High-throughput homogenous assay for the direct detection of Listeria monocytogenes DNA.

The Amplified Luminescent Proximity Homogenous Assay-linked Immunosorbent Assay (AlphaLISA) is known for detecting various protein targets; however, its ability to detect nucleic acid sequences is not well established. Here, the capabilities of the AlphaLISA technology were expanded to include direct detection of DNA (aka: oligo-Alpha) and was applied to the detection of Listeria monocytogenes. Parameters were defined that allowed the newly developed oligo-Alpha to differentiate L. monocytogenes from other Listeria species through the use of only a single nucleotide polymorphism within the 16S rDNA region. Investigations into the applicability of this assay with different matrices demonstrated its utility in both milk and juice. One remarkable feature of the oligo-Alpha is that greater sensitivity could be achieved through the use of multiple acceptor oligos compared to only a single acceptor oligo, even when only a single donor oligo was employed. Additional acceptor oligos were easily incorporated into the assay and a tenfold change in the detection limit was readily achieved, with detection limits of 250 attomole of target being recorded. In summary, replacement of antibodies with oligonucleotides allows us to take advantage of genotypic difference(s), which both expands its repertoire of biological markers and furthers its use as a diagnostic tool.

Detection of Escherichia coli O157:H7 in Ground Beef Using Long-Read Sequencing.

Foodborne pathogens are a significant cause of illness, and infection with Shiga toxin-producing Escherichia coli (STEC) may lead to life-threatening complications. The current methods to identify STEC in meat involve culture-based, molecular, and proteomic assays and take at least four days to complete. This time could be reduced by using long-read whole-genome sequencing to identify foodborne pathogens. Therefore, the goal of this project was to evaluate the use of long-read sequencing to detect STEC in ground beef. The objectives of the project included establishing optimal sequencing parameters, determining the limit of detection of all STEC virulence genes of interest in pure cultures and spiked ground beef, and evaluating selective sequencing to enhance STEC detection in ground beef. Sequencing libraries were run on the Oxford Nanopore Technologies' MinION sequencer. Optimal sequencing output was obtained using the default parameters in MinKNOW, except for setting the minimum read length to 1 kb. All genes of interest (eae, stx1, stx2, fliC, wzx, wzy, and rrsC) were detected in DNA extracted from STEC pure cultures within 1 h of sequencing, and 30× coverage was obtained within 2 h. All virulence genes were confidently detected in STEC DNA quantities as low as 12.5 ng. In STEC-inoculated ground beef, software-controlled selective sequencing improved virulence gene detection; however, several virulence genes were not detected due to high bovine DNA concentrations in the samples. The growth enrichment of inoculated meat samples in mTSB resulted in a 100-fold increase in virulence gene detection as compared to the unenriched samples. The results of this project suggest that further development of long-read sequencing protocols may result in a faster, less labor-intensive method to detect STEC in ground beef.

Detection and Isolation of Campylobacter spp. from Raw Meat.

This article presents a rapid yet robust protocol for isolating Campylobacter spp. from raw meats, specifically focusing on Campylobacter jejuni and Campylobacter coli. The protocol builds upon established methods, ensuring compatibility with the prevailing techniques employed by regulatory bodies such as the Food and Drug Administration (FDA) and the U.S. Department of Agriculture (USDA) in the USA, as well as the International Organization for Standardization (ISO) in Europe. Central to this protocol is collecting a rinsate, which is concentrated and resuspended in Bolton Broth media containing horse blood. This medium has been proven to facilitate the recovery of stressed Campylobacter cells and reduce the required enrichment duration by 50%. The enriched samples are then transferred onto nitrocellulose membranes on brucella plates. To improve the sensitivity and specificity of the method, 0.45 µm and 0.65 µm pore-size filter membranes were evaluated. Data revealed a 29-fold increase in cell recovery with the 0.65 µm pore-size filter compared to the 0.45 µm pore-size without impacting specificity. The highly motile characteristics of Campylobacter allow cells to actively move through the membrane filters towards the agar medium, which enables effective isolation of pure Campylobacter colonies. The protocol incorporates multiplex quantitative real-time polymerase chain reaction (mqPCR) assay to identify the isolates at the species level. This molecular technique offers a reliable and efficient means of species identification. Investigations conducted over the past twelve years involving retail meats have demonstrated the ability of this method to enhance recovery of Campylobacter from naturally contaminated meat samples compared to current reference methods. Furthermore, this protocol boasts reduced preparation and processing time. As a result, it presents a promising alternative for the efficient recovery of Campylobacter from meat. Moreover, this procedure can be seamlessly integrated with DNA-based methods, facilitating rapid screening of positive samples alongside comprehensive whole-genome sequencing analysis.

Magnetic capture device for large volume sample analysis.

Immunomagnetic separation (IMS) techniques employing superparamagnetic particles can successfully isolate various components from mixtures. However, their utility can be limited for large-volume samples, viscous samples, or those containing a high density of particulate matter because of the need to generate high field gradients for particle recovery. Therefore, a new class of immunomagnetic particles was devised utilizing a single, macroscopic Pyrex spinbar conjugated with biorecognition elements to address these limitations. Advantages include an inherent capacity for effective mixing, an almost instantaneous recovery of the spinbar that can be performed without expensive equipment and with no loss of magnetic particles during processing, and reduced transfer of sample matrix. As a result, spinbars can provide an effective means for IMS with large-volume assays composed of complex matrices.

Use of a commercial tissue dissociation system to detect Salmonella-contaminated poultry products.

Successful detection of bacterial pathogens in food can be challenging due to the physical and compositional complexity of the matrix. Different mechanical/physical and chemical methods have been developed to separate microorganisms from food matrices to facilitate detection. The present study benchmarked a commercial tissue digestion system that applies both chemical and physical methods to separate microorganisms from tissues against stomaching, a standard process currently utilized by commercial and regulatory food safety laboratories. The impacts of the treatments on the physical properties of the food matrix were characterized along with the compatibility of the methods with downstream microbiological and molecular detection assays. The results indicate the tissue digestion system can significantly reduce the average particle size of the chicken sample relative to processing via a stomacher (P < 0.001) without adversely affecting either real-time PCR (qPCR) or plate counting assays, which are typically used to detect Salmonella. Furthermore, inoculated chicken treated with the GentleMACS resulted in a significant increase (P < 0.003) in the qPCR's detection capabilities relative to stomached controls. Cohen kappa (κ) coefficient and McNemar's test indicate the plating assays and PCR results agree with measurements obtained via the 3 M Molecular Detection System as defined in the MLG standard (κ > 0.62; P > 0.08). Collectively, the results demonstrate that the technique enables detection of pathogens in meat at lower levels of contamination using current industry standard technologies.

Complete Genome Sequence of Campylobacter jejuni BSD5, a Multidrug-Resistant Isolate from a Poultry Processing Facility in the United States.

Raw poultry can harbor microbial pathogens. Campylobacter jejuni BSD5, isolated from a critical control point within a poultry production plant, was sequenced. Genome annotation revealed several virulence genes including antibiotic resistance genes in agreement with the phenotypic results, indicating a potential risk of this strain to public health.

Identification of a Chromosomal Deletion Mutation and the Dynamics of Two Major Populations of 'Candidatus Liberibacter asiaticus' in Its Hosts.

'Candidatus Liberibacter asiaticus' (Las) is the prominent species of Liberibacter associated with huanglongbing, a devastating disease of citrus worldwide. In this study, we report the identification of an ∼8.3-kb DNA region of the Las genome containing eight putative open reading frames flanked by two inverted repeats, which was not present in the Las str. psy62 genome. Comparisons with other genome sequences established this region as a unique genetic element associated with genome plasticity/instability. Primers specific for both the presence (Las wild type) and absence (Las mutant) of this region were designed to study the population dynamics and host adaptation of the two strains. Las populations with and/or without the wild-type strain were detected and differentiated in >2,300 samples that included psyllids, periwinkle, and several species of citrus. In psyllids, although a mixed population of the wild type and mutant was observed in most samples (88%), the wild-type Las was detected alone at a rate of 11%. In contrast, none of the infected citrus plants were positive for the wild type alone, which harbored either the mutant strain alone (8%) or a mixed population of the mutant and wild type (92%). Furthermore, the dynamics of these two major Las populations varied with different citrus hosts, whereas an in-depth study on grapefruit that did not rapidly succumb to disease revealed that the population of mutant alone increased with time, indicating that the absence of this genetic element is associated with the fitness of Las in planta under the selection pressure of its host.

Flow-Through Electrochemical Biosensor for the Detection of Listeria monocytogenes Using Oligonucleotides.

Consumption of food contaminated by Listeria monocytogenes can result in Listeriosis, an illness with hospitalization rates of 94% and mortality rates up to 30%. As a result, U.S. regulatory agencies governing food safety retain zero-tolerance policies for L. monocytogenes. However, detection at such low concentrations often requires strategies such as increasing sample size or culture enrichment. A novel flow-through immunoelectrochemical biosensor has been developed for Escherichia coli O157:H7 detection in 1 L volumes without enrichment. The current work further augments this biosensor's capabilities to (1) include detection of L. monocytogenes and (2) accommodate genetic detection to help overcome limitations based upon antibody availability and address specificity errors in phenotypic assays. Herein, the conjugation scheme for oligo attachment and the conditions necessary for genetic detection are laid forth while results of the present study demonstrate the sensor's ability to distinguish L. monocytogenes DNA from L. innocua with a limit of detection of ~2 × 104 cells/mL, which agrees with prior studies. Total time for this assay can be constrained to <2.5 h because a timely culture enrichment period is not necessary. Furthermore, the electrochemical detection assay can be performed with hand-held electronics, allowing this platform to be adopted for near-line monitoring systems.

Transcriptomics of Listeria monocytogenes Treated With Olive Leaf Extract.

Listeria monocytogenes is a regulated foodborne pathogen that is known to cause listeriosis, a disease associated with high mortality rates in humans. Olive leaf extract (OLE) has been shown to act as a plant antimicrobial and inhibit the growth of pathogens, such as L. monocytogenes, although its mode of action has not been defined. To help identify the cellular mechanisms important for conveying these beneficial traits, RNA-Seq was used to study the transcriptome of L. monocytogenes upon exposure to a sublethal level of OLE. Results obtained from cells cultured both with and without OLE at two different time points (3.5-h and 24-h) revealed 661 genes that were differentially expressed. Of the differentially expressed genes (DEGs) identified, transcription was altered for 171 genes in response to the 3.5-h OLE treatment while 490 genes were altered in response to the 24-h OLE treatment. These DEGs included but were not limited to genes encoding for signal transduction, ATP-binding cassette (ABC) transporters, and the phosphotransferase system. Interestingly, several virulence-related genes were downregulated including an ABC transporter permease previously shown to negatively regulate biofilm formation, genes involved in flagella assembly and binding/entry into host cells as well as those regulating acid resistance suggesting that OLE may decrease the virulence potential of L. monocytogenes. Furthermore, quantitative reverse-transcription PCR was used to validate the data obtained via RNA-Seq. Our study provides insight into the mode of action of OLE treatment against L. monocytogenes and may aid in identifying synergetic strategies to inhibit L. monocytogenes in food.

Detection of Shiga toxin-producing Escherichia coli (STEC) in beef products using droplet digital PCR.

Many of the current accredited methods for the molecular detection of Shiga toxin-producing Escherichia coli (STEC) in foods rely on a PCR-based screen for the pathotype-specific genetic markers stx and eae. Unfortunately, these methods can inaccurately conclude the presence of E.coli containing both stx and eae because of the inability of the methods to determine if the two genes originated from a single organism as opposed to a mixture of organisms. This study was undertaken to evaluate if a droplet digital PCR (ddPCR)-based method that does not require DNA isolation could reliably identify the presence of an STEC containing eae in beef samples by confirming that both genes reside within the same cell, even when present in a mixed culture. The ddPCR system used in this study, dd-Check STEC Solution (Bio-Rad), works without the need for DNA isolation by partitioning intact cells into emulsion droplets, where they are lysed, and subsequently undergo multiplexed endpoint PCR. This enables the assay to differentiate between samples where a single organism contains both stx and eae from samples in which stx and eae reside in different organisms. Comparisons were made between the dd-Check STEC Solution, the BAX System Real-Time PCR STEC assay suite (Hygiena), and the iQ-Check STEC PCR detection kit (Bio-Rad) using 37 unique simulations of E. coli contamination in ground beef. While no single platform was consistently superior at detecting eae and stx across all pathogens tested, the results indicated that the dd-Check STEC Solution has the potential to reduce the number of inaccurately identified samples when screening for E. coli with a stx+, eae+ genotype because it can identify the co-existence of multiple virulence genes within a cell even when in the presence of a mixed microbial population containing identical genes. Ultimately, incorporation of this system could result in substantial cost savings by reducing the expenses incurred when product samples are incorrectly classified as containing E. coli with a stx+, eae+ genotype.

Comparative Methylome Analysis of Campylobacter jejuni Strain YH002 Reveals a Putative Novel Motif and Diverse Epigenetic Regulations of Virulence Genes.

Campylobacter jejuni is a major cause of foodborne gastroenteritis worldwide inflicting palpable socioeconomic costs. The ability of this pathogen to successfully infect its hosts is determined not only by the presence of specific virulence genes but also by the pathogen's capacity to appropriately regulate those virulence genes. Therefore, DNA methylation can play a critical role in both aspects of this process because it serves as both a means to protect the integrity of the cellular DNA from invasion and as a mechanism to control transcriptional regulation within the cell. In the present study we report the comparative methylome data of C. jejuni YH002, a multidrug resistant strain isolated from retail beef liver. Investigation into the methylome identified a putative novel motif (CGCGA) of a type II restriction-modification (RM) system. Comparison of methylomes of the strain to well-studied C. jejuni strains highlighted non-uniform methylation patterns among the strains though the existence of the typical type I and type IV RM systems were also observed. Additional investigations into the existence of DNA methylation sites within gene promoters, which may ultimately result in altered levels of transcription, revealed several virulence genes putatively regulated using this mode of action. Of those identified, a flagella gene (flhB), a RNA polymerase sigma factor (rpoN), a capsular polysaccharide export protein (kpsD), and a multidrug efflux pump were highly notable.

Impacts of Clarification Techniques on Sample Constituents and Pathogen Retention.

Determination of the microbial content in foods is important, not only for safe consumption, but also for food quality, value, and yield. A variety of molecular techniques are currently available for both identification and quantification of microbial content within samples; however, their success is often contingent upon proper sample preparation when the subject of investigation is a complex mixture of components such as foods. Because of the importance of sample preparation, the present study employs a systematic approach to compare the effects of four different separation techniques (glass wool, 50 µm polypropylene filters, graphite felt, and continuous flow centrifugation (CFC)) on sample preparation. To define the physical effects associated with the use of these separation methods, a multifactorial analysis was performed where particle size and composition, both pre- and post- processing, were analyzed for four different food matrices including lean ground beef, ground pork, ground turkey and spinach. Retention of three important foodborne bacterial pathogens (Escherichia coli O157:H7, Salmonella enterica, and Listeria monocytogenes) was also examined to evaluate the feasibility of the aforementioned methods to be utilized within the context of foodborne pathogen detection. Data from the multifactorial analysis not only delineated the particle size ranges but also defined the unique compositional profiles and quantified the bacterial retention. The three filtration membranes allowed for the passage of bacteria with minimal loss while CFC concentrated the inoculated bacteria. In addition, the deposition and therefore concentration of food matrix observed with CFC was considerably higher for meat samples relative to spinach. However, filtration with glass wool prior to CFC helped clarify meat samples, which led to considerably lower amounts of solids in the CFC vessel post processing and an increase in the recovery of the bacteria. Overall, by laying a framework for the deductive selection of sample preparation techniques, the results of the study can be applied to a range of applications where it would be beneficial to scientifically guide the pairing of the criteria associated with a downstream detection method with the most advantageous sample preparation techniques for complex matrices such as foods.

Rapid detection of Salmonella enterica serotype Typhimurium in large volume samples using porous electrodes in a flow-through, enzyme-amplified immunoelectrochemical sensor.

Foodborne illness is a common yet preventable public health concern generating significant costs for the healthcare system, making systems to accurately detect this pathogen a topic of current research. Enzyme-based immunoassays are highly desirable because they offer shorter response times compared to traditional culture-based methods. Biosensors employing the electrochemical and optical detection of a substrate oxidized by horseradish peroxidase (HRP) have been used to successfully detect biomolecules; however, their inability to handle large sample volumes severely limits their application to food safety despite their accuracy and reliability. Here, we describe a biosensor with the capacity to process a large sample volume by utilizing an Ag/AgCl reference electrode, a platinum counter electrode, and a porous working electrode made from graphite felt coated with antibodies specific for Salmonella common structural antigens. This design allows samples to flow-through the electrode while capturing target pathogens. Following sample exposure, HRP-conjugated antibodies facilitate pathogen detection that culminates in an oxidation reaction with the output analyzed via Osteryoung square wave voltammetry. Detection limits of 1000 Salmonella enterica serotype Typhimurium cells were achieved using this newly devised flow-through, enzyme-amplified, electrochemical biosensor in samples as large as 60 mL. The low cost of the sensor allows for incorporation into disposable detection devices while its design not only broadens its applicability in sample processing but also permits the detection of various microbes by simply exchanging the antibodies.

Detection of Shiga Toxin 2 Produced by Escherichia coli in Foods Using a Novel AlphaLISA.

Amplified luminescent proximity homogenous assay-linked immunosorbent assay (AlphaLISA) is comprised of a bead-based immunoassay that is used for small molecule detection. In this study, a novel AlphaLISA was developed and optimized for the detection of Shiga-toxin 2 (Stx2). Efficacy and sensitivity trials showed the AlphaLISA could detect ≥0.5 ng/mL of purified Stx2, which was comparable to the industry-standard enzyme-linked immunosorbent assay (ELISA) tests for Stx2 detection. In addition, evaluation of Shiga toxin-producing Escherichia coli (STEC)-inoculated Romaine lettuce and ground beef samples demonstrated that both the AlphaLISA and the ELISA were able to discern uninoculated samples from 1× and 10× diluted samples containing ~10 CFU/mL of STEC enriched in modified tryptic soy broth with mitomycin C for 16 h. Overall, the increased signal-to-noise ratios indicated a more robust signal was produced by the AlphaLISA compared to the ELISA and the delineation of higher toxin concentrations without the need for sample dilution implied a greater dynamic range for the AlphaLISA. Implementation of the newly developed AlphaLISA will allow for more rapid analysis for Stx2 with less manual manipulation, thus improving assay throughput and the ability to automate sample screening while maintaining detection limits of 0.5 ng/mL.

Solar thermotherapy reduces the titer of Candidatus Liberibacter asiaticus and enhances canopy growth by altering gene expression profiles in HLB-affected citrus plants.

Huanglongbing (HLB), a systemic and destructive disease of citrus, is associated with 'Candidatus Liberibacter asiaticus' (Las) in the United States. Our earlier work has shown that Las bacteria were significantly reduced or eliminated when potted HLB-affected citrus were continuously exposed to high temperatures of 40 to 42 °C for a minimum of 48 h. To determine the feasibility and effectiveness of solar thermotherapy in the field, portable plastic enclosures were placed over commercial and residential citrus, exposing trees to high temperatures through solarization. Within 3-6 weeks after treatment, most trees responded with vigorous new growth. Las titer in new growth was greatly reduced for 18-36 months after treatment. Unlike with potted trees, exposure to high heat did not eradicate the Las population under field conditions. This may be attributed to reduced temperatures at night in the field compared to continuous high temperature exposure that can be maintained in growth chambers, and the failure to achieve therapeutic temperatures in the root zone. Despite the presence of Las in heat-treated commercial citrus, many trees produced abundant flush and grew vigorously for 2 to 3 years after treatment. Transcriptome analysis comparing healthy trees to HLB-affected citrus both before and after heat treatment demonstrated that post-treatment transcriptional expression patterns more closely resembled the expression patterns of healthy controls for most differentially expressed genes and that genes involved with plant-bacterium interactions are upregulated after heat treatment. Overall, these results indicate that solar thermotherapy can be an effective component of an integrated control strategy for citrus HLB.

Molecular mechanisms behind the accumulation of ATP and H2O2 in citrus plants in response to 'Candidatus Liberibacter asiaticus' infection.

Candidatus Liberibacter asiaticus (Las) is a fastidious, phloem-restricted pathogen with a significantly reduced genome, and attacks all citrus species with no immune cultivars documented to date. Like other plant bacterial pathogens, Las deploys effector proteins into the organelles of plant cells, such as mitochondria and chloroplasts to manipulate host immunity and physiology. These organelles are responsible for the synthesis of adenosine triphosphate (ATP) and have a critical role in plant immune signaling during hydrogen peroxide (H2O2) production. In this study, we investigated H2O2 and ATP accumulation in relation to citrus huanglongbing (HLB) in addition to revealing the expression profiles of genes critical for the production and detoxification of H2O2 and ATP synthesis. We also found that as ATP and H2O2 concentrations increased in the leaf, so did the severity of the HLB symptoms, a trend that remained consistent among the four different citrus varieties tested. Furthermore, the upregulation of ATP synthase, a key enzyme for energy conversion, may contribute to the accumulation of ATP in infected tissues, whereas downregulation of the H2O2 detoxification system may cause oxidative damage to plant macromolecules and cell structures. This may explain the cause of some of the HLB symptoms such as chlorosis or leaf discoloration. The findings in this study highlight important molecular and physiological mechanisms involved in the host plants' response to Las infection and provide new targets for interrupting the disease cycle.

Transient Expression of Candidatus Liberibacter Asiaticus Effector Induces Cell Death in Nicotiana benthamiana.

Candidatus Liberibacter asiaticus "Las" is a phloem-limited bacterial plant pathogen, and the most prevalent species of Liberibacter associated with citrus huanglongbing (HLB), a devastating disease of citrus worldwide. Although, the complete sequence of the Las genome provides the basis for studying functional genomics of Las and molecular mechanisms of Las-plant interactions, the functional characterization of Las effectors remains a slow process since remains to be cultured. Like other plant pathogens, Las may deliver effector proteins into host cells and modulate a variety of host cellular functions for their infection progression. In this study, we identified 16 putative Las effectors via bioinformatics, and transiently expressed them in Nicotiana benthamiana. Diverse subcellular localization with different shapes and aggregation patterns of the effector candidates were revealed by UV- microscopy after transient expression in leaf tissue. Intriguingly, one of the 16 candidates, Las5315mp (mature protein), was localized in the chloroplast and induced cell death at 3 days post inoculation (dpi) in N. benthamiana. Moreover, Las5315mp induced strong callose deposition in plant cells. This study provides new insights into the localizations and potential roles of these Las effectors in planta.

Bismerthiazol Inhibits Xanthomonas citri subsp. citri Growth and Induces Differential Expression of Citrus Defense-Related Genes.

Citrus canker, caused by Xanthomonas citri ssp. citri, is a serious disease that causes substantial economic losses to the citrus industry worldwide. The bactericide bismerthiazol has been used to control rice bacterial blight (X. oryzae pv. oryzae). In this paper, we demonstrate that bismerthiazol can effectively control citrus canker by both inhibiting the growth of X. citri ssp. citri and triggering the plant's host defense response through the expression of several pathogenesis-related genes (PR1, PR2, CHI, and RpRd1) and the nonexpresser of PR genes (NPR1, NPR2, and NPR3) in 'Duncan' grapefruit, especially at early treatment times. In addition, we found that bismerthiazol induced the expression of the marker genes CitCHS and CitCHI in the flavonoid pathway and the PAL1 (phenylalanine ammonia lyase 1) gene in the salicylic acid (SA) biosynthesis pathway at different time points. Moreover, bismerthiazol also induced the expression of the priming defense-associated gene AZI1. Taken together, these results indicate that the induction of the defense response in 'Duncan' grapefruit by bismerthiazol may involve the SA signaling pathway and the priming defense and that bismerthiazol may serve as an alternative to copper bactericides for the control of citrus canker.

Rapid screening for citrus canker resistance employing pathogen-associated molecular pattern-triggered immunity responses.

Citrus canker, caused by the bacterial pathogen Xanthomonas citri ssp. citri (Xcc), has been attributed to millions of dollars in loss or damage to commercial citrus crops in subtropical production areas of the world. Since identification of resistant plants is one of the most effective methods of disease management, the ability to screen for resistant seedlings plays a key role in the production of a long-term solution to canker. Here, an inverse correlation between reactive oxygen species (ROS) production by the plant and the ability of Xcc to grow and form lesions on infected plants is reported. Based on this information, a novel screening method that can rapidly identify citrus seedlings that are less susceptible to early infection by Xcc was devised by measuring ROS accumulation triggered by a 22-amino acid sequence of the conserved N-terminal part of flagellin (flg22) from X. citri ssp. citri (Xcc-flg22). In addition to limiting disease symptoms, ROS production was also correlated with the expression of basal defense-related genes such as the pattern recognition receptors LRR8 and FLS2, the leucine-rich repeat receptor-like protein RLP12, and the defense-related gene PR1, indicating an important role for pathogen-associated molecular pattern-triggered immunity (PTI) in determining resistance to citrus canker. Moreover, the differential expression patterns observed amongst the citrus seedlings demonstrated the existence of genetic variations in the PTI response among citrus species/varieties.

Genetic diversity of Candidatus Liberibacter asiaticus based on two hypervariable effector genes in Thailand.

Huanglongbing (HLB), also known as citrus greening, is one of the most destructive diseases of citrus worldwide. HLB is associated with three species of 'Candidatus Liberibacter' with 'Ca. L. asiaticus' (Las) being the most widely distributed around the world, and the only species detected in Thailand. To understand the genetic diversity of Las bacteria in Thailand, we evaluated two closely-related effector genes, lasAI and lasAII, found within the Las prophages from 239 infected citrus and 55 infected psyllid samples collected from different provinces in Thailand. The results indicated that most of the Las-infected samples collected from Thailand contained at least one prophage sequence with 48.29% containing prophage 1 (FP1), 63.26% containing prophage 2 (FP2), and 19.38% containing both prophages. Interestingly, FP2 was found to be the predominant population in Las-infected citrus samples while Las-infected psyllids contained primarily FP1. The multiple banding patterns that resulted from amplification of lasAI imply extensive variation exists within the full and partial repeat sequence while the single band from lasAII indicates a low amount of variation within the repeat sequence. Phylogenetic analysis of Las-infected samples from 22 provinces in Thailand suggested that the bacterial pathogen may have been introduced to Thailand from China and the Philippines. This is the first report evaluating the genetic variation of a large population of Ca. L. asiaticus infected samples in Thailand using the two effector genes from Las prophage regions.