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This project has received funding from the European Union's Seventh Framework Programme for research, technological development and demonstration under grant agreement no 613678

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Strategies to develop effective, innovative and practical approaches to protect major european fruit crops from pests and pathogens

Bacterial pathogen on-site detection tools

A compact, lightweight and robust instrument suitable for use in the laboratory or in the field thanks to its embedded rechargeable Lithium-Polymer battery. The device was specifically designed to run any isothermal amplification method (up to 16 samples per run) that employs target detection by fluorescence measurement.

Diagnostic assays have been developed for all three bacterial pathogens (Pseudomonas syringae pv. actinidiae (Psa), Xanthomonas arboricola pv. pruni (Xap) and Xanthomonas fragariae (Xf)) based on loop-mediated amplification (LAMP). A rapid diagnostic can be achieved within 30 min on the field. These assays are currently being validated before transfer to stakeholders for on-site pathogen detection.

The LAMP technology offers practical advantages compared with PCR-based assays in terms of:

Greater specificity for discriminating pathogen subtypes that are of most economic or phytosanitary concern,
eliminating false-positives from related bacteria of little/no disease relevance and
suitability for rapid on-site diagnostics directly in the field

Additionally LAMP does not require expensive and bulky thermocycler equipment and can be done at body-temperature (incubation underarm is sometime used as a practical hack for field work).

LAMP primers design

LAMP primers design

Location of the specific LAMP target regions (PSA3 and PSA LAMP region) on the whole genome BLAST comparison of
Pseudomonas syringae pv. actinidiae (Psa) subpopulations and closely related members of the P. syringae species complex

LAMP is based on a set of six primers (rather than two typically for PCR) allowing for multiple genome sequence regions to be included as specificity targets.

A primer set includes: Forward Inner Primer (FIP), Backward Inner Primer (BIP), Forward Outer Primer (F3), Backward Outer Primer (B3), and Loop Primers F and B. Loop primers contain complementary sequences to the single stranded loop region, and this provides an increased number of starting points for DNA synthesis (e.g. copying sample DNA fragments) that delivers the improved specificity and sensitivity compared to most PCR based detection assays.
Pathogen specific 500 bp-long regions were obtained using a comparative genomics approach (“dual-BLAST”). Several sets of LAMP primers were designed and further tested for in vitro validation.

Specificity and sensitivity validation

Specificity and sensitivity validation

Sensitivity test of the PSA LAMP and PSA3 LAMP assays. Serial dilutions of Psa3 strain CFBP 7287 (black lines) and bacteria with plant extracts from kiwi leaves (green lines) were tested. Both assays have a detection limit of ~125 CFU per reaction and detection time of 9-17 min.

In order to assess the specificity of the designed LAMP assays, geographically and genetically representative collections of targeted bacteria were analyzed. To ensure high specificity of the assays, closely related bacteria, as well as bacteria commonly found in the host plant’s phyllosphere, were also included. An amplification was only observed for all the tested strains of Psa (n = 31) and Xf (n = 37) whereas no amplification could be observed with the non-target bacteria tested (n = 56, for Psa; n = 77, for Xf).

For Psa, two assays were designed in order to constitute specific diagnostic markers for general detection of non-pathogenic/weakly virulent Psa subtypes (Psa LAMP assay) and also for discriminative detection of the actual pandemic Psa3 subpopulation (Psa3 LAMP assay).

The Psa assays showed the ability to detect the presence of Psa and Psa biovar 3 with quantitative amplification down to 105CFU ml-1 (corresponding to 125 CFU or 80 fg per reaction). Similarly, a consistent lower detection of a range of 105 CFU ml-1 (corresponding to 500 CFU or 500 fg per reaction) was reached with the Xf assay.

The presence of plant extracts slightly delayed the amplification but did not affect the sensitivity of the LAMP assay. Although the time needed for amplification was dependent on the concentration of the target pathogen present in the samples, a strong positive reaction was produced within 30 minutes.

Field validation and technology transfer

Field validation and technology transfer

An example of commercialized user-friendly kit for on-site diagnostic of the fungal pathogen Phytophthora kernoviae together with an isothermal amplification portable instrument used for target detection by fluorescence measurement. The kit contents all the material, reagents and controls required to perform the run in the field.

These diagnostic tools will be applied to collect additional isolates for other tasks of the project. Technology transfer of detection assays to DROPSA partners and stakeholders will continue as needed in the remaining period of the project. Methods for the Xap LAMP assay have been published (Bühlmann et al., 2013), whilst those for the Psa and Xf LAMP assays are in the process of being published.

Training involving about 15 kiwi growers will be held in Auckland (New Zealand) on 19thFebruary 2016 by Plant & Food Research. A demonstration of the LAMP technology is also planned during a stakeholders meeting to be held in Girona (Spain) on 10thMarch 2016 after the 4thDROPSA project meeting.

The consortium is currently exploring possibilities to commercially develop these LAMP assays for Xf, Psa and Xap as user-friendly kits for on-site diagnostics with a SME industry partner.



Joël F. Pothier
ZHAW Zurich University of Applied Sciences