Impact Of Host Plant Xylem Fluid On Xylella Fastidiosa Multiplication ...

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Specific Aim 2: Identify specific phage with potentially useful properties within our collection. 2A) Screen the collection to identify virulent phages. 2B) Screen the collection to identify generalizing transducing phages. RESULTS AND CONCLUSIONS The first goal of this project is to generate a collection of Xf-specific phages that exhibit different biological properties. To increase our chances of obtaining a diverse set of phages, we have collected samples from PD-infected grapevines growing in different vineyards in Northern California. Using infected grapevines as a source seems particularly promising based on the work of Dr. Lauzon and her colleagues (5). Our strategy has been to collect sap from infected vines and samples from the tissue of symptomatic plants. We have also collected soil samples from around infected grapevines to determine if the soil is a good source of Xf-specific phage. The next step in our analysis will be to determine if any of these samples contain phage that can infect Xf. As a starting point, we will use previously published protocols that have successfully been used to isolate phages from environmental samples for other Gram-negative bacteria. REFERENCES 1. Ackerman, H., and M. DuBow 1987. Viruses of Prokaryotes, vol. I and II. CRC Press, Boca Raton. 2. Brussow, H., and R. W. Hendrix 2002. Phage genomics: small is beautiful. Cell. 108:13-6. 3. Hendrix, R. W. 2003. Bacteriophage genomics. Curr Opin Microbiol. 6:506-11. 4. Hopkins, D. L., and A. H. Purcell 2002. Xylella fastidiosa: Cause of Pierce's disease of grapevine and other emergent diseases. Plant Disease. 86:1056-1066. 5. Lauzon, C. R. 2001. A survey of insect vectors of Pierce's Disease (PD) and PD-infected Plants for the presence of bacteriophages that infect Xylella fastidiosa, p. 62. Proceedings Pierce's Disease Research Symposium. California Department of Food and Agriculture, Coronado, California. 6. Purcell, A. H. 1997. Xylella fastidiosa, a regional problem or global threat? Journal of Plant Pathol. 79:99-105. 7. Purcell, A. H., and D. L. Hopkins 1996. Fastidious xylem-limited bacterial plant pathogens. Annu Rev Phytopathol. 34:131-51. 8. Smith, M., and C. Rees 1999. Exploitation of Bacteriophages and their Components. Methods Microbiol. 29:97- 132. FUNDING AGENCIES Funding for this project was provided by the CDFA Pierce’s Disease and Glassy-winged Sharpshooter Board. - 199 -
Project Leader: Michele M. Igo Section of Microbiology Division of Biological Sciences and California Agricultural Experimental Station University of California Davis, CA 95616 THE XYLELLA FASTIDIOSA CELL SURFACE Cooperators: Andrew Walker Dept. of Viticulture and Enology University of California Davis, CA 95616 Bruce Kirkpatrick Dept. of Plant Pathology University of California Davis, CA 95616 Linda Bisson Dept. of Viticulture and Enology University of California Davis, CA 95616 Reporting Period: The results reported here are from work conducted from October 1, 2003 to September 30, 2004. ABSTRACT A common response of Gram-negative bacteria to environmental stress is to change the composition of their cell surface, particularly the protein composition of their outer membrane. These changes are known to have a profound effect on the sensitivity of Gram-negative bacteria to detergents, antibiotics, and bacteriophages. The goal of this project is to determine how environmental changes influence the protein composition of the Xylella fastidiosa (Xf) outer membrane. Our strategy has been to isolate the outer membrane fraction from Xf cells grown under different environmental conditions. The proteins in this fraction are then separated by one- or two-dimensional gel electrophoresis and their identity established by peptide mass fingerprinting. In this report, I have focused on experiments that examine the Xf outer membrane protein profile using one-dimensional gel electrophoresis. This analysis has allowed us to assign three outer membrane proteins to specific genes on the Xf chromosome. These gels have also allowed us to examine how the composition of the Xf outer membrane changes in response to environmental signals and the physiological state of the bacterial cell. INTRODUCTION Pierce’s disease (PD) is a devastating disease of grapevines that is caused by the Gram-negative, endophytic bacterium Xylella fastidiosa (Xf). Although the specific details of the disease process are not fully understood, an important feature is the ability of this pathogen to colonize the xylem tissue of plants and the foregut of insect vectors (for a recent review, see 5). As with most pathogenic bacteria, successful colonization is dependent on the ability of planktonic Xf cells to adhere to the host cell surface and to form a microcolony (3, 4, 7). This surface-associated growth commonly leads to the formation of a biofilm. Biofilm-associated Xf bacteria constitute a major component of the bacterial biomass in the host tissue. In contrast, planktonic bacteria are less prevalent and are seen primarily as a mechanism for the bacteria to translocate from one surface to another. The transition of bacteria from the planktonic to the biofilm-associated state involves profound physiological changes (3). The most obvious change is the production of an exopolysaccharide matrix, one of the distinguishing characteristics of a bacterial biofilm. However, the matrix-enclosed mode of bacterial growth requires many other changes, including changes in the protein composition of the bacterial cell envelope. In Gram-negative bacteria, these changes include differences in both the relative abundance of some major outer membrane proteins and the appearance or disappearance of specific highaffinity receptor proteins. This differential expression allows the bacteria to cope with the new environmental condition and with alterations in the nutrient supply. Changes in the protein composition of the outer membrane are known to have a profound effect on the sensitivity of Gramnegative bacteria to detergents, antibiotics, and bacteriophages (8). As a result, strategies designed to attack planktonic cells are usually not effective against biofilm-associated cells (3). Therefore, in order to develop effective methods for controlling the spread of Xf, it is important to obtain information concerning the protein composition of the Xf outer membrane and how the composition of this membrane changes in response to environmental signals and the physiological state of the bacterial cell. OBJECTIVES The goal of this project is to analyze the outer membrane proteome of Xf and to determine how the outer membrane protein profile changes in response to various physiological and environmental conditions. Our experiments are designed to address two objectives: 1. Identify the major outer membrane proteins of Xf and assign them to a specific gene on the Xf chromosome. 2. Determine how the protein composition of the Xf outer membrane is influenced by environmental signals and signals from the infected grapevine. - 200 -
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IMPACT OF HOST PLANT XYLEM FLUID ON
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0.18 600 Optical density 0.16 0.14
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OPTIMIZING MARKER-ASSISTED SELECTIO
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esistant and susceptible genotypes
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MAP BASED IDENTIFICATION AND POSITI
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esistant genotypes are in process a
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BREEDING PIERCE’S DISEASE RESISTA
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Progeny from crosses of field resis
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a = (1=low, 4= high); b = (1=green,
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v.8) for differences due to the pla
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SHARPSHOOTER FEEDING BEHAVIOR IN RE
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correlated with all other findings
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EFFECTS OF FEEDING SUBSTRATE ON RET
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REFERENCES Bextine, B. and T. Mille
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will also provide insights into the
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OBJECTIVES 1. Determine glassy-wing
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GWSS per 30 s sweep (seasonal avera
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ELISA for the presence of GWSS egg
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Project Leader: Thomas P. Freeman E
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Freeman, T. P., R. A. Leopold, D. R
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pathogen, when they move into viney
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A NOVEL IMMUNOLOGICAL APPROACH FOR
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1.6 GWSS Adults That Fed on Protein
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Hagler, J.R. & S.E. Naranjo. 2004.
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RESULTS: Oviposition Survey Wild gr
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Host specificity testing: No-choice
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currently employed morphological ch
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increase our present understanding
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BIOLOGY AND MORPHOMETRIC ANALYSIS O
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Table 1. Mean a developmental durat
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EFFECTS OF USING CONSTANT AND CYCLI
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REFERENCES Gautam, R. D. 1986. Effe
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PARASITISM OF THE GLASSY-WINGED SHA
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Table 1. Parasitism by G.ashmeadi o
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Project Leader: Robert F. Luck Dept
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A more interesting analysis using t
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MYCOPATHOGENS AND THEIR EXOTOXINS I
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POPULATION DYNAMICS AND INTERACTION
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No egg masses were recorded on olea
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EXPLORATION FOR FACULTATIVE ENDOSYM
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Although Baumannia and Wolbachia we
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EFFECTS OF SUBLETHAL DOSES OF IMIDA
Page 89 and 90: Table 2. Mortality and flight perfo
Page 91 and 92: A NOVEL METHOD TO INDUCE OVIPOSITIO
Page 93 and 94: REFERENCES Brodbeck, B. V., P. C. A
Page 95 and 96: Cohorts H. coagulata neonates were
Page 97 and 98: REFERENCES Blua, M. J. and D. J. W.
Page 99 and 100: Figure 2 shows the average numbers
Page 101 and 102: REPRODUCTIVE BIOLOGY AND PHYSIOLOGY
Page 103 and 104: REFERENCES Blua, M. J., Phillips, P
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Page 109 and 110: RESULTS Some plant families had no
Page 111 and 112: Table 5. Winter, spring and summer
Page 113 and 114: 16S rDNA is by far the most sequenc
Page 115 and 116: DNA MICROARRAY AND MUTATIONAL ANALY
Page 117 and 118: Inhibition of biofilm is BSA concen
Page 119 and 120: CULTURE-INDEPENDENT ANALYSIS OF END
Page 121 and 122: Borneman, J., M. Chrobak, G. Della
Page 123 and 124: P (CFU P OBJECTIVE 1. Determine the
Page 125 and 126: Purcell, AH and SR Saunders. 1999a.
Page 127 and 128: Figure 1: Southern blot of tolC::ap
Page 129 and 130: Project Leader: David Gilchrist Dep
Page 131 and 132: III. Production of transgenic plant
Page 133 and 134: RESULTS Construction of cDNA Librar
Page 135 and 136: CONCLUSIONS Genetic resistance and
Page 137 and 138: Mutagenesis of Xylella The EZ::TN T
Page 139: ISOLATION OF BACTERIOPHAGES SPECIFI
Page 143 and 144: outer membrane fractions using two-
Page 145 and 146: 1995; Purcell and Saunders, 1999).
Page 147 and 148: DEVELOPMENT OF SSR MARKERS FOR GENO
Page 149 and 150: Strain Name Host of Origin County o
Page 151 and 152: ROLE OF ATTACHMENT OF XYLELLA FASTI
Page 153 and 154: wild-type cells was not conclusive
Page 155 and 156: DETERMINATION OF GENES CONFERRING H
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Page 159 and 160: MULTILOCUS SEQUENCE TYPING TO IDENT
Page 161 and 162: GENOME-WIDE IDENTIFICATION OF RAPID
Page 163 and 164: Americas and since most of the plan
Page 165 and 166: EFFECTS OF CHEMICAL MILIEU ON ATTAC
Page 167 and 168: CONCLUSIONS Our overall objective i
Page 169 and 170: RESULTS Objective 1. We conducted t
Page 171 and 172: Redak, R. A., Purcell, A. H., Lopes
Page 173 and 174: In parallel, an “activating trans
Page 175 and 176: PATTERNS OF XYLELLA FASTIDIOSA INFE
Page 177 and 178: % of vessels 100 80 60 40 20 Mugwor
Page 179 and 180: DOCUMENTATION AND CHARACTERIZATION
Page 181 and 182: Magnolia002 showed more identity (9
Page 183 and 184: PLASMID ADDICTION AS A NOVEL APPROA
Page 185 and 186: GENETIC VARIABILITY OF XYLELLA FAST
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Page 189 and 190: probing activities). In preliminary
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the slow release valve was opened a
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12. Hopkins DL (1977) Diseases caus
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The almost complete absence of info
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QUANTIFYING LANDSCAPE-SCALE MOVEMEN
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Table 1. The mean (±SD) ELISA read
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EPIDEMIOLOGICAL ASSESSMENTS OF PIER
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multiply to relatively high (easily
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SPATIAL DATABASE CREATION AND MAINT
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Project Leader: Thomas M. Perring D
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Figure 2. Individual leaves from a
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The state variables, process functi
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DEVELOPMENT OF A FIELD SAMPLING PLA
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Figure 1. Three main dispersion pat
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OBJECTIVES 1. Track the movement of
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REFERENCES 1. Beard, C.B., Cordon-R
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cases, positive phloem samples were
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EXPLOITING XYLELLA FASTIDIOSA PROTE
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Figure 2. Polymer disk accumulation
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CHARACTERIZATION OF NEONICOTINOIDS
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EVALUATION OF RESISTANCE POTENTIAL
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Project Leader: Doug Cook Dept. of
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Based on the in silico analysis, de
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CONTROL OF PIERCE’S DISEASE THROU
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Figure 1. Oleander ‘White’ afte
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RESULTS During the reporting period
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DEVELOPMENT OF AN ARTIFICIAL DIET A
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DESIGN OF CHIMERIC ANTI-MICROBIAL P
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Neutrophil elastase Cecropin B Chim
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and WTXb is very low (0.00-0.40%) a
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100 100 0.056 North America 100 0.0
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GENETIC DIFFERENTIATION AMONG GEOGR
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Table 1. Single-populations descrip
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MOLECULAR DISTINCTION BETWEEN POPUL
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These novel observations strongly s
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SEQUENCE DIVERGENCE IN TWO MITOCHON
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Table 3. Pairwise sequence distance
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DEVELOPMENT OF MOLECULAR DIAGNOSTIC
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A. B. Relative Density of SCAR Band
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THE ALIMENTARY TRACK OF GLASSY-WING
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We have dissected and identified al
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REALIZED LIFETIME PARASITISM AND TH
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REPRODUCTIVE AND DEVELOPMENTAL BIOL
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Mean adult longevity (days) 25 20 1
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the proposed exploratory work; thes
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SEARCHING FOR AND COLLECTING EGG PA
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REFERENCES Hoddle, M. S. and S. V.
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some Gram(+) bacteria, but are inac
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7. Hultmark, D., et al., Insect Imm
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Isolation of Fungal Pathogens Soil
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IDENTIFICATION OF MECHANISMS MEDIAT
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concentrations of 1.5 x 10 7 bacter
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anchor it in the outer membrane (sh
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SYMBIOTIC CONTROL OF PIERCE’S DIS
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MANAGEMENT OF PIERCE’S DISEASE OF
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pfF mutants are taken up by insects
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Simpson, A. J. G., F. C. Reinach, P
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Al-Wahaibi, A.K., Owen, and J. G. M
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patterns differed among the lines,
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Punja, Z.K. 2001. Genetic engineeri
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mind, we conducted an additional st
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REFERENCES Toscano, N., Byrne, F.,
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RESULTS AND CONCLUSIONS The program
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COMPATIBILITY OF INSECTICIDES WITH
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More tests are in progress to addre
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