Modeling cold curing of Pierce's disease in Vitis vinifera 'Pinot Noir' and 'Cabernet Sauvignon' grapevines in California.

Pierce's disease (PD) of Vitis vinifera grapevines is caused by the bacterium Xylella fastidiosa, a pathogen with a wide plant host range. Exposure of X. fastidiosa-infected plant tissue to cold temperatures has been shown to be effective at eliminating the pathogen from some plant hosts such as grapevines. This "cold curing" phenomenon suggests itself as a potential method for disease management and perhaps control. We investigated cold therapy of PD-affected 'Pinot Noir' and 'Cabernet Sauvignon' grapevine. In the fall, inoculated plants and controls of each cultivar were transported to each of four field sites in California (Foresthill, McLaughlin, Hopland, and Davis) that differed in the magnitude of cold winter temperatures. A model for progression of the elimination of plant disease in relation to temperature was conceptualized to be a temperature-duration effect, where temperatures below a particular threshold kill X. fastidiosa with increasing efficacy as the temperature decreases to some value <6?C. The temperature effect was modeled as a likelihood of a particular temperature killing the pathogen and is termed the ?killing index?. We developed a mathematical model for cold curing of grapevines inoculated with X. fastidiosa and calibrated the model with cold-curing data collected in a field study. Parameter estimation resulted in lowest sum of squared differences across all 10 trials to be low temperature below which the organism is killed (T(0)) = 6°C, number of hours to achieve 100% cure (N(100)) = 195 h, number of hours to achieve 10% cure (N(10)) = 20 h, and killing index (K(x)) = 0.45 for Pinot Noir and T(0) = 6°C, N(100) = 302 h, N(10) = 170 h, and K(x) = 0.41 for Cabernet Sauvignon. With the parameter estimates optimized by model calibration, the simulation model was effective at predicting cold curing in four locations during the experiment, although there were some differences between Hopland for Pinot Noir and Davis for Cabernet Sauvignon. Using historical temperature data, the model accurately predicted the known severity of PD in other grape-growing regions of California, suggesting that it may have utility in assessing the relative risk of developing PD in proposed new vineyard sites.

Cloning and Detection of DNA from a Nonculturable Plant Pathogenic Mycoplasma-like Organism.

The ability to detect, quantify, and differentiate nonculturable mycoplasma-like organisms (MLOs) would greatly facilitate epidemiological and taxonomical studies of this unique group of plant and insect pathogens. DNA isolated from extracts of insects infected with the Western X-disease MLO was cloned in Escherichia coli. X-disease-specific clones, when labeled and used as probes, readily detected X-disease MLOs in infected plants and insects but did not hybridize with DNA from healthy plants or insects, or from several other plant pathogenic MLOs or spiroplasmas. These methods provide both a sensitive diagnostic tool and a basis for genetically differentiating MLOs.

Transposon mutagenesis of Xylella fastidiosa by electroporation of Tn5 synaptic complexes.

Pierce's disease, a lethal disease of grapevine, is caused by Xylella fastidiosa, a gram-negative, xylem-limited bacterium that is transmitted from plant to plant by xylem-feeding insects. Strains of X. fastidiosa also have been associated with diseases that cause tremendous losses in many other economically important plants, including citrus. Although the complete genome sequence of X. fastidiosa has recently been determined, the inability to transform or produce transposon mutants of X. fastidiosa has been a major impediment to understanding pathogen-, plant-, and insect-vector interactions. We evaluated the ability of four different suicide vectors carrying either Tn5 or Tn10 transposons as well as a preformed Tn5 transposase-transposon synaptic complex (transposome) to transpose X. fastidiosa. The four suicide vectors failed to produce any detectable transposition events. Electroporation of transposomes, however, yielded 6 x 10(3) and 4 x 10(3) Tn5 mutants per microg of DNA in two different grapevine strains of X. fastidiosa. Molecular analysis showed that the transposition insertions were single, independent, stable events. Sequence analysis of the Tn5 insertion sites indicated that the transpositions occur randomly in the X. fastidiosa genome. Transposome-mediated mutagenesis should facilitate the identification of X. fastidiosa genes that mediate plant pathogenicity and insect transmission.

Expression of chloramphenicol acetyltransferase in Bacillus subtilis under the control of a phytoplasma promoter.

A cloned putative promoter region upstream of the 16S rRNA gene of the western X-disease phytoplasma was inserted behind the promoterless chloramphenicol acetyltransferase gene of plasmid pPL603. The DNA construct was used to transform Bacillus subtilis cells. The transformants were assayed for chloramphenicol acetyltransferase activity, showing that the phytoplasma promoter is efficiently expressed in a B. subtilis background.

A small-scale procedure for extracting nucleic acids from woody plants infected with various phytopathogens for PCR assay.

The complexity of most nucleic acid extraction procedures limits the number of samples that can be easily processed for analysis by polymerase chain reaction (PCR). A simple, small-scale procedure was developed which can be carried out entirely in 1.5-ml microfuge tubes whereby the container and contents are frozen with liquid nitrogen, tissue is pulverized, and targeted nucleic acids are extracted. DNA of bacterial and phytoplasmal plant pathogens was extracted in hot CTAB buffer followed by chloroform clarification. Following centrifugation, the DNA in the aqueous fraction was precipitated with isopropanol and resuspended in water. RNA originating from viruses and viroids was extracted from triturated tissue using STE buffer and phenol. The nucleic acid fraction was purified using CF-11 cellulose. All purified preparations were used as PCR or RT-PCR templates to detect DNA or RNA, respectively. These procedures were used to detect Xylella fastidiosa, peach yellow leaf roll phytoplasma, sour cherry green ring mottle virus, and peach latent mosaic viroid by agarose gel electrophoresis.

Frequency and seasonal distribution of pear psylla infected with the pear decline phytoplasma in california pear orchards.

ABSTRACT Pear decline (PD) is an important disease of Pyrus communis fruiting cultivars in Europe, Asia, and the Americas. PD is caused by a phloem-limited phytoplasma that, in California, is transmitted from diseased to healthy trees by pear psylla, Cacopsylla pyricola. The percentage of phytoplasma-infected pear psylla has never been assessed in the United States in field-collected insects. Pear psylla were collected monthly from PD-infected trees from three orchards in northern California. Individual psylla were tested for the presence of PD phytoplasma, using both a quantitative DNA hybridization and PD phytoplasma-specific polymerase chain reaction (PCR) assays. The percentage of infected psylla ranged from 0 to 45% depending on the orchard, the month and year collected, and the method of detection. The PD phytoplasma was detected in both the winterform and summerform pear psylla. Significantly more infected psylla were detected with PCR than with DNA hybridization analysis in two of the three orchards. The number of PD phytoplasma per pear psylla was estimated to range from 1 x 10(6) to 8.2 x 10(7). The percentage of PD-infected pear psylla found in the three northern California pear orchards suggests that both winterform and summerform pear psylla could be important in the transmission of PD.

Influence of stem diameter, water content, and freezing-thawing on bacterial canker development in excised stems of dormant stone fruit.

ABSTRACT In excised dormant stems of peach (Prunus persica), prune (Prunus domestica), and almond (Prunus dulcis), stem diameter, stem hydration, and freezing-thawing influenced the extent of infection caused by Pseudomonas syringae pv. syringae. Bacterial lesion length increased with increasing stem diameter, demonstrating the need to account for the effects of stem diameter when lesion length data are analyzed. Lesion length increased or decreased with stem hydration or dehydration, respectively. However, tissue water content was not a good indicator of tissue susceptibility to infection by P. syringae pv. syringae, as larger diameter stems had larger lesions and lower water content than did smaller diameter stems. After freezing at -5 degrees C for 12 to 24 h, inoculations made during the thawing process produced significantly larger lesions than did inoculations performed before freezing or after thawing. These results support the hypothesis that the increased susceptibility to bacterial canker that is associated with noninjurious freezing is a result of the increased passive spread of bacteria through water redistribution when inoculation is performed during the thawing process. Plant tissue water relationship characteristics that can influence water movement during freezing and thawing may be an important component of bacterial canker development in stone fruit trees.

A Mycoplasma gallisepticum strain-specific DNA probe.

Total DNA from the vaccine F strain (K810) and the reference S6-strain of Mycoplasma gallisepticum (MG) was cloned in Escherichia coli using the plasmid pUC8. A 6-kilobase fragment, specific for the vaccine strain, was identified by colony dot and Southern hybridization analyses. When labeled and used as a probe, this fragment hybridized with the homologous and one other vaccine F-strain (F2F10), but it did not hybridize with other MG strains (Fg38, S6, A5969, V503) or with three other species of avian mycoplasmas.

Identification of Phytoplasma Taxa and Insect Vectors of Peach Yellow Leaf Roll Disease in California.

Two peach diseases in California, western X-disease (WX) and peach yellow leaf roll (PYLR) are caused by two genetically distinct phytoplasmas. Based on symptoms alone, WX and PYLR cannot be reliably distinguished. The objectives of this study were to determine which phytoplasma was causing PYLR disease in peach orchards planted near pear orchards and which phytoplasmas were present in potential insect vectors captured in both peach and pear orchards. Leafhoppers and pear psylla were collected from yellow sticky traps placed in three pear orchards and four peach orchards located in the Sacramento Valley from 1994 to 1996. DNA was extracted from potential vectors and suspect diseased trees, and analyzed for the presence of the WX and PYLR phytoplasmas using strain-specific DNA hybridization or polymerase chain reaction (PCR) assays. The most abundant phytoplasma detected in diseased peach trees was the PYLR phytoplasma. In 1994 and 1995, 10 to 25% of groups of pear psylla tested positive for the PYLR phytoplasma, while no psylla groups tested positive for the WX phytoplasma. Only one captured leafhopper tested positive for the WX phytoplasma. These results indicate the pear psylla is the primary vector of PYLR in northern California.

Analysis of Double-Stranded RNAs from Cherry Trees with Stem Pitting in California.

Five distinct dsRNA species were recovered from Bing sweet cherry (Prunus avium (L.) L.) trees with stem pitting symptoms. A 4.7-kilobase pair (kbp) dsRNA was isolated from mahaleb rootstock (P. mahaleb L.); an unrelated 4.7-kbp dsRNA, always co-purified with a 1.3-kbp dsRNA, and a 9-kbp dsRNA were from Bing cherry. In addition, an 8.5-kbp dsRNA found in diseased Shirofugen flowering cherry and in Bing cherry was identified as sour cherry green ring mottle virus (CGRMV). The larger, 8.5- and 9.0-kbp dsRNA species were graft-transmissible, while the smaller ones were non-transmissible and appeared cryptic in nature. Reverse transcription-polymerase chain reaction (RT-PCR) assays were developed for each dsRNA species by cloning and sequencing cDNA synthesized from the dsRNA templates. When several diseased collections were assayed by RT-PCR, approximately 14% reacted positively with primers for the 9.0-kbp dsRNA or CGRMV. Although CGRMV and the 9.0-kbp dsRNA caused wood-marking symptoms in graft-inoculated Mazzard (P. avium) seedling trees, no xylem or canopy symptoms developed in grafted Bing cherry. The causal agent or agents of cherry stem pitting have not been identified.

Effects of Rootstock and Budding Height on Bacterial Canker in French Prune.

Bacterial canker is one of the most economically important diseases of stone fruit trees, including 'French' prune (Prunus domestica). Field trials were conducted to evaluate the effect of rootstock selection and budding height on the incidence and severity of bacterial canker in four orchards with low to high disease pressure. Treatments included French prune scions low-grafted on 'Lovell' peach (Prunus persica) rootstocks as well as Myrobalan 29C (Prunus cerasifera) plum rootstocks grafted at 15, 50, and 90 cm above the rootstock crown. Another treatment consisted of growing Myrobalan 29C plum rootstocks in the field for one growing season, then field-grafting French prune buds onto rootstock scaffolds. Lovell peach rootstock provided the greatest protection from bacterial canker as measured by disease incidence and tree mortality in all orchards. Field-budded rootstocks and rootstocks grafted at the highest budding height provided moderate levels of resistance to bacterial canker. These treatments reduced the incidence but not the severity of disease.

Phylogenetic relationships between the western aster yellows mycoplasmalike organism and other prokaryotes established by 16S rRNA gene sequence.

Restriction fragments containing the 16S rRNA gene of the western aster yellow mycoplasmalike organism (SAY-MLO) were identified in Southern blots probed with cloned fragments of the western X-disease mycoplasmalike organism 16S rRNA gene. Two fragments which contained the entire SAY-MLO 16S rRNA gene and flanking DNA were cloned in M13 and sequenced. The SAY-MLO 16S rRNA gene is approximately 1,535 bp long, has a G+C content of 47 mol%, and has an overall secondary structure similar to that proposed for Escherichia coli. Putative rRNA promoter sequences and sequences involved in processing of the primary rRNA transcript were similar in the SAY-MLO, two Mycoplasma species, and Bacillus subtilis, suggesting that these prokaryotes and the mycoplasmalike organisms may have similar transcriptional and processing enzymes. We identified two tRNA genes, a tRNA(Tyr) (GTA) gene upstream from the 16S rRNA gene and a tRNA(Ile) (GAT) gene in the spacer region between the 16S and 23S rRNA genes. Comparisons of the SAY-MLO 16S rRNA nucleotide sequence with 16S rRNA sequences of other organisms indicated that the SAY-MLO is phylogenetically related most closely to other plant-pathogenic mycoplasmalike organisms, followed by Anaeroplasma species, Acholeplasma species, and some Mycoplasma species.

Identification and characterization of plasmids from the western aster yellows mycoplasmalike organism.

Supercoiled double-stranded DNA molecules (plasmids) were isolated from plants infected with three laboratory strains of western aster yellows mycoplasma-like organism (AY-MLO) by using cesium chloride-ethidium bromide density gradients. Southern blot analysis, using plasmids from the severe strain of AY-MLO (SAY-MLO) as the probe, identified at least four plasmids in celery, aster, and periwinkle plants and in Macrosteles severini leafhopper vectors infected with either the dwarf AY-MLO, Tulelake AY-MLO, or SAY-MLO strain. Plasmids were also detected in two California field isolates of AY-MLO but not in plants infected with the beet leafhopper-transmitted virescence agent, western X, or elm yellows MLOs. SAY-MLO plasmids were 5.2, 4.9, 3.4, and 1.7 kilobase pairs in size. Plasmids isolated from dwarf AY- and Tulelake AY-MLOs were 7.4, 5.1, 3.5, and 1.7 kilobase pairs in size. No evidence was obtained for integration of SAY-MLO plasmids into the MLO chromosome.

Isolation and characterization of full-length chromosomes from non-culturable plant-pathogenic Mycoplasma-like organisms.

We describe the isolation and characterization of full-length chromosomes from non-culturable plant-pathogenic, mycoplasma-like organisms (MLOs). MLO chromosomes are circular and their sizes (640 to 1185 kbp) are heterogeneous. Divergence in the range of chromosome sizes is apparent between MLOs in the two major MLO disease groups, and chromosome size polymorphism occurs among some related agents. MLO chromosome sizes overlap those of culturable mycoplasmas; consequently, small genome size alone cannot explain MLO non-culturability. Hybridization with cloned MLO-specific chromosomal and 16S rRNA probes detected two separate chromosomes in some MLO 'type' strains. Large DNA molecules that appear to be MLO megaplasmids were also demonstrated. The ability to characterize full-length chromosomes from virtually any non-culturable prokaryote should greatly facilitate the molecular and genetic analysis of these difficult bacteria.

Identification of Phytophthora citrophthora with Cloned DNA Probes.

Two different DNA fragments, one of 2.9 kilobases and the other of 5.1 kilobases, were cloned from Phytophthora citrophthora and showed no homology with DNA from plants and other related fungi. These DNA probes hybridized with DNA from 12 different P. citrophthora isolates obtained from a variety of hosts but did not hybridize with DNA from 6 P. citrophthora isolates obtained from cacao. Southern blot analysis revealed that the probes contained repetitive DNA, and restriction fragment length polymorphisms were identified among several P. citrophthora isolates. Of the isolates tested, two major groups were observed whose genetic similarity correlated with geographical distribution. One of the DNA probes was used to detect P. citrophthora growing from infected citrus roots incubated on semiselective medium. P. citrophthora was not detected by a hybridization assay of total DNA extracted directly from infected roots.

Mycoplasma gallisepticum species and strain-specific recombinant DNA probes.

Genomic libraries of vaccine (F-K810) and wild type (S6) Mycoplasma gallisepticum were constructed in Escherichia coli (strain JM83) using the plasmid vector pUC8. Recombinant clones were screened by colony, dot and Southern hybridisations using 32P-labelled genomic DNA from M. gallisepticum strains K810 and S6. Eight clones were identified which contained DNA sequences specific to M. gallisepticum and one clone was identified which contained a DNA fragment unique to the vaccine strain (F-K810) of M. gallisepticum. When labelled and used as a probe in dot hybridisation assays, one of the M. gallisepticum species-specific recombinant plasmids differentiated standard reference cultures, atypical strains and wild type isolates of M. gallisepticum from other avian Mycoplasma species. In similar assays, the plasmid containing vaccine strain-specific sequences differentiated vaccine strains of M. gallisepticum from several other wild type strains of M. gallisepticum. Recombinant DNA probes provided sensitive and specific detection of M. gallisepticum strains and the vaccine-specific probe will be useful for determining if the vaccine strain can replace wild type M. gallisepticum in commercial layer facilities.

Physical map of the Western X-disease phytoplasma chromosome.

A physical map of the chromosome of the western X-disease phytoplasma was constructed and represents the first physical map of a phytoplasma chromosome. The western X-disease phytoplasma is a nonculturable, plant-pathogenic member of the class Mollicutes and is the causal agent of a severe disease of fruit trees in North America. The map was generated by performing restriction digests of the chromosome and resolving the restriction fragments by pulsed-field gel electrophoresis. Southern blot analysis using cloned phytoplasma probes confirmed the arrangement of contiguous restriction fragments. The locations of 20 restriction sites for the enzymes SalI, XhoI, BssHII, RsrII, SmaI, and NotI were mapped on the chromosome, which is circular and comprises approximately 670 kb. The locations or the two rRNA operons and of four previously cloned fragments of chromosomal DNA were also placed on the map.

cDNA cloning and molecular characterization of cherry green ring mottle virus.

The complete nucleotide sequence of the cherry green ring mottle virus (CGRMV) genome was determined to be 8372 nt excluding a 3' poly(A) tail. Based on computer analysis and sequence comparison, five open reading frames (ORFs) were identified on the virion strand encoding: a putative RNA-dependent RNA polymerase, a triple gene block and a coat protein. Two other ORFs with Mr values over 10,000 and internal to the helicase and coat protein genes, but of unknown function, were also identified. Sequence and genome structure comparisons with other filamentous viruses indicated that CGRMV is most similar to apple stem pitting virus, some carlaviruses and potexviruses. However, it is different from members of any of these virus groups in regard to sequence homology and genome organization. A chimeric fusion coat protein was expressed in E. coli and antibodies specific for the CGRMV coat protein were raised in rabbits. The antibody was used in Western blot analyses to detect the CGRMV coat protein in infected cherry tissue.

Phytoplasma-specific PCR primers based on sequences of the 16S-23S rRNA spacer region.

In order to develop a diagnostic tool to identify phytoplasmas and classify them according to their phylogenetic group, we took advantage of the sequence diversity of the 16S-23S intergenic spacer regions (SRs) of phytoplasmas. Ten PCR primers were developed from the SR sequences and were shown to amplify in a group-specific fashion. For some groups of phytoplasmas, such as elm yellows, ash yellows, and pear decline, the SR primer was paired with a specific primer from within the 16S rRNA gene. Each of these primer pairs was specific for a specific phytoplasma group, and they did not produce PCR products of the correct size from any other phytoplasma group. One primer was designed to anneal within the conserved tRNA(Ile) and, when paired with a universal primer, amplified all phytoplasmas tested. None of the primers produced PCR amplification products of the correct size from healthy plant DNA. These primers can serve as effective tools for identifying particular phytoplasmas in field samples.