Rapid real-time quantitative colorimetric LAMP methodology for field detection of Verticillium dahliae in crude olive-plant samples

Abstract

Background: Verticilium dahliae is the most important wilt pathogen of olive trees with a broad host range causing devastating diseases currently without any effective chemical control. Traditional detection methodologies are based on symptoms-observation or lab-detection using time consuming culturing or molecular techniques. Therefore, there is an increasing need for portable tools that can detect rapidly V. dahliae in the field.

Results: In this work, we report the development of a novel method for the rapid, reliable and on-site detection of V. dahliae using a newly designed isothermal LAMP assay and crude extracts of olive wood. For the detection of the fungus, LAMP primers were designed targeting the internal transcribed spacer (ITS) region of the rRNA gene. The above assay was combined with a purpose-built prototype portable device which allowed real time quantitative colorimetric detection of V. dahliae in 35 min. The limit of detection of our assay was found to be 0.8 fg/μl reaction and the specificity 100% as indicated by zero cross-reactivity to common pathogens found in olive trees. Moreover, detection of V. dahliae in purified DNA gave a sensitivity of 100% (Ct < 30) and 80% (Ct > 30) while the detection of the fungus in unpurified crude wood extracts showed a sensitivity of 80% when multisampling was implemented. The superiority of the LAMP methodology regarding robustness and sensitivity was demonstrated when only LAMP was able to detect V. dahliae in crude samples from naturally infected trees with very low infection levels, while nested PCR and SYBR qPCR failed to detect the pathogen in an unpurified form.

Conclusions: This study describes the development of a new real time LAMP assay, targeting the ITS region of the rRNA gene of V. dahliae in olive trees combined with a 3D-printed portable device for field testing using a tablet. The assay is characterized by high sensitivity and specificity as well as ability to operate using directly crude samples such as woody tissue or petioles. The reported methodology is setting the basis for the development of an on-site detection methodology for V. dahliae in olive trees, but also for other plant pathogens.

Keywords: Verticillium wilt; Defoliating pathotype; Field-based detection; Loop mediated isothermal amplification; Molecular diagnostics; Soilborne pathogens.

Fig. 1

Schematic representation of the (A) complete sample-to-result protocol and (B) qcLAMP assay and detection with a novel 3D-printed, purpose-built portable device

Fig. 2

A Real time colorimetric LAMP detection of 20 ng from two pathotypes of V. dahliae (63 V: defoliating isolate, 25 V: non-defoliating isolate), using the newly ITS-region designed primers compared to the RAPD-published primer set [40]. All experiments were performed in triplicates; B Real time colorimetric LAMP using as template serial dilutions of DNA extracted from V. dahliae culture ranging from 2 ng to 20 fg per reaction C Calibration curve derived from six tenfold dilutions starting from 2 ng of gDNA; the x axis depicts the amount of DNA in the reaction and the y axis the time-to-positive derived from the real time graph shown in B. Error bars represent deviation of at least triplicates; D Specificity of the real time colorimetric LAMP assay. At 20 ng fungal DNA only V. dahliae was detected at 16 min. No detection was observed when the same amount of DNA from other pathogens was used

Fig. 3

A Real time colorimetric LAMP using as template serial dilutions of DNA extracted from an olive tree naturally infected with V. dahliae of a reported Ct value at 19.6 in real time PCR assays; B Correlation (R² = 0.99) between the qcLAMP time-to-positive results and six tenfold serial dilutions of infected plant using 20 ng of extracted plant DNA as template

Fig. 4

A Scatter plot of the Ct values (ranging from 19.6 to ~ 31.5) for 19 positive samples of DNA extracted from naturally infected trees versus the qcLAMP time-to-positive (ranging from 14.0 to 28.8 min) using 20 ng extracted plant DNA as template; (B) comparison of qcLAMP to qPCR for various Ct cut off values