Abstract
Citrus Huanglongbing (HLB, also known as “citrus greening”), an important disease worldwide, is associated with three species of phloem-limited Candidatus liberibacter, of which Candidatus L. asiaticus (CLas) is the predominant one that has severely affected citrus production. TaqMan real-time polymerase chain reaction (PCR) (TM) has been the standard and very efficient method to diagnose several strains of Candidatus Liberibacter in citrus; however, it detects total bacteria and is unable to differentiate dead from live Liberibacter. The detection of only live bacteria is essential for testing methods of control for this important citrus disease. It is well known that ethidium monoazide and propidium monoazide (PMA) are compounds that supposedly enter only dead or membrane-damaged bacteria, intercalate the DNA strand, and make the DNA unavailable for amplification by PCR. These compounds are widely used when extracting the plant DNA to detect only live bacteria. In this research, we tested primers amplifying products from 79 to 1160 bp in TM and SYBR Green real-time PCR (SG) and PMA as DNA intercalating compound. Specifically, primers amplifying a 500-bp amplicon in SG provided the most reliable live-only detection, whereas those producing a smaller amplicon were unable to distinguish between live and dead. This is the first report of testing primers amplifying various amplicon sizes for the detection of only live CLas cells in citrus.
Candidatus Liberibacter asiaticus (CLas), the supposed causal agent of Huanglongbing (HLB), is a deadly bacterial pathogen responsible for devastation of the Florida citrus industry. CLas has been detected in nearly all citrus-producing areas in the United States, and the prognosis is for the loss of many thousands of trees as the disease advances. CLas is a hard-to-culture, phloem-inhabiting bacterium vectored by the Asian Citrus Psyllid insect [Diaphorina citri Kuwayama (Hemiptera: Liviidae)], which facilitates disease spread (Capoor et al., 1974). The bacteria have an uneven distribution in the canopy of the trees (Tatineni et al., 2008); however, they are evenly distributed in feeder roots, with a preference for horizontal distribution (Louzada et al., 2016). Roots may also serve as repository for the bacteria when conditions are not favorable in the canopy (Johnson et al., 2014). No cure has been found for HLB, so only disease management strategies (e.g., removal of infected trees, insect control) have been applied in to reduce inoculum or to prevent further dissemination of the disease.
These strategies increased dramatic grove care costs in addition to crippling the industry. Control strategies are needed to protect existing trees and new planting because no resistant commercial varieties are known.
Testing for compounds able to control the HLB disease meets with limitations because real-time PCR, which has been efficiently used as a diagnostic tool for CLas, cannot differentiate live from dead bacteria, and therefore it is not possible to determine the efficacy of potential controlling compounds. Ethidium monoazide (EMA) and propidium monoazide (PMA) are DNA intercalating compounds that upon photoactivation crosslink with the DNA strands (Fittipaldi et al., 2012), leading to DNA breakage (Soejima et al., 2007). EMA and PMA has been widely used in conjunction with quantitative (qPCR) and viability quantitative PCR (vqPCR) to the viability of estimate bacteria (Exterkate et al., 2014; Moyne et al., 2013) and other microorganisms (Crespo-Sempere et al., 2013; Leifels et al., 2015) because these dyes selectively penetrate dead and membrane-compromised cells (Bae and Wuertz, 2009; Soejima et al., 2007; Zhang et al., 2015).
Although vqPCR has been widely used, the method is not standardized and needs to be optimized for each specific situation (Fittipaldi et al., 2012). Many parameters affect the reliability of vqPCR. Any factor affecting sample turbidity will affect the efficiency of cross-linking of the dyes, which is promoted by photoactivation (Fittipaldi et al., 2011; Gedalanga and Olson, 2009). The concentration of dye, the time of sample exposure, and photoactivation time are important parameters (Desneux et al., 2015; Fittipaldi et al., 2012; Seinige et al., 2014). The type of dye used will also determine the number of false positives that will be produced. EMA overestimates the number of dead cells because it penetrates living cells (Flekna et al., 2007; Kobayashi et al., 2009), mainly in high concentration (Fittipaldi et al., 2012). PMA does not penetrate living cells and is therefore more reliable to differentiate between live and dead cells (Cawthorn and Witthuhn, 2008; Nocker et al., 2006; Pan and Breidt, 2007); however, PMA does not completely remove signals from dead cells, although this depends on the experimental design (Barbau-Piednoir et al., 2014). Care must also to be taken with the concentration of PMA to be used because residual non-crosslinked PMA may affect the efficiency of the reaction, leading to a reduction in amplification without photoactivation (Schnetzinger et al., 2013). There are a few reports on the use of sodium deoxycholate (NaDC) to improve vqPCR signals of PMA-treated samples (Canh et al., 2019; Wang et al., 2015; Yang et al., 2011); however, Nkuipou-Kenfack et al. (2013) observed that NaDC caused PMA to be uptaken by live Listeria cells. Wang et al. (2014) reported that sodium lauroyl sarcosinate (sarkosyl) is more efficient than NaDC in increasing PMA signals of vqPCR; however, no reports are available on how sarkosyl increases the signals of PMA-treated cells.
One of the most significant parameters to be considered, but that it has been neglected in most experiments, is the primer design related to the amplicon size. In most cases, small amplicon size, normally used in real-time PCR, cannot detect dead bacteria.
McCarty and Atlas (1993) were the first to report the influence of amplicon size on bacterial cell viability detection. They observed that even after 24-h exposure of Legionella pneumophila to 10 mg⋅L−1 chlorine, conventional PCR using primers amplifying a 168-bp amplicon could detect the bacteria by hybridization assay. However, primers amplifying an amplicon of 650 bp could not detect live bacteria after 32 min of exposure under the same conditions and detection analysis. Ditommaso et al. (2015) reported similar results when primers amplifying a 400-bp amplicon size were able to suppress more signal from dead Legionella spp. cells than primers amplifying 100 bp. Martin et al. (2013) observed the same effect of amplicon size in experiments to differentiate dead from live Salmonella bacteria, where suppression of amplification signals was very efficient with 417-bp amplicon size but not with 95- or 285-bp amplicon sizes. Other studies (Banihashemi et al., 2012; Contreras et al., 2011; Schnetzinger et al., 2013) confirm that amplicon size plays a very important role in the efficiency of vqPCR to differentiate live from dead bacteria.
In the case of CLas in citrus, an additional layer of complication is the fact that this bacterium has not yet been cultured, and it is a phloem-inhabiting pathogen. There are a few reports of using vqPCR in citrus to detect viable CLas (Hu et al., 2013, 2014; Trivedi et al., 2009); however, in all these studies, the primers used amplify a fragment of less than 100 bp. Trivedi et al. (2009) used EMA, which can also penetrate live cells (Flekna et al., 2007; Kobayashi et al., 2009), and Hu et al. (2013) and Hu et al. (2014) used PMA and primers that amplify a small fragment of 75 bp. The small fragments are desirable for conventional TaqMan qPCR (TM) but are not suitable for vqPCR.
The objectives of this research were to evaluate primers amplifying different amplicon sizes, to compare TM with real-time PCR using SYBR green (SG), and to verify the efficacy of enhancers [Biotium, sodium dodecyl sulfate (SDS), and NaDC] for the detection of only live CLas bacteria. Afterward, the best procedure was used to estimate live CLas population in grapefruit leaves at various stages of development.
Materials and Methods
Leaf sampling.
Leaves for optimization of vqPCR (Expt. 1) were collected from HLB-infected, 9-year-old ‘Rio Red’ grapefruit (Citrus ×aurantium) trees on sour orange rootstock from a commercial grove in San Juan, TX. The leaves with the most prominent symptoms of HLB were sampled randomly throughout the canopies of the infected trees. The midribs and petioles were separated from the leaves, thoroughly chopped into pieces ≈2 mm long with a razor blade, pooled into a single sample, and stored in a magenta GA-7-3 vessel (Sigma-Aldrich, St. Louis, MO) at 4 °C. For each treatment described in Table 1, 50 mg of samples were used in six replications per treatment using TM and SG.
Propidium monoazide (PMA) pretreatment combinations. Heat treatment at 60 °C for 1 h in a ThermoStat Plus heating block (Eppendorf North America, Inc., Hauppauge, NY). A combination of treatments includes nonheated samples, heated samples, presence or absence of enhancers [25 µM PMA enhancer for Gram-negative bacteria (Biotium), Sodium deoxycholate (5% W/V NaDC), or sodium dodecyl sulfate (5% W/V SDS)], and presence or absence of PMA.
A second experiment was set up (Expt. 2) to use the best vqPCR method from Expt. 1 to analyze live and dead CLas status in grapefruit leaves under normal field conditions. Leaves classified in groups from 1 to 10 according to their developmental stage, physical similarity, and levels of HLB symptoms (Fig. 1) were randomly collected from symptomatic branches of six mature (10-year-old), field-grown, naturally infected HLB-positive grapefruit trees grafted onto sour orange (Citrus ×aurantium) rootstock from an orchard in San Juan, TX. Groups 1–7 were nonsymptomatic leaves.
Leaves randomly collected from symptomatic branches of mature field-grown, naturally infected Huanglongbing (HLB)-positive grapefruit trees grafted onto sour orange rootstock were classified into groups 1–10 according to their developmental stage, physical similarity, and levels of HLB symptoms. Groups 1–7 were non-symptomatic leaves.
Citation: HortScience 57, 6; 10.21273/HORTSCI16600-22
Leaves randomly collected from symptomatic branches of mature field-grown, naturally infected Huanglongbing (HLB)-positive grapefruit trees grafted onto sour orange rootstock were classified into groups 1–10 according to their developmental stage, physical similarity, and levels of HLB symptoms. Groups 1–7 were non-symptomatic leaves.
Citation: HortScience 57, 6; 10.21273/HORTSCI16600-22
Leaves randomly collected from symptomatic branches of mature field-grown, naturally infected Huanglongbing (HLB)-positive grapefruit trees grafted onto sour orange rootstock were classified into groups 1–10 according to their developmental stage, physical similarity, and levels of HLB symptoms. Groups 1–7 were non-symptomatic leaves.
Citation: HortScience 57, 6; 10.21273/HORTSCI16600-22
Sample preparation.
For Expt. 1 (inactivation of CLas), samples of leaf midrib (samples 1–4 on Table 1) were performed by heat treatment at 60 °C for 1 h in a ThermoStat Plus heating block (Eppendorf North America, Inc., Hauppauge, NY). A combination of treatments, including nonheated samples, heated samples, presence or absence of enhancers [PMA enhancer for Gram-negative bacteria (Biotium, Hayward, CA), NaDC, or SDS)], and the presence or absence of PMA, as detailed on Table 1, were performed in six replications. For Expt. 2, groups 1, 4, and 5 consisted of four biological replications; groups 2, 3, and 10 had three biological replications; and groups 6, 7, 8, and 9 had 8, 12, 13, and 2 biological replications, respectively. The number of replications depended on the availability of leaves in each particular group. Each biological replication consisted of 10 leaves from which midribs were collected and finely chopped using a sterile blade. Three hundred milligrams of chopped midribs were pulverized in the presence of liquid nitrogen and divided in six samples of 50 mg (three samples for PMA treatment and three samples for control).
PMA and enhancers pretreatment application.
For Expt. 1, 10 µL of 5 mm PMA solution was added to 50-mg ground leaf samples, and the volume completed to 1 mL (final 50 µm). The samples were incubated in the dark for 10 min at room temperature with occasional flicking and exposed to bright blue light for 15 min using a PMA-Lite LED photolysis device (Biotium Inc.). For samples where enhancers were used, the PMA concentration was reduced to 25 µm, and 200 µL of 5x PMA-e, 5% (W/V) NaDC, or 5% (W/V) SDS (Sigma Aldrich) was added. The tubes were centrifuged at 5000 gn for 10 min, and 800 µL of supernatant was discarded. Total genomic DNA was extracted from the remaining pellet and supernatant with the DNeasy Plant Mini Kit (Qiagen) according to the manufacturer’s instructions. Total DNA (50 µL) was eluted in the final step. DNA from control samples (no chemicals added) were isolated also using DNeasy Plant Mini Kit (Qiagen). All treatments were performed in six replications.
In Expt. 2, DNA extracts consisted of samples treated with PMA (Biotium Inc.) and a control without PMA treatment (no enhancers or heat treatment was used). DNA was extracted in triplicate from PMA-treated and control leaf samples in each group of leaves as described for Expt. 1.
Primer screening for detection of live CLas bacterium based on amplicon size.
To verify the best amplicon size for CLas vqPCR, the primers HLBaspr (Li et al., 2006) (79bp), LJfpr (Morgan et al., 2012) (100 bp), CQULA04F/CQULAP10/CQULA04R (CQULA, Wang et al., 2006) (87 bp), Las606/LSS (Fujikawa and Iwanami, 2012) (500 bp), and OI1/OI2c (Jagoueix et al., 1996) (1160 bp) and respective probes were used in TaqMan and SYBR Green real-time PCR reaction to evaluate their performance for the detection of only live CLas. Conventional PCR was also performed with all primers, and the amplicons were sequenced to confirm that they were from CLas.
Real-time PCR.
For Expt. 1, real-time PCR assays were performed using 2 µL of DNA extract in a 25-µL reaction. The TaqMan assay contained 2.5 µL 10X PCR buffer (Mg2+) (Invitrogen, Carlsbad, CA), 2.5 mm MgCl2, 0.2 mm of each dNTP, 0.2 µm each primer, and 1 unit of Platinum Taq DNA polymerase. SYBR Green qPCR assay was performed using the Bio-Rad CFX96 Real-Time system ThermoCycler. The reaction mixture consisted of 12.5 µL of 2X SYBR Premix Ex Taq II (Tli RNase H Plus; Clontech, Takara, Kusatsu, Shiga, Japan), 2 µL of DNA template, 0.25 µm each of sense and antisense primers, and nuclease-free water for a total reaction mixture of 25 µL.
For Expt. 2, SYBR Green qPCR assay was performed using the Bio-Rad CFX96 Real-Time system ThermoCycler and primers Las606/LSS (Fujikawa and Iwanami, 2012) to amplify the 16S ribosomal DNA sequences of CLas. The reaction mixture consisted of 12.5 µL of 2X SYBR Premix Ex Taq II (Tli RNase H Plus; Clontech), 2 µL of DNA template, 0.25 µm of Las606/LSS primers, and nuclease-free water, for a total reaction mixture of 25 µL. The reaction conditions included initial denaturation at 95 °C for 30 s followed by 95 °C for 10 s, 56 °C for 30 s, and 72 °C for 2 min in a 40-cycle reaction. Each PCR reaction was performed in duplicate. To verify the sensitivity of the assay in estimating CLas concentration within leaf tissue, the Las606/LSS PCR fragment of ≈500 bp was cloned into the pCR 4-TOPO Vector (Thermo Fisher Scientific) according to the manufacturer’s instructions, transformed into Escherichia coli bacterial cells, and sequenced. The cloned plasmid was used to produce a 10-fold serial dilution and a melt curve, and the data were analyzed using the CFX Manager software (Bio-Rad). The concentrations for 10-fold serial diluted recombinant plasmid ranged from 2.633 ng⋅µL−1 (5.42 × 108 copies per µL) to 2.633 fg⋅µL−1 (5.42 × 101 copies per µL). PCR was performed using three replicates for each dilution.
Conventional PCR.
Conventional PCR was performed in a 25-µL reaction containing 2.5 µl 10X PCR buffer [buffer contains 200 mm Tris-HCl (pH 8.4) and 500 mm KCl], 6 mm MgCl2, 0.240 mm dNTPs, 1.2 µm of each primer Las606/LSS (Fujikawa and Iwanami, 2012) or OI1/OI2c (Jagoueix et al., 1996), and 1 unit of Platinum Taq DNA polymerase (Invitrogen). Twelve microliters of the PCR products were analyzed with a 1% agarose gel electrophoresis and stained with ethidium bromide.
The details of the primers tested in competitive PCR, SG, and TM assays are listed in Table 2.
Description of different primers and probes used in this study.
Statistical analysis.
A two-way ANOVA was used to evaluate the effects of heat, PMA, and their interaction on CLas detection in leaf tissue for each of the methods tested. The qPCR cycle threshold (Ct) value of leaf tissue was used as dependent variable. The Ct values were transformed into log-CLas genome equivalents for total and live bacteria. One-way ANOVA was used to test whether total and live bacterial cell counts of leaf tissues varied with the developmental stage or leaf age. Whenever significant F values were obtained using the ANOVA, and treatment means were compared using the Student–Newman–Keuls test. All analyses were done using SAS (SAS Institute Inc., Cary, NC).
Results
Comparison of primers for CLas DNA amplification.
To evaluate the adequacy of primers HLBaspr, LJfpr, CQULA, Las606/LSS, and OI1/OI2c to amplify DNA from only live CLas bacteria using TM and SG v-qPCR, a combination of treatments was performed with nonheated, heated, and heated plus PMA samples (Table 1). Enhancers were also tested. Nonheated samples lacking chemical treatments were used as control.
The primer pairs HLBaspr, LJfpr, and CQULA (Table 2) were all efficient in detecting total CLas (live and dead) by both TM and SG using control samples. Primers Las606/LSS did not produce the 500-bp amplicon using TM, but it was efficient using SG, as verified by a melting curve and a serial dilution series (Fig. 2A and B). Melting and a serial dilution curves were obtained for primer set Las606/LSS using SG. The melting curve revealed a single peak with a melt temperature of 87.5 °C (Fig. 2A). In the serial dilution, there was an approximate difference of three Ct values between each dilution (Fig. 2B). The regression equation Y = 12.491 – 0.3094X was used to quantify CLas genome equivalent cells per gram of tissue; in the equation, Y is the log copy number, and X is the Ct value (Fig. 2C). The log concentrations of the plasmid and Ct values showed a linear relationship (R2 = 0.998, slope = −3.24, and efficiency of the reaction = 103.5%). Primer OI1/OI2c did not amplify the 1160-bp product with TM or SG and therefore was excluded from all experiments.
Sensitivity and specificity of the LAS606/LSS primers (Fujikawa and Iwanami, 2012) in SYBR Green real-time polymerase chain reaction (PCR) to detect Candidatus Liberibacter asiaticus from grapefruit leaves. (A) Melt curve analysis showing a single peak at melt temperature of 87.5 °C. (B) Amplification plot generated for 10-fold serial diluted plasmid DNA. RFU = relative fluorescence units. (C) Standard curve generated using a 10-fold serial diluted recombinant plasmid containing a 500-bp 16S ribosomal DNA fragment. The concentrations for 10-fold serial diluted recombinant plasmid ranged from 2.633 ng⋅µL−1 (5.42 × 108 copies per µL) to 2.633 fg⋅µL−1 (5.42 × 101 copies per µL). PCR was performed using three replicates for each dilution. The log concentrations of the plasmid and quantitative PCR threshold cycle Ct values showed a linear relationship with R2 = 0.998, slope of −3.24, and the efficiency of the reaction, E = 103.5%.
Citation: HortScience 57, 6; 10.21273/HORTSCI16600-22
Sensitivity and specificity of the LAS606/LSS primers (Fujikawa and Iwanami, 2012) in SYBR Green real-time polymerase chain reaction (PCR) to detect Candidatus Liberibacter asiaticus from grapefruit leaves. (A) Melt curve analysis showing a single peak at melt temperature of 87.5 °C. (B) Amplification plot generated for 10-fold serial diluted plasmid DNA. RFU = relative fluorescence units. (C) Standard curve generated using a 10-fold serial diluted recombinant plasmid containing a 500-bp 16S ribosomal DNA fragment. The concentrations for 10-fold serial diluted recombinant plasmid ranged from 2.633 ng⋅µL−1 (5.42 × 108 copies per µL) to 2.633 fg⋅µL−1 (5.42 × 101 copies per µL). PCR was performed using three replicates for each dilution. The log concentrations of the plasmid and quantitative PCR threshold cycle Ct values showed a linear relationship with R2 = 0.998, slope of −3.24, and the efficiency of the reaction, E = 103.5%.
Citation: HortScience 57, 6; 10.21273/HORTSCI16600-22
Sensitivity and specificity of the LAS606/LSS primers (Fujikawa and Iwanami, 2012) in SYBR Green real-time polymerase chain reaction (PCR) to detect Candidatus Liberibacter asiaticus from grapefruit leaves. (A) Melt curve analysis showing a single peak at melt temperature of 87.5 °C. (B) Amplification plot generated for 10-fold serial diluted plasmid DNA. RFU = relative fluorescence units. (C) Standard curve generated using a 10-fold serial diluted recombinant plasmid containing a 500-bp 16S ribosomal DNA fragment. The concentrations for 10-fold serial diluted recombinant plasmid ranged from 2.633 ng⋅µL−1 (5.42 × 108 copies per µL) to 2.633 fg⋅µL−1 (5.42 × 101 copies per µL). PCR was performed using three replicates for each dilution. The log concentrations of the plasmid and quantitative PCR threshold cycle Ct values showed a linear relationship with R2 = 0.998, slope of −3.24, and the efficiency of the reaction, E = 103.5%.
Citation: HortScience 57, 6; 10.21273/HORTSCI16600-22
Detection of live CLas bacteria.
For each primer, a two-way ANOVA was conducted to determine the effect of heat and PMA and their interaction on qPCR Ct values. There was no significant difference in Ct values among PCR replications for either the TM or the SG method. Heating the sample for 1 hour did not affect significantly the Ct values obtained with any primer for TM or SG, with less than one cycle average increase indicating that real-time PCR, regardless of the method, amplifies DNA from dead cells.
PMA added to the samples during DNA extraction penetrates only dead cells and intercalates the DNA strand, making it unavailable for amplification by real-time PCR; therefore, it is expected that amplification would occur only from live cells. TM assay using DNA from heated samples treated with PMA had a statistically significant increase in Ct values from 2.7 to 3.17 cycles for primers HLBaspr and LJfpr, but their interaction with heat plus PMA was not significant (Table 3). TM using CQULA primers for the same samples showed a maximum increase of Ct value of only 1.37 and was statistically nonsignificant (Table 3). When using SG for the heated plus PMA sample, there was a significant difference for HLBaspr, LJfpr, and CQULA primers, but the maximum Ct difference was 4.0, with no significance in the interaction between heat and PMA. The Las606/LSS primer set proved to be the best when using SG, showing a significant Ct difference of 6.56 after heating the samples for 1 h, in addition to showing a significant increase of Ct value of 5.19 when PMA was added to heated samples, demonstrating a substantial interaction between heat and PMA.
Analysis of variance showing the effects of heat (60 °C for 1 h) and propidium monoazide (PMA; 25 µM) and their interaction on the quantitative polymerase chain reaction threshold cycle values of leaf tissue using different primers and probe to detect Candidatus Liberibacter asiaticus.
The total Ct increase of heat plus PMA was 11.75 when compared with nonheated and with the non–PMA-treated control (Table 3). Because of these observations, we concluded that LAS606/LSS was the only primer set able to detect only live CLas. It was reported that the addition of enhancers improves the amplification of only live cells. We studied three enhancers (Biotium, SDS, and NaDC) in an attempt to improve the detection of only live bacteria; no benefit was observed with any of the enhancers used (unpublished data).
Because the primers Las606/LSS coupled with SG was able to detect only live CLas as demonstrated above, we used this method to screen ‘Rio Red’ grapefruit leaf samples of various developmental stages and HLB symptoms status (groups 1–10; Fig. 1) for the presence of only live CLas bacteria. Each group consisted of biological replications, each derived from 10 leaves. qPCR and vqPCR in each biological replication were performed in triplicate. Group 1 consisted of very young leaflets, equivalent to V1 stage (Cifuentes-Arenas et al., 2018). The Ct value in all biological replications in this group for total CLas was close to the cutoff value of 37, and the presence of live CLas by vqPCR was not observed. Group 2 comprised young leaves equivalent to V1 stage in Cifuentes-Arenas et al. (2018) but had well-defined petioles. In this group Ct values ranged from the low 20s to 33 for total CLas among the three biological replications. Measurement of only live CLas by vqPCR showed Ct values for two biological replications close to the cutoff value of 37 and one biological replication with Ct values of ≈25. Group 3 leaves were equivalent to V2 leaves from Cifuentes-Arenas et al. (2018), but margins were opened. There were three biological replications in this group, and total CLas Ct values varied from ≈25 to 27, and only live CLas varied from non-amplification (only dead CLas in the total bacteria) to Ct values in the range of 32. Group 4 total CLas Ct values were very homogeneous among the biological replications, varying from ≈23 to ≈24. Live CLas also did not vary among biological replications, with Ct values of ≈27, indicating a reasonable number of live bacteria. Biological replications from group 5 had total CLas Ct values ranging from ≈25 to ≈31. Among the four replications, two showed no amplification with vqPCR, indicating no live bacteria, one had Ct close to the cutoff of 37, and one Ct 33. Group 6 was comprised of eight biological replications, with a variation in total CLas Ct values from ≈19 to ≈25, but the majority were in the low 20s. Live CLas Ct values ranged from 25 to 32, with the majority above 30. In group 7 all five biological replications had total Ct values of 30 and above and no live CLas. Groups 8–10 were CLas symptomatic leaves. Group 8 had 12 biological replications, with 10 of them being very consistent with total CLas Ct values in the low 20s. The maximum and minimum live CLas Ct values for this group were 35.2 and 26.8, respectively. Groups 9 and 10 had only two and three biological replications, respectively; they were very similar, and the average total CLas Ct value of 21.7 was the highest. The highest Ct value for live CLas was 30.9, indicating that even though there were many dead CLas, there were still good numbers of live bacteria.
The total CLas genome equivalents (F = 42.38; df = 9, 136; P < 0.0001) and live CLas genome equivalents (F = 7.91; df = 9, 107; P < 0.0001) varied significantly with leaf age. For both parameters, leaf group 1 had the lowest bacterial load, whereas mature leaves (stages 9 and 10) had the highest total CLas genome equivalents. Leaf group 4 had the highest live CLas genome equivalents, followed by groups 8, 9, and 10.
Discussion
The objective of this study was to establish a reliable vqPCR methodology to diagnose only live CLas bacteria in citrus based on PMA, a DNA intercalating dye. We evaluated five sets of primers and respective probes in qPCR (HLBaspr, LJfpr, CQULA, OI1/OI2c, and Las606/LSS) using TM and five sets of primers with no probes (HLBaspr, LJfpr, CQULA, OI1/OI2c, and Las606/LSS) using SG (Expt. 1). Amplification products for primers HLBaspr, LJfpr, CQULA were ≈100 bp and below, whereas OI1/OI2c and Las606/LSS were 1160 and 500 bp, respectively. The logic for testing these primers is the fact that there are several reports (Banihashemi et al., 2012; Contreras et al., 2011; McCarty and Atlas, 1993; Schnetzinger et al., 2013) indicating that primers amplifying small product are not efficient in identifying only live bacteria. Our study confirms these reports. Using TaqMan real-time PCR, none of the primers was efficient in discriminating live from dead CLas bacteria. OI1/OI2c and Las606/LSS did not amplify products with the control sample (no heat, no PMA), most likely because of the large product size. HLBaspr, LJfpr, and CQULA were all efficient in amplifying control samples, but there was no significant difference in Ct value when the samples were heated for 1 h, confirming that these primers amplify products from dead bacterial cells. The CLas Ct values obtained after the addition of PMA to the heated samples was significant (Table 3), but the maximum increase was 3.17, and there was no significant difference in the heat–PMA interaction; therefore, these primers were not efficient in quantifying only live CLas by TM (Table 3).
When using SYBR Green real-time PCR, OI1/OI2c did not amplify any product from control samples (no heat, no PMA), so it was removed from further tests. HLBaspr, LJfpr, and CQULA followed the same tendency as in TM, being efficient in identifying total CLas cells from control samples, but they were unable to amplify only live CLas when samples were heated. The addition of PMA to heated samples induced a CLas Ct value increase to a maximum of 4, which was significant but not enough to be used as a vqPCR method. Las606 was the when using SG and was as efficient as HLBaspr, LJfpr, and CQULA when using TM or SG.
The Las606 primer set also amplifies products when the samples were heated, without PMA treatment, meaning that by itself it cannot distinguish between live and dead bacteria naturally; however, the addition of PMA to heated samples makes this primer an effective tool to amplify DNA from only live CLas cells. Only the addition of PMA to nonheated samples provoked an increase of 6.74 to the CLas Ct value, indicating that the control sample had naturally good numbers of dead cells. When PMA was added to heated samples, the Ct value increased to 11.75 from a Ct value of 25.75–37.50, which is above the cutoff Ct value of 37 (USDA, 2015) and indicates no quantifiable CLas. Barbau-Piednoir et al. (2014) reported that PMA does not completely remove signals from dead cells, depending on the experimental design, which could explain why Las606/LSS amplified some products after the heated samples were treated with PMA.
Based on the results above, we analyzed by SG 10 groups of leaf samples (control and PMA treated) using primer pair Las606/LSS; each group had different numbers of biological replications depending on the availability of leaves of similar characteristics at the time (Fig. 1). Each biological replication was composed of 10 leaves. The objective in this study was not to correlate population dynamic of CLas related to leaf age or with symptoms but to evaluate the efficacy of the vqPCR in various leaf types collected randomly and under different loads of bacteria. Leaves varied from very young and asymptomatic (groups 1–4) to old and highly symptomatic (groups 9 and 10) (Fig. 1). Total CLas load varied from high (6.14 log CLas genome equivalent, group 9) to low (1.1474 log CLas equivalent, group 1). Groups 9 and 10 were the oldest leaves and the most symptomatic and also contained the highest total CLas load. Out of the total CLas in groups 9 and 10 (6.14 and 6.02 log genome equivalent, respectively), almost 50% were live bacteria, with log CLas genome equivalent of 3.23 and 3.66, respectively. Psyllid may still occasionally feed in these two groups of leaves, but acquisition rates are low compared with young leaves (Sétamou et al., 2020). Leaf groups 1, 3, 5, and 7 grouped into the lowest in live CLas log genome equivalent, with group 1 (the youngest leaf group) having the lowest amount of live CLas (0.793 log CLas genome equivalent). Group 2 leaves (the second youngest leaf group) had 64% of their total CLas alive. The highest amount of live CLas was in leaf group 4, with 4.065 log genome equivalent. Groups 1–4 are preferred by the Asian Citrus Psyllid.
This study demonstrated that TM or SG, using primers producing small amplicon size, are not efficient in distinguishing between live and dead CLas bacteria using PMA intercalating dye. Primers Las606/LSS produce a 500-bp amplicon shown to be effective in distinguishing live from dead CLas bacteria when used in SG. To further confirm the efficacy of these last primers we analyzed close to 500 leaves, divided into 10 groups (Fig. 1) as previously discussed. We confirmed that primer Las606/LSS was very efficient in excluding dead CLas from amplification.
This is the first report about using SG to amplify only live CLas bacteria using primers producing longer amplicons in citrus.
For an easy-to-follow protocol for amplification of only live CLas bacteria our recommendation are as follows:
To 50 mg ground leaf samples, add 10 μL of 5 mm PMA solution and complete the volume to 1 mL (final 50 µm).
Incubate the samples in the dark for 10 min at room temperature with occasional flicking and exposs to bright blue light for 15 min using a PMA-Lite LED photolysis device (Biotium).
Centrifuge the tubes at 5000 gn for 10 min and remove 800 µL of supernatant.
Extract DNA from remaining supernatant plus pellet using DNeasy Plant Mini Kit (Qiagen) according to manufacturer instructions.
Perform SG using 0.25 µm of Las606/LSS primers, 2 µL template DNA, and 2X SYBR Premix Ex Taq II in a 25-µL reaction.
Reaction conditions should be an initial denaturation at 95 °C for 30 s followed by 95 °C for 10 s, 56 °C for 30 s, and 72 °C for 2 min in a 40-cycle reaction.
The above protocol is very efficient, but it may require few adjustments if other enzymes and/or other real-time thermal cycler are used.
Literature Cited
Bae, S. & Wuertz, S. 2009 Discrimination of viable and dead fecal Bacteroidales Bacteria by quantitative PCR with propidium monoazide Appl. Environ. Microbiol. 75 2940 2944 https://doi.org/10.1128/AEM.01333-08
Banihashemi, A., Van Dyke, M.I. & Huck, P.M. 2012 Long-amplicon propidium monoazide-PCR enumeration assay to detect viable Campylobacter and Salmonella J. Appl. Microbiol. 113 863 873 https://doi.org/10.1111/j.1365-26 72.2012.05382.x
Barbau-Piednoir, E., Mahillon, J., Pillyser, J., Coucke, W., Roosens, N.H. & Botteldoorn, N. 2014 Evaluation of viability-qPCR detection system on viable and dead Salmonella serovar Enteritidis J. Microbiol. Methods 103 131 137 https://doi.org/10.1016/j.mimet.2014.06.003
Canh, V.D., Kasuga, I., Furumai, H. & Katayama, H. 2019 Viability RT-qPCR combined with sodium deoxycholate pre-treatment for selective quantification of infectious viruses in drinking water samples Food Environ. Virol. 11 40 51 https://doi.org/10.1007/s12560-01368-2
Capoor, S.P., Rao, D.G. & Vishwanath, S.M. 1974 Greening disease of citrus in the Deccan trap country and its relationship with the vector, Diaphorina citri Kuwayama Weathers, L.G. & Cohen, M. Proceedings of the 6th Conference of the International Citrus Virology, University of California, Division of Agricultural sciences https://escholarship.org/uc/item/6rm6x1tw.
Cawthorn, D.M. & Witthuhn, R.C. 2008 Selective PCR detection of viable Enterobacter sakazakii cells utilizing propidium monoazide or ethidium bromide monoazide J. Appl. Microbiol. 105 1178 1185 https://doi.org/10.1111/j.1365-2672.2008.03851.x
Cifuentes-Arenas, J.C., de Goes, A., de Miranda, M.P., Beattie, G.A.C. & Lopes, S.A. 2018 Citrus flush shoot ontogeny modulates biotic potential of Diaphorina citri PLoS One 13 1 e019056 https://doi.org/10.1371/journal.pone.0190563
Contreras, P.J., Urrutia, H., Sossa, K. & Nocker, A. 2011 Effect of PCR amplicon length on suppressing signals from membrane-compromised cells by propidium monoazide treatment J. Microbiol. Methods 87 89 95 https://doi.org/10.1016/j.mimet.2011.07.016
Crespo-Sempere, A., Estiarte, N., Marín, S., Sanchis, V. & Ramos, A.J. 2013 Propidium monoazide combined with real-time quantitative PCR to quantify viable Alternaria spp. contamination in tomato products Int. J. Food Microbiol. 165 214 220 https://doi.org/10.1016/j.ijfoodmi cro.2013.05.017
Desneux, J., Chemaly, M. & Pourcher, A.-M. 2015 Experimental design for the optimization quantify viable and non-viable bacteria in piggery effluents BMC Microbiol. 15 164 https://doi.org/10.1186/s12866-015-0505-6
Ditommaso, S., Giacomuzzi, M., Ricciardi, E. & Zotti, C.M. 2015 Viability-qPCR for detecting Legionella: Comparison of two assays based on different amplicon lengths Mol. Cell. Probes 29 237 243 https://doi.org/10.1016/j.mcp.2015.05.011
Exterkate, R.A., Zaura, E., Buijs, M.J., Koopman, J., Crielaard, W. & ten Cate, J.M. 2014 The effects of propidium monoazide treatment on the measured composition of polymicrobial biofilms after treatment with chlorhexidine Caries Res. 48 291 298 https://doi.org/10.1159/000356869
Fittipaldi, M., Nocker, A. & Codony, F. 2012 Progress in understanding preferential detectionof live cells using viability dyes in combination with DNA amplification J. Microbiol. Methods 91 276 289 https://doi.org/10.1016/j.mimet.2012.08.007
Fittipaldi, F., Codony, B., Adrados, B., Camper, A.K. & Morato, J. 2011 Viable real-time PCRin environmental samples: Can all data be interpreted directly? Microb. Ecol. 61 7 12 https://doi.org/10.1007/s00248-010-9719-1
Flekna, G., Stefanic, P., Wagner, M., Smulders, F.J., Mozina, S.S. & Hein, I. 2007 Insufficient differentiation of live and dead Campylobacter jejuni and Listeria monocytogenes cells by ethidium monoazide (EMA) compromises EMA/real-time PCR Res. Microbiol. 158 405 412 https://doi.org/10.1016/j.resmic.2007.02.008
Fujikawa, T. & Iwanami, T. 2012 Sensitive and robust detection of citrus greening (huanglongbing) bacterium “Candidatus Liberibacter asiaticus” by DNA amplification with new 16S rDNA-specific primers Mol. Cell. Probes 26 194 197 https://doi.org/10.1016/j.mcp.2012.06.001
Gedalanga, P.B. & Olson, B.H. 2009 Development of a quantitative PCR method to differentiate between viable and nonviable bacteria in environmental water samples Appl. Microbiol. Biotechnol. 82 587 596 https://doi.org/10.1007/s00253-008-1846-y
Hu, H., Roy, A. & Brlansky, R.H. 2014 Live population dynamics of ‘Candidatus Liberibacter asiaticus’, the bacterial agent associated with citrus Huanglongbing, in Citrus and non-Citrus Hosts Plant Dis. 98 876 884 https://doi.org/10.1094/PDIS-08-13-0886-RE
Hu, H., Davis, M.J. & Brlansky, R.H. 2013 Quantification of the live ‘Candidatus Liberibacter asiaticus’ populations using real-time PCR and propidium monoazide Plant Dis. 97 1158 1167 https://doi.org/10.1094/PDIS-09-12-0880-RE
Jagoueix, S., Bové, J.M. & Garnier, M. 1996 PCR detection of the two ‘Candidatus’ Liberobacter species associated with greening disease of citrus Mol. Cell. Probes 10 43 50 https://doi.org/10.1006/mcpr.1996.0006
Johnson, E.G., Wu, J., Bright, D.B. & Graham, J.H. 2014 Association of ‘Candidatus Liberibacter asiaticus’ root infection, but not phloem plugging with root loss on huanglongbing-affected trees prior to appearance of foliar symptoms Plant Pathol. 63 290 298 https://doi.org/10.1111/ppa.12109
Kobayashi, H., Oethinger, M., Tuohy, M.J., Hall, G.S. & Bauer, T.W. 2009 Unsuitable distinction between viable and dead Staphylococcus aureus and Staphylococcus epidermidis by ethidium bromide monoazide Lett. Appl. Microbiol. 48 633 638 https://doi.org/10.1111/j.1472-765X.2009.02585.x
Leifels, M., Jurzik, L., Wilhelm, M. & Hamza, I.A. 2015 Use of ethidium monoazide and propidium monoazide to determine viral infectivity upon inactivation by heat, UV-exposure and chlorine Int. J. Hyg. Environ. Health 218 686 693 https://doi.org/10.1016/j.ijheh.2015.02.003
Li, W., Hartung, J.S. & Levy, L. 2006 Quantitative real-time PCR for detection and identification of Candidatus Liberibacter species associated with citrus huanglongbing J. Microbiol. Methods 66 104 115 https://doi.org/10.1016/j.mimet.2005.10.018
Louzada, E. S., Vazquez, O.E., Braswell, W.E., Yanev, G., Devanaboina, M., Kunta, M. & M. 2016 Distribution of “Candidatus Liberibacter asiaticus” above and below ground in Texas citrus Phytopathology 106 702 709 https://doi.org/10.1094/PHYTO-01-16-0004-R
Martin, B., Raurich, S., Garriga, M. & Aymerich, T. 2013 Effect of amplicon length in propidium monoazide quantitative PCR for the enumeration of viable cells of Salmonella in cooked ham Food Anal. Methods 6 683 690 https://doi.org/10.1007/s12161-012-9460-0
McCarty, S.C. & Atlas, R.M. 1993 Effect of amplicon size on PCR detection of bacteria exposed to chlorine PCR Methods Appl. 3 181 185 https://doi.org/10.1101/gr.3.3.181
Morgan, J.K., Zhou, L., Li, W., Shatters, R.G., Keremane, M. & Duan, Y.-P. 2012 Improved real-time PCR detection of ‘Candidatus Liberibacter asiaticus’ from citrus and psyllid hosts by targeting the intragenic tandem-repeats of its prophage genes Mol. Cell. Probes 26 90 98 https://doi.org/10.1016/j.mcp.2011.12.001
Moyne, A.-L., Harris, L.J. & Marco, M.L. 2013 Assessments of total and viable Escherichia coli O157:H7 on field and laboratory grown lettuce PLoS One 8 7 E70643 https://doi.org/10.1371/journal.pone.0070643
Nkuipou-Kenfack, E., Engel, H., Fakih, S. & Nocker, A. 2013 Improving efficiency of viability-PCR for selective detection of live cells J. Microbiol. Methods 93 20 24 https://doi.org/10.1016/j.mimet.2013.01.018
Nocker, A., Cheung, C.Y. & Camper, A.K. 2006 Comparison of propidium monoazide with ethidium monoazide for differentiation of live vs. dead bacteria by selective removal of DNA from dead cells J. Microbiol. Methods 67 310 320 https://doi.org/10.1016/j.mimet.2006.04.015
Pan, Y. & Breidt, F. 2007 Enumeration of viable Listeria monocytogenes cells by real-time PCR with propidium monoazide and ethidium monoazide in the presence of dead cells Appl. Environ. Microbiol. 73 8028 8031 https://doi.org/10.1128/AEM.01198-07
Schnetzinger, F., Pan, Y. & Nocker, A. 2013 Use of propidium monoazide and increased amplicon length reduces false-positive signals in quantitative PCR for bioburden analysis Appl. Microbiol. Biotechnol. 97 2153 2162 https://doi.org/10.1007/s00253- 013-4711-6
Sétamou, M., Alabi, O.J., Kunta, M., Dale, J. & da Graça, J.V. 2020 Distribution of Candidatus Liberibacter asiaticus in citrus and the Asian citrus psyllid in Texas over a decade Plant Dis. 104 1118 1126 https://doi.org/10.1094/PDIS-08-19-1779-RE
Soejima, T., Iida, K., Qin, T., Taniai, H., Seki, M., Takade, A. & Yoshida, S. 2007 Photoactivated ethidium monoazide directly cleaves bacterial DNA and is applied to PCR for discrimination of live and dead bacteria Microbiol. Immunol. 51 763 775 https://doi.org/10.1111/j.1348-0421.2007.tb03966.x
Seinige, D., Krischek, C., Klein, G. & Kehrenberg, C. 2014 Comparative analysis and limitations of ethidium monoazide and propidium monoazide treatments for the differentiation of viable and nonviable Campylobacter cells Appl. Environ. Microbiol. 80 2186 2192 https://doi.org/10.1128/AEM.03962-13
Tatineni, S., Sagaram, U.S., Gowda, S., Robertson, C.J., Dawson, W.O., Iwanami, T. & Wang, N. 2008 In planta distribution of ‘Candidatus Liberibacter asiaticus’ as revealed by polymerase chain reaction (PCR) and real-time PCR Phytopathology 98 592 599 https://doi.org/10.1094/PHYTO-98-5-0592
Trivedi, P., Sagaram, U.S., Kim, J.S., Brlansky, R.H., Rogers, M.E., Stelinski, L.L., Oswalt, C. & Wang, N. 2009 Quantification of live ‘Candidatus Liberibacter asiaticus’ in hosts using quantitative PCR with the aid of ethidium monoazide (EMA) Eur. J. Plant Pathol. 124 553 563 https://apsjournals.apsnet.org/doi/pdf/10.1094/PDIS-09-12-0880-RE
USDA 2015 Plant sample extraction for use in Citrus Greening (Huanglongbing, HLB) molecular diagnostic assays Revision 2. Document control number: WI-B-T-1–17
Wang, L., Ye, C., Xu, H., Aguilar, Z.P., Xiong, Y., Lai, W. & Wei, H. 2015 Development of an SD-PMA-mPCR assay with internal amplification control for rapid and sensitive detection of viable Salmonella spp., Shigella spp. and Staphylococcus aureus in food products Food Control 57 314 320 https://doi.org/10.1016/j.foodcont.2015.04.016
Wang, L., Li, P., Zhang, Z., Chen, Q., Aguilar, Z., Xu, H., Yang, L., Xu, F., Lai, W., Xiong, Y. & Wei, H. 2014 Rapid and accurate detection of viable Escherichia coli O157:H7 in milk using a combined IMS, sodium deoxycholate, PMA and real-time quantitative PCR process Food Control 36 119 125 https://doi.org/10.1016/j.foodcont.2013.08.011
Wang, Z., Yin, Y., Hu, H., Yuan, Q., Peng, G. & Xia, Y. 2006 Development and application of molecular-based diagnosis for ‘Candidatus Liberibacter asiaticus’, the causal pathogen of citrus huanglongbing Plant Pathol. 55 630 638 https://doi.org/10.1111/j.1365-3059.2006.01438.x
Yang, X., Badoni, M. & Gill, C.O. 2011 Use of propidium monoazide and quantitative PCR for differentiation of viable Escherichia coli from E. coli killed by mild or pasteurizing heat treatments Food Microbiol. 28 1478 1482 https://doi.org/10.1016/j.fm.2011.08.013
Zhang, Z., Liu, W., Xu, H., Aguilar, Z.P., Shah, N.P. & Wei, H. 2015 Propidium monoazide combined with real-time PCR for selective detection of viable Staphylococcus aureus in milk powder and meat products J. Dairy Sci. 98 1625 1633 https://doi.org/10.3168/jds.2014-8938
