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Title: Novel tetraplex qPCR assays for simultaneous detection and identification of Xylella fastidiosa subspecies in plant tissues
Abstract
Xylella fastidiosa is an insect-borne bacterium confined to the xylem vessels of plants. This plant 15 pathogen has a broad host range estimated to 560 plant species. Five subspecies of the pathogen with 16 different but overlapping host ranges have been described, but only three subspecies are widely 17 accepted, namely subspecies fastidiosa, multiplex and pauca. Initially limited to the Americas, Xf has 18 been detected in Europe since 2013. As management of X. fastidiosa outbreaks in Europe depends on 19 the identification of the subspecies, accurate determination of the subspecies in infected plants as early 20 as possible is of major interest. Thus, we developed various tetraplex and triplex qPCR assays for 21
Xylella fastidiosa detection and subspecies identification in planta in a single reaction. We designed 22
primers and probes using SkIf, a bioinformatics tool based on k-mers, to detect specific signatures of 23 the species and subspecies from a dataset of 58 genome sequences representative of X. fastidiosa 24 diversity. We tested the qPCR assays on 39 target and 30 non-target strains, as well as on 13 different 25 plant species spiked with strains of the different subspecies of X. fastidiosa, and on samples from 26 various environmental and inoculated host plants. Sensitivity of simplex assays was equal or slightly 27
better than the reference protocol on purified DNA. Tetraplex qPCR assays had the same sensitivity 28 than the reference protocol and allowed X. fastidiosa detection in all spiked matrices up to 10 3 cells.mL -29 1 . Moreover, mix infections of two to three subspecies could be detected in the same sample with 30 tetraplex assays. In environmental plant samples, the tetraplex qPCR assays allowed subspecies 31 identification when the current method based on multilocus sequence typing failed. The qPCR assays 32 described here are robust and modular tools that are efficient for differentiating X. fastidiosa subspecies 33 directly in plant samples. 34
Xylella fastidiosa (Xf) is a worldwide insect-transmitted plant pathogenic bacterium that presents a very 36 large host range. Altogether, 563 plant species grouped into 82 botanical families have been reported 37
as Xf hosts (EFSA, 2018a). Plants with a major socio-economic interest such as grapevine, citrus, 38 coffee, and olive trees are hosts of Xf (EFSA, 2018a). Forest trees, shade trees, ornamentals and 39 landscape species are included in the host plant database making this pathogen a potential worldwide 40 threat (EFSA, 2018a). Disease management of Xf is impeded by its asymptomatic period that can last 41 several years (EFSA, 2018b) . 42
This bacterial species is genetically diverse as five subspecies including fastidiosa, morus, multiplex, 43 pauca and sandyi are currently described (EFSA, 2018b) . Although this subspecies delineation was 44 initially associated to Xf host range and places of occurrence, more and more observations report specific regions at the species or subspecies level are available. Among these tests, the qPCR assay 84 developed by Harper et al. (2010) has been identified as one of the most appropriate for the detection 85
of Xf, as it has shown a high diagnostic sensitivity compared to others qPCR assays, detects all 86 subspecies, has no cross-reactivity with any other bacterial species and has been successfully used on 87 a wide range of plants ( Modesti et al., 2017; Reisenzein, 2017) . Several tests have been proposed to 88 identify one or more subspecies but no test is currently available to identify all subspecies. The 89
subspecies identification is then routinely performed by MLST, but this method while accurate and 90 portable is time consuming, labor intensive and expensive. From 2018, sequences of only two 91 housekeeping genes (rpoD and cysG or rpoD and malF) are required for subspecies identification in 92
France, while other sets of gene pairs are recommended by EPPO (EPPO, 2018b). 93
In recent years, multiplexed Taqman qPCR has become a useful tool for the identification and 94 quantification of pathogens in different areas such as food safety (Köppel et In this study, we described the development and evaluation of six multiplex qPCR assays for the 106 detection and identification of Xf subspecies. These tests have been designed and tested in silico on a 107 wide range of target and non-target genomic sequences, in vitro on target and non-target bacterial 108 strains, on Xf-spiked plant extracts, and finally in planta on samples from environmental or inoculated 109 plants. These assays allowed the detection of Xf subspecies up to 10 pg.mL -1 of DNA, 1x10 3 CFU. (Wells et al., 1981) or modified PWG media (agar 12 g.L -1 ; soytone 4 g.L -1 ; bacto tryptone 1 g.L -123 1 ; MgSO4.7H2O 0,4 g.L -1 ; K2HPO4 1.2 g.L -1 ; KH2PO4 1 g.L -1 ; hemin chloride (0.1% in NaOH 0.05 124 M) 10 ml.L -1 ; BSA (7.5%) 24 ml.L -1 ; L-glutamine 4 g.L -1 ) at 28°C for one to two weeks. Other strains 125
were grown at 25°C for one to two days on: MG media (Mougel et al., 2001) for Agrobacterium and 126
Rhizobium, TSA (tryptone soy broth 30 g.L -1 ; agar 15 g.L -1 ) for Clavibacter, Ensifer, 127
Stenotrophomonas, Xanthomonas and Xylophilus and King's B medium (KH2PO4 1.5 g.L -1 ; MgSO4, 128 7H2O 1.5 g.L -1 ; protease peptone 20 g.L -1 , glycerol 10 mL.L -1 ; agar 15 g.L -1 ) for Dickeya, Erwinia, 129
Pantoea and Pseudomonas. For qPCR assays, bacterial suspensions were prepared from fresh cultures 130 in sterile distilled water, adjusted at OD600 nm = 0.1. To evaluate assay specificity bacterial suspensions 131
were boiled for 20 min, followed by a thermal shock on ice and a centrifugation at 10,000 g during 10 132
min. 133 pauca (XFP primers) ( Table 3 ). The parameters were set up with an optimal size of 20 bp (sizing 199 between 18-27 bp), an optimal product size of 85 to 150 bp; a Tm of 60°C (± 3°C) and 70°C (± 3°C) 200
for primers and probes, respectively. Then, the individual primer and probe combinations and the six 201
sets of four combinations were tested using Amplify to check the absence of dimer and cross-202 amplification (Engels, 1993) . The specificity of all primers and probes was tested in silico using 203
PrimerSearch The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/699371 doi: bioRxiv preprint in planta identification of Xylella fastidiosa subspecies 6 Four others primer and probe combinations previously published were used in this study. The first 208 targets the rimM gene of Xf (Harper et al., 2010) and was used as reference protocol. The second targets 209 the eukaryotic rRNA18S gene (Ioos et al., 2012) and was used as internal control. The remaining two 210 tests target fastidiosa or multiplex subspecies (Burbank and Ortega, 2018). 211
The tetraplex qPCR assays designed in this study were optimized for: i) primer and probe hybridization 213 temperature that was checked individually by PCR using a gradient ranging from 57.5 to 61. ng.µl -1 of BSA (ThermoFisher) and 1 µL of extracted DNA. The optimal thermocycling conditions 226 selected were: 3 min at 95°C, followed by 40 cycles of 15 s at 95°C and 30 s at 60°C. The qPCR assays 227 results were analyzed, with expert verification, using Bio-Rad CFX Manager 3.1 software and its 228 regression mode. The reaction efficiency was calculated using serial dilutions with the formula: E = 229 10 (-1/slope) . 230
The specificity of the newly designed primer and probe combinations was validated using the 232 optimized protocol on the boiled bacterial suspensions of the 69 strains listed in the The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/699371 doi: bioRxiv preprint in planta identification of Xylella fastidiosa subspecies 7 3
Results 250
Species-specific and subspecies-specific long-mers were identified with SkIf ( for this study (Table 2) . For the subspecies fastidiosa sl specific long-mers were searched for on our 255 58 genome sequences of Xf, using the subspecies fastidiosa, morus and sandyi genomes as ingroups 256 and the multiplex and pauca genomes as outgroups. In total, 3,345 long-mers were identified, ranging 257 from 22 bp to 235 bp (Supplemental data 1). 258
Primers and probes were designed within specific long-mers (Table 3) . Specific amplifications were 259 obtained in silico on XF genome sequences and WGS bacterial sequences from NCBI at the expected 260 amplification size, without any mismatch for the five primer and probe combinations (XFF, XFFSL, 261 XFM, XFMO and XFP). Only two mismatches were observed and concerned the XF primer and probe 262 combination. One mismatch was on the eighth nucleotide on the XF probe for the Xfm Dixon, Griffin1, 263 M12, Sycamore, CFBP 8416, CFBP 8417, CFBP 8418 strains and the second one was on the sixth 264 nucleotide of the forward XF primer of the Ann-1 Xfs strain. As there were not many possible 265
combinations of primers and probes for the XF set, this combination was nevertheless retained, and 266 subsequent in silico checks proved the specificity of all primer and probe combinations. 267
The specificity of each newly designed primer and probe combination was validated in simplex qPCR 269 assays on 39 Xf strains and on 30 plant associated-bacterial strains (Table 1) . These strains were 270 selected as they potentially share the same niche than Xf or for being phylogenetically closely related. 271
No amplification was detected on non-target strains or healthy host plant species and the primer and 272
probe combinations allowed amplification of all strains or subspecies of Xf, for which they were 273 designed (XF: 39/39, XFF: 10/10, XFM: 16/16, XFMO: 1/1, XFP: 7/7, XFFSL: 16/16). 274
In simplex qPCR assays, the LODs of the new primer and probe combinations designed in this study 275 were as good as the LODs obtained with the Harper's qPCR assay or 10 times better for XFM primers 276
( Table 4 ). The efficiency of each combination was evaluated on serial dilutions of calibrated DNA 277
solutions. The XF, XFM, XFMO, XFP, and XFFSL primers and probes allowed detection of Xf up to 278 10 pg.mL -1 (4 copies/reaction). XFF primers were slightly less sensitive with a threshold up to 100 279 pg.mL -1 (40 copies/reaction primers (XFMO) the LOD of tetraplex qPCR assays was usually 10 times higher than the LOD of the 289 simplex test on DNA (Table 4 and Supplemental data 2). In addition, it should be noted that the closer 290 the Ct value was to the detection limit, the higher the SEM was. In tetraplex qPCR assays set n°1, XF, 291 . CC-BY-NC-ND 4.0 International license is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/699371 doi: bioRxiv preprint in planta identification of Xylella fastidiosa subspecies 8 XFM and XFP primers allowed a detection up to 100 pg.mL -1 . The XFFSL primers allowed the 292 detection of Xff up to 10 pg.mL -1 and of Xfmo up to 100 pg.mL -1 . The set n°2 allowed detection up to 293 100 pg.mL -1 using XFF and XFM primers and up to 10 pg.mL -1 with XFP primers. The XF primers 294 allowed the detection of Xff and Xfm up to 100 pg.mL -1 and of Xfp up to 10 pg.mL -1 . The set n°3, 295 allowed a detection up to 100 pg.mL -1 with XF, XFF and XFM primers and up to 10 pg.mL -1 with 296 XFMO primers. 297
A triplex qPCR assay for the simultaneous detection of subspecies fastidiosa and multiplex has recently 298 been published (Burbank and Ortega, 2018). In order to analyze the potential of their targets and 299
potentially introduce them into our sets to improve Xf detection, we tested their specificity in silico and 300
in vitro on selected bacterial strains. sequencing was feasible to confirm the identification. 317
After validation of the efficiency and specificity of the primers and probe, the three sets of tetraplex 319 qPCR assays n°1, 2 and 3, were tested on spiked samples. As the three sets gave similar results, this 320 section is focused on the tetraplex set n°1: XF -XFFSL -XFM -XFP, which covers the full known 321 diversity of Xf (Table 5 ). The results of the other two tetraplex assays are provided in Supplemental 322
Data 5 and Supplemental data 6. This tetraplex qPCR assay (set n°1) was tested on 29 combinations of 323 plant petioles and midribs spiked with one to three strains of the different subspecies. (The full results 324 of the dilution ranges are available in Supplemental data 7). This tetraplex allowed the detection and 325
correct identification of all subspecies in all combinations without false positive result. Although the 326 detection limit was expected to be similar for all plants, since they were all enriched with the same 327 bacterial suspensions, different LODs were observed ranging from 1x10 3 to 1x10 5 CFU.mL -1 (5 to 328 5x10 3 CFU/reaction) depending on the matrix for plants spiked with only one strain. An independent 329
repetition of this test was performed two months after the first one. For O. europaea, P. myrtifolia, P. 330 cerasus, P. dulcis and Q. ilex the LOD was either identical between the two assays or 10 time higher. 331
The LOD of Xf in V. vinifera was 100 times higher in the second assay highlighting a potential 332 accumulation of qPCR inhibitors between the two experiments. Moreover, on 11 combinations out of 333 46, XF primers had a LOD 10 times higher in planta than the one obtained for the subspecies. Xf 334 subspecies could be identified until a Ct value of 35.08 using Harper's qPCR assay in a spiked sample 335 . CC-BY-NC-ND 4.0 International license is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/699371 doi: bioRxiv preprint in planta identification of Xylella fastidiosa subspecies 9 of P. dulcis. In other matrices the LOD of the tetraplex qPCR assay corresponded usually to a Ct value 336 ranging from 30 to 34 using Harper's qPCR. 337
Moreover, the tetraplex qPCR assay set n°1 allowed the detection and identification of mix infections 338 with two to three subspecies simultaneously. On N. oleander, O. europaea, P. myrtifolia and P. dulcis 339 the LOD for the two or three inoculated subspecies is similar of the one obtained for single inoculations 340 (Table 5 ). 341
To demonstrate that our multiplex qPCR assays are modular tools, which can be adapted to one's needs, 342 three other primer and probe sets were evaluated. In one set, we removed the primers and probe 343 targeting the species (set n°4: XFFSL-XFM-XFP). In a second one, we replaced it by the Harper's 344 primers and probe as this test is known to be highly sensitive (set n°5: Harper-XFFSL-XFM-XFP), and 345
we also tested the use of primers and probes targeting the 18S rRNA as an internal control (set n°6: 346
18S-XFFSL-XFM-XFP). Evaluation of these three sets on calibrated DNA suspensions of the Xff strain 347 CFBP 7970 indicated that the LOD for the XFFSL primers was the same than the one found previously 348
for the sets n°1, 4, 5 and 6 (10 pg.mL -1 ) (Supplemental data 8). In Q. robur and C. monspeliensis 349 samples spiked with the Xfm strain CFBP 8416, the LOD obtained for the primers detecting the 350 multiplex subspecies (XFM) was the same for the three sets (1x10 5 CFU.mL -1 ) (Supplemental data 9). 351
The use of Harper's primers and probe in set n°5 allowed the detection of Xf strain at the same LOD 352 than for XF primers and probe in spiked Q. robur samples, but the detection was slightly better (a gain 353 of one Log unit) in the spiked C. monspeliensis samples. A Ct value was obtained for all spiked samples 354 with the 18s rRNA primers, highlighting that these primers and probe were reliable internal 355 amplification controls. 356
plants by tetraplex qPCR assays 358 Ten plant samples from Corsica, France (Table 6 ) and ten samples from inoculated plants (Table 7) 359 were tested using the tetraplex set n°1. Our tetraplex qPCR assay was able to detect the bacterium in 360 samples declared contaminated with Harper's qPCR assay up to Ct =34.97. However, this LOD was 361 variable depending on the matrices (Table 7) . While the bacterium was detected at the subspecies level 362 with one or the other primer and probe combinations in eight environmental plant samples, the XF 363 primers and probe was less efficient and allowed the detection in only five samples ( In this context, on the basis of a large dataset of in-house and publicly available genome sequences of 384
Xf and mix-suspensions these assays display high efficiency (i.e. low LOD), even if, as Ito and Suzaki 429
(2017) have shown, multiplexing increases the LOD by up to one log unit. With a LOD of 10 to 100 430 pg.mL -1 (i.e. 4x10 3 to 4x10 4 copies.mL -1 ), these multiplex qPCR assays still present a sensitivity that 431
is similar to the one of the reference protocol, on single bacterial suspensions (Harper et al., 2010) . 432
In spiked and environmental plant samples, the benefit from the use of our tetraplex assays is obvious. 433
The tetraplex qPCR assays developed here are able to identify Xf subspecies up to 10 3 CFU. 2009). These variations could explain the 10 to 100 fold higher LOD obtain for the second repetition 460 that was performed with grapevine and olive tree sampled two months after the first sample set. 461
While we added a sonication step to improve DNA extraction, we did not test here other ways to 462
improve per se the DNA extraction step and improve the LOD of our assays. Various options are 463 available. A phenol-chloroform step could be added to the DNA extraction method to reduce the level 464 of extracted proteins (Schrader et al., 2012) . Reagents such as Tween 20, DMSO, polyethylene glycol 465 or active carbon could be used to precipitate the polysaccharides before DNA precipitation (Schrader 466 et al., 2012) . Phenol levels may be reduced with the use of polyvinyl-pyrrolidone or the addition of 467 . CC-BY-NC-ND 4.0 International license is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/699371 doi: bioRxiv preprint borate (Wilkins and Smart, 1996) . Drying plant samples at 65°C for 2 days, prior to DNA extraction, 468
could also help to cancel out the effect of phenolic inhibitors (Sipahioglu et al., 2006) . 469
One of the great advantages of the multiplex qPCR assays we developed is that they are modular and 470
reliable. Combinations of primers and probe can be adapted to include sets aiming at detecting 471 infections at the species and/or only at the subspecies level, and having internal controls for each 472 reaction. We showed here as proofs of concept, that replacing our XF primers and probe with the ones 473 from Harper's test is feasible and leads to highly susceptible test, as using 18S rRNA primers and probe 474 as internal control is efficient. 475
In addition, unlike with identification relying on MLST scheme, the qPCR tetraplex assays allow the 476 simultaneous identification of several subspecies in one sample, as demonstrated with spiked samples. 477
In The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/699371 doi: bioRxiv preprint 8
The authors declare that the research was conducted in the absence of any commercial or financial 532 relationships that could be construed as a potential conflict of interest. The present work reflects only 533 the authors' view and no analysis has been made in the French Reference Lab; in particular ED is not 534 authorized to perform any official tests at Anses. 535
Funding 536 ED salary was funded by INRA SPE division and Anses. This work received support from the 537 European Union's Horizon 2020 research and innovation program under grant agreement 727987 538 XF_ACTORS (Xylella Fastidiosa Active Containment Through a multidisciplinary-Oriented 539
Research Strategy). The present work reflects only the authors' view and the EU funding agency is 540 not responsible for any use that may be made of the information it contains. 541 The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/699371 doi: bioRxiv preprint Table 1 :
List of strains used in this study and signals obtained with the primers and probe 744 combinations in simplex qPCR assays on DNA suspensions calibrated at OD600nm = 0.1. 745 Table 2 :
Description and composition of the longest specific long-mers obtained using SkIf for 746 the various targets. 747 Table 3 :
Primers and probes designed in this study for Xf detection at the species and subspecies 748 level. 749 . CC-BY-NC-ND 4.0 International license is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/699371 doi: bioRxiv preprint
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