## 4 Discussion  
As in other _Xf_ epidemic areas, the rapid, accurate, and cost-effective detection of the bacterium is required for timely eradication and containment measures that prevent further spread of the disease in geographical areas still free from the pathogen, especially in small territories, such as the Balearic Islands, with a broad genetic diversity of _Xf_ subspecies that affect a wide host range. The LAMP assay is an innovative, cost-effective, and user-friendly method for plant pathogen detection [32]. While several reports apply optical readers for fluorescent [33; 34; 35] or even colorimetric LAMP results detection [36; 37], in the work herein described, it has been demonstrated that, without requiring prior DNA purification or thermal denaturation of the extracts, and by naked-eye observation only, a rapid test for _Xf_ detection can be established for almond trees. The range of positively detected concentrations borders on what it is possible to detect in symptomless plants by the most sensitive qPCR. Similar to the performance of the same assay applied to the olive matrix [24], some incongruity cases between cLAMP vs. qPCR results were observed at lower target concentrations (Cq \(\geq\) 27) and could be ascribed to the different analytical sensitivities of the two methods, as well as to the stochastic target copy distribution in very low concentrated solutions [38; 39]. The cLAMP diagnostic specificity on the truly negative samples was greater than 90%, with only one false positive result, which makes the assay reliable. In the cLAMP experiment using crude extracts both in olive and almond matrices, the tiny amount of tissues from a tree pooled in a sample and the very small volume of extract added to the reaction should account, mainly in a symptomless condition, for the highly erratic distribution of the pathogen, which produces a false-negative more frequently than a false-positive test. It is quite common that sampling from different quadrants or heights of a tree results in different pathogen concentrations [40; 41]. It should also be noted that when samples from an 'undetermined' qPCR status were analyzed in digital droplet PCR for the presence of _Xf_, the samples were infected by an absolutely low copy number of genome \begin{table}
\begin{tabular}{c c c c} \hline \hline \multirow{2}{*}{**qPCR Value**} & **cLAMP Positives/Nr Analyzed** & **Diagnostic** & **Diagnostic** \\  & **Plants (Per Cq Value)** & **Sensitivity \%** & **Specificity \%** \\ \hline
18–24 & 4/4 & 100 & / \\
25 & 4/4 & 100 & / \\
26 & 5/9 & 55.55 & / \\
27 & 2/5 & 40 & / \\
28 & 1/4 & 25 & / \\
29 & 1/5 & 20 & / \\
30–33 & 2/10 & 20 & / \\
33.01-NA & 1/12 & & 92.30 \\ \hline Total & 20/53 & & \\ \hline \hline \end{tabular}
\end{table} 
Table 2: Comparative performance of cLAMP vs. qPCR assay for the detection of _Xf_ in almond alkaline crude extracts. Positive samples in cLAMP are indicated for each average Cq group obtained from qPCR assay. Percentage of diagnostic sensitivity and specificity generated by cLAMP assay are also reported. copies [31]. The average values of pathogen titers obtained by qPCR were used in our research as a standard reference to set a reliability range and not as an absolute demarcation. A large-scale application of cLAMP certainly could suffer from the relatively low diagnostic sensitivity when compared to the standard qPCR. However, its strength and innovative impact reside in its being a fully field-operative practice that is almost instrument-free and could be used in a new or presumptive infection focus in perennial host orchards. This new method of application, which offers the opportunity to positively test around 45% of symptomless infected trees in an orchard, can be added to the tools currently used for _Xf_ diagnosis. Moreover, it will improve pathogen identification without the need to move plant material from the contaminated territory to centralized laboratories, thus limiting the risk of pathogen dissemination in areas that are pest-free.