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 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, C_t 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.     

Differential colonization patterns of Xylella fastidiosa infecting citrus genotypes

Xylella fastidiosa is a phytopathogenic bacterium that causes disease in many different crops worldwide. In Brazil, X. fastidiosa subsp. pauca causes citrus variegated chlorosis (CVC), which is a disease responsible for economic losses in the citrus agribusiness. Variable host responses to bacterial colonization and disease development have been observed. This work studies the colonization processes of a pathogenic GFP‐labelled X. fastidiosa citrus strain in sweet orange (susceptible) and tangor (resistant) parents and two resulting hybrids that exhibited contrasting responses to CVC. Xylella fastidiosa showed increased populations and movement in the susceptible genotypes, but slower compared to other hosts such as grapevine. Scanning electron microscopy revealed that the predominant pitted stem morphology in citrus makes the bacterial movement difficult. In susceptible genotypes X. fastidiosa can move from the primary to the secondary xylem, whilst it is confined to the primary xylem in resistant plants. Associated with this is an induction of lignification that occurs earlier in the resistant genotypes when in the presence of the pathogen, and represents a genetic mechanism that leads to formation of a physical barrier, impairing bacterial colonization.     

Seven years of negative detection results confirm that Xylella fastidiosa,the causal agent of CVC,is not transmitted from seeds to seedlings

Knowledge about the mechanism of transmission of systemic pathogens of citrus species is highly important for the safe movement of citrus germplasm, management of citrus mother trees, and also production of young plants. Among systemic pathogens of citrus, Xylella fastidiosa the causal agent of the citrus variegated chlorosis (CVC), is one the most important pathogens causing decline in tree vigour and productivity. Seven-year experiments were conducted to evaluate the hypothesis of seed-to-seedling transmission of X. fastidiosa. This bacterium was found colonizing the fruit (exocarp, central axis and mesocarp) and the seed parts (seed coat and endosperm plus embryo). After 7 years of PCR assay, no positive PCR detection of X. fastidiosa was confirmed in seedlings propagated from the seeds infected with X. fastidiosa. This result demonstrates the lack of seed-to-seedling transmission of this bacterium.     

Coffee bacterial diseases: a plethora of scientific opportunities

Coffee is a very important crop for several tropical countries across different continents. The diseases bacterial halo blight (BHB), bacterial leaf spot (BLS), bacterial leaf blight (BLB) and coffee leaf scorch (CLS), caused by the bacterial pathogens Pseudomonas syringae pv. garcae (Psgc), P. syringae pv. tabaci (Psta), Pseudomonas cichorii (Pch) and Xylella fastidiosa subsp. pauca (Xfp), respectively, cause significant reductions in coffee production, although other minor bacterial diseases have also been reported in some countries. Little research progress has been made on aspects that are relevant for control and management of these diseases. In all cases, there is an urgent need to develop rapid and more reliable methods for early detection of the pathogens in order to minimize their negative impact on coffee production. Because of the high rate of intra- and intersubspecific recombination occurring in X. fastidiosa, a permanent revision of the detection methods is necessary. Greater efforts should be made to understand the genetic and virulence diversity of Psgc, Psta and Pch populations. Early studies reported the identification of potential sources of resistance against Psgc and Psta, but, to date, no resistance gene has been isolated. Little effort has been made to understand the biology and molecular mechanisms underlying the interaction between Coffea spp. and these pathogenic bacteria. This review discusses the recent progress on the molecular mechanisms used by these bacteria to cause diseases on other plant species, in order to provide a guideline for the establishment of future research programmes.     

A combined agar-absorption and BIO-PCR assay for rapid, sensitive detection of Xylella fastidiosa in grape and citrus

Application of the polymerase chain reaction (PCR) to disease diagnosis is limited in part by the presence of PCR inhibitors. Inhibition can be overcome and sensitivity increased by culturing bacteria on agar media prior to PCR (termed BIO-PCR). However, Xylella fastidiosa grows slowly, requiring 10–14 days for visible colonies to appear. In this study an agar-absorption BIO-PCR method for detecting X. fastidiosa in grape and citrus plants was developed. Optimum lengths of time for absorption of inhibitors by the agar medium or enrichment of bacteria on the medium were determined for Pierce's disease of grape and citrus variegated chlorosis. When petioles of grape and citrus leaves with symptoms were spotted onto agar media, the spots washed after various time intervals and assayed for X. fastidiosa by real-time PCR, 97% (31 out of 32) and 100% (six out of six) of spots were positive after 2 days and 4 h for grape and citrus, respectively. With direct PCR, only 12·5% (four out of 32) and 33% (two out of six) of spots were positive, respectively, and visible X. fastidiosa colonies were evident after 10 and 14 days, respectively. In a separate experiment with samples from a different vineyard, 46% (13 out of 28) of the grape samples (agar spots) were positive after 1 day and 93% (26 out of 28) after 5 days using agar-absorption PCR. In contrast, all samples were negative by direct PCR. Viable X. fastidiosa were recovered from all samples after 14 days. Further tests with eight randomly selected grape petioles from three Texas vineyards known to have Pierce's disease resulted in 50% being positive by a simple 24 h agar-absorption PCR assay, whereas none was positive by direct PCR. Overall, 10 out of 16 (63%) vines from five vineyards (two in California and three in Texas) were positive after the 24 h agar-absorption PCR assay. In contrast, only one vine was positive by direct PCR. This simple agar absorption-based PCR assay protocol should prove useful for the routine detection of X. fastidiosa and other slow-growing bacteria in the presence of PCR inhibitors.     

Specific characters of 16S rRNA gene and 16S–23S rRNA internal transcribed spacer sequences of Xylella fastidiosa pear leaf scorch strains

Pear leaf scorch, the only Xylella fastidiosa-induced disease reported from Taiwan, was found in area where the variety Hengshan (Pyrus pyrifolia) was grown. Strains of pear leaf scorch Xyl. fastidiosa (XF-PLS) shared similarities to strains of other host origins in the requirement of complex medium and the exhibition of rippled cell walls, however, recent serological and molecular biology studies showed difference among them. Five strains of XF-PLS were compared with 20 other strains originally isolated from almond, oleander, pecan, plum, peach, mulberry, grapes, citrus, coffee, and sycamore by sequence analyses of the 16S rRNA gene and 16S–23S rRNA internal transcribed spacer region (ITS). When sequences of 16S rRNA gene based on fragment size of 1,537–1,540 bp were compared, the similarity index among 5 XF-PLS strains was 99.3–99.8%, whereas it was 97.8–98.6% between XF-PLS strains and strains from other hosts. When sequences of 16S–23S rRNA ITS based on fragment size of 510–540 bp were compared, the similarity index among 5 XF-PLS strains was 99.0–100%, whereas it was 80.7–82% between XF-PLS strains and strains from other hosts. Multiple sequence alignments led to the identification of 5 polymorphic nucleotides in the 16S rRNA gene among the 25 Xyl. fastidiosa strains, and there were considerable variations in the nucleotide sequences of 16S–23S rRNA ITS between XF-PLS and the other 20 Xyl. fastidiosa strains. The phylogenetic trees revealed that XF-PLS strains were separated from strains of other hosts. Strains of other hosts were divided into four subgroups: strains from (1) oleander, (2) grape, almond M23 and mulberry, (3) citrus and coffee, and (4) pecan, peach, plum, sycamore and almond M12. Results indicate that XF-PLS strains were not closely related to the above-mentioned strains from other hosts and could possibly belong to a new subspecies of Xyl. fastidiosa.     

Assessment of the genetic diversity of Xylella fastidiosa in imported ornamental Coffea arabica plants

A study was performed in order to assess the presence of Xylella fastidiosa in imported ornamental plants, among them Olea europaea, Coffea arabica and Nerium oleander. Positive results were only obtained from C. arabica, where 15 plant samples tested positive for X. fastidiosa by PCR, nine from Costa Rica and six from Honduras. Transmission electron microscopy observations indicated that rod‐shaped bacterial cells exhibiting the characteristics of X. fastidiosa cells were present in the xylem vessels of leaf petioles obtained from the infected C. arabica plants. Diversity of X. fastidiosa in C. arabica plants was assessed through a multilocus sequence typing (MLST) analysis of seven housekeeping genes (leuA, petC, lacF, cysG, holC, nuoL and gltT) and compared with X. fastidiosa infecting different host plants worldwide. Based on this MLST analysis, the prevalence of different sequence types (STs) of X. fastidiosa in the C. arabica ornamental plants was demonstrated and related to different X. fastidiosa subspecies, underlining the risk of introducing additional genetic diversity for X. fastidiosa to Europe. ST53, related to X. fastidiosa subsp. pauca, was frequently found in these C. arabica samples. A second ST related to X. fastidiosa subsp. pauca, ST73, has been assessed in coinfection with ST53 in one individual plant. Additionally, ST72 and ST76, related to X. fastidiosa subsp. fastidiosa, have been recorded. Next to these previously described STs, a novel ST, namely ST77 has been revealed, related to X. fastidiosa subsp. fastidiosa. Isolation of X. fastidiosa from leaf petioles and midribs of infected C. arabica plants was successfully performed only after the application of an additional ultrasonication step during the extraction procedure. Based on this approach, a number of X. fastidiosa isolates were obtained and further characterized.     

Several subspecies and sequence types are associated with the emergence of Xylella fastidiosa in natural settings in France

Xylella fastidiosa is a plant pathogenic bacterium emerging in Europe. In France its emergence has been demonstrated through interceptions of contaminated coffee plants and, in 2015, by a survey of natural settings. The first French focus of contamination was detected in 2015 in Corsica; since then, almost 300 foci have been found and nearly 30 plant species have been declared contaminated, with Polygala myrtifolia remaining the principal host, suffering from severe leaf scorch. This study reports on the diversity of X. fastidiosa identified in France in 2015. Multilocus sequence analysis/typing revealed the presence of mainly X. fastidiosa subsp. multiplex sequence types (STs) ST6 and ST7. A focus of X. fastidiosa subsp. pauca ST53 was identified in mainland France; one sample contaminated by X. fastidiosa subsp. sandyi ST76, one novel recombinant, and coinfections of different isolates in individual samples were also identified, but could not be confirmed by successive samplings, indicating limited or transient contamination. Koch's postulates were fulfilled for two isolates of X. fastidiosa subsp. multiplex on P. myrtifolia, one being ST6 and the other ST7. Comparative genomics of the genome sequences of three French isolates (one ST6 and two ST7) with available sequences revealed that, unlike the American Dixon strain, the French ST6 and ST7 strains are devoid of a plasmid encoding a complete type IV secretion system. Other differences regarding phage sequences were highlighted. Altogether, the results suggest that the emergence of X. fastidiosa in France is linked to several introduction events of diverse strains from different subspecies.     

The chemotaxis regulator <Emphasis Type="Italic">pilG</Emphasis> of <Emphasis Type="Italic">Xylella fastidiosa</Emphasis> is required for virulence in <Emphasis Type="Italic">Vitis vinifera</Emphasis> grapevines

Type IV pili of X. fastidiosa are regulated by pilG, a response regulator protein putatively involved in chemotaxis-like operon sensing stimuli through signal transduction pathways. To elucidate the roles of pilG in pathogenicity of X. fastidiosa, the pilG-deletion mutant XfΔpilG and complemented strain XfΔpilG-C were generated. While all strains had similar growth curves in vitro, XfΔpliG showed significant reduction in cell-matrix adherence and biofilm production compared with wild-type X. fastidiosa and XfΔpilG-C. The genes pilE, pilU, pilT, and pilS were down-regulated in XfΔpliG when compared with its complemented strain and wild-type X. fastidiosa. Finally, no Pierce’s disease symptoms were observed in grapevines inoculated with XfΔpilG, whereas grapevines inoculated with the wild-type X. fastidiosa and complemented strain of XfΔpilG-C developed typical Pierce’s Disease (PD) symptoms. The results indicate that pilG has a role in X. fastidiosa virulence in grapevines.     

Intercepted isolates of <Emphasis Type="Italic">Xylella fastidiosa</Emphasis> in Europe reveal novel genetic diversity

After the first confirmed outbreak of Xylella fastidiosa in the European Union (EU), associated with an olive disease denoted olive quick decline syndrome, mandatory surveys are now carried out in the member States and inspections increased at EU entry points such as ports. Such activities led to the interception of X. fastidiosa-infected coffee plants in consignments originating from Central America. Similarly, the geographic expansion of the olive decline epidemic area of the Apulia region (southern Italy) prompted investigations to identify new host plants. Here we report the interception of three novel bacterial sequence types in Italy, based on multi-locus sequence typing, that cluster with different X. fastidiosa subspecies, illustrating the risk of the introduction of additional pathogen genetic diversity into Europe. In the epidemic area of Apulia, new foci as well as host plant species positive with X. fastidiosa, including cherry, myrtleleaf and rosemary, were found to be all infected with the same sequence type of this bacterium (ST53, or CoDiRO strain). This work highlights the limited knowledge of X. fastidiosa phylogenetic and phenotypic diversity, the risk of novel X. fastidiosa introductions via contaminated plant material, and corroborates other studies indicating that the Apulia epidemic emerged from a single introduction of this pathogen into the region.     

Strain-specific alfalfa water stress induced by <Emphasis Type="Italic">Xylella fastidiosa</Emphasis>

Differences in the virulence of a pathogen among host species can occur because hosts differ in their resistance or tolerance to infection or because of underlying genetic variation in the pathogen. The xylem-limited bacterium Xylella fastidiosa is pathogenic to dozens of plant species throughout the Americas, and is structured into genetically and biologically distinct strains. In some plants X. fastidiosa causes striking leaf scorch symptoms and in others, such as alfalfa, stunting is the primary symptom. The mechanism by which these symptoms occur has been debated. We tested the hypothesis that symptoms result from X. fastidiosa-induced water stress, and that the magnitude of water stress is strain-dependent. We mechanically inoculated alfalfa plants with one of 14 isolates (5 identified genetically as “almond” and 9 as “grape” isolates), and compared stable carbon isotope ratios among isolates. Infected plants showed significant isotopic shifts (up to 2% on average) relative to healthy plants that were consistent with water stress. Moreover, there were significant differences in water stress among isolates, with a tendency for grape isolates to cause more severe water stress than almond isolates. There was also a positive relationship between plant infection level and isotopic shift (slope ± SE = 0.273 ± 0.068), which supports the hypothesis that X. fastidiosa symptoms result from bacterial multiplication and vessel occlusion. Unexpectedly, however, water stress was not correlated with measures of alfalfa stunting. These results suggest X. fastidiosa induces strain-specific levels of water stress, but factors other than water stress alone are responsible for stunting.     

Identification by suppression subtractive hybridization and expression analysis of Medicago truncatula putative defence genes in response to Orobanche crenata parasitization

Crenate broomrape (Orobanche crenata) is the major constraint for pea and faba bean production in the Mediterranean region. In this study, a systematic sequencing of expressed sequence tags (ESTs) was chosen to obtain a first global picture of the assembly of genes involved in defence response. A cDNA-library was established by suppression subtractive hybridization in the model legume Medicago truncatula infected by O. crenata in order to identify a large number of host plant ESTs. Eighty-one presumably up-regulated genes were identified and classified in functional categories. EST-annotations showed homologies to a number of well-characterized genes. Most of the proteins encoded by these genes, are already known in defence in M. truncatula, such as genes related to the JA pathway or involved in cell wall modifications. A notable number of the ESTs, however, were derived from novel genes not matching entries of the large-scale M. truncatula sequences collections. Expression analyses by quantitative RT-PCR of 20 genes corresponding to different functional categories showed high expression levels, supporting their involvement in the defence response.     

Effect of xylem fluid from susceptible and resistant grapevines on developmental biology of Xylella fastidiosa

Xylella fastidiosa causes Pierce??s disease (PD), a serious disease in grapevines, and grapevine cultivars vary in susceptibility to X. fastidiosa in the field. The mechanism(s) by which this occurs has not been clearly elucidated. To explore possible mechanisms, X. fastidiosa cells from a PD strain were grown in pure xylem fluid of PD-susceptible grapevines, Vitis vinifera and V. labrusca, versus PD-resistant grapevines, V. champinii and V. smalliana. When grown in xylem fluid from the susceptible species, X. fastidiosa cells formed a heavier biofilm compared to those in xylem fluid from the resistant species. Differential expression of selected genes of X. fastidiosa cultured in the xylem fluids of V. vinifera and V. smalliana was analyzed using a DNA macroarray. Compared with xylem fluid of V. smalliana, xylem fluid of V. vinifera stimulated the expression of X. fastidiosa genes involved in virulence regulation, such as rpfC, gacA, xrvA, gcvR, and cysB, and genes involved in biogenesis of pili and twitching motility, such as pilI, pilU, pilE and pilG. Increased expression of virulence genes likely contributes to the expression of PD symptom in the susceptible grapevines, whereas reduced expression of these genes may lead to limitation of symptoms in resistant grapevines.     

小麦条锈菌cDNA文库构建和表达序列标签(ESTs)分析

 小麦条锈菌(Puccinia striiformis f. sp. tritici)是全世界范围内小麦生产上的重要病原真菌, 但是对小麦条锈菌的基因组和基因功能却了解甚少。为了促进小麦条锈菌基因组学的发展和大规模基因发现, 我们以噬菌体λTrip1Ex2为载体, 采用SMART技术构建了小麦条锈菌萌发夏孢子的cDNA文库。原始文库的滴度为1.1×10⁶pfu/mL, 平均插入片段长度为750 bp。从文库中随机挑取279个cDNA克隆测序, 分析发现这些ESTs的平均GC含量为45.08%。通过聚类分析, 279个ESTs拼接成31个contigs和80 singletons。BLASTx分析表明, 47%的ESTs与GenBank中报道的功能已知或未知蛋白具有相似性。tBLASTx分析表明12个uniseqs与EST数据库中的序列具有相似性, 其中9个是来自担子菌的cDNA文库。几个EST与已知的真菌致病相关基因具有高度相似性。RT-PCR分析了几个基因在小麦条锈菌侵染过程中的表达水平。这些结果为小麦条锈菌夏孢子萌发以及侵染寄主过程中的基因表达研究奠定了很好的分子生物学基础。     

Effect of cultural practices on the development of arabica coffee berry disease,caused by <Emphasis Type="Italic">Colletotrichum kahawae</Emphasis>

In the high altitude regions of Africa, coffee berry disease (CBD), caused by Colletotrichum kahawae, is the main constraint for arabica coffee (Coffea arabica) production. However, certain agricultural practices can reduce losses caused by the disease and thereby promote optimum production. On small family farms in Cameroon, mixed cropping with fruit trees, intercropping with food crops and maintenance pruning of coffee trees are very widespread agricultural practices that can affect CBD epidemics. Consequently, an epidemiological study was conducted to assess how cultural practices affected the disease in an arabica coffee smallholding in Cameroon. The disease was monitored on a weekly basis over four successive years (2002–2005) on coffee trees in diverse cultural situations. Cultural practices likely to reduce losses due to CBD were identified. The infection rate was significantly lower on coffee trees grown intensively than on coffee trees grown in the traditional manner. Coffee trees located under the shade of fruit trees were significantly less attacked than those located in full sunlight. In addition, berries on the leafless parts of branches, near the main trunk of the coffee tree, were less infected than those on leafy sections. These results show that maintenance pruning, removal of mummified berries, and mixed cropping with shade plants are cultural practices which create environmental conditions that limit CBD development.     

Expressed sequence tags from the phytopathogenic fungus Botrytis cinerea

A large set of genes was identified in the phytopathogenic fungus Botrytis cinerea by using an expressed sequence tag approach. The fungus was grown in axenic culture and a cDNA library was produced. From this library, 6559 ESTs were obtained. The combined sequences represent 3026 unisequences that corresponds to approximately one-quarter of the estimated total number of genes in B. cinerea. Approximately 18% of the ESTs showed significant similarities with genes coding for proteins with known functions,~56% were similar to genes coding for proteins with unknown functions and ~26% were orphans. A substantial B. cinerea gene inventory including putative virulence factors was therefore obtained and is now available at the Génoplante-Info Database interface (http://urgi.infobiogen.fr///Projects/GPiDB/Interface/).     

Cloning of a Genomic DNA Encoding Caffeoyl-coenzyme A 3-<Emphasis Type="Italic">O</Emphasis>-methyltransferase of Citrus

In recent studies, we found that Apll (a PthA homologue) bound to three citrus proteins. Amino acid sequence analysis revealed that one of the target proteins was homologous to that of S-adenosyl-l-methionine : trans-caffeoyl-coenzyme A 3-O-methyltransferase (CCoAMT), an enzyme which is specific for the substrate trans-caffeoyl-CoA and catalyzes the synthesis of trans-feruloyl-CoA. From the consensus nucleotide sequences of CCoAMT genes, primers were chosen for PCR amplification of this gene from citrus total DNA. Two selected DNA fragments of 1.0 kb and 2.0 kb were obtained. These fragments were used as the probe to screen a citrus library. One clone, pCCl00, contained a 1.0-kb SalI fragment that hybridized to the probes. The nucleotide sequence of this fragment was determined in both directions. In this fragment, there was an open reading frame of 232 amino acids interrupted by an intron of 106 nt, and the deduced amino acid sequence had 95.9% homology to tobacco CCoAMT. Southern blot analysis of total citrus DNA showed that four EcoRI fragments hybridized to the probes, suggesting the presence of more than one copy of CCoAMT in the citrus DNA. Received 4 November 1999/ Accepted in revised form 26 November 1999     

柑橘大实蝇低覆盖度基因组及其分析

为探究柑橘大实蝇Bactrocera minax的全基因组信息,以海关口岸截获的柑橘大实蝇样本为材料,提取DNA后构建350 bp短片段文库,采用Illumina二代测序平台开展全基因组测序,进行基因组组装、完整性评估和注释分析。结果表明,柑橘大实蝇的基因组大小为368.14 Mb,重复序列占比为16.27%,杂合率为0.79%,属于中等杂合度基因组。基因组组装得到43 124条contigs,contig N50长度为94 994 bp。BUSCO评估显示,组装的基因组可完整覆盖98.80%昆虫保守的单拷贝直系同源基因,表明该组装的完整性很高,可以满足后续分析。通过EVM注释流程整合了从头预测、同源预测和基于转录组预测等不同方法的注释结果,共预测到35 655个蛋白编码基因,其中24 343个基因有功能注释。