| Grasslands are an important “green” protective barrier of the ecological environment,and also serve for the development of animal husbandry.In recent years,ecoservice functions of grasslands in China have declined seriously.A main signature of such decline is the increase of toxic and harmful grassland weeds,which are detrimental to grazing livestock.Among those weeds,the toxic Astragalus and Oxytropis plants,collectively known as locoweeds,are the most widely distributed and influential ones.Spread of locoweeds result in decreased diversity of grassland vegetation and the loss of grazing function.Locoweed toxicity is caused by the biosynthesis of the toxic alkaloid swainsonine(SW)by the symbiotic endophytic fungus Alternaria sect.Undifilum in the plant hosts,as a result causing livestock poisoning.The previous studies of locoweeds mainly focused on the synthetic pathway,influencing factors and the animal poisoning mechanism of SW.At present,the research on the symbiotic relationship between locoweeds and endophytic fungi are still scarce.Exploring the biological significance and ecological effects of the symbiosis in locoweed,will help understand the nature of expansion and spread of locoweeds.Therefore,in this study I focused on how endophytic fungi and locoweed symbiosis affect the growth of locoweeds.Using the symbiotic endophyte,Alternaria sect.Undifilum,from the typical locoweed Oxytropis ochrocephala as the main material,I established its cocultivation system with its natural host and Arabidopsis thaliana,and carried out experiments on the mechanism of the plant and fungal interaction.1.I explored the influence of A.sect.Undifilum on the growth and development of the host plant O.ochrocephala by determining the growth characteristics,nutrient accumulation and phytohormone levels of the plant in the symbiotic/non-symbiotic states.2.By establishing the co-cultivation system between A.sect.Undifilum and Arabidopsis,I studied the mechanism of how the endophytic fungi promotes plant growth and development.Furthermore,concentrating on the regulatory role of auxin,the growth-promoting mechanism of A.sect.Undifilum on Arabidopsis at the genetic and protein levels was verified by using the mutants and transgenic lines in auxin synthesis,signaling and transport.3.Through the detection of the volatile metabolites of A.sect.Undifilum,the material basis of plant growth promotion was analyzed.The main findings are as follows:(1)Using O.ochrocephala seeds as the experimental material,the endophytic fungi were isolated and purified using PDA medium containing 100 ?g/m L chloramphenicol.The fungi were identified as A.sect.Undifilum by colony morphology,ITS sequencing and phylogenetic analysis,and was named A.sect.Undifilum strain OoNwu521.By removing the seed coat,the sterile seedlings of O.ochrocephala were obtained,and then a uniendophyte symbiosis between O.ochrocephala and A.sect.Undifilum OoNwu521 was established in the laboratory.Analyses showed that A.sect.Undifilum OoNwu521 mainly distributed in the intercellular space and was closely connected with the cell wall of the host.The mycelia arranged longitudinally along the medulla cells,uniform in thickness and small in curvature.The colonization of A.sect.Undifilum OoNwu521 had a promoting effect on the O.ochrocephala host seedlings,and the fresh weight and root length of the Oxytropis E+ were notably higher than E-seedlings.(2)The properties of E+(endophyte-infected)and E-(endophyte-free)seedlings of O.ochrocephala under different phosphorus concentrations were analyzed.Results showed that A.sect.Undifilum OoNwu521 stimulated the root development and promoted the growth and development of O.ochrocephala seedlings post germination.At the later developmental stage,the fresh weight of the aboveground part of the seedlings increased significantly.Measurement of carbon and nitrogen content of the E+ and E-plants showed that with the increase of phosphorus concentration,the accumulation of nitrogen content in O.ochrocephala E+ seedlings increased significantly,the carbon content did not change significantly and the C:N ratio decreased dramatically.IAA content was notably higher in the E+ than the E-plants.From the above results it was speculated that A.sect.Undifilum OoNwu521 may have the effect in promoting the growth and development in lowphosphorus environment by regulating the auxin content in O.ochrocephala seedlings.(3)On the Illumina Hi Seq 4000 sequencing platform,RNA-seq was performed in 12 O.ochrocephala samples as E+ and E-plants treated with different phosphorus concentrations for 20 days.A total size of 85.71 Gb clean data and 40,249 unigenes were obtained,of which36,267(90.11%)received functional annotations.Those unigenes were annotated to the homologues from Cicer arietinum?Medicago truncatula?Glycine max?Cajanus cajan and Trifolium pratense as the top five species hits.In total,1,178 differentially expressed genes(DEGs)were identified through gene expression analysis.These DEGs are mainly enriched in phenylalanine metabolism,isoflavone biosynthesis,and phytohormone signaling pathway,revealing that these response pathways may play a key role in the symbiosis to react to low phosphorus.(4)To further study the mechanism of influence of the endophytic symbiosis on growth promotion,the model plant Arabidopsis was cultivated with A.sect.Undifilum OoNwu521.Results showed that: A.sect.Undifilum OoNwu521 could significantly promote the growth of Arabidopsis,manifested by increased fresh weight,inhibited taproot length,and increased lateral roots and denser root hairs.Endogenous auxin levels in Arabidopsis seedlings also increased significantly.Further gene expression analysis revealed that A.sect.Undifilum OoNwu521 enhanced the expression of key auxin biosynthetic genes CYP79B2 and CYP79B3,auxin transport genes AUX1 and EIR1,auxin response factors AXR3 and ARF1,and auxin transport related genes family the PIN genes.Use auxin receptor mutant tir1-1,auxin signal mutant axr3-1,axr4-1,it was determined that A.sect.Undifilum plays an important role in in the regulation of Arabidopsis growth and development via the auxin signal pathway;using auxin transport mutant pin2 and aux1-7,it was demonstrated that A.sect.Undifilum OoNwu521 also influenced auxin polar transport.In addition,A.sect.Undifilum OoNwu521 affected the expression and protein abundance of the auxin polar transport gene family of PINs.This indicates that the endophytic fungus A.sect.Undifilum OoNwu521 regulates the growth and development of Arabidopsis by manipulating auxin biosynthesis,signaling and polar transport.(5)Using the liquid fermentation of A.sect.Undifilum OoNwu521,the effects of different extraction fractions on the growth of Arabidopsis seedling were investigated,e.g.,the petroleum ether and chloroform fractions,etc.Results indicated that the petroleum ether extract was able to increase the fresh weight of Arabidopsis significantly,revealing that the growth-promoting components were mainly in this fraction.Further analysis found when Arabidopsis and A.sect.Undifilum OoNwu521 were co-cultured on Petri dishes with septum,in the plants the principal root was significantly inhibited,however,the fresh weight,lateral root number and physiological measurements related to photosynthesis increased significantly.This indicates that volatile substances have a significant promoting effect on Arabidopsis growth.The results of headspace collection & GC-MS analysis suggested that these volatile organic compounds were essentially esters,alkanes,ketones,alcohols,ethers,aldehydes,etc. |
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