Ostryopsis Davidiana Facilitate Formation Of Mycorrhizae On Pinus Tabulaeformis And Functional Analysis Of Phosphate Transporter Gene From Ectomycorrhizal Fungi

We found that when outplanted in association with Ostryopsis davidiana plants on reforestation sites, Pinus tabulaeformis seedlings become mycorrhizal and survival and growth are enhanced; without O. davidiana, pines often remain without mycorrhizae and performance is poorer. To better understand this relationship, we initiated an experiment using rhizoboxes that restricted root and tested the hypoth esisthat O. davidiana seedlings facilitated ectomycorrhizae formation on P.tabulaeformis seedlings as mycorrhizal helper plant. Five species of ectomycorrhizal fungi were selected for the mycorrhizal inoculation. The mycorrhizal colonization,biomass, total N and P contents, the pH of soil and the phosphatase of two plants species were determined in the process of experiment. And two excellent ectomycorrhizal fungi of P. tabulaeformis were screened at the same time.The P deficiency in North China negatively affects the growth, development, and spatial distribution of P. tabulaeformis trees, resulting in significant losses. In addition,P. tabulaeformis is a typical mycorrhizal plant and shows high dependence on the ectomycorrhizae. The previous study indicated that inoculated with ectomycorrhizal fungi significant improve the growth status and P absorption of plants. So we want to explain this promotion from the P uptake. The P acquisition of mycorrhizal plants is highly depended on the mycorrhizal fungal phosphate transporters, but a reference gene must be selected before we analyzed the phosphate transporters of two excellent ectomycorrhizal fungi of P. tabulaeformis. Then we identified these two species fungi by the ITS sequences. Based on the above basis, two high-affinity PT genes were cloned, identified, and functionally characterized from two fungi.The research results indicated that:(1) O. davidiana seedlings colonized with ECM fungi grew better than non-mycorrhizal seedlings. Both colonized and non-colonized O. davidiana plants facilitated subsequent mycorrhizae formation on P. tabulaeformis seedlings,manifested by production of dichotomous root tips on the pine root systems and improved seedling growth. After inoculated with five species of ectomycorrhizal fungi, the soil pH of O. davidiana and P. tabulaeformis were not occur significantchanges, but the content of soil acid phosphatase and alkaline phosphatase were increased significantly. In addition, total N and P content of two plants seedlings improved at the same tiome. Compared to the treatment which absence of O.davidiana, the presence of O. davidiana improved mycorrhizal P. tabulaeformis all index more effectively. Combined with all index, Rhizopogon luteolus and Leucocortinarius bulbiger were the excellent ectomycorrhizal fungi in the 5 species of ectomycorrhizal fungi at the present study.(2) The γ-actin genes of Rhizopogon luteolus(Rl-act) and Leucocortinarius bulbiger(Lb-act) were isolated by using homology based method and RACE technique. These two genes have 3 highly conserved regions of γ-actin gene of fungi.In different culture conditions of carbon and phosphorus level, the quantity expression of these two genes were almost same,which validated the reliability of Rl-act and Lb-act as reference gene in quantitative analysis of mRNA expression.(3) The phylogeny relationship between the ITS sequence of R. luteolus and the ITS sequences of Tricholoma scalpturatum, T. cingulatum and T. myomyces was closely, which was different from the traditional morphological classification; while the phylogeny relationship between the ITS sequence of L. bulbiger and the ITS sequences of 5 species fungi, which belongs to Hebeloma genus was closely, this results coincided with the traditional morphological classification because these 6species fungi belonged to Cortinariaceae.(4) In this study, two phosphate transporter 1(Pht1) highly homologous genes which named RlPT and LbPT, were isolated from two ectomycorrhizal fungi of P.tabulaeformis, Rhizopogon luteolus and Leucocortinarius bulbiger, respectively. The results of protein sequences phylogenetic analysis showed that RlPT have the closest relationship with the phosphate transporter of Boletus edulis(BePT), and LbPT closed to the HcPT1.The optimal growth pH values of the 2 kinds of MB192 transformed yeast were 6 in the 5 determined pH values. The subcellular localization of RlPT and LbPT indicated that these two genes were expressed in the plasma membrane. The complementation assay in yeast showed that both RlPT and LbPT partially compensated for the absence of phosphate transporter activity in yeast strain MB192.The estimated Km of RlPT was 57.90 μmol/L Pi, LbPT was 35.87 μmol/L Pi.Real-time quantitative PCR analysis revealed that RlPT and LbPT were significantly up-regulated at lower P availability, which may enhance P uptake and transport under Pi starvation. Our results suggest that RlPT and LbPT play a key role in Pi acquisitionof the P. tabulaeformis by the ectomycorrhizal fungi.