| Orchid plant is a typical mycorrhizal symbiosis plants, the mycorrhizal fimgi is nutrition source for orchid plant life cycle. However, rnycorrhizal symbiosis not only depends on mycorrhizal fungi but also includes endophytic bacteria. Endophytie bacteria can affect the formation of the mycorrhiza and stabifty of symbiotic relationship, they pky an important role in the Bfe cycle of orchids. In this study we use Phalaenopsis pulcherrima (LindL) JJ Sm., an endemic species in Southeast Asa. At first wM plants were collected from different habitats and isolated endophytic bacteria from fresh roots to do the morphological and molecular identification and analysis diversity of endophytic bacteria in pulcherrima in different habitats. Then we used pulcherrima mature seeds and sterile tissue culture as tieback object to screen the growth-promoting strains, and enrich sources of endophytic bacteria in Pulcherrima. Meanwhile, in order to explore the mechanism of growth-promoting endophytic bacteria in orchid plants, this study has also launched activity assay of plants which were promoted growth by endophytie bacteria promoting-growth strains. Finally, in order to study interaction between fungi and endophytic bacteria on pulcherrima growth, in combmation with four pubherrima growth promoting fungi strains:Chaemmium (B03G1-7), Mymthecium (29r-9), Pestalotia (021-4) and Guignardia (111-7), we inoculated the mixture in sterile tissue culture plants after the non-antagonistic screening step. The main results are as follows:(1) In total59strains of entophytic bacteria were isolated from roots of P. pulcherrima in different habitats. More specifically, among the P. pulcherrima,45strains of entophytic bacteria were isolated from land-type populations and14strains of entophytic bacteria were isolated from stone-type populations, which account for76.27%and23.73%respectively.(2) After morphological and molecular identification analysis, the results showed that these isolated entophytic bacteria strains’sequence were divided into10otu, and they belong to seven genera, which are Bacillus, Burkholderia, Pandoraea, Agrobacterium, Paenibacillus, Pantoea, Erwinia, respectively. Among those entophytic bacteria strains, Bacillus, Burkholderia and Pantoea are dominant genera in P. pulcherrima, which account for84.74%in total. The AMOVA result shows that the diversity of endophytic bacteria in P. pulcherrima are affected by habitat For instance, the diversity level of land-type strains are higher than stone-type strains and understory litter layer is most favorable place for the growth of endophytic bacteria in P. pulcherrima.(3)15strains were selected and inoculated into seed and plantlets separately and incubated for90days. The results show that there are3endophytic bacteria species (Bacillus megaterium■> Bacillus mycoides■Bacillus cereus) that have promoted effect on seed germination of P. pulcherrima; S endophytic bacteria species (Paenibacillus gram mis, Pantoea agghmerans, Bacillus megaterium, Bacillus mycoides, Pandoraea pnomenusa, Agrobacterium radiobacter, Burkholderia sp., Erwinia tasmaniensis) have growth-promoting effect on P. pidcherrima plantlets; B. megaterium (TW1-1) and B. mycoides (SW2-3) can not only significantly promote seed germination but also significantly promote the growth of tissue culture, these two strains play a significant role in the life cycle of P. pidcherrima’, P. graminis has most significant growth-promoting effect in life cycle of P. pidcherrima.(4)13strains were processed PGPA analyses. The results showed that9strains have potential nitrogen fixation abiliry;8strains have ability to produce IAA, IAA production ranges from5.673to29.256u.g/mL;7strains has abiliry to dissolve phosphorus, the dissolved phosphorus content ranges from13.311to77.023mg/L, the ability of Phosphate-solubilizJng in endophytic bacteria is positively correlated with their abiliry to produce acids;9strains have abiliry to produce siderophore; Burkholderia sp.(TW3-1), B. mycoides(SVJ2-3) and P. graminis(TW2-l) had all the biological characteristics; High concentrations of IAA has positive effect on P. pidcherrima seed germination but not conducive to the subsequent growth and development; The strains which have both nitrogen fixation abiliry and IAA secretion ability have better growth-promoting effect(5)4P. pidcherrima growth promoting fungi strains and8P. pulcherrima promoting bacteria were processed antagonistic combination experiments. The results show there were15antagonistic combinations and17non-antagonistic combinations. By co-culturing17non-antagonistic combinations with seedlings of P. pulcherrima for120days,9combinations were screened step by step with positive effect on the weight net growth rate and dry weight Then those9combinations were compared with single inoculation, the results showed that B03G1-7+TW3-2,021-4+TW2-1,021-4+TW3-2and111-7+SW2-3were advantage combinations, they all showed a significant enhancement when compared with singly inoculation, thus they had synergistic effect or additive effect in seedlings of P. pulcherrima. |
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