Background: Real-time RT-PCR has become a powerful technique to monitor low-abundance mRNA expression and is a useful tool when examining bacterial gene expression inside infected host tissues. However, correct evaluation of data requires accurate and reliable normalisation against internal standards. Thus, the identification of reference genes whose expression does not change during the course of the experiment is of paramount importance. Here, we present a study where manipulation of cultural growth conditions and in planta experiments have been used to validate the expression stability of reference gene candidates for the plant pathogen Pectobacterium atrosepticum, belonging to the family Enterobacteriaceae. Results: Of twelve reference gene candidates tested, four proved to be stably expressed both in six different cultural growth conditions and in planta. Two of these genes (recA and ffh), encoding recombinase A and signal recognition particle protein, respectively, proved to be the most stable set of reference genes under the experimental conditions used. In addition, genes proC and gyrA, encoding pyrroline-5-carboxylate reductase and DNA gyrase, respectively, also displayed relatively stable mRNA expression levels. Conclusion: Based on these results, we suggest recA and ffh as suitable candidates for accurate normalisation of real-time RT-PCR data for experiments investigating the plant pathogen P. atrosepticum and potentially other related pathogens.

Lactobacillus plantarum is a normal, potentially probiotic, inhabitant of the human gastrointestinal (GI) tract. The bacterium has great potential as food-grade cell factory and for in situ delivery of biomolecules. Since protein secretion is important both for probiotic activity and in biotechnological applications, we have carried out a genome-wide experimental study of signal peptide (SP) functionality.

Pathogenic soft rot Enterobacteriaceae (SRE) belonging to the genera Pectobacterium and Dickeya cause diseases in potato and numerous other crops. Seed potatoes are the most important source of infection, but how pathogen-free tubers initially become infected remains an enigma. Since the 1920s, insects have been hypothesized to contribute to SRE transmission. To validate this hypothesis and to map the insect species potentially involved in SRE dispersal, we have analyzed the occurrence of SRE in insects recovered from potato fields over a period of 2 years. Twenty-eight yellow sticky traps were set up in 10 potato fields throughout Norway to attract and trap insects. Total DNA recovered from over 2,000 randomly chosen trapped insects was tested for SRE, using a specific quantitative PCR (qPCR) TaqMan assay, and insects that tested positive were identified by DNA barcoding. Although the occurrence of SRE-carrying insects varied, they were found in all the tested fields. While Delia species were dominant among the insects that carried the largest amount of SRE, more than 80 other SRE-carrying insect species were identified, and they had different levels of abundance. Additionally, the occurrence of SRE in three laboratory-reared insect species was analyzed, and this suggested that SRE are natural members of some insect microbiomes, with herbivorous Delia floralis carrying more SRE than the cabbage moth ( Plutella xylostella ) and carnivorous green lacewing larvae ( Chrysoperla carnea ). In summary, the high proportion, variety, and ubiquity of insects that carried SRE show the need to address this source of the pathogens to reduce the initial infection of seed material. IMPORTANCE Soft rot Enterobacteriaceae are among the most important pathogens of a wide range of vegetables and fruits. The bacteria cause severe rots in the field and in storage, leading to considerable harvest losses. In potato, efforts to understand how soft rot bacteria infect and spread between healthy plants have been made for over a century. Early on, fly larvae were implicated in the transmission of these bacteria. This work aimed at investigating the occurrence of soft rot bacteria in insects present in potato fields and at identifying the species of these insects to better understand the potential of this suspected source of transmission. In all tested potato fields, a large proportion of insects were found to carry soft rot bacteria. This suggests a need to give more weight to the role of insects in soft rot ecology and epidemiology to design more effective pest management strategies that integrate this factor.