Assessment of the diagnostic potential of Immmunocapture-PCR and Immuno-PCR for Citrus Variegated Chlorosis

Xylella fastidiosa causes significant losses in many economically important crops. An efficient pathogen detection system is critical for epidemiology studies, particularly when large sample size is involved. In this study we report the development of immunomolecular assays like Immmunocapture-PCR and Immuno-PCR for direct detection of X. fastidiosa without DNA isolation. Whereas the reactivity of ELISA and PCR ranged from 10(6) to 10(4) bacterial cells, the IC-PCR sensitivity was up to 10(3) and the detection limit of I-PCR was up to 10(1) bacterial cells. These methods can use either plant sample extracts or cultivated media, and show no cross reaction for any other endophytic citrus-bacteria. Therefore, IC-PCR and I-PCR assays provide an alternative for quick and very sensitive methods to screening X. fastidiosa, with the advantage of not requiring any concentration or DNA purification steps while still allowing an accurate diagnosis of CVC.     

A Triply Cloned Strain of Xylella fastidiosa Multiplies and Induces Symptoms of Citrus Variegated Chlorosis in Sweet Orange

Xylella fastidiosa isolate 8.1.b obtained from a sweet orange tree affected by citrus variegated chlorosis in the state of S?o Paulo, Brazil, and shown in 1993 to be the causal agent of the disease, was cloned by repeated culture in liquid and on solid PW medium, yielding triply cloned strain 9a5c. The eighth and the 16th passages of strain 9a5c were mechanically inoculated into sweet orange plants. Presence of X. fastidiosa in sweet orange leaves of shoots having grown after inoculation (first-flush shoots) was detected by DAS-ELISA and PCR. Thirty-eight days after inoculation, 70% of the 20 inoculated plants tested positive, and all plants gave strong positive reactions 90 days after inoculation. Symptoms first appeared after 3 months and were conspicuous after 5 months. X. fastidiosa was reisolated from sweet orange leaves, 44 days after inoculation. These results indicate that X. fastidiosa strain 9a5c, derived from pathogenic isolate 8.1.b by triply cloning, is also pathogenic. Strain 9a5c is now used for the X. fastidiosa genome sequencing project undertaken on a large scale in Brazil. Received: 1 February 1999 / Accepted: 1 April 1999     

Potential vectors of Xylella fastidiosa: a study of leafhoppers and treehoppers in citrus agroecosystems affected by Citrus Variegated Chlorosis

This study investigated the predominant leafhopper and treehopper (Hemiptera, Auchenorrhyncha) species in Citrus Variegated Chlorosis (CVC)‐affected citrus agroecosystems in Argentina, their seasonal fluctuation, and their potential role as vectors of Xylella fastidiosa Wells et al., using molecular methods for detection. More than 6 000 Auchenorrhyncha were collected from three citrus agroecosystems over a period of 3 years using yellow sticky traps and entomological nets. Cicadellidae and Membracidae were the most abundant families. Of the 43 species identified, five were predominant in citrus orchards, and three were predominant in weeds surrounding citrus plants. All predominant species and another four non‐predominant species tested positive for X. fastidiosa in PCR and real‐time PCR assays. In a transmission assay, Dechacona missionum (Berg), Tapajosa rubromarginata (Signoret), and Cyphonia clavigera (Fabricius) transmitted X. fastidiosa successfully. Scaphytopius bolivianus Oman and Frequenamia spiniventris (Linnavuori) populations increased once (during the summer), possibly due to favorable weather conditions, and Bucephalogonia xanthophis (Berg), Molomea lineiceps Young, and T. rubromarginata populations increased twice a year: once in summer and once in winter, coinciding with the increase in early citrus shoots (flush). Among the X. fastidiosa‐positive species, those with the higher population densities during the sprouting period, where trees are highly susceptible to infection, must be considered as most relevant vectors of CVC in the citrus‐growing areas in Argentina.     

Sterols of Mulberry Leaves and Small Leaf Curl Disease

Free and bound sterols of leaves of five mulberry cultivars differing in their susceptibility to small leaf curl disease have been studied. The total content of sterols in all samples is similar and is not correlated with the resistance of the cultivars. The qualitative composition of particular sterols is also identical. They are represented by cholesterol, campesterol, stigmasterol, sitosterol, and two 4α-methylsterols. The leaves of the most sensitive cultivar are characterized by high cholesterol content. The ratio sitosterol : stigmasterol decreased in proportion to the resistance level of a cultivar.__________Translated from Prikladnaya Biokhimiya i Mikrobiologiya, Vol. 41, No. 4, 2005, pp. 460–462.Original Russian Text Copyright © 2005 by Zambakhidze, Sulaberidze, Mzhavanadze, Tsiklauri.     

Enhanced Reliability and Accuracy for Field Deployable Bioforensic Detection and Discrimination of Xylella fastidiosa subsp. pauca,Causal Agent of Citrus Variegated Chlorosis Using Razor Ex Technology and TaqMan Quantitative PCR

A reliable, accurate and rapid multigene-based assay combining real time quantitative PCR (qPCR) and a Razor Ex BioDetection System (Razor Ex) was validated for detection of Xylella fastidiosa subsp. pauca (Xfp, a xylem-limited bacterium that causes citrus variegated chlorosis [CVC]). CVC, which is exotic to the United States, has spread through South and Central America and could significantly impact U.S. citrus if it arrives. A method for early, accurate and sensitive detection of Xfp in plant tissues is needed by plant health officials for inspection of products from quarantined locations, and by extension specialists for detection, identification and management of disease outbreaks and reservoir hosts. Two sets of specific PCR primers and probes, targeting Xfp genes for fimbrillin and the periplasmic iron-binding protein were designed. A third pair of primers targeting the conserved cobalamin synthesis protein gene was designed to detect all possible X. fastidiosa (Xf) strains. All three primer sets detected as little as 1 fg of plasmid DNA carrying X. fastidiosa target sequences and genomic DNA of Xfp at as little as 1 - 10 fg. The use of Razor Ex facilitates a rapid (about 30 min) in-field assay capability for detection of all Xf strains, and for specific detection of Xfp. Combined use of three primer sets targeting different genes increased the assay accuracy and broadened the range of detection. To our knowledge, this is the first report of a field-deployable rapid and reliable bioforensic detection and discrimination method for a bacterial phytopathogen based on multigene targets.     

Pierce's Disease of Grapevines in Taiwan: Isolation,Cultivation and Pathogenicity of Xylella fastidiosa

Characteristic symptoms of Pierce's disease (PD) in grapevines (Vitis vinifera L.) were observed in 2002 in the major grape production fields of central Taiwan. Disease severity in vineyards varied, and all investigated grape cultivars were affected. Diseased tissues were collected from fields for subsequent isolation and characterization of the causal agent of the disease (Xylella fastidiosa). Koch's postulates were fulfilled by artificially inoculating two purified PD bacteria to grape cultivars Kyoho, Honey Red and Golden Muscat. The inoculated plants developed typical leaf‐scorching symptoms, and similar disease severity developed in the three cultivars from which the bacterium was readily re‐isolated, proving that the leaf scorch of grapevines in Taiwan is caused by the fastidious X. fastidiosa. This confirmed PD of grapevines is also the first report from the Asian Continent. Phylogenetic analyses were performed by comparing the 16S rRNA gene and 16S‐23S rRNA internal transcribed spacer region (16S‐23S ITS) of 12 PD strains from Taiwan with the sequences of 13 X. fastidiosa strains from different hosts and different geographical areas. Results showed that the PD strains of Taiwan were closely related to the American X. fastidiosa grape strains but not to the pear strains of Taiwan, suggesting that the X. fastidiosa grape and pear strains of Taiwan may have evolved independently from each other.     

Roditis Leaf Discoloration - A New Virus Disease of Grapevine: Symptomatology and Transmission to Indicator Plants

The symptoms and some characteristics of an unreported disease of grapevine, which was observed during the last years on the cv. ‘Roditis’ in central Greece, are described. The disease is transmissible to V. vinifera‘Mission’ and to some herbaceous test plants. The constant association of a virus to naturally infected plants cv. ‘Roditis’ and chip-budding infected indicator plants cv. ‘Mission’ supports the evidence that the disease is caused by a virus.     

Whole Genome Sequences of Two Xylella fastidiosa Strains (M12 and M23) Causing Almond Leaf Scorch Disease in California

Xylella fastidiosa is a Gram-negative plant-pathogenic bacterium causing many economically important diseases, including almond leaf scorch disease (ALSD) in California. Genome information greatly facilitates research on this nutritionally fastidious organism. Here we report the complete genome sequences of two ALSD strains of this bacterium, M12 and M23.Xylella fastidiosa is a Gram-negative and nutritionally fastidious plant-pathogenic bacterium that causes almond leaf scorch disease (ALSD) and Pierce''s disease (PD) of grapevine. In 2003, we isolated two ALSD strains of X. fastidiosa from almond trees in Kern County in the San Joaquin Valley of California. Strain M12 caused only ALSD, and strain M23 caused both ALSD and PD. 16S rRNA gene sequences were analyzed; strain M12 was regarded as A genotype and strain M23 as G genotype (1), corresponding to X. fastidiosa subsp. multiplex and X. fastidiosa subsp. fastidiosa (4), respectively.Genomic DNAs of X. fastidiosa strains M12 and M23 were extracted from pure culture in PW medium (1). The random shotgun method was used for genome sequencing. Large-insert (40-kb), medium-insert (8-kb), and small-insert (3-kb) random libraries were partially sequenced, and sequences were assembled with parallel Phrap (High Performance Software, LLC). Possible misassemblies were corrected with the Dupfinisher software program (2). Gaps between contigs were closed by custom primer walking through PCR amplification. Annotation of the assembled genome sequence was carried out with the genome annotation system Oak Ridge Genome Annotation and Analysis (ORGAA) Pipelines and JGI Integrated Microbial Genomes (IMG) server (3). A combined gene prediction strategy was applied by means of the GLIMMER 2.0 system and the CRITICA program suite, along with postprocessing by the RBSfinder tool. tRNA genes were identified using the tRNAscan-SE server. The deduced proteins were functionally characterized by automated searches in public databases, including SWISS-PROT and TrEMBL, Pfam, TIGRFAM, InterPro, and KEGG. Additionally, the SignalP, helix-turn-helix, and TMHMM software programs were applied. Finally, each gene was functionally classified by assigning a clusters of orthologous groups (COG) number and corresponding COG category and gene ontology numbers. Detailed information about the genome properties, genome annotation, and its related references can be obtained from the JGI Integrated Microbial Genomes website at http://img.jgi.doe.gov/pub/.The genome of X. fastidiosa M12 consists of a single, circular, 2,475,130-nucleotide (nt) chromosome that has a GC content of 51.9%. A total of 2,368 protein-encoding genes are predicted, 2,104 of which have been assigned a tentative function. The genome of X. fastidiosa M23 consists of a single, circular, 2,535,690-nt chromosome that has a GC content of 51.7%. A total of 2,280 protein-encoding genes are predicted, 2,161 of which have been assigned a tentative function. In addition, a circular plasmid of 38,297 nt, pXFAS01, with a GC content of 49%, was also identified in strain M23 but was absent in strain M12. Both strains had two identical rRNA operons in their chromosomes.     

Diurnal Changes in Citrus Leaf Thickness, Leaf Water Potential and Leaf to Air Temperature Difference

Diurnal changes in leaf water potential and leaf thickness ofwell-watered citrus trees were found to be highly correlated.Midday decreases in leaf thickness of about 30–35 µm reflected midday decreases in leaf water potential of about1.1–1.3 MPa from predawn values. Leaf water potentialwas also correlated with changes in leaf-to-air temperaturedifference and ambient vapour pressure deficit. Leaf thicknessas well as leaf to air temperature difference could possiblybe used to monitor leaf water status continuously as an indicatorof citrus tree water stress.     

Development and Application of Monoclonal Antibodies Against Xylella fastidiosa, the Causal Bacterium of Pear Leaf Scorch

Ten stable hybridoma cell lines, M As l -10, secreting monoclonal antibodies specific to the causal bacterium of pear leaf scorch (PLS), Xylella fastidiosa. were produced. The monoclonal antibodies can detect 3 × 10⁵ PLS-bacterium cells by indirect enzyme-linked immunosorbent assay (ELISA). In the antibody titer determination by indirect ELISA, hybridoma-culture supernatant from clone MA4 had the highest titer of 20480. In the antibody specificity tests, nine of the 10 monoclonal antibodies did not cross-react with 14 other bacterial strains belonging to nine genera. Only the antibody from hybridoma clone MAI cross-reacted with Xanthomonas campestris pv. cam-pestris and X. campestris pv. vesicatoria. In western blot analysis, all the monoclonal antibodies recognized the major 46.9-kDa polypeptide from all 12 X. fastidiosa strains and a distinct 21.5-kDa polypeptide only from PLS bacterium. In tissue-blotting detection, the PLS bacteria were specifically detected in blots of tissue sections from infected pear with the antibodies developed.     

Genetic Diversity of Pierce's Disease Strains and Other Pathotypes of Xylella fastidiosa

Strains of Xylella fastidiosa isolated from grape, almond, maple, and oleander were characterized by enterobacterial repetitive intergenic consensus sequence-, repetitive extragenic palindromic element (REP)-, and random amplified polymorphic DNA (RAPD)-PCR; contour-clamped homogeneous electric field (CHEF) gel electrophoresis; plasmid content; and sequencing of the 16S-23S rRNA spacer region. Combining methods gave greater resolution of strain groupings than any single method. Strains isolated from grape with Pierce's disease (PD) from California, Florida, and Georgia showed greater than previously reported genetic variability, including plasmid contents, but formed a cluster based on analysis of RAPD-PCR products, NotI and SpeI genomic DNA fingerprints, and 16S-23S rRNA spacer region sequence. Two groupings of almond leaf scorch (ALS) strains were distinguished by RAPD-PCR and CHEF gel electrophoresis, but some ALS isolates were clustered within the PD group. RAPD-PCR, CHEF gel electrophoresis, and 16S-23S rRNA sequence analysis produced the same groupings of strains, with RAPD-PCR resolving the greatest genetic differences. Oleander strains, phony peach disease (PP), and oak leaf scorch (OLS) strains were distinct from other strains. DNA profiles constructed by REP-PCR analysis were the same or very similar among all grape strains and most almond strains but different among some almond strains and all other strains tested. Eight of 12 ALS strains and 4 of 14 PD strains of X. fastidiosa isolated in California contained plasmids. All oleander strains carried the same-sized plasmid; all OLS strains carried the same-sized plasmid. A plum leaf scald strain contained three plasmids, two of which were the same sizes as those found in PP strains. These findings support a division of X. fastidiosa at the subspecies or pathovar level.     

Investigations on Transmission of Grapevine Leafroll, Yellow Speckle and Fleck Diseases by Dodder

Attempts were made to transmit the infective agents of leafroll, yellow speckle, and fleck diseases from infected grapevmes to several herbaceous species and to healthy vines using dodder (Cuscuta campestris). Dodder transmitted leafroll to all and fleck to half of the respective receptor vines, but none of the three dieases were transmitted to herbaceous plants including Chenopodium, Gomphrena and Nicotiana species, Cowpea, Bean, Cucumber, Squash and Petunia. Suggested transmission of yellow speckle to vine was inconclusive because this disease may have been spread naturally.     

Structural,Functional, and Evolutionary Analysis of the Unusually Large Stilbene Synthase Gene Family in Grapevine

Stilbenes are a small family of phenylpropanoids produced in a number of unrelated plant species, including grapevine (Vitis vinifera). In addition to their participation in defense mechanisms in plants, stilbenes, such as resveratrol, display important pharmacological properties and are postulated to be involved in the health benefits associated with a moderate consumption of red wine. Stilbene synthases (STSs), which catalyze the biosynthesis of the stilbene backbone, seem to have evolved from chalcone synthases (CHSs) several times independently in stilbene-producing plants. STS genes usually form small families of two to five closely related paralogs. By contrast, the sequence of grapevine reference genome (cv PN40024) has revealed an unusually large STS gene family. Here, we combine molecular evolution and structural and functional analyses to investigate further the high number of STS genes in grapevine. Our reannotation of the STS and CHS gene families yielded 48 STS genes, including at least 32 potentially functional ones. Functional characterization of nine genes representing most of the STS gene family diversity clearly indicated that these genes do encode for proteins with STS activity. Evolutionary analysis of the STS gene family revealed that both STS and CHS evolution are dominated by purifying selection, with no evidence for strong selection for new functions among STS genes. However, we found a few sites under different selection pressures in CHS and STS sequences, whose potential functional consequences are discussed using a structural model of a typical STS from grapevine that we developed.Plants produce a vast array of secondary metabolites, many of them being restricted to specific groups of plant species. This extraordinary chemical diversity is believed to have evolved from a limited number of ubiquitous biosynthetic pathways through gene duplication followed by functional divergence (Pichersky and Gang, 2000). The phenylpropanoid pathway, derived from Phe, illustrates perfectly this phenomenon, as it gives rise to a large diversity of phenolic compounds playing key roles in plants, including participation in structural polymers, defense against herbivores and pathogens, protection from abiotic stress, and important functions in plant-pollinator interactions. Stilbenes are a small family of phenylpropanoids produced in a number of unrelated plant species, including dicotyledon angiosperms such as grapevine (Vitis vinifera), peanut (Arachis hypogaea), and Japanese knotweed (Fallopia japonica, formerly Polygonum cuspidatum), monocotyledons like sorghum (Sorghum bicolor), and gymnosperms such as several Pinus and Picea species. In addition to their participation in both constitutive and inducible defense mechanisms in plants, several stilbenes display important pharmacological properties. Since resveratrol (3,5,4′-trihydroxy-trans-stilbene) was postulated to be involved in the health benefits associated with a moderate consumption of red wine (Renaud and de Lorgeril, 1992), plant stilbenes have received considerable interest. Nowadays, resveratrol ranks among the most extensively studied natural products, and hundreds of studies have shown that it can slow the progression of a wide variety of illnesses, including cancer and cardiovascular disease, as well as extend the life spans of various organisms (Baur and Sinclair, 2006). Stilbene synthases (STSs) are characteristic of stilbene-producing plants and catalyze the biosynthesis of the stilbene backbone from three malonyl-CoA and one CoA-ester of a cinnamic acid derivative. STSs are members of the type III polyketide synthases family, chalcone synthases (CHSs), which catalyze the first step of flavonoid biosynthesis, being the most ubiquitous polyketide synthase in plants. Both CHS and STS use p-coumaroyl-CoA and malonyl-CoA as substrates and synthesize the same linear tetraketide intermediate. However, STS uses a specific cyclization mechanism involving a decarboxylation to form the stilbene backbone. STS proteins share extensive amino acid sequence identity with CHS, and phylogenetic analysis of the STS and CHS gene families has shown that STS genes may have evolved from CHS genes several times independently (Tropf et al., 1994). In most stilbene-producing plants, STS genes form small families of closely related paralogs. For example, two STS cDNAs have been cloned from peanut (Schröder et al., 1988), the genome of Scots pine (Pinus sylvestris) has been shown to contain a small family of four STS genes (Preisig-Müller et al., 1999), and three STS genes have been characterized in Japanese red pine (Pinus densiflora; Kodan et al., 2002). Only one STS gene has been isolated from Japanese knotweed to date (Liu et al., 2011), and the sequencing of sorghum genome has shown that SbSTS1 was the only STS gene in this plant species (Yu et al., 2005; Paterson et al., 2009). Grapevine is a noteworthy exception among stilbene-producing plants, as its genome has been shown to contain a large family of putative STS genes. Early Southern-blot experiments suggested that the grapevine genome contained more than 20 STS genes (Sparvoli et al., 1994). Analyses of the first drafts of the grapevine genome sequence confirmed the large size of this multigene family, with an estimated number of STS genes ranging from 21 to 43 (Jaillon et al., 2007; Velasco et al., 2007). However, these relatively low-coverage sequence drafts did not allow a precise analysis of large families of highly similar genes. The more recently released 12× genome sequence of grapevine inbred Pinot Noir cultivar PN40024 offered an improved sequence quality, allowing an accurate analysis of the STS gene family. In this work, we take advantage of the improved 12× sequence of the grapevine ‘PN40024’ genome to analyze the grapevine STS gene family. Furthermore, we combine molecular evolution to structural and functional analyses to gain more insight into the significance of the remarkable amplification of the STS family in grapevine.