Diversity Of AMF Communities In Mangrove Near By Pearl River Estuary And Their Responses To Environmental Factors

Arbuscular mycorrhizal fungi （AMF） are probably the most widespread symbiotic microbes in the nature ecosystems, which can form symbiosis with most of the terrestrial plants. They have shown great application perspectives in the areas of environment, agriculture and forestry. Therefore, there has been a great concern in the research of these fungi. During these years, there has been an increasing concern on the exploitaion of marine resources and the protection of the marine environment. The present study systematically investigated the AMF species compostion, community structure, diversity level and their responses to environmental factors in the mangrove swamps near by the estuary of the Pearl River, employing both the traditional methods in ecological and environmental research and the cutting-edge methods in molecular biology. The main contents and results from this research are as follows:1. The symbiosis between AMF and mangrove plant species was investigated in two mangrove swamps near by the Pearl River estuary in south China. AMF were mostly found in the form of hyphae and were commonly associated with all the mangrove species we investigated. The colonization intensities of vesicle and arbuscule were relatively low, though these structures were detected in most of the investigated mangrove species. Six AMF morphological species belonging to the genera Glomus or Acaulospora were identified after a trap culture procedure. The colonization intensity of AMF was significantly higher in the high and middle tide levels than that in the low tide level （p < 0.05） in both swamps. Multiple step-wise linear regression analyses showed that hydrological conditions and soil P levels in the rhizosphere were the main environmental factors affecting the colonization of mangrove species by AMF.2. A greenhouse experiment was conducted to evaluate the effects of AMF inoculation, which is trap-cultured from a manrove swamp, on the growth and nutrient uptake of a true mangrove plant species, Sonneratia apetala B. Ham. The inoculated AMF significantly improved growth, resulting in greater plant height, diameter at ground level and plant biomass （p < 0.05）. The AMF colonization also increased the absorption of N, P and K of the host plants （p < 0.05）. These findings suggest that AMF play important roles in mangrove ecosystems.3. The colonization of AMF to four semi-mangrove species and the AMF spore density and species composition in the rhisosphere soil were investigated in the landward part of the Zhuhai Mangrove forests. A relatively high AMF colonization rate （total colonization rate: 33.3%⁶3.7%） was detected in the investigated plants. Although vesicle （13.8%³8.2%） and arbuscule （14.7%²9.8%） were also commonly detected, hyphae had the highest colonization intensity （27.9%⁵4.5%）. A relatively low AMF spore density was detected in the rhisosphere （0.1 - 1.5 spores g-1 soil）. All the AMF spores were designed to seven phylotypes （five Glomus phylotypes and two Acaulospora phylotypes） according to their morphological features and the phylogenetic analysis based on an 800 bp rDNA sequences. Two to four spore phylotypes were detected from the rhizosphere soil of each semi-mangrove species. No obvious differences were found in the distribution pattern and phylotype diversity of AMF spores from different semi-mangrove species. The results of correlation analysis showed that the soil organic matter content could be the most important environmental factor affecting the symbiosis between AMF and semi-mangrove species.4. To evaluate the effects of host plant and tide level on the AMF colonization to mangrove plants in mangrove ecosystems, the colonization intensity of AMF within the roots of three semi-mangrove species （Heritiera littoralis Ait., Acrostichum aureum L. and Acanthus ilicifolius L.）, which are located in all of the high, middle and low tide levels in Zhuhai Mangrove forest, were investigated. The AMF structures were detected within the roots of all investigated species. AMF hyphae had the highest colonization intensity, but vesicle and arbuscular structures were also commonly detected. The results of Two-way ANOVA showed that the host plants had significant effects on the vesicle and arbuscular colonization intensity （P < 0.05）, but no significant effects on either total or hyphal colonization intensity. The tide level significantly affected all types of AM colonization （P < 0.01）: the AMF colonization intensities in the roots from high and middle tide zones are higher than that from the low tide level, and AMF colonization intensity in the roots of middle tide level is generally higher than that at the high tide level. The interactions between plant species and tide level also significantly affected all types of AM colonization （P < 0.05）.5. To further investigate the diversity and species composition of AMF in mangrove ecosystems, the communities of arbuscular mycorrhizal fungi （AMF） colonizing the roots of three semi-mangrove species were characterized along a tidal gradient in ZH Mangrove forest, using a“Nested PCR– Clone– Sequencing”procedure. A fragment, designated SSU-ITS-LSU, including part of the small subunit （SSU, c. 230 bp）, the entire internal transcribed spacer （ITS, c. 480 bp） and part of the large subunit （LSU, c. 830 bp） of rDNA from samples of AMF-colonized roots was amplified, cloned and sequenced using AMF-specific primers. A total of 761 AMF sequences were obtained.1) AMF diversity within the roots: The results of phylogenetic trees based on the neighbor-joining and MrBayesian analyses indicated that the 761 obtained AMF sequences were grouped into 37 operational taxonomic units （OTUs） based on SSU-ITS-LSU sequence similarities of 97-100%, and finally assigned to 23 AMF phylotypes. Two to seven AMF phylotypes were detected from each individual plant sample. The diversity levels of AMF detected from these three species were much higher than that reported from other wetland ecosystems and most terrestrial ecosystems, indicating that the AMF diversities in mangrove ecosystems could at least be comparable to those in most terrestrial ecosystems, and that the AMF diversity level in wetland ecosystem is not necessarily low.2) AMF species composition: Twenty-two （759 sequences） of the 23 phylotypes detected in the roots belong to the genus Glomus, only one phylotype （2 sequences） belongs to the genus Acaulospora. Eleven （367 sequences） of the 23 phylotypes detected in this study were novel. Four of the other 12 phylotypes were preliminarily identified to G. intraradices,“G. intraradices DAOM197198”, G. mosseae and G. sinuosum. The G. intraradices sequences detected in this study are the first true record of G. intraradices outside Florida. The other phylotypes were related to the AMF sequences that have not yet been identified to species level.3) Effects of host plants and flooding intensity: Plant species had no significant effects on the AMF diversity （based on the phylotype richness and Shannon’s index） （P > 0.05）, but significantly affected the AMF phylotype communities within the roots of three semi-mangrove species （P < 0.05）, indicating the presence of host preference between AMF and host species. The tide level （flooding） has a highly significant effect （P < 0.01） on AMF diversity and phylotype composition, and the effects are dependent on the degree of flooding: intensive flooding (7^-10 hr day^-1) was clearly associated with decreased AMF diversity in the roots of all three mangrove species; while moderate flooding (2-4 hr day^-1) showed a promotion effects on the diversity of AMF in any of the three plant species investigated. The hydrologic conditions should be the most important factors affecting the diversity and community structure of AMF in mangrove ecosystems.6. To investigate the heavy metal contamination level in Shenzhen mangrove swamp, one of the most vulnerable mangrove ecosystems in China, concentrations of six heavy metals （Cu, Ni, Zn, Cd, Cr and Pb） in sediments, and fine roots, thick roots, branches and leaves of six mangrove plant species collected from this site were measured. The results show that the Shenzhen mangrove sediments are moderately contaminated by heavy metals, Zn and Cu being the main contaminants. All investigated metals showed very similar distributions in the sediments, and had relatively high proportions of DTPA extractable heavy metals, implying that they had the same anthropogenic source（s）. High accumulations of the heavy metals were observed in the root tissues, especially the fine roots, and much lower concentrations in the other organs, indicating that the roots strongly immobilize them and （hence） that mangrove plants possess mechanisms that limit the upward transport of heavy metals and exclude them from sensitive tissues. There were significant differences in the accumulation ability of each investigated heavy meatl between different mangrove species （P < 0.05）. The accumulation abilities of the same species to different heavy metals were also significantly different （P < 0.01）. 7. To further evaluate the potential risk of heavy metals posed to mangrove ecosystems, the growth performance of propagules and six-month-old seedlings of Bruguiera gymnorhiza in the presence of contaminating Cd (c. 1 mg kg^-1, 3 mg kg^-1) and Cu (c. 100 mg kg^-1, 200 mg kg^-1) was examined. The results show that the heavy metal contamination set in this study showed no significant effects on the growth and nutrient uptake in both 0³ month seedling and 6⁹ month seedling （P > 0.05）, indicating that this plant is not sufficiently sensitive to heavy metals after its propagule stage for its regeneration and growth to be significantly affected by the heavy metal contamination in the Shenzhen mangrove ecosystem. However, the high Cd (c. 3 mg kg^-1) treatment decreased leaf chlorophyll a contents and chlorophyll a/b ratios in the three-month seedlings （p < 0.05）, but not in the nine-month seedlings. In addition, older mangrove seedlings accumulated much higher Cd level in plants in both the low Cd (c. 1 mg kg^-1) and high Cd (3 mg kg^-1) treatments. These results indicated that the older seedlings are more metal-tolerant than the younger seedlings, due to their more efficient exclusion mechanism. Therefore, it is necessary to assess the effects of metal contamination on young seedlings when evaluating the risks posed by heavy metals in an ecosystem.8. A greenhouse experiment was conducted to evaluate the effects of heavy metal contamination on the symbiosis between AMF and mangrove species, and to study the roles of AMF in the responses of mangrove species to heavy metal contaminants, taking the trap cultures of AMF from mangrove swamp as the fungal materials. Instead of the mangrove plants, maize （Zea mays L.）, a typical sensitive mycorrhizal plant, was used as the test plant. The results show that both the Cd (c. 1 mg kg^-1, 3 mg kg^-1) and Cu (c. 100 mg kg^-1, 200 mg kg^-1) treatments significantly inhibited the symbiosis between AMF and host species （p < 0.05）, and decreased the plant height and biomass of the tested plants （p < 0.05）. AMF colonization significantly increased the concentrations of Cu and Cd in the shoots and roots of the maize plants （p < 0.05）, and improved the heavy metal resistance of plants, partly by increasing the proline levels in host plant （p < 0.05）, resulting in greater plant height and biomass of the host plants （p < 0.05）. These results indicate that the mangrove AMF could decrease the potential risks of heavy metal contaminants through improving the heavy metal tolerance of host plants. In mangrove habitat, heavy metal contamination can inhibite the symbiosis between AMF and host species, posing a risk to mangrove ecosystems.9. A medium-sized mangrove constructed wetland and a small-scale mangrove constructed wetland were set up for the purpose of evaluating the effects of domestic sewage discharge on the symbiosis between AMF and mangrove plants. The colonization intensity in the roots and spore density from the rhisosphere soil of mangrove species locating within varied distances from the sewage inlet in the medium-sized wetlands were tested after approxiamately three years growth of the plants. The colonization intensity and spore density of AMF from the small-scale wetland were also examined after the treatments of sewage and running water （CK） for nine months. The results show that the sewage discharge had no significant effect on the spore density of AMF in both medium-sized and small-scale mangrove constructed wetlands （P > 0.05）. However, it obviously decreased the colonization intensity of the AMF （p < 0.01）, and the inhibition effect of sewage is largely depended on the concentration of the sewage. The vesicle and arbuscular structures were more sensitive than the haphal structure to the sewage dischage. Because the Aegiceras corniculatum had a higher radial oxygen loss ability than the Kandelia candel, combined with some other unknown reasons, AMF colonization intensity in the roots of Aegiceras corniculatum were always significantly higher that that of Kandelia candel （p < 0.05） under the same conditions, providing a solid evidence showing the effects of host plants on the symbiosis between AMF and plants. Given that AMF play important roles in the mangrove ecosystems, the results of the presented research show that sewage discharge to mangrove ecosystems could lead a harmful effect to these habitats through destroying or inhibiting the symbiosis between AMF and mangrove plants.