Development of a new technique to detect living cells

Development of a new technique to detect living cells of Xanthomonas campestris pv. campestris in crucifers seeds: the seedqPCR Samia Laala, Zouaoui Bouznad & Charles Manceau

European Journal of Plant Pathology Published in cooperation with the European Foundation for Plant Pathology ISSN 0929-1873 Eur J Plant Pathol DOI 10.1007/s10658-014-0532-4

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Author's personal copy Eur J Plant Pathol DOI 10.1007/s10658-014-0532-4

Development of a new technique to detect living cells of Xanthomonas campestris pv. campestris in crucifers seeds: the seed-qPCR Samia Laala & Zouaoui Bouznad & Charles Manceau

Accepted: 9 September 2014 # Koninklijke Nederlandse Planteziektenkundige Vereniging 2015

Abstract Xanthomonas campestris pv. campestris is a seed-borne bacterium that causes black rot on Brassicaceae. Ensuring seed lot sanitary quality is the most efficient control strategy against bacterial diseases. Currently, the procedures adopted in the control of seed lots are mainly based on microbiological techniques combined with PCR or plant inoculation which is time and money consuming. The aim of this study was to propose a reliable and rapid detection technique of living X. c. pv. campestris in cabbage seeds. We have shown that not all cells of X. campestris is able to grown on rich medium after washing and soaking seeds as no colony of X. campestris was detected on inoculated seeds, whereas plantlets develop symptoms 7–14 days after germination. The PCR technique used alone does not address the viability of bacteria in samples. We set up a technique named seed-qPCR for the detection of living X. c. pv. campestris bacterial cells in seed lots. This technique is based on an enrichment of bacterial population associated with infected seeds by seed S. Laala CNCC, Centre National de Contrôle et de Certification des semences et plants, BP 119 Hassen Badi, 16200 El Harrach, Alger, Algeria Z. Bouznad ENSA, Ecole Nationale Supérieure d’Agriculture, 1 Avenue Pasteur, Hassan Badi - El Harrach - Alger, 16000 El Harrach, Alger, Algeria C. Manceau (*) Anses, Laboratoire de la santé des végétaux, 7 rue Jean Dixméras, 49044 Angers, France e-mail: [email protected]

germination coupled with real-time Taq-man PCR after extraction of the target DNA. It is an inexpensive technique that allow the detection of down to 1 contaminated seed among 10 000 healthy seeds. The seed-qPCR method combines an efficient extraction based on bacterial multiplication on seedlings with a sensitive technique qPCR for the detection of bacteria in seed lots. Keywords Xanthomonas campestris . Living bacteria . Seed . Detection . Seed-qPCR

Introduction Xanthomonas campestris pv. campestris is an important seed-borne bacterium which causes black rot of Brassicaceous plants. It is worldwide distributed and considered as the most destructive disease of crucifers (Williams 1980). Low level contaminated seed lots may lead to severe crop losses as only one contaminated seed among 10 000 healthy seeds may be enough to initiate an outbreak after planting (Schaad et al. 1980; Cook et al. 1952). The new classification of species within the genus Xanthomonas was based on DNA-DNA hybridization (Crosa et al. 1973). Vauterin et al. (1995) described the Xanthomonas campestris species and assigned six pathovars X. c. pv. aberrans, X. c. pv. armoraciae, X. c. pv. barbarae, X. c. pv. raphani, X.c. pv. campestris and X. c. pv. incanae. All pathovars are pathogenic on cruciferous plants. Fargier and Manceau (2007) and Young et al. (2008) showed that X. campestris species is in fact restricted to three

Author's personal copy Eur J Plant Pathol

pathovars X. c. pv. campestris, X. c. pv. raphani and X. c. pv. incanae and included non pathogenic strains. Methods recommended by ISTA (International Seed Testing Initiative) and adopted by official laboratories for the detection of this pathogen is based on plating assays followed by a pathogenicity or a PCR test on suspect suspected colonies (Koenraadt et al. 2005; Roberts and Koenraadt 2014). These methods are time consuming and labour intensive (Chitarra et al. 2002; Tsygankova et al. 2004; Park et al. 2004; Berg et al. 2005, 2006). Molecular techniques provide alternatives to methods that include pathogenicity tests, which improve the rapidity and reliability of detection. Several procedures based on PCR (Polymerase Chain Reaction) have been developed for the detection of X. campestris in seed-lots (Rijlaarsdam et al. 2004; Tsygankova et al. 2004; Park et al. 2004; Berg et al. 2005) including real time PCR (Berg et al. 2006). However, techniques based only on DNA amplification do not reflect the viability of the pathogen in the sample. This may lead to obtain false positives by amplification of DNA from dead cells (Josephson et al. 1993; Masters et al. 1994). Other techniques have been developed for the detection of living cells that are based on an enrichment of bacteria on a semi-selective medium combined with a PCR amplification of specific DNA locus (BIO-PCR) (Schaad et al. 1995; Ito et al. 1998; Weller et al. 2000; Fargier and Manceau 2007). In this paper we described a new method to detect living cells of X. c. pv campestris in crucifer seeds: The Seed-qPCR based on an enrichment of bacteria by germinating seeds on pleated blotting paper placed in germination cabinet, followed by real-time PCR amplification of a specific DNA fragment from macerated seedlings. This technique has been optimized for lots of 30 000 seeds naturally infected and disinfected seed-lots. The efficiency of the seed-qPCR was shown on batches containing one seed infected with X. c. pv. campestris among 10 000 healthy seeds.

(CFBP), Angers, France (http://www-intranet.angers. inra.fr/cfbp/). Bacteria were routinely cultured on YPGA medium (yeast extract 7 g l−1, peptone 7 g l−1, glucose 7 g l−1, agar 15 g l−1, pH 7). Cultures were stored in sterile water at - 20 °C for short periods (less than 1 month). YPGA medium was supplemented with cephalexine (25 mg l−1), vancomycine (0.5 mg l−1) and cycloheximide (100 mg l−1) to form YPGACv medium and was used to grow bacteria from plant and seed samples. Seed lots Six batches of healthy and naturally infected cabbage seeds (Brassica oleracea cv. capitata) were used for this study. These seeds were provided by the GEVESSNES, Angers, France. Their sanitary status had previously been established using a standard procedure (ISTA Seed Health Method 7–109) and by BIO-PCR (this study). Seed inoculation X. c. pv. campestris, either CFBP 1121 strain or CFBP 5241, strain was inoculated into batches of 3,000 seeds of Brassica oleracea cv. capitata. Batches of 3,000 seeds were soaked in 300 ml of bacterial suspensions in sterile distilled water at different concentrations (1× 108, 1×107, 1×106 and 1×105 colony-forming units ml−1) and in sterile distilled water to be used as control. In order to allow bacterial cells to penetrate into seeds, vials containing the seeds were placed in desiccators and a vacuum equal to −0.098 bars was applied for 1 min and bluntly broken. The liquid was removed and seeds were dried on blotting paper for 2 h in a BioSafety cabinet. Seeds were weighted before inoculation and after drying to assess the amount of inoculum uploaded per seed. Then seed were stored at room temperature in sterile Petri dishes until used. Seed germination

Material and methods Bacterial strains and culture media Two strains of X. c. pv. campestris used in this study (CFBP 1121 and CFBP 5241) were obtained from the Collection Française de Bactéries associées aux Plantes

Four types of germination processes were conducted according to the purpose of investigation. In order to assess the seed inoculation method, batches of 100 seeds were sown on accordion pleated paper with 50 pleats (Schleicher and Schull), two seeds per pleat. The pleated strips were kept into 25×25 cm transparent closed containers and moistened with 40 ml


of sterile water. Germination tests were carried out at a temperature of 20 °C in darkness for 8 h and 30 °C in light for 16 h per day. Observation of symptoms was achieved 7 and 14 days after sowing time. Three independant experiments were run for each treatment. Three methods of germination were also evaluated using a batches of 10 000 seeds. Geves-SNES provided us with a seed lot (ADAR 3) naturally infected with X. c. pv. campestris The infection rate of this seed lot had been assessed to 1.4 ×102 cfu per seed by colony counting on YPGA medium. The minimum sample size recommended by ISTA is equal to 30 000 seeds separated in three batches of 10 000 seeds (Roberts and Koenraadt 2014). In order to set up a method to extract X.c. pv. campestris cells from seed lots with a size compatible with international rules for seed testing (Roberts and Koenraadt 2014), three sub-samples of seeds (10 000 seeds) were sown under three different conditions. Sub-samples were either sown in 25×25 cm transparent plastic containers with a lid containing blotting paper (Schleicher and Schull Catalog No. 426994), moistened with sterile distilled water, or into a 500-ml conical flask containing 100 ml of sterile distilled water. The seeded boxes were then directly placed in a germination cabinet at 20–30 °C with a photoperiod 8 h light and 16 h darkness per day (method 1). The conical flasks were shaken at 150 rpm for 1 h and the washing liquid was removed; the flasks were sealed and incubated in a germination cabinet at 25 °C 8 h/16 h day/night (method 2); or in an incubator at 27 °C in the darkness (method 3). In all cases the incubation time lasted 3 days. Germinated seeds washing Seedlings were washed in 3 ml per g of sterile saline (0.85 % NaCl) containing Tween 20 (0.02 % v/v) in sterile plastic bags (Bioreba) by shaking for 2 h on an orbital shaker at 150–200 rpm at room temperature. One ml of each extract was than pipetted into sterile 1.5-ml microtubes for later analysis. DNA preparation and PCR amplification DNA extraction was performed according to two protocols: The first was described by Llop et al. (1999). This protocol is based on precipitation of nucleic acid with isopropanol. The second one was based on alkaline

lysis. Aliquot of 1 ml of soaking liquid was centrifuged at 8,000 rpm for 5 min. The supernatant was removed and the pellet was re-suspended with 500 μl of NaOH 0.5 N. The tubes were incubated at 65 °C with shaking at 100 rpm for 10 min. Five μl of the solution were transferred into a 1.5 ml micro-tube containing 495 μl Tris HCl (20 mM) and stored at −20 °C before performing PCR. DNA extracted according to the both protocols was amplified with real time PCR. Best results were obtained with extraction protocol described by Llop et al. (1999) as the Cts were lower whatever the concentration of DNA target in the sample (data not shown). Even, the negative check samples displayed a difference in the Ct that would indicate that either the pH or the Tris concentration could affect the Taqpolymerase activity in the case of alkaline lysis based protocol. The primers set used in this study were previously described in the literature (Eden et al. 1991; Berg et al. 2005, 2006). The amplifications were carried out by multiplex PCR in a final volume of 20 μl. Contaning 1X Green GoTaq Flexi Buffer (Promega), 1.5 mM MgCl 2, 0.2 mM dNTP, 0.5 μM of each specific primer DLH 120 (5′-CCGTAGCACTTAGTGCAATG-3′) et DLH125 (5′-GCATTTCCATCGGTCACGATTG-3′), 0.05 μM of each universal primer : 1052-F (5′-GCAT GGTTGTCGTCAGCTCGT-3′); and Bac-R (5′TACG GCTACCTTGTTACGACT 3 ′), 0.05 U of GoTaq Flexi DNA polymerase and 3 μl of template DNA. The PCR reaction conditions were: a denaturation step of 95 °C for 3 min followed by 35 cycles of 40 s at 95 °C, 40 s at 63 °C (touchdown to 58 °C over the first 6 cycles) and 40 s at 72 °C. The amplification products were separated on 2 % agarose gel in TBE buffer 0.5 X, strained with ethidium bromide, and visualized using UV transilluminator on a GelDoc 2000 Bio-RAD. Real-time PCR reactions including a fluorescently labelled probe were carried out in final volume of 20 μl. It contained 1x Real Master Mix Probe (Eurogentec), 400 nM of each primer DLH 153 (5′-GTAATTGATACCGCACTGCAA-3′) and DLH 154 (5′-CACCGCTCCAGCCATATT-3′) and 200 nM of the Taq-Man probe P7 (5 ′ATGCCGGCGAGTTTCCAACG-3′) and 2 μl of template DNA. The reaction was run for 35 cycles of two steps: 20 s at 94 °C, and 40 s at 60 °C with initial denaturation for 2 min 30 s at 94 °C. Results were evaluated using the software Icycler iQ Real Time (BIORAD).


Comparison of method suitable for germination lots of 10 000 seeds

Results Inoculation of cabbage seeds

Bacterial concentration in each seed lot was assessed by plating tenfold dilutions of concentrated extract on YPGA medium and colony counting. After 3 days of germination, the seedlings were macerated in Bioreba extraction bag containing 3 ml of sterile saline solution (0.85 %, 0.02 % Tween 20) per g and shaken at 150– 200 rpm for 2 h. One ml of washing liquid was collected in a 1.5-ml microtube and aliquots were used to perform bacterial DNA extraction. Comparison between BIO-PCR method and seedqPCR for the detection of X. c. pv campestris in naturally infected crucifer seeds. A comparative analysis of BIO-PCR and seed-qPCR was carried out on batches of cabbage seeds naturally infected by X. c. pv campestris and healthy lot. Each batch was subdivided into three lots of 10 000 seeds and tested by BIO-PCR. BIO-PCR was performed according to Fargier and Manceau (2007). 100 μl from seeds extract were speared with sterile bent glass over the surface of YPGACvc medium. Plates were incubated for 45 h at 28 °C. Agar surface was washed with 3 ml sterile saline (0.85 % NaCl). The bacterial suspension obtained was homogenized using a pipette and an aliquot (1 ml) collected in 1.5 ml for DNA extraction according to the alkaline lysis protocol. Seed-qPCR using the protocols of DNA extraction and amplification previously described.

Sensitivity limit of seed-qPCR Cabbage seeds were inoculated with 8×108 cfu ml−1 (8×104 bacteria per seed) of X. c. pv. campestris added to batches of healthy seeds, to achieve the infection rate of 1:10, 1:100, 1:1000 and 1: 10000 with three replicates per batch. The lots were planted on blotting paper and placed in germination cabinets. After three days (20 °C in darkness for 8 h and 30 °C in light for 16 h per day), the seedlings formed were collected in extraction bag (Stomacher Bioreba) with sterile saline (0.85 % NaCl) containing 0.020 % of Tween 20 and shaken at 150 rpm for 2 h. Three 1-mlaliquots of each extract were pipetted in three sterile 1.5-ml microtubes and analyzed in real-time PCR after DNA extraction.

The purpose of this study was to develop a method to inoculate seeds with bacterial cells by soaking healthy seeds in bacterial suspensions of X. c. pv. campestris. Extraction of bacteria was carried out by soaking the seed samples and the seedlings in sterile saline solution. Bacterial extraction was conducted on three samples of each batch of contaminated seed. The concentration of X. c. pv campestris per seed was assessed by plating on YPGACv medium and colony counting. The results are presented in Table 1. The seeds inoculated with a bacterial suspension assessed to 1×108 cfu ml−1of X. c. pv. campestris were recovered to be infected with a bacterial population of 1 103 cfu per seed (strain CFBP 1121) and 1.6×103 cfu per seed (strain CFBP 5241) in average. No colony of X. c. pv. campestris was isolated from seeds inoculated with all suspensions of lower concentrations (107, 106 and 105 cfu ml−1) and with sterile water as well. The volume of inoculum absorbed by seeds was assessed to 0.1 μl per seed as the weight of 1,000 seeds increased by about 100 μg after infiltration and drying. Therefore either only a portion of bacterial cells survived to the inoculation or only a portion of inoculated bacteria was removed from seeds by washing, or more than one bacterial cell is required to form a colony on the YPGACv medium. Three sets of 100 seeds of each batch were sown on filter paper to check the growth of X. c. pv. campestris on seedlings. The seedlings were observed daily and first symptoms of rotting were observed and counted 7 days after sowing. At that time, the rate of diseased seedlings was about 40 % for seed batches inoculated with bacterial suspension of 1×108,1×107 and 1× 106 cfu ml−1 and 20 % for seed batch inoculated with 1×105 cfu ml−1 of X. c. pv. campestris. After 14 days, all the seedlings of all batches were infected, whereas the negative control showed no symptom of disease (Table 1, Fig. 1). Assessment of the sensitivity of the detection of X. c. pv. campestris in seedlings by seed-qPCR Cabbage seeds were inoculated with 10fold dilutions series (from 1×108 to 1×102 cfu ml−1) of bacterial suspension (CFBP 1121). Seeds were incubated at

Author's personal copy Eur J Plant Pathol Table 1 Bacterial infection on seeds after inoculation by vaccum infiltration Inoculum concentration (cfu/ml)

1×108

Strains

CFBP1121

1×107

1×106

1×105

0 (water)

CFBP5241 CFBP1121 CFBP5241 CFBP1121 CFBP5241 CFBP1121 CFBP5241

≤0.4 ≤0.4 ≤0.4 ≤0.4 ≤0.4 ≤0.4 X. c. pv. 1000 +/− 2a 1600 +/− 3 ≤0.4b campestris per seed (cfu/seed) assessed one day after inoculation Percentage of 7c 45 +/− 1.72 40 +/− 2.26 38 +/− 2.99 42 +/− 2.84 42 +/− 2.35 40 +/− 1.13 25 +/− 1.72 15 +/− 1.95 0 seedling 14c 100 100 100 100 100 100 100 100 0 with symptoms The concentration of X. c. pv. campestris per seed were assessed 1 day after seed inoculation by vaccum infiltration, by plating on YPGACv medium and colony counting (a average number of cfu/seed after inoculation, b limit of detection of cfu per seed). The percentages of rotting seedling were assessed in each set of 100 seeds of each batch 7 and 14 days after inoculationc . Three replicates were made for each treatment

25 °C (8 h/16 h light/darkness). Seedlings were collected 3 days after sowing for detection of X. c. pv. campestris. X. c. pv. campestris was detected in the samples whatever the used DNA extraction protocol, e.g. seedling batches inoculated with inoculum ranged from 1× 108 to 1×104 cfu ml−1 and both techniques led to the same conclusion (Fig. 2). Theoretically, this technique that we have called Bseed-qPCR^, allows the detection of as low as one cfu of X. c. pv. campestris per single seed because it is the number of cfu infiltrated per seed according to the volume absorbed by single seeds under vaccum infiltration (0.1 μl) when the inoculum concentration is equal to 1×104 cfu ml−1.

Dynamics growth of X.c. pv campestris (CFBP 1121) was assessed on eight seed batches of 300 seeds each inoculated by a 10-fold dilutions series (from 1×108 to 10 cfu ml−1) of bacterial suspension (CFBP 1121) (Fig. 3). The Ct values equal to 18 were observed in lots inoculated with high concentrations of bacteria reflecting a strong contamination of seedlings. Bacterial concentrations were extrapolated using regression lines calculates with known concentrations of bacterial suspensions from 1×108 to 1×102 cfu ml−1 at each sampling time. Three days after planting, the

Fig. 1 Cabbage seedlings on accordion pleated paper 14 days after sowing. a Healthy seedlings wetted with sterile water seeds were infiltrated with sterile distilled water. b Diseased seedling

from seeds infected by vaccum infiltration with a bacterial suspension (105 cfu ml−1) of the strain CFBP 5241. Fungi developed after the bacterial infection occurred

Dynamics of bacterial growth during germination assessed by seed-qPCR


Fig. 2 Assessment of the sensitivity of the seed-qPCR for the detection of X. c. pv. campestris strain CFBP1121 in seedlings according to two DNA extraction protocols : a protocol described by Llop et al. (1999) based on the use of isopropanol for nucleic acid extraction, and a protocol based on alkaline lysis. Positive detection signals (Ct < Ct water) were observed for seed samples inoculated with bacterial suspensions at concentrations equal or superior to 1×104 cfu ml−1. According to the volume of inoculated

suspension (0.1 μl per seed), seeds inoculated with bacterial suspension equal to 1×104 cfu ml−1 was inoculated with one bacterial cell. Three replicated were performed, the Ctwater was not significantly different to Cts of samples inoculated with suspension equal and inferior to 1×103 cfu ml−1 but superior to Cts of seed samples inoculated with bacterial suspensions at concentrations equal or superior to 1×104 cfu ml−1

contamination of seedlings was proportional to the bacterial concentration used for inoculation of seeds. For all batches inoculated with a bacterial concentration less than 1×107 cfu ml−1, the bacterial concentration increased over time, reflecting a growth of the bacterial population in seedlings. However, we noted at 6 and 7 days after germination, that bacterial concentrations were lower in seeds inoculated with a bacterial suspension of 101 and 102 cfu ml−1, possibly caused by the heterogeneity of seed infection in samples inoculated by low inoculum concentrations.

Testing Association). Three days later, 100 % of sown seeds germinated with a development of each seedling (root, stem and leaves) (Fig. 4a). In the cases of method 2 (seeds in flask incubated in light/darkness daily period) and method 3 [seeds in flask incubated darkness (incubator)], the seeds germinated but rootlets did not develop (Fig. 4b and c). A seed-qPCR was carried out on DNA extracted from seeds germinated following the three methods tested. The results are shown on Table 2. The Ct values obtained by seed-qPCR on plantlets according to method 1 reflected an increase by a factor of 100 of the bacterial population. It rose 1, 4 104 to 1.15×106 cfu ml−1. Bacteria can also multiply in seed placed in Erlenmeyer flasks but only by a factor of 3. We chose method 1 to grow seeds for the further seed-qPCR assays. Comparison between YPGACvc / BIO-PCR and seed-qPCR methods for the detection X. c. pv. campestris in naturally infected crucifers seeds. A comparative study was performed on seed lots naturally infected by X. c. pv campestris. Seed lots were

Development of a method suitable to test batches of 10 000 seedlings Three seed lots (10 000 seeds) of cabbage (ADAR 3) naturally infected by X. c. pv campestris (1.4×102 cfu per seed) were assessed by colony counting on YPGA medium. Seeds were germinated for 3 days following three methods previously described. Method 1 (germination cabinet) is based on the method of germination reference ISTA (International Seed


Fig. 3 Dynamics of bacterial growth during germination assessed by seed-qPCR. Bacterial concentrations were extrapolated using regression lines calculates with known concentrations of bacterial suspensions from 1×108 to 1×102 cfu ml−1 at each sampling time. Each line corresponds to a seed lot inoculated with a bacterial suspension at the concentration indicated in the panel. Adar 2,

Adar 3 and EN are naturally infected seed lots. The low bacterial concentration at 6 and 7 days in seeds inoculated with bacterial suspensions equal to 101 and 102 cfu ml−1 is probably caused by the heterogeneity of seed infection in these seed samples. Only low percentages of seeds were infected in these seed lots

tested by BIO-PCR (Fargier and Manceau 2007) and seed-qPCR as described above. The results show the efficiency of seed-qPCR compared to BIO-PCR (Table 3) The Ct values obtained by seed-qPCR revealed the sensitivity of this technique, X. c. pv campestris was detected by this technique in some samples when no detection was obtained by BIOPCR. The E16-725 lot which had been disinfected during the process factory was found infected, putting into questions the efficiency of the treatment. The detection of bacteria in the batch cannot be attributed to the presence of DNA from dead cells in the seed because the detection was even positive by BIO-PCR which is a technique in which the culture on agar medium hydrolyzes free DNA.

Limit of sensitivity of seed-qPCR

Fig. 4 Germinated seeds (10 000 seeds) 3 days after sowing according the three methods tested. a Method 1 : Germination of seeds in a plastic container fitted with a filter paper and incubated in a germination cabinet with light/darkness daily period). b

The sensitivity of the seed-qPCR was assessed on seeds naturally and artificially infected by X. c. pv campestris. Duplicate seed batches carrying infection rate of 1:100, 1: 1000, 1: 10 000 seeds were analysed for the presence of X. campestris by seed-qPCR. The bacteria were detected on all batches neither of 1:10 000 samples. Similar Ct values were observed for the duplicate batches of each infection rate (Table 4).

Discussion The detection method of X. c. pv campestris adopted by International Seed Testing Association (ISTA) is mainly

Method 2 : Germination of seeds in a conical flask and incubated in germination cabinet with light/darkness daily period. c Method 3 : Germination of seeds in a conical flask and incubated in an incubator (in darkness)

Author's personal copy Eur J Plant Pathol Table 2 Comparison of the sensitivity of the seed-qPCR according to three methods of seed germination process Initial concentration (cfu / seed)

1,4 X 102 a

Bacterial concentration on seedlings 3 days after sowing Method 1 ISTA germination process

Method 2 In conical flask with light period

Method 3 In conical flask in darkness

23.84a

28.11 a

27.96 a

1.15×104b

3.42×102 b

3.26×102 b

Ct values on seedling samples collected 3 days after sowing

b

Bacterial concentration (cfu/seedling) assessed with the regression curve y=− 2.727 x+40 calculated with known concentrations of bacterial suspensions (strain CFBP 1121) of 10fold dilution from 1×108 to 10 to 1×102 cfu ml−1

based on plating assays followed by pathogenicity and PCR test of the suspect bacteria to confirm the diagnosis (Roberts and Koenraadt 2006), Grimault et al. 2013; Roberts and Koenraadt 2014) This technique has been considered to be reliable and efficient but also time consuming and laborious for routine use (Berg et al. 2005, 2006). The objective of this study was to develop a rapid and reliable method for the detection of living cells of X. campestris in Brassica seed. This technique is based on a bacterial enrichment by germination followed by an amplification of target DNA by real time PCR. It makes use of the natural process of colonization of seedlings by bacteria during germination since bacteria multiply in

living plant tissue which leads to an increase from the initial bacteria concentration. Berg et al. (2006) have developed a primer pair combined with a Taq-man probe identified in the hrpF gene (hypersensitive response and pathogenicity) coding the protein that formed the translocon Xanthomonas type III secretion system. Enrichment of bacteria by germinating seeds conjugate with a Taq-Man real-time PCR with the DLH primer pair provides a rapid and reliable protocol to detect up to two equivalent cfu of X.c. pv. campestris per seed. Germination period is proposed for 3 days, the time required for a regular germination of all seed sown and a multiplication of X. c. pv. campestris to achieve a

Table 3 Comparison of seed-qPCR with BIO-qPCR and classical BIO-PCR Seed lot

E41660 (ADAR 2) E 41–661 (ADAR 3)

seed-qPCR

Lot N° 1

30.55 a

YPGACvc / BIO-PCR real time

2.84×103 b 6

nt c

23.4

1.76×10

35.8

37.5

Lot N° 2

27.6

3.26×104

27.9

6.6×103

Lot N° 3

20.28

5.68×10

nt

ADAR 5

Lot N° 1

–

–

–

E41-667 (ADAR 9)

Lot N° 1

25.96

1.54×105

nt

Lot N° 2

35.20

26.41

nt

E 16–725 disinfected lot

++

Lot N° 1

6

3

YPGACvc /BIO-PCR Conventionnal

+− +− −−

–

−− ++ ++ 2

Lot N° 1

29.7

4.76×10

34.3

1.47×10

++

Lot N° 2

27.9

2.50×104

25.2

7.1×105

++

Lot N° 3

28.2

1.91×104

26.3

2.48×105

++

Positive control

19.4

2.20×108

Negative control

–

–

–

–

−−

a

++

Ct value obtained by real time PCR using primers (DLH 153 / DLH154/Taq-Man P7)

b

Bacterial concentration in cfu / seed assessed with the regression curve y=− 2.426 x+38.593 Calculated with a known concentration of bacterial suspension of CFBP 1121 from 1×108 to 10 cfu ml−1

Detection of an expected bp-long DNA fragment by agarose gel electrophoresis (two repetitions per sample) c

nt : not tested

Author's personal copy Eur J Plant Pathol Table 4 Study of the sensitivity threshold of the seed–qPCR Samples

Infection rate

Seed-qPCR (Ct value)

1.1

1 : 100

+ (22.7)

1.2

1 : 100

+ (22.5)

1.3

1 : 100

+ (21.4)

2.1

1 : 1000

+ (22.3)

2.2

1 : 1000

+ (20.5)

2.3

1 : 1000

+ (20.0)

3.1

1 : 10 000

+ (24.2)

3.2

1 : 10 000

+ (25.2)

3.3

1 : 10 000

+ (22.4)

Positive control

100 : 100

+ (29.0)

Bacterial suspension CFBP 1121 Negative control

1×108 cfu/ml

+ (18.5) − (39.1)

One infected seed (8×104 bacterial cells per seed) was added to healthy seed lots containing 100, 1000 and 10000 seeds, respectively

detectable number of bacterial DNA molecules by PCR. In addition, the DNA of dead cells is hydrolyzed by ribonucleases released during seed germination and multiplication of bacteria. The effectiveness of the seed-qPCR was compared to YPACvc real-time BIO-PCR and conventional YPACvc BIO-PCR on seed lots naturally contaminated, disinfected and healthy. Results showed that both techniques are effectives, and allow the detection of up to 10 cfu ml−1. The seed-qPCR has the advantage of being inexpensive and requires no preparation of medium. The sensitivity of the seed-qPCR was assessed on artificially infected B. oleracea seed batches at different rates 1: 100, 1: 1000 and 1: 10 000. Seed-qPCR can detect a single X. c. pv. campestris infected seed among 10 000 clean seeds. The observation of disease symptoms on all seedling batches inoculated by vaccum infiltration indicates that the inoculation by this method was very effective as the infiltration of 10 bacterial cells per seed (0.1 μl at 1× 105 cfu/ml) led to an infected seedling 14 days after sowing. It also indicates that the plating tests carried out on seeds before sowing has dramatically underestimated the level of seed infection as X. c. pv. campestris was detected in the batches inoculated with the highest concentration (1×108 cfu ml−1) only. Such seeds had been

theorically inoculated with 1×104 bacteria per seed. The first hypothesis proposed above (bacterial mortality at inoculation time) can be discarded because the symptoms appeared on all plantlets 14 days after inoculation even in the seed lot inoculated with 105 cfu ml−1. Inoculation of seeds by this method is very effective and can be used to infect seedlings to assess the colonization of plants via seed infection and to assess plant susceptibility to seed borne bacteria. We have shown that X. c. pv. campestris cells inside the seeds were not all able to be extracted from seed by washing, as no colony of X. c. pv. campestris was detected on inoculated seed; whereas plantlets developed symptoms 7–14 days after inoculation. The seedqPCR method combines the most efficient extraction with the most sensitive technique for the detection of bacteria in extracted samples to date. Acknowledgments We thank Valérie Grimault, Geves-SNES, France, for her kind provision of seeds.

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