Correction: Development and validation of X-ComEC qPCR, a novel assay for accurate universal detection of both Xylella fastidiosa and Xylella taiwanensis

Xylella fastidiosa is a devastating plant pathogenic bacteria known for its broad host range, in contrast to the related species Xylella taiwanensis , which is only known to cause disease in Asian pears. Despite the potential threats they pose to Australian agriculture, diagnostic assays capable of detecting both Xylella species are scarce. Bridging this critical gap, this study presents the development of the X- ComEC qPCR assay that targets a genus-specific DNA sequence, enabling accurate generic detection of all Xylella species. Benchmarking this novel qPCR assay against other published Xylella qPCR assays demonstrated its superior performance. The X- ComEC qPCR assay stands out as the only assay that can accurately detect both X. fastidiosa and X. taiwanensis without cross-reactivity with related bacteria. We have also carried out a comprehensive inter-laboratory test performance study, which demonstrated that the X- ComEC qPCR and the qPCR described by Harper et al. (Development of LAMP and real-time PCR methods for the rapid detection of Xylella fastidiosa for quarantine and field applications; erratum 2013) are highly robust and ready to use in Australia. Combining these two assays into a duplex qPCR enables simultaneous detection and species-level identification of X. fastidiosa and X. taiwanensis. The findings of this study have been incorporated into the Australian National Diagnostic Protocol for Xylella detection, arming diagnostic laboratories with critical knowledge to combat these globally significant pathogens. Overall, the collaborative and systematic approach employed in this study provides a model for developing and validating assays for all plant pathogens.

Probe-based real-time PCR method for multilocus melt typing of Xylella fastidiosa strains

Xylella fastidiosa is a regulated plant pathogen in many parts of the world. To increase diagnostic capability of X. fastidiosa in the field, a loop-mediated isothermal amplification (LAMP) and real-time polymerase chain reaction (PCR) assay were developed to the rimM gene of X. fastidiosa and evaluated for specificity and sensitivity. Both assays were more robust than existing published assays for detection of X. fastidiosa when screened against 20 isolates representing the four major subgroups of the bacterium from a range of host species. No cross-reaction was observed with DNA from healthy hosts or other bacterial species. The LAMP and real-time assays could detect 250 and 10 copies of the rimM gene, respectively, and real-time sensitivity was comparable with an existing published real-time PCR assay. Hydroxynapthol blue was evaluated as an endpoint detection method for LAMP. When at least 500 copies of target template were present, there was a noticeable color change indicating the presence of the bacterium. Techniques suitable for DNA extraction from plant tissue in situ were compared with a standard silica-column-based laboratory extraction method. A portable PickPen and magnetic bead system could be used to successfully extract DNA from infected tissue and could be used in conjunction with LAMP in the field.

Development of single chain variable fragment (scFv) antibodies against Xylella fastidiosa subsp. pauca by phage display

The American elm (Ulmus americana) is a highly desirable deciduous shade tree with moderately dense foliage and a symmetrical broad or upright vase-shaped crown. This plant is susceptible to various diseases because of rapid terminal growth when new springwood vessels are fully functional. The American elm, along with many other shade tree species, can be affected by Xylella fastidiosa (Hearon et al. 1980), and X. fastidiosa has been previously confirmed on American elm in Ontario (Goodwin and Zhang 1997), Washington, D.C. (Harris and Balci 2015), Oklahoma (Olson et al. 2006), and Alabama (Parker et al. 2012). X. fastidiosa colonizes the xylem vessels of plant hosts and can cause bacterial leaf scorch disease (Pearson et al. 1998). On elm, X. fastidiosa causes leaf discoloration and browning, marginal scorching, and dieback. In the summer of 2019, chlorosis and marginal leaf scorch symptoms consistent with those of bacterial leaf scorch disease were observed on an American elm tree in a nursery in Pulaski County, Georgia. This elm tree was propagated from cuttings taken from a mature tree in Houston County, Georgia. Initial identification of the disease was performed by assessing characteristic symptoms of infection like leaf scorch, dieback signs, and so on, as described in Hernandez-Martinez et al. (2006). X. fastidiosa was verified via molecular and serological methods. Genomic DNA was extracted from the petioles and midribs of symptomatic leaves using the DNeasy Plant Kit (Qiagen, Valencia, CA), and real-time PCR was performed in a Cepheid smart cycler II (Sunnyvale, CA) using iQ SYBR Green Supermix (BioRad Laboratories, Hercules, CA). A 25-µl reaction was prepared according to the manufacturer’s protocol with primers XF-F/XF-R, which target the 16s rRNA processing protein (Harper et al. 2010). Real-time PCR results confirmed the presence of X. fastidiosa in all symptomatic leaves. The recombinase-polymerase-amplification technology-based AmplifyRP Acceler8 end-point detection assay (Agdia, Elkhart, IN) was carried out on symptomatic tissue using according to the manufacturer’s instructions, and this assay also confirmed the presence of X. fastidiosa in all symptomatic leaves (Waliullah et al. 2019). For further confirmation, the remaining petiole tissue was tested for X. fastidiosa using the DAS-ELISA Reagent Set for X. fastidiosa (Agdia) with minor modifications to the manufacturer’s protocol. In addition to tests of the symptomatic plant, testing of leaves from five additional young elm trees from the same nursery was also carried out according to the same procedures. These five visually healthy young elms tested negative for X. fastidiosa based upon all three testing methods. In total, three independent tests confirmed the presence of X. fastidiosa in symptomatic elm tissues, whereas asymptomatic elm trees from the same nursery tested negative. Attempts to isolate the bacteria from symptomatic leaf tissue on periwinkle wilt media (Davis et al. 1981) were not successful; however, the strong association of symptoms with the thrice-confirmed presence of X. fastidiosa in symptomatic tissue clearly indicates the role of X. fastidiosa in the observed symptoms. Although X. fastidiosa is most commonly transmitted by xylem-feeding insects, cuttings taken from infected hosts can produce infected plants; however, the mature tree used for propagation in this case could not be located for testing. To the best of our knowledge, this is the first report of X. fastidiosa associated with the American elm in Georgia. The presence of X. fastidiosa in American elm has the potential to impact homeowners, landscapers, and nursery producers within Georgia, and our findings suggest that Georgia tree nursery growers should monitor their nursery stock for bacterial leaf scorch disease.

The U.S. agricultural sector is vulnerable to intentionally introduced microbial threats because of its wide and open distribution and economic importance. To investigate such events, forensically valid assays for plant pathogen detection are needed. In this work, real-time PCR assays were developed for three model plant pathogens: Pseudomonas syringae pathovar tomato, Xylella fastidiosa, and Wheat streak mosaic virus. Validation included determination of the linearity and range, limit of detection, sensitivity, specificity, and exclusivity of each assay. Additionally, positive control plasmids, distinguishable from native signature by restriction enzyme digestion, were developed to support forensic application of the assays. Each assay displayed linear amplification of target nucleic acid, detected 100fg or less of target nucleic acid, and was specific to its target pathogen. Results obtained with these model pathogens provide the framework for development and validation of similar assays for other plant pathogens of high consequence.

Bacterial leaf scorch (BLS), caused by Xylella fastidiosa (Xf), is a prevalent disease of blueberries in the southeastern United States. Initially, this disease was reported to be caused by X. fastidiosa subsp. multiplex (Xfm). However, a recent survey revealed the presence of another subspecies, X. fastidiosa subsp. fastidiosa (Xff), within naturally infected blueberry plantings in Georgia. Since knowledge regarding the origins of isolates causing Xf outbreaks can impact management recommendations, a routine method for identifying the pathogen at the subspecies level can be beneficial. Several detection strategies are available to identify Xf infection at the subspecies level. However, none of these have been developed for the routine and rapid differentiation of the blueberry-infecting Xf subspecies. Here, we developed two separate straightforward and rapid detection techniques, a cleaved amplified polymorphic sequence (CAPS) marker, and a loop-mediated isothermal amplification (LAMP) assay, targeting the RNA polymerase sigma-70 factor (rpoD) gene sequence of Xfm to discriminate between the two Xf subspecies infecting blueberry. With the CAPS marker, specific detection of Xfm isolates was possible from pure cultures, inoculated greenhouse-grown plant samples, and field infected blueberry samples by restriction digestion of the rpoD gene PCR product (amplified with primers RST31 and RST33) using the BtsI enzyme. The LAMP assay allowed for specific real-time amplification of a 204-bp portion of the XfmrpoD gene from both pure bacterial cultures and infected plant material using the Genie® III system, a result further affirmed by gel electrophoresis and SYBR™ Green I DNA staining for visual observation. These detection strategies have the potential to greatly aid existing diagnostic methods for determining the distribution and prevalence of these Xf subspecies causing bacterial leaf scorch (BLS) in blueberries in the southeastern United States.

Phytoplasmas and Xylella spp. are bacteria that cause many economically important plant diseases worldwide. TaqMan probe-based quantitative real-time polymerase chain reaction (qPCR) assays have been utilized to universally detect phytoplasmas or Xylella fastidiosa. To develop a superior universal qPCR method, we used a dual priming oligonucleotide (DPO) with two annealing sites as a reverse primer to target the well-conserved bacterial 16S rDNA. The new qPCR assays universally detected various species of phytoplasmas and subspecies of X. fastidiosa as well as Xylella taiwanensis, and generally showed superior threshold cycle values when amplifying specific or non-specific products compared to current universal qPCR assays. The proposed qPCR assays were integrated to develop a multiplex qPCR assay that simultaneously detected phytoplasmas, Xylella spp., and an internal plant DNA positive control within 1 hour. This assay could detect a minimum of ten bacterial cells and was compatible with crude extractions used in the rapid screening of various plants. The amplicons were of sufficient lengths to be directly sequenced for preliminary identification, and the primers could be used in universal conventional PCR assays. Additionally, reverse DPO primers can be utilized to improve other probe-based qPCR assays.

Dickeya solani one of the most aggressive pectinolytic phytopathogens, causes blackleg disease in potatoes, resulting in significant economic losses and adversely impacting one of the world's most important food crops. The diagnostics methods are critical in monitoring the latent infection for international trade of potato seed tubers and in implementation of control strategies. Our study employed a whole-genome comparative approach, identifying unique target gene loci (LysR and TetR family of transcriptional regulators gene regions) and designing loop-mediated isothermal amplification (LAMP) and a multi-gene-based multiplex TaqMan qPCR assays for specific detection and differentiation of D. solani. Both methods underwent meticulous validation with extensive inclusivity and exclusivity panels, exhibiting 100% accuracy and no false positives or negatives. The LAMP method demonstrated the detection limit of 100 fg and 1 CFU per reaction using pure genomic DNA and crude bacterial cell lysate, respectively. The qPCR detection limit was 1 pg, 100 fg and 10 fg with quadplex, triplex, and singleplex, respectively. None of the assays were impacted by any inhibitory or competitive effects after adding host DNA (in qPCR) or crude lysate (in LAMP). The assays proved robust and reproducible in detecting the target pathogen in infected samples, with the LAMP assay being field-deployable due to its simplicity and rapid results acquisition within approximately 9 min. The reproducibility was confirmed by performing the assay in two independent laboratories. These assays offer a robust, rapid, and reliable solution for routine testing, with applications in phytosanitary inspection, disease diagnosis, and epidemiological studies.IMPORTANCEDickeya solani, one of the most aggressive soft rot causing bacteria and a quarantine pathogen, poses a severe threat to food security by causing substantial economic losses to the potato industry. Accurate and timely detection of this bacterium is vital for monitoring latent infections, particularly for international trade of potato seed tubers, and for implementing effective control strategies. In this research, we have developed LAMP and multi-gene-based multiplex TaqMan qPCR assays for specific detection of D. solani. These assays, characterized by their precision, rapidity, and robustness, are crucial for distinguishing D. solani from related species. Offering unparalleled sensitivity and specificity, these assays are indispensable for phytosanitary inspection and epidemiological monitoring, providing a powerful tool enabling management of this threatening pathogen.

Xylella fastidiosa is a plant pathogenic bacterium that causes many economically important agricultural diseases and is transmitted by the glassy-winged sharpshooter, Homalodisca vitripennis (Hemiptera: Cicadellidae). Efficient detection of X. fastidiosa in field collected H. vitripennis in an area-wide management program can contribute to risk assessment associated with insect presence in vineyards. Prior to conducting molecular assays for detection of X. fastidiosa in individual insects, H. vitripennis must be removed from yellow sticky traps with a solvent such as orange oil. In this study, we determined the effect of orange oil concentration on extraction of individual H. vitripennis following trap removal on detection of X. fastidiosa by qRT-PCR. In a ten-fold dilution series of orange oil, increasing amounts of orange oil caused decreasing levels of X. fastidiosa detection in standardized positive samples. Additionally, tests on the effects of Stickem® brand trap adhesive on qRT-PCR and development of methods which lowered the concentration of orange oil often present in field samples determined the point where detection of X. fastidiosa was negatively impacted. These results benefit the monitoring and screening for Xylella fastidiosa from leafhoppers collected on sticky cards used in regulatory area-wide management.

Xylella fastidiosa is a fastidious Gram-negative bacterium that is associated with several important plant diseases, and is regulated as a quarantine pest in many countries where strategies are implemented to prevent its introduction and spread. To enact efficient quarantine measures, effective and early detection of the pathogen are essential, especially because global trade of goods increases the risks of introduction of alien pathogens. this study aimed to adapt two qPCR-based diagnostic methods (SYBR Green and Probe based qPCR), already in use to detect X. fastidiosa, for use with a nanoplate based digital PCR assay. Detection of the pathogen using the two digital PCR assays (EvaGreen- and Probe-based) was similar to standard qPCR, giving 100% sensitivity, specificity, and accuracy, while providing accurate absolute quantification of the pathogen when using experimental samples that had low concentrations of host DNA. Using undiluted plant DNA added with low concentrations of X. fastidiosa, only the TaqMan method maintained satisfactory performance and quantification, and is therefore preferred. These results are a first step demonstrating the usefulness of nanoplate-based digital PCR for detection of plant pathogens, which allows greater throughput than qPCR, reducing the time and cost of diagnostic assays.

Xylella fastidiosa subsp. pauca (Xfp) infects olive trees and other hosts in Southern Apulia (Italy), devastating agriculture and landscape. A containment strategy of the disease requires quick and sensitive detection tools. Therefore, a colorimetric LAMP protocol was developed using as a template a crude alkaline sap obtained from incubation of 50–60 mg of thin slices of olive twigs in a NaOH-containing buffer. This rapid molecular assay can be performed directly in the field, as it needs only a portable isothermal block. Tissues of the same olive trees analysed by this technique were also compared to qPCR (using purified total plant DNA as template) as well as digital droplet PCR (on the same crude alkaline extracts used in cLAMP). A titration of the cLAMP reaction with healthy olive sap, spiked with dilutions of in vitro cultivated Xfp cells and plasmid DNA containing the target sequence, gave positive detection results as low as 102 CFU/mL and up to 169.2 target copies/µL, equivalent to about 0.9 pg of the genomic DNA. A portable, sensitive and target-specific Xfp field test was developed, which has a 40 min sample-to-answer time and does not require any DNA isolation procedure or laboratory equipment. The application of this detection assay could help the monitoring and containment of the disease spread.

High-throughput DNA isolation method for detection of Xylella fastidiosa in plant and insect samples

Two new superior primer pairs for universal detection of Xylella spp. in conventional PCR and TaqMan quantitative real-time PCR

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