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.

ABSTRACT 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.

Abstract Pathogens circumvent host immune defense to propagate their lifecycle. We have been studying the mechanisms of pathogen-induced host cell death to develop a robust strategy for pathogen clearance. The strategy involves designing and developing a protein chimera with one domain for recognizing conserved pathogen membrane elements and another for lysing the pathogen membrane. This dualistic synergy of recognition and lysis permits rapid pathogen clearance thereby preventing host cell death and disease development. This strategy has been successfully applied to counteract bacterial infections and diseases in grape, citrus, and tobacco. We have engineered a chimera consisting of protease (recognition domain) and cecropin (lysis domain) to prevent Pierce’s disease, a deadly disease in grape caused by Xylella fastidiosa. We have shown that transgenic plants expressing the chimera of protease and cecropin effectively clear Xylella fastidiosa from sites of colonization. We have also designed a protein chimera of thionin (with both recognition and lysis domains) and a synthetic lytic peptide. We have shown that this same chimera prevents citrus canker which is caused by Xanthamonas axonopodis citri and wildfire disease in tobacco which is caused by Pseudomonas syringe pv. tobaci. This protein engineering strategy appears to be an effective therapy against a broad spectrum of plant pathogens. A similar approach is now being evaluated against multiple human pathogens.

“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.

Hong Lin,Helvecio D.Coletta-Filho,CliffS.Han,Binghai Lou,EdwinL.Civerolo,Marcos A.Machado,and Goutam Gupta.Draft genome sequence of"Candidatus Liberibacter americanus" bacterium associated with citrus huanglongbing in Brazil.Genome Announcements,2013,1(3):e00275-13.在此,我们报道“Candidatus Liberibacter americanus" PW_SP株系基因组草图序列.大小为1,176,071bp的基因组,G+C含量为31.6％,包含948个开放阅读框,38个tRNA,3个完整的rRNA.

ABSTRACT 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.

Abstract A few years ago, we introduced a concept that a protein chimera of pathogen recognition and lysis domains would be able to rapidly clear a broad-spectrum of pathogens [Crit Rev Immunol 2007;27(3):233-245]. For a wide variety of viral, bacterial, and fungal pathogens, appropriate recognition and lysis domains can be chosen from the host innate immune repertoire. A chimera of the recognition and lysis domains would be designed with the aid of a flexible linker to ensure synergy of the two functions and therefore, the rapid clearance of the targeted pathogen. In this work, we demonstrate the design of such a chimera and the efficacy of this chimera in clearing a plant pathogen Xylella fastidiosa (Xf) that causes diseases in multiple plants of economic importance. The most notable ones are Pierce’s disease (PD) in grape and variegated chlorosis (CVC) in citrus. Specifically, we show the construction of the transgenic grapevines expressing a protein chimera of recognition and lysis domains specific for Xf. This chimera clears Xf from the xylem (the site of colonization) and blocks the development of PD [Proc Natl Acad Sci U S A. 2012;109(10):3721-3725]. The same chimera can be applied to block CVC. Finally, we indicate how such chimeras of recognition and lysis domains can be developed to target multiple human pathogens.

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.

Xylella fastidiosa (Xf) causes diseases in many agricultural, horticultural, and landscape crops, including Pierce's disease (PD) of grapevines. PD has been a serious problem in scattered table, wine and raisin grape growing regions throughout California, as well as in Texas for more than 100 years. In this study, we evaluated the genetic variation in diversity and structure of Xf associated with PD of grapevines in California and Texas using simple sequence repeat (SSR) markers. The analysis revealed a high level of genetic diversity across the populations of Xf isolated from the grapevines from different viticulture locations or counties in California and Texas. Overall genetic diversity of Xf was detected comparatively high in Texas (0.835) than that in California (0.662). Hierarchical analysis of molecular variance (AMOVA) showed a significant genetic structure of Xf population between California and Texas. UPGMA clustering analysis and the Bayesian clustering algorithm using STRUCTURE consistently placed California and Texas Xf populations into two major genetic groups with no clear population structure within each state.

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 16S rDNA sequences identified bacteria belonging to 16 genera. Seven (Brachybacterium, Enterobacter, Microbacterium, Paenibacillus, Staphylococcus, Stenotrophomonas and Variovorax) were only isolated from ZCD-affected potato tubers, whereas four (Bosea, Nocardia, Sphingomonas and Sphingopyxis) were only isolated from non-ZCD potato tubers. However, a more sensitive PCR analysis diminished the specific association of the bacteria to either ZCD or non-ZCD tubers, suggesting that infection by Ca. L. solanacearum' influenced the titres of other endophytic bacteria in potato tubers.

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.

'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.

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.

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.

trees in California (Blua et al., 1999; Purcell et al., 1999; Purcell & Saunders, 1999).This insect is an invasive species in California that was first detected in the state in 1989 (Sorensen & Gill, 1996).This sharpshooter is a key vector of Xylella fastidiosa and has changed the epidemiology of X. fastidiosa (Xf) diseases affecting important agronomic and horticultural crops as well as landscape, ornamental and native trees in California based on the infection of these crops by the bacteria (Blua et al., 1999; Purcell et al., 1999; Purcell & Saunders, 1999) (Fig. 1).It is not clear if management strategies for Pierce's Disease (PD) developed in GWSS-free regions of California are suitable to manage the disease in vineyards where GWSS has become established.Data from earlier studies suggest that GWSS has continued to increase in number (population density) and geographical range in California (Purcell & Saunders, 1999;Tubajika et al., 2004).GWSS populations are widely distributed over a large number of hosts including perennial agronomic crops, ornamental plantings, and weedy plant species (Hill & Purcell, 1995; Purcell et al., 1999;Raju et al., 1980).Previous studies by Blua et al. (1999) and Perring et al. (2001) showed that GWSS populations utilize citrus plants as their primary over-wintering host (other plants are available) when grapes, stone fruits, ornamental hosts, www.intechopen.com How to referenceIn order to correctly reference this scholarly work, feel free to copy and paste the following:

BACKGROUND:Huanglongbing (HLB) is one of the most destructive citrus diseases in the world. The disease is associated with the presence of a fastidious, phloem-limited α- proteobacterium, 'Candidatus Liberibacter asiaticus', 'Ca. Liberibacter africanus' or 'Ca. Liberibacter americanus'. HLB-associated Liberibacters have spread to North America and South America in recent years. While the causal agents of HLB have been putatively identified, information regarding the worldwide population structure and epidemiological relationships for 'Ca. L. asiaticus' is limited. The availability of the 'Ca. L. asiaticus' genome sequence has facilitated development of molecular markers from this bacterium. The objectives of this study were to develop microsatellite markers and conduct genetic analyses of 'Ca. L. asiaticus' from a worldwide collection. Two hundred eighty seven isolates from USA (Florida), Brazil, China, India, Cambodia, Vietnam, Taiwan, Thailand, and Japan were analyzed.RESULTS:A panel of seven polymorphic microsatellite markers was developed for 'Ca. L. asiaticus'. Microsatellite analyses across the samples showed that the genetic diversity of 'Ca. L. asiaticus' is higher in Asia than Americas. UPGMA and STRUCTURE analyses identified three major genetic groups worldwide. Isolates from India were genetically distinct. East-southeast Asian and Brazilian isolates were generally included in the same group; a few members of this group were found in Florida, but the majority of the isolates from Florida were clustered separately. eBURST analysis predicted three founder haplotypes, which may have given rise to three groups worldwide.CONCLUSIONS:Our results identified three major genetic groups of 'Ca. L. asiaticus' worldwide. Isolates from Brazil showed similar genetic makeup with east-southeast Asian dominant group, suggesting the possibility of a common origin. However, most of the isolates recovered from Florida were clustered in a separate group. While the sources of the dominant 'Ca. L. asiaticus' in Florida were not clearly understood, the less-pervasive groups may have been introduced directly from Asia or via Brazil. Notably, the recent outbreak of HLB in Florida probably occurred through multiple introductions. Microsatellite markers developed in this study provide adequate discriminatory power for the identification and differentiation of closely-related isolates, as well as for genetic studies of 'Ca. L. asiaticus'.

Huanglongbing (HLB) is a lethal disease of citrus and the bacterium, ‘Ca. Liberibacter asiaticus’ (Las) is the most prevalent etiological agent of the disease worldwide. Here, proteomic analyses via 2‐DE and mass spectrometry were employed to elucidate the protein expression profiles in leaves of Las‐infected greenhouse‐grown citrus grapefruit and lemon plants that are pre‐symptomatic or symptomatic for HLB. We also applied ICP spectroscopy to identify the effect of the disease on the nutrient status of greenhouse‐grown citrus grapefruit, lemon, and trifoliate plants. 2‐DE analysis revealed over 200 and 70 protein spots in grapefruit and lemon leaves, respectively, that were differentially expressed due to Las infection compared to uninfected plants. Interestingly, LC‐MS analysis showed that chitinase and starch synthase are among a subset of proteins precociously induced in the leaves of Las‐infected grapefruit and lemon plants. Furthermore, nutrient status analysis of leaves suggests that active accumulations of Zn and Ca in lemon and trifoliate plants, respectively, are associated with citrus response to Las infection. In summary, our results provide novel insights into potential host‐specific response mechanisms associated with HLB, which is important for early detection and better nutritional management of the disease. This project was supported by the USDA, Agricultural Research Service.