Abstract
The pathogen Xylella fastidiosa is a xylem-restricted, gram-negative bacterium that is known to cause diseases of many cultivated plant species. Recent outbreaks of X. fastidiosa diseases in Europe have brought attention to the impact of this pathogen, especially to perennial crops. Among the Prunus genus, X. fastidiosa is known to have a wide range of hosts, including plum, almond, peach, cherry, and apricot. Infected trees have reduced fruit quality, possibly resulting in unmarketable fruits, as well as reduced lifespan. There are no curative management options for X. fastidiosa diseases in Prunus; therefore, development of resistant or tolerant cultivars through breeding represents an efficient option to reduce the impact of this pathogen. In this context, the main objective of this study was to determine the occurrence of X. fastidiosa in germplasm of the Stone Fruit Breeding Program at the University of Florida located in Gainesville, FL, USA, under natural infection conditions. A total of 43 individuals representing 10 different genotypic groups within the Prunus genus were tested for the presence of X. fastidiosa. Additionally, we report a novel and easy sampling method using sawdust collected from tree trunks for the detection of this pathogen in Prunus and the development of an endogenous control for improving the diagnosis of this pathogen using real-time polymerase chain reactions. Our results showed a high incidence of X. fastidiosa in the germplasm tested, with more than 65% of the samples positive for the presence of the bacterial pathogen. However, X. fastidiosa was not detected in most of the P. mume samples tested, whereas almost all the P. mume × P. armeniaca hybrids were positive. Negative individuals were also identified in P. avium, P. campanulata, P. umbellata, and P. salicina × P. ceracifera. These trees have been planted in the field, exposed to natural infection for 4 to 11 years, and are considered to show field resistance. Finally, primers and probes based on the Prunus COX gene developed in this study can be used as an internal amplification control to enhance the interpretation of results of X. fastidiosa detection assays using sawdust samples.
Xylella fastidiosa is a bacterial pathogen native to the Americas that was first reported in 1892, by Newton Pierce, to cause a disease in grapevine, which became known as Pierce’s disease (Pierce 1892). Since then, several studies have shown that this pathogen causes important diseases of many cultivated crops such as citrus, coffee, plum, and almond (He et al. 2000; Li et al. 2001; Mizell et al. 2004; Wells et al. 1981). In the past decade, this pathogen has been given more attention because of its recent and quickly spreading outbreaks in Europe (Saponari et al. 2016). There are six well-accepted subspecies of this pathogen with differences in host ranges, symptomatology, distributions, and genetics: X. fastidiosa ssp. fastidiosa, X. fastidiosa ssp. morus, X. fastidiosa ssp. multiplex, X. fastidiosa ssp. pauca, X. fastidiosa ssp. sandyi, and X. fastidiosa ssp. tashke (European Food Safety Authority et al. 2023; Schuenzel et al. 2005; Trkulja et al. 2022). Two of these subspecies have been reported in North America and are associated with disease in Prunus spp., X. fastidiosa ssp. fastidiosa, and X. fastidiosa ssp. multiplex. A third subspecies was described in the southwestern United States, but not in Prunus (Randall et al. 2009). Four subspecies, , X. fastidiosa ssp. fastidiosa, X. fastidiosa ssp. multiplex, X. fastidiosa ssp. pauca, and X. fastidiosa ssp. sandyi, have been reported in Europe in a variety of crops (Greco et al. 2021; Trkulja et al. 2022); however, the last one (X. fastidiosa ssp. sandyi) seems to have been successfully eradicated.
The occurrence of this bacterial pathogen is widespread in the southeastern United States, and the diseases can limit tree longevity and reduce fruit yield and quality of Prunus trees (Mizell et al. 2004; Wells et al. 1981). X. fastidiosa is known to infect many cultivated Prunus spp., such as almond (P. dulcis), plum (P. salicina), cherry (P. avium), apricot (P. armeniaca), and peach (P. persica) (Mizell et al. 2004; Nunney et al. 2013; Olmo et al. 2017; Wells et al. 1981), causing phony peach disease, plum leaf scald, and bacterial leaf scorch in the remaining stone fruit species. Other common symptoms in Prunus spp. are twig dieback, stunted growth, and irregular leaf abscission (Rapicavoli et al. 2018). X. fastidiosa is transmitted by xylem-feeding sharpshooters belonging to the order Hemiptera, including Oncometopia orbona and Homalodisca vitripennis (Greco et al. 2021; Mizell et al. 2004), in the southeastern United States.
There are no curative management options for diseases caused by X. fastidiosa (Mizell et al. 2004). Therefore, there are limited disease management strategies for Prunus that are mainly focused on excluding the pathogen from the orchard. When the pathogen is established, pruning and rogueing symptomatic branches and plants, respectively, could reduce the inoculum source (Mizell et al. 2004). In this context, development of resistant or tolerant cultivars through breeding is a more efficient option to reduce its impact in commercial groves. The first step toward this goal is identifying potential sources of resistance/tolerance to be used as parents in breeding pipelines. The main objective of this study was to determine the occurrence of X. fastidiosa in germplasm of the Stone Fruit Breeding Program at the University of Florida located in Gainesville, FL, USA. We also developed and validated a novel and easy sampling method for the detection of this pathogen in Prunus that includes an endogenous amplification control to improve the dependability of the diagnosis using real-time polymerase chain reactions (PCRs).
Materials and Methods
Plant material.
Forty-three individuals including Prunus spp. and interspecific hybrid trees that are part of the germplasm collection of the Stone Fruit Breeding Program at the University of Florida located in Gainesville, FL, USA (29°38′02.9″N, 82°21′52.0″W), were surveyed for the presence of X. fastidiosa using a real-time PCR. The included species were P. avium, P. campanulata, P. dulcis, P. mume, P. persica, and P. umbellata, and the hybrids P. geniculata × P. salicina, P. mume × P. armeniaca, P. persica × P. dulcis, and P. salicina × P. ceracifera. All trees were part of the University of Florida stone fruit breeding program and planted at the program’s orchard located on campus in Gainesville, FL, USA (Fig. 1), where they were exposed to natural infection. The plants in the field were 4 to 11 years old, except for the ‘Flordaguard’ #1 peach, which was 2 years old. The following plants, grown under greenhouse conditions, were used as negative controls: P. dulcis ‘Jeffries × Nonpareil’ #7 and Fla.31–2; P. persica ‘Flordaguard’ #0 and ‘UFSun’ #0; P. mume #0; P. armeniaca #0; and P. campanulata #0. A DNA sample from a ‘UFSun’ peach tree collected at the University of Florida Plant Science and Research and Education Unit (Citra, FL, USA) that had tested positive for X. fastidiosa at the University of Florida Institute of Food and Agricultural Sciences Plant Diagnostic Center of the University of Florida (Gainesville, FL, USA) was used as a positive control.
Symptoms of Xylella fastidiosa infection in the Prunus germplasm collection at the University of Florida. (A) Leaf scorch symptom caused by X. fastidiosa in P. persica × P. dulcis hybrids similar to that of almond leaf scorch (left) and asymptomatic P. persica (right). (B) Leaf scorch symptoms caused by X. fastidiosa in P. angustifolia hybrid ‘Sharpe’ (left) and asymptomatic P. umbellata (right). (C) Comparison between a peach tree infected with X. fastidiosa (left) and an asymptomatic tree (right). The infected peach tree presents a compact canopy and shorter internodes.
Citation: HortScience 58, 8; 10.21273/HORTSCI17199-23
Sample collection.
Plant tissue was collected in Fall 2020 from 43 trees. Sawdust from tree trunks was collected using an electric drill and a 7/32-inch-diameter drill bit. We aimed to sample only the xylem tissue where X. fastidiosa is found in the plant host. Therefore, the sawdust collected from the initial 1 to 2 cm was discarded; then, the drill bit was cleaned with a brush and re-introduced into the same hole to collect sawdust from the xylem tissues. A funnel was used to capture the sawdust in a 15-mL falcon tube. Each sample weighed ∼300 mg and was composed of sawdust collected from two holes in the tree trunk located in opposing sections (north/south or east/west). The holes were drilled 10 to 15 cm above the soil level or above the graft union. Samples were stored in a cooler with ice immediately after collection and transferred to a refrigerator (4 °C) until processing. All tools were sterilized between sample collections; they were dipped in 10% bleach followed by a 70% ethanol solution and either air-dried or dried with a paper towel. DNA was extracted from the samples within 48 h after collection.
DNA extraction.
Two DNA extraction procedures were compared for the sawdust samples. During the first procedure, DNA extractions were performed using the Qiagen DNeasy® Plant Mini Kit following the manufacturer’s instructions with modifications. First, DNA was extracted from 100 mg of sawdust homogenized in a Tissuelyzer (PowerLyzer 24 Homogenizer) at 2900 rpm for 1.5 min in a 1.5-mL tube containing extraction buffer and two 4.5-mm steel beads. The final elution of the DNA from the column was performed with 50 μL of AE buffer instead of 100 μL to obtain higher DNA concentrations.
The second DNA extraction method combined initial DNA extraction using DNAzol ES plant genomic DNA isolation reagent (Molecular Research Center Inc, Cincinnati, OH, USA) followed by purification of total DNA using the Qiagen DNeasy® Plant Mini Kit. First, 50 mg of sawdust and 1 mL of DNAzol were added to a 1.5-mL screw-cap tube containing two 4.5-mm steel beads. The plant tissue was finely ground using a Tissuelyzer (PowerLyzer® 24 Homogenizer 24) at 2900 rpm for 1.5 min. The homogenized samples were incubated at room temperature for 5 min; then, 1 mL of chloroform was added and vigorously shaken until an emulsion was formed (approximately 20 s). The samples were incubated for 10 min, followed by centrifugation for 10 min at 12,000 gn at room temperature. The supernatant (600 µL) was transferred to a clean 1.5-mL tube, and 500 µL of 100% ethanol was added by mixing thoroughly by inversion; it was incubated at room temperature for 5 min. The DNA was precipitated by centrifugation at 5000 gn for 4 min at room temperature, and the supernatant was carefully discarded. The pellet was resuspended in 100 μL of 10 mm EDTA pH 8.0 and 1 mL of a solution containing both 70% DNAzol and 30% ethanol was added to the sample, incubated for 5 min at room temperature, and centrifuged at 5000 gn for 4 min. Then, the supernatant was removed without disturbing the pellet. Then, 1 mL of 75% ethanol was added to the sample, vortex-mixed, and incubated for 5 min before centrifugation at 5000 gn for 4 min. Once again, the supernatant was carefully removed without disturbing the pellet; then, the pellet was air-dried for 2 to 8 min. The DNA was solubilized in 200 μL of sterile water, and the samples were stored in the refrigerator overnight. The next day, the samples were centrifuged at 12,000 gn for 10 min to precipitate any insoluble materials. A 100-µL aliquot was transferred to a clean 1.5-mL tube without disturbing the pellet, and the purification procedure continued with the Qiagen DNeasy® Plant Mini Kit as described by the manufacturer’s instructions starting with step 6, with the addition of 1.5 volumes (150 μL) of AW1 buffer, mixing, and transferring to a DNeasy® mini spin column.
To compare the two DNA extraction procedures, 10 sawdust samples from the 43 trees to be examined for the presence of X. fastidiosa were selected and DNA was extracted using both methods. DNA concentrations and A260/280 ratios (purity index) were subsequently obtained using a NanoDrop spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA), and means and SDs of the 10 extractions for each method were calculated. To test whether the means of the DNA concentration and A260/280 ratio obtained with each extraction procedure were the same, Student’s t test was performed at a significance level (α) of 0.05 using JMP Statistical Software. DNA concentration values were adjusted to a normal distribution using a square root transformation; no transformation was required for the A260/280 purity index.
Real-time PCR, primer selection, and design for X. fastidiosa detection.
The presence of X. fastidiosa in the samples was tested by performing a real-time PCR assay using the TaqMan probe and primer set XF16Sfpr as previously described (Li et al. 2013). To determine whether contaminants in the DNA extracts had any effect on the real-time PCR amplification, two complementary oligonucleotides containing the target sequence for the Xf16S primers and probe (Table 1) were synthesized. The oligonucleotide pair was added to DNA extracts obtained from greenhouse plants that were never exposed to X. fastidiosa or its vector. Six two-fold serial dilutions in DNA extracts ranging from 10,000 to 312 double-stranded molecules per reaction were tested using a real-time PCR. The species analyzed were P. armeniaca, P. campanulata, P. dulcis, P. mume, and P. persica ‘Flordaguard’ and ‘UFSun’. The other species and hybrids were not available as uninfected samples. The oligonucleotide pair mixed with the Qiagen DNeasy elution buffer without DNA extract was used as the control. There were two replicates per dilution per extract, and the experiment was performed three times. JMP statistical software was used to perform an analysis of variance (ANOVA) to compare the effect of the DNA extracts and buffer on the cycle threshold (Ct) values.
Oligonucleotides used for real-time polymerase chain reaction (PCR) detection of Xylella fastidiosa in Prunus spp.
Additionally, to improve the interpretation of results, we designed and evaluated two probe and primer sets to be used as endogenous controls for our reactions (Table 1). The Prunus mitochondrial cytochrome c oxidase subunit I (PruCOX) gene and Prunus chloroplast NADH dehydrogenase subunit I (PruNdhI) gene sequences were collected from the National Center for Biotechnology Information database (GenBank accessions in Supplemental Table S2). MEGA-X: Molecular Evolutionary Genetics Analysis software (Kumar et al. 2018) was used to perform alignments and identify highly conserved regions for the probe and primer design.
Real-time PCRs were performed using a StepOnePlus real-time PCR system (Applied Biosystems, Waltham, MA, USA) and TaqMan MGB probes (Table 1). The amplification parameters were 95 °C for 20 s, 40 cycles of 95 °C for 1 s, and 60 °C for 20 s. The reactions were composed of 10 μL of Taqman™ Fast Universal PCR Master Mix (2×) (Applied Biosystems), 1 μL of TaqMan MGB Probe And Primer Assay Mix (20×) (Applied Biosystems), 12 ng of DNA, and water to a final volume of 20 μL. The final concentrations of each primer and probe were 900 nM and 250 nM, respectively. Samples were considered positive for the presence of X. fastidiosa when the average Ct value between the two technical replicates was 35 or less.
Results
DNA extraction from sawdust.
DNA was successfully extracted from the sawdust samples using the Qiagen DNeasy® Plant Mini Kit alone or in combination with an initial extraction using DNAzol ES Plant Genomic DNA isolation reagent. In general, DNA extractions using only the Qiagen DNeasy® Plant Mini Kit produced less consistent quantities of DNA (i.e., there were instances when multiple attempts of DNA extraction were required for certain samples to obtain enough DNA for the real-time PCR analysis). An average of two to three of the 10 samples failed to produce DNA. Conversely, the DNA extractions performed using the combined methodology of the DNAzol® Genomic DNA isolation reagent and DNA purification steps of the Qiagen DNeasy® Plant Mini Kit were considerably more consistent. A subset of 10 Prunus samples was initially used to compare the results of both DNA extraction methods (Supplemental Table S3). There were no statistically significant differences between the mean yields obtained with either method (P = 0.5182 and t ratio = −0.663 and P = 0.3547 and t ratio = −0.95261 for the DNA concentration and A260/280 ratio, respectively); however, the combined method (DNAzol followed by purification using the Qiagen DNeasy® Plant Mini Kit) resulted in higher average yields with smaller SDs and higher average purity ratios.
Effect of the DNA isolation procedure on real-time PCR amplification.
The purity of the extracted DNA, represented by the A260/A280 ratio, was less than 1.7 (Supplemental Table S3) for all but one extraction, indicating the potential presence of contaminants that could include PCR amplification inhibitors.
All uninfected samples spiked with target oligonucleotides had an average of two technical replicates with Ct ≤35 (a positive result) in the dilution range assayed (Supplemental Table S4). Negative controls of the DNA extract or buffer not containing the target oligonucleotides were negative (no amplification). A one-way ANOVA indicated no statistical differences between the mean Ct values of the buffer (no DNA extract) and DNA extracts from any Prunus species spiked with the target DNA in any of the dilutions tested (Supplemental Table S4). Therefore, real-time PCR from DNA extracts obtained using the combined protocol showed no indication of inhibition that affected the positive and negative results and generated the same mean Ct values as the spiked extraction buffer.
Evaluation of endogenous control genes.
We evaluated two endogenous genes (one mitochondrial and one plastid) as positive controls for the DNA extraction and amplification steps to improve the interpretation of results. The ideal endogenous control would be amplified in all samples and Prunus species tested. We assessed the amplification of the organellar endogenous controls, PruNdhI and PruCOX, in 20 samples. This study showed that the primer and probe combination used for the plastid gene, PruNdhI, failed to amplify in some samples (P. persica Fla. 13–06 and Fla. 13–07, and P. mume × P. armeniaca Mumecot #1) (Supplemental Table S5). Conversely, the primers and probe designed for the mitochondrial gene, PruCOX, showed amplification in all the samples tested (Supplemental Table S5). Based on these data, we decided to move forward with the X. fastidiosa detection survey using PruCOX as our positive endogenous control.
Xylella fastidiosa detection using real-time PCR.
The survey of 43 Prunus from the trees from the Stone Fruit Breeding Program at the University of Florida (Gainesville, FL, USA) indicated that 28 trees (65%) tested positive for the presence of X. fastidiosa using a real-time PCR (Table 2). The ‘UFSun’ peach sample previously diagnosed as positive for the presence of X. fastidiosa (positive control) was also confirmed as positive in our experiments, and negative controls grown under greenhouse conditions showed no amplification (undetected). At least one negative individual was observed in P. persica (1/5; 1 of 5 trees tested negative), P. mume (6/8), P. mume × P. armeniaca (1/13), P. avium (2/3), P. campanulata (2/3), P. umbellata (2/2), and P. salicina × P. ceracifera (2/2). At the time of the survey, the negative P. avium and P. salicina × P. ceracifera trees had been planted in the field for 4 years and 6 years, respectively. The other negative trees were planted 8 years to 11 years previously (Supplemental Table S1). Thus, these trees have been in the field long enough to be exposed to and acquire the pathogen (Hill and Purcell 1997). The P. persica ‘Flordaguard’ #1 negative tree had been in the field for only 2 years, and it will need to be tested again to corroborate the results.
Presence of Xylella fastidiosa in field Prunus trees at the University of Florida Stone Fruit Breeding Program (Gainesville, FL, USA).
Discussion
The main goal of this study was to determine the presence and incidence of X. fastidiosa infection among different genotypes in the Stone Fruit Breeding Program germplasm collection at the University of Florida (Gainesville, FL, USA). This information aids field evaluations and the identification of elite material resistant or tolerant to this important pathogen to further the breeding program. The species and hybrids included in this study were chosen because they have been evaluated for adaptability and commercial potential in Florida and the southeastern United States. Therefore, we examined sawdust samples collected near the base of the tree as the source of DNA and standardized DNA extraction and real-time PCR procedures. Other studies demonstrated that root samples can be used for the diagnosis of X. fastidiosa in Prunus because of high titers of the pathogen in that organ compared with other parts of the tree (Chen et al. 2019). However, root sampling is labor-intensive, time-consuming, prone to contamination from soil microbes, and can result in insufficient tissue, thus reducing efficiency and leading to erroneous results. Xylem samples in the form of sawdust from the main tree trunk taken close to the root system but above the soil line can be collected quickly and easily with a power drill within seconds.
DNA extraction from woody tissues can be difficult, yielding low concentrations and poor quality (Marsal et al. 2013). Therefore, two protocols were tested for their suitability to produce DNA for the diagnosis of X. fastidiosa. Final DNA yields were similar between the two protocols; however, the two-step procedure, with an initial DNAzol extraction, produced an average of 10% more DNA from most samples and resulted in less failures than Qiagen column purification alone. Therefore, this procedure was selected for this study because it was more cost-effective. Furthermore, no inhibition in the real-time PCR reactions was observed using this DNA extraction procedure, and we were able to detect as little as 312 copies of X. fastidiosa target per sample (Supplemental Table S4). Inclusion of the COX endogenous control verified the presence of DNA and success of the amplification reactions, thus validating the results and facilitating the interpretation of negative results. Other studies have used endogenous controls derived from Prunus nuclear genes (Chen et al. 2019); however, we chose to test two organellar target genes as endogenous controls for the real-time PCR. Our reasoning was that the smaller organellar genomes (≈157 Kbp and ≈508 Kbp chloroplast and mitochondria, respectively) (Fang et al. 2021; Pervaiz et al. 2015) would serve as better DNA purification controls for X. fastidiosa (≈2679 Kbp) (Simpson et al. 2000) than nuclear DNA. It is worth mentioning that after we conducted our experiments, works were published that also used COX as the endogenous control in Prunus spp. for the detection of Xylella fastidiosa (Hodgetts 2022; Hodgetts et al. 2021); however, the primers and probe sequences were different from those reported here. The COX Ct values of samples of P. campanulata, P. avium, P. umbellate, and P. salicina × P. ceracifera hybrids did not show much variation (<2 Ct) (Table 2) between biological replicates when using the same amounts of total DNA, suggesting this gene could potentially be used as a standard for the relative quantification of X. fastidiosa in these genotypes. However, we had a limited number of samples; these results will have to be confirmed with more biological replications. A previous study indicated that X. fastidiosa Ct values from peach root tissue were lower (higher titers) than those in stems and considered more reliable (Chen et al. 2019). The study, however, used different sets of primers and chemistry (SYBER green vs TaqMan-based fluorogenic probe in the present study). TaqMan assays are considered more specific and sensitive, and aboveground tissue is often used for the diagnosis of this pathogen in other species; therefore, the methodology described was regarded as appropriate.
The results of the survey indicated a high incidence of X. fastidiosa at the Gainesville location, with 65% of the samples analyzed testing positive. A recent survey of commercial peach orchards in Alabama, Florida, Georgia, and South Carolina indicated that 74% of the orchards had at least one symptomatic tree, and the incidence varied from 0% to 35% (Johnson et al. 2023). Removal of the infective trees and vector control are not part of the management strategy in the collection, which may explain, at least in part, the almost doubled incidence. Other factors such as climate may also have a role (Feil and Purcell 2001; Johnson et al. 2023). Of note, X. fastidiosa was not detected in several individuals despite being exposed to the risk of potential infection for several years or, for some of them, a decade or more. Although further corroboration is necessary, these trees may represent individuals with field resistance because they are resistant to the bacterial strains present in the area or to their insect vectors or their transmission is blocked. There is also a chance, although remote, that these individuals represent disease escapes; however, the trees grow in close proximity, and the incidence of symptomless individuals certainly varies by species. The three mechanisms mentioned have been demonstrated to occur in Prunus against X. fastidiosa (Amanifar and Luvisi 2022; Dalbó et al. 2016; Kleina et al. 2020; Ledbetter and Rogers 2009).
Our field evaluations indicated that P. mume trees tend to be long-living and asymptomatic for leaf scorching under our growing conditions. However, P. armeniaca trees have historically performed poorly at this location and do not survive after a few years. The P. mume × P. armeniaca crosses were made with the intention of improving the longevity and performance of P. armeniaca. These hybrid trees have also survived, and certain individuals perform adequately, including some that develop symptoms. The results of the survey showed a large proportion of negative X. fastidiosa trees in P. mume, with six of eight trees testing negative, thus supporting the field observations. The P. mume trees in the collection have various origins, such as the following: the United States Department of Agriculture/Agricultural Research Service National Clonal Germplasm Repository; Tree Fruit & Nut Crops & Grapes (Davis, CA, USA), which is a California commercial orchard; and the Department of Agriculture and Fisheries in Queensland, Australia. However, only one of the 13 P. mume × P. armeniaca hybrids was negative for X. fastidiosa. The P. mume parent of these hybrid trees was from Thailand and is not currently part of the collection. These results suggest that genetic diversity within this species exists in relation to the observed field resistance; however, some of the hybrids show tolerance because they are infected with X. fastidiosa but have survived and performed well. Field resistance was also observed in individuals of P. avium, P. campanulata, P. umbellate, and P. salicina × P. ceracifera hybrids.
We have no information about the pathogenicity, virulence, or transmissibility of the X. fastidiosa subspecies and strains present at this location. Therefore, the observed field resistance may not be durable in other locations and requires further investigation. This underscores the importance of surveying Prunus populations for their reaction to local pathogen strains. Finally, the results of the survey help us understand the impact of this pathogen on the performance and survival of Prunus species and hybrids in the germplasm collection and will help guide the breeding of improved stone fruit cultivars adapted to the high-disease environment of the southeastern United States.
References Cited
Amanifar N, Luvisi A. 2022. Resistance of almond (Prunus dulcis) to Xylella fastidiosa: A comparative study on cultivars. Plant Dis. 106(10):2625–2630. https://doi.org/10.1094/Pdis-02-22-0336-Re.
Chen C, Bock CH, Brannen PM. 2019. Novel primers and sampling for PCR detection of Xylella fastidiosa in peach. Phytopathology. 109(2):307–317. https://doi.org/10.1094/phyto-11-18-0439-fi.
Dalbó MA, Bruna ED, Nodari RO, Saifert L. 2016. Plum selections with total resistance to leaf scald (Xylella fastidiosa). Acta Hortic. 1127:61–64. https://doi.org/10.17660/ActaHortic.2016.1127.11.
European Food Safety Authority (EFSA), Delbianco A, Gibin D, Pasinato L, Boscia D, Morelli M. 2023. Update of the Xylella spp. host plant database – systematic literature search up to 30 June 2022. EFSA J. 21(1):e07726. https://doi.org/10.2903/j.efsa.2023.7726.
Fang B, Li J, Zhao Q, Liang Y, Yu J. 2021. Assembly of the complete mitochondrial genome of Chinese Plum (Prunus salicina): Characterization of genome recombination and RNA editing sites. Genes (Basel). 12(12):1970. https://doi.org/10.3390/genes12121970.
Feil H, Purcell AH. 2001. Temperature-dependent growth and survival of Xylella fastidiosa in vitro and in potted grapevines. Plant Dis. 85(12):1230–1234. https://doi.org/10.1094/PDIS.2001.85.12.1230.
Greco D, Aprile A, De Bellis L, Luvisi A. 2021. Diseases caused by Xylella fastidiosa in Prunus genus: An overview of the research on an increasingly widespread pathogen. Front Plant Sci. 12:712452. https://doi.org/10.3389/fpls.2021.712452.
He CX, Li WB, Ayres AJ, Hartung JS, Miranda VS, Teixeira DC. 2000. Distribution of Xylella fastidiosa in citrus rootstocks and transmission of Citrus variegated chlorosis between sweet orange plants through natural root grafts. Plant Dis. 84(6):622–626. https://doi.org/10.1094/PDIS.2000.84.6.622.
Hill BL, Purcell AH. 1997. Populations of Xylella fastidiosa in plants required for transmission by an efficient vector. Phytopathology. 87(12):1197–1201. https://doi.org/10.1094/phyto.1997.87.12.1197.
Hodgetts J. 2022. A panel of real-time PCR assays for the direct detection of all of the Xylella fastidiosa subspecies, p 201–230. In: Luchi N (ed). Plant pathology: method and protocols. Springer, New York, NY, USA. https://doi.org/10.1007/978-1-0716-2517-0_13.
Hodgetts J, Glover R, Cole J, Hall J, Boonham N. 2021. Genomics informed design of a suite of real‐time PCR assays for the specific detection of each Xylella fastidiosa subspecies. J Appl Microbiol. 131(2):855–872. https://doi.org/10.1111/jam.14903.
Johnson KA, Bock CH, Vinson EL, Brannen PM. 2023. Prevalence and distribution of phony peach disease (caused by Xylella fastidiosa) in the United States. Plant Dis. 107(2):326–334. https://doi.org/10.1094/pdis-03-22-0653-re.
Kleina HT, Kudlawiec K, Esteves MB, Dalbó MA, de Paula Oliveira T, Maluta N, Lopes JRS, May-De-Mio LL. 2020. Settling and feeding behavior of sharpshooter vectors of Xylella fastidiosa on plum genotypes resistant to leaf scald disease. Eur J Plant Pathol. 158(3):633–644. https://doi.org/10.1007/s10658-020-02104-8.
Kumar S, Stecher G, Li M, Knyaz C, Tamura K. 2018. MEGA X: Molecular Evolutionary Genetics Analysis across Computing Platforms. Mol Biol Evol. 35(6):1547–1549. https://doi.org/10.1093/molbev/msy096.
Ledbetter CA, Rogers EE. 2009. Differential susceptibility of Prunus germplasm (Subgenus Amygdalus) to a California isolate of Xylella fastidiosa. HortScience. 44(7):1928–1931. https://doi.org/10.21273/Hortsci.44.7.1928.
Li W-B, Pria WD, Teixeira DC, Miranda VS, Ayres AJ, Franco CF, Costa MG, He C-X, Costa PI, Hartung JS. 2001. Coffee leaf scorch caused by a strain of Xylella fastidiosa from Citrus. Plant Dis. 85(5):501–505. https://doi.org/10.1094/PDIS.2001.85.5.501.
Li WB, Teixeira DC, Hartung JS, Huang Q, Duan YP, Zhou LJ, Chen JC, Lin H, Lopes S, Ayres AJ, Levy L. 2013. Development and systematic validation of qPCR assays for rapid and reliable differentiation of Xylella fastidiosa strains causing citrus variegated chlorosis. J Microbiol Methods. 92(1):79–89. https://doi.org/10.1016/j.mimet.2012.10.008.
Marsal G, Boronat N, Canals JM, Zamora F, Fort F. 2013. Comparison of the efficiency of some of the most usual DNA extraction methods for woody plants in different tissues of Vitis vinifera L. J Int Sci Vigne Vin. 47(4):227–237. https://doi.org/10.20870/oeno-one.2013.47.4.1559.
Mizell R, Andersen P, Tipping C, Brodbeck B. 2004. Xylella fastidiosa diseases and their leafhopper vectors, University of Florida. IFAS Extension, EDIS. ENY-683. https://doi.org/10.32473/edis-in174-2003.
Nunney L, Vickerman DB, Bromley RE, Russell SA, Hartman JR, Morano LD, Stouthamer R. 2013. Recent evolutionary radiation and host plant specialization in the Xylella fastidiosa subspecies native to the United States. Appl Environ Microbiol. 79(7):2189–2200. https://doi.org/10.1128/Aem.03208-12.
Olmo D, Nieto A, Adrover F, Urbano A, Beidas O, Juan A, Marco-Noales E, López MM, Navarro I, Monterde A, Montes-Borrego M, Navas-Cortés JA, Landa BB. 2017. First detection of Xylella fastidiosa infecting cherry (Prunus avium) and polygala myrtifolia plants, in Mallorca Island, Spain. Plant Dis. 101(10):1820. https://doi.org/10.1094/PDIS-04-17-0590-PDN.
Pervaiz T, Sun X, Zhang Y, Tao R, Zhang J, Fang J. 2015. Association between Chloroplast and Mitochondrial DNA sequences in Chinese Prunus genotypes (Prunus persica, Prunus domestica, and Prunus avium). BMC Plant Biol. 15(1):4. https://doi.org/10.1186/s12870-014-0402-4.
Pierce NB. 1892. The California vine disease: A preliminary report of investigations. Published by authority of the Secretary of Agriculture. Bulletin of the US Department of Agriculture Division of Vegetable Pathology No 2.
Randall JJ, Goldberg NP, Kemp JD, Radionenko M, French JM, Olsen MW, Hanson SF. 2009. Genetic analysis of a novel Xylella fastidiosa subspecies found in the Southwestern United States. Appl Environ Microbiol. 75(17):5631–5638. https://doi.org/10.1128/AEM.00609-09.
Rapicavoli J, Ingel B, Blanco-Ulate B, Cantu D, Roper C. 2018. Xylella fastidiosa: An examination of a re-emerging plant pathogen. Mol Plant Pathol. 19(4):786–800. https://doi.org/10.1111/mpp.12585.
Saponari M, Boscia D, Altamura G, D’Attoma G, Cavalieri V, Zicca S, Morelli M, Tavano D, Loconsole G, Susca L, Potere O, Savino V, Martelli GP, Palmisano F, Dongiovanni C, Saponari A, Fumarola G, Carolo MD. 2016. Pilot project on Xylella fastidiosa to reduce risk assessment uncertainties. EFSA Supporting Publications. 13(3):1013E. https://doi.org/10.2903/sp.efsa.2016.EN-1013.
Schuenzel EL, Scally M, Stouthamer R, Nunney L. 2005. A multigene phylogenetic study of clonal diversity and divergence in North American strains of the plant pathogen Xylella fastidiosa. Appl Environ Microbiol. 71(7):3832–3839. https://doi.org/10.1128/AEM.71.7.3832-3839.2005.
Simpson AJG, Reinach FC, Arruda P, Abreu FA, Acencio M, Alvarenga R, Alves LMC, Araya JE, Baia GS, Baptista CS, Barros MH, Bonaccorsi ED, Bordin S, Bové JM, Briones MRS, Bueno MRP, Camargo AA, Camargo LEA, Carraro DM, Carrer H, Colauto NB, Colombo C, Costa FF, Costa MCR, Costa-Neto CM, Coutinho LL, Cristofani M, Dias-Neto E, Docena C, El-Dorry H, Facincani AP, Ferreira AJS, Ferreira VCA, Ferro JA, Fraga JS, França SC, Franco MC, Frohme M, Furlan LR, Garnier M, Goldman GH, Goldman MHS, Gomes SL, Gruber A, Ho PL, Hoheisel JD, Junqueira ML, Kemper EL, Kitajima JP, Krieger JE, Kuramae EE, Laigret F, Lambais MR, Leite LCC, Lemos EGM, Lemos MVF, Lopes SA, Lopes CR, Machado JA, Machado MA, Madeira AMBN, Madeira HMF, Marino CL, Marques MV, Martins EAL, Martins EMF, Matsukuma AY, Menck CFM, Miracca EC, Miyaki CY, Monteiro-Vitorello CB, Moon DH, Nagai MA, Nascimento ALTO, Netto LES, Nhani A Jr, Nobrega FG, Nunes LR, Oliveira MA, de Oliveira MC, de Oliveira RC, Palmieri DA, Paris A, Peixoto BR, Pereira GAG, Pereira HA Jr, Pesquero JB, Quaggio RB, Roberto PG, Rodrigues V, de M. Rosa AJ, de Rosa VE Jr, de Sá RG, Santelli RV, Sawasaki HE, da Silva ACR, da Silva AM, da Silva FR, Silva WA, da Silveira JF, et al. 2000. The genome sequence of the plant pathogen Xylella fastidiosa. Nature. 406(6792):151–157. https://doi.org/10.1038/35018003.
Trkulja V, Tomić A, Iličić R, Nožinić M, Milovanović TP. 2022. Xylella fastidiosa in Europe: From the introduction to the current status. Plant Pathol J. 38(6):551–571. https://doi.org/10.5423/PPJ.RW.09.2022.0127.
Wells JM, Raju BC, Thompson JM, Lowe SK. 1981. Etiology of Phony peach and plum leaf scald diseases. Phytopathology. 71(11):1156–1161. https://doi.org/10.1094/Phyto-71-1156.
Supplemental Table S1. List and age of Prunus species and hybrids surveyed for the presence of Xylella fastidiosa.
Supplemental Table S2. Prunus spp. GenBank accession numbers for the DNA sequences used to design the endogenous control primers and probes.
Supplemental Table S3. DNA concentration and purity (A260/A280 ratio) obtained with the two extraction procedures, Qiagen DNeasy Plant Mini Kit and the DNAzol combined with the Qiagen DNeasy Plant Mini Kit.
Supplemental Table S4. Real-time polymerase chain reaction (PCR) inhibition test. Cycle threshold (Ct) values of Prunus DNA extracts spiked with Xylella fastidiosa 16S target oligonucleotides.
Supplemental Table S5. Real-time polymerase chain reaction (PCR) amplification (Ct) of Prunus spp. organellar endogenous controls.
