Real-Time Polymerase Chain Reaction for One-Hour On-Site Diagnosis of Pierce's Disease of Grape in Early Season Asymptomatic Vines

113

The bacterial pathogen, Xylella fastidiosa, infects many plant species in the Americas, making it a good model for investigating the genetics of host adaptation. We used multilocus sequence typing (MLST) to identify isolates of the native U.S. subsp. multiplex that were largely unaffected by intersubspecific homologous recombination (IHR) and to investigate how their evolutionary history influences plant host specialization. We identified 110 "non-IHR" isolates, 2 minimally recombinant "intermediate" ones (including the subspecific type), and 31 with extensive IHR. The non-IHR and intermediate isolates defined 23 sequence types (STs) which we used to identify 22 plant hosts (73% trees) characteristic of the subspecies. Except for almond, subsp. multiplex showed no host overlap with the introduced subspecies (subspecies fastidiosa and sandyi). MLST sequences revealed that subsp. multiplex underwent recent radiation (<25% of subspecies age) which included only limited intrasubspecific recombination (/ ‫؍‬ 0.02); only one isolated lineage (ST50 from ash) was older. A total of 20 of the STs grouped into three loose phylogenetic clusters distinguished by nonoverlapping hosts (excepting purple leaf plum): "almond," "peach," and "oak" types. These host differences were not geographical, since all three types also occurred in California. ST designation was a good indicator of host specialization. ST09, widespread in the southeastern United States, only infected oak species, and all peach isolates were ST10 (from California, Florida, and Georgia). Only ST23 had a broad host range. Hosts of related genotypes were sometimes related, but often host groupings crossed plant family or even order, suggesting that phylogenetically plastic features of hosts affect bacterial pathogenicity.T he genetic typing of bacterial isolates is an increasingly important tool for understanding the epidemiology of pathogenic bacteria. It permits us to track biogeographical patterns, host specificity, and the evolutionary changes occurring within taxa. To achieve these goals, one of the most useful and widely used typing methodologies is multilocus sequence typing (MLST), introduced in 1998 (1, 2). The spread of this methodology has been rapid in the study of human bacterial pathogens; however, MLST schemes are equally important in the study of plant pathogens (3), and schemes for plant bacteria are beginning to become established, e.g., Pseudomonas syringae (4), Xylella fastidiosa (5, 6), fruit tree phytoplasmas (7), Ralstonia solanacearum (8), Xanthomonas spp. (9), and Acidovorax citrulli (10).The genetic typing of plant pathogens provides a critical first step in answering the important question of what genetic factors determine host specificity. Genome comparison among species and pathovars of Xanthomonas suggest specificity is determined by a complex set of genetic differences involving both gene content and gene sequence (11). Of these two kinds of difference, content and sequence, gene content differences are more easily studied, since it...

Section: Methodsmentioning

confidence: 99%

Recent Evolutionary Radiation and Host Plant Specialization in the Xylella fastidiosa Subspecies Native to the United States

Nunney

Vickerman

Bromley

et al. 2013

113

“…A nested real-time PCR assay for P. ramorum using SYBR green (a double-stranded DNAbinding dye [as DNA is amplified, more dye is bound and thus fluoresces]) has been described (14), and an assay using molecular beacons (probes containing reporter and quencher dyes which hybridize to the amplified product, resulting in increased fluorescence) is in development (5). Real-time PCR methods based on TaqMan chemistry (amplification-dependent cleavage of probes incorporating reporter and quencher dyes, resulting in increased fluorescence) have the particular advantage of requiring no postamplification steps and therefore involve a reduced risk of cross-contamination, and they have been described for a wide range of plant pathogens (26,30,32,33,34). A single-round TaqMan PCR assay for the detection of P. ramorum has recently been developed which compares extremely favorably with morphological methods of identification (K. J. D. Hughes, R. L. Griffin, J.…”

mentioning

confidence: 99%

“…system and RAZOR instrument (Idaho Technologies, Salt Lake City, UT), and the BioSeeq instrument (Smiths Detection, Edgewood, MD), which are designed for on-site molecular testing. Use of the SmartCycler has been described for on-site detection of the bacterium Xylella fastidiosa in grape plants (30) directly from sap and macerated chips of xylem. The molecular detection of fungal pathogens in plant material, however, requires the extraction of DNA (29), so on-site molecular testing demands not only a portable realtime PCR platform and suitable assay but also a simple and robust DNA extraction method which can be performed in the field.…”

mentioning

confidence: 99%

On-Site DNA Extraction and Real-Time PCR for Detection of Phytophthora ramorum in the Field

Tomlinson¹,

Boonham²,

Hughes³

et al. 2005

119

Phytophthora ramorum is a recently described pathogen causing oak mortality (sudden oak death) in forests in coastal areas of California and southern Oregon and dieback and leaf blight in a range of tree, shrub, and herbaceous species in the United States and Europe. Due to the threat posed by this organism, stringent quarantine regulations are in place, which restrict the movement of a number of hosts. Fast and accurate diagnostic tests are required in order to characterize the distribution of P. ramorum, prevent its introduction into pathogen-free areas, and minimize its spread within affected areas. However, sending samples to a laboratory for testing can cause a substantial delay between sampling and diagnosis. A rapid and simple DNA extraction method was developed for use at the point of sampling and used to extract DNAs from symptomatic foliage and stems in the field. A sensitive and specific single-round real-time PCR (TaqMan) assay for P. ramorum was performed using a portable real-time PCR platform (Cepheid SmartCycler II), and a costeffective method for stabilizing PCR reagents was developed to allow their storage and transportation at room temperature. To our knowledge, this is the first description of a method for DNA extraction and molecular testing for a plant pathogen carried out entirely in the field, independent of any laboratory facilities.Phytophthora ramorum is the causal agent of extensive oak mortality (commonly known as sudden oak death) in coastal forests in California (27) and southern Oregon (12,25). This pathogen also causes ramorum leaf blight and dieback on a range of other plant species (9) and can have a profound and devastating effect on forest ecosystems. A distinct population of the same pathogen (6, 35) is found in a number of European countries (10,20,23,37), mostly causing dieback and leaf blight on a range of ornamental plants in nurseries and landscaped areas (2). There have also recently been a number of incidences of lethal bark cankers caused by P. ramorum in native and nonnative trees in Europe (7). P. ramorum has a broad and expanding host range (3,4,10,11,19,20,28,31), and as a result of the threat posed to forest ecosystems, the movement of a variety of its host species is subject to restrictions in Europe and the United States. Emergency European Community phytosanitary measures for P. ramorum were introduced in 2002 (1), and in the United States quarantine restrictions at both the federal and state levels control the movement of a variety of plant species from infested areas in California and Oregon (31). The availability of rapid and accurate detection methods for P. ramorum is critical to allow its prevalence to be monitored and to expedite management or eradication steps to prevent its introduction and minimize its spread.The identification of P. ramorum is not possible based on host symptoms alone due to the considerable variation in their expression and because a range of other causes can produce similar symptoms. These include infections by several other Phyt...

“…Tomlinson and coworkers (4, 10) developed on-site, loop-mediated isothermal amplification for phytoplasmas and SmartCycler protocols for Phytophthora ramorum. The SmartCycler has been used also for Xylella fastidiosa detection (12). Razor Ex BioDetection system-based methods are more rapid than those employing the SmartCycler (10,12,14).…”

Section: Discussionmentioning

confidence: 99%

“…Simple on-site DNA purification and PCR protocols for accurate pathogen identification would facilitate diagnosis and regulation, as well as disease monitoring and management. Ideally, these protocols would use a portable, battery-operated real-time qPCR platform designed for on-site molecular testing, which allows plant pathogen detection by minimally trained operators in the absence of laboratory facilities and conditions, including electricity, centrifuges, liquid nitrogen, water (12) and for diagnosis of foot-and-mouth disease (13). These methods take about 2 h and detect a single target gene.…”

mentioning

confidence: 99%

Development of a Rapid, Sensitive, and Field-Deployable Razor Ex BioDetection System and Quantitative PCR Assay for Detection of Phymatotrichopsis omnivora Using Multiple Gene Targets

Arif

Fletcher

Marek

et al. 2013

A validated, multigene-based method using real-time quantitative PCR (qPCR) and the Razor Ex BioDetection system was developed for detection of Phymatotrichopsis omnivora. This soilborne fungus causes Phymatotrichopsis root rot of cotton, alfalfa, and other dicot crops in the southwestern United States and northern Mexico, leading to significant crop losses and limiting the range of crops that can be grown in soils where the fungus is established. It is on multiple lists of regulated organisms. Because P. omnivora is difficult to isolate, accurate and sensitive culture-independent diagnostic tools are needed to confirm infections by this fungus. Specific PCR primers and probes were designed based on P. omnivora nucleotide sequences of the genes encoding rRNA internal transcribed spacers, beta-tubulin, and the second-largest subunit of RNA polymerase II (RPB2). PCR products were cloned and sequenced to confirm their identity. All primer sets allowed early detection of P. omnivora in infected but asymptomatic plants. A modified rapid DNA purification method, which facilitates a quick (ϳ30-min) on-site assay capability for P. omnivora detection, was developed. Combined use of three target genes increased the assay accuracy and broadened the range of detection. To our knowledge, this is the first report of a multigene-based, field-deployable, rapid, and reliable identification method for a fungal plant pathogen and should serve as a model for the development of field-deployable assays of other phytopathogens.