Plant-associated Microbiota From Saline Areas And Its Potential Phytobeneficial Effects

Plants(trees)have coevolved with numerous groups of microbes to adapt to the complex environmental conditions.Apart from the typical rhizobial and mycorrhizal mutualists in the root,a novel and diverse array of symbiotic microorganisms,including fungal endophyte and plantgrowth-promoting rhizobacteria(PGPR),have been harbored by plants under various environment and different soil structure,which can participate in many physiological process of plant and play a positive role in the host phenotypic traits(nutrient absorption,growth and stress tolerance).Recently,with the rapid progress in high-throughput DNA sequencing technology,we gain insights into the structure and core microbiota of plant-associated microbes,however,it is generally poorly understood how plant-associated microbes are structured and to what extent they contribute to improved tree productivity and adaptation.In this work,we mainly studied on the beneficial microorganisms isolated from several halophytes grown in saline areas of China,and aimed to identify a novel type of plant-microbes symbiosis between the microbiota and trees(eg.Populus tomentosa,P.deltoides× P.euramericana and Liquidambar styraciflua).Using the forward screening(microbe-based)approaches and the reverse screening(holobiont-based)approaches,we revealed the potential roles of functional fungi,bacteria and the total microbiome in enhancing plant growth and stree tolerance.The main results are as follows:(1)Diverse pleosporalean fungi and their roles on plant growth: here,we investigate the community composition of root,rhizosphere and seed-associated fungi from different halophytes growing in saline areas,and found that the pleosporalean taxa(Ascomycota)were most frequently isolated across samples.A total of twenty-seven representative isolates were selected for construction of the phylogeny based on the multi-locus data(partial 18 S rDNA,28 S rDNA,and transcription elongation factor 1),which classified them into seven families,one clade(nine isolates)potentially representing a novel lineage.Subsequent growth response assays suggested that these fungi had a wide pH tolerance,while most isolates showed a variable degree of sensitivity to increasing concentration of either salt or sorbitol.Co-culture assays indicated that most isolates had only neutral or even adverse effects on plant growth in the presence of inorganic nitrogen,but when provided with organic nitrogen,most isolates can enhance plant growth,especially aboveground biomass.Microscopy revealed that some pleosporalean fungi can successfully colonize roots and form melanized hyphae and/or microsclerotia-like structures within cortical cells,suggesting a phylogenetic assignment as dark septate endophytes.This work reveals that these fungi can mineralize the organic compounds(nitrogen)in the soil and take part in the process of nutrient uptake.Moreover,Curvularia sp.can significantly promote the growth and salt tolerance of poplars and American sweetgum seedlings,which can be used as superior fungal resource in the forest ecosystems.(2)Seed endophytic microbiota in a coastal plant Suaeda salsa and phytobeneficial properties of Cladosporium cladosporioides: we characterized the seed fungal endophytes present in S.salsa using 454 pyrosequencing.The fungal community present in its seeds exhibited extremely low species richness and the genus Cladosporium sp.dominated.In parallel,we recovered one isolate of C.cladosporioides from the seeds.Subsequent inoculation test revealed its role in improving host seed germination rates,meanwhile,scanning electronic microscopy analysis of its colonization pattern during seed germination revealed that C.cladosporioides can transfer from the belowground to the aboveground and become the dominated fungus in the seed.Furthermore,significant growth enhancement was recorded in American sweetgum seedlings inoculated with an optimal conidia concentration(1×105 ml-1),suggesting C.cladosporioides exhibit low host specificity.(3)Isolation and characterization of bacteria from root and rhizosphere and rapid detection of bacteria containing ACC deaminase: we investigated the community composition of root and rhizosphere bacteria from several halophytes,and identified 247 bacteria species in total.Based on the polymerase chain reaction(PCR),we amplified the ACC deaminase structural gene(acdS)and identified 25 bacteria containing ACC deaminase,which classified into the following five groups: Pseudomonas sp.,Variovorax sp.,Curiavidus sp.,Achromobacter sp.,Halomonas sp.and Burkholderia sp.In this study,we proved that various bacteria containing ACC deaminase can gather in the root and rhizosphere,and built a efficient and reliable approach to select the functional symbiotic bacteria which can improve salt tolerant of the plants.(4)A comparative study of several methods for extraction of soil microbiome: based on 16 S rRNA and ITS high-throughput sequencing,BIOLOG analysis(including ECO plate and FF plate)as well as colony-forming unit(cfu)assay of culturable fungi and bacteria,we synthetically evaluated the difference of community structure and diversity,viable counts and the community level physiological profiling of the extracted fungi and bacteria under the different four treaments compared with the natural soil microbiome.,including warling blender,ultrasonic wave,sodium pyrophosphate and MES monohydrate.The results showed that,compared to the natural microbiome,different treatments can have a negative infludence in a certain degree.When treated with pyrophosphate or the warling blender,biodiversity and metabolic activity of extracted microbiome were better.Therefore,we suggest that it is better to combine these two methods to extract the soil microbiome and applied the following experiments of the rhizosphere microbiome engineering.(5)Developing the co-culture systems of rhizosphere microbiome engineering to confer salt-tolerance to trees: in this work,sterile and clone poplar seedlings were cultivated as material.We extracted the microbiome from sympatric soils(collected from the poplar forest)and allopatric soils(collected from saline areas),built the closed,semi-open,and open experimental systems to reveal the effects of different soil microbiome on the plant growth and salt-tolerace of poplar seedlings.Preliminary results showed that the co-culture systems were feasible and there were no adverse effects on plant growth in the presence of the soil microbiome,which can confer salt tolerance to some extent.And on this basis,we re-extracted the rhizosphere microbiome of the seedlings with desirable phenotype(salt-tolerant)and developed a multigenerational selection to obtain a beneficial microbiome,which would open a new avenue for building synthetic microbial communities,as well as generating a robust and powerful mixed microbial inoculants used for trees.