Pecan bacterial leaf scorch (PBLS), caused by Xylella fastidiosa has been reported in Georgia, Louisiana, Texas, New Mexico, Arizona, and California. Accurate methods are critical for the early detection of X. fastidiosa, but the validation of current diagnostic tools for pecan has yet to be investigated. Collected petioles, leaflets, and shoots from pecan and other Carya relatives in Texas, Indiana, and Georgia were used as tissue samples, and to isolate crude xylem sap and gDNA for side-by-side testing using immunological (ELISA) and molecular-based assays [traditional PCR and real-time quantitative PCR (qPCR)]. Isolated crude sap was found to be the most reliable template for ELISA diagnostics. X. fastidiosa-specific genes were amplified with previously published PCR primer sets; however, they revealed non-specific binding. New Xylella-specific primers were subsequently generated and validated using infected tissue from pecan and related Carya species. Two new primer sets (NMU3 and 5) produced expected amplicons specific to X. fastidiosa but did not amplify any non-specific bands of the pecan gDNA. When compared to that of total gDNA as the template in PCR reactions, diluted crude sap was found to be an efficient way to detect X. fastidiosa in pecan petioles. A novel TaqMan qPCR assay was also developed for the detection of X. fastidiosa. The results of the qPCR experiments were equivalent to the traditional PCR amplification when crude sap was used as the template. Comparative PCR analysis confirms that the PCR protocol outlined in this study can be replicated across different laboratories.
Pecan (Carya illinoinensis) is a native tree to North America. The US produced approximately 227,400 metric tons of in-shell pecans in 2017, and the annual revenue on the cultivated crop now exceeds $650 million (National Agriculture Statistics Service 2018). Global demands for pecan have continued to increase annually: the USDA Foreign Agricultural Service (FAS) has reported a 30% increase in the United States’ global exports of shelled pecans since 2012 (Global Agricultural Trade System 2017). Nations now cultivating pecan outside the native range include China, Korea, South Africa, Australia, Israel, Uruguay, Argentina, Peru, and Brazil, and the production of pecan nuts are expected to increase over the next 30 years (Zhang et al. 2015; Wood et al. 1990; Wakeling et al. 2001; Lazarotto et al. 2014).
Pecan bacterial leaf scorch (PBLS) is an economically important disease of pecan and has been confirmed to occur in the southern US, including the states of Louisiana, Georgia, Texas, New Mexico, Arizona, and California (Hilton et al. 2017; Sanderlin and Heyderich-Alger 2000; Bock et al. 2018; Su et al. 2012). In a previous study, Sanderlin and Heyderich-Alger (2003) investigated the impacts of PBLS on infected pecan tree cultivar ‘Cape Fear’ for a 3-year period and a significant reduction in nut and kernel weight equating to an average 12% yield loss was found, a value that could lead to economic losses greater than $450/ha. PBLS can also cause over 50% premature defoliation of the tree canopy. Foliage loss has ramifications for return bloom and alternate bearing, which may impact yields in subsequent years as well (Wood et al. 2002). PBLS has been cited as an emerging issue for the propagation and dissemination of pecan worldwide (Grauke et al. 2016).
X. fastidiosa subsp. multiplex was demonstrated to be the causal agent of PBLS through Koch’s postulates and phylogenetic analyses using a combination of multi-primer PCR, 16S–23S rRNA ITS and pg1A sequencing, REP-PCR, and ERIC-PCR (R. Sanderlin and Heyderich-Alger 2000; Melanson et al. 2012). X. fastidiosa is a fastidious, gram-negative, xylem-limited bacterium, of which six different subspecies have been identified (Schaad et al. 2004; Randall et al. 2009; Chen et al. 2014; Nunney et al. 2014). X. fastidiosa subsp. multiplex is endemic to North America and can colonize a wide range of woody hosts, including species of oak (Quercus spp.), American sycamore (Platanus occidentalis), peach/plum/almond (Prunus spp.), maple (Acer spp.), grape (Vitis spp.), and more recently olive (Olea europaea) (Nunney et al. 2013; Baldi and La Porta 2017; Krugner et al. 2014). Symptoms of X. fastidiosa on pecan include uniform tan to light brown necrotic lesions on leaflets, which eventually result in abscission from the rachis (Sanderlin and Heyderich-Alger 2003, 2000). However, scorch-like symptoms similar to those of PBLS have been attributed to other conditions, including fungal anthracnose, insect damage, imbalances of nitrogen and potassium, or excessive salinity (Worley 1990; Deb et al. 2013; Latham et al. 1995; Pierce 1953). Thus, basing the diagnosis of PBLS on symptoms alone is unreliable, and there is a need for immunological or molecular methods to diagnose the pathogen accurately. As demonstrated in many plant hosts, including grape, citrus, and pecan, X. fastidiosa causes chronic infections from year-to-year and can colonize the host without presenting symptoms (R. Sanderlin and Heyderich-Alger 2003; Hopkins and Purcell 2002; R. Sanderlin and Heyderich-Alger 2000). Infections can be localized to individual branches or systemic across the entire canopy of the tree. Due to the unreliability of symptom identification and the likelihood for latent and/or localized infections in which the bacterium is present in low concentrations, correct diagnosis of X. fastidiosa has been challenging, and the likelihood of false negatives is prominent in many crop systems (Baldi and La Porta 2017). Furthermore, early detection of X. fastidiosa prior to symptom development is desirable to implement new or existing strategies for the management of PBLS.
Current trends in globalization have resulted in anthropogenic introductions of Xylella subspecies, which has increased the genetic diversity of the pathogen through homologous intersubspecific and intrasubspecific recombination (Nunney et al. 2013). Consequently, invasive subspecies of X. fastidiosa have undergone host shifts that threaten vulnerable agricultural crops and ecosystems that carry little to no tolerance to the pathogen. The USDA-ARS National Collection of Genetic Resources for Pecans and Hickories (NCGR-Carya) is responsible for the distribution of pecan germplasm internationally. Distribution of pecan and related species of Carya falls under regulation outlined by Plant Import Permits of cooperating nations, which requires an inspection for and certification that samples are free of X. fastidiosa. The European and Mediterranean Plant Protection Organization (EPPO) has recommended quarantine identities of A1 and A2, depending on the presence of X. fastidiosa within the region, which outlines the handling of X. fastidiosa as a quarantined pest in more than forty member countries, including those in the European Union, southwest Asia, southern Africa, and South America. This further emphasizes the need to improve screening for Xylella in pecan through the development of reliable detection methods to prevent its further dissemination (Pooler and Hartung 1995).
The purpose of this study was to validate and determine the optimal conditions for serological and DNA-based diagnostic methods for X. fastidiosa in pecan and related species of Carya. Our goal was to develop a robust and efficient pipeline to detect X. fastidiosa directly from plant tissue, with or without the expression of symptoms. We address sample selection and sample processing prior to testing, as well as the reliability of various immunological and molecular-based platforms. The reproducibility of our diagnostic approach was confirmed through the exchange of sample materials between labs and subsequent comparison of results.
Materials and methods
Pecan cultivars and other Carya germplasm were selected for sampling based on known susceptibilities to PBLS and/or prior symptom expression of X. fastidiosa from USDA-ARS pecan orchards in Texas, native stands of Carya in Indiana, and in commercial orchards in Georgia (Table 1). Samples varied from asymptomatic to moderate scorching appearing on leaflet tips and margins (Fig. 1). Sanitized pruners were used to excise shoots from mature trees or seedlings, which were subsequently sealed individually in plastic bags. For samples used for crude sap extraction, a moist paper towel was wrapped around shoots to prevent excess water loss. Plant specimens were processed immediately after collection or stored for up to 3 days at 4 °C prior to sap extraction. For DNA isolation, pecan petioles or leaflets were harvested and frozen in liquid nitrogen. Samples were processed immediately or stored at −80 °C for up to one month.
Crude sap extraction
Crude sap samples from pecan seedlings and mature shoots were collected using a Scholander-type 3000 series plant water status console (Soilmoisture Equipment Corp., Goleta, CA) (Scholander et al. 1965). Pecan shoots were fitted into the pressure chamber, and pneumatic pressure was applied at approximately 150 to 300 psi. The pressure needed to exude sap samples was variable depending on the hydrostatic pressure potential, disease severity, and sample type. Pneumatic pressure was applied at a 5 to 10 psi/s rate of flow, according to the manufacturer’s protocols. Once samples began exuding sap, a 10-μl pipette was used to collect the sap by means of capillary action and a slow rate of suction to prevent cross-contamination. The specimen holder was thoroughly rinsed with 70% ethanol prior to processing the following sample.
Genomic DNA (gDNA) isolation
Two methods of total gDNA isolation were used prior to downstream analysis by PCR and qPCR. gDNA was isolated from pecan and other Carya leaves or petioles using the Qiagen DNeasy Plant Mini kit (#69104, Qiagen, Germantown, MD) or the ZR Fungal/Bacterial DNA MiniPrep kit (#D6005, Zymo Research, Irvine, CA) based on manufacturers’ protocols. The DNA pellets were eluted in 50 μl TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0), and the concentration was estimated using a NanoDrop 2000 ultraviolet (UV)-Vis spectrophotometer (ThermoFisher Scientific, Waltham, MA). The DNA concentration was adjusted to 10 ng/μl in sterile deionized water (dH2O) prior to the downstream application.
Enzyme-linked immunosorbent assay (ELISA)
A lyophilized positive control of X. fastidiosa supplied by Agdia Inc. (#LPC 34503, Agdia Inc., Elkhart, IN) was serially diluted (0.1, 1, 10, 25, and 50 μl per 100 μl 1× PBST) to create a standard curve. A set of negative controls included were six 1× PBST (8 mM Na2HPO4, 150 mM NaCl, 2 mM KH2PO4, 3 mM KCl, 0.5% Tween 20, pH 7.4) samples used as template. Using the Agdia Inc. ELISA reagent set (DAS ELISA, Peroxidase label, formerly #SRP 34501 Agdia Inc.) for X. fastidiosa, a 100 μl volume of diluted capture antibody in carbonate coating buffer (1:200) was used to coat a 96-well high-bind microtiter plate for 4 h at room temperature (RT) and subsequently washed 8 times with 1× PBST. A 100 μl volume of each sample was then added to individual wells and incubated overnight in the coated microtiter plate in a humid box at 4 °C. Following the sample incubation period, the microtiter plate was washed 8 times with 1× PBST, and 100 μl of the peroxidase enzyme conjugate antibody diluted (1:200) in a 1× PBST solution containing 20% MRS component was added and incubated for 2 h at RT. The microtiter plate was then washed 8 times with 1× PBST. Finally, 100 μl of the TMB substrate was added to each well and incubated for 20 min at RT under observation. Immediately following the last incubation step, the optical density (OD) of the ELISA test wells was measured at 650 nm by an Epoch 2 microplate spectrophotometer (BioTek Instruments, Inc., Winooski, VT), as previously described (Hernandez-Martinez et al. 2009). An arbitrary threshold value of at least twice the average OD measurement of the negative control samples were used to determine a positive reading (Purcell et al. 1999).
Pecan shoots were harvested from cultivars, VC1–68 (CSV 3–4), Curtis (CSV 16–2), and Cape Fear (CSV 18–11) and processed by three different sample preparation methods for comparison by ELISA (Fig. 2a). Four biological replicates per cultivar were included for each sample preparation treatment to account for variation. An additional fifth replicate from VC1–68 was included for incubated petioles and homogenized tissue. Sample preparation was performed as follows: first, 50 μl crude sap extracted from pecan shoots using a plant water status console plus 50 μl 1× PBST buffer, as previously described, was directly used for the assay. The second method was to incubate plant petiole tissue in General Extraction Buffer (GEB) (Agdia Inc.). A 10 mg sample of petiole tissue (enough to fit inside the microtiter plate well) was cut from sample shoots and immediately placed in individual wells of a pre-coated microtiter plate. The plate wells were filled with 50 μl GEB and incubated at RT for 2 h. Following incubation, the volume of each well was increased to 100 μl with GEB. The third method was to use homogenized plant tissue. Sample petioles and leaflets (1.5 g) were placed in individual mesh bags. Procedures for tissue homogenization were based on manufacturer’s protocols (Agdia Inc.). The GEB was added to the sample bags in a 1:5 ratio, and a mallet was used to macerate the plant tissue. A 100-μl volume of the tissue slurry was added to the wells of a pre-coated microtiter plate. Sample testing was conducted concurrently with approximately equal incubation times and followed manufacturer’s protocols as previously described. Significant differences between the three sample preparation methods were determined by analysis of variance (ANOVA), followed by a post-hoc Tukey’s HSD test using SAS V9.4 M6 University Edition (p < 0.05).
In a separate experiment, two blocking agents and hydrogen peroxide (H2O2) quenching were tested to determine whether they improved the outcome of the ELISA reactions. Eight cultivars that were previously tested for the presence of X. fastidiosa were selected, their crude sap was isolated and directly used for ELISA testing. Due to limited sap volume, sap samples were composited per cultivar, and equal 100 μl volumes were used for each treatment by the different blocking agents. Pretreatment wells were blocked with bovine albumin serum (PBS with 5% BSA, 0.1% Tween 20) or dried nonfat milk (PBS with 5% milk, 0.1% Tween 20) and incubated at RT for 2 h. In an attempt to block potential endogenous hydrogen peroxidase activity by the plant, a subset of sample wells was quenched with 3% H2O2 in the last 15 min of the sample incubation step. An ANOVA followed by the post-hoc Tukey’s HSD test (α = 0.05) was performed using SAS V9.4 M6 University Edition to determine the significance between OD values of pretreated, quenched, and non-treated wells.
Polymerase chain reaction (PCR) detection
Three previously designed X. fastidiosa-specific primer sets (Table 2), S-S-X.fas-0838-a-S-21/S-S-X.fas-1439-a-A-19, RST31/RST33 and HL-5/HL-6 (Francis et al. 2006; C. Chen et al. 2019; Rodrigues et al. 2003) were used to amplify X. fastidiosa target DNA from total gDNA isolated from petioles of a pecan cv. Cape Fear tree where the presence of X. fastidiosa had been confirmed. A Phire Plant Direct PCR Master Mix (#F170, ThermoFisher Scientific) was used for the PCR assays. The PCR reaction components included 2 μl nuclease-free dH2O, 5 μl 2× Phire Plant Direct PCR Master Mix, 1 μl of each forward and reverse primer (0.5 μM), and 10 ng of sample template. Preliminary results of PCR amplification using the annealing temperatures that were reported for the previously designed primer sets indicated non-specific binding (Francis et al. 2006; C. Chen et al. 2019; Rodrigues et al. 2003). As a result, the PCR profile was optimized based on annealing temperature for the three designed primers. Three different annealing temperatures, 55 °C, 58 °C, and 60 °C, were compared for PCR yield and specificity. Additional cycling conditions were based on the manufacturer’s protocols.
Non-specific amplification produced by the previously designed and tested primer sets led to the development of novel primer sets, which were designed based on the sequence of the X. fastidiosa HL (hypothetical protein) amplicon (NCBI GenBank #KY628016) from pecan cv. Pawnee. Conserved regions within the HL amplicon were aligned with the draft genome of pecan genotype 87MX3–2.11 (Jenkins et al. 2013). The HL region that was not homologous to any pecan genome sequences was used to design three new primer sets (assigned as NMU3 to NMU5) (Table 2). Following primer design, the NMU primers were also submitted to NCBI BLASTn analysis to confirm specificity to X. fastidiosa. Two DNA polymerases, iTaq SYBR Green Supermix (#1725120, Bio-Rad, Hercules, CA) and SsoAdvanced Universal SYBR Green Supermix (#1725270, Bio-Rad) were evaluated for amplification effectiveness. Reactions consisted of master mix reagents corresponding to each polymerase, 0.2 μM forward primer (NMU3-F, NMU4-F, or NMU5-F), 0.2 μM reverse primer (NMU-3-R, NMU4-F, or NMU5-R), and DNA template. A gradient PCR was performed to determine the optimal annealing and extension temperature between 55 °C to 62 °C. Two-step PCR was utilized for all reactions; this consisted of denaturation at 95 °C followed by the annealing and extension temperature at 62 °C for 39 cycles. A positive control (purified X. fastidiosa DNA) and negative control (nuclease-free dH2O) were included. Amplification of verified X. fastidiosa-positive pecan gDNA was used for primer comparison.
The NMU3 primer set was further validated for specificity by screening against the purified DNA of 13 bacterial species, selected based on ubiquity within the environment and availability from other labs (Table 3). The Phire Plant Direct PCR Master Mix (ThermoFisher Scientific) was used for the PCR assay with components as previously described. The PCR profile consisted of an initial denaturation step at 98 °C for 5 min, 40 cycles of 98 °C for 5 s, 62 °C for 5 s, and 72 °C for 20 s, and a final extension step at 72 °C for 1 min. PCR products were resolved on a 2% agarose gel and visualized under UV light. The Low Range O’GeneRuler Express DNA ladder (#SM1203, Thermofisher Scientific) was used to estimate band size.
Additionally, direct PCR using crude sap from pecan cv. Cape Fear (CSV 18–11) as a template was performed. Three shoot samples were collected and immediately processed for sap extraction using a plant water status console, as previously described. Isolated crude sap was prepared in serial dilutions (100%, 2.5%, 1.3%, 1%, 0.7% v/v) using the dilution buffer supplied with the Phire Plant Direct PCR Master Mix. Crude sap diluent (1 μl) was used as the template for direct testing. PCR was performed using the NMU3 primer set. The optimal working dilution was selected for further PCR testing using crude sap as the template.
Four different polymerases, KAPA3G Plant PCR Kit (#07961723001, Roche, Basel, Switzerland), EconoTaq DNA Polymerase (#30031–1, Lucigen, Middleton, WI), GoTaq DNA Polymerase (#M3001, Promega, Madison, WI), and Phire Plant Direct PCR Master Mix (ThermoFisher Scientific), were compared for PCR amplification when directly amplifying X. fastidiosa target DNA from crude sap isolated from pecan shoots. Crude sap was diluted to 1.3% in nuclease-free dH2O or dilution buffer respective to each kit and used as the template in the PCR amplification with the HL-5/HL-6 primer set. Cycling conditions were as previously described, with an annealing temperature of 60 °C, and following manufacturers’ protocols for each respective PCR kit.
Total gDNA and crude sap were isolated from the same twelve symptomatic and asymptomatic pecan shoots, as previously described. Three biological replicates were taken from each tree to account for the potential variability in X. fastidiosa concentration within the canopy. Amplification of X. fastidiosa-specific target DNA was performed using three previously designed primer sets, S-S-X.fas-0838-a-S-21/S-S-X.fas-1439-a-A-19, RST31/RST33 and HL-5/HL-6. Results of the PCR were visualized under UV by gel electrophoresis, and then analyzed for the presence of X. fastidiosa, amplicon intensity, and the consistency between sample replicates and the primers used for amplification.
A final unknown sample concentration of 1 ng of gDNA as template was used per PCR reaction. Non-template, negative controls consisting of nuclease-free dH2O (1 μl) as the template, as well as a positive control (1 μl) with a stock solution consisting of lyophilized X. fastidiosa eluted in 2 mL 1× PBST supplied by Agdia Inc. (#LPC 34503, Agdia Inc.) diluted (1 × 10−3) in Dilution Buffer (ThermoFisher Scientific), were prepared and included in each PCR experiment. All PCR products were resolved on a 2% agarose electrophoresis gel and visualized under UV light. The O’GeneRuler Express DNA ladder (#SM1553, Thermofisher Scientific) or the Low Range GeneRuler DNA ladder (#SM1193, ThermoFisher Scientific) was used to estimate band size.
To verify the reproducibility of the PCR assay for the detection of X. fastidiosa, PCR amplification using a novel primer set (NMU3), as described previously, was performed at two different laboratories (USDA-ARS-SEFTNRL, Byron, GA and USDA-ARS Pecan Breeding and Genetics, College Station, TX) and the results were compared. A total of twenty-three shoots were collected from symptomatic and asymptomatic trees in a commercial pecan orchard in Berrien Co., GA. gDNA was isolated from petioles using the ZR Fungal/Bacterial DNA MiniPrep kit (Zymo Research, Irvine, CA), and the concentration was measured using a NanoDrop 2000 UV spectrophotometer (ThermoFisher Scientific). The DNA samples were shared between the two laboratories for independent PCR detection. PCR analysis from both laboratories utilized the Phire Plant Direct PCR Master Mix (ThermoFisher Scientific) for amplification. The PCR cycling conditions were the same across each lab, with an initial denaturation step at 98 °C for 5 min, 35 cycles of 98 °C for 10 s, 60 °C for 10 s, 72 °C for 20 s, and a final extension step at 72 °C for 1 min. PCR amplification was performed through the use of two different thermal cyclers: the USDA-ARS Pecan Breeding and Genetics lab in College Station used an Applied Biosystems SimpliAmp thermal cycler (ThermoFisher Scientific) and the USDA-ARS-SEFTNRL in Byron used a Bio-Rad C1000 Touch thermal cycler (Bio-Rad). PCR product visualization was performed as previously described.
Sequencing and sequence analysis
PCR amplicons of twenty geographically distinct Carya samples were selected for sequencing. Sap, endosperm, or gDNA was used as the template for PCR amplification using the HL-5/HL-6, RST31/RST33, S-S-X.fas-0838-a-S-21/S-S-X.fas-1439-a-A-19, and NMU3 primer sets. The resulting amplicons were gel purified using the Zymoclean Gel DNA Recovery kit (Zymo Research, Irvine, CA), according to the manufacturer’s instructions, for direct Sanger sequencing by Eton Biosciences, Inc. (San Diego, CA). Sequences with a trace score of <20 were omitted from subsequent analysis. Sequences were analyzed by NCBI BLASTn, and global alignments of HL and 16S rRNA PCR sequence fragments were performed using Geneious 9.15 with free end gaps and a cost matrix of 65% (Kearse et al. 2012), as previously described (Hilton et al. 2017).
Real-time quantitative PCR (qPCR)
The HL sequences generated from the twenty geographically distinct Carya samples (Table 1) were examined to determine conserved regions by multiple sequence comparison by log-expectation (MUSCLE) using the online software provided by EMBL-EBI (https://www.ebi.ac.uk/Tools/msa/muscle/) (Edgar 2004). Conserved regions were identified within the sequence encoding for the HL protein and used for designing a Taqman probe and primer set (Table 2). An additional probe and primer set were designed to be specific to pecan actin to serve as the internal positive control (IPC). Primer sequences and the fluorescent probes were generated using the IDT DNA PrimerQuest tool (Integrated DNA Technologies). The newly designed probe and primers for X. fastidiosa were also submitted to NCBI BLASTn analysis to confirm specificity. qPCR reactions were prepared on optical 96-well plates using the TaqPath ProAmp Master Mix (#A30865, ThermoFisher Scientific). Fluorescent intensities in each reaction well were determined by an Applied Biosystems StepOne Real-Time PCR system (ThermoFisher Scientific). Data acquisition and analysis were performed using the StepOne Software v2.3 (ThermoFisher Scientific) with default call settings (Ct threshold = 0.2) and a confidence value of 95%. Determination of ΔRn, the signal strength generated in the qPCR reaction, was calculated as previously described (Weller and Stead 2002). A positive control of lyophilized X. fastidiosa (Agdia) was serial diluted (10−1 to 10−10) to create a standard curve. The standard curve was analyzed to determine qPCR efficiency and specificity of the primer set. Three HL-blocked IPCs and three non-template controls consisting of dH2O and qPCR components were also prepared. Total gDNA isolated from twelve previously tested samples (PCR results presented in Fig. 7) was used to determine the presence or absence of X. fastidiosa. A final DNA concentration of 1 ng/μl was used for each qPCR reaction. The qPCR profile using standard cycling was performed based on manufacturer’s protocols specific for plant genotyping.
The threshold OD value for a positive determination was 0.74 nm. The thirteen sap samples tested by ELISA were all positive based on the determined threshold value (Fig. 2). When the samples were prepared by petiole incubation, nine samples were positive, but four samples were found to be negative (< 0.74 threshold OD). When pecan samples were homogenized in mesh bags prior to testing, only four of the thirteen samples were found to be positive, and nine samples were negative for the presence of X. fastidiosa.
The blocking agents, dried nonfat milk and BSA, or the quenching agent, H2O2, did not significantly impact the mean or variation of OD measurements in comparison to the standard, non-treated control group, nor was there a significant impact on the sensitivity for determining the presence of X. fastidiosa (Fig. 3). All samples tested with or without the blocking agents or quenching agent were found to be positive for X. fastidiosa.
PCR reactions using the different primer sets resulted in an amplicon of the expected size for X. fastidiosa when compared to the positive control (Supp Fig. 1). However, each of the previously reported primer sets revealed non-specific binding and produced similar amplification specificity when purified gDNA of the X. fastidiosa-positive pecan samples was used as the template at the three annealing temperatures. Thus, an annealing temperature of 58 or 60 °C was used for all subsequent PCR reactions. The PCR profile for amplification using the HL-5/HL-6 primer set was an initial denaturation step at 98 °C for 5 min, 35 cycles of 98 °C for 10 s, 60 °C for 10 s, 72 °C for 20 s, and a final extension step at 72 °C for 1 min. Amplification using the RST31/RST33 and the S-S-X.fas-0838-a-S-21/ S-S-X.fas-1439-a-A-19 primer sets followed a PCR profile of an initial denaturation step at 98 °C for 5 min, 35 cycles of 98 °C for 10 s, 58 °C for 10 s, 72 °C for 20 s, and a final extension step at 72 °C for 1 min.
New primers specific to HL of X. fastidiosa (NMU3 to NMU5) were designed and validated. Isolated gDNA from pecan tissue that had previously tested positive for X. fastidiosa was used as the template for PCR. Separation on an agarose gel revealed single amplicon bands for primers NMU3 and NMU5, at the expected amplicon size (Fig. 4). Direct sequencing of the NMU3 and NMU5 amplicons confirmed the identity as X. fastidiosa. Amplification using the NMU4 primer set resulted in multiple bands that were due to non-specific binding to pecan chloroplast DNA (data not included) and not specific to X. fastidiosa. PCR amplification using the NMU3 primer set did not result in any non-specific binding to the DNA of the thirteen bacterial isolates tested and was thus selected for subsequent studies (Supp Fig. 2). BLASTn analysis of the NMU3 primer set resulted in 100% identity to each of the X. fastidiosa subspecies, as well as the NCBI Refseq representative genome, X. fastidiosa 9a5c (NC_002488.3).
Serial dilutions (100%, 2.5%, 1.3%, 1%, 0.7% v/v) of the crude sap from cv. Cape Fear was used to determine the optimal concentration for direct PCR with the NMU3 primer set (Fig. 5). Dilution of crude sap was necessary for PCR amplification of X. fastidiosa DNA due to the presence of PCR inhibitors. A 1.3% v/v dilution of crude sap was optimal for PCR amplification in two of the three biological replicates tested. Four different polymerases were compared for PCR yield when directly amplifying X. fastidiosa target DNA from isolated crude sap from pecan petioles (Fig. 6). The KAPAG3 Plant PCR Kit (Roche) and the Phire Plant Direct PCR Master Mix (ThermoFisher Scientific) were the optimum chemistries for increased yield of PCR bands specific to X. fastidiosa when using crude sap as the template.
A comparison of PCR amplification of X. fastidiosa using total gDNA or crude sap as the template was performed (Fig. 7). Total gDNA or crude sap was isolated from the same symptomatic and asymptomatic pecan shoots and amplification of X. fastidiosa-specific target DNA was performed using the three previously designed primer sets, HL-5/HL-6, S-S-X.fas-0838-a-S-21/ S-S-X.fas-1439-a-A-19 and RST31/RST33. A PCR template of total gDNA revealed greater sensitivity but less consistency in the amplification between the different primers when compared to crude sap. Increased non-specific binding was also found in amplicons when gDNA was used.
Purified amplicons from reactions using the NMU3, HL-5/HL-6, S-S-X.fas-0838-a-S-21/ S-S-X.fas-1439-a-A-19 and RST31/RST33 primer sets were submitted for sequencing (Fig. 8; results of HL-5/HL-6 not shown). An analysis of the sequences of the amplicons using NCBI BLASTn revealed global alignments of highly conserved regions (Supp File 1) between amplicon sequences and the NCBI Refseq representative genome, X. fastidiosa 9a5c (NC_002488.3). HL amplicons of the X. fastidiosa hypothetical gene (WP_004087362.1) had 94 to 99% identity to X. fastidiosa M23 (gb:CP001011.1) and MUL0034 (gb:CP006740.1). The 16S rRNA amplified fragments aligned with 99 to100% identity to X. fastidiosa sub. Multiplex Dixon ctg92 (NZ_AAAL02000001.1). The RNA Polymerase Sigma Factor − 70 amplified fragments aligned with 93 to100% identity to X. fastidiosa strain DSM 10026 (NZ_FQWN01000008.1).
PCR reproducibility assay
The established PCR protocol was conducted independently in two USDA-ARS laboratories (Fig. 9). The NMU3 primer set was used for the PCR amplification of the X. fastidiosa HL target gene. There was good agreement in the detection between the two labs, showing 100% congruence in the PCR results.
Real-time PCR (qPCR)
A TaqMan qPCR assay was developed to determine the presence or absence of X. fastidiosa in isolated total gDNA from pecan tissue (Fig. 10). BLASTn analysis of the newly designed probe and primer set resulted in 100% identity to each of the X. fastidiosa subspecies, as well as the NCBI Refseq representative genome, X. fastidiosa 9a5c (NC_002488.3). The standard curve was robust (R2 = 0.985), with a qPCR efficiency of 99.939%. The 12 gDNA samples (Fig. 7) used for the comparison of gDNA and crude sap as the template by conventional PCR were used for the qPCR assay, and the results were compared. Results were comparable to that of crude sap as the conventional PCR template, with six samples testing positive for X. fastidiosa at a default Ct threshold of 0.2 (P < 0.05). Two additional samples tested positive, but experimental replicates were inconsistent. An IPC was run in multiplex with each unknown sample reaction, all of which resulted in successful amplification.
Our study evaluated currently available diagnostic tools, as well as improved methods for the detection of X. fastidiosa in pecan and related Carya species. Previous reports of PBLS in pecan have primarily relied on the use of ELISA to determine the presence or absence of X. fastidiosa in macerated plant tissue, with only one recent study utilizing PCR for diagnostics of the bacterium in pecan (R. Sanderlin and Heyderich-Alger 2000; R. Sanderlin and Melanson 2006, 2010; R. S. Sanderlin 2015; R. Sanderlin 2017). Comparisons of different sample templates for ELISA testing have been performed for the detection of X. fastidiosa in oleander and grapevines, providing evidence for increased sensitivity when crude xylem sap was used as the template (Bextine and Miller 2004). Results from this study verified that crude sap is the most reliable template for ELISA testing when compared to macerated tissue and excised petioles. The detection limit of ELISA for X. fastidiosa is approximately 2 × 104 to 1 × 105 colony forming units (CFUs) per mL (Norman W Schaad et al. 2003; Minsavage et al. 1994). As X. fastidiosa is xylem-limited, it can be inferred that higher concentrations of the bacterium are found in crude sap when compared to other sources that are diluted by plant material and other microorganisms (Hopkins 1989).
The potential for false positives has been discussed in relation to using ELISA to diagnose X. fastidiosa. (Carbajal et al. 2004). Non-specific binding of the microtiter wells by sample reactants, such as antigens or unspecified proteins, can lead to higher background noise, thereby necessitating the use of blocking agents to effectively coat any non-saturated binding sites (Steinitz 2000; Xiao and Isaacs 2012). Two common blocking agents, BSA and dried nonfat milk, were used to treat test wells prior to sample incubation. The overall OD averages for wells treated with the blocking agents were slightly elevated but not significantly different when compared to the OD mean of samples from standard, untreated test wells. False positives can also be linked to the potential endogenous activity of plant-produced peroxidases, which can interact with the TMB substrate, a critical component of the DAS ELISA (peroxidase) platform utilized for X. fastidiosa detection. To address this, we also attempted sample quenching by H2O2 in the last 20 min of the sample incubation step to reduce peroxidase enzyme activity. Sample quenching appeared to have the greatest impact on the overall OD mean, and the variation between samples was reduced. Yet, no significant difference was found in OD values when compared to standard, untreated test wells.
Advances in DNA-based diagnostic methods have improved our ability to detect smaller quantities of plant pathogens in plant tissue when compared to ELISA. PCR has the sensitivity to detect approximately 100 bacterial CFU/mL (Minsavage et al. 1994). This study provided the optimal conditions for PCR reactions for detection of X. fastidiosa in pecan by evaluating previously reported primer sets (HL-5/HL-6, RST31/RST33, S-S-X.fas-0838-a-S-21/ S-S-X.fas-1439-a-A-19), and by the development of novel primers sets, NMU3 and NMU5. Each of the previously reported primer sets revealed non-specific binding to pecan gDNA and/or other unknown biological contaminants present in the sample at three different annealing temperatures tested. This necessitated the development of the new primers specific to X. fastidiosa that would not bind to the gDNA of pecan or other sample contaminants. Two primer sets, NMU3 and NMU5, were found to be specific to X. fastidiosa and did not reveal non-specific binding to other biological contaminants in total gDNA or crude sap samples. A PCR screen using the NMU3 primer set against 13 bacterial isolates also did not result in non-specific bands, indicating this primer set has a high specificity to X. fastidiosa. Furthermore, the BLASTn analysis revealed 100% identity to X. fastidiosa subspecies, indicating the possible use for diagnostics of other strains of Xylella.
Crude sap was successfully used as the template for direct PCR analysis, and improper dilution of sap increased the risk of false negatives, likely due to the presence of phenolic compounds in pecan that act as PCR inhibitors during amplification (Kays and Payne 1982; Wetzstein and Sparks 1983; Levi et al. 1992). An optimal dilution factor of 1.3% v/v of sap was determined appropriate for PCR amplification. However, a dilution series should be prepared to optimize sample concentration before using crude sap as the template for PCR as seasonal changes in transpiration of pecan influences the content of sap and amount of dilution required for PCR amplification (data not shown). A comparison of PCR detection of X. fastidiosa from crude sap revealed that chemistry is crucial to successful amplification. Three commercial PCR reagent systems were used for the amplification of markers specific to X. fastidiosa gDNA present in the crude sap. The KAPAG3 Plant PCR Kit and the Phire Plant Direct PCR Master Mix were the most sensitive for X. fastidiosa detection and featured increased resistance to PCR inhibition caused by plant secondary metabolites. Isolation of crude sap was a more cost-effective and expedient method for sample preparation (taking approximately 5 min per sample) when compared to DNA extraction from plant tissue.
When comparing the amplification of X. fastidiosa DNA using the three previously designed primer sets, we found that there was an increase in non-specific binding when using total gDNA versus crude sap as the template. A gDNA template revealed increased sensitivity but decreased consistency between primers when testing the same set of samples. Crude sap provided more consistency, despite the risk for inhibition, given the need to properly dilute crude sap prior to PCR analysis. The sequencing of PCR amplicons verified the specificity of primer sets to the target DNA of X. fastidiosa. However, these primers should not be used as a standalone reference to establish X. fastidiosa nomenclature, as the resolution is limited to the species level, and cannot be used to determine subspecies. As there are multiple bacterial endophytes that inhabit plants, it is not surprising that other bacterial sequences may be amplified if the primers are not species-specific. Comparative PCR analysis across the two different laboratories confirmed the PCR protocol developed with the novel NMU3 primer set in this study is reproducible.
A new qPCR probe and primer set was developed for presence/absence experiments of X. fastidiosa in pecan. Compared to conventional PCR, qPCR has an increased dynamic range for detection, is less susceptible to cross-contamination, and does not require downstream electrophoresis and amplicon visualization by UV (Norman W Schaad and Frederick 2002). qPCR can also be optimized for quantification of the target organism, allowing for greater understanding of the epidemiology of the pathogen within its specific host (for example, estimates of bacterial titer). A standard curve based on lyophilized material of X. fastidiosa indicated a high efficiency of the qPCR reaction. However, not all samples that were tested in the comparison of gDNA and crude sap resulted in successful amplification of the target region of the X. fastidiosa genome by qPCR. gDNA was used for the qPCR analysis; however, the results were more consistent with that of conventional PCR amplification using crude sap as the template. One of the major limitations of qPCR systems is the susceptibility to inhibitors contained within the sample (Demeke and Jenkins 2010). Pecan tissue is a commercial source of tannins and other polymers, and these could inhibit qPCR reactions when attempting to detect X. fastidiosa (McGraw et al. 1992). Thus, additional purification attempts may be necessary to ensure accurate results in subsequent qPCR experiments.
Total DNA can be extracted from pecan shoots to serve as the template for PCR. Multiple methods used in this study were successful for DNA isolation and subsequent downstream detection of X. fastidiosa. This allows for a wide range of sample preparation options for researchers based on budget, time, and ease of use. However, not all samples originating from the same tree tested positive in all instances. In a recent study, PCR was utilized to diagnose X. fastidiosa in pecan with primers RST31/RST33; however, false negatives were reported to be a major limitation due to inconsistencies between biological replicates from the same tree, and between different sampling times (R. Sanderlin 2017). This may be a function of the localization of X. fastidiosa within the host since the bacterium does not evenly colonize individual limbs nor distributed evenly across a canopy (R. Sanderlin and Heyderich-Alger 2000). Therefore, despite improved diagnostic methods, there is still the likelihood that X. fastidiosa can exist within plant germplasm below the detection limit. It is recommended that multiple samples be taken per individual tree to ensure proper diagnosis of PBLS, although optimal sample size per tree and sampling methodologies remain to be determined. Using multiple methods of detection, such as performing ELISA and PCR concurrently, can also help to improve reliability of diagnostic results.
The purpose of this research was to evaluate and develop reliable methods for the detection of X. fastidiosa in pecan and related species of Carya. Early detection of PBLS is critical for the development of appropriate integrated pest management approaches. It is important that techniques used in field diagnostics be accurate, rapid, and simple in application (Baldi and La Porta 2017). The development of a single test that can be used in all conditions can be especially challenging, but different approaches can be combined to balance sensitivity, specificity, and ease of use. This report demonstrates that serological and DNA-based PCR methods can be used for the diagnosis of PBLS. It will be important for researchers to use combined approaches with multiple samples to reduce the chance of false negatives or false positives. No source of resistance has yet been established in native pecan or improved cultivars (R. Sanderlin 2005), so other management options must be used. For example, heat treatment can be used to minimize vertical transmission through pecan scions (R. Sanderlin and Melanson 2008).
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This article reports the results of research only. Mention of a trademark or proprietary product is solely for the purpose of providing specific information and does not constitute a guarantee or warranty of the product by the US Department of Agriculture or Texas A&M AgriLife Extension and does not imply its approval to the exclusion of other products that may also be suitable.
This work was supported by USDA-ARS CRIS 3091–21000-042-00D “Management of the National Collection of Carya Genetic Resources and Associated Information”; USDA-ARS CRIS 6042–21220–012-00-D “Mitigating Alternate Bearing of Pecan”; National Plant Germplasm System Grant 58–3091–6-022 “Screening Xylella fastidiosa in the USDA ARS National Collection of Genetic Resources for Carya”; and the Southern Integrative Pest Management (IPM) Center Program (project #1702922).
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be constructed as a potential conflict of interest. This research did not involve human participants or animals.
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Hilton, A., Wang, X., Zhang, M. et al. Improved methods for detecting Xylella fastidiosa in pecan and related Carya species. Eur J Plant Pathol 157, 899–918 (2020). https://doi.org/10.1007/s10658-020-02050-5
- Pecan bacterial leaf scorch