Impact Of An Invasive Plant, Ageratina Adenophora Sprengel (Compositae), On The Communities Of Free-living Nitrogen Fixing Microorganism And Arbuscular Mycorrhizal Fungi

Belowground part of an ecosystem links closely to its upper ground. In the processes of soil ecological cycling, soil microorganisms play very important roles in direct decomposition of plant residue, substance circulation and energy flow. The structure of a soil microbe community can be changed when the plant community is altered through invasion by exotic plants. In particular, a far-reaching influence will be exerted on soil ecological processes when the structure of some functional soil microorganisms which contribute substantially to plant nutrition is changed. Arbuscular mycorrhizal fungi (AMF) are of interest to researchers because of their wide distribution and diversity in function. These fungi have been recently reported as a feedback factor of plant invaders. In a natural ecosystem, biological nitrogen fixation and nitrogen deposition are major pathways for N input into environments. For those plants which are unable to form root nodules with symbiotic azotobacter in arid soil, A. adenophora for example, free-living nitrogen-fixing microorganisms (FNFM) may be an important problem solver.Ageratina adenophora, an invasive plant known to cause great economical losses and damage to ecosystems in southwest China, has received extensive attention. Uncovering the mechanisms of successful invasion by this noxious weed is crucial not only because it can provide a theoretical basis for control, but also because it will hopefully enrich the theory of plant community succession. In this study, effects of the invasive plant A. adenophora on community structure of FNFM in soil of the invaded region were determined by a combination of traditional cultivation method and the optimized method of T-RFLP. Meanwhile, effects of A. adenophora invasion on community composition and structure of AMF from native soil were also examined through a cloning library. The general aim of this study was to reveal feedbacks of these two functional microorganisms to A. adenophora invasion. The main results were as follows:1. Optimization of T-RFLPT-RFLP techanique is widely used to analyze a certain functional microorganism in different environments rencently. In order to determine an optical method to minimize the systemitic error in our study, comparison of T-RFLP reproducibility of the process was used to illustrate the impact of the systematic errors arising from each process on the experimental results.Results suggested that the accuracy of T-RFLP analysis was nearly unaffected by capillary electrophoresis (CE), which confirmed that it was reliable to conduct T-RFLP analysis using ABI3730. We found that during the sample handling process, errors were introduced by PCR even for samples in the same conditions. The PCR results varied among different samples. Results of T-RFLP were also affected greatly by restriction enzymes. The similarity among genetic linkage maps was low when the same samples were treated with different restriction enzymes. It was found that T-RFLP retained its unparalleled advantage on condition that the same enzyme was used throughout the whole study. In terms of evaluation parameters, Hellinger distance analysis was superior to the Sorenson coefficient due to its better detection of differences among samples and repeatability among replicates.In conclusion, intermixing of several PCR outcomes, selecting suitable and consistent restriction enzymes and using Hellinger distance analysis were found to be the optimal combination for successful application of T-RFLP in this study.2. Effects of A. adenophora invasion on the content of C and N in invaded soilContents of total N, NH⁴⁺-N, NO₃^--N and AOC in rhizosphere soil in the A. adenophora dominant area (DA), native plant dominant area (DN) and area of co-occurrence of A. adenophora and native plants (T) were measured and compared. The results showed that contents of soil total N, NH⁴⁺-N, NO₃^--N and AOC in DA were significantly higher than in DN and T; the content of soil total N in DA was 2.6 and 2 times that of in DN and T, respectively, and soil NH⁴⁺-N 1.3 and 1.6 times, soil NO₃^--N 1.4 and 1.5 times, soil AOC 3.06 and 3.43 times, respectively. In terms of T and DN, contents of soil total N and AOC in T were remarkably higher than in DN while the content of soil NH⁴⁺-N demonstrated an opposite trend, and NO₃^--N was not obviously affected.In addition, soil nitrogen-fixing capacity in DA was much stronger than in T and DN, and the capacity value in DA was 1.2 and 1.4 times higher, respectively. Compared with DN, the capacity value in T was 1.2 times higher.In conclusion, after the invasion by A. adenophora, contents of soil total N, ammonium nitrogen (NH⁴⁺-N), nitric nitrogen (NO₃^--N) and available organic carbon (AOC) were increased significantly and activity of soil nitrogenase which links to N cycling was noticeably improved. The increases in soil fertility and enzyme activity will contribute a positive feedback to the growth of A. adenophora and enable its further expansion.3. Effects of A. adenophora invasion on the structure of FNFM in invaded soilBased on the above conclusion, we used cultural and molecular biological methods to analysize the FNFM community structure in the soil before and after A. adenophora invasion.Results from traditional cultivation method suggested that the number of soil FNFM in DA was significantly higher than that of T and DN. But, according to initial colonial morphology classification results, there was only one dominant strain identified in DN, while there were two and three dominant strains in T and DA, respectively.Result from T-RFLP was similar to the former result. The structure of soil FNFM was not obviously changed after A. adenophora invasion because the differences in relative frequency of dominant strains were not significant, although there were slight differences in structure of FNFM among the three soil samples when types of soil FNFM were marked by terminal restricted fragments (T-RFs). Similarity comparison results of T-RFLP fingerprints among three soil samples indicated that the Hellinger distance between DA and T (68.5) was much larger than that between DA and DN (51.5), DN and T (51.1) respectively. The differences among the three soil samples did not show a tendency of linear gradient arrangement. In conclusion, it appears that a great number of FNFM appeared in rhizosphere soil in response to the presence of A. adenophora, and were available to provide sufficient N for continuous growth and successful invasion.4. Effects of A. adenophora invasion on soil AMFBesides samples from the three soil zones mentioned above, bare soil was added to make a total of 4 types that were sampled. Five areas of each type were sampled and pooled for analysis with the molecular methods. From the resulting 4 samples, we obtained 314 AMF sequences all together, and 28 OTUs were then identified. An OTU was defined based on more than 97% homology of the sequences. Thereafter, a dominant OTU was also determined when its content was higher than 50%. In all, 10 OTUs were found in DA soil, among which 3 were evaluated dominant. Alignment results from NCBI suggested that they were Glomus constrictum and two kinds of uncultured species in Glomus. In T, one out of 10 OTUs was also determined as dominant and belonged to uncultured species in Glomus. In addition, 12 and 5 OTUs in DN and bare soil were found, respectively, among which they both had 2 dominant OTUs. The 2 dominant OTUs in DN belonged to uncultured species in Glomus while in the bare soil sample; they were Glomus macrocarpum and Glomus eburneum.The original composition and structure of AMF community were altered after A. adenophora invasion, mainly by promoting the reproduction of some species, resulting in numerical dominance, while inhibiting the growth of some other species. However, the distribution of some AMF species in different types of soils was almost same, and this resulted in insensitivity of those AMF species to the structure of plant community.In conclusion, the structure of soil AMF community was changed at the species level after A. adenophora invasion. Three types of Glomus species of AMF were found to be enhanced in soil, including Glomus constrictum.5. ConclusionThe content of soil AOC and N input (total N, NH⁴⁺-N and NO₃^--N ) were increased significantly in the rhizosphere after A. adenophora invasion. Soil nitrogenase activity and the number of FNFM were also greatly increased at the same time. Thereby, A. adenophora had created a favorable environment for itself to strengthen its competitiveness and realize further expansion. With respect to AMF, their community structures were obviously changed and several dominant AMF species were enriched in the rhizosphere by A. adenophora. The detailed mechanisms are not known, and need further study. The results illustrate new invasion mechanisms of alien plants mediated by soil microorganisms and provide practical guidance for soil and plant management strategies to prevent invasion by A. adenophora.