Detection Of Bacterial Wilt Of Tomato By Biolum-inescent Reporter And Hyperspectral Imaging Technology

Ralstonia solanacearum is one of the most destructive bacterial pathogens,causing bacterial wilt disease on more than 250 plant species worldwide.The bacterium can live for a long time in water and soil,which forms a reservoir source of inoculum.R.solanacearum infects roots of host plants,and then proliferates massively in the plant xylems,leading to host plant wilting and death.However,the pathogenesis mechanism of R.solanacearum is still unclear,and no resistant plant cultivars were reported yet.In order to assist the research on the molecular mechanism of plant resistance to R.solanacearum and the rapid breeding of disease-resistant cultivars,systematic and high-throughput methods for monitoring bacterial wilt were established using bioluminescent system and hyperspectral imaging in this study,and the results obtained were as follows:(1)A luminescent strain of R.solanacearum expressing luxCDABE operon was constructed.The luxCDABE operon encodes luciferase and substrate synthetase,which can emit light autonomously and has been widely used in biological detection.We constructed a recombinant plasmid containing the luxCDABE operon and transformed it into R.solanacearum FJ1470 and named FJ1470-LUX.Compared with FJ1470,FJ1470-LUX had no significant difference in growth rate,biofilm formation and motility;luminescent signals of FJ1470-LUX were positively related to the bacterial concentrations;luminescent signals were not impaired by the temperature and p H suitable for bacterial growth;luminescent signals of FJ1470-LUX were not significantly reduced after successive subculturing for 10 times.After FJ1470-LUX inoculation,there was no significant difference in pathogenicity compared with FJ1470.Luminescent signals started to be observed at tomato stems 2 d after FJ1470-LUX inoculation,and levels were linearly related to the bacterial concentrations.In addition,the luxCDABE recombinant plasmid in this research can be applied to generate other luminescent strains,such as Pectobacterium carotovorum,Escherichia coli and Agrobacterium tumefaciens.In summary,the bioluminescent reporter system could be used to detect tomato bacterial wilt non-destructively,systematically and in real-time.(2)A high-throughput detection model for tomato bacterial wilt was constructed based on hyperspectral imaging technology.Hyperspectral imaging of R.solanacearum infected and uninfected tomatoes to obtain the raw spectral information.After being normalized by the standard normal variate(SNV)method,the successive projections algorithm(SPA)was used to extract the featured wavelengths,and then the detection models were established based on the featured wavelengths.Among all the models,the accuracy of linear discriminant analysis(LDA)was more than 90%,and the non-linear anthocyanin reflectance index(ARI)could also distinguish the inoculated plants from the control.Therefore,LDA and ARI could be used as high-throughput detection models for tomato bacterial wilt based on hyperspectral imaging technology.In summary,we generated a luminescent strain of R.solanacearum which could monitor tomato bacterial wilt systematically,and established a high-throughput detection model for tomato bacterial wilt based on hyperspectral imaging technology.These methods provided new tools for the research of tomato bacterial wilt.