Use of loop-mediated isothermal amplification (LAMP) as diagnosis tool to identify Psa in open field

Pseudomonas syringae pv. actinidiae (Psa) is the causal agent of bacterial canker kiwifruit. This disease spread very rapidly, also due to the circulation of infected plant material, till becoming pandemic. In order to prevent local spread and future introduction into Psa-free area, the early diagnosis of asymptomatic material is essential. Moreover, Psa symptoms in open filed are represented, from spring to autumns, mainly by leaf spots which may be caused also by other pathogens (i.e. P. syringae pv. syrinagae). Thus a fast, reliable, easy to use an inexpensive diagnostic method may represent a key tool screen nursery material or to tailor control treatments, orchard management inputs. Due to its plasticity, cost effective, rapidity and sensitivity, loop-mediated isothermal amplification (LAMP) assay is an ideal approach to develop such diagnostic tool. LAMP primers were designed on the AvrPT01 protein, a virulence factor specific of Psa and tested for specificity with genomic DNA from different Pseudomonas syringae pathovars (tomato, tobacco, glycinea, syringae and theae), other bacteria commonly found on Actinidia species (Pseudomonas viridflava, Pantoea agglomerans, Pantoea vagans, Paenibacillus spp, Pseudomonas fluorescens) and other bacteria not related to kiwifruit (Erwinia amylovora, Escherichia coli). Positive diagnosis, which is indicated in a clear color change or the reaction medium, was found only on Psa pathovars 1, 2 and 3. The method was also tested using crude extracts from infected Actinidia deliciosa and Actinidia chinensis plants. The sensibility of Lamp-based methodology here described was 1000 CFU/ml, comparable to other molecular tools commonly used for Psa identification such as end-point PCR and QPCR