his Bachelor of Science

degree in biology from Florida State University in 1998. He has received his Master of Science

from the University of Florida in horticultural science in 2006. He is planning on pursuing a

PhD in environmental microbiology.