Micro-Ecosystem Associated With The Rhizosphere Of Pinus Tabulaeformis On The Loess Plateau And Resistance Of Ectomycorrhizal Fungi To Damping-Off Disease

The plate dilution method and denaturing gradient gel electrophoresis (DGGE) were combined to study microflora, bacterial and fungal communities in the rhizosphere of Pinus tabulaeformis on the Loess Plateau. The relationship between edaphic indicators and soil microorganisms in the rhizosphere of P. tabulaeformis was analyzed. In addition, ectomycorrhizal (ECM) fungi were isolated from the fruiting bodies collected in the study area. After been isolated, identified and cultured, the interaction between ECM fungi and Fusarium solani was conducted by plate confrontation assays which was as the basis of preliminary screening and then effects of ECM fungal inoculation on palnt growth and resistant to damping-off of P. tabulaeformis seedings were studied. The main results as follows:1. Effects of different restoration patterns and tree age on soil properties in the rhizosphere of P. tabulaeformis on the Loess PlateauThe present study investigated that soil properties in the rhizosphere of P. tabulaeformis in the process of plantation and natural secondary forest restorations on the Loess Plateau. Results indicated that effects of two restoration patterns on soil pH, total P, organic matter, total N, alkali-hydrolyzale N, available P, the quantity of fungi, microbial biomass C, microbial biomass N and the activities of invertase, urease, alkaline phosphatase, catalase and polyphenol oxidase were significant, while they were not significant effect on quantities of bacteria and actinomyces; soil pH and polyphenol oxidase activity were higher in plantation of P. tabulaeformis than in natural secondary forest and other soil parameters were higher in natural secondary forest than in plantation. In the present study, soil properties in the rhizosphere of P. tabulaeformis were all significantly affected by tree age; soil pH and polyphenol oxidase activity were highest in3-year-old site of plantation while other soil parameters were lowest. Moreover, the interaction between restoration pattern and tree age had a significant effect on organic matter, total N, alkali-hydrolyzale N, total P, available P, microbial biomass C and the activities of urease, alkaline phosphatase, catalase and polyphenol oxidase, while their interaction had no significant influence on soil pH, invertase activity, microbial biomass N and the quantities of bacteria, fungi and actinomyces.2. The diversity and community structure of bacteria in the rhizosphere of P. tabulaeformis and their related affecting factors on the Loess PlateauThe values of bacterial Shannon-Weiner index and species richness were significantly positively related to organic matter, total N, available P and C/P ratio; Simpson index had significant positive correlation with organic matter, total N and C/P ratio; however, Shannon-Weiner index, species richness, Evenness index and Simpson index had no correlation with soil pH, alkali-hydrolyzale N, total P and C/N ratio. RDA analysis revealed that available P, total N, alkali-hydrolyzale N and organic matter had significant effects on bacterial community structure, and their eigenvalues were35.34%,33.47%,31.19%and30.66%, respectively. Phylogenetic analysis showed that bacteria in the rhizosphere of P. tabulaeformis belonged to Proteobacteria, Acidobacteria and Firmicute and they accounted for57.89%,26.32%and15.79%of the total bacterial species, respectively.3. The diversity and community structure of fungi in the rhizosphere of P. tabulaeformis and their related affecting factors on the Loess PlateauThe values of fungal Shannon-Weiner index, species richness, Evenness index and Simpson index had no correlation with organic matter, total N, available P, C/P ratio, soil pH, alkali-hydrolyzale N, total P and C/N ratio. RDA analysis revealed that available P and C/P ratio were affected RDA1and RDA2of the fungal community mostly, respectively; effects of available P, total N, organic matter, C/P ratio, alkali-hydrolyzale N, total P, pH and C/N ration fungal community structure weaken successively and their eigenvalues were26.56%,25.62%,23.12%,21.52%,21.06%,19.46%,19.29%and18.72%. Phylogenetic analysis showed that fungi in the rhizosphere of P. tabulaeformis belonged to Basidiomycota, Ascomycota and Zygomycota and they accounted for66.67%,27.78%and5.56%of the total fungal species, respectively.4. Screening for antagonistic ECM fungal strains against damping-off disease of P. tabulaeformisThere was one ECM fungus isolated from ECM fungal fruiting bodies and it colonized in the root system of P. tabulaeformis by mycorrhizal synthesis in pot culture. Based on morphological characteristics and molecular identification, the isolate was identified as Handkea utriformis. Meanwhile, we found that the best culture conditions for H.utriformis were25℃and pH6.0, and the best carbon and nitrogen source were sucrose and proline, respectively. The interactions between four different ECM fungi(Suillus lactifluus, S. bovinus,5. tomentosus and H. utriformis) and Fusarium solani was conducted by plate confrontation assays, and results showed that all four ECM fungi exhibited inhibitory effect on the growth of the pathogenic fungus of damping-off, and their inhibitory rates were26.3%,59.6%,38.4%and35.1%; the difference of diameters between co-culture of F. solani and S. bovinus as well as S. tomentosus were not significant and there had a inhibitory zone when co-culture of F. solani and S. tomentosus. Hyphal interactions between ECM fungi and F. solani in plate confrontation assays were study by scanning electron microscope, and the results indicated that the hyphae of F. solani were coiling by hyphae of S. lactifluus and S. tomentosus and the hyphae of H. utriformis exhibited coiling around and penetration of the hyphae into F. solani. Hence, according to inhibitory effect of ECM fungi on F. solani and their hyphal interactions, S. lactifluus, S. tomentosus and H. utriformis were selected.5. Biological control efficiency and growth-promoting of P. tabulaeformis seedings by ECM fungiInoculation of S. lactifluus, S. tomentosus and H. utriformis were all formed mycorrhizae with P. tabulaeformis and H. utriformis had the highest colonization rate by59%. Compared to non-inoculation control, inoculation of H. utriformis、S. tomentosus and S. lactifluus on P. tabulaeformis seedings improved survival rate by21%,16%and9%; plant height, basal diameter, shoot dry weight, root dry weight, total dry weight, root activity, total root length, root surface area, root volume and root tips were increased, of which shoot dry weight, root activity and root surface area were significantly increased by40.27%～118.66%,12.63%～72.91%, and64.64%～246.82%, respectively. The three ECM fungi were all exhibited control efficiencies to damping-off of P. tabulaeformis seedings; inoculation of H. utriformis had the best control effect by60%, followed by S. tomentosus (53%) and finally S. lactifluus (44%). Correlation analysis indicated that control efficiency was significant positive correlation with colonization rate, shoot dry weight, root dry weight and total dry weight, suggesting that the higher of colonization rate and accumulation of dry weight, the higher of ECM fungal inhibitory effect on pathogen.