Pectolytic bacteria were isolated from potato tubers and stems showing tuber soft rot and blackleg symptoms. Approximately half (52%) of the isolates could grow at both 27 and 37 °C while another half (48%) failed to grow at 37 °C. All isolates could be amplified with primers specific to the pectate lyase (pel) gene. Carbon utilisation profiles could not conclusively identify these isolates. PCR amplification using primers specific for Pectobacterium carotovorum subsp. brasiliensis was positive for all isolates that grew at 37°C. However, the group that did not grow at 37 °C failed to amplify with P. atrosepticum specific primers. To characterise this group of isolates, the intergenic transcribed spacer region (ITS) was amplified and PCR products digested with two restriction enzymes (RsaI and CfoI) to generate ITS-PCR-RFLP profiles. The profiles of these new isolates were compared to those of the type strains of other pectolytic bacteria. Profiles of five of the selected atypical strains generated with the enzyme CfoI appeared to be most similar to those of P. wasabiae type strain. Phylogenetic analysis using concatenated partial gene sequences of housekeeping genes mdh and gapA clustered these isolates together with those of P.wasabiae reference strains thus confirming their identity. These strains were virulent on potato tubers and stems but did not elicit hypersensitive response on tobacco plants. This is the first report of P. wasabiae causing soft rot and blackleg of potatoes in South Africa.

The efficacy of three elicitors, DL-β-aminobutyric acid (BABA), Acibenzolar-S-methyl (Bion) and Messenger (Harpin) on potato plants inoculated with Meloidogyne incognita second stage juveniles (J2s) was investigated in this study. The efficacy of each elicitor was evaluated based on galling index, egg numbers, total root knot nematode (RKN) population numbers, number of juveniles and the percentage of females in respective extracted populations per treatment at 28 and 56 dpi. There was a significant decrease in galling indices of all treated compared to untreated control plants. The highest percentage reduction was observed for 20 mM BABA-treated plants (33%) followed by 5mM BABA (23%), 50mg/L Bion (18%), 100mg/L Bion (15%) and Messenger (8%). The number of eggs extracted from plants treated with 20mM BABA was significantly lower (66%) compared to untreated controls. Furthermore, treating plants with 20mM BABA decreased the total RKN population numbers by 55% whilst the decrease observed for 2 Bion and Messenger was between 24-39%. Of the three tested elicitors, 20mM BABA was found to be the most effective elicitor at both 28 and 56 dpi. Hence, it was selected for further testing against RKN and Pectobacterium carotovorum subsp. brasiliense (Pcb) disease complex. Efficacy was tested by comparing the colony forming units (cfu) of Pcb from treated and untreated control plants inoculated with M. incongita and Pcb simultaneously. The concentration of Pcb from untreated plants inoculated with both pathogens was significantly higher (3.3 × 10 3 cfu/ml) compared to their treated counterparts (8.71 × 10 2 cfu/ml). This suggests that BABA has the potential to decrease soft rot bacterial infections in RKN disease complexes by decreasing the rate of RKN infection. This is the first report where BABA is shown to decrease the infection rate of RKN as a primary causal agent in combination with a secondary pathogen.

Significance and Impact of the Study: This study uses fluorescent reporter protein tagging as a tool to demonstrate the interaction between root-knot nematode (Meloidogyne incognita) and the soft rot Enterobacteriacea (Pectobacterium carotovorum subsp. brasiliensis). Introduction of Pectobacterium through wounds generated by second-stage juveniles (J2) into potato tubers was demonstrated. These results suggest that RKN juveniles can facilitate latent infection of potato tubers in the soil. These findings have important implications in the management of RKN and SRE in seed potato production. Furthermore, this tool can be used to study other nematode-bacteria interactions that have not been previously studied.Keywords soil-borne pathogens, nematode-bacteria complexes, pectolytic bacteria, potato. AbstractThe study of plant parasitic nematodes such as Meloidogyne spp. and their interactions with phytopathogenic bacteria remains underexplored. One of the challenges towards establishing such interactions is the dependence on symptom development as a measure of interaction. In this study, mCherry was employed as a reporter protein to investigate the interaction between the soft rot Enterobacteriaceae (SRE) Pectobacterium carotovorum subsp. brasiliensis (Pcb) and root-knot nematode (M. incognita). Pectobacterium carotovorum subsp. brasiliensis was transformed with pMP7604 generating Pcb_mCherry strain. This strain was shown to attach to the surface coat of M.incognita J2 at the optimum temperature of 28°C. This suggests that RKN juveniles may play a role in disseminating Pcb in soils that are heavily infested with Pcb. The presence of RKN juveniles was shown to play a role in introducing Pcb_mCherry into potato tubers potentially acting as a source of latent tuber infections.