Potato blackleg caused by pectinolytic Pectobacterium and Dickeya species is a bacterial disease creating serious economic losses in (seed)potato production worldwide. Effective management to... Show morePotato blackleg caused by pectinolytic Pectobacterium and Dickeya species is a bacterial disease creating serious economic losses in (seed)potato production worldwide. Effective management to control blackleg is absent and validated, cost-effective detection protocols for blackleg bacteria do not exist. This situation results partially from the lack of knowledge of the ecology of blackleg pathogens in general, and Dickeya spp. in particular. Since 2000 an increasing frequency of Dickeya spp. in seed potatoes in Europe has been observed which is connected to emergence of a new species belonging to biovar 3 but unclassified inside known and described six Dickeya species. This species was provisionally named __D. solani__ and isolated from potato in The Netherlands, Finland, Poland, Germany, Belgium, France, United Kingdom and Israel. The objectives of the study described in this thesis were to acquire knowledge on the ecology (i. e. distribution of Dickeya spp. in seed potato tubers, role of the soil-borne inoculum in disease incidence, role of the aerial haulm infection for the blackleg incidence in progeny tubers) of a new genetic clade of biovar 3 Dickeya spp.; and to find and characterize in detail the valuable biocontrol agent(s) (isolated from rotten potato tissue) to cure seed potato tubers from blackleg caused by biovar 3 Dickeya spp. (__D. solani__). Direct isolation of viable bacterial cells using (selective) plating techniques combined with molecular (16S rDNA and Rep-PCR), serological (DAS-ELISA) and biochemical (biovar determination) characterization of the isolates were used to evaluate the presence of Dickeya and Pectobacterium spp. in different plant parts and tissues. Bacterial population dynamics in planta was studied in greenhouse experiments. For the studies on colonization of potato tissues by biovar 3 Dickeya spp. (__D. solani__) and Serratia plymuthica A30, bacterial strains were transformed with plasmid-based genes coding for red or green fluorescent proteins (DsRed and GFP, respectively) that were constitutively expressed in bacterial cells. Epifluorescence stereo microscopy and confocal laser scanning microscopy were used to visualize bacterial presence in different tissues of potato tubers and plants. Studies on the distribution of Dickeya ( and __D. solani__) and Pectobacterium species in seed potatoes revealed that the pathogens were distributed unevenly inside tubers and that the stolon end tuber part harbored the highest bacterial inoculum, whereas deeper located tissues were usually not colonized. Studies on the importance of soil-borne biovar 3 Dickeya sp. (__D. solani__) inoculum for systemic colonization of potato plants and latent infection of progeny tubers unveiled that D. solani is able to systemically colonize progeny tubers from soil via the roots and to cause true blackleg symptoms in infected plants after soil infestation. Studies on the role of haulm infections for latent contamination of progeny tubers by D. solani presented that the injection of bacteria into stems can result in the downward vascular translocation and a colonization of underground plant parts and finally, latent contamination of progeny tubers. Potato leaves inoculation with a biovar 3 Dickeya sp., showed degradation of the inoculated plant material and spreading of the internal inoculum to the petiole and axil and finally to the main stem but bacteria were not detected in the underground plant parts. Studies on characterization of the bacteria antagonistic to biovar 3 Dickeya sp. (__D. solani__) allowed to obtain knowledge of the presence of such strains in rotten potato tissue and on possibility of their used in the biocontrol of Dickeya spp. in potato. Selection of a candidate strain (Serratia plymuthica A30) that was effective in protecting potato plant tissue from blackleg was performed on the basis of in vitro and greenhouse tests. In vitro tests proved that S. plymuthica A30 possess different mechanisms by which it can control Dickeya spp. (i. e. production of antibiotics and biosurfactants). The interaction of D. solani and S. plymuthica A30 under greenhouse conditions was investigated in detail using artificially co-inoculated tubers and by application of A30 to the soil. It was showed that both micro-organisms interact in situ and that S. plymuthica A30 is able to decrease or eradicate Dickeya sp. inoculum from infected potato plants by competition un the same niches. In conclusion, a new biovar 3 Dickeya spp. strain was present in high densities in stolon end of naturally infected progeny tubers. The bacterium efficiently colonized various plant tissues after artificial inoculation of soil or haulms and was able to systemically colonize entire potato plant in relatively short time. For biocontrol of biovar 3 Dickeya spp. in potato, a bacterial strain characterized as S. plymuthica A30 tested in in vitro and under greenhouse conditions proved to have great potential for controlling blackleg in conditions favorable for disease development and when Dickeya sp. was applied in the high inoculum. The A30 strain effectively protected potato plants against blackleg in greenhouse experiments, both in vacuum infiltrated tuber and when applied as superficial treatment during planting. Treatment with A30 reduced the disease incidence to 0%. S. plymuthica A30 was able to colonize the plants internally, and combat internal infections with D. solani. Show less
