The exopolysaccharide of Xylella fastidiosa is essential for biofilm formation, plant virulence, and vector transmission.

Exopolysaccharides (EPS) synthesized by plant-pathogenic bacteria are generally essential for virulence. The role of EPS produced by the vector-transmitted bacterium Xylella fastidiosa was investigated by knocking out two genes implicated in the EPS biosynthesis, gumD and gumH. Mutant strains were affected in growth characteristics in vitro, including adhesion to surfaces and biofilm formation. In addition, different assays were used to demonstrate that the mutant strains produced significantly less EPS compared with the wild type. Furthermore, gas chromatography-mass spectrometry showed that both mutant strains did not produce oligosaccharides. Biologically, the mutants were deficient in movement within plants, resulting in an avirulent phenotype. Additionally, mutant strains were affected in transmission by insects: they were very poorly transmitted by and retained within vectors. The gene expression profile indicated upregulation of genes implicated in cell-to-cell signaling and adhesins while downregulation in genes was required for within-plant movement in EPS-deficient strains. These results suggest an essential role for EPS in X. fastidiosa interactions with both plants and insects.

Leaf Scorch of Purple-Leafed Plum and Sweetgum Dieback: Two New Diseases in Southern California Caused by Xylella fastidiosa Strains with Different Host Ranges.

Sweetgum dieback and leaf scorch of purple-leafed plum are 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.

Differentiation of Strains of Xylella fastidiosa Infecting Grape, Almonds, and Oleander Using a Multiprimer PCR Assay.

Xylella fastidiosa is a xylem-limited, nutritionally fastidious bacterium that causes several plant diseases including Pierce's disease (PD) in grape and leaf scorch in almond (ALS) and oleander (OLS). OLS strains belong to X. fastidiosa subsp. sandyi, PD strains belong to X. fastidiosa subsp. fastidiosa, and strains from almond designated as ALS strains are of two general types belonging either to X. fastidiosa subsp. multiplex or X. fastidiosa subsp. fastidiosa. The ALS strains assigned to X. fastidiosa subsp. multiplex belong to two different genotypes (ALSI and ALSII) below the subspecies level. The OLS strains do not infect grape or almond. PD strains produce diseases in grape, alfalfa, almond, and some weeds, but they do not infect oleander, oak, peach, or citrus. ALS strains that belong to X. fastidiosa subsp. multiplex do not produce disease on grape. In this study, a relatively simple polymerase chain reaction (PCR) based method was developed to distinguish among PD, OLS, and ALS strains. PCR performed with primers XF1968-L and XF1968-R amplified a 638-bp fragment from OLS strains but not from PD strains or ALS strains that belong to X. fastidiosa subsp. fastidiosa. PCR with primers XF2542-L and XF2542-R amplified a 412-bp fragment from PD strains, but not from OLS strains. PCR with primers ALM1 and ALM2 produced a fragment of 521 bp from strains isolated from almond that belong to X. fastidiosa subsp. multiplex. The combination of the three primer sets allowed the distinction of the two ALS genotypes of X. fastidiosa subsp. multiplex. These results are in agreement with those obtained from analysis of sequences of 16S-23S rDNA intergenic spacer regions sequence analysis and with previous results based on randomly amplified polymorphic DNA analysis.

Discovery and Characterization of Xylella fastidiosa Strains in Southern California Causing Mulberry Leaf Scorch.

Mulberry leaf scorch (MLS), caused by Xylella fastidiosa, is a disease of mulberry trees in the United States that has largely been documented from locations in the eastern and central areas of the country. MLS was recently detected for the first time in white mulberry (Morus alba) trees in southern California. 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 RST31-33. Isolated strains were characterized by the sequencing of their 16S-23S rDNA intergenic spacer regions (ISR) 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. The ISR sequences of two MLS-strains, MLS063 and MLS059, were 100% identical to that of the Mulberry-VA sequence, whereas MLS012 and MLS024 were 99.8 and 99.6% identical to the Mulberry-VA-strain and 99.4% identical among themselves. Genomic analysis using RAPD revealed no differences among the four 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.

Detection and differentiation of Xylella fastidiosa strains acquired and retained by glassy-winged sharpshooters (Hemiptera: Cicadellidae) using a mixture of strain-specific primer sets.

Xylella fastidiosa Wells is a bacterial pathogen that causes a variety of plant diseases, including Pierce's disease (PD) of grapevine, almond leaf scorch, alfalfa dwarf, citrus variegated chlorosis, and oleander leaf scorch (OLS). Numerous strains of this pathogen have been genetically characterized, and several different strains occur in the United States. The dominant vector in southern California is the glassy-winged sharpshooter, Homalodisca coagulata (Say) (Hemiptera: Cicadellidae). The high mobility of this insect, and its use of large numbers of host plant species, provides this vector with ample exposure to multiple strains of X. fastidiosa during its lifetime. To learn more about the ability of this vector to acquire, retain, and transmit multiple strains of the pathogen, we developed a polymerase chain reaction (PCR)-based method to detect and differentiate strains of X. fastidiosa present in individual glassy-winged sharpshooter adults. Insects were sequentially exposed to plants infected with a PD strain in grapevine and an OLS strain in oleander. After sequential exposure, a few insects tested positive for both strains (7%); however, in most cases individuals tested positive for only one strain (29% PD, 41% OLS). In transmission studies, individual adults transmitted either the PD or OLS strain of the pathogen at a rate (39%) similar to that previously reported after exposure to a single strain, but no single individual transmitted both strains of the pathogen. PD and OLS strains of X. fastidiosa remained detectable in glassy-winged sharpshooter, even when insects were fed on a plant species that was not a host of the strain for 1 wk.

Molecular mechanisms of copper resistance and accumulation in bacteria.

An unusual mechanism of metal resistance is found in certain plant pathogenic strains of Pseudomonas syringae that are exposed to high levels of copper compounds used in disease control on agricultural crops. These bacteria accumulate blue Cu2+ ions in the periplasm and outer membrane. At least part of this copper sequestering activity is determined by copper-binding protein products of the copper resistance operon (cop). Potential copper-binding sites of the periplasmic CopA protein show conservation with type-1, type-2, and type-3 copper sites of several eukaryotic multi-copper oxidases. In addition to compartmentalization of copper in the periplasm, two components of the cop operon, copC and copD, appear to function in copper uptake into the cytoplasm. Copper resistance operons related to cop have been described in the related plant pathogen Xanthomonas campestris and in Escherichia coli, but these resistance systems may differ functionally from the Pseudomonas syringae system.

A New Leaf Blotch Disease of Sudangrass Caused by Pantoea ananas and Pantoea stewartii.

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. A 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 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.

Plant Hosts of Xylella fastidiosa In and Near Southern California Vineyards.

Xylella fastidiosa is a xylem-limited bacterium that causes Pierce's disease (PD) of grapevines. A variety of plant species found near a severe outbreak of PD in vineyards 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 (Spartium junceum) and wild mustard (Brassica spp). 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 and should be removed or monitored for signs of infection. Sequences from oleander isolates from Temecula formed another cluster with a previously published oleander strain sequence. Oleander strains do not infect grapevines and thus do not appear to cause a direct threat to grapevines. Two additional isolates from almond were determined to be genetically different from PD strains, and the ability of these strains to infect grapevine is not known. Greenhouse transmission studies indicate that the glassy-winged sharpshooter was able to transmit a PD strain of X. fastidiosa to Spanish broom, black mustard, and other hosts.

Pseudomonas putida 06909 genes expressed during colonization on mycelial surfaces and phenotypic characterization of mutants.

AIMS: The main focus of this study was to gain an overall view of Pseudomonas putida 06909 genes involved in the Pseudomonas-Phytophthora interaction as a biological control mechanism, and to understand the roles of these genes. METHODS AND RESULTS: Sixteen Ps. putida genes with increased expression on Phytophthora mycelial surfaces were identified using in vivo expression technology (IVET) screening. Sequence analysis of these Phytophthora mycelium-induced (pmi) genes revealed that many of them 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 pmi genes encoding succinate semialdehyde dehydrogenase, a dicarboxylic acid transporter, and glyceraldehyde-3-phosphate dehydrogenase showed significant phenotypic differences involved in the colonization processes, including motility, biofilm formation on abiotic surfaces, colony morphology, and competitive colonization of fungal mycelia. All three of these pmi genes were induced by glycogen and other substances, such as organic acids and amino acids utilized by Ps. putida. CONCLUSIONS: The IVET screening and mutant characterization can be used to identify bacterial genes that are induced on the mycelial surface and provide insight into the possible mechanisms of mycelial colonization by this bacterium. SIGNIFICANCE AND IMPACT OF THE STUDY: The IVET screening through a bacterial genome library might be a huge task. However, because the genes involved in direct interaction with Phytophthora and in bacterial adaptation can be identified, the IVET system will be a valuable tool in studying biocontrol bacteria at the molecular and ecological levels.

Effect of host plant Xylem fluid on growth, aggregation, and attachment of Xylella fastidiosa.

In the Temecula Valley of California the proximity of citrus groves to vineyards influences the incidence and severity of Pierce's disease (PD) in grapes, a disease caused by the gram-negative bacterium, Xylella fastidiosa. Although the glassy-winged sharpshooter (GWSS), the major insect vector of the bacterium, feeds on and moves back and forth between citrus groves and vineyards, there are no visible symptoms of disease caused by X fastidiosa in citrus. Previous evidences suggested that while grapevines are susceptible to the PD strain of X. fastidiosa, citrus trees are resistant or tolerant but could be a reservoir to harbor the pathogen for the GWSS acquisition. We investigated the mechanisms of host plant resistance/susceptibility 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. Our results revealed that 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. The densities of X. fastidiosa cells in grapefruit xylem fluid treatment were significantly higher at 6, 8, and 9 d postinoculation compared with those in grape xylem fluid treatment. The cell densities of X. fastidiosa cultures in orange or lemon xylem fluid were generally lower than those in grape xylem fluid treatment, whereas citrus xylem fluid significantly inhibited X. fastidiosa biofilm formation compared to grape xylem fluid.

In vitro activities of antibiotics and antimicrobial peptides against the plant pathogenic bacterium Xylella fastidiosa.

AIMS: The objective of this study was to evaluate the effectiveness of antibiotics and antimicrobial peptides against 10 strains of Xylella fastidiosa. METHODS AND RESULTS: The minimal inhibitory concentration (MIC) of 12 antibiotics and 18 antimicrobial peptides were determined by agar dilution tests and growth inhibition assays. 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. CONCLUSION: All X. fastidiosa strains were sensitive to several groups of antibiotics, and minor differences in sensitivity to several antimicrobial peptides were observed among strains. SIGNIFICANCE AND IMPACT OF THE STUDY: This study shows that antibiotics and antimicrobial peptides have some activity against the pathogen, X. fastidiosa and may have application in protecting plants from developing Pierce's disease.

A spontaneous insertion in the agrocin sensitivity region of the Ti-plasmid of Agrobacterium tumefaciens C58.

A spontaneous agrocin-resistant mutant of Agrobacterium tumefaciens strain C58 was shown to have an insertion of 1.2 kb in the agrocin-sensitivity region of pTiC58. The insertion was cloned from the Ti-plasmid, and a subclone containing only DNA internal to the insertion was used to probe the Ti-plasmid and chromosomal DNA of the wild-type strain C58. The probe showed homology to chromosomal sequences but showed no homology to wild-type pTiC58. Homology was also detected with chromosomal sequences of A. tumefaciens strains, B6, K24, and T37. These results suggest that an indigenous insertion sequence of 1.2 kb transposed from the chromosome to the agrocin-sensitivity region of the Ti-plasmid in this spontaneous mutant of C58.

Plasmid-Determined Copper Resistance in Pseudomonas syringae from Impatiens.

A strain of Pseudomonas syringae was recently identified as the cause of a new foliar blight of impatiens. The bacterium was resistant to copper compounds, which are used on a variety of crops for bacterial and fungal disease control. The bacterium contained a single 47-kilobase plasmid (pPSI1) that showed homology to a copper resistance operon previously cloned and characterized from P. syringae pv. tomato plasmid pPT23D (D. Cooksey, Appl. Environ. Microbiol. 53:454-456, 1987). pPSI1 was transformed by electroporation into a copper-sensitive P. syringae strain, and the resulting transformants were copper resistant. A physical map of pPSI1 was constructed, and the extent of homology to pPT23D outside the copper resistance operon was determined in Southern hybridizations. The two plasmids shared approximately 20 kilobases of homologous DNA, with the remainder of each plasmid showing no detectable homology. The homologous regions hybridized strongly, but there was little or no conservation of restriction enzyme recognition sites.

Characterization of a Copper Resistance Plasmid Conserved in Copper-Resistant Strains of Pseudomonas syringae pv. tomato.

A 35-kilobase plasmid was conserved among 12 copper-resistant strains of Pseudomonas syringae pv. tomato. Restriction patterns of this plasmid from each strain were identical, and a cloned copper resistance gene from 1 strain hybridized to the same location on the 35-kilobase plasmid of all 12 strains.

Copper uptake and resistance in bacteria.

Copper ions are essential for bacteria but can cause a number of toxic cellular effects if levels of free ions are not controlled. Investigations of copper-resistant bacteria have revealed several mechanisms, mostly plasmid-determined, that prevent cellular uptake of high levels of free copper ions. However, these studies have also revealed that bacteria apparently have efficient chromosomally encoded systems for uptake and management of trace levels of copper. This review will explore the relationship of copper uptake systems to resistance mechanisms and the possibility that copper resistance has evolved directly through modification of chromosomal copper uptake genes.

Induction of the copper resistance operon from Pseudomonas syringae.

Cupric sulfate induced mRNA specific to the copper resistance gene cluster previously cloned from Pseudomonas syringae pv. tomato PT23. mRNA from each of the four genes of this cluster responded in a similar manner to induction over time and with different concentrations of cupric sulfate. Promoter fusion constructs indicated the presence of a single copper-inducible promoter upstream from the first open reading frame.

Nucleotide sequence and organization of copper resistance genes from Pseudomonas syringae pv. tomato.

The nucleotide sequence of a 4.5-kilobase copper resistance determinant from Pseudomonas syringae pv. tomato revealed four open reading frames (ORFs) in the same orientation. Deletion and site-specific mutational analyses indicated that the first two ORFs were essential for copper resistance; the last two ORFs were required for full resistance, but low-level resistance could be conferred in their absence. Five highly conserved, direct 24-base repeats were found near the beginning of the second ORF, and a similar, but less conserved, repeated region was found in the middle of the first ORF.

Development of Host Range Mutants of Xanthomonas campestris pv. translucens.

Xanthomonas campestris pv. translucens is the causal agent of bacterial leaf streak of cereal grains and grasses, and individual strains within the pathovar differ in their host range among the cereals. Coinoculation of a wide-host-range and a narrow-host-range strain resulted in the wide-host-range reaction. Transposon and chemical mutagenesis of the wide-host-range strain Xct4, pathogenic on barley, wheat, rye, and triticale, resulted in variants with reduced host range. When pathogenicity was inactivated independently for barley, wheat, triticale, and rye, wild-type symptoms were retained on the other members in the host range. Testing of some host range mutants on additional varieties of the cereals indicated some cultivar specificity. In addition, mutants nonpathogenic on combinations of the hosts or on all hosts were isolated. This suggests that there are independent positive factors determining host range in this species, rather than an avirulence gene system such as those determining race specificity in other plant pathogens.

Indigenous plasmids in Pseudomonas syringae pv. tomato: conjugative transfer and role in copper resistance.

Twenty strains of Pseudomonas syringae pv. tomato were examined for the presence of plasmid DNA. P. syringae pv. tomato plasmids were grouped into five size classes: class A ranged from 95 to 103 kilobases (kb); class B ranged from 71 to 83 kb; class C ranged from 59 to 67 kb; class D ranged from 37 to 39 kb; and class E was 29 kb. All strains contained at least two plasmids in classes A and B. The conjugative ability of P. syringae pv. tomato plasmids in three strains was demonstrated by mobilization of the nonconjugative plasmid RSF1010 into Pseudomonas syringae pv. syringae recipients. Plasmids from the three conjugative strains were labeled with Tn5. Four conjugative plasmids were identified by their repeated transfer to P. syringae pv. syringae recipients. P. syringae pv. tomato strains varied in sensitivity to copper sulfate (CuSO4): MICs were 0.4 to 0.6 mM for sensitive strains, 1.2 mM for moderately resistant strains, and 1.6 to 2.0 mM for very resistant strains. One very resistant strain, PT23, functioned as a donor of copper resistance. Recipient P. syringae pv. syringae strains PS51 and PS61 were inhibited by 0.1 mM CuSO4, whereas the CuSO4 MICs for transconjugant strains PS51(pPT23A) and PS61(pPT23C) were 1.8 and 2.6 mM, respectively. P. syringae pv. tomato strains PT12.2 and PT17.2 were inhibited by 0.6 mM copper sulfate, but their copper sulfate MICs were 2.6 and 1.8 mM, respectively, when they acquired pPT23C. Therefore, copper resistance in PT23 was controlled by two conjugative plasmids, designated pPT23A (101 kb) and pPT23C (67 kb).

Characterization of chromosomal homologs of the plasmid-borne copper resistance operon of Pseudomonas syringae.

Copper-resistant and copper-sensitive strains of Pseudomonas syringae, as well as many other pseudomonads, contain chromosomal DNA homologous to the plasmid-borne copper resistance operon (copABCD). cop homologs were cloned from the chromosome of P. syringae pv. tomato PT12.2, which had an elevated level of resistance to copper compared with typical copper-sensitive strains of other P. syringae pathovars and showed an unusually high frequency of spontaneous mutation to high levels of copper resistance. Two chromosomal cop homolog regions were cloned. Homolog 1 hybridized with copA and copB, and homolog 2 hybridized with copA, copB, copC, and the copper-responsive regulatory genes copRS. Homolog 1 had no detectable function when transferred to a copper-sensitive strain of P. syringae. However, homolog 2 conferred the low level of copper resistance observed with PT12.2 and produced proteins related to CopA and CopC. In addition, homolog 2 conferred a high frequency of mutation to full copper resistance. In a spontaneously mutated derivative of the cloned homolog 2 (pCOPH2R) that conferred copper resistance, an increased level of CopA was observed. pCOPH2R also supported a higher level of transcriptional activity of the cop promoter that was fused to lacZ and provided in trans (pCOP38), suggesting that the spontaneous mutation was regulatory, probably involving the copRS homologs. Homolog 2 was similar but not identical to the plasmid-borne cop operon, and it did not complement site-specific mutations in cop genes.