Progress 01/01/12 to 12/31/12
Outputs
OUTPUTS: Xylella fastidiosa causes Pierce's disease (PD), a serious disease in grapevines, and grapevine cultivars vary in susceptibility to X. fastidiosa in the field. The mechanism(s) by which this occurs has not been clearly elucidated. To explore possible mechanisms, X. fastidiosa cells from a PD strain were grown in pure xylem fluid of PD-susceptible grapevines, Vitis vinifera and V. labrusca, versus PD-resistant grapevines, V. champinii and V. smalliana. When grown in xylem fluid from the susceptible species, X. fastidiosa cells formed a heavier biofilm compared to those in xylem fluid from the resistant species. Differential expression of selected genes of X. fastidiosa cultured in the xylem fluids of V. vinifera and V. smalliana was analyzed using a DNA macroarray. Compared with xylem fluid of V. smalliana, xylem fluid of V. vinifera stimulated the expression of X. fastidiosa genes involved in virulence regulation, such as rpfC, gacA, xrvA, gcvR, and cysB, and genes involved in biogenesis of pili and twitching motility, such as pilI, pilU, pilE and pilG. Increased expression of virulence genes likely contributes to the expression of PD symptom in the susceptible grapevines, whereas reduced expression of these genes may lead to limitation of symptoms in resistant grapevines. PARTICIPANTS: Xiangyang Shi received training as a postdoctoral researcher on this project. Hamid Azad received further training as a staff research associate on this project. TARGET AUDIENCES: The agricultural industry, particularly the grape industry, is the primary target audience for our efforts to find long-term solutions to the problem of Pierce's Disease in grapevines. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Understanding the basis for differential regulation of virulence genes in response to differences in plant xylem could lead to strategies to reduce virulence gene expression for practical control. The reduced expression of virulence genes that we observed in a PD strain in xylem fluid from resistant vs. susceptible grapevine cultivars may be due to substances that repress expression of these genes. Such substances could then be directly tested for controlling disease expression in susceptible grapevine cultivars with the substance used much like a pesticide or antimicrobial drug.
Publications
- Shi, X., Bi, J., Morse, J.G., Toscano, N.C., and Cooksey, D.A. (2013). Effect of xylem fluid from susceptible and resistant grapevines on developmental biology of Xylella fastidiosa. Eur. J. Plant Pathol. 135:127-135.
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Progress 01/01/11 to 12/31/11
Outputs
OUTPUTS: Xylella fastidiosa causes Pierce's disease (PD) in grapevine. In previous work, we began to look at the effect of different host xylem fluids on expression of virulence genes in Xylella fastidiosa. In a Pierce's disease (PD) strain of Xf, several virulence genes were more highly expressed in xylem fluid of grapevine vs. xylem fluid of citrus, a non-host plant for the PD strain. This finding suggested that host range of Xf may be influenced by differential expression of virulence genes in response to different host xylem chemistry. During the reporting period, we further explored that hypothesis by culturing a PD strain into grapevine, mulberry and citrus in vitro, with inoculated PD3 medium and non-inoculated xylem fluid as controls, to detect differential growth and expression patterns. The PD strain grew similarly in pure xylem fluid of grapevine, mulberry and citrus. Transcriptional profiles based on macroarray analysis of 110 pathogeneticity-related genes showed that 27 genes had higher expression, and three lower, in grape xylem fluid compared with that of mulberry and citrus. Expression of these genes in mulberry vs. citrus was not significantly different. Although the PD strain grew similarly in xylem fluid from all three hosts, the increased expression of pathogenicity genes likely contributes to disease development in grape by the PD strain, whereas no symptoms are produced by this strain in mulberry and citrus. These results were presented to researchers and growers at an annual PD symposium organized by the California Department of Food and Agriculture. PARTICIPANTS: PARTICIPANTS: Xiaoli Yuan received training as a postdoctoral researcher on this project. Hamid Azad received further training as a staff research associate on this project. TARGET AUDIENCES: TARGET AUDIENCES: The agricultural industry, particularly the grape industry, is the primary target audience for our efforts to find long-term solutions to the problem of Pierce's Disease in grapevines. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Understanding the basis for differential regulation of virulence genes in response to differences in plant xylem could lead to strategies to reduce virulence gene expression for practical control. The reduced expression of virulence genes that we observed in a PD strain in citrus and mulberry xylem fluid may be due to substances that repress expression of these genes. Such substances could then be directly tested for controlling disease expression in susceptible grapevine cultivars with the substance used much like a pesticide or antimicrobial drug.
Publications
- Cooksey, D. A., X. Yuan, and H. Azad . 2011. Influence of host xylem chemistry on regulation of Xylella fastidiosa virulence genes and host specificity. Pages 55-58 IN: Proceedings of the Pierces Disease Research Symposium, December 13-15, 2011, Sacramento, CA, California Department of Food and Agriculture.
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Progress 01/01/10 to 12/31/10
Outputs
OUTPUTS: Xylella fastidiosa causes a serious Pierce's disease (PD) in grapevine. Xylella fastidiosa cells from a PD strain were grown in a pure xylem fluid of a susceptible grapevine cultivar vs. xylem fluid from citrus, which is not a host for this strain of X. fastidiosa. When grown in grapevine xylem fluid, cells of the PD strain formed clumps and biofilm formed to a greater extent than in citrus xylem fluid, although the PD strain did grow in xylem fluid of three citrus varieties. The differential expression of selected genes of a PD X. fastidiosa strain cultured in the two xylem fluids was analyzed using a DNA macroarray. Compared with citrus xylem fluid, grapevine xylem fluid stimulated the expression of X. fastidiosa genes involved in virulence regulation, such as gacA, algU, xrvA, and hsq, and also genes involved in the biogenesis of pili and twitching motility, such as fimT, pilI, pilU, and pilY1. Increased gene expression likely contributes to PD expression in grapevine, whereas citrus xylem fluid did not support or possibly suppressed the expression of these virulence genes. These results were reported in the publication listed below and presented to researchers and growers at an annual PD symposium organized by the California Department of Food and Agriculture. PARTICIPANTS: XiangYang Shi received training as a postdoctoral researcher on this project. Jianlong Bi is a collaborator from the University of California Cooperative Extension office in Salinias, CA. Joseph Morse and Nick Toscano are collaborators in the Department of Entomology at the University of California, Riverside. TARGET AUDIENCES: The agricultural industry, particularly the grape industry, is the primary target audience for our efforts to find long-term solutions to the problem of Pierce's Disease in grapevines. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Understanding the basis for differential regulation of virulence genes in response to differences in plant xylem could lead to strategies to reduce virulence gene expression for practical control. The reduced expression of virulence genes that we observed in a PD strain in citrus xylem fluid may be due to substances that repress expression of these genes. Such substances could then be directly tested for controlling disease expression in susceptible grapevine cultivars with the substance used much like a pesticide or antimicrobial drug.
Publications
- Shi, X.Y, J. Bi, J. G. Morse, N.C. Toscano, and D.A. Cooksey. 2010. Differential expression of genes of Xylella fastidiosa in xylem fluid of citrus and grapevine. FEMS Microbiol. Lett. 304:82-88.
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Progress 01/01/09 to 12/31/09
Outputs
OUTPUTS: Sweetgum dieback and leaf scorch of purple-leafed plum were described as two new diseases of southern California landscape ornamentals. Samplings were conducted in 2003 and 2004 and 28 of 105 sweetgum (Liquidambar styraciflua) and 38 of 62 purple-leafed plum (Prunus cerasifera) plants tested positive for Xylella fastidiosa by enzyme linked immunosorbent assay. In all, 3 strains of X. fastidiosa were isolated from sweetgum and 13 from purple-leafed plum. All sweetgum strains and some purple-leafed plum strains grew on PW but not PD3 media. Strain PC045 from purple-leafed plum and strain LS022 from sweetgum were inoculated into their original hosts in addition to almond, oleander, and grapevine plants. Sweetgum plants also were inoculated with strains causing Pierce's disease, almond leaf scorch, and oleander leaf scorch. Strain PC045 caused symptoms in purple-leafed plum and almond plants within 6 months, and the pathogen was recovered from 93 and 100% of inoculated plants, respectively. Inoculation of grapevine and oleander plants with PC045 did not result in disease or recovery of the pathogen. In all, 5 of 25 sweetgum plants inoculated with LS022 showed symptoms after 9 months, and the pathogen was recovered from 3 of these plants. Inoculation of grapevine, oleander, and almond with LS022 resulted in no disease or recovery of the pathogen from the plants. A strain of Pierce's disease, a strain of oleander leaf scorch, and two strains from almond did not cause disease in sweetgum. These results confirm the role of X. fastidiosa strains as pathogens of purple-leafed plum and sweetgum, and that strains from sweetgum are unique in their host range. Dickeya dadantii (Erwinia chrysanthemi) is a Gram-negative plant pathogen that invades a wide range of plant species to cause diseases. Hypersensitive response and pathogenicity genes (hrp) are important virulence factors in D. dadantii. However, few hrpL up-regulated genes of D. dadantii have been reported. In this study, a green fluorescence protein (GFP)-based Escherichia coli promoter-probe system was used to identify hrpL up-regulated genes in D. dadantii 3937. From approximate 20,000 library clones screened, hrpA, hrpK, dspE, yijC (a DNAbinding transcriptional regulatory protein) and yecF (unknown function) were identified to be up-regulated by hrpL in D. dadantii 3937. Expression pattern fluorescence of five genes was observed in the wildtype strain and a hrpL deletion mutant strain of D. dadantii 3937 in hrp-inducing minimal medium. Mutants with hrpA, hrpK, dspE, yijC, and yecF genes mutated, respectively, were confirmed, and most of the mutants showed virulence reduction when infecting the plant host African violet (Saintpaulia ionantha). PARTICIPANTS: Two former graduate students work on these projects, Xiangyang Shi (now a postdoctoral researcher at Cornell University) and Rufina Hernandez-Martinez (now an assistant professor at the Center for Scientific Research and Higher Education of Ensenada (CICESE), Mexico), as part of their Ph.D. training at the University of California, Riverside. TARGET AUDIENCES: Outreach to growers on the new information about Xylella fasitiosa host range reported here was accomplished through reports and presentations at the annual Pierce's Disease Research Symposium organized by the California Department of Food and Agriculture. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Identification of the causal agent for two new diseases of landscape ornamentals in southern California has increased our understanding of the host range of the plant pathogen Xylella fastidiosa, which helps us to predict which plants can carry inoculum of this pathogen that may pose threats to agriculturally-important crop species. The identification of genetic factors that enable Dickeya dadantii to express plant disease symptoms should lead to new disease management strategies including new targets for disruption of the disease process.
Publications
- Shi, X.Y., and D. A. Cooksey. 2009. Identification of hrpL up-regulated genes of Dickeya dadantii. Eur. J. Plant Pathol. 124:105-116.
- Hernandez-Martinez, R., D. A. Cooksey, and F. P. Wong. 2009. Leaf scorch of purple-leafed plum and sweetgum dieback: two new diseases in Southern California caused by Xylella fastidiosa strains with different host ranges. Plant Dis., 93:1131-1138.
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Progress 01/01/08 to 12/31/08
Outputs
OUTPUTS: The xylem-limited, insect-transmitted bacterium Xylella fastidiosa causes Pierce's disease in grapes and related leaf scorch diseases in many other plants. GacA is a regulatory protein that controls various physiological processes and pathogenicity factors in many gram-negative bacteria, including biofilm formation in Pseudomonas syringae pv. tomato (Pst) DC3000. The cloned gacA gene of X. fastidiosa was found to restore the hypersensitive response and pathogenicity in gacA mutants of Pst DC3000 and Erwinia amylovora. A gacA mutant of X. fastidiosa (DAC1984) had significantly reduced abilities to adhere to a glass surface, form biofilm, and incite disease symptoms on grapevines compared with the parent A05. cDNA microarray analysis identified seven genes that were positively-regulated by GacA, including xadA and hsf, predicted to encode outer membrane adhesion proteins, and 20 negatively-regulated genes, including gumC and an antibacterial polypeptide toxin gene, cvaC. These results suggest that GacA of X. fastidiosa regulates many factors, which contribute to attachment and biofilm formation, as well as some physiological processes that may enhance the adaptation and tolerance of X. fastidiosa to environmental stresses and the competition within the host xylem. PARTICIPANTS: X. Y. Shi was a graduate student who earned a Ph.D. in Plant Pathology from the University of California, Riverside while working on this project. He is now a postdoctoral researcher at Cornell University. C. K. Dumenyo received postdoctoral training at UC Riverside during this project and is now an assistant professor at Tennesse State University. R. Hernandez-Martinez was a graduate student who earned a Ph.D. in Plant Pathology from UC Riverside during this project and is now an assistant professor at the Center for Scientific Research and Higher Education of Ensenada (CICESE), Mexico. TARGET AUDIENCES: This work was supported by the California Department of Food and Agriculture on behalf of the California wine grape growers who provided funding as part of an effort to find solutions to Pierce's disease of grape. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
The identification of genetic factors that enable X. fastidiosa to express Pierce's disease symptoms should lead to new disease management strategies including new targets for disruption of the disease process.
Publications
- Shi, X. Y., C. K. Dumenyo, R. Hernandez-Martinez, H. Azad, and D. A. Cooksey. 2009. Characterization of regulatory pathways in Xylella fastidiosa: genes and phenotypes controlled by gacA. Appl Environ. Microbiol. 75: In Press.
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Progress 01/01/07 to 12/31/07
Outputs
OUTPUTS: In Vivo Expression Technology (IVET) was used to gain an understanding of genes involved in a Pseudomonas-Phytophthora interaction as a biological control mechanism for root rot diseases. Sixteen Pseudomonas putida genes with increased expression on Phytophthora mycelial surfaces were identified. Sequence analysis of these genes revealed many of them to display similarity to genes known or predicted to be involved in carbohydrate catabolism, energy metabolism, amino acid/nucleotide metabolism, and membrane transport processes. Disruption of three of the induced genes encoding succinate semialdehyde dehydrogenase, a dicarboxylic acid transporter, and glyceraldehyde-3-phosphate dehydrogenase showed significant phenotypic differences involved in colonization processes, including motility, biofilm formation on abiotic surfaces, colony morphology, and competitive colonization of fungal mycelia. All three of these genes were induced by glycogen and other substances, such as
organic acids and amino acids utilized by P. putida. We also genetically characterized strains of Xylella fastidiosa isolated from ornamental hosts in southern California to understand their distribution and identity. Strains of X. fastidiosa isolated from ornamentals were characterized using a multiprimer PCR system, random amplified polymorphic (RAPD)-PCR and sequence analysis of the 16S-23S rDNA intergenic spacer regions (ISR). Based on RAPD-PCR and 16S-23S rDNA ISR, strains isolated from daylily, jacaranda and magnolia clustered with members of X. fastidiosa subsp. sandyi and caused oleander leaf scorch but not Pierce's disease symptoms. This demonstrated both that our groupings based on genetic characterization were valid and that strains of X. fastidiosa subsp. sandyi are present in hosts other than oleander. Strains isolated from spanish broom, cherry, and one strain isolated from western redbud, clustered with X. fastidiosa subsp. fastidiosa members. Strains isolated from
purple-leafed plum, olive, peach, plum, sweetgum, maidenhair tree, crape myrtle and another western redbud strain, clustered with members of X. fastidiosa subsp. multiplex. All strains isolated from mulberry and one from heavenly bamboo formed a separate cluster that has not yet been defined as a subspecies. We also investigated a "global" regulatory gene in X. fastidiosa and identified potential virulence genes coordinately regulated by this gene. The role of algU in Xylella fastidiosa, the cause of Pierce's disease of grapevines, was analyzed by mutation and whole-genome microarray analysis to define its involvement in aggregation, biofilm formation, and virulence. In this study, an algU::nptII mutant had reduced cell-cell aggregation, attachment, biofilm formation, and lower virulence in grapevines. Microarray analysis showed that 42 genes had significantly lower expression in the algU::nptII mutant than the wild type. Among these are several genes that could contribute to cell
aggregation and biofilm formation, as well as other physiological processes such as virulence, competition, and survival.
PARTICIPANTS: Rufina Hernandez-Martinez, received Ph.D. in Plant Pathology working on this project. Xianyang Shi, received Ph.D. in Plant Pathology working on this project. Sang-Joon Ahn, recieved Ph.D. in Plant Pathology working on this project. C. Korsi Dumenyo, received postdoctoral training working on this project.
TARGET AUDIENCES: Information from the reported work on Xylella fastidiosa was communicated to growers at an annual symposium on Pierce's disease. Results from this project were also used in lecture and laboratory instruction at the University of California, Riverside in a graduate course on bacterial plant pathogens and in an undergraduate course on pathogenic microbiology. Lectures in the undergraduate course reached a racially diverse student body. UCR is now formally a Hispanic Serving Institution (HSI).
Impacts
IVET screening and mutant characterization can be used to identify bacterial genes that are induced on the mycelial surface and provided insight into possible mechanisms of mycelial colonization by this bacterium that might be used in improving biological control of Phytophthora root rot. Characterization of hosts of Xylella fastidiosa in southern California, and characterization of the host range and genotype of new strains from those hosts will help us to predict the threat of such strains to agricultural crops in the area and to manage inoculum reservoirs of the pathogen. The identification of genetic factors that enable X. fastidiosa to express Pierce's disease symptoms should lead to new disease management strategies including new targets for disruption of the disease process.
Publications
- S.-J. Ahn, C.-H. Yang, and D. A. Cooksey. 2007. Pseudomonas putida 06909 genes expressed during colonization on mycelial surfaces and phenotypic characterization of mutants. J. Appl. Microbiol., 103:120-132.
- Hernandez-Martinez, R., K. de la Cerda, H. S. Costa, D. A. Cooksey, and F. P. Wong. 2007. Phylogenetic relationships of Xylella fastidiosa strains isolated from landscape ornamentals in southern California. Phytopathology, 97:857-864.
- Shi, X.Y., C. K. Dumenyo, R. Hernandez-Martinez, H. Azad, and D. A. Cooksey. 2007. Characterization of regulatory pathways in Xylella fastidiosa: genes and phenotypes controlled by algU. Appl. Environ. Microbiol. 73:6748-6756.
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Progress 01/01/06 to 12/31/06
Outputs
Mulberry leaf scorch (MLS), caused by Xylella fastidiosa, was detected for the first time in white mulberry (Morus alba) trees in southern California in this work. Four MLS-strains were isolated from two locations and confirmed as X. fastidiosa by enzyme-linked immunosorbent assay (ELISA), direct isolation of the pathogen, and use of the X. fastidiosa-specific PCR primers. Isolated strains were characterized by the sequencing of their 16S-23S rDNA intergenic spacer regions and random amplified polymorphic DNA (RAPD) analysis and subsequent comparison with a previously characterized MLS-strain (Mulberry-VA) and representatives of X. fastidiosa subsp. fastidiosa, X. fastidiosa subsp. multiplex, and X. fastidiosa subsp. sandyi. MLS strains isolated from California were distinct from strains causing almond leaf scorch, oleander leaf scorch, and Pierce's disease and similar to the Mulberry-VA-strain. Genomic analysis using RAPD revealed no differences among the four MLS
strains. The pathogenicity of one strain, MLS063, was confirmed by inoculation of glasshouse-grown white mulberry plants. Three months after inoculation, the pathogen was recovered from 21 of 25 inoculated plants, and 5 of 25 plants were dead within a year of inoculation. Inoculation of grapevines and oleanders with MLS063 did not result in any disease or recovery of the pathogen up to 1 year later, showing that this strain was not cross-infective to these hosts. In a second project, differences in susceptibility to Pierce's disease in several plants that are frequent hosts of the insect vector, the glassy-winged sharpshooter, were investigated by examining the in vitro effect of xylem fluid from grapefruit, orange, lemon, and grape on the growth, aggregation, and attachment of a X. fastidiosa strain isolated from grape. Xylem fluid from grapefruit, orange, and lemon trees caused the bacterial cells to form aggregations of large whitish clumps, whereas the xylem fluid from grape vines
created a visible thick biofilm, which has been associated with the disease process in grapevines. Citrus xylem fluid significantly inhibited X. fastidiosa biofilm formation compared to grape xylem fluid. In another project, the role of production of the plant hormone indole-3-acetic acid (IAA) was examined in the phytopathogen Erwinia chrysanthemi 3937, which possesses iaaM and iaaH genes. A knockout iaaM mutant was constructed, and IAA production was reduced. Compared with wild-type, this mutant exhibited reduced ability to produce local maceration. The production of exoenzymes including the cellulases, proteases, and pectate lyases of the iaaM mutant was diminished in measurements using a GFP reporter system combined with a fluorescence activated cell sorter. In addition, expression of type III secretion system genes was reduced. Further evidenced suggested that reduced exoenzyme production of the mutant might be due to the GacA-Rsm regulatory pathway.
Impacts
Characterization of new hosts of Xylella fastidiosa in southern California, and characterization of the host range and genotype of new strains from those hosts will help us to predict the threat of such strains to agricultural crops in the area and to manage inoculum reservoirs of the pathogen. The finding that xylem fluid from citrus (a non-host of the Pierce's disease strain) inhibits biofilm formation helps to explain why this pathogen does not cause disease in this host and emphasizes the importance of biofilm formation as a potential target for disease control strategies. Understanding the role of IAA in virulence of Erwinia chrysanthemi helps us to understand how this bacterium causes soft rot diseases and provides a potential target for disruption of the disease process.
Publications
- Hernandez-Martinez, R., T. Pinckard, H. S. Costa, D. A. Cooksey, and F. P. Wong. 2006. Characterization of mulberry leaf scorch strains isolated in Southern California, discovery of a new Xylella fastidiosa strain infecting shade trees in the urban landscape. Plant Dis., 90:1143-1149.
- Yang, S., Q. Zhang, J. Guo, A. O. Charkowski, B. R. Glick, A. M. Ibekwe, D. A. Cooksey, and C.-H. Yang. 2007. Global effect of Indole-3-acetic acid (IAA) biosynthesis on multiple virulence factors of Erwinia chrysanthemi 3937. Appl. Environ. Microbiol. 73: 1079-1088.
- Bi, J. L., C. K. Dumenyo, R. Hernandez-Martinez, D. A. Cooksey, and N. C. Toscano. 2007. Effect of host plant xylem fluid on growth, aggregation and attachment of Xylella fastidiosa. J. Chem. Ecol., 33: 493-500.
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Progress 01/01/05 to 12/31/05
Outputs
A multi-primer polymerase chain reaction (PCR) based diagnostic method was developed to distinguish among three main subspecies of the bacterium, Xylella fastidiosa, that causes Pierce's disease in grape, almond leaf scorch, and oleander leaf scorch is a. Three PCR primer sets were developed to amplify DNA fragments specifically from each group, and then the three primer sets were combined to allow the simultaneous distinction between the groups with one PCR reaction. We were also able to distinguish between two groups within of the almond leaf scorch subspecies. We also used the multi-primer PCR assay to detect and differentiate strains of X. fastidiosa present in individual glassy-winged sharpshooter adults. The dominant vector in southern California is the glassy-winged sharpshooter, Homalodisca coagulata. The high mobility of this insect, and its utilization of large numbers of host plant species, provides this vector with ample exposure to multiple strains of X.
fastidiosa during its lifetime. Insects were sequentially exposed to plants infected with a Pierce's disease strain in grapevine and an oleander leaf scorch strain in oleander. After sequential exposure, a few insects tested positive for both strains. However, in most cases individuals tested positive for only one strain. In transmission studies, individual adults transmitted either strain of the pathogen at a rate similar to that previously reported after exposure to a single strain, but no single individual transmitted both strains of the pathogen. We also conducted a study to evaluate the effectiveness of antibiotics and antimicrobial peptides against 10 strains of Xylella fastidiosa. The minimal inhibitory concentration (MIC) of 12 antibiotics and 18 antimicrobial peptides were determined by agar dilution tests and growth inhibition assays. This study shows that antibiotics and antimicrobial peptides have some activity against the pathogen, X. fastidiosa. Antibiotics with the
lowest MIC for X. fastidiosa strains were gentamicin, tetracycline, ampicillin, kanamycin, and novobiocin, chloramphenicol, and rifampin. Plate growth inhibition assays showed that four of the antimicrobial peptides (Magainin 2, Indolicidin, PGQ, and Dermaseptin) were toxic to all X. fastidiosa strains. Minor differences in toxicity of these proteins were detected for different strains of these bacteria.
Impacts
The development of improved diagnostic methodology for the Pierce's disease bacterium, Xylella fastidiosa, allows us to more rapidly characterize strains from new hosts of Xylella fastidiosa that we are encountering and to predict whether the strains from these new hosts pose a threat to important agronomic crop species. This lead to vegetation management strategies to reduce the impact of this disease on grapevine or other economic crops.
Publications
- Hernandez-Martinez, R., H. S. Costa, C. K. Dumenyo, and D. A. Cooksey. 2006. Differentiation of strains of Xylella fastidiosa infecting grape, almonds and oleander using a multiprimer PCR assay. Plant Dis., In Press.
- Costa, H. S., A. Guzman, R. Hernandez-Martinez, C. Gispert, and D. A. Cooksey. 2006. Detection and differentiation of Xylella fastidiosa strains acquired and retained by glassy-winged sharpshooters using a mixture of strain-specific primer sets. J. Econ. Entomol., In Press.
- Kuzina, L. V., T. A. Miller, and D. A. Cooksey. 2006. In vitro activities of antibiotics and antimicrobial peptides against the plant pathogenic bacterium Xylella fastidiosa. Lett. Appl. Microbiol., In Press.
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Progress 01/01/04 to 12/31/04
Outputs
Copper-resistant strains of Xanthomonas axonopodis pv. vesicatoria were previously shown to carry plasmid-borne copper resistance genes related to the cop and pco operons of Pseudomonas syringae and Escherichia coli, respectively. However, instead of the two-component (copRS and pcoRS) systems determining copper-inducible expression of the operons in P. syringae and E. coli, a novel open reading frame, copL, was found to be required for copper-inducible expression of the downstream multicopper oxidase copA in X. axonopodis. copL encodes a predicted protein product of 122 amino acids that is rich in histidine and cysteine residues, suggesting a possible direct interaction with copper. Deletions or frameshift mutations within copL, as well as an amino acid substitution generated at the putative start codon of copL, caused a loss of copper-inducible transcriptional activation of copA. A nonpolar insertion of a kanamycin resistance gene in copL resulted in copper
sensitivity in the wild-type strain. Analysis of the genomic sequence databases shows that there are copL homologs upstream of copAB genes in X. axonopodis pv. citri, X. campestris pv. campestris, and Xylella fastidiosa. The cloned promoter area upstream of copA in X. axonopodis pv. vesicatoria did not function in Pseudomonas syringae or in E. coli, nor did the P. syringae cop promoter function in Xanthomonas. However, a transcriptional fusion of the Xanthomonas cop promoter with the Pseudomonas copABCDRS was able to confer resistance to copper in Xanthomonas, showing divergence in the mechanisms of regulation of the resistance to copper in phytopathogenic bacteria. In work on the soft-rotting bacterium, Erwinia chrysanthemi, a green-fluorescent protein-based in vivo expression technology leaf array was used to identify genes in the bacterium that were specifically upregulated in plants compared with growth in a laboratory culture medium. Of 10,000 cones, 61 were confirmed as plant
upregulated. Putative functions were inferred from the nucleotide sequences of these genes when compared to genome databases. Mutations were constructed in five of the plant upregulated genes, and the mutants were tested for virulence. Several of the genes were shown to be important in virulence. In work on Xylella fastidiosa, a variety of plant species found near a severe outbreak of Pierce's disease (PD) of grapevines in the Temecula Valley of California were tested using enzyme-linked immunosorbent assay, culture on media, and polymerase chain reaction to identify potential inoculum sources in the area. Species that consistently tested positive for X. fastidiosa were the known hosts, grape, almond, and oleander, and two new hosts, Spanish broom and wild mustard. Sequence analysis of the 16S-23S rRNA spacer region found that strains isolated from grapevine, Spanish broom, wild mustard, and almond clustered with previously sequenced PD strains. Thus, these species could serve as
sources of inoculum for infection of grapevines. Greenhouse transmission studies indicated that the glassy-winged sharpshooter was able to transmit a PD strain of Xylella fastidiosa to Spanish broom, black mustard, and other hosts.
Impacts
Our work on copper resistance gene regulation helps us to understand the diversity of copper resistance systems in plant pathogens and the threat of this resistance to the continued success of copper bactericides for control of bacterial diseases on a variety of crops. The identification of virulence genes in Erwinia chrysanthemi is expected to provide new targets for strategies to intervene in the infection process for this and related plant pathogens. The identification of inoculum sources for Xylella fastidiosa has already led to changes in weed management practices in Temecula vineyards, to reduce the spread of the pathogen from weeds to grapevines by the glassy-winged sharpshooter.
Publications
- Voloudakis, A. E., Reignier, T. M. and Cooksey, D. A.. 2005. Regulation of Resistance to Copper in Xanthomonas axonopodis pv. vesicatoria. Appl. Environ. Microbiol. 71:782-789.
- Yang, S., Perna, N.T., Cooksey, D. A., Okinaka, Y., Lindow, S. E., Ibekwe, A. M., Keen, N. T., and Yang, C.-H. 2004. Genome-wide identification of plant-upregulated genes of Erwinia chrysanthemi 3937 using a GFP-based IVET leaf array. Mol. Plant-Microbe Interact. 17:999-1008.
- Costa, H. S., Raetz, E., Pinckard, T. R., Gispert, C., Hernandez-Martinez, R., Dumenyo, C. K., and Cooksey, D. A. 2004. Plant hosts of Xylella fastidiosa in and near southern California vineyards. Plant Dis. 88:1255-1261.
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Progress 01/01/03 to 12/31/03
Outputs
We are investigating several approaches to better understand the nature of Pierce's disease of grapevines and develop potential disease management strategies. One is to understand the role of xanthan gum production by the pathogen, XYLELLA FASTIDIOSA, and to develop methods of degrading it inside of the plant with xanthan-degrading endophytes. Mutants for production of xanthan gum were constructed from a parental strain of X. FASTIDIOSA. These mutants produced less of a slime layer and did not adhere as well to various surfaces as the wild type, but they formed significantly more biofilm in a quantitative polystyrene microtiter assay. The effects of the loss of xanthan gum production on virulence of X. FASTIDIOSA are still under way. Of several xanthan-degrading endophytes that were selected on media containing purified xanthan gum as the sole carbon source, we selected one with the greatest potential for degradation. Complete degradation of xanthan gum by this
bacterium was measured in vivo, and colonization of plants was studied with and without the presence of XYLELLA. Interestingly, colonization of plant xylem by the xanthan degrader was enhanced by co-inoculation with XYLELLA, suggesting that it was benefiting from xanthan gum or other substances produced by the pathogen in the plant. We also completed a 3-year study to identify potential inoculum sources of XYLELLA in invasive weeds, native plants, and cultivated plant species near vineyards of southern California. In addition to the finding of this pathogen in several previously-described hosts, two new hosts were found: Spanish broom and wild mustard. Greenhouse transmission studies also indicated that the glassy-winged sharpshooter can vector the pathogen from these hosts to grapevines. Vineyard managers are now controlling these weeds more diligently for reduction of inoculum. Manuscripts have been submitted on both of the above projects.
Impacts
Identification of new inoculum sources for XYLELLA FASTIDIOSA, the pathogen that causes Pierce's disease of grapevines, has led to improved disease management practices through removal of weed species that we demonstrated to carry signficant populations of the pathogen that were transmitted to grapevines in experimental transmission studies with the glassy-winged sharpshooter.
Publications
- No publications reported this period
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Progress 01/01/02 to 12/31/02
Outputs
As part of ongoing collaborations with University of California county advisors, we identified and characterized a new bacterial disease of spinach, caused by a non-fluorescent strain of PSEUDOMONAS SYRINGAE. The disease occurred in the Salinas Valley of California in 2000 and 2001 and consisted of leaf spots that coalesced to cause death of large areas of leaves. The causal organism was isolated, and Koch's postulates were completed to prove its pathogenicity on spinach. Fatty acid methyl ester analysis indicated that the spinach strain was most closely related to P. SYRINGAE pv.MACULICOLA, although there were differences in carbon source utilization between that pathovar and the spinach strain. Inoculations of the spinach pathogen to hosts of P. SYRINGAE pv.MACULICOLA (cabbage and cauliflower) were negative, and only the spinach strain caused leaf spots of spinach. In addition to this study, the the majority of our efforts were directed toward Pierce's disease of
grapevine. Pierce's disease of grapevine and other leaf scorch diseases caused by XYLELLA FASTIDIOSA are associated with a gummy matrix, thought to be xanthan gum, and subsequent blockage of water uptake. We began to isolate endophytic bacteria that produce xanthan-degrading enzymes to target this specific virulence factor of X. FASTIDIOSA. For enrichment of xanthan-degrading bacteria, it was not feasible to produce enough xanthan gum for our studies from XYLELLA. Instead, we genetically modified a strain of XANTHOMONAS CAMPESTRIS to produce xanthan gum with the same chemical structure as that from XYLELLA. We used the modified xanthan gum from the XANTHOMONAS mutant as the sole carbon source for enrichment culture from surface-disinfected diseased grapevine stems and oleander with leaf scorch disease. Eleven strains were confirmed to effectively degrade xanthan gum. Six of these had low or non-detectable cellulase activity and will be further tested for biological control efficacy in
plants. Another approach we are pursuing for control of Pierce's disease is to construct non-pathogenic mutants of the Pierce's disease pathogen to be used in a competitive displacement strategy. We are employing a systematic process of identifying which virulence genes are important for disease expression but are not essential for colonization of plants in a nonpathogenic state. Knock-out mutations in selected genes were created through homologous recombination in XYLELLA, and these are being tested in grapevines. Another project has been to identify the alternative sources of inoculum for Pierce's disease in the Temecula Valley by repeatedly sampling other plant species in agricultural, landscape, and natural areas adjacent to vineyards. In addition to grapevine, several plant species harbored significant populations of XYLELLA, and studies to characterize these strains and investigate insect transmission to and from these plants are underway.
Impacts
Knowledge of the cause of a new disease, such as the spinach leaf spot disease, is essential for the design of control strategies, such as seed treatments or other means of reducing the introduction of a bacterial pathogen into a field. The development of biological control agents for Pierce's disease, through isolation of xanthan-degrading endophytes or the construction of non-pathogenic derivatives of XYLELLA, offers the potential for a relatively near-term strategy for reducing the damage that the bacterium causes to grapevines. Knowledge of the source of disease inoculum for Pierce's disease is critical to the choice and management of plant species surrounding vineyards.
Publications
- Koike, S.T., H.R. Azad, and D.A. Cooksey. 2002. First Report of Bacterial Leaf Spot of Spinach Caused by a Pseudomonas syringae Pathovar in California. Plant Dis. 86:921.
- Cooksey, D.A. 2002. Control of Pierce's disease through degradation of xanthan gum. Pages 25-26 IN: Proceedings of the Pierce's Disease Research Symposium, December 15-18, 2002, San Diego, CA, California Department of Food and Agriculture.
- Cooksey, D.A. 2002. Biological control of Pierce's disease with non-pathogenic strains of Xylella fastidiosa. Pages 123-24 IN: Proceedings of the Pierce's Disease Research Symposium, December 15-18, 2002, San Diego, CA, California Department of Food and Agriculture.
- Cooksey, D.A. and H. S. Costa. 2002. Epidemiology of Pierce's disease in Southern California; identifying inoculum sources and transmission pathways. Pages 65-66 IN: Proceedings of the Pierce's Disease Research Symposium, December 15-18, 2001, San Diego, CA, California Department of Food and Agriculture.
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Progress 01/01/01 to 12/31/01
Outputs
A 3-kb chromosomal fragment encoding cadmium resistance was cloned from PSEUDOMONAS PUTIDA 06909, a rhizosphere bacterium, and complete sequence analysis revealed two divergently transcribed genes, CADA and CADR. CadA showed strong similarity to cadmium-transporting ATPases known mostly from gram-positive bacteria, and to ZntA, a lead, zinc, and cadmium-transporting ATPase from ESCHERICHIA COLI. CadR was related to the MerR family of response regulators that normally control mercury detoxification in other bacterial systems. A related gene, ZNTR, regulates ZNTA in E. COLI, but it is not contiguous with ZNTA in the E. COLI genome as CADA and CADR were in P. PUTIDA. In addition, unlike ZntA and other CadA homologs, but similar to the predicted product of gene PA3690 in the P. AERUGINOSA genome, the P. PUTIDA CadA sequence had a histidine-rich N-terminal extension. CadR and the product of PA3689 of P. AERUGINOSA had histidine-rich C-terminal extensions not found in other
MerR family response regulators. Mutational analysis indicated that CADA and CADR are fully responsible for cadmium resistance and partially for zinc resistance. However, unlike ZNTA, they did not confer significant levels of lead resistance. The CADA promoter (PCADA ) was responsive to Cd(II), Pb(II) and Zn(II) while the CADR promoter (PCADR) was only induced by Cd(II). Insertional inactivation of CADR resulted in constitutive expression from PCADR, suggesting that it is a repressor of its own gene expression. However, CadR apparently does not repress PCADA. Southern blot hybridization analysis detected homologs of the cadmium-transporting ATPase in many other PSEUDOMONAS species. In another study, a new pathovar of XANTHOMONAS CAMPESTRIS was described as the cause of a new disease of catnip.
Impacts
Pseudomonads from environmental sources vary widely in their sensitivity to cadmium, with many displaying high levels of resistance, but the basis for this resistance is largely uncharacterized. The finding of the cadmium-transporting ATPase in this biological control agent and in many other species of PSEUDOMONAS helps to explain the mechanisms by which these bacteria adapt to toxic metals in their environment. The identification of the cause of a new catnip disease is an important step in developing strategies to control disease on this plant that is grown commercially in California for use as herbs, seasonings, and tea.
Publications
- Koike, S. T., H. R. Azad, and D. A. Cooksey. 2001. Xanthomonas leaf spot of catnip: A new disease caused by a pathovar of XANTHOMONAS CAMPESTRIS. Plant Disease 85:1157-1159.
- Lee, S.W., E. Glickmann, and D. A. Cooksey. 2001. Chromosomal locus for cadmium resistance in PSEUDOMONAS PUTIDA consisting of a cadmium-transporting ATPase and a MerR family response regulator. Appl. Environ. Microbiol. 67:1437-1444.
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Progress 01/02/00 to 12/31/00
Outputs
An unreported disease of sudangrass (SORGHUM SUDANENSE) was observed in commercial fields in Imperial Valley of California. Symptoms included light-colored necrotic streaks, and white or tan irregular blotches, often associated with reddish purple to dark brown margins. PANTOEA ANANAS was consistently isolated from the blotches with reddish margins, while PANTOEA STEWARTII or mixtures of both species were isolated from necrotic streaks without reddish margins. Fourteen seed samples harvested in different locations were assayed and found to be 0.0 to 3.6% infested with P. ANANAS. Seed transmission may be a means by which the pathogen is introduced. Symptoms in inoculated plants appeared as early as 2 and as late as 20 days after inoculation, depending on the inoculum level, methods of inoculation, temperature, and available moisture. The initial symptoms caused by inoculations with both bacteria were similar, but as symptoms progressed, P. ANANAS was associated with
white streaks or irregular necrotic blotches often surrounded by a reddish or purplish hue. P. STEWARTII was associated with light-colored necrotic streaks. Synergistic or antagonistic relationship was not observed between the two pathogens in co-inoculations. In host range studies, both bacteria caused disease on sorghum and sudangrass at similar levels of severity. P. ANANAS was also pathogenic on corn and oat. P. STEWARTII from sudangrass was pathogenic on corn but did not cause wilting that was observed with Stewart's wilt strains of P. STEWARTII from corn. The sudangrass strains of P. STEWARTII also infected oat and triticale, while the Stewart's wilt strains did not. Both P. ANANAS and P. STEWARTII from sudangrass grew at relatively high temperatures (43 C and 37 C, respectively) and caused disease at elevated temperatures and conditions of relative humidity similar to those in the Imperial Valley during late summer when epidemics of the disease were common.
Impacts
Sudangrass production in the Imperial Valley of California was valued at nearly $34 million in 1998, when there were 70,000 acres produced. The new bacterial disease has caused severe losses in recent years. Understanding its cause and the source of the bacterial pathogens is the first step toward controlling this important disease.
Publications
- Lee, S.-W., and D. A. Cooksey. 2000. Genes expressed in PSEUDOMONAS PUTIDA during colonization of a plant-pathogenic fungus. Appl. Environ. Microbiol. 66:2764-2772.
- Azad, H. R., G. J. Holmes, and D. A. Cooksey. 2000. A new leaf blotch disease of sudangrass caused by PANTOEA ANANAS and PANTOEA STEWARTII. Plant Dis. 84:973-979.
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Progress 01/01/99 to 12/31/99
Outputs
PSEUDOMONAS PUTIDA 06909 is being investigated as a potential biological control agent for citrus root rot. To better understand mechanisms of inhibition of PHYTOPHTHORA PARASITICA by PSEUDOMONAS PUTIDA, we adapted in vivo expression technology (IVET) to clone genes from P. PUTIDA that are expressed during colonization of PHYTOPHTHORA. The system was based on a promoterless transcriptional fusion between pyrBC', encoding aspartate transcarbamoylase, and lacZ. Random genomic fragments of P. PUTIDA were cloned in front of the pyrBC'-lacZ fusion and placed in a pyrB mutant strain of P. PUTIDA, which was defective for pyrimidine biosynthesis. Pools of such clones were then inoculated to hyphae of PHYTOPHTHORA PARASITICA. The pyrB mutant strain did not survive on PHYTOPHTHORA hyphae, but clones containing genomic fragments containing active promoters in the appropriate orientation survived by expressing pyrBC'-lacZ. Several clones were recovered that contained promoters
active during colonization of PHYTOPHTHORA hyphae but not expressed during growth on media without the fungus. Sequencing of three of the clones revealed similarities to bacterial ABC transporters and outer membrane porins. The possible function of the genes in fungal colonization and suppression is being investigated. Additional experiments demonstrated that the pyrB mutant did not survive in the rhizosphere of citrus, suggesting that this IVET system will also be useful for isolation of clones containing promoters expressed in the citrus rhizosphere. In another project, we continued our investigation of the mechanisms of resistance to copper bactericides in bacterial pathogens. Sequence analysis of the region upstream of the copper resistance operon (cop) in XANTHOMONAS CAMPESTRIS pv. vesicatoria revealed an open reading frame, copL, and other different sequence features that were not present in previously characterized cop operons in other plant pathogens. Mutagenesis of copL
indicated that it was important in regulation of the cop operon in this bacterium. CopL was expressed as a fusion to the maltose binding protein, and the copper-binding and DNA-binding properties of the protein are being investigated to determine its role in copper-inducible gene regulation.
Impacts
Understanding mechanisms of biological control of fungal diseases of plants with beneficial bacteria should lead to improvements in this alternative to chemical control and improve agricultural productivity. Understanding mechanisms and regulation of bacterial pathogen resistance to copper bactericides can lead to more effective copper formulations to overcome resistance.
Publications
- Lee, S. W., and Cooksey, D.A. 1999. Expression of two chromosomal loci of PSEUDOMONAS PUTIDA during colonization of PHYTOPHTHORA PARASITICA. Abstracts of the General Meeting of the American Society for Microbiology, 99:365 (Abstr.).
- Reignier, T. M.. and Cooksey, D.A. 1999. Regulation of the copper resistance gene expression in XANTHOMONAS CAMPESTRIS pv. vesicatoria. Abstracts of the General Meeting of the American Society for Microbiology, 99:360 (Abstr.).
- Seon-Woo Lee. 1999. Identification of chromosomal loci of PSEUDOMONAS PUTIDA induced during colonization of PHYTOPHTHORA PARASITICA. Ph.D. Thesis, University of California, Riverside, 150 pp.
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Progress 01/01/98 to 12/01/98
Outputs
Bacterial blight was described as a new disease of broccoli raab or rappini (BRASSICA RAPA subsp. RAPA) that developed on commercially grown crops in the Salinas Valley of California. Symptoms included small, angular, water-soaked flecks on lower foliage that expanded and became surrounded by bright yellow borders. With time, multiple leaf spots coalesced and resulted in large, irregular necrotic areas, extensive leaf yellowing, and eventual leaf death. If symptoms developed on the uppermost leaves attached to the inflorescence, the shoots lost market quality and were not harvested. PSEUDOMONAS SYRINGAE was consistently isolated from symptomatic plants, and selected strains caused similar symptoms when inoculated onto broccoli raab test plants. They were shown to produce the phytotoxin coronatine, which is the likely cause of the yellowing symptoms. Broccoli raab strains caused leaf spot symptoms on nine other cruciferous plants, as well as on three grass species
(California brome, oat, and common timothy). Conversely, broccoli raab was not infected by P. SYRINGAE pathovars coronafaciens, maculicola, and tomato, which contain strains that can infect other cruciferous plants. Fatty acid analysis indicated that the strains from broccoli raab were closely related to pathovars coronafaciens and maculicola, but their distinct host range suggests that they belong to a separate pathovar.
Impacts
(N/A)
Publications
- KOIKE, S.T., D.M. HENDERSON, H.R. AZAD, D.A. COOKSEY and E.L. LITTLE. 1998. Bacterial blight of broccoli raab: A new disease caused by a pathovar of PSEUDOMONAS SYRINGAE. Plant Disease 82:727-731.
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Progress 09/01/92 to 06/01/97
Outputs
Within the broader goal of this project to enhance our knowledge of bacterial pathogens as threats to agricultural crops of California, specific objectives related to our understanding of bactericide resistance were met. Copper compounds are the most commonly used bactericides for disease control, and during the past five years, we have enhanced our understanding of how bacterial pathogens develop resistance to copper. We have shown that related plasmid-encoded copper resistance operons exist in plant pathogens of different genera, such as PSEUDOMONAS and XANTHOMONAS, and that these genes are related to common chromosomal genes in bacteria. However, DNA sequence analysis suggests that the origin of plasmid-borne copper resistance was in ancient times, rather than the result of a recent bacterial adaptation. Our evidence does suggest, however, that the resistance plasmids have spread recently among plant pathogenic bacteria, and that the regulation of the related
copper resistance operons is specifically adapted to the host genus. A novel two-component system for recognizing copper and inducing expression of the copper resistance genes was defined. In addition, genes involved in the biogenesis of cytochrome c were shown to play a role in both copper resistance and competitive fitness in a soil bacterium, PSEUDOMONAS FLUORESCENS, that is being tested as a biological control agent against a fungal root rot disease of citrus. Other factors important to the interaction of the biological control agent and fungal pathogens were also identified, including the ability to attach.
Impacts
(N/A)
Publications
- COOKSEY, D. A. 1996. Molecular genetics and evolution of copper resistance in bacterial plant pathogens. Pages 79-88 in: T. M. Brown, ed., Molecular Genetics and Ecology of Pesticide Resistance, ACS Symposium Series, American Chemical Soc.
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Progress 01/01/96 to 12/30/96
Outputs
Two new bacterial plant diseases were described in California ornamental crops. Commercial nursery growers in Santa Cruz County observed a leaf disease of French and English lavender. The disease symptoms were associated with the bacterial pathogen XANTHOMONAS CAMPESTRIS, and this pathogen was proven to be the cause of the disease by reinoculation and fulfillment of Koch's postulates. Analysis of carbon source utilization patterns suggested relatedness of this pathogen to X. CAMPESTRIS pv. VESICATORIA, but its pathovar status has not yet been confirmed through host range tests. A new bacterial disease of the bulb and cut flower crop, ranunculus, was observed on several cultivars in commercial fields in San Diego and Riverside Counties. XANTHOMONAS CAMPESTRIS was shown to be the cause of leaf spot and blight symptoms, as well as necrosis of root tubers. The pathogen was shown to spread systemically in infected plants and was recovered from commercial seed and tubers.
Genetic analyses indicated that all ranunculus strains of the pathogen were closely related to each other and to X. CAMPESTRIS pv. CAMPESTRIS, but pathogenicity tests suggested that the ranunculus strains represent a new pathovar.
Impacts
(N/A)
Publications
- KOIKE, S. T., TJOSVOLD, S. A., COOKSEY, D. A. and AZAD, H. R. 1995. A bacterial leaf disease of lavender caused by XANTHOMONAS CAMPESTRIS.
- AZAD, H. R., VILCHEZ, M., PAULUS, A. O. and COOKSEY, D. A. 1996. A new ranunculus disease caused by XANTHOMONAS CAMPESTRIS. Plant Dis.
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Progress 01/01/95 to 12/30/95
Outputs
An improved method for detecting the oleander knot pathogen was developed for the California nursery industry. Existing selective media for isolating the pathogen, PSEUDOMONAS SAVASTANOI, were found to be inadequate, and a new medium was therefore developed. The new medium, called OKA, was highly selective and allowed detection of the pathogen not only from diseased (galled) plant materials, but also from the surface and interior of symptomless plants. The study showed that this bacterial pathogen invades and moves systemically in oleander plants. The new medium should help efforts to screen oleander plants for the presence of the pathogen before vegetative propagation. An additional project investigated the variation in pathogenicity and other characteristics of a recently-described pathogen of avocado. XANTHOMONAS CAMPESTRIS was shown to be the cause of bacterial canker of avocado in several locations in California. A series of pathogenicity tests, coupled with
molecular biology methods, such as restriction fragment length polymorphisms and isozymes, showed that there was considerable diversity among strains of the pathogen recovered from several different locations. The results suggested that the disease was not the result of a single recent introduction, but may have been present in California for many years.
Impacts
(N/A)
Publications
- AZAD, H. R., COOKSEY, D. A. 1995. A semiselective medium for detecting epiphyticand systemic populations of PSEUDOMONAS SAVASTANOI from oleander. Phytopathology85:740-745.
- COOKSEY, D. A., AZAD, A. R. 1994. Pathogenicity and variability of XANTHOMONAS CAMPESTRIS from avocado canker in California. Pages 355-360 in: M. Lemattre, S. Freigoun, K. Rudolph, and J. G. Swings (eds), Plant Pathogenic Bacteria, INRA,.
- COOKSEY, D. A., OHR, H. D., KORSTEN, L. 1994. Bacterial canker. Page 75 in: R. C. Ploetz, G. A. Zentmyer, W. T. Nishijima, K. G. Rohrbach, and H. D. Ohr (eds),Compendium of Tropical Fruit Diseases, APS Press, St. Paul.
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Progress 01/01/94 to 12/30/94
Outputs
The mechanism by which bacteria sense copper ions, which are used for bacterial disease control, was further investigated by purifying and characterizing a copper-responsive transcriptional activator protein. DNA/protein mobility-shift assays were used to show that the protein binds specifically to the promoter/operator region of copper resistance genes in PSEUDOMONAS SYRINGAE. DNaseI footprinting was performed to define the binding site, which is conserved in chromosomal and plasmid-borne copper-inducible promoters of this species. In addition, the transcriptional start site and other characteristics of the copper-inducible promoters were defined. This work helps us to understand how bacterial plant pathogens adapt to copper exposure in the agricultural environment and may lead to novel copper formulations that circumvent the resistance mechanism or induction of its expression.
Impacts
(N/A)
Publications
- MILLS, S. D., LIM, C.-K., COOKSEY, D. A. 1994. Purification and characterization of CopR, a transcriptional activator protein that binds to a conserved domain (cop box) in copper-inducible promoters of PSEUDOMONAS SYRINGAE. Mol. Gen. Genet.
- COOKSEY, D. A. 1994. Molecular mechanisms of copper resistance and accumulation in bacteria. FEMS Microbiol. Rev. 14:381-386.
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Progress 01/01/93 to 12/30/93
Outputs
A bacterial strain that is being tested for biological control of citrus root rot was shown to be copper resistant. Through transposon mutagenesis, the copper resistance genes were shown to be important in survival in copper-contaminated soils, such as those found in citrus groves after many years of copper fungicide application. Surprisingly, these genes were also important for competitive survival in nonsterile soil without high levels of copper. In other work on the copper resistance system previously defined in the plant pathogen, PSEUDOMONAS SYRINGAE, two genes required for copper-inducible expression were cloned and sequenced. The structural copper resistance genes from this species were also shown to be related to resistance genes in another genus, suggesting that copper resistance genes have been exchanged between plant pathogens. The mechanism of resistance was further defined by identification of an apparent copper transport system linked to expression of the
previously-described copper sequestration mechanism for preventing copper entry into the cell. The evolution of plasmid-determined copper resistance was studied by investigating spontaneous mutants in chromosomal homologs of the copper resistance genes.
Impacts
(N/A)
Publications
- YANG, C.-H., MENGE, J. A., and COOKSEY, D. A. 1993. Role of copper resistance in competitive survival of PSEUDOMONAS FLUORESCENS in soil. Appl. Environ. Microbiol. 59:580-584.
- MILLS, S. D., JASALAVICH, C. A., and COOKSEY, D. A. 1993. A two-component regulatory system required for copper-inducible expression of the copper resistance operon of PSEUDOMONAS SYRINGAE. J. Bacteriol. 175:1656-1664.
- VOLOUDAKIS, A. E., BENDER, C. L., and COOKSEY, D. A. 1993. Similarity between copper resistance genes from XANTHOMONAS CAMPESTRIS and PSEUDOMONAS SYRINGAE. Appl. Environ. Microbiol. 59:1627-1634.
- CHA, J.-S., and COOKSEY, D. A. 1993. Copper hypersensitivity and uptake in PSEUDOMONAS SYRINGAE containing cloned components of the copper resistance operon. Appl. Environ. Microbiol. 59:1671-1674.
- LIM, C.-K., and COOKSEY, D. A. 1993. Characterization of chromosomal homologs of the plasmid-borne copper resistance operon of PSEUDOMONAS SYRINGAE. J. Bacteriol. 175:4492-4498.
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Progress 01/01/87 to 09/30/92
Outputs
The genetics and mechanisms of copper resistance in plant pathogenic bacteria were defined, and copper resistance was shown to be an important component of pathogen fitness in the tomato pathogen PSEUDOMONAS SYRINGAE pv. tomato. Copper is still the most commonly-used bactericide on agricultural crops. The copper resistance operon, COP, encodes a unique mechanism of metal resistance, in which the bacteria sequester copper in the outer membrane and periplasmic space with copper-binding proteins. This mechanism is specifically induced by copper; a single, copper-inducidle promoter controls the COP operon. Since several copper-resistant species of PSEUDOMONAS accumulated copper, sequestration of copper appears to be a common resistance mechanism in pseudomonads isolated from the agricultural environment. Evidence suggests that the plasmid-borne copper resistance genes may have evolved recently from indigenous chromosomal genes that are present in many species of
PSEUDOMONAS. Two different pathovars of PSEUDOMONAS SYRINGAE, from tomato and impatiens, contained closely-related copper resistance plasmids, suggesting that transmission of copper resistance has occurred between different bacterial pathogens by conjugation.
Impacts
(N/A)
Publications
- COOKSEY, D.A. 1992. Copper uptake and resistance in bacteria. Mol. Microbiol., in press.
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Progress 01/01/91 to 12/30/91
Outputs
The mechanism of resistance to copper bactericides was determined for the tomatopathogen PSEUDOMONAS SYRINGAE pathovar TOMATO. This bacterium prevents toxic levels of copper ions from entering the cytoplasm by sequestering copper in the outer membrane and periplasmic space. Copper sequestration is accomplished by the production of copper-binding proteins encoded by the plasmid-borne copper resistance operon, COP. One of the characterized proteins binds one copper atom, and the other binds about eleven copper atoms. Accumulation outside of the cytoplasm is an unusual mechanism of metal resistance for bacteria; most other metal-resistant microbes detoxify the metal by transformation or prevent its entry by an active efflux mechanism. The levels of accumulation of copper and other metals were determined for several copper-resistant pseudomonads. This work expanded our previous study and suggested that sequestration of copper is a common resistance mechanism in
pseudomonads isolated from the agricultural environment. Some other metals were also accumulated by these bacteria, but the other metals did not generally appear to be accumulated by the same mechanism as copper.
Impacts
(N/A)
Publications
- CHA, J.S. and COOKSEY, D.A. 1991. Copper resistance in PSEUDOMONAS SYRINGAE mediated by periplasmic and outer membrane proteins. Proc. Natl. Acad. Sci. USA 88:8915-8919.
- COOKSEY, D.A. and AZAD, H.R. 1992. Accumulation of copper and other metals by copper-resistant plant-pathogenic and saprophytic pseudomonads. Appl. Environ. Mirobiol. 58:274-278.
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Progress 01/01/90 to 12/30/90
Outputs
We previously identified and characterized genes encoding resistance to copper bactericides in the tomato pathogen PSEUDOMONAS SYRINGAE. This year we found that several pathogenic and saprophytic species of PSEUDOMONAS contain genes that are similar to the copper resistance genes from P. SYRINGAE. These copper resistance gene homologs may represent ancestral genes that the resistance genes evolved from, and they are therefore of interest to our understanding of how resistance developed in P. SYRINGAE from tomato and the possibility of resistance developing in other plant pathogenic pseudomonads. Proteins related to those produced from copper resistance genes were also detected by immunoblot analysis of the different species of PSEUDOMONAS. In another study, a leaf spot disease of cilantro was found for the first time in the United States. The causal agent, PSEUDOMONAS SYRINGAE, was unusual, because it was always nonfluorescent, unlike most other strains of this
species.
Impacts
(N/A)
Publications
- COOKSEY, D. A., AZAD, H. R., CHA, J.-S., and LIM, C.-K. 1990. Copper resistance gene homologs in pathogenic and saprophytic bacterial species from tomato. Appl. Environ. Microbiol. 56:431-435.
- COOKSEY, D. A. 1990. Genetics of bactericide resistance in plant pathogenic bacteria. Annu. Rev. Phytopathol. 28:201-219.
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Progress 01/01/89 to 12/30/89
Outputs
A new bacterial pathogen of Impatiens was identified and characterized. The disease was found in a commercial nursery in Santa Barbara County, and the pathogen was identified as Pseudomonas syringae. The bacterium caused rapid water-soaking of leaf tissues, followed by necrosis in large sectors of leaves. The pathogen did not infect any other plants that were inoculated, suggesting that it may represent a new pathovar of P. syringae. The pathogen of Impatiens carried a 47-kilobase copper resistance plasmid (pPSI1); the role of pPSI1 in copper resistance was demonstrated by electroporation of the plasmid into a copper-sensitive strain of P. syringae. Electroporation of this plasmid occurred at a high frequency into P. syringae, but the plasmid was not conjugative in mating experiments. About 20 kilobases of pPSI1 was homologous with copper resistance plasmid pPT23D, which we previously isolated and characterized from the tomato pathogen P. syringae pv. tomato.
Impacts
(N/A)
Publications
- COOKSEY, D.A., and KOIKE, S.T. 1990. A new foliar blight of Impatiens caused by Pseudomonas syriangae. Plant Disease 74: (in-press).
- COOKSEY, D.A. 1990. Plasmid-determined copper resistance in Pseudomonas syringae from Impatiens. Appl. Environ. Microbiol. 56: (In press).
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Progress 08/01/88 to 12/30/88
Outputs
We previously cloned and sequenced a copper resistance gene cluster from a conserved plasmid in copper-resistant strains of Pseudomonas syringae pv. tomato. This plant pathogen is resistant to copper bactericides that are used commercially for disease control. Recent work on gene regulation in this system showed that the genes controlling copper resistance are inducible by copper. Analysis of mRNA demonstrated that regulation is at the transcriptional level. Using gene probes specific for each of the four genes of the cluster, mRNA levels for each gene were shown to be coordinately induced over time and with increasing concentrations of copper. A single, copper-inducible promoter was cloned from the region 5' to the first gene of the cluster, suggesting that the four genes controlling copper resistance are transcribed as an operon.
Impacts
(N/A)
Publications
- MELLANO, M.A., and COOKSEY, D.A. (1988). Induction of the copper resistance operon from Pseudomonas syringae pv. tomato. J. Bacteriol. 170:4399-4401.
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