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Almond leaf scorch (ALS) disease has been present in California's almond-growing regions for over 60 years. This disease is caused by the bacterium Xylella fastidiosa and the pathogen is vectored by xylem-feeding sharpshooters and spittlebugs. Currently, there are no effective management techniques that prevent trees from becoming infected. Within affected orchards throughout California's Central Valley, disease incidence and the risk of tree-to-tree spread appears to be low. Consequently, the decision to remove or keep infected trees depends on lost productivity. We compared yield and vitality between infected and uninfected almond for cvs. Sonora and Nonpareil. Sonora was examined at three sites over 3 years and Nonpareil was examined at one site over 2 years. Yields of ALS-affected trees were significantly lower for both cultivars, although yield losses of Sonora were proportionally greater than those of Nonpareil. Yields of infected trees did not decline incrementally over years; rather, they fluctuated similarly to those of uninfected trees. In addition, no infected trees died during the course of the study. These results are in direct contrast to previous anecdotal reports which suggest that yields of infected trees incrementally decline and infected trees eventually die. A simple economic model was developed to determine conditions under which rouging infected trees would increase returns. Based on the model, orchard age, yield loss due to infection, and the value of a maximally producing almond tree should be considered when deciding to remove ALS-affected trees.

"Candidatus Liberibacter asiaticus" (CaLas) is associated with citrus Huanglongbing (HLB, yellow shoot disease), which is highly destructive to world citrus production. Understanding the relationships of CaLas isolates from different geographical regions is important for HLB research and development of disease management strategies. In this study, 301 CaLas isolates [85 Brazil, 132 China, and 84 U.S. (83 Florida and 1 California)] were collected, and genomic variations among them were evaluated based on the analyses of two genomic loci: trn1, characteristic of variable tandem repeat numbers (TRNs), and snp1, characteristic of single nucleotide polymorphisms (SNPs). Locus trn1 revealed the homogeneity of all Brazilian isolates, and locus snp1 revealed the homogeneity of most Florida isolates. When the two loci were analyzed simultaneously, i.e., double-locus (DL) analyses, CaLas isolates were clustered mostly according to geographical origins. DL genotype 1 included 97 % of the Chinese isolates, DL genotype 2 included all Brazilian isolates, and DL genotype 3 included 93 % of the U.S. isolates. DL analyses successfully revealed inter-continental overlapping or movement pattern of CaLas isolates. The isolate recently found in California belonged to Asiatic DL genotype 1.

Zebra chip disease (ZCD) of potato (Solanum tuberosum L.) has caused significant economic losses to the potato industry in the USA. ‘Candidatus Liberibacter solanacearum’ Liefting et al. has been associated with ZCD. This non-culturable bacterium dwells within the phloem of plants where other endophytic bacteria may also live. Knowledge concerning phloem colonization by ‘Ca. L. solanacearum’ and other endophytic bacteria may provide a better understanding about disease biology and could facilitate the development of novel disease management strategies. In this study, endophytic bacteria in potato tubers afflicted with ZCD were evaluated by scanning electron microscopy (SEM), in vitro cultivation, and PCR analyses. Bacillus, coccus, and filamentous bacterial cells were observed by SEM in phloem tissue of ZCD-diseased tubers, but were absent in healthy potato tubers. Fifty-one bacterial isolates were obtained in vitro from ZCD-affected tubers and 34 isolates were from non-ZCD tubers. Comparison of ...

Little is known about specific host chemistry effects on zebra chip disease symptom development in potatoes (Solanum tuberosum). This research compared chemical profiles and defense-related enzyme levels between non-symptomatic and zebra chip-symptomatic potato tubers. Levels of phenolics, five amino acids, peroxidases, polyphenol oxidases, chitinases, and β-1,3-glucanases were greater in symptomatic tubers than non-symptomatic tubers, and many of these compounds also were positively correlated with zebra chip disease severity. ‘Candidatus Liberibacter solanacearum’ was consistently present in symptomatic tubers. However, the lack of associations between titers and tuber chemistry suggests a complicated relationship between this bacterium and zebra chip symptoms.

‘Candidatus Liberibacter solanacearum’ is associated with the Zebra Chip (ZC) disorder of potatoes. A panel of eight simple sequence repeat (SSR) markers was developed and used to genetically characterize ‘Ca. L. solanacearum’ strains obtained from ZC-affected potato plants in the United States and Mexico. The multilocus SSR markers in this study effectively differentiated genotypes and estimated genetic diversity of ‘Ca. L. solanacearum’ strains. Genotype assignment analyses identified two major lineages of ‘Ca. L. solanacearum’ in the North American populations while only one lineage type was identified in Mexican population. No clear genetic structure was found among haplotypes based on geographical proximity or host. The high resolution power of the SSR marker system developed in this study provides a useful tool for genotyping closely related strains and tracking sources of the pathogen. Genotype information combined with epidemiological data will advance knowledge of ZC disease and will facilitate development of effective disease management.

Xylella fastidiosa ( Xf ) is the bacterial causal agent of Pierce’s disease (PD) as well as other economically important diseases in a number of agronomic, horticultural and ornamental plants. The objective of this research was to tentatively identify proteins that are differentially expressed in grapevines and involved in disease development or defense responses to Xf -inoculation. We comparatively analyzed proteins differentially expressed in Xf- inoculated grape stems using a pair of siblings of 9621-67 (highly susceptible) and 9621-94 (highly resistant) from a cross of Vitis rupestris × Vitis arizonica . Total proteins were extracted from the stems of uninoculated controls and Xf- inoculated plants at 1, 6, and 12 weeks after inoculation, separated by a 2D-PAGE system, and spots representing differentially expressed proteins were analyzed and tentatively identified using LC/MS/MS. Protein identification was performed using BLASTp and tBLASTn against NCBI non-redundant protein databases and EST databases, respectively. Ten tentatively identified proteins were differentially expressed at different time points after inoculation. A thaumatin-like protein and the pathogenesis-related protein 10 from both genotypes, and the 40S ribosomal protein S25 from the susceptible genotype were up-regulated in response to Xf -inoculation. Furthermore, the expression of the thaumatin-like protein increased sharply 12 weeks post-inoculation in the PD-resistant genotype only. Three heat shock proteins, 17.9 kDa class II, protein 18 and 21 were highly expressed in healthy tissues compared with those in tissues infected with Xf , and heat shock protein 21 was not detectable in the Xf -inoculated PD-susceptible genotype. In addition, a down-regulated putative ripening related protein was found in the Xf -inoculated PD-susceptible genotype. Glycoprotein and formate dehydrogenase were identified in the PD-resistant genotype and their expression was constant during plant development. A putative GTP-binding protein was down-regulated in the PD-susceptible genotype. Our results revealed that differential expression of proteins in response to Xf -inoculation was genotype and tissue development stage dependent. The specific roles of these candidate proteins in alleviation or aggravation of this disease are under investigation. The information obtained in this study will aid in the understanding of the mechanisms related to the host–pathogen interactions involved in PD.

Zebra chip (ZC) is an established and highly destructive disease of potato (Solanum tuberosum L.) that occurs in several southwestern states of the United States, Mexico, Central America, and New Zealand. The causal agent for this disease has not been identified. However, the bacterium “Candidatus Liberibacter solanacearum” and the potato psyllid, Bactericera cockerelli (Sulc), its insect vector, are associated with the disease. Tubers from ZC-affected potato plants exhibit dramatic browning of vascular tissue concomitant with “necrotic flecking” both of which can affect the entire tuber. Upon frying, these tubers develop a characteristic striped pattern of discoloration rendering them unmarketable. These characteristic ZC symptoms in the tubers have been suggested to be associated with general cell death, though no evidence to confirm this hypothesis has been shown. In order to determine if cell death is associated with ZC disease, a series of experiments were undertaken. Cell death was initially quantified by comparing cellular ion leakage from ZC-affected and ZC-free tubers. Levels of ion leakage were found to be significantly higher in ZC-affected tubers compared to ZC-free tubers. To examine further the association of cell death with ZC disease, ZC-affected and ZC-free tubers were compared using classical histochemical staining methods in conjunction with optical microscopy, which revealed layers of dead cells surrounding numerous, small, irregularly-shaped lesions throughout the parenchymatic medullary region, vascular ring and cortex of ZC-affected tubers. This cell death was confirmed using high-resolution, field-emission scanning electron microscopy (FE-SEM) of fresh-cut tuber tissue.

article i nfo An ultra-sensitive and quantitative diagnostic system by combining nested PCR and TaqMan® PCR in a single tube was developed for detection of "Candidatus Liberibacter asiaticus". The procedure involves two PCR steps using the species-specific outer and inner primer pairs. Different annealing temperatures allow both the first and the second rounds of PCR to be performed sequentially in the same closed tube. The first PCR with outer primers was performed at a higher annealing temperature and with limited amount of primers to prevent interference with the inner primers during the second round of PCR. The specificity of the dual primer TaqMan® is high because the fluorescent signal can only be generated from the TaqMan® probes that are homologous to the product amplified by the outer and inner primers. This new detection system can reliably detect as few as single copies of target DNA. The sensitivity of the dual primer system is comparable to the conventional two-tube nested PCR, but it eliminates the potential risk of cross contamination commonly associated with conventional nested PCR. This one-tube dual primer TaqMan® PCR method is gel- free with reduced handling time and is cost effective. At the same time, this system provides significantly increased sensitivity, improved reliability and high through-put capability suitable for routine, large scale diagnoses of clinical plant tissue and insect samples. The technique described here is generic and can be applied to the detection of other plant pathogenic bacteria. Published by Elsevier B.V.

Almond leaf scorch disease (ALSD) caused by Xylella fastidiosa is potentially a serious threat to the almond industry in San Joaquin Valley of California. Knowledge of X. fastidiosa behaviour in the plant host under field conditions is important for disease control and this issue is being addressed in this project. Occurrence of ALSD is strongly influenced by environmental factors. In 2006, the earliest leaf scorching symptoms were observed in June, whereas in 2007, the earliest occurrence of leaf scorching symptoms was in July, a delay of 1 month. In both years, PCR detected X. fastidiosa 1 month before of symptom expression. PCR was slightly more sensitive than cultivation method for early bacterial detection. However, uneven bacterial distribution and random sampling errors may have contributed to the differences among the assays. Correlation between cultivation and PCR detection was greater than 90%. During the processing of a large number of samples, we noticed occasional failures in PCR amplifications of some samples, interfering result interpretation. We developed an array-PCR protocol using primers from seven housekeeping genes to correct the deficiency.

Pierce’s disease (PD) of grapevines is caused by the xylem-limited bacterium Xylella fastidiosa (Xf). All Vitis vinifera-based cultivars are susceptible to Xf infection; however, many grape species from the southern United States (such as V. arizonica, V. shuttleworthii, V. simpsonii, V. smalliana, and Muscadinia rotundifolia) are resistant. In this study, the effects of xylem sap from PD-resistant and PD-susceptible grapes, as well as several free cell wall constituents, on in vitro bacterial growth, biofilm formation, and cellular aggregation were investigated. Media containing xylem sap from PD-susceptible plants provided better support for bacterial growth and biofilm formation than media supplemented with xylem sap from PD-resistant plants. Culturing Xf on media containing various purified cell wall constituents demonstrated that CM-cellulose, xylan, β-D-glucan, k-carrageenan, cello-oligosaccharide and laminarin promoted bacterial growth whereas lichenan suppressed growth. However, only laminarin, xylan, and k-carrageenan promoted biofilm formation in vitro. Lichenan, oligosaccharide, k-carrageenan, laminarin, xylan and β-D-glucan all significantly decreased Xf cellular aggregation in vitro. This study suggests that differences in xylem sap composition and cell wall properties among PD-resistant and PD-susceptible grapes may affect characteristics of Xf growth, biofilm formation and cellular aggregation involved in pathogenesis.

Citrus huanglongbing (HLB) or yellow shoot disease (i.e., greening disease) is highly destructive to citrus production worldwide. Understanding the etiology of HLB is critical for managing the disease. HLB is currently associated with infection by ‘Candidatus Liberibacter spp.’ around the world, including China. However, Koch's postulates have not been fulfilled. In addition, other plant pathogens also may be involved in HLB. In a survey performed in Guangdong Province, P. R. China in 2006 and 2007, 141 citrus samples showing typical symptoms of HLB from 11 different cities were collected. Polymerase chain reaction (PCR) using phytoplasma-specific primer sets fU5/rU3 nested with primer set P1/P7 identified 110 (78.0%) positive samples. A 1,785-bp amplicon was obtained with primer set P1/P7. Analysis showed a 100% identity of this sequence in the region of 16S rDNA and 16S-23S rRNA intergenic transcribed spacer to three strains of ‘Candidatus Phytoplasma asteris’ (onion yellows [Japan], aster yellows ‘watercress’ [Hawaii], and valeriana yellows [Lithuania]). Of the 141 samples, 89 (63.1%) samples were positive for ‘Ca. Liberibacter asiaticus’. When mixed infection was considered, 69 (48.9%) samples were positive for both ‘Ca. P. asteris’ and ‘Ca. L. asiaticus’. Transmission electron microscopy (TEM) showed low titers of both walled and wall-less bodies in the phloem sieve tubes of HLB citrus. When transmission from symptomatic citrus to periwinkle (Catharanthus roseus) via dodder (Cuscuta campestris) was conducted, both phytoplasma and ‘Ca. L. asiaticus’ were detected from the affected periwinkle. In addition to yellowing/mottling, the infected periwinkle showed symptoms of virescence and phyllody which are commonly associated with phytoplasmal diseases. TEM analysis of affected periwinkle revealed pleomorphic and wall-less organisms, characteristic of phytoplasmas, filling some phloem sieve tubes. In contrast, walled bacteria were at low titer. This study showed that in addition to ‘Ca. L. asiaticus’, a phytoplasma related to ‘Ca. P. asteris’ could also be detected in citrus showing HLB symptoms in Guangdong.

The grass sharpshooter, Draeculacephala minerva Ball (Hemiptera: Cicadellidae), is a very common and often abundant grass-feeding leafhopper in California. Its population dynamics and ability to transmit Xylella fastidiosa were monitored over a 2-yr period in California's San Joaquin Valley. Collections of individuals from natural populations in irrigated pastures and alfalfa, Medicago savita L. fields adjacent to X. fastidiosa-infected almond (Prunus spp.) orchards indicated the occurrence of three discrete generations per year that peaked during the summer. Population densities varied significantly among experimental field survey sites. Insects captured on intercepting mesh traps, yellow sticky cards, and UV-light traps indicated local movement of these insects into and surrounding X. fastidiosa-infected, almond orchards. Local movement and seasonal transmission of X. fastidiosa from infected almonds to Catharanthus roseus (L.) G. Don indicated that this insect may be partly responsible for the slow spread of almond leaf scorch now recently observed in California's San Joaquin Valley.

Deng, X., Chen, J., Feng, Z., Shan, Z., Guo, H., Zhu, J., Li, H., and Civerolo, E. L. 2008. Identification and characterization of the Huanglongbing bacterium in pummelo from multiple locations in Guangdong, P. R. China. Plant Dis. 92:513-518. Huanglongbing (HLB, yellow shoot disease, ex. citrus greening disease), caused by Candidatus Liberibacter spp., is highly destructive to citrus production in Asia, Africa, and South America. Although primarily affecting sweet orange and mandarin, HLB has long been observed in pummelo in Guangdong Province, People’s Republic of China; however, the disease in pummelo has received little research attention. Accordingly, it remains unclear how closely related the strains of Ca. Liberibacter in pummelo are to those in other citrus species. In this study, the loci of 16S rDNA, rplAJ (β-operon of ribosomal protein), and an outer membrane protein (omp) gene were analyzed and characterized among strains of Ca. Liberibacter in pummelo samples from six different locations in Guangdong. Sequence comparisons indicated that ‘Candidatus Liberibacter asiaticus’, but not ‘Ca. Liberibacter africanus’ or ‘Ca. Liberibacter americanus’, was exclusively associated with HLB symptoms in pummelo. The pummelo strains of ‘Ca. Liberibacter asiaticus’ from Guangdong were highly homogeneous. Analyses of single-nucleotide polymorphisms in the omp locus showed that the Guangdong pummelo strains grouped with ‘Ca. Liberibacter asiaticus’ strains from Thailand, Nepal, and an unspecified location in China but differed from the Philippine and China-Behai strains. Based on the sequence homogeneity at the omp locus, the history of pummelo culture and the means by which HLB is known to be spread, we believe that, likely, the pummelo strain of ‘Ca. Liberibacter asiaticus’ recently was spread to pummelo in the study areas from infected sweet orange or mandarin trees by insect vectors or by propagation of pummelo infected elsewhere. Additional keywords: Citrus grandis, β-operon

Xylella fastidiosa, the causal agent of almond leaf scorch disease (ALSD), is currently re-emerging as a serious concern in California. Efficient pathogen detection is critical for ALSD epidemiological studies, particularly when a large sample size is involved. We here report a PCR procedure to detect X. fastidiosa directly from infected almond tissue without the laborious DNA extraction. Plant samples were prepared by freeze-drying and pulverized. Appropriate dilutions of the pulverized freeze-dried tissue (PFT) were determined to minimize the effect of enzyme inhibitors from plant tissue and retain PCR detection of X. fastdiosa cells at a single digit number level. This PFT-PCR procedure was evaluated by comparing to the in vitro cultivation method using 102 symptomatic samples and resulted in a predictive value of 90.8%. PFT-PCR was further applied to monitor the seasonal occurrence of X. fastidiosa from four selected almond trees in two orchards in 2005. The results matched with those of the cultivation method at 92.3%. Considering the simplicity and reliability, we conclude that PFT-PCR is a valuable option for high throughput rapid detection of X. fastidiosa.

An effective PCR-based genomic walking approach is described to discover previously unknown flanking genomic DNA sequences from Candidatus Liberibacter asiaticus, an unculturable, phloem-limited bacterium. Using this technique, 8564 bp of new DNA sequences were obtained from three genomic loci; tufB-secE-nusG-rplKAJL-rpoBC gene cluster, omp gene (outer membrane protein, Omp) and 16/23S rRNA gene in Ca. L. asiaticus. These, together with publicly available Ca. Liberibacter sequences, are clustered into five contigs and two singlets representing 24,477 non-redundant base pairs. BLAST annotation predicts 12 full-length genes, two partial genes and one pseudogene among these sequences. The sequences obtained in this study provide new genome information about Ca. Liberibacter that will facilitate development of new genome-based detection tools. The technique described here can also be employed to acquire new genomic information for other unculturable or fastidious organisms for which available sequences are limited or for filling sequence gaps between known flanking genomic DNA sequences.

Readily transformable Nicotiana tabacum cv. SR1 (Petite Havana) was evaluated as a host for the bioassay of Xylella fastidiosa strains. Plant growing conditions and inoculation methods were optimized to enhance symptom expression 4 to 6 weeks post inoculation. Tobacco plants were inoculated with X. fastidiosa strains associated with almond leaf scorch disease (ALSD) and Pierce's disease (PD) of grapevine in California. All PD strains and the ALSD strain Dixon caused characteristic leaf scorch symptoms, whereas two other ALSD-associated strains (M12 and M23) caused severe leaf chlorosis followed by necrosis, leaf death, and drooping of older leaves. Symptoms began to develop 10 to 14 days post inoculation and proceeded to resemble those of X. fastidiosa-infected grape and almond. The presence of X. fastidiosa in affected plants was confirmed by reisolation of the pathogen, enzyme-linked immunosorbent assay, quantitative polymerase chain reaction (QPCR), and observation of X. fastidiosa cells by transmission and scanning electron microscopy, as well as by confocal laser scanning microscopy, in the xylem cells of inoculated plants. The pathogenicity of selected reisolated strains was confirmed by inoculation of grape plants in the greenhouse. The average levels of X. fastidiosa cells/g of tissue, estimated by QPCR, were higher for PD strains than for ALSD strains and reflected the relative titers of these strains in economic hosts. No symptoms were observed and bacteria were not detected in untreated tobacco or in tobacco inoculated with Xanthomonas campestris pv. campestris or water. Symptoms induced by Xylella fastidiosa in this bioassay were fully expressed within 2 months following inoculation. The described bioassay, under optimized environmental conditions, provides a useful system for studying X. fastidiosa strains (e.g., confirmation of pathogenicity and differentiation of PD and ALSD pathotypes) and for investigating X. fastidiosa–host interactions. N. tabacum cv. SR1 tobacco was a better bioassay host for X. fastidiosa than N. tabacum cvs. Havana, RP1, and TNN described previously.

Xylella fastidiosa is the causal agent of almond leaf scorch disease (ALSD), which is currently reemerging in California as a potential threat to almond (Prunus dulcis) production. We previously reported the presence of different colony morphotypes of X. fastidiosa ALSD strains on periwinkle wilt medium solidified with Gelrite and their association with genotypes or pathotypes after a low number of serial transfers. The morphotypes could be differentiated by single nucleotide polymorphisms and were classified as A- and G-genotypes. The stability of colony morphology was not studied. Yet, it is an important issue in the bacterial characterization. In this project, we evaluated the variations of colony morphology through 14 subculture passages. The G-genotype strains were consistently (>99%) associated with smooth colony morphotypes. Similarly, A-genotype strains were consistently associated with rough colony morphotypes. Rough A-genotype strains reversed to smooth colony variants at a low frequency suggest...

We report here the complete genome sequence of “ Candidatus Liberibacter africanus” strain PTSAPSY. The 1,192,232-bp genome with 34.5% G+C content comprises 1,017 open reading frames, 44 tRNAs, and three complete rRNAs in a circular chromosome.

Lin, H., Islam, M. S., Rosa, J. C. C.-L., Civerolo, E. L., and Groves, R. L. 2015. Population structure of Xylella fastidiosa associated with almond leaf scorch disease in the San Joaquin Valley of California. Phytopathology 105:825-832. Xylella fastidiosa causes disease in many commercial crops, including almond leaf scorch (ALS) disease in susceptible almond (Prunus dulcis). In this study, genetic diversity and population structure of X. fastidiosa associated with ALS disease were evaluated. Isolates obtained from two almond orchards in Fresno and Kern County in the San Joaquin Valley of California were analyzed for two successive years. Multilocus simple-sequence repeat (SSR) analysis revealed two major genetic clusters that were associated with two host cultivars, ‘Sonora’ and ‘Nonpareil’, respectively, regardless of the year of study or location of the orchard. These relationships suggest that host cultivar selection and adaptation are major driving forces shaping ALS X. fastidiosa population structure in the San Joaquin Valley. This finding will provide insight into understanding pathogen adaptation and host selection in the context of ALS disease dynamics. Additional keywords: annual variation.

Xylella fastidiosa is an important pathogen of many commercial crops. Detection of X. fastidiosa is difficult due to low concentrations of the bacteria in insects and asymptomatic plant tissue, and non-uniform distribution in infected plants. A dual purpose conventional PCR and quantitative PCR (TaqMan™) system was developed for the generic detection of X. fastidiosa strains. Primers HL5 and HL6, designed to amplify a unique region common to the sequenced genomes of four Xylella strains, amplified a 221 bp fragment from strains associated with Pierce’s disease of grapes, almond leaf scorch, and oleander leaf scorch disease and from DNA from an Xf strain associated with citrus variegated chlorosis. Standard curves were obtained using concentrations of Xylella ranging from 5 to 105 cells per reaction in water and grape extracts and 10–105 cells in insect DNA. Regression curves were similar, with correlation coefficients of r 2 > 0.97. In quantitative PCR, Ct values ranged between 20 and 36 cycles for 5–105 bacterial cells per reaction. No amplicons were obtained with several non-Xf bacterial strains tested including related plant pathogenic, grape endophytic bacteria and endosymbiotic bacteria isolated from glassy-winged sharpshooters. The method was evaluated for clinical diagnosis of Xf in grapes, almonds and insect vectors. The procedure described is reliable for detection of the pathogen with a high degree of sensitivity and specificity.

A genome-wide search was performed to identify simple sequence repeat (SSR) loci among the available sequence databases from four strains of Xylella fastidiosa (strains causing Pierce's disease, citrus variegated chlorosis, almond leaf scorch, and oleander leaf scorch). Thirty-four SSR loci were selected for SSR primer design and were validated in PCR experiments. These multilocus SSR primers, distributed across the X. fastidiosa genome, clearly differentiated and clustered X. fastidiosa strains collected from grape, almond, citrus, and oleander. They are well suited for differentiating strains and studying X. fastidiosa epidemiology and population genetics.

The gamma-proteobacterium Xanthomonadales groups two closely related genera of plant pathogens, Xanthomonas and Xylella. Whole genome sequencing and comparative analyses disclosed a high degree of identity and co-linearity of the chromosome backbone between species and strains. Differences observed are usually clustered into genomic islands, most of which are delimited by genetic mobile elements. Focus is given in this paper to describe which groups of mobile elements are found and what is the relative contribution of these elements to Xanthomonas and Xylella genomes. Insertion sequence (IS) elements have invaded the Xanthomonas genome several times, whereas Xylella is rich in phage-related regions. Also, different plasmids are found inhabiting the bacterial cells studied here. Altogether, these results suggest that the integrative elements such as phages and transposable elements as well as the episomal plasmids are important drivers of the genome evolution of this important group of plant pathogens.

Groves, R. L., Chen, J., Civerolo, E. L., Freeman, M. W., and Viveros, M. A. 2005. Spatial analysis of almond leaf scorch disease in the San Joaquin Valley of California: Factors affecting pathogen distribution and spread. Plant Dis. 89:581-589. Almond leaf scorch (ALS) disease has emerged as a serious threat to almond (Prunus amygdalus) production areas throughout California’s San Joaquin Valley. This disease is caused by the xylem-limited bacterium Xylella fastidiosa, and this pathogen is transmitted by xylophagous insects including sharpshooter leafhoppers (Hemiptera: Cicadellidae) and spittlebugs (Hemiptera: Cercopidae). Among four orchards surveyed, enzyme-linked immunosorbent assay (ELISA) and bacterial isolation followed by polymerase chain reaction (PCR) were equally effective in detecting X. fastidiosa from ALS-symptomatic trees. Disease incidence varied among almond cultivars in each orchard, with the highest mean incidence and most severe symptoms frequently encountered in ‘Sonora’. X. fastidiosa isolates consisted of mixtures of grape or “G-genotype” and almond or “A-genotype” strains present in surveyed orchards. The X. fastidiosa G-genotypes characterized from each orchard were associated with the most severely affected ‘Sonora’ trees in three of the four orchards. Both ordinary runs and simple randomization analyses revealed aggregations of ALS in three of the four orchards. Clusters of ALSaffected trees frequently occurred in the outermost orchard rows. Plots of semivariance in ALS incidence over distance varied in shape and magnitude among cultivars. Semivariance increased over distance in ‘Sonora’ and ‘Carmel’, indicating spatial dependence or aggregations of incidence best fit by a combination of spherical and linear models. These results document both random and aggregate patterns of ALS spatial distribution in selected orchards and further illustrate how cultivar susceptibility influences the distribution patterns of ALS incidence. Following the recent introduction and establishment of the glassy-winged sharpshooter, Homalodisca coagulata, the impact upon the epidemiology and spread of ALS is unknown. Additional keywords: Pierce’s disease

Xylella fastidiosa causes many important plant diseases including Pierce's disease (PD) in grape and almond leaf scorch disease (ALSD). DNA-based methodologies, such as randomly amplified polymorphic DNA (RAPD) analysis, have been playing key roles in genetic information collection of the bacterium. This study further analyzed the nucleotide sequences of selected RAPDs from X. fastidiosa strains in conjunction with the available genome sequence databases and unveiled several previously unknown novel genetic traits. These include a sequence highly similar to those in the phage family of Podoviridae. Genome comparisons among X. fastidiosa strains suggested that the "phage" is currently active. Two other RAPDs were also related to horizontal gene transfer: one was part of a broadly distributed cryptic plasmid and the other was associated with conjugal transfer. One RAPD inferred a genomic rearrangement event among X. fastidiosa PD strains and another identified a single nucleotide polymorphism of evolutionary value.

Submitted by Guilherme Lemeszenski (guilherme@nead.unesp.br) on 2014-02-26T17:15:29Z No. of bitstreams: 1 WOS000180834300035.pdf: 1288581 bytes, checksum: c80b0ab1625eeba1cd15e9da6e465b26 (MD5) Made available in DSpace on 2014-02-26T17:15:29Z (GMT). No. of bitstreams: 1 WOS000180834300035.pdf: 1288581 bytes, checksum: c80b0ab1625eeba1cd15e9da6e465b26 (MD5) Previous issue date: 2003-02-01 Submitted by Vitor Silverio Rodrigues (vitorsrodrigues@reitoria.unesp.br) on 2014-05-20T15:25:38Z No. of bitstreams: 1 WOS000180834300035.pdf: 1288581 bytes, checksum: c80b0ab1625eeba1cd15e9da6e465b26 (MD5) Made available in DSpace on 2014-05-20T15:25:38Z (GMT). No. of bitstreams: 1 WOS000180834300035.pdf: 1288581 bytes, checksum: c80b0ab1625eeba1cd15e9da6e465b26 (MD5) Previous issue date: 2003-02-01 Xylella fastidiosa is a xylem-dwelling, insect-transmitted, gamma-proteobacterium that causes diseases in many plants, including grapevine, citrus, periwinkle, almond, oleander, and coffee. X. fastidiosa has an unusually broad host range, has an extensive geographical distribution throughout the American continent, and induces diverse disease phenotypes. Previous molecular analyses indicated three distinct groups of X.fastidiosa isolates that were expected to be genetically divergent. Here we report the genome sequence of X. fastidiosa (Temecula strain), isolated from a naturally infected grapevine with Pierce's disease (PD) in a wine-grape-growing region of California. Comparative analyses with a previously sequenced X.fastidiosa strain responsible for citrus variegated chlorosis (CVC) revealed that 98% of the PD X.fastidiosa Temecula genes are shared with the CVC X. fastidiosa strain 9a5c genes. Furthermore, the average amino acid identity of the open reading frames in the strains is 95.7%. Genomic differences are limited to phage-associated chromosomal rearrangements and deletions that also account for the strain-specific genes present in each genome. Genomic islands, one in each genome, were identified, and their presence in other X.fastidiosa strains was analyzed. We conclude that these two organisms have identical metabolic functions and are likely to use a common set of genes in plant colonization and pathogenesis, permitting convergence of functional genomic strategies. Univ São Paulo, Inst Biociencias, Dept Bot, BR-05508900 São Paulo, Brazil Univ São Paulo, Inst Quim, BR-05508900 São Paulo, Brazil Univ São Paulo, Inst Ciências Biomed, BR-05508900 São Paulo, Brazil Univ São Paulo, Escola Super Agr Luiz de Queiroz, BR-13418900 Piracicaba, Brazil Univ São Paulo, Ctr Energia Nucl Agr, BR-13400970 Piracicaba, Brazil Inst Agron Campinas, Ctr Citricultura Sylio Moreira, BR-13490970 Cordeiropolis, Brazil Univ Estadual Paulista, Fac Ciências Agr & Vet, BR-14884900 Jaboticabal, Brazil Univ Estadual Campinas, Ctr Biol Mol & Engn Genet, BR-13083970 Campinas, SP, Brazil Univ Estadual Campinas, Inst Biol, BR-13083970 Campinas, SP, Brazil Univ Estadual Campinas, Inst Comp, BR-13083970 Campinas, SP, Brazil Univ São Paulo, Fac Ciências Farmaceut, BR-14040901 Ribeirao Preto, Brazil Univ São Paulo, Fac Filosofia Ciências & Letras, BR-14040901 Ribeirao Preto, Brazil Inst Ludwig Pesquisa Canc, BR-01509010 São Paulo, Brazil Univ Mogi das Cruzes, Nucleo Integrado Biotecnol, BR-08780911 Mogi das Cruzes, Brazil Inst Agron Campinas, BR-13001970 Campinas, SP, Brazil Inst Biol, BR-04014002 São Paulo, Brazil Univ Estadual Paulista, Fac Med Vet & Zootecnia, BR-18603970 Botucatu, SP, Brazil Univ Estadual Paulista, Fac Ciências Agron, BR-18603970 Botucatu, SP, Brazil Univ Estadual Paulista, Inst Biociencias, BR-18618000 Botucatu, SP, Brazil Univ Fed Sao Carlos, BR-13600970 São Paulo, Brazil Univ Fed Mato Grosso do Sul, Dept Comp & Estatist, BR-79070900 Campo Grande, MS, Brazil USDA ARS, Parlier, CA 93648 USA Univ Estadual Paulista, Fac Ciências Agr & Vet, BR-14884900 Jaboticabal, Brazil Univ Estadual Paulista, Fac Med Vet & Zootecnia, BR-18603970 Botucatu, SP, Brazil Univ Estadual Paulista, Fac Ciências Agron, BR-18603970 Botucatu, SP, Brazil Univ Estadual Paulista, Inst Biociencias, BR-18618000 Botucatu, SP, Brazil

SUMMARY Agrobacterium tumefaciens tumorigenesis is initiated by the horizontal transfer of a suite of oncogenes that alter hormone synthesis and sensitivity in infected plant cells. Transgenic plants silenced for the iaaM and ipt oncogenes are highly recalcitrant to tumorigenesis, and present a unique resource to elucidate fundamental questions related to Agrobacterium biology and post-transcriptional gene silencing (PTGS). The oncogene-silenced transgenic tomato line 01/6 was used to characterize A. tumefaciens growth in planta and to screen for iaaM and ipt sequence variants. Even in the absence of macroscopic and microscopic indications of tumorigenesis, A. tumefaciens is capable of long-term survival in the hypocotyl tissues of the 01/6 line. A. tumefaciens growth, however, is significantly reduced in the 01/6 line, with populations decreased by 96% relative to wild-type at 52 days post-inoculation. In addition, the 01/6 line displayed suppression of tumorigenesis against all 35 tested strains of A. tumefaciens. High target homology is an absolute requirement of PTGS, therefore this result suggests that regions of the iaaM and ipt oncogenes are very highly conserved across most A. tumefaciens strains. Finally, graft transmissibility of oncogene silencing was assessed by grafting various non-silenced tomato genotypes on to the 01/6 line. Phenotypic and molecular evidence (tumorigenesis and absence of small interfering RNAs, respectively) suggest that oncogene silencing is not graft-transmissible, at least to wild-type and antisense iaaM-over-expressing genotypes.

The pathogenic ‘Candidatus Liberibacter’ species have caused tremendous economic losses to agricultural crops. Since these bacteria are, generally, not yet unculturable, information regarding genetics and pathogenesis of the pathogens is limited. Comparative analyses of multiple Liberibacter genomes provide unprecedented insights into the evolutionary history, phylogenetic diversity, and metabolomic capacities among pathogenic bacteria. In addition, genomic analyses of plant disease-associated Liberibacters have led to the identification of potential or putative pathogenicity and virulence factors, as well as biomarkers associated with Liberibacter–host plant interactions. The availability of specific biomarkers associated with Liberibacter–host plant interactions will aid in the development of improved, reliable diagnostic protocols for early (i.e., presymptomatic), rapid Liberibacter detection as part of disease management strategies. Since no host resistance has been found in Liberibacter-associated diseases, knowledge of specific Liberibacter genes and their products associated with pathogenicity and virulence, as well as interactions with psyllid vectors, will lead to the identification of potential targets for mitigating Liberibacter acquisition and transmission by psyllids, host plant infection, and disease development. Finally, genomics-based research will facilitate improved understanding of the complex and diverse mechanisms of Liberibacter–plant interactions.

Crown gall disease, caused by the soil bacterium Agrobacterium tumefaciens , results in significant economic losses in perennial crops worldwide. A. tumefaciens is one of the few organisms with a well characterized horizontal gene transfer system, possessing a suite of oncogenes that, when integrated into the plant genome, orchestrate de novo auxin and cytokinin biosynthesis to generate tumors. Specifically, the iaaM and ipt oncogenes, which show ≈90% DNA sequence identity across studied A. tumefaciens strains, are required for tumor formation. By expressing two self-complementary RNA constructions designed to initiate RNA interference (RNAi) of iaaM and ipt , we generated transgenic Arabidopsis thaliana and Lycopersicon esculentum plants that are highly resistant to crown gall disease development. In in vitro root inoculation bioassays with two biovar I strains of A. tumefaciens , transgenic Arabidopsis lines averaged 0.0–1.5% tumorigenesis, whereas wild-type controls averaged 97.5% tumorigenesis. Similarly, several transformed tomato lines that were challenged by stem inoculation with three biovar I strains, one biovar II strain, and one biovar III strain of A. tumefaciens displayed between 0.0% and 24.2% tumorigenesis, whereas controls averaged 100% tumorigenesis. This mechanism of resistance, which is based on mRNA sequence homology rather than the highly specific receptor–ligand binding interactions characteristic of traditional plant resistance genes, should be highly durable. If successful and durable under field conditions, RNAi-mediated oncogene silencing may find broad applicability in the improvement of tree crop and ornamental rootstocks.

We report here the complete genome sequence of “ Candidatus Liberibacter asiaticus” (strain Guangxi-1). The 1,268,237-bp genome with a 36.5% G+C content comprises 1,141 open reading frames, 44 tRNAs, and 3 complete rRNAs in a circular chromosome.

We report here the draft genome sequence of “ Candidatus Liberibacter americanus” strain PW_SP. The 1,176,071-bp genome, with 31.6% G+C content, comprises 948 open reading frames, 38 tRNAs, and three complete rRNAs.

Background Huanglongbing (HLB) is a highly destructive citrus disease which threatens citrus production worldwide and ‘Candidatus Liberibacter asiaticus’ (Las), a non-culturable phloem-limited bacterium, is an associated causal agent of the disease. To better understand the physiological and molecular processes involved in host responses to Las, 2-DE and mass spectrometry analyses, as well as ICP spectroscopy analysis were employed to elucidate the global protein expression profiles and nutrient concentrations in leaves of Las-infected grapefruit plants at pre-symptomatic or symptomatic stages for HLB. Results This study identified 123 protein spots out of 191 spots that showed significant changes in the leaves of grapefruit plants in response to Las infection and all identified spots matched to 69 unique proteins/peptides. A down-regulation of 56 proteins including those associated with photosynthesis, protein synthesis, and metabolism was correlated with significant reductions in the concentrations of Ca, Mg, Fe, Zn, Mn, and Cu in leaves of grapefruit plants in response to Las infection, particularly in symptomatic plants. Oxygen-evolving enhancer (OEE) proteins, a PSI 9 kDa protein, and a Btf3-like protein were among a small group of proteins that were down-regulated in both pre-symptomatic and symptomatic plants in response to Las infection. Furthermore, a Las-mediated up-regulation of 13 grapefruit proteins was detected, which included Cu/Zn superoxide dismutase, chitinases, lectin-related proteins, miraculin-like proteins, peroxiredoxins and a CAP 160 protein. Interestingly, a Las-mediated up-regulation of granule-bound starch synthase was correlated with an increase in the K concentrations of pre-symptomatic and symptomatic plants. Conclusions This study constitutes the first attempt to characterize the interrelationships between protein expression and nutritional status of Las-infected pre-symptomatic or symptomatic grapefruit plants and sheds light on the physiological and molecular mechanisms associated with HLB disease development.

We postulated that a synergistic combination of two innate immune functions, pathogen surface recognition and lysis, in a protein chimera would lead to a robust class of engineered antimicrobial therapeutics for protection against pathogens. In support of our hypothesis, we have engineered such a chimera to protect against the Gram-negative Xylella fastidiosa (Xf), which causes diseases in multiple plants of economic importance. Here we report the design and delivery of this chimera to target the Xf subspecies fastidiosa (Xff), which causes Pierce disease in grapevines and poses a great threat to the wine-growing regions of California. One domain of this chimera is an elastase that recognizes and cleaves MopB, a conserved outer membrane protein of Xff. The second domain is a lytic peptide, cecropin B, which targets conserved lipid moieties and creates pores in the Xff outer membrane. A flexible linker joins the recognition and lysis domains, thereby ensuring correct folding of the individual domains and synergistic combination of their functions. The chimera transgene is fused with an amino-terminal signal sequence to facilitate delivery of the chimera to the plant xylem, the site of Xff colonization. We demonstrate that the protein chimera expressed in the xylem is able to directly target Xff, suppress its growth, and significantly decrease the leaf scorching and xylem clogging commonly associated with Pierce disease in grapevines. We believe that similar strategies involving protein chimeras can be developed to protect against many diseases caused by human and plant pathogens.

The Xylella fastidiosa comparative genomic database is a scientific resource with the aim to provide a user-friendly interface for accessing high-quality manually curated genomic annotation and comparative sequence analysis, as well as for identifying and mapping prophage-like elements, a marked feature of Xylella genomes. Here we describe a database and tools for exploring the biology of this important plant pathogen. The hallmarks of this database are the high quality genomic annotation, the functional and comparative genomic analysis and the identification and mapping of prophage-like elements. It is available from web site http://www.xylella.lncc.br.

Cellular fatty acids of 80 strains of Xanthomonas campestris, representing 9 different pathovars, were analyzed by gas-liquid chromatography and mass spectrometry. A total of 48 fatty acids were identified, the most important being the 16:0 (averaging at least 4.5 % of the total), the cis- and trans- 9 16 : 1 (over 14.4 %), and the iso and anteiso 15 : 0 (over 30 %). Other major fatty acids (averaging over 1 % of total) were the saturated 14 : 0 and 15 : 0, the hydroxy-substituted iso 3-OH 11 : 0, 3-OH 12 : 0 and iso 3-OH 13 : 0, and the branch-chained iso 11 : 0, iso 16 : 0, iso 17 : 1, iso 17 : 0 and anteiso 17 : 0. Of 33 minor fatty acids detected and identified, only 7 have been previously reported in the xanthomonads. Significant differences in mean percentages of 5 major fatty acids and 4 (chemical) class totals were detected among pathovars, which statistically segregated into three groups by rank analysis. X. campestris pv. dieffenbachiae was in a group by itself; pvs. campestris, citri (pathotypes A and B), manihotis, phaseoli, pruni and vesicatoria were in a seond group, and pvs. glycines, begonia and citri (pathotype E) were in a third.

Zebra Chip (ZC) is an emerging plant disease that causes aboveground decline of potato shoots and generally results in unusable tubers. This disease has led to multi-million dollar losses for growers in the central and western United States over the past decade and impacts the livelihood of potato farmers in Mexico and New Zealand. ZC is associated with ‘Candidatus Liberibacter solanacearum’, a fastidious alpha-proteobacterium that is transmitted by a phloem-feeding psyllid vector, Bactericera cockerelli Sulc. Research on this disease has been hampered by a lack of robust culture methods and paucity of genome sequence information for ‘Ca. L. solanacearum’. Here we present the sequence of the 1.26 Mbp metagenome of ‘Ca. L. solanacearum’, based on DNA isolated from potato psyllids. The coding inventory of the ‘Ca. L. solanacearum’ genome was analyzed and compared to related Rhizobiaceae to better understand ‘Ca. L. solanacearum’ physiology and identify potential targets to develop improved treatment strategies. This analysis revealed a number of unique transporters and pathways, all potentially contributing to ZC pathogenesis. Some of these factors may have been acquired through horizontal gene transfer. Taxonomically, ‘Ca. L. solanacearum’ is related to ‘Ca. L. asiaticus’, a suspected causative agent of citrus huanglongbing, yet many genome rearrangements and several gene gains/losses are evident when comparing these two Liberibacter. species. Relative to ‘Ca. L. asiaticus’, ‘Ca. L. solanacearum’ probably has reduced capacity for nucleic acid modification, increased amino acid and vitamin biosynthesis functionalities, and gained a high-affinity iron transport system characteristic of several pathogenic microbes.

BackgroundPlant cytochrome P450 monooxygenases (CYP) mediate synthesis and metabolism of many physiologically important primary and secondary compounds that are related to plant defense against a range of pathogenic microbes and insects. To determine if cytochrome P450 monooxygenases are involved in defense response toXylella fastidiosa(Xf) infection, we investigated expression and regulatory mechanisms of the cytochrome P450 monooxygenaseCYP736Bgene in both disease resistant and susceptible grapevines.ResultsCloning of genomic DNA and cDNA revealed that theCYP736Bgene was composed of two exons and one intron with GT as a donor site and AG as an acceptor site.CYP736Btranscript was up-regulated in PD-resistant plants and down-regulated in PD-susceptible plants 6 weeks afterXfinoculation. However,CYP736Bexpression was very low in stem tissues at all evaluated time points. 5'RACE and 3'RACE sequence analyses revealed that there were three candidate transcription start sites (TSS) in the upstream region and three candidate polyadenylation (PolyA) sites in the downstream region ofCYP736B. Usage frequencies of each transcription initiation site and each polyadenylation site varied depending on plant genotype, developmental stage, tissue, and treatment. These results demonstrate that expression ofCYP736Bis regulated developmentally and in response toXfinfection at both transcriptional and post-transcriptional levels. Multiple transcription start and polyadenylation sites contribute to regulation ofCYP736Bexpression.ConclusionsThis report provides evidence that the cytochrome P450 monooxygenaseCYP736Bgene is involved in defense response at a specific stage ofXfinfection in grapevines; multiple transcription initiation and polyadenylation sites exist forCYP736Bin grapevine; and coordinative and selective use of transcription initiation and polyadenylation sites play an important role in regulation ofCYP736Bexpression during growth, development and response toXfinfection.

Huanglongbing (HLB) (yellow shoot disease) is a highly destructive disease that threatens citrus production worldwide. The disease was first observed in Guangdong, P.R. China, over 100 years ago, and was found in Florida, United States, in 2005. ‘Candidatus Liberibacter asiaticus’ has been associated with HLB in many citrus-growing regions around the world, including Guangdong and Florida. The global epidemiology of HLB, as well as management of the disease, relies on knowledge of ‘Ca. L. asiaticus’ populations in different geographical regions around the world. In this study, we identified a genetic marker containing small tandem repeats in the genome of ‘Ca. L. asiaticus’ and comparatively analyzed the tandem repeat numbers (TRNs) in ‘Ca. L. asiaticus’ populations from Guangdong and Florida. Analyses of TRNs showed that the bacterial population in Guangdong was different from that in Florida. The Guangdong population consisted predominately of strains with a TRN of 7 (TRN7) at a frequency of 47.6%. The Florida population consisted predominately of strains with a TRN of 5 (TRN5) at a frequency of 84.4%. TRNs ranged from 3 to 16. The apparent absence of TRNs of 9, 10, 11, and 12 separated the bacterial strains into two groups: TRNs < 10 (TRN) and TRNs > 10 (TRN>10). In Florida, TRN strains (103/109, or 94.5%) were widely distributed in all HLB-affected counties. TRN>10 strains (6/109, or 5.5%) were found in central Florida. This is the first report documenting the differentiation of ‘Ca. L. asiaticus’ populations between Asia and North America and the possible presence of two differentially distributed genotypes of ‘Ca. L. asiaticus’ in Florida.

Although citrus stubborn disease (CSD), caused by the phloem resident mollicute Spiroplasma citri, is a significant threat to California citrus industry, our knowledge of its epidemiology is mostly anecdotal. We optimized multiple pathogen-detection protocols, measured disease incidence in two plots of commercial California groves, assessed pathogen impact on fruit quality and yield, and evaluated genetic diversity among S. citri isolates. Fruit columellas and receptacles were more suitable than leaves or bark for bacterial cultivation. Using cultivation and S. citri-specific PCR for detection, the incidence of CSD in two orchards, respectively, ranged from 46 to 85% and 1 to 4%, depending on the sampling technique. Yield and quality of fruits produced by trees that were mildly or severely CSD-symptomatic were compared to those of S. citri-free trees in one California orchard in 2006 and 2007. These infected trees had reduced fruit quality and up to 32% lower yield relative to S. citri-free trees. Using RAPD markers to compare 35 S. citri isolates collected 20 years ago from the United States and Mediterranean region with 34 isolates recently collected from California, significant genetic diversity was identified but was not correlated with the time or location of collection. Our findings suggest that CSD incidence in the commercial groves evaluated could be as high as 85% and its impact on yield and fruit quality are significant. Accepted for publication 1 March 2010. Published 26 May 2010.

The activities of catalase (EC 1.11.1.6; CAT), peroxidase (EC 1.11.1.7; POD), polyphenoloxidase (EC 1.10.3.1; PPO), glucose-6-phosphate dehydrogenase (EC 1.1.1.49; G6PDH), 6-phosphogluconate dehydrogenase (EC 1.1.1.44; 6PGDH), isocitrate dehydrogenase (EC 1.1.1.42; ICDH), and the total phenolic content in citrus leaves differing in susceptibility to infection by strains of Xanthomonas campestris pv. citri (Xcc) were determined. The levels of oxidative enzymes (CAT, POD and PPO), pentose-phosphate cycle enzymes (G6PDH and 6PGDH), and the total phenolic contents were higher in Xcc resistant plants (Yuzu, Dancy, Cleopatra, and Kawano Wase) than in susceptible plants (citrumelo, lemon, Rough lemon and grapefruit). However, the glycolysis-tricarboxylic acid cycle enzyme, ICDH, was lower in Xcc resistant plants than in susceptible plants.

Chen, J., Groves, R., Civerolo, E. L., Viveros, M., Freeman, M., and Zheng, Y. 2005. Two Xylella fastidiosa genotypes associated with almond leaf scorch disease on the same location in California. Phytopathology 95:708-714. Almond leaf scorch disease (ALSD) has recently reemerged in the San Joaquin Valley of California threatening almond production. ALSD is caused by Xylella fastidiosa, a nutritionally fastidious bacterium. Single nucleotide polymorphisms (SNPs) in the 16S rRNA gene (16S rDNA) of X. fastidiosa strains were identified to characterize the bacterial population in infected trees. Genotype-specific SNPs were used to design primers for multiplex polymerase chain reaction assays of early passage cultures. Two genotypically distinct types of X. fastidiosa strains, G-type and A-type, coexist simultaneously in the same infected almond orchard. This was substantiated by restriction fragment length polymorphism analysis of a different genetic locus, RST31-RST33, which has previously been used to identify and differentiate X. fastidiosa strains. Furthermore, unique bacterial colony morphology was consistently associated with the A-type X. fastidiosa strains. To our knowledge, this is the first report of a mixed genotype infection of X. fastidiosa disease on the same location under natural environmental conditions. The concept of mixed genotype infection could affect the current epidemiological study based on the assumption that one genotype causes ALSD on one location and, therefore, the disease management strategy.

Made available in DSpace on 2013-08-28T14:11:31Z (GMT). No. of bitstreams: 1 WOS000260747900001.pdf: 308056 bytes, checksum: b5a13200d1c91c0a124c5e9072f49ef3 (MD5) Made available in DSpace on 2013-09-30T19:22:43Z (GMT). No. of bitstreams: 2 WOS000260747900001.pdf: 308056 bytes, checksum: b5a13200d1c91c0a124c5e9072f49ef3 (MD5) WOS000260747900001.pdf.txt: 29000 bytes, checksum: 8548b2485cf1b52be3b4ba0e1e65b54b (MD5) Previous issue date: 2008-10-20 Submitted by Vitor Silverio Rodrigues (vitorsrodrigues@reitoria.unesp.br) on 2014-05-20T15:33:55Z No. of bitstreams: 2 WOS000260747900001.pdf: 308056 bytes, checksum: b5a13200d1c91c0a124c5e9072f49ef3 (MD5) WOS000260747900001.pdf.txt: 29000 bytes, checksum: 8548b2485cf1b52be3b4ba0e1e65b54b (MD5) Made available in DSpace on 2014-05-20T15:33:55Z (GMT). No. of bitstreams: 2 WOS000260747900001.pdf: 308056 bytes, checksum: b5a13200d1c91c0a124c5e9072f49ef3 (MD5) WOS000260747900001.pdf.txt: 29000 bytes, checksum: 8548b2485cf1b52be3b4ba0e1e65b54b (MD5) Previous issue date: 2008-10-20 Citrus Research Board (CRB) University of California (IUCRP) Background: The increasing number of genomic sequences of bacteria makes it possible to select unique SNPs of a particular strain/species at the whole genome level and thus design specific primers based on the SNPs. The high similarity of genomic sequences among phylogenetically-related bacteria requires the identification of the few loci in the genome that can serve as unique markers for strain differentiation. PrimerSNP attempts to identify reliable strain-specific markers, on which specific primers are designed for pathogen detection purpose.Results: PrimerSNP is an online tool to design primers based on strain specific SNPs for multiple strains/species of microorganisms at the whole genome level. The allele-specific primers could distinguish query sequences of one strain from other homologous sequences by standard PCR reaction. Additionally, PrimerSNP provides a feature for designing common primers that can amplify all the homologous sequences of multiple strains/species of microorganisms. PrimerSNP is freely available at http://cropdisease.ars.usda.gov/similar to primer.Conclusion: PrimerSNP is a high-throughput specific primer generation tool for the differentiation of phylogenetically-related strains/species. Experimental validation showed that this software had a successful prediction rate of 80.4 - 100% for strain specific primer design. USDA ARS, San Joaquin Valley Agr Sci Ctr, Parlier, CA 93648 USA Citrus Res Board, Visalia, CA 93291 USA Univ Calif Davis, Seed Biotechnol Ctr, Davis, CA 95616 USA Univ Calif Davis, Dept Viticulture & Enol, Davis, CA 95616 USA Univ Estadual Paulista, Fac Ciencias Agr & Vet, Dept Tecnol, BR-14884900 São Paulo, Brazil Univ Estadual Paulista, Fac Ciencias Agr & Vet, Dept Tecnol, BR-14884900 São Paulo, Brazil CRB: 5300-05F University of California (IUCRP): 06-002845

Presumptive phage particles associated with Xylella fastidiosa strain Temecula-1 grown in PW broth were observed by transmission electron microscopy (TEM) in ultrathin sections of bacterial cell-containing low speed centrifugation pellets and in partially purified preparations from CsCl equilibrium centrifugation density gradients. Ultrathin-sectioned cell pellets contained icosahedral particles of about 45 nm in diameter. Samples collected from CsCl density gradients revealed mostly non-tailed icosahedral but also tailed particles. The icosahedral particles could be divided into two types: a large type (about 45 nm) and a small type (about 30 nm). Filamentous phage-like particles (17 × 120 to 6,300 nm) were also observed. The presence of different types of phage-like particles resembling to those in several bacteriophage families provides new physical evidence, in addition to X. fastidiosa genomic information, that X. fastidiosa possesses active phages. This is the first report of phage particles released in X. fastidiosa cultures.

Xylella fastidiosa is a gram-negative plant pathogenic bacterium that causes almond leaf scorch disease (ALSD) and Pierce's disease (PD) of grape in many regions of North America and Mexico. Of the two 16S rRNA gene genotypes described in California, A genotype strains cause ALSD only and G genotype strains cause both PD and ALSD. While G genotype strains cause two different diseases, little is known about their genetic variation. In this study, we identified a putative protease locus, PD0218 ( pspB ), in the genome of X. fastidiosa and evaluated the variation at this locus in X. fastidiosa populations. PD0218 contains tandem repeats of ACDCCA, translated to threonine and proline (TP), upstream of the putative protease conserved domain. Among 116 X. fastidiosa ALSD and PD strains isolated from seven locations in California, tandem repeat numbers (TRNs) varied from 9 to 47, with a total of 30 TRN genotypes, indicating that X. fastidiosa possesses an active mechanism for contracting and expanding tandem repeats at this locus. Significant TRN variation was found among PD strains (mean = 29.9), which could be further divided into two TRN groups: PD-G small (mean = 17.3) and PD-G large (mean = 44.3). Less variation was found in ALSD strains (mean = 21.7). The variation was even smaller after ALSD strains were subdivided into the A and G genotypes (mean = 13.3, for the G genotype; mean = 27.1, for the A genotype). Genetic variation at the PD0218 locus is potentially useful for sensitive discrimination of X. fastidiosa strains. However, TRN stability, variation range, and correlation to phenotypes should be evaluated in epidemiological applications such as pathotype identification and delineation of pathogen origin.

Background The family Vitaceae consists of many different grape species that grow in a range of climatic conditions. In the past few years, several studies have generated functional genomic information on different Vitis species and cultivars, including the European grape vine, Vitis vinifera. Our goal is to develop a comprehensive web data source for Vitaceae. Description VitisExpDB is an online MySQL-PHP driven relational database that houses annotated EST and gene expression data for V. vinifera and non-vinifera grape species and varieties. Currently, the database stores ~320,000 EST sequences derived from 8 species/hybrids, their annotation (BLAST top match) details and Gene Ontology based structured vocabulary. Putative homologs for each EST in other species and varieties along with information on their percent nucleotide identities, phylogenetic relationship and common primers can be retrieved. The database also includes information on probe sequence and annotation features of the high density 60-mer gene expression chip consisting of ~20,000 non-redundant set of ESTs. Finally, the database includes 14 processed global microarray expression profile sets. Data from 12 of these expression profile sets have been mapped onto metabolic pathways. A user-friendly web interface with multiple search indices and extensively hyperlinked result features that permit efficient data retrieval has been developed. Several online bioinformatics tools that interact with the database along with other sequence analysis tools have been added. In addition, users can submit their ESTs to the database. Conclusion The developed database provides genomic resource to grape community for functional analysis of genes in the collection and for the grape genome annotation and gene function identification. The VitisExpDB database is available through our website http://cropdisease.ars.usda.gov/vitis_at/main-page.htm.