MinION Nanopore-based detection of Clavibacter nebraskensis, the corn Goss's wilt pathogen, and bacteriomic profiling of necrotic lesions of naturally-infected leaf samples

The Goss’s bacterial wilt pathogen,Clavibacter nebraskensis, of corn is a candidate A1 quarantine organism; and its recent re-emergence and spread in the USA and Canada is a potential biothreat to the crop. We developed and tested an amplicon-based Nanopore detection system forC.nebraskensis(Cn), targeting a purine permease gene. The sensitivity (1 pg) of this system in mock bacterial communities (MBCs) spiked with serially diluted DNA ofC.nebraskensisNCPPB 2581Tis comparable to that of real-time PCR. Average Nanopore reads increased exponentially from 125 (1pg) to about 6000 reads (1000 pg) after a 3-hr run-time, with 99.0% of the reads accurately assigned toC.nebraskensis. Three run-times were used to process control MBCs, Cn-spiked MBCs, diseased and healthy leaf samples. The mean Nanopore reads doubled as the run-time is increased from 3 to 6 hrs while from 6 to 12 hrs, a 20% increment was recorded in all treatments. Cn-spiked MBCs and diseased corn leaf samples averaged read counts of 5,100, 11,000 and 14,000 for the respective run-times, with 99.8% of the reads taxonomically identified asC.nebraskensis. The control MBCs and healthy leaf samples had 47 and 14 Nanopore reads, respectively. 16S rRNA bacteriomic profiles showed thatSphingomonas(22.7%) andClavibacter(21.2%) were dominant in diseased samples whilePseudomonashad only 3.5% relative abundance. In non-symptomatic leaf samples, however,Pseudomonas(20.0%) was dominant withClavibacterat 0.08% relative abundance. This discrepancy inPseudomonasabundance in the samples was corroborated by qPCR using EvaGreen chemistry. Our work outlines a new useful tool for diagnosis of the Goss’s bacterial wilt disease; and provides the first insight onPseudomonascommunity dynamics in necrotic leaf lesions.