Persistence, population dynamics and competitiveness for nodulation of marker gene-tagged Rhizobium galegae strains in field lysimeters in the boreal climatic zone

Abstract

A non-indigenous wild-type strain Rhizobium galegae HAMBI 540, which specifically nodulates perennial goat's rue (Galega orientalis), and its marker gene-tagged derivatives R. galegae HAMBI 2363(luc), R. galegae HAMBI 2368(gusA21) and R. galegae HAMBI 2364(gusA30) were used to evaluate the persistence, population dynamics and competitiveness for nodulation of rhizobia under field conditions in Finland. The lysimeters were filled with clean or diesel oil-polluted (3000 Wg g 31 ) agricultural soil. During the first 2 years of the field release luc-and gusA21-tagged strains could be effectively detected by cultivation, reinforced with colony polymerase chain reaction. The population densities remained relatively stable from 10 4 to 10 5 cfu g 31 dry soil from spring until late autumn. Replicate limiting dilution polymerase chain reaction analysis gave comparable results with cultivation with strain HAMBI 2363 until 49 weeks after inoculation. GUS activity of strain HAMBI 2368 could be stably detected in nodules and soil. On the other hand, luc activity weakened clearly in cold conditions along with decreased metabolic activity of rhizobia. The competitive ability for nodulation of the gusA30-tagged strain decreased slowly with time compared to the wild-type strain. Moderate soil pollution did not have significant effects on target bacteria or plant growth. Limited vertical movement of target bacteria outside the rhizosphere was detected from percolated water.

Rhizobium Impacts on Seed Productivity, Quality, and Protection of Pisum sativum upon Disease Stress Caused by Didymella pinodes: Phenotypic, Proteomic, and Metabolomic Traits

“…The resulting milky suspension was streaked on CR-YMA and YEM Agar as selective Rhizobium media ( Kneen and LaRue, 1983 ; Agrawal and Choure, 2011 ; Neo et al, 2012 ; Zohra and Mourad, 2016 ) and incubated at 28 ± 1°C for 48–72 h ( Deshwal and Chaubey, 2014 ; Datta et al, 2015 ). To enumerate the total number of viable cells, standard plate count method and turbidimetric measurement by spectrophotometry were used ( Somasegaran and Hoben, 1985 ; Pitkäjärvi et al, 2003 ). Single colonies were picked and restreaked on CR-YMA and YEM Agar plates for preparation of pure cultures ( Datta et al, 2015 ).…”

Release of transgenic bacterial inoculants - rhizobia as a case study

“…They can improve soil texture and fertility (especially N availability) for other microoganisms that degrade recalcitrant organic com- Pitkäjärvi et al (2003) investigated the survival of R. galegae after it was applied as an inoculant to its host plant goat's rue (Galega orientalis) in uncontaminated and oilcontaminated soils in lysimeters in the field. The rhizobia were marked by insertion of luc or gusA to facilitate monitoring survival of the inoculant.…”

“…However, together these methods have allowed a comparison of the long-term fluctuations in the indigenous R. leguminosarum biovar viciae population and the survival of introduced inoculant strains (Hirsch, 1996). The use of PCR with marker gene-specific primers enables sensitive detection of inoculant rhizobia and has revealed no evidence for the existence of significant numbers of non-culturable rhizobial cells surviving in field soils (Cullen et al, 1998;Corich et al, 2000;Pitkäjärvi et al, 2003).…”

Long term evaluation of field-released genetically modified rhizobia

“…For example, a recA-minus derivative of S. meliloti showed a stability similar to that of its wild-type, as it declined from 10 6 to 10 4 cfu.g −1 of soil in two years time (Schwieger and Tebbe, 2000). Climatic conditions such as heavy rain after release negatively affect persistence (Hirsch, 1996), and so do cold winters, as shown in the boreal zone for GM R. galegae (Pitkajarvi et al, 2003). Besides survival, in terms of nodulation, released GM rhizobia, like all symbiotic inoculants, face the competition of indigenous members of the same species, as shown quantitatively for GM S. meliloti (Miethling and Tebbe, 2004).…”