Colonization of potato plants by soilborne, green fluorescent protein (GFP)-tagged Dickeya sp. IPO2254 was investigated by selective plating, epifluorescence stereo microscopy (ESM), and confocal laser scanning microscopy (CLSM). Replicated experiments were carried out in a greenhouse using plants with an intact root system and plants from which ca. 30% of the lateral roots was removed. One day after soil inoculation, adherence of the pathogen on the roots and the internal colonization of the plants were detected using ESM and CLSM of plant parts embedded in an agar medium. Fifteen days post-soil inoculation, Dickeya sp. was found on average inside 42% of the roots, 13% of the stems, and 13% of the stolons in plants with undamaged roots. At the same time-point, in plants with damaged roots, Dickeya sp. was found inside 50% of the roots, 25% of the stems, and 25% of the stolons. Thirty days postinoculation, some plants showed true blackleg symptoms. In roots, Dickeya sp. was detected in parenchyma cells of the cortex, both inter- and intracellularly. In stems, bacteria were found in xylem vessels and in protoxylem cells. Microscopical observations were confirmed by dilution spread-plating the plant extracts onto agar medium directly after harvest. The implications of infection from soilborne inoculum are discussed.

Summary1. Blackleg and soft rot disease of potatoes Solanum tuberosum L., mainly caused by the bacterial pathogen Erwinia carotovora ssp. atrospetica (Eca), lead to enormous yield losses world-wide. Genetically modified (GM) potatoes producing anti-bacterial agents, such as cecropin/attacin and T4 lysozyme, may offer effective future pathogen control strategies. Because of concerns about undesirable ecological side-effects of GM crops, it is important to analyse the potential environmental impact of GM crops carefully. The objective of this study was to investigate the effect of GM potatoes with anti-bacterial activity on the diversity and functional abilities of bacteria colonizing the intercellular spaces and vascular tissues (endosphere) of potato plants. 2. A greenhouse experiment was performed to analyse the effect of GM potatoes expressing either attacin/cecropin or T4 lysozyme on endophytic bacterial communities. Endophytic bacteria colonizing the GM potato lines as well as their nearly isogenic wild types were analysed at two vegetation stages. In order to compare GM-related variations with impacts caused by changing environmental conditions, potatoes were cultivated in two different soil types, and challenged with the pathogen Eca. Endophytic diversity was assessed by 16S rRNA-based terminal-restriction fragment length polymorphism (T-RFLP) analysis. Cultivated community members were identified by 16S rRNA gene analysis and screened for a range of plant growth-promoting and plant pathogenantagonistic abilities. 3. Both genetic transformation events induced a differentiation in the community structures of associated bacterial populations and in the related functional abilities of cultivated bacterial endophytes. In comparison with the other factors analysed, the impact of both genetic modification types was minor or comparable with the variations caused by plant genotype, vegetation stage, pathogen exposure and soil type. 4. Synthesis and applications. This study has shown that the expression of anti-bacterial proteins may affect bacterial endophytes; however, the impacts were no greater than those of other factors analysed. Future risk assessment studies of GM crops should consider different environmental factors. This study contributes to the ongoing risk assessment of GM crops and provides valuable baseline information for prospective GM crop assays.

In western Europe, Pectobacterium carotovorum subsp. brasiliense is emerging as a causal agent of blackleg disease. In field experiments in the Netherlands, the virulence of this pathogen was compared with strains of other Dickeya and Pectobacterium species. In 2013 and 2014, seed potato tubers were vacuum infiltrated with high densities of bacteria (10 6 CFU mL À1 ) and planted in clay soil. Inoculation with P. carotovorum subsp. brasiliense and P. atrosepticum resulted in high disease incidences (75-95%), inoculation with D. solani and P. wasabiae led to incidences between 5% and 25%, but no significant disease development was observed in treatments with P. carotovorum subsp. carotovorum, D. dianthicola or the water control. Co-inoculations of seed potatoes with P. carotovorum subsp. brasiliense and D. solani gave a similar disease incidence to inoculation with only P. carotovorum subsp. brasiliense. However, coinoculation of P. carotovorum subsp. brasiliense with P. wasabiae resulted in a decrease in disease incidence compared to inoculation with only P. carotovorum subsp. brasiliense. In 2015, seed potatoes were inoculated with increasing densities of P. carotovorum subsp. brasiliense, D. solani or P. atrosepticum (10 3 -10 6 CFU mL À1 ). After vacuum infiltration, even a low inoculum density resulted in high disease incidence. However, immersion without vacuum caused disease only at high bacterial densities. Specific TaqMan assays were evaluated and developed for detection of P. carotovorum subsp. brasiliense, P. wasabiae and P. atrosepticum and confirmed the presence of these pathogens in progeny tubers of plants derived from vacuum-infiltrated seed tubers.

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Gliocladium roseum was found to be the most common and probably the most effective mycoparasite in potato fields in the northern parts of the Netherlands. It is able to parasitize and kill living hyphae at temperatures of 12~ and higher. Sclerotia of R. solani are often infected and killed by this fungus under suitable conditions, i.e. at temperatures of 16 ~ and more. Killing of sclerotia by other antagonistic organisms was also observed. It is also shown by not parasitic fungi and is caused by toxins produced by the antagonist.The development of the G. roseum population was studied during the growth of a potato crop in two soils. In both soils its initial level was very low. In both a slightly acid sandy soil and a neutral sandy loam, suppression of R. solani can occur; G. roseum accumulated in the former mainly under continuous potato crops, Colletotrichum coccodes was the main antagonist in the latter.

Inoculation of seed potatoes with the mycoparasite Verticillium biguttatum, isolate M73 (combined with Gliocladium roseum in 1981, either alone or mixed with isolate M180 plus antibiotics-producing isolates of Azotobacter chroococcum in 1982) repeatedly proved successful in reducing Rhizoctonia solani on stolons and stems. In field experiments, this ultimately led to a reduced formation of sclerotia on new tubers, particularly in neutral sandy loam and clay loam soils. In 1981 inoculation with antagonists led, when compared with no inoculation, to average reductions of 22 and 42% for the harvest from clean, and 15 and 26% for the harvest from infected seed tubers grown on slightly acid sandy soils and on neutral loam soils, respectively. The harvest from clean, inoculated seed tubers had the lowest sclerotium index. In 1982 inoculation of seed tubers planted in slightly acid sandy soils gave reductions of the sclerotium index of up to 22%. In the neutral marine loam soils considerable reductions were often achieved, viz., in slightly infected loams 51-68% and in rather heavily infected ones 4-43%. Chemical disinfection of seed tubers proved effective only in loam soils that were slightly infested with R. solani. In both years inoculation of seed tubers with antagonists led to significantly lower sclerotium indices of the harvest (p = 0.1% in 1981; p = 50/0 in 1982).V. biguttatum was present more frequently and in greater densities on stems and stolons of plants from inoculated than from non-inoculated seed tubers. The latter were colonized by wild V. biguttatum strains from the soil, apparently less effective antagonists.Early in the season, the soil temperature was too low for growth of V. biguttatum. Nevertheless, inoculation of tubers that were planted early resulted in a considerable control of R. solani.