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

Abstract

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.

Detection and Control of Pantoea agglomerans Causing Plum Bacterial Shot-Hole Disease by Loop-Mediated Isothermal Amplification Technique

“…The LAMP method established in this test showed high sensitivity. The minimum detection limit of DNA was 5 fg·μl −1 , which was 10 times more sensitive than the LAMP detection method established by Kini et al ( 2021a , b ) and 1,000 times higher than that of conventional PCR.…”

“…stewartii accurately in plants with a detection limit of 2 pg/μl genomic DNA. Kini et al ( 2021a , b ) designed multiplex PCR primers based on the whole genome sequence of Pantoea and detected P. agglomerans, P. ananatis , and P. stewartii growing in rice seeds with a detection threshold at 0.5 ng/μl genomic DNA. Tambong et al ( 2010 ) established a TaqMan-based real-time PCR assay for the detection and identification of P. stewartii in maize, which targets the cpsD gene and could specifically detect P .…”

Development of a Genome-Informed Protocol for Detection of Pseudomonas amygdali pv. morsprunorum Using LAMP and PCR

“…Comparison between different techniques allows the selection of the best method performed under specific conditions. The most commonly used routine method today is PCR, but nowadays there are other detection methods such as LAMP, which is specifically used for the detection of plant pathogenic bacterial genera, which is the case of Pectobacterium [ 16 ], Pantoea [ 17 ], Xanthomonas [ 18 ], Pseudomonas [ 19 , 20 , 21 , 22 , 23 ], and other pathogens such as Venturia carpophila [ 24 ], Bipolaris oryzae [ 25 ], or the set of phytoplasmas associated with sesame-phyllody disease [ 26 ]. Additional features such as low equipment requirements considering that it could be performed without a PCR machine, amplification under isothermal conditions, and the use of 4 to 6 primers targeting more genomic regions ensure high operational efficiency and are highly specific even against the co-presence of non-target DNA [ 27 ].…”