Edwin L. Civerolo

Genome-based PCR primers for specific and sensitive detection and quantification of Xylella fastidiosa

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 221xa0bp 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 r2u200a>u200a0.97. 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.

The effect of ‘Candidatus Liberibacter asiaticus’ infection on the proteomic profiles and nutritional status of pre-symptomatic and symptomatic grapefruit (Citrus paradisi) plants

BackgroundHuanglongbing (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.ResultsThis 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 9xa0kDa 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.ConclusionsThis 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.

A new diagnostic system for ultra-sensitive and specific detection and quantification of Candidatus Liberibacter asiaticus, the bacterium associated with citrus Huanglongbing

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.

Evidence that Cell Death is Associated with Zebra Chip Disease in Potato Tubers

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 (Šulc), 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.ResumenZebra chip (ZC) es una enfermedad establecida y altamente destructiva de papa (Solanum tuberosum L.) que se presenta en varios estados del suroeste de los Estados Unidos, México, América Central y Nueva Zelandia. El agente causal de esta enfermedad no ha sido identificado. No obstante, la bacteria “Candidatus Liberibacter solanacearum” y el psílido de la papa Bactericera cockerelli (Šulc), su insecto vector, están asociados con la enfermedad. Los tubérculos de plantas afectadas por ZC presentan oscurecimiento dramático del tejido vascular concomitante con “pecas necróticas” que en ambos casos pueden afectar al tubérculo completo. Al freírse, estos tubérculos desarrollan un patrón característico de rayado haciéndolos no comerciales. Se ha sugerido que estos síntomas característicos en los tubérculos estén asociados con muerte general de las células, aún cuando no se ha mostrado evidencia para confirmar esta hipótesis. Se llevaron a cabo varios experimentos a fin de determinar si la muerte de las células está asociada con la enfermedad de ZC. La muerte celular se cuantificó inicialmente comparando el lixiviado iónico celular de tubérculos con y sin ZC. Se encontró que los niveles de iones lixiviados fueron significativamente más altos en tubérculos afectados con ZC comparados con los libres de ZC. Para examinar mas la asociación de la muerte de la célula con la enfermedad ZC, a tubérculos infectados y a libres de ZC se les comparó usando los métodos de la clásica tinción histoquímica, junto con microscopía óptica, lo cual reveló capas de células muertas rodeando a numerosas lesiones pequeñas, de forma irregular, a través de la región medular parenquimatosa, el anillo vascular y el cortex de tubérculos afectados por ZC. Esta muerte celular se confirmó usando microscopía electrónica de barrido de alta resolución (FE-SEM) de tejido de cortes de tubérculo fresco.

Multilocus Simple Sequence Repeat Markers for Differentiating Strains and Evaluating Genetic Diversity of Xylella fastidiosa

ABSTRACT 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 Pierces 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.

Analysis of the genome-wide variations among multiple strains of the plant pathogenic bacterium Xylella fastidiosa

BackgroundThe Gram-negative, xylem-limited phytopathogenic bacterium Xylella fastidiosa is responsible for causing economically important diseases in grapevine, citrus and many other plant species. Despite its economic impact, relatively little is known about the genomic variations among strains isolated from different hosts and their influence on the population genetics of this pathogen. With the availability of genome sequence information for four strains, it is now possible to perform genome-wide analyses to identify and categorize such DNA variations and to understand their influence on strain functional divergence.ResultsThere are 1,579 genes and 194 non-coding homologous sequences present in the genomes of all four strains, representing a 76. 2% conservation of the sequenced genome. About 60% of the X. fastidiosa unique sequences exist as tandem gene clusters of 6 or more genes. Multiple alignments identified 12,754 SNPs and 14,449 INDELs in the 1528 common genes and 20,779 SNPs and 10,075 INDELs in the 194 non-coding sequences. The average SNP frequency was 1.08 × 10-2 per base pair of DNA and the average INDEL frequency was 2.06 × 10-2 per base pair of DNA. On an average, 60.33% of the SNPs were synonymous type while 39.67% were non-synonymous type. The mutation frequency, primarily in the form of external INDELs was the main type of sequence variation. The relative similarity between the strains was discussed according to the INDEL and SNP differences. The number of genes unique to each strain were 60 (9a5c), 54 (Dixon), 83 (Ann1) and 9 (Temecula-1). A sub-set of the strain specific genes showed significant differences in terms of their codon usage and GC composition from the native genes suggesting their xenologous origin. Tandem repeat analysis of the genomic sequences of the four strains identified associations of repeat sequences with hypothetical and phage related functions.ConclusionINDELs and strain specific genes have been identified as the main source of variations among strains, with individual strains showing different rates of genome evolution. Based on these genome comparisons, it appears that the Pierces disease strain Temecula-1 genome represents the ancestral genome of the X. fastidiosa. Results of this analysis are publicly available in the form of a web database.

Genetic diversity of ‘Cadidatus Liberibacter solanacearum’ strains in the United States and Mexico revealed by simple sequence repeat markers

Abstract‘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.

Multilocus microsatellite analysis of 'Candidatus Liberibacter asiaticus' associated with citrus Huanglongbing worldwide

BackgroundHuanglongbing (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.ResultsA 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.ConclusionsOur 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.

Transcriptional regulation of the grape cytochrome P450 monooxygenase gene CYP736B expression in response to Xylella fastidiosa infection

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 to Xylella fastidiosa (Xf) infection, we investigated expression and regulatory mechanisms of the cytochrome P450 monooxygenase CYP736B gene in both disease resistant and susceptible grapevines.ResultsCloning of genomic DNA and cDNA revealed that the CYP736B gene was composed of two exons and one intron with GT as a donor site and AG as an acceptor site. CYP736B transcript was up-regulated in PD-resistant plants and down-regulated in PD-susceptible plants 6 weeks after Xf inoculation. However, CYP736B expression was very low in stem tissues at all evaluated time points. 5RACE and 3RACE 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 of CYP736B. 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 of CYP736B is regulated developmentally and in response to Xf infection at both transcriptional and post-transcriptional levels. Multiple transcription start and polyadenylation sites contribute to regulation of CYP736B expression.ConclusionsThis report provides evidence that the cytochrome P450 monooxygenase CYP736B gene is involved in defense response at a specific stage of Xf infection in grapevines; multiple transcription initiation and polyadenylation sites exist for CYP736B in grapevine; and coordinative and selective use of transcription initiation and polyadenylation sites play an important role in regulation of CYP736B expression during growth, development and response to Xf infection.

Complete Genome Sequence of a Chinese Strain of “Candidatus Liberibacter asiaticus”

ABSTRACT 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.