| According to the different growth stages of cottons, we collected soil samples (0-20 cm) at seedling, budding, boll forming and boll opening stage during the year 2009 and 2010, and studied the population of culturable microorganisms, the diversity of bacterial community, the functional diversity of microbial metabolism, the community structure of invertebrates as well as soil nematode in the three different cotton fields at a cotton farm in China Cotton Institution of Agricultural Scientific Academy (Anyang, Henan Province) where non-transgenic field had been planted all along and other two cotton fields where transgenic insect-resistant cotton had been planted since 1997,2002 respectively. Two kinds of transgenic insect-resistant cottons and their parental cottons as the experimental materials, the composition of root exudates from transgenic cottons and conventional cottons and the effects on the growth of Fusarium oxysporum were studied, the changes of physiological and biochemical characteristics of transgenic cottons and conventional cottons after infection by the pathogen were analyzed and the SSH cDNA libraries were also constructed to study the type and quantity of disease resistance related genes of transgenic cotton induced by Fusarium oxysporum. The results show that:(1) Effects of transgenic insect-resistant cottons on the population and diversity of soil microbial communityThere were no significant differences in the number of bacterial, actinomycetes, fungal, azotobacter, denitrobacteria, nitrosobacteria and the diversity indices of soil microbial in two transgenic insect-resistant cotton fields compared with that of the non-transgenic cotton field, but significantly seasonal variation in the number of each microbial and diversity indices of microbial in all three cotton fields. The populations and diversity indices of microbial have the similar tendency of changes during the whole growing period in three cotton fields, while change rules of different microbial population differs in the growth period.The diversity of soil bacterial in the three cotton fields was analyzed by PCR-DGGE method. The results showed that the different planting years of transgenic insect-resistant cottons have no significant effects on the composition and diversity of soil bacterial community compared to the cultivation of conventional cotton, but different sampling time has significant effects on the composition and diversity of soil bacterial community.The metabolic characteristics of soil microbial communities in the cotton fields were determined using Biolog-ECO plate. The results showed that long-term cultivation of transgenic insect-resistant cottons has no significant effects on the functional diversity of microbial activity, but different sampling time has some effects. Principal component analysis showed that the sugars and esters were the main carbon sources of soil microbial in these fields, but the use of sugars and esters by soil microbial were different at different sampling time, which can be used as basis to distinguish the effects of different cotton growth stages.(2) Effects of transgenic insect-resistant cottons on the population and diversity of soil fauna communitySoil meso-and microarthropodas were collected from the soil samples with the modified Tullgren method to monitor environmental impact of long-term cultivation of transgenic insect-resistant cotton in field conditions. The data from 8 sampling times during the 2 years showed that it was similar for community composition of soil meso-and microarthropodas in two transgenic cotton fields and one conventional cotton field, and there were no significant variations. The generalized linear mixed model (GLMM) analysis of the 2-year data on soil arthropods showed that no significant difference was found between cotton fields in individual number and the community diversity indices of the soil meso-and microarthropodas of the main soil arthropod groups, but significant seasonal variation was observed in the individual numbers and the diversity indices of the main soil meso-and microarthropodas. The principal component analysis suggested that Collembola, Acarina and Opisthopora were relatively higher in value and could be cited as important indicator organisms to monitor environmental impacts of transgenic plants in the future in this region.The soil nematodes were collected from soil using sugar flotation and centrifugation method. The ecological index such as species richness, abundance, diversity index and trophic group index were analyzed. Results showed that there were no significant difference in the individual density and diversity indices of soil nematode in two transgenic cotton fields compared with that of the non-transgenic cottons, but had significant seasonal variation in the individual density of main trophie groups and diversity indices of nematode in all three cotton fields. There were no significant changes for community composition of soil nematodes in two transgenic cotton fields and one conventional cotton field. In addition, no significant difference was found between cotton fields in the indices of the functional structure of the nematode communities (Maturity Index, Plant Parasite Index and EMI index), which indicated no significant effect of continuously planting transgenic insect-resistant cottons on free-living marine nematodes and plant parasite nematodes.(3) The mechanism for attenuation of disease resistance in transgenic insect-resistant cotton to Fusarium wiltThe resistance of the two insect-resistant cotton lines to F. oxysporum was inferior to the parental lines, and that their root exudates promoted fungal spore germination and mycelial growth. Alkanes, alcohols, acids, esters, phenols, aldehydes, ketones, olefins and heterocyclic were detected in root exudates of cottons. Compared with the parent conventional cottons, the kinds and relative contents of root exudates decreased, some hydrocarbons appeared and some specific substances reduced in transgenic insect-resistant cottons.The structure of leaf pore in cotton seedling stage was studied by scanning electron microscopy. Compared with the parent conventional cottons, the stomatal density significantly reduced, and the pore size significantly increased in transgenic insect-resistant cottons. There were significant changes of physiological and biochemical indicators in transgenic insect-resistant cottons inoculated with cotton F.oxysporum, in which SOD activity, MDA and soluble protein content were significantly higher, and POD enzymes, PAL enzymes and soluble sugar content decreased significantly compared with the parents.Many genes of transgenic insect-resistant cottons and parental cottons have significantly changed when inoculated with cotton F.oxysporum and mostly were up-regulated genes. There were significant differences in the types and numbers of response genes of transgenic insect-resistant cottons inoculated with cotton F.oxysporum compared to conventional cottons. Some types of up-regulated genes in parental cottons infected by pathogen mainly referred to the function of biological processes and molecular, and they were inhibited in the expression of transgenic insect-resistant cottons after infected by pathogen. The expressions of catalase, alcohol dehydrogenase,β-galactosidase and chlorophyll AB binding protein gene were significantly increased in transgenic insect-resistant cottons and conventional cottons infected by pathogen. The expression of the above genes in conventional cotton infected by pathogens was significantly higher than that of transgenic insect-resistant cottons.(4) Technical guide for monitoring effect of transgenic insect-resistant plants on soil ecosystemThis technical guide covers the distribution of sampling, monitoring indicators, monitoring techniques, mathematical statistics and assessment of results in monitoring effect of transgenic insect-resistant plants on soil ecosystems. It can be used for monitoring the effect of transgenic insect-resistant plants on soil ecosystems. |
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