Eur J Plant Pathol

https://doi.org/10.1007/s10658-018-01606-w

Identification, intra- and inter-laboratory validation

of a diagnostic protocol for ‘Candidatus Liberibacter
solanacearum’ in carrot seeds
Vincenza Ilardi & Valentina Lumia & Elisa Di Nicola &
Mario Tavazza

Accepted: 25 September 2018

# Koninklijke Nederlandse Planteziektenkundige Vereniging 2018

Abstract ‘Candidatus Liberibacter solanacearum’
(CaLsol) is a phloem-limited, unculturable, Gram-
negative bacterium associated with emerging diseases
in crops of the Solanaceae and Apiaceae families. As it
has been shown to be seed-transmitted in carrot, emer-
gency measures for exportation require carrot seed to be
heat-treated or tested by PCR and found CaLsol free.
Therefore, the identification and harmonization of a
protocol for CaLsol diagnosis in carrot seed are becom-
ing of socio-economic priority. We initially set up an
improved DNA extraction method for Apiaceae seeds
and identified, among the widely used PCR tests to
detect and identify CaLsol, the real-time PCR developed
by Li et al. (Journal of Microbiological Methods, 78(1),
59–65, 2009) and the end-point PCR by Ravindran et al.
(Plant Disease, 95(12), 1542–1546, 2011) to be the
most sensitive ones. The two PCR methods were ini-
tially intra-laboratory validated followed by a BTest
Performance Study^ involving 11 Italian laboratories
that received both the samples and the material neces-
Electronic supplementary material The online version of this
article (https://doi.org/10.1007/s10658-018-01606-w) contains
supplementary material, which is available to authorized users.
V. Ilardi (*) : V. Lumia : E. Di Nicola
Consiglio per la ricerca in agricoltura e l’analisi dell’economia
agraria-Centro di ricerca difesa e certificazione sede di Roma
(CREA-DC-RM), Via C. G. Bertero, 22, 00156 Rome, Italy
e-mail: vincenza.ilardi@crea.gov.it
M. Tavazza
ENEA CR Casaccia, SSPT-BIOAG, Via Anguillarese, 301,
00123 Rome, Italy
sary to carry out the experiments. The results indicated
that the improved DNA extraction method was robust
and that the real-time PCR showed the highest analytical
sensitivity in the intra-laboratory validation tests. Simi-
larly, the real-time PCR outperformed the end-point
PCR in the inter-laboratory comparison assay showing
a higher percentage of accuracy, accordance, and con-
cordance. The overall obtained data could be used for
the appropriate application of phytosanitary measures
against CaLsol.
Keywords Ring test . Molecular detection . Apiaceae .
Plant-pathogen . EPPO standards
Introduction
‘Candidatus Liberibacter solanacearum’ (CaLsol) is a
phloem-limited, unculturable, Gram-negative α
proteobacterium associated with several detrimental
emerging diseases (Janse 2012; Ilardi and Catara 2013;
Haapalainen 2014; Munyaneza 2015a).
CaLsol was first identified as the causal agent of
zebra chip disease in potato (Solanum tuberosum) and
then associated with diseases in many other solanaceous
crops, causing significant economic losses in North and
Central America, and Oceania (Munyaneza et al. 2007;
Hansen et al. 2008; Liefting et al. 2008).
In Europe and the Mediterranean area CaLsol was
associated with vegetative disorders in species of the
Apiaceae family, mainly in carrot (Daucus carota) (Fin-
land, Munyaneza et al. 2010a, b; Norway, Munyaneza

et al. 2012a; Sweden, Munyaneza et al. 2012b; Spain
Canary Islands, Alfaro-Fernández et al. 2012a; Spain
Mainland, Alfaro-Fernández et al. 2012b; France,
Loiseau et al. 2014; Morocco, Tahzima et al. 2014;
Germany, Munyaneza et al. 2015b; Greece, Holeva
et al. 2017; Austria, EUPHRESCO 2017; Israel, EPPO
reporting service 2017; Italy, Catara et al. 2017; Tunisia,
Ben Othmen et al. 2018). Also, CaLsol was recently
detected in commercial Apiaceae seeds sold in Italy and
the United Kingdom (Ilardi et al. 2016a, b; Monger and
Jeffries 2016) as well as in the United Kingdom
Apiaceae historical seed (Monger and Jeffries 2017).
The genetic diversity of CaLsol has been studied, and
five haplotypes (A-E) have so far been described by
single nucleotide polymorphisms (SNPs) across portions
of 16S ribosomal rRNA gene, 16S/23S intergenic spacer
region (ISR), and 50S rpIJ and rpIL ribosomal protein
genes (Nelson et al. 2011, 2013; Teresani et al. 2014).
Haplotypes A and B are associated with diseases in potato
and other solanaceous crops in America and Oceania and
are included in the EPPO A1 quarantine list (European
Plant Protection Organization 2016). Haplotypes C, D,
and E, are associated with Apiaceae disorders in Europe
and the Mediterranean area (Hajri et al. 2017; Nelson
et al. 2013; Teresani et al. 2014). Very recently, a novel,
sixth haplotype of CaLsol, closely related to A and D,
was found in stinging nettle (Urtica dioica, Urticaceae),
and named haplotype U (Haapalainen et al. 2018).
CaLsol is spread from infected to healthy plants by
psyllid (Family Psyllidae) insect vectors. Also, Bertolini
et al. (2015) showed that CaLsol is seed-borne in carrot.
This last finding, although debated (Loiseau et al. 2017),
prompted the activation of FAO/IPPC Emergency ac-
tion: BNotification of phytosanitary measures to reduce
the risk of introduction of CaLsol through the importa-
tion of carrot seed …^ (Food and Agriculture Organi-
zation of the United Nations - International Plant Pro-
tection Convention, Emergency actions, 2015). These
measures require carrot seed to be tested by PCR and
found CaLsol free or heat-treated to inactivate the bac-
terium. Therefore, establishing a diagnostic protocol to
control the presence of CaLsol in carrot seed lots as-
sumes strategic importance from agricultural-social-
economical points of view. For the appropriate applica-
tion of phytosanitary measures, using detection methods
able to produce reliable analytical results is the prereq-
uisite. Thus, after the identification of the methods, it is
necessary to evaluate and test them to define how valid
and reliable they are. Validation, as defined by the ISO/
Eur J Plant Pathol
IEC 17025, EPPO 7/122 (1) (2014) and EPPO 7/98 (2)
standards, is a rigorous process that includes both single
laboratory validation and an inter-laboratory test perfor-
mance study (TPS), the latter being also referred to as
ring test, a form of inter-laboratory comparison.
Due to the inability of CaLsol to be cultured in vitro
and the low titre in which it occurs in the host plants,
especially in the seeds, CaLsol detection and identifica-
tion are based on molecular tests. Several PCR-based
methods have been developed, mainly targeting the
CaLsol genome regions used for haplotypes determina-
tion (16S ribosomal rRNA gene, 16S/23S ISR, and 50S
rpIJ and rpIL ribosomal protein genes). The most used
end-point PCRs are those targeting: i) the 16S ribosomal
rRNA, Li et al. (2009) and Liefting et al. (2008); ii) the
50S rpIJ and rpIL ribosomal protein genes, Munyaneza
et al. (2009); and iii) the 16S/23S ISR, Ravindran et al.
(2011). For the real-time PCR, the most widely used
protocol (e.g., by International Seed Federation- ISF
2016; and International Plant Protection Convention
–IPPC 2017) is the one developed by Li et al.
(2009). The method of Teresani et al. (2014) could
also be performed (IPPC/ISPM 2017). Both assays
target the same region of the 16S ribosomal rRNA
gene. Importantly, these PCR-based tests were not
yet inter-laboratory validated as a complete protocol
starting from the DNA extraction from seed to the
final detection and identification of CaLsol.
This work presents an implemented DNA extrac-
tion method for the detection of CaLsol in Apiaceae
seeds, the identification and intralaboratory valida-
tion of one sensitive end-point and one real-time
PCR test to detect CaLsol in Apiaceae seeds follow-
ed by a TPS, involving 11 laboratories.
Materials and methods
Samples: Apiaceae seed lots, CaLsol control DNA,
other pathogens, and bacteria
A total of 72 samples were used in this work. They are
listed and described in Online Resource 1 and can be
classified as follows: a) 37 CaLsol infected Apiaceae
seed lots, of which 21 for gardening and 16 for agricul-
ture; b) the pTXZC18 plasmid (kindly provided by
Wenbin Li) containing the 16S CaLsol real-time PCR
target sequence; c) 14 CaLsol free Apiaceae seed lots, of
which nine for gardening and five for agriculture; d) 13
Eur J Plant Pathol

characterized bacteria from CREA-DC-RM and CREA- using Platinum Taq polymerase (Thermo Fisher) or Go
DC-BAT collections (kindly provided by Stefania Loreti Taq G2Flexi DNA polymerase (Promega).
and Loredana Sigillo, respectively); e) 1 phytoplasma
(kindly provided by Luca Ferretti); and f) 6 unidentified
Real-time PCR
bacterial isolates from carrot seeds.
To isolate unknown bacteria from carrot seeds, 0.5 g
The real-time PCR methods tested in this work were
of seeds were washed and soaked in PBS-Tween buffer
those of Li (Li et al. 2009) and Teresani (Teresani et al.
O/N at 4 °C, then they were crushed in a stomacher bag
2014) following the authors’ instructions and using
in PBS buffer and decanted for 5 min. Two ml were
Platinum Taq polymerase (Thermo Fisher) or TaqMan
centrifuged at 200×g for 5 min and the supernatant
Universal Master Mix II (Thermo Fisher). For both
centrifuged at 9000×g for 20 min. The pellet was resus-
methods, 45 cycles were applied. A sample was consid-
pended in PBS buffer and plated on the nutrient agar
ered positive if an exponential amplification curve was
glucose (NAG) substrate. Among the different grown
produced with a cycle threshold (Ct) value of <40.
colonies, six were selected and propagated.
The bacterial DNA was extracted using Gentra®
Puregene® (QIAGEN.com) according to the Intralaboratory validation of the PCR-based methods
manufacturer’s instructions.
The intra-laboratory validation of the PCR-based
methods was performed by following the EPPO
DNA extraction from Apiaceae seeds
7/98 (2) and 7/76 (4) standards. Since CaLsol is
not in vitro cultivable, analytical sensitivity
The International Seed Federation (ISF 2016) recom-
(smallest amount of target that can be detected
mends testing samples of 20 g of Apiaceae seeds divided
reliably), analytical specificity (performance of the
into two sub-samples of 10 g each. In this work samples
test with regard to cross-reactions with non-target),
of 20 g (about 20.000 seeds) and/or 0.5 g (about 500
selectivity (extent to which variations in the matrix
seeds) were tested. The ISF DNA extraction protocol
affect the test performance), repeatability (level of
(2016) was used with some modification. Briefly, seeds
agreement between replicates of a sample tested
were washed by shaking them for 30 min in 0.5% Triton
under the same conditions), and reproducibility
X-100 and, after several rinses, they were left to soften in
(ability of the test to provide consistent results
water overnight. The seeds were crushed with a mechan-
when applied to aliquots of the same sample tested
ical homogenizer in heavy plastic bags (Bioreba) in 1:10
under different conditions such as time, persons,
(w/v) of a modified Trimethylammonium bromide
equipment etc.) were evaluated according to
(CTAB) buffer (2,5% CTAB, NaCl 1.4 M, Tris-HCl
phytoplasmological guidance. In particular, the
1 M pH 8.0, EDTA 0.5 M, pH 8.0, PVP-40 1%,
EPPO7/98 (2) standard requires the following key
30 mM ascorbic acid). 400 μg of RNase A was added
parameters for each performance criteria: a) analyt-
to 500 μl of homogenate (corresponding to 50 seeds),
ical sensitivity: determine the maximum dilution of
and after incubation at 65 °C for 30 min, total genomic
DNA detected by performing at least three experi-
DNA was extracted using a DNeasy Plant Mini Kit
ments with serial dilution; b) analytical specificity:
(Qiagen, Germany) following the manufacturer’s instruc-
analyse a range of targets and relevant non-targets
tions. DNA was eluted in 100 μl of AE buffer provided
(the targets and non-targets used in this work are
by the kit.
listed in Online Resource 1); c) selectivity: deter-
mine whether variations of the sample material
End-point PCR (e.g. by using different cultivars of the host plant)
affect the test performance; d) repeatability: analyse
The end-point PCR methods tested in this work were at least three replicates of sample extracts with a
those of: i) Li (Li et al. 2009); ii) Liefting (Liefting et al. low (relative) concentration; and e) reproducibility:
2008); iii) Munyaneza (Munyaneza et al. 2009); and iv) as for repeatability, but with different operator(s) if
Ravindran (Ravindran et al. 2011). The methods were possible, on different days, and with different
performed according to the authors’ instructions and equipment when relevant.
 Eur J Plant Pathol

Interlaboratory comparison: test performance study
(TPS)
Participants
TPS was performed by ten laboratories of the Italian
Regional Plant Protection Service (IRPPS), widespread
throughout the country, and CREA-DC-Rome laborato-
ry. One laboratory did not participate in the real-time
(laboratory code 9) and another in the end-point (labo-
ratory code 7) PCR tests.
provided together with the buffers and the DNeasy
Plant Mini Kit (Qiagen, Germany).
To assess the stability of the samples and reagents
during the delivery a similar package containing one kit
was left out on a balcony (facing towards the sun in the
afternoon) for eight days, which corresponds to the most
prolonged period of delivering to the participants. In that
period (December 2016) the maximum and minimum
temperatures were 18 °C and − 2 °C, respectively. After
that, the kit was stored at 4 °C (as requested to the
participants) and later tested by the organizers.

Evaluation of the performance criteria

Material provided to the participants
The blind samples sent to the TPS participants are listed
in Table 1. For real-time and end-point PCR, the oligo-
nucleotides and reagents, including water, were sent to
the participants. For the end-point PCR and real-time
PCR, Go Taq G2Flexi DNA polymerase (Promega) and
TaqMan Universal Master Mix II (Thermo Fisher) were
provided, respectively. Each sample was tested by the
participants in triplicate (technical replicates).
To test the DNA extraction protocol, CaLsol in-
fected and CaLsol free seeds (Table 1), were
The methods were evaluated by the qualitative re-
sults received by each laboratory. The results pre-
sented by a laboratory were excluded for a given
method when a false positive result was obtained for
not-template control (NTC), indicating contamina-
tion in the reaction mixture.
Diagnostic sensitivity (ability of the test to detect the
target in infected samples), diagnostic specificity (ability
of the test not to detect the target in uninfected samples)
and relative accuracy (degree of correspondence be-
tween the responses obtained and those expected) were

Table 1 Blind samples sent to the test performance study (TPS) participants

Samples for end-point PCR Samples for real-time PCR Samples for DNA extraction
from Apiaceae seeds

DNA of CaLsol infected C4 carrot seed CaLsol infected C4 carrot seed DNA CaLsol infected seeds sub sample A*
10−2 diluted DNA (LOD) of CaLsol 10−3 diluted DNA (LOD) of CaLsol CaLsol infected seeds sub sample B*
infected C4 carrot seed infected C4 carrot seed
DNA of CaLsol free carrot seed 104 DNA target copies° CaLsol Free seeds*
DNA of CaLsol infected DNA of CaLsol infected
ISPAVE_VIb_1 carrot seed ISPAVE_VIb_1 carrot seed
X. campestris DNA 102 DNA target copies°
10−2 diluted DNA (LOD) of CaLsol 5 DNA target copies (LOD)°
infected C4 carrot seed
10−2 diluted DNA (LOD) of CaLsol DNA of CaLsol free carrot seed
infected C4 carrot seed
DNA of CaLsol free fennel seed X. campestris DNA
DNA of CaLsol free fennel seed 10−3 diluted DNA (LOD) of CaLsol
infected C4 carrot seed
not-template control (NTC) 10−3 diluted DNA (LOD) of CaLsol
infected C4 carrot seed
Pseudomonas sp. DNA not-template control (NTC)

*The DNA of these seed samples was extracted by each participant using the protocol set up in this work. The obtained DNAs were tested by
participants by end-point and real-time PCR
° pTXZC18 plasmid DNA containing the 16S CaLsol real-time PCR target sequence (kindly provided by Wenbin Li)
LOD = Limit of detection
Eur J Plant Pathol
used, expressed in percent, to assess the performance of
each method (EPPO standards PM 7/76 (4) 2017 and
PM7/98 (2) 2014; ISO 16140: 2003). In addition, ac-
cordance [Bthe percentage chance of finding the same
result (i.e. both negative or both positive) from two
identical test portions analysed in the same laboratory,
under repeatability conditions^], concordance (Bthe per-
centage chance of finding the same result for two iden-
tical samples analysed in two different laboratories B),
and concordance odds ratio (COR) [COR = accordance
× (100 – concordance)/concordance × (100 – accor-
dance)], to address the variability of the method within
and between laboratories, were calculated as indicated
by ISO 16140:2003.
Results and discussion
DNA extraction from Apiaceae seeds
CaLsol is not yet cultivable in axenic medium, it is
confined to the phloem sieve tubes of seed coat
(Bertolini et al. 2015), and its DNA can only be extract-
ed from infected plant tissues, thus co-purifying with
that of the host genome. A DNA extraction procedure,
to be widely applicable, should not require the use of
chemical hoods. To this end, as a starting point, the
DNA extraction method described by ISF (2016) was
adopted, which, differently from that of Bertolini et al.
(2015), does not make use of organic solvents. Then,
some modifications to improve it were introduced.
First, seeds were left to soften in water overnight,
facilitating seed coat crushing while avoiding
overheating of the sample and stomacher bag breakage.
Second, a modified CTAB extraction buffer was used
instead of PBS (ISF 2016), obtaining a significant re-
duction (P < 0.02) in the Ct values from 28.5 ± 0.61 to
26.5 ± 0.37. In the modified CTAB buffer, the 2-
mercaptoethanol (Murray and Thompson 1980) was
replaced by the harmless ascorbic acid. Third, the first
centrifugation step at low speed, performed to remove
the plant debris produced during crushing, was skipped,
increasing significantly (P < 0.05) the DNA throughput
(Ct values decreased from 28.1 ± 0.59 to 26.3 ± 0.51).
This last result is in accord with the presence of CaLsol
in the seed coat (Bertolini et al. 2015). Thus, an im-
proved protocol of DNA extraction from Apiaceae
seeds without the use of organic solvent was set up. It
should be noticed that both protocols, ISF (2016) and
the one presented here, make use of kits based on silica
spin columns, which were shown to reduce the cross-
contamination among samples (Williams et al. 2015).
Comparison of two real-time PCR methods to detect
CaLsol in infected seeds
The methods developed by Li (Li et al. 2009) and
Teresani (Teresani et al. 2014) were compared for their
ability to detect CaLsol in carrot seed. Both are TaqMan
real-time PCR methods that detect the 5′ end region of
the 16S rRNA gene.
The real-time PCR proposed by Li is a multiplex
PCR simultaneously targeting CaLsol and the plant
COX gene as internal control. However, in our
hands, the presence of COX oligos interfered with
CaLsol detection, giving false negative results
(Fig. 1). This finding could be due to the competi-
tion between the two amplification products and/or
not-optimal primer sets balance especially when the
CaLsol target is at a low concentration. For this
reason, in the present work, the method by Li was
applied as simplex PCR with the primers-probe set
LsoF/HLBr/HLBp for CaLsol detection.
The comparison of the sensitivity of the real-time
PCR methods of Li and Teresani was performed with
total DNA extracted from CaLsol infected carrot seeds.
The method of Li showed the best sensitivity with Ct
values significantly lower (2.98 ± 0.24 cycles, P < 0.05)
than those obtained by Teresani’s method. This result
was confirmed in parsley seeds where CaLsol was de-
tected, with a Ct value of 35.04 ± 0.176, in the P4SCS
samples (Online Resource 1) only by the method of Li.
Looking at the two methods, the forward primer of
Teresani starts at nucleotide 18 while the one of Li starts
at 37 (calculated on the representative CaLsol sequence
accession number EU 812559) and the amplicons are of
111 and 78 bp in length, respectively. For the reverse
primer and probe, Li used those previously designed (Li
et al. 2006) to detect the three citrus-infecting
‘Candidatus Liberibacter spp.’, while those used by
Teresani partly overlap the ones of Li. The different
sensitivity showed by the two methods could be due to
the ability of the primers to anneal to the targets and/or
to the type of quencher used. In fact, Teresani uses
TAMRA which, having its own emission, contributes
to a background fluorescence signal, thus reducing the
sensitivity of the method (Johansson 2006).
 Eur J Plant Pathol

Fig. 1 Real-time PCR (Li et al. a

2009) of carrot seed samples. a Multiplex
multiplex with the primer-probe
sets for CaLsol (black) and the
positive internal control COX
gene (gray); b simplex with the
primer-probe set for CaLsol
(black). Circle = negative CaLsol
free seeds control; Triangle =
positive CaLsol infected seeds COX (TET)
control; cross = negative NTC
control (water); FAM and TET are
the reporter dyes used. (Bio-Rad
CaLsol (FAM)
CFX Manager graphics program)

b
Simplex

CaLsol (FAM)

The results obtained in the present work are in con-
trast with those shown on celery plants by Teresani et al.
(2014) that found their method to be the most sensitive.
This discrepancy indicates the necessity of inter-
laboratory comparison tests to define and harmonize
the detection protocols.
Comparison of four end-point PCR methods to detect
CaLsol in infected seed
Several end-point PCR methods were proposed to detect
CaLsol. In particular, the most often used are those of: i)
Li (Li et al. 2009) and Liefting (Liefting et al. 2008),
targeting the 16S ribosomal rRNA; ii) Munyaneza
(Munyaneza et al. 2009), targeting the 50S rpIJ and
rpIL ribosomal protein genes; and iii) Ravindran
(Ravindran et al. 2011) targeting the 16S/23S ISR. The
above methods were compared to identify the most
sensitive one in detecting CaLsol. The comparison was
performed using carrot seed lots previously identified,
by real-time PCR, as CaLsol-infected (Online
Resource 1). DNA was extracted at least two times from
each seed lot, and each PCR test was performed at least
two times for a total of 198 analyses (data not shown).
The method by Ravindran (primers set LsoTX F/R) was
able to detect CaLsol in 88.4% of the performed exper-
iments, while those of Munyaneza (primers set CL514
F/R), Li (primers set LsoF/OI2c) and Liefting (primers
Eur J Plant Pathol
set OA2/OI2c) were able to detect CaLsol in 40.5%,
37%, and 27.5% of the experiments, respectively. There
were three seed lots (Online Resource 1, numbers 11, 12
and 13) in which none of the end point-PCR methods
was able to detect CaLsol, and two others (Online
Resource 1, numbers 3 and 8), in which only the
Ravindran method detected CaLsol. That the Ravindran
end-point PCR method outperformed the other ones
agrees with the previous results obtained in potato and
celery plants (Teresani et al. 2014). In particular, the
primers LsoTX F/R, out-performed LsoF/OI2c in de-
tecting CaLsol in potato samples (Ravindran et al.
2011). Similarly, in celery leaf, LsoTX F/R was the most
sensitive primer pair compared with OA2/OI2c and
CL514 F/R (Teresani et al. 2014). The sensitivity dif-
ference observed among the methods was probably due
to the length of the amplicons, that is of 1173
(LsoF/OI2c), 1168 (OA2/OI2c), 669 (CL514 F/R) and
383 bp (LsoTX F/R), respectively; where the shorter
ones are more easily amplified.
The two most sensitive methods (Ravindran et al.
2011; Munyaneza et al. 2009), were tested to detect
CaLsol also in infected parsley seed lots (Online
Resource 1 numbers 21÷35). The method by
Munyaneza was not only, as expected, less sensitive
(58% of success in detecting CaLsol vs. 100%
showed by Ravindran), but also resulted in the am-
plification of unspecific bands (Fig. 2).
Since the Ravindran method resulted as the most
effective and efficient in detecting CaLsol in carrot
and parsley seeds, it was chosen to perform the
following studies.
Regardless of which is the most sensitive method, it
is inadvisable to use one common primer between the
end-point and the real-time PCRs (as proposed by Li
et al. 2009) because it introduces the risk of contamina-
tion between the two amplification products. As the
CaLsol detection methods mostly rely on PCR based
Carrot
H2O C1 C2 C3 C4 C5
LsoTX 16/23S
383 bp →
CL514
669 bp →
Fig. 2 End-point PCR amplification of carrot (C1÷ C6) and
parsley (P1÷ P4) CaLsol Infected seed samples performed with:
a) CL514 primers set (Munyaneza et al. 2009); and LsoTX 16/23S
assays, this aspect is even more severe than for the
detection of in vitro cultivable bacteria.
Intra-laboratory validation of the PCR-based methods
To detect and identify CaLsol in carrot seeds, the
Ravindran end-point PCR (Ravindran et al. 2011), with
the primers LsoTX F/R, and the Li TaqMan real-time
PCR (Li et al. 2009), with the primers-probe set
LsoF/HLBr/HLBp, were chosen as the most sensitive
among the methods evaluated. In addition, the two tests
target different CaLsol genomic regions allowing a
more reliable detection and identification and more im-
portantly, avoiding the risk of contamination between
the PCR-derived products.
The limit of detection (LOD), calculated with the
total DNA extract of CaLsol infected seeds, was of
10−2 and 10−3 dilution for the end-point and real-time
PCR, respectively; indicating that, real-time PCR was
the most sensitive for CaLsol detection in seeds. Our
results are in accord with previously performed compar-
ative PCR assays for CaLsol detection in different plant
species and tissues, in which real-time PCR was 10- to
100-fold more sensitive than the end-point one (Li et al.
2009; Teresani et al. 2014). For the Li real-time PCR,
the LOD was also evaluated with purified pTXZC18
diluted with water. Five copies of the target were detect-
ed with Ct values of 34.57 ± 0.428 in 100% of the
experiments (24/24), and for this reason, this concentra-
tion was chosen as LOD. In fact, 2 copies were detected
(Ct 35.76 ± 0.774) in 94.4% of cases (17/18) and 1 copy
in 85.71% (18/21) with a Ct value of 38.03 ± 0. 60.
Ten and one copy of pTXZC18 were also diluted in
healthy Apiaceae seed DNA extracts obtaining Ct
values of 34.23 ± 0.23 and 37.46 ± 0.10 (carrot), 34,14
± 0,11 and 38.57 ± 0.86 (parsley), 34.50 ± 0.51 and
36.46 ± 0.44 (celery), 33.09 ± 0.16 and 36.56 ± 1.32
(fennel), respectively, indicating that the presence of
Parsley
C6 H2O P1 P2 P3 P4 H2O C- C+ M H2O
primers set (Ravindran et al. 2011). C- = negative CaLsol free
seeds control; C+ = positive CaLsol infected seeds control; M =
100 bp DNA Molecular Weight Marker

the seed matrices did not affect the amplification reac-
tion. Similar results were also obtained with different
spiked Apiaceae matrices such as carrot taproot and leaf,
potato tuber, leaf and sprout, parsley and fennel leaves
(data not shown). For both methods, the intralaboratory
repeatability and reproducibility were of 100% at the
respective LOD.
Both methods showed good analytical specificity and
selectivity performances. In fact, Li and Ravindran tests
were able to detect 37 and 34 CaLsol isolates, respec-
tively, from different host species (carrot, parsley, and
celery) and cultivars (at least 15 among carrot, parsley,
and celery) (Online Resource 1). Regarding the analyt-
ical specificity with non-target prokaryotes (Online
Resource 1), both methods did not show false positives.
Thus, our data increased by 20 the number of non-target
microorganisms for which no specific amplification oc-
curred, reaching a total of 25 and 34 microorganisms for
end-point PCR (Ravindran et al. 2011) and real-time
PCR (Li et al. 2009), respectively.
Interlaboratory comparison: test performance study
(TPS)
Table 2 reports the laboratory diagnostic sensitivity,
diagnostic specificity, relative accuracy and the
means obtained for real-time PCR and end-point
PCR. The accordance, concordance, and concor-
dance odds ratio values, evaluated for each method,
are also shown. The results obtained by laboratory
n. 11 were excluded for the end-point PCR because
of the false positive result obtained for the blind not-
template control (NTC) sample.
The best overall performance in detecting CaLsol in
Apiaceae seeds was obtained by the real-time PCR. In
fact, although the diagnostic sensitivity (ability of the
test to detect the target in infected samples) of the real-
time PCR was lower than the end-point PCR, this dif-
ference was not significant and it was actually only due
to the failure of a single laboratory in detecting five
DNA target copies, the sample at limit of detection
(LOD). Instead, the real-time PCR showed the best
statistically significant ability not to detect the target in
uninfected samples and generally, the best degree of
correspondence between the responses obtained and
those expected. Looking at accordance and concordance
values, not only real-time PCR showed the higher per-
centages, but the difference between accordance and
concordance values was very low (0.2%). On the
Eur J Plant Pathol
contrary, end-point PCR concordance is smaller than
the accordance of 2.2 percentage points, indicating that
identical samples are more likely to give the same result
when analysed by the same laboratory than by different
ones. This finding suggests that there can be perfor-
mance variability between laboratories and/or that the
method is less robust to reproduce the same results in
different laboratories. Accordingly, the concordance
odds ratio (COR), that should be 1.00 if accordance
and concordance are equal, is higher for the end-point
PCR indicating that the inter-laboratory variation is
slightly predominant.
Regarding the uniformity of CaLsol infection in
the seed lot C4 delivered to the participants we
evaluated it analyzing six subsamples of 0.5 g and
four of 10 g by real-time PCR (Li et al. 2009). The
average Ct value was of 26.12 ± 0.87, indicating the
uniformity of CaLsol infection throughout the dif-
ferent subsamples of lot C4. In addition, we quanti-
fied CaLsol in the C4 lot by standard curves obtain-
ed with DNA extract of healthy carrot seeds spiked
with a tenfold serial dilution of the plasmid
pTXZC18 (Fig. 3). The results showed that there
was an average of 3370 ± 311 copies of the target
per seed. As in the CaLsol genome there are three
copies of rRNA operon (Lin et al. 2011), there was
an average of 1123 CaLsol cells per seed in the C4
lot. This finding was consistent with the results
obtained by Loiseau et al. (2017) that showed that
the total number of CaLsol cells in seed lots ranged
from 1.61 × 103 to 1.11 × 104 cells/seed.
Regarding results obtained in the TPS about the
DNA extraction, all laboratories gave positive feedback,
as they were able to detect CaLsol by real-time PCR in
all provided infected seed samples, reaching 100% of
diagnostic sensitivity (Table 2). One laboratory obtained
a false positive for the healthy sample, and in fact, the
diagnostic specificity and accuracy values did not reach
the maximum score albeit remaining at high levels. On
the other hand, as the end-point PCR was less sensitive
than real-time PCR, CaLsol was not always detected in
the DNA extracted from the infected seeds. Two labo-
ratories, which detected CaLsol by real-time PCR, were
not able to obtain the same results from the same sam-
ples by end-point PCR. In addition, one laboratory
detected CaLsol by end-point PCR only in DNA ex-
tracted from one sub-sample. As for the molecular
methods, also for the DNA extraction method, the
values of accordance were higher than those of
 Eur J Plant Pathol

Table 2 Results of TPS obtained for real-time PCR, end-point PCR and DNA extraction method evaluated by both real-time and end-point PCR

Laboratory code real-time PCR end-point PCR seed DNA extract evaluated by real-time PCR seed DNA extract evaluated by end-point PCR

Sensitivity Specificity Accuracy Sensitivity Specificity Accuracy Sensitivity Specificity Accuracy Sensitivity Specificity Accuracy

1 100.0 100.0 100.0 100.0 83.3 90.0 100.0 100.0 100.0 100.0 100.0 100.0
2 100.0 100.0 100.0 100.0 50.0 70.0 100.0 100.0 100.0 66.66 100.0 77.77
3 100.0 100.0 100.0 100.0 66.6 80.0 100.0 100.0 100.0 100.0 100.0 100.0
4 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0
5 85.7 100.0 90.0 100.0 83.3 90.0 100.0 100.0 100.0 100.0 100.0 100.0
6 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0
7 100.0 100.0 100.0 np np np 100.0 50.0 87.5 np np np
8 100.0 100.0 100.0 100.0 83.3 90.0 100.0 100.0 100.0 100.0 100.0 100.0
9 np np np 100.0 100.0 100.0 np np np 66.66 100.0 77.77
10 100.0 100.0 100.0 100.0 66.6 80.0 100.0 100.0 100.0 83.33 100.0 88.88
11 100.0 100.0 100.0 e e e 100.0 100.0 100.0 e e e
Mean 98.6 100.0 99.0 100.0 81.5 88.9 100.0 95.0 98.75 90.74 100.0 93.82

Accordance 98.2 82.2 97.81 90.12

Concordance 98.0 80.0 97.5 88.20
COR* 1.11 1.15 1.14 1.22

Diagnostic sensitivity, diagnostic specificity and relative accuracy, expressed in percent, are indicated for each laboratory (the mean of each performance criteria is indicated in bold).
Accordance, Concordance (expressed in percent) and Concordance odds ratio are reported for each method (in bold)
np
laboratory not participating
e
laboratory excluded because of false positive for the not-template control (NTC) sample
*Concordance odds ratio

Fig. 3 Standard curves obtained
with DNA extract of healthy
carrot seeds spiked with a tenfold
serial dilution of the plasmid
pTXZC18 (o), and results
obtained for DNA extracts of the
C4 lot (x); two biological
replicates performed in triplicate.
FAM is the reporter dye used.
(Bio-Rad CFX Manager graphics
program)
concordance, and the COR values were higher than 1
(Table 2). This finding pointed out once again that
greater inter-laboratories rather than intra-laboratory
variability. Unlike other inter-laboratory comparison
studies where each laboratory used their reagents and
extraction kit (e. g. Olivier et al. 2016; Chabirand et al.
2017), in the present work, we provided the participants
with all that was required to perform the experiments,
eliminating this kind of variable in the evaluation of the
methods. Although the materials and the reagents were
the same for all laboratories, greater inter-laboratories
rather than intra-laboratory variability was reported,
highlighting the importance of the operator effect.
Conclusion
This work, which is meant to fulfill the requests from the
Plant Protection Services and FAO/IPPC Emergency ac-
tion (2015, 2016), has achieved a validated and harmo-
nized diagnostic protocol to detect CaLsol in carrot seeds.
The DNA extraction method was specifically set
up considering CaLsol location in the phloem of the
Apiaceae seed coat and allowing its broad applica-
bility even in those laboratories that do not have
chemical hoods.
For the molecular detection and identification of
CaLsol, two PCR-based methods, the real-time by
Li et al. (2009) and the end-point by Ravindran
et al. (2011), were chosen as the most sensitive and
specific among the widely used ones. Significantly,
the two PCR tests target different CaLsol genome
regions (16S rDNA gene and 16/23 S ISR, respec-
tively), thus avoiding the risk of contamination
between the amplified products. The real-time
Eur J Plant Pathol
PCR was performed only with CaLsol oligonucle-
otides set because the analytical sensitivity of
CaLsol detection decreased significantly and false
negative results occurred under the multiplex real-
time PCR condition (CaLsol 16S rDNA and COX
genes) (Fig. 1).
As expected, between the two methods, the real-time
PCR was 10-fold more sensitive than end-point PCR in
detecting CaLsol in seed extract DNA. In addition, real-
time PCR was always able to detect five copies of the
target and until one copy in the 86% of the experiments
carried out showing good sensitivity performance.
Regarding analytical specificity and selectivity, both
tests were able to detect different CaLsol isolates and
haplotypes in different Apiaceae host species (carrot,
parsley, and celery), cultivars (at least 15 among carrot,
parsley, and celery) and matrices. In addition, both tests
did not show false positive results using twenty non-
target microorganisms (Online Resource 1).
The test performance study performed by 11 lab-
oratories, confirmed that the DNA extraction method
was well suited for the detection of CaLsol in carrot
seeds and that the best overall performance, regard-
ing the molecular tests, was obtained by the real-time
PCR by Li et al. (2009). Nevertheless, also the end-
point PCR by Ravindran et al. (2011) showed good
performance results (values equal or higher than
80%), indicating this test as another valid CaLsol
detection tool. In fact, as for CaLsol detection, spe-
cific molecular tests are required, so it is advisable to
apply both methods to detect and identify the bacte-
rium. As the participants received both the samples
and all that was required to perform the experiments,
the variability among the laboratories was reduced
making the concordance values more reliable.
Eur J Plant Pathol
The obtained results could be advantageous for the
appropriate application of phytosanitary measures
against CaLsol.
Acknowledgments This work was supported by the Ministero
per le Politiche Agricole Alimentari e Forestali (project Azioni a
supporto della protezione delle piante ASPROPI: Linea di ricerca
BSviluppo protocollo analitico per ‘Candidatus Liberibacter
solanacearum’^).
Compliance with ethical standards The manuscript complies
with the rules of good scientific practice and ethical rules of
European Journal of Plant Pathology, as reported in the BEthical
Responsibilities of Authors^ of the BInstructions for Authors^
section. There are no potential conflicts of interest, and the re-
search does not involve human participants and/or animals. All
authors have approved the manuscript and agreed with its submis-
sion to European Journal of Plant Pathology.
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