Importance of celery in Spain• Spanish production: approx. 74.000 t (MAGRAMA, 2012)

• Approx. 37.000 t are exported to UK (70%), France (12%) and about 18% to other countries (Italy, Sweden and Germany)

• Three cycles of cultivation along the year(120 - 180 days/cycle)

Main areas of production

Murcia

Alicante

Barcelona

New symptomatology in celery crops in 2008

�Abnormal number of shoots �Curling of stems �Yellowing

Commercial production losses were up to 70%

Research related to the celery problem:

• Design a detection method for ‘Ca. Liberibactersolanacearum’ (CaLsol) based on real-time PCR

• Comparison of conventional DNA extraction and directmethods of target preparation for CaLsol detection

• Association of symptoms with CaLsol detection

• Electron microscopy assays

• Comparison of partial sequences of CaLsol with thoseavailable

Real-time PCR

+ celeryextract 1/10 1/102 1/103 1/104 1/105 1/106

Conv

entio

nal

Liefting et al., 2009 + - - - - - -Li et al., 2009

(Lsof/OI2c) + - - - - - -Ravindran et al., 2011

(adk) + - - - - - -Ravindran et al., 2011

(TX) + + + + - - -

Real

tim

e PC

R (C

t) Li et al. 2009 + (22,82) + (25,78) + (28,49) + (33,71) + (34,57) - -IVIA + (23,66) + (26,53) + (28,31) + (31,68) + (32,53) - -

• 16 species of other pathogenic bacteria• 50 species of potato saprophytic microbiota• 21 species of carrot saprophytic microbiota

Sensitivity

Specificity

Methods of sample preparation

Conventional - DNA extraction

Samples are prepared into individual plastic bags

using HOMEX 6

Direct method - SPOT

Comparison DNA extraction vs. SPOT

DNA Extraction

+ -

SPOT+ 151 1

- 100 250 502

502 samples were analysed by real-time PCR for detection of CaLsolby both methods of sample preparation

We considered the 151 positive samples by both techniques as TRUE POSITIVES and the 250 negatives samples by both techniques as

TRUE NEGATIVES

Estimated prevalence 30,07%

Coincidence 79,88%

Cohen Kappa Index (κ) 0,597± 0,041

Mc Nemar Index 0,970 < 0,001

BAK Index0,581 ±

0,0446

Sensitivity Specificity

SPOT 0,60 (0,54-0,66)* 0,99 (0,98-1,00)

Calculation of diagnostic parameters

If we consider DNA extraction as a “gold

stardard” (sensitivity and specificity = 1,00) the

specificity of the SPOT is practically the same, but the sensitivity is lower *Average (confidence intervals at 95%)

Scale for Cohen Kappa Index (κ)

Comparison DNA extraction vs. SPOT

Kappa (k) Degree of agreement< 0,00 without agreement

0,00 - 0,20 insignificant0,21 - 0,40 medium0,41 - 0,60 moderate0,61 - 0,80 substantial0,81 - 1,00 almost perfect

Advantages of the SPOT method of sample preparation

• Is a feasible method for analyses of large number ofsamples in a rapid way

• Is much more economic than DNA extraction

• Is as specific as DNA extraction (high confidence forpositive detections) in spite of being less sensitive thanDNA extraction (risk of false negatives)

• Has lower risk of contamination than DNA extraction

• Routine screening of samples by spot– Positive samples: considered true positives– Negative samples: perform DNA extraction for key samples

+++Clear symptomsNot marketable

-SymptomlessMarketable

+ Some symptoms

Marketable

Association of symptoms with detection of ‘Ca. Liberibacter solanacearum’ and/or phytoplasmas

(Scale of symptoms severity)

Presence of symptoms and universal detection of phytoplasmas

(Hren, et al., 2007)

Presence of symptoms and specific detectionof ‘Ca. Liberibacter solanacearum’

(CaLsppf/r)

Analysis of variance

This results show the statistical association of the symptoms with the presence of‘Ca. Liberibacter solanacearum’ and not with phytoplasmas

Symptoms Real time PCR(DNA + SPOT)

Positive Negative Total+++ 150 24 174

+ 63 87 150- 38 140 178

Total 251 251 502

Symptoms Real time PCR(DNA + SPOT)

Positive Negative Total+++ 28 146 174

+ 25 125 150- 26 152 178

Total 79 423 502

Association of symptoms with the detection of ‘Ca. Liberibacter solanacearum’ and/or phytoplasmas

The SEM studies revealed the presence of bacteria-like organisms (BLOs) in celery phloem cellsof positive real-time PCR tested plants (possible ‘Ca. Liberibacter solanacearum’)

Scanning Electron Microscopy (SEM)

Celery phloem cell containing BLOs

0,5 µm

Transmission Electron Microscopy (TEM)

Negative control

Absence of BLOs in phloem cells of negative real-time PCR tested plants

Analysis of 16S rDNA gene sequence(1166 bp)

The 16S sequences of the Spanish Bactericera , carrot and celery samples are very similar.They cluster close to those of

other countries and hosts

Analysis of 50S rDNA gene sequence(659 bp)

The 50S sequences of the Spanish Bactericera , carrot and celery samples are very similar.They cluster close to those of

other countries and hosts

Conclusions

• With the results obtained we conclude that the vegetative disorders observed in celery are

associated with the presence of ‘Ca. Liberibactersolanacearum’ and not with phytoplasmas

• The sequences of 16S and 50S rDNA genes of the celery samples are quite similar to those of

other hosts and vector species

Thank you foryour attention