Folia Microbiol (2013) 58:269–276
DOI 10.1007/s12223-012-0206-6
Multiplex PCR to detect four different tomato-infecting pathogens
Gabriela Alejandra Quintero-Vásquez &
María Luisa Bazán-Tejeda & Eva Martínez-Peñafiel &
Luis Kameyama-Kawabe & Rosa María Bermúdez-Cruz
Received: 12 April 2012 / Accepted: 23 October 2012 / Published online: 8 November 2012
# Institute of Microbiology, Academy of Sciences of the Czech Republic, v.v.i. 2012
Abstract This work was aimed to develop a multiplex
PCR assay to detect infectious agents such as Clavibacter michiganensis subsp. michiganensis, Fusarium
sp, Leveillula taurica, and begomoviruses in tomato
(Solanum lycopersicum) plants. Specific primer sets of
each pathogen were designed based on intergenic ribosomal RNA sequences for the first three, whereas for
begomoviruses, primers were designed based on conserved regions. The design also considered that the
length (200–800 bp) of the PCR products was resolvable by electrophoresis; thus 296, 380, 457, and 731 bp
fragments for Clavibacter, Fusarium, Leveillula, and
begomoviruses, respectively, were considered. PCR conditions were optimized to amplify all the products in a
single tube from genomic DNA and circumvent PCR
inhibitors from infected plants. Finally, when the multiplex PCR assay was tested with tomato plants infected
with any of the four pathogens, specific PCR products
confirmed the presence of the pathogens. Optimized
PCR multiplex allowed for the accurate and simultaneous detection of Clavibacter, Fusarium, Leveillula,
and begomoviruses in infected plants or seeds from
tomato.
Electronic supplementary material The online version of this article
(doi:10.1007/s12223-012-0206-6) contains supplementary material,
which is available to authorized users.
G. A. Quintero-Vásquez : M. L. Bazán-Tejeda :
E. Martínez-Peñafiel : L. Kameyama-Kawabe :
R. M. Bermúdez-Cruz (*)
Genetics and Molecular Biology Department,
Centro de Investigaciones y Estudios Avanzados del IPN,
Av IPN 2508. Delegación Gustavo A,
Madero, Federal District 07360, México
e-mail: [email protected]
Introduction
Fungal, bacterial, and viral diseases are important constraints
in tomato agriculture, and their identification is crucial in
order to apply specific solutions. There are molecular methods available aimed to identify pathogens causing disease in
tomato, however, they mostly focus on one organism. Different pathogens may produce similar symptoms in the plant,
for example wilting is observed in plants infected by Clavibacter or Fusarium or Verticillium (Jones 2008). Therefore
tools enabling differential detection of infecting organisms in
the same plant matrix are desirable.
Clavibacter michiganensis subsp. michiganensis, Fusarium oxysporum sp. lycopersici, Leveillula taurica, and
begomoviruses are important pathogens of tomato and cause
economic losses worldwide (Senasica 2008, Strider 1969;
Katan et al. 1997; Correll et al. 2005; Torres-Pacheco et al.
1996). Various PCR methods have been developed for these
pathogens alone or in combination with others (Dreier et al.
1995; Sousa-Santos et al. 1997; Bach et al. 2003; Hirano
and Arie 2006; Ozdemir 2009; Kokoskova et al. 2010;
Inami et al. 2010; Davino et al. 2008; Accotto et al. 2000;
Lefeuvre et al. 2007). Here, we report a method by which all
four pathogens can be identified from a single PCR reaction.
The bacterial canker is caused by C. michiganensis
subsp. michiganensis CMM, a Gram (+) bacterium, being
one of the most frequent diseases (Strider 1969; Gartemann
et al. 2003). Contaminated seeds are the primary source of
infection; they can survive during long periods of time in the
soil and plant residues. The bacterium penetrates in the plant
by the roots displaying vascular necrosis.
Fusarium oxysporum f. sp. lycopersici FO caused wilting
in tomatoes is a disease distributed worldwide (Takken and
Rep 2010; Michielse and Rep 2009). The fungus has the
ability to survive from one season to another, misdiagnosis
leads to increased persistence in the field. Symptoms on
270
leaves are yellow and irregular areas, on the abaxial leaf
they appear like hairs that correspond to the mycelium and
fungus spores (Michielse and Rep 2009).
Geminiviruses are phytopathogens that infect an ample variety of cultures causing great economic losses in the tropical
and subtropical regions of the world. The members of the
family Geminiviridae have genomes composed of one or two
circular single-stranded DNA (ssDNA) molecules of about 2.6–
3.0 kb, that are packed into twinned capsids (Varma and Malathi
2003). Begomoviruses belong to the genus Begomovirus BV,
Geminiviridae and are comprised for more than 200 species
(Fauquet et al. 2008) causing severe diseases to dicotyledonous
plants and are transmitted by the whitefly (Bemisia tabaci),
which is considered a worldwide agricultural pest (Varma and
Malathi 2003). Tomato has been severely affected by various
begomovirus species such as Sinaloa tomato leaf curl virus,
Tomato chino La Paz virus, Tomato severe leaf curl virus,
Chino del tomate virus, Tomato chlorotic leaf distortion virus,
Tomato yellow leaf distortion virus, Tomato mottle virus, Tomato golden vein virus, Tomato severe rugose virus, Tomato
mild mosaic virus, Tomato mosaic Havana virus, Tomato mottle
Taino virus, etc. (Fauquet et al. 2008).
Powdery mildew of tomato is mainly caused by L. taurica
LT, among others. This disease can be injurious in
greenhouse-grown tomatoes where the losses may exceed
50 % in infected crops (Jones and Thomson 1987). The extent
of loss depends on environmental conditions, date of disease
onset, and effectiveness of fungicide control. Hot, dry days
with an occasional rainstorm lead to disease development.
In this work, we designed and optimized a multiplex PCR
assay to identify four tomato pathogens, C. michiganensis
subsp. michiganensis, Fusarium sp., L. taurica, and begomoviruses, in plants and seeds. The method was optimized by
making genomic DNA suitable for PCR amplification through
genomic DNA linearization and also by eliminating PCR inhibitors during both DNA extraction and PCR amplification.
Folia Microbiol (2013) 58:269–276
Center (kindly donated by Dr. Angel Gabriel Alpuche Solís)
strains were used.
Genomic DNA extraction
The genomic DNA (gDNA) was extracted from healthy or
naturally infected tomato plants and seeds (these infections
were confirmed by commercial diagnostic laboratories). DNA
extraction was carried out with either DNeasy Plant Mini Kit
(Qiagen, Germany) or with the method reported by Murray
and Thompson (1980) and modified in this report by the
addition of 0.5 % charcoal. Briefly, a volume of 1.5 mL of
CTAB-activated charcoal extraction solution (50 mmol/L
Tris–HCl pH08.0, 10 mmol/L EDTA, 0.7 mol/L NaCl, 1 %
CTAB cetyl trimethyl ammonium bromide, 2 % polyvinylpyrrolidone PVP, 1 % beta-mercaptoethanol and 0.5 % activated charcoal) was added to nitrogen frozen and mortarcrushed 100-mg tomato leaves or 200 mg of seeds. One sixth
volumes of 5 % sarkosyl were added and mixed then incubated for 1 h at 60 °C and inverted occasionally. One milliliter of
chloroform–isoamylic alcohol mix (24:1, v/v) was added and
mixed. After samples were centrifuged at 14,000×g for
15 min, and aqueous phase was transferred to a clean tube,
and 300 μL of isopropanol were added. The tube was placed
at −20 °C for 1 h and spun at maximum speed for 15 min and
supernatant was removed. Five hundred μL of 70 % ethanol
with 10 mmol/L NH4–acetate were added and tube was
incubated overnight at 4 °C. Finally, tube was spun at
14,000×g for 15 min and the supernatant was removed,
DNA was resuspended in 50 μL of TE buffer and heated to
65 °C for 30 min and stored at −20 °C. Also, genomic DNA
was extracted from Bacillus subtillis, Pseudomonas syringae
pv. syringae, Pseudomonas syringae pv. tomato, Verticillium
dahliae, and Alternaria solani using a commercial kit (ZR
Fungal/Bacterial DNA Kit, Zymo Research, Lithuania).
Plasmid DNA extraction
Materials and methods
Strains and primers
Three strains of Escherichia coli DH5α, TOP 10, or TOP
10 F′ were used for plasmid transformation. This was carried out by either electroporation (Cell-Porator-GIBCO
BRL) or chemical transformation (Hanahan 1983). Primers
Clav-F/Clav-R, Fus-F/Fus-R, Lt-F/Lt-R, Bv-F/Bv-R, and
ITS5/ITS4 (Table 1) were purchased from Sigma-Aldrich,
Mexico. For specificity tests, bacterial Bacillus subtillis
PY79, Pseudomonas syringae pv. syringae B728A, Pseudomonas syringae pv. tomato DC 3000 (kindly donated by
Dr. Olmedo) and fungal Verticillium dahliae strain 9765,
Alternaria solani strain 5924 from Nite Biological Resource
To obtain plasmid DNA, easy preps technique was used
(Berghammer and Auer 1993). Briefly, cells were pelleted in
a microcentrifuge at maximun speed for 30 s. Pellet was resuspended with a pipette in 40 μL of lysis buffer (10 mmol/L Tris–
HCl pH08.0, 1 mmol/L EDTA, BSA 0.1 mg/mL, RNase
0.2 mg/mL, sucrose 15 %w/v, and lysozyme 2 mg/mL) and
placed on ice for 5 min. This mix was boiled for 60 s, then
placed on ice for another 60 s and centrifuged at maximun
speed for 15 min. Without touching the pellet, supernatant
was removed and transferred to a clean tube for posterior use.
Single PCR, cloning, and sequencing
Each PCR product was obtained by using specific primers
(Table 1) and genomic DNA from either corresponding
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271
Table 1 Species-specific primers used for the identification of C. michiganensis subsp. michiganensis, Fusarium sp, L. taurica, and Begomovirus
isolated from tomato plants
Primers
Sequence
Ann temp and length
Reference
Clav-F
Clav-R
Fus-F
Fus-R
Lt-F
Lt-R
Bv-F
Bv-R
ITS1a
ITS4
RepQEW-fora
CP450-rev
5′- TGGATCACCTCCTTTCTAAG-3′
5′- CACCACCATCCACAACAGGA-3′
5′- ACAACTCCCAAACCCCTGT-3′
5′- TATGGAAGCTCGACGTGACC-3′
5′- GTGTCGACTCGTCTCCTGTT-3′
5′- TGGGGACTTTGTGGTTGCTG-3′
5′- AAGGTGACAGGTGGACAGTA-3′
5′- CACATCCRCCCTCTATCAAG -3′
5′-TCCGTAGGTGAACCTGCGG-3
5′-TCCTCCGCTTATTGATATGC-3′
5′-CCRAARTAAGMATCRGCCCAYTCTTG-3′
5′-GTCCTCGAGTAGACGGCATAGCCTGACC-3′
55.9 °C, 296 bp
This study
55.2 °C, 380 bp
This study
55.9 °C, 457 bp
This study
53 °C, 731 bp
This study
55 °C, ~600 bp
White et al. (1990)
55 °C, ~1.75 Kbp
Velázquez-Valle et al. (2012)
27Fa
1492R
ITS5
ITS4
5′-AGAGTTTGATCCTGGCTCAG-3′
5′-GGTTACCTTGTTACGACTT-3′
5′-GGAAGTAAAAGTCGTAACAAGG-3′
5′-TCCTCCGCTTATTGATATGC-3′
55 °C, ~1.4 kbp
Lane et al. (1991)
58.2 °C, 713 pb
Gardes and Bruns (1993)
a
These primers were used as positive control for non-target organisms
Ann temp PCR product annealing temperature
pathogen or naturally infected plant. Briefly, PCR amplification was carried out in a volume of 50 μL containing:
1× PCR reaction buffer [100 mmol/L Tris–HCl pH08.8 and
50 mmol/L KCl], 0.6 μmol/L each primer, 1.5 mmol/L
MgCl2, 0.2 mmol/L each dNTP, 4 or 100 ng of plasmid
DNA or genomic DNA (digested with restriction enzymes)
as indicated, 1.5 units Taq Altaenzymes Polymerase
(IBMOL, Mexico), 2 % PVP (polyvinylpyrrolidone), 1 μg/
μL BSA (bovine serum albumin), and water. PCR reactions
were carried out in a Perkin Elmer 9600 thermocycler (Applied Biosystems, USA) with an initial denaturation at 95 °C
for 5 min followed by 35 cycles of the following segments:
95 °C for 30 s, specific annealing temperature for each
region to be amplified was used for 30–45 s (Table 1), and
an extension at 72 °C for 30 s. These were followed by an
extension at 72 °C for 7 min. PCR products were separated
by 1–1.5 % agarose gel electrophoresis. To confirm PCR
products identity, they were cloned in pCR2.1-TOPO vector
(TOPO TA cloning kit, Invitrogen USA) and then sequenced. 713bp PCR product (ribosomal intergenic sequence from Solanum lycopersicum) was also cloned and
sequenced. Transformation reactions were placed onto LB
100 μg/mL ampicillin and 20 μg/mL X-Gal supplemented
plates. Sequencing (Perkin Elmer automatic sequencer ABI
Prism 310) on PCR products was performed as recommended by suppliers (Applied Biosystems, USA; data not
shown). The PCR fragments cloned into pCR2.1-TOPO
vector generated the plasmids: TOPO TA-CB, TOPO
TA-FO, TOPO TA-LT, TOPO TA-BV, and TOPO TAPL for CMM, FO, LT, BV, and Solanum lycopersicum,
respectively.
Multiplex PCR optimization
The plasmids bearing the 296, 380, 457, and 731 bp (TOPO
TA-CB, TOPO TA-FO, TOPO TA-LT, and TOPO TA-BV,
respectively) fragments were mixed in a final concentration
10–15 ng in a 50-μL reaction to generate the positive
control (C+) plasmid cocktail. To establish optimal conditions, various MgCl2 and primer concentrations as well as
annealing temperatures were determined by gradient PCR
using as a template the plasmid cocktail. The optimal conditions obtained were the following: 0.6 μmol/L primers
each (Table 1), 2 mmol/L MgCl2, 0.4 mmol/L dNTPs, with
an annealing temperature of 55 °C for 40 cycles. Whenever
indicated, genomic DNA was used as a HindIII-digested
template, enzymatic digestion with HindIII does not cleave
any of the PCR products. Additionally, to circumvent the
remaining PCR inhibitors present in plants, 2 % PVP and
0.1 μg/μL BSA were included in the PCR reaction.
Results and discussion
Design of primers
The design of specific primers was achieved using Oligo
version 4.1 program based on the alignment of ribosomal
intergenic sequences for different subpsecies of C. michiganensis, and different species of Fusarium and Leveillula
genus (see Electronic supplementary material (ESM) S1A,
S1B, and S1C) within regions that were specific for each
pathogen while for the Begomovirus genus, complete
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genome sequences of different infecting tomato species
were aligned and primers were designed within conserved
sequences such as genes av1 and ac1 (data S1D). Alignment
analysis was carried out by ClustalW program to identify
conserved regions (or segments).
Primers design was carried out by selecting 18–20 bp segments that contained 40–60 % of GC content within conserved
areas and an annealing temperature between 55–60 °C for each
PCR product. To accomplish PCR products with different
sizes, primers within ribosomal intergenic sequences (CMM,
FO, and LT) were designed first then BV primer design within
ac1 and av1 gene sequences was done accordingly to sizes
considered for the first three. For primers designed for CMM,
alignment among the different subspecies within C. michiganensis revealed that while Clav-F primer is generic for all of
them, Clav-R primer is specific for subspecies michiganensis
(ESM S1A). For L. taurica, a similar case was obtained,
alignment among different species within Leveillula genus
showed that Lt-R primer is specific to species taurica while
Lt-F primer is generic (S1B). For Fusarium sp, alignment
using different species indicated that both primers Fus-F/R
cannot distinguish among species (S1C). For Begomovirus
genus, 12 different infecting tomato virus genome sequences
were aligned (S1D), this allowed us to identify conserved
regions, among these ac1 and av1 were appropriate to design
degenerate primers in a convenient size range. Since the aim
was to test for multiple products in a single PCR reaction,
primer pairs that would yield products of different sizes were
designed. Thus final PCR proposed products were 296, 380,
457, and 731 bp (Table 1).
Diverse primers have been previously designed for CMM
(Dreier et al. 1995; Sousa-Santos et al. 1997; Bach et al. 2003;
Kokoskova et al. 2010) to amplify specific fragments ranging
from 223 to 645 pb (annealing temperatures, 65–69 °C). Also,
for Begomovirus primers whose amplified products range
from 400 bp to 2.6 kb (annealing temperatures, 50–60 °C)
have been set up earlier (Rojas, et al. 1993; Accotto et al.
2000; Rampersad and Umaharan 2003; Lefeuvre et al. 2007;
Davino et al. 2008). However, since a Multiplex PCR requires
specific fragments from several organisms to be amplified
simultaneously, the annealing temperature of each PCR product has to be within a small range of temperature. Therefore, in
order to have a working Multiplex PCR with amplified products resolvable by agarose gel, new primers were designed
and a new multiplex PCR was established.
Optimization of genomic DNA extraction
To compare the quality of the genomic DNA (gDNA)
extracted by DNAeasy Plant mini Kit and the modified protocol, we amplified plant genomic DNA with generic ITS4/
ITS5 primers (Gardes and Bruns 1993). The modified extraction method yielded a genomic DNA with good quality
Folia Microbiol (2013) 58:269–276
suitable for amplification (Fig. 1B) as compared to that
obtained by the commercial kit (Qiagen, Germany; Fig. 1A).
However, frequently the gDNA had to be diluted 1:25 (4 ng)
due to the presence of inhibitors since these interfere with the
PCR amplification reaction (Fig. 1A and B) as compared to
that with undiluted gDNA (100 ng; Fig. 1A and B). The
modified method produced a genomic DNA with a better
quality since a faint amplified product can be observed even
at 1:10 dilution (10 ng; Fig. 1B) whereas no product was
obtained with same dilution for the gDNA obtained by commercial kit (Fig. 1A). Also, it is important to remark that the
product yield obtained by both methodologies differs. It is
more abundant for the modified method (Fig. 1B) than for the
commercial kit (Fig. 1A). Since an equal mass of leaf tissue
was processed in each procedure, these data indicate that the
addition of PVP and charcoal during DNA extraction in the
modified method resulted in a better gDNA yield and quality
than that obtained by the commercial kit.
Plants contain diverse contaminants such as polysaccharides and phenolic compounds which are difficult to separate
from the DNA (Katterman and Shattuck 1983; Murray and
Thompson 1980). These compounds often inhibit polymerases and other enzymes (Varma et al. 2007). Though the
Murray and Thompson procedure already had the nonionic
detergent CTAB (Saghaimaroof et al. 1984) and PVP to
overcome this unwanted effect, in this study the addition of
0.5 % activated charcoal removed remaining contaminants
(Krizman et al. 2006; Vroh et al. 1996).
Multiplex PCR optimization
Single PCR allows the amplification of only one DNA template whereas Multiplex PCR reduces time and work since
one can amplify more than one template in a single tube.
However, often the use of several primers and templates
may result in preferential binding of some primers to their
templates producing a lower yield for some products, thus
affecting the outcome of the assay (Elnifro et al. 2000).
Despite all this, several multiplex PCR assays have been set
up and optimized for detecting numerous pathogens (Bertolini
et al. 2003; Uga and Tsuda 2005; Ozdemir 2009).
To establish a multiplex PCR, primers are required allowing
their corresponding PCR products to be resolved in a gel and
templates to be amplified efficiently by their primers at a
specific annealing temperature (Markoulatos et al. 2003). Additionally, there are other factors that affect a multiplex assay,
such as gDNA quality, PCR inhibitors present in the gDNA
extracted, etc.
For this, we tested all the primers with corresponding
templates (gDNA from naturally infected plants) in a single
PCR tube at temperatures indicated in Table 1. As expected,
PCR amplified products of 296, 380, 457, and 731 bp fragments were obtained for CMM, FO, LT, and BV (Fig. 2; CB,
Folia Microbiol (2013) 58:269–276
A
1636
1018
506
396
273
B
800
700
600
500
400
300
200
Fig. 1 Agarose migration profile from amplicons obtained by PCR
using ITS4/ITS5 primers and plant genomic DNA obtained by either A
DNAeasy plant mini Kit (Qiagen); 100, 10, and 4ng (from left to right)
or B modified Murray and Thompson method; 100, 10, and 4ng (from
right to left). The amplified product is indicated by an arrow and
molecular weight markers (1-kb DNA ladder in panel A and 100 bp
DNA ladder in panel B, in basepairs) by an M
FO, LT, and BV), respectively. To amplify them all at the
same time, a gradient PCR, with temperatures increasing from
50 to 60 °C was applied to each template of the pathogens
under study. A common annealing temperature for all templates was determined as 55±1 °C (data not shown).
To facilitate Taq polymerase access to DNA templates,
gDNA was digested with a restriction enzyme that did not
digest the PCR product fragments. Also, to eliminate PCR
inhibitors 2 % PVP and 0.1 μg/μL BSA were used in multiplex PCR reaction. Our findings show that expected amplified
products 296, 380, 457, and 731 bp were successfully
obtained for naturally infected plants with C. michiganensis
subsp. michiganensis, Fusarium oxysporum f. sp. lycopersici,
L. taurica, and begomoviruses (Fig. 3, lanes CB, FO, LT, and
BV). For the positive control, the plasmid cocktail was used as
template (indicated as C+). Identity for CMM, FO, LT, and
Begomovirus genus was confirmed by sequencing.
Despite having established a method to obtain genomic
DNA with good yield and quality, oftentimes and despite of
all these approaches to eliminate PCR inhibitors, there was a
remaining unwanted effect. Therefore, PVP and BSA were
added during the PCR reaction. In order to further optimize
this reaction, the genomic DNA was digested with an enzyme
that did not cleave any of the amplified fragments of the
multiplex assay. All these approaches taken helped to amplify
any genomic DNA. As previously observed (Henegariu et al.
1997), the most critical factor in optimization of the multiplex
PCR assay was the relative concentration of the primer sets.
To obtain simultaneous amplification of all four targets it was
necessary to titrate primer concentrations. Often, when there is
more than one template some sensitivity is lost. Fortunately
our modifications may have improved the DNA quality to
such an extent that sensitivity with multiple templates was not
reduced (data not shown).
Specificity and sensitivity of the assay
To discard PCR amplification of other pathogens, each primer
sequence was analyzed by BLAST (http://blast.ncbi.nlm.nih.
gov/) and the primer specificity for each pathogen targeted
was confirmed except for Fusarium oxysporum as other Fusarium species (100 % match with Fus-F/R primers set) were
also found (data no shown). However, within Fusarium genus, mostly oxysporum species has been shown to cause
severe losses in tomato crops (Michielse and Rep 2009).
To further verify the specificity of primers obtained in the
Multiplex assay, we tested each individual primer pair (see
ESM S2A–E, indicated as CB, FO, LT, and BV) and the primer
mix with gDNA from pure cultures of non-target organisms
700
600
500
400
300
200
100
Fig. 2 Agarose gel electrophoresis of single PCR using HindIII
digested-gDNA from infected plants. Lanes CB, FO, LT, and BV
indicate PCR carried out with primers specific for C. michiganensis
subsp. michiganensis, Fusarium sp., L. taurica, and Begomovirus,
respectively. M stands for molecular weight markers (100 bp DNA
ladder in basepairs)
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Folia Microbiol (2013) 58:269–276
Fig. 3 A Multiplex PCR
electrophoretic pattern of
naturally infected plants. Either
CB, FO, LT, and BV in A or FO,
LT, BV in B correspond to
Fusarium oxysporum, L.
taurica, and Begomovirus
HindIII digested-genomic
DNA, using a plasmid cocktail
as positive control (C+). Molecular weight markers (100 bp
DNA ladder) are shown in
basepairs (M)
A
B
FO
M CB FO LT BV
700
600
500
400
300
LT
BV
M
C+
700
600
500
400
300
200
200
but also pathogens such as bacteria (Pseudomonas and Bacillus,
indicated as “+” under Oligos cocktail), fungi (Verticillium and
Alternaria, indicated as “+” under Oligos cocktail) and viral
(Beet curly top virus) that are related to tomato plants (see ESM
S2A–E). Our findings indicate that the individual primer pairs
or primer mix are specific since no products were obtained for
any of them. This result is validated as gDNAs from each
pathogen were amplified with either generic or specific primers
27F and 1492R for Bacillus and Pseudomonas (Lane 1991; see
ESM S2A and S2B, respectively) and ITS1/ITS4 primers for
Alternaria and Verticillium (White et al. 1990; see ESM S2C
and S2D, indicated as gDNA) and RepQEW-for CP450-rev
primers for Beet curly top virus (see ESM S2F). Identity was
confirmed by sequencing CMM, FO, LT, and BV PCR products. The Begomovirus identity was revealed by using the
obtained sequence in a Blast search: Sinaloa Tomato leaf curl
virus (data not shown). To evaluate extent of multiplex assay,
other two Begomovirus gDNAs were amplified: Pepper huasteco yellow vein virus PHYVV and Tomato Chino La Paz virus
TCHLPV (S2F). These results show that these begomoviruses
100
were also specifically amplified by Bv-F/R primers as confirmed by sequencing. However, Tomato yellow leaf curl virus
TYLV may not be amplified in this assay since there are not
perfect match for Bv-F/R primers sequence in TYLCV genome
sequences as shown by alignment (data not shown).
During the last two decades, begomoviruses have increased
in terms of their number, prevalence, and distribution throughout the world with the identification and characterization of
more begomoviruses as time goes by (Navas-Castillo et al.
2011), surely recombination playing a major role. Having said
that, the multiplex PCR assay established in this work may not
include in the future as many begomoviruses as it does today.
The sensitivity of the assay was also examined by defining
the detection limit of each primer set in the multiplex format.
For this, serial tenfold dilutions of a known amount of each
genomic DNA were tested. Thus, the sensitivity for C. michiganensis subsp. michiganensis, Fusarium sp., L. taurica was
found to be 100 pg, and for 1 ng for Begomovirus.
Despite the optimization approaches, some remaining PCR
inhibition was observed frequently. Therefore, we added
1500
1000
750
500
250
Fig. 4 Optimized multiplex assay electrophoretic pattern of genomic
DNA from an infected plant with C. michiganensis subsp. michiganensis using PVP and BSA. From left to right, first half, PCR amplification with generic primers ITS4/ITS5 is shown for a healthy and a C.
michiganensis subsp. michiganensis-infected plant using 4 or 100 ng
of HindIII digested-genomic DNA in presence or absence of PVP +
BSA (indicated by P + B). The genomic DNA samples were also
amplified using the primer mix as shown in the second half. M stands
for molecular weight markers (1-kb DNA ladder in basepairs) and C+
for positive control (plasmids cocktail and primer mix)
Folia Microbiol (2013) 58:269–276
A
700
600
400
300
200
100
275
B
500
300
200
100
Fig. 5 Multiplex assay electrophoretic pattern of genomic DNA
extracted from an infected seed with C. michiganensis subsp. michiganensis. PCR amplification using A generic ITS4/ITS5 primers or B
primer mixtures and seed genomic DNA. Positive controls used were
plasmid TOPO TA-PL in A and plasmids cocktail in B. Molecular
weight markers (100-bp DNA ladder) are indicated in basepairs (M).
An arrow is pointing at amplified product
polyvinylpyrrolidone PVP and bovine serum albumin BSA
during PCR since this has previously been reported to be
helpful (Koonjul et al. 1999) to circumvent inhibitory effects
of polyphenols (Varma et al. 2007). Our results demonstrate
that PVP+BSA were helpful as no dilution of gDNA (100 ng)
was required to obtain a PCR product with generic ITS4/ITS5
primers whenever PVP+BSA were present (Fig. 4, healthy and
infected plants under ITS4/ITS5). The relative abundance of
amplified product in the presence of PVP+BSA for 4 ng of
DNA is comparable to that obtained when using 100 ng of
gDNA (25 times more PCR inhibitors present; Fig. 4, healthy
and infected plants under ITS4/ITS5). These same gDNAs
were challenged with the primer mix and we found the specific
PCR product (296 bp) expected for a plant infected with CMM
(Fig. 4, infected plant under primer mix). In this case, the 296
bp product was obtained with 4 and 100 ng of gDNA. For the
latter, this was only achieved when using PVP+BSA (Fig. 4,
infected plant under primer mix) since no product was obtained
in their absence. As a negative control, no PCR product
was observed at any DNA concentration for the healthy
plant when using primer mix (Fig. 4, healthy plant
under primer mix).
Finally, it was important to challenge this optimized
multiplex assay in gDNA obtained from seeds since there
are seedborne pathogens such as CMM. gDNA from tomato
seeds naturally infected with CMM was analyzed and a
specific 296 bp product was obtained (Fig. 5B), thus indicating the accuracy of the assay. As a control, this gDNA
was also amplified with generic ITS4/ITS5 primers and a
713 bp product was obtained (Fig. 5A).
Since tomato crops are susceptible to infection by various
pathogens (bacteria, fungi, and viruses) there is a need to
develop rapid, specific, and cost-effective assays for their
detection. In this study, we developed a multiplex assay to
allow the simultaneous detection of C. michiganensis subsp.
michiganensis, Fusarium sp., L. taurica, and begomoviruses in plants and seeds. This assay is much faster. Finally,
the developed assay is robust and readily adaptable to plant
diagnostic laboratories.
At the time when this study was undertaken, the method
established here allowed us to avoid an economic loss in the
greenhouse where the samples were obtained since detecting the presence of C. michiganensis subsp. michiganensis
in tomato seeds on time prevented their posterior culture.
Thus, this method is a useful alternative for detection of
some pathogens with phytosanitary importance in tomato in
a fast, simple, and suitable way.
Acknowledgments We thank Hidroponicos Especializados de
Chihuahua S.A. de C.V., Dr. Josefina León-Félix from Laboratorio de
Microbiología Ambiental y de Alimentos, Centro de Investigación en
Alimentación and Desarrollo, Dr. Angel Gabriel Alpuche Solís from
Instituto Potosino de Investigación Científica and Tecnológica División
de Biología Molecular, and Dr. Gabriela Olmedo Alvarez from Departamento de Ingeniería Genética Cinvestav-IPN for kindly donating infected
plants and/or genomic DNA from different organisms. The authors also
thank Dr. Gerardo Rafael Arguello-Astorga for critically reviewing the
manuscript and Biol. Salvador Ambriz-Granados for providing begomoviruses DNA. We also acknowledge Adalberto Herrera and David Israel
Vazquez Montiel for technical assistance.
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