Development of two loop-mediated isothermal amplification (LAMP) genomics-informed diagnostic protocols for rapid detection of Pantoea species on rice

At least three species of Pantoea are responsible for bacterial blight disease and grain discoloration of rice in Sub-Saharan Africa. Thus, measures need to be taken to limit the pathogens' dispersion and robust diagnostic tools are required for rapid and cheap diagnosis in the field as well as for routine seed certification or control. Therefore, several diagnostic tools such as simplex and multiplex PCR schemes and a semi-selective medium have been developed and are being used. However, the use of these tools is time-consuming, expensive and therefore limited to laboratories that can afford the chemicals. We have therefore developed two isothermal loop amplification (LAMP) protocols, one of which detects all Pantoea species in the genus and another one that is specific for P. ananatis.• The novel LAMP assays allow rapid and sensitive detection of these bacteria.• They will help plant protection services in routine field and laboratory tests especially for monitoring the phytosanitary status of rice seeds.

Pantoea spp. are seed-transmitted and thus pose a significant threat to interstate and continental seed exchanges [7 , 15 , 16] . Thus, measures need to be taken to limit the pathogens' dispersion and robust diagnostic tools are required for rapid and cheap diagnosis that can be used in the field as well as in routine screening of seed lots.
New diagnostic tools have been developed, such as simplex and multiplex PCR schemes able of identifying the three major Pantoea species affecting rice plants [12] . A semi-selective medium for their isolation and identification has also been developed [13] . However, their use is complex, time consuming, expensive and cannot be deployed outside the laboratory.
Isothermal loop-mediated amplification (LAMP) is a relatively simple technique that facilitates rapid amplification of DNA and has a high level of sensitivity and specificity at low cost [17 , 18] . The technique uses the thermostable Bst DNA polymerase for reliable and fast polymerization and operates under isothermal conditions at relatively high temperature [19 , 20] . The results of LAMP reactions can be detected without electrophoretic separation but by visualization of turbidity, fluorescence or a color change thanks to a metal ion indicator [17] . LAMP has several advantages, such as portability, simplicity and cost-efficiency because the reaction can be carried out in a water bath or heating block. It has been used for highly specific and sensitive DNA amplification to detect various pathogens, including viruses, bacteria, protozoa and fungi [21] . However, LAMP has so far not been applied to rice/ Pantoea spp., neglected pathosystems that are wide-spread and important in Africa (12,14).
The goal of this work was to overcome the above difficulties and facilitate a quick and costeffective diagnosis of the bacteria in rice leaves and paddy fields. Two diagnostic tools have been therefore developed: one that detects all known Pantoea species and another one that is specific for P. ananatis . These tools will help monitor the phytosanitary status of rice seeds to avoid the dissemination of Pantoea spp.

Loop-mediated isothermal amplification (LAMP) reaction
NB: Use either colorimetric LAMP or regular LAMP reactions  2.5 μl of pre-mixed LAMP primers and 9 μl of nuclease-free water ( Table 2 ), vortex briefly and centrifuge the reaction mix. 3. Then, add 1 μl of one of the target templates (20 ng/μl genomic DNA, heat-killed bacterial cells or bacterial ooze from leaf tissue or rice seeds) to the 24-μl reagent mixes. Seal, vortex and centrifuge the reaction tubes that will provide a solution of bright pink color, which indicates the initial high pH required for successful LAMP reaction. 4. Incubate the reaction tubes at 65 °C for 30 min and examine by eye, with positive reactions turning into yellow while negative controls remaining pink. 5. In case of an orange color suggestive of a positive reaction, return the tubes to 65 °C for an additional 10 min. In order to intensify the color of positive reactions, cool the reaction tubes to room temperature. 6. Finally, take a photography of the tubes to record the colorimetric result.  ( Table 3 ).  2. Add 1 μl of the template from one of the four types of templates: genomic DNA (20 ng/μl), heat-killed bacterial cells, the bacterial ooze from the leaf tissue or rice seeds. 3. Use 20 μl of mineral oil to cover the surface of the LAMP mixture in each reaction.

Data analysis
For the colorimetric LAMP assay, the results can be judged by naked eye. Negative (-) reactions remain pink while positive ( + ) reactions change to yellow. ( Fig. 1 ).
For the regular detection of LAMP products, reaction tubes were placed under UV light. Positive reactions, i.e. DNA amplification, were identified by the bright green fluorescent color and could be seen by naked eye ( Fig. 2 ). Green fluorescent tubes are positive ( + ) while the others are negative (-).

Notes
1. The LAMP reaction is very sensitive. Attention should be paid to avoid contamination during the operation, and a stringent laboratory compartmentalization is strongly recommended for LAMP and other amplification assays. 2. To prevent cross-contamination, different sets of pipettes and different work areas must be used for DNA template preparation, PCR mixture preparation and DNA amplification. Gloves must be changed regularly and sterile pipetting techniques must be applied during the entire LAMP experiment. 3. To avoid potential contamination, agarose gel electrophoresis of LAMP products is not encouraged. 4. For bacterial colonies (lysates), the detection limit of the two LAMP assays was 10 4 colony-forming units/ml. Using purified DNA, the thresholds were 0.5 fg for the Pantoea -genus-specific tool and 50 fg for the P. ananatis -specific tool.

Recipes
• Peptone sucrose agar (PSA): To 1 l of sterile distilled water, add 10 g peptone, 10 g sucrose, 16 g agar, and 1 g glutamic acid. Adjust pH to 7.1 ± 0.2 using 1 M KOH or NaOH solution. Autoclave at 121 °C for 20 min. Cool the bottle but let not solidify the medium, then pour into Petri dishes.
To 1 l of sterile distilled water (pH 7.1 ± 0.2); add 65 g NaCl; 0.001 g crystal violet; 8.5 g sodium thiosulfate; 13.5 g agar; 10 g peptone; and 10 g sucrose. Autoclave at 121 °C for 20 min. Cool the bottle but don't let to solidify the medium, then pour into Petri dishes. To prepare a solution of 1 M HCl, add 83 ml of concentrated HCl (37% w/w) to 1l of water.
• Preparation of 70% ethanol Following the Gay-Lussac dilution table, add 40.85 ml of 96 °ethanol to 100 ml of distilled sterile water.

Declaration of Competing Interests
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.