Abstract
Conifer trees are of great ecological importance and represent a significant source of primary production in the Northern Hemisphere. Among conifers, the genus Abies, which comprises about 40 species distributed in boreal and subalpine forest zones, have a great commercial value, with many Abies spp. being grown for the production of wood, pulp and paper and for use as Christmas trees. Abies nordmanniana (Stev.) Spach is a major Christmas tree species grown throughout Europe, with a yearly production of 50-60 million trees. However, the natural slow growth habit of A. nordmanniana represents a challenge to Christmas tree growers as the trees only reach their harvesting stage after several years. Furthermore, after initial nursery growth, A. nordmanniana seedlings are transplanted to the field where tree growth is often stunned for a few years after transplantation before optimal growth is regained, causing an important economic loss. To overcome the slow growth, commercial chemical plant growth regulators have been applied, but many growth regulators are currently being out-phased due to concerns over their impact on human health and the environment. As an alternative, microbiological solutions have attracted considerable attention, with the rhizosphere community representing a valuable resource for plant growth promoting microorganisms. Rhizosphere microbial communities generally impact the growth of the host plant, however, the dynamics of these communities during nusery plant growth, and during transplanting of A. nordmanniana has not been addressed. Nevertheless, it is assumed that the maintenance of beneficial rhizosphere microbial communities during early growth stages, and during transplanting of seedlings from the nursery to the field, is a prerequisite for the development of microbial inoculants for more efficient and sustainable production systems for A. nordmanniana. Overall this thesis focused on the belowground microbial communities associated with roots of A. nordmanniana, and how the composition of microbial communities is affected by plant phenotypic traits and development, as well as by production systems, climate and soil type. This thesis thus contributes increasing the knowledge and understanding of the effects of beneficial belowground microbial interactions for approaches in plant inoculation. Furthermore, this thesis presents bacterial strains that are able to promote A. nordmanniana seed germination and initial growth under laboratory, as well as in greenhouse conditions. The experimental approach for this thesis consisted of two main work packages; the first work package relied on amplicon sequencing of the 16S and 18S ribosomal rRNA gene region, to determine the community compositions of the bacterial and fungal taxa in rhizosphere or corresponding bulk soil samples. Additionally, a qPCR approach targeting selected genes involved in biogeochemical cycling was used to determine the potential functionality of rhizosphere bacterial communities. The second work package included traditional microbiological cultivation techniques, which were used to establish culture strain collections, and testing the effect of selected bacteria on seedling germination and seedling growth, under laboratory and greenhouse conditions. Further, the effect of microbial communities as well as individual bacterial strains, on plant physiology was determined through the activity of a panel of antioxidative defense enzymes. The outcomes of this thesis are separated into four manuscripts; Manuscript I report a first characterization of the A. nordmanniana rhizosphere microbial communities through analyses of field grown three-year-old plants, collected from two nurseries in Denmark and Germany, that displayed differences in growth phenotype (small versus tall plants). The findings indicate differences in the relative abundance and composition of microbial taxa between samplings sites, supporting the notion that geographic location and local climate are important factors in shaping the composition and dynamics of root-associated microbial communities in A. nordmanniana. Furthermore, correlations between the relative abundances of specific taxa of the microbiome and the plant antioxidative enzyme profiles were established. The main result of these correlations was that many more bacterial taxa correlated positively than negatively with one or more antioxidative enzyme activity. This finding may suggest that the ability of bacteria to increase plant antioxidative enzyme defenses is widespread. Manuscript II focused on the dynamics of bacterial rhizosphere communities during plant growth in nurseries, and during transplanting of A. nordmanniana from greenhouse to the field. Overall, a rhizosphere bacterial core microbiome of A. nordmanniana, comprising 30% of the taxa at order level, was maintained across plant age, nursery production systems and even during the transplantation of plants from greenhouse to field. The core microbiome was dominated by bacterial orders harboring several nitrogen (N) fixing and plant growth promoting taxa. However, both plant age and production system caused significant changes in rhizosphere bacterial communities. Further, the N-cycling potential of rhizosphere bacterial communities was dynamic, showing an increase in the relative abundance of N-genes involved in nitrogen fixation and denitrification by plant age. Interestingly, different community structures seem to lead to increased potential for N-fixation and denitrification in field versus greenhouse nurseries. Manuscript III addressed the plant growth promoting potential of bacterial isolates from the rhizosphere of A. nordmanniana. For this part of the thesis work, rhizosphere bacteria were isolated and characterized. From a strain collection dominated by the genera Bacillus, Pseudomonas and Paenibacillus, strains able to produce the phytohormone indole-3-acetic acid (IAA) in pure cultures were selected for a greenhouse study. Seed germination was promoted significantly by a selected strain Bacillus sp. s50 under both laboratory and greenhouse conditions. Further, seedling growth was enhanced by a selected strain Paenibacillus sp. s37 under greenhouse conditions. Additionally, we reported that the selected strains of Bacillus and Paenibacillus had different impacts on the seedlings’ antioxidative enzyme profiles under optimal growth conditions. Moreover, the persistence of the strains, added by seed coating, in the A. nordmanniana rhizosphere was further documented by a 16S rRNA
gene targeted amplicon sequencing approach, where relative abundances of the relevant genera were used as a proxy for bacterial persistence. It was found that Paenibacillus strain 37, persisted well in the rhizosphere, while Bacillus sp. strain 50 apparently declined after one month of growth in the greenhouse. Finally, in Manuscript IV, the complete genome sequence of the plant growth promoting bacterium Paenibacillus sp. strain 37, isolated from the rhizosphere of A. nordmanniana, is reported. Mining of the genome revealed the presence of several genes potentially involved in plant growth promotion and biocontrol. The complete genome of this Paenibacillus strain will assist in future studies of the mechanisms behind plant growth stimulation mediated by this bacterial strain. In conclusion, the research performed in this Ph.D. thesis provides new insights into the interactions between the tree crop A. nordmanniana and its root-associated microorganisms. Findings from this thesis can lead to future research in both fundamental and applied science and to new business applications, based on use of microbial inoculants for the promotion of increased sustainable production in the European Christmas tree industry.
gene targeted amplicon sequencing approach, where relative abundances of the relevant genera were used as a proxy for bacterial persistence. It was found that Paenibacillus strain 37, persisted well in the rhizosphere, while Bacillus sp. strain 50 apparently declined after one month of growth in the greenhouse. Finally, in Manuscript IV, the complete genome sequence of the plant growth promoting bacterium Paenibacillus sp. strain 37, isolated from the rhizosphere of A. nordmanniana, is reported. Mining of the genome revealed the presence of several genes potentially involved in plant growth promotion and biocontrol. The complete genome of this Paenibacillus strain will assist in future studies of the mechanisms behind plant growth stimulation mediated by this bacterial strain. In conclusion, the research performed in this Ph.D. thesis provides new insights into the interactions between the tree crop A. nordmanniana and its root-associated microorganisms. Findings from this thesis can lead to future research in both fundamental and applied science and to new business applications, based on use of microbial inoculants for the promotion of increased sustainable production in the European Christmas tree industry.
| Original language | English |
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| Publisher | Department of Plant and Environmental Sciences, Faculty of Science, University of Copenhagen |
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| Publication status | Published - 2019 |