Study On The Mechanism Of Endophytic Actinobacteria CoT10 And CoH27 In Regulating Phosphorus Absorption And Utilization Of Camellia Oleifera

Camellia oleifera is a woody oil crop species endemic to China,which is mainly cultivated in the acidic red soil regions in southern China.Due to the iron-rich and alumina-rich characteristics and strong oxidative fixation ability of red soil,the phosphorus in it is easily fixed with Ca²⁺,Fe³⁺,Al³⁺,and other metal ions to form insoluble phosphates,leading to the lack of available phosphorus in red soil,which is not conducive to the absorption and utilization of C.oleifera.Therefore,improving the content of available phosphorus in red soil in C.oleifera cultivation areas is of great significance to reduce the amount of phosphorus fertilizer and solve the problem of“low yield and low efficiency”of C.oleifera.Endophytes play an important role in plant growth and nutrient absorption,and the interaction between endophytes and plant roots can be enhanced under nutrient stress,which in turn promotes the uptake of phosphorus and other nutrients by the host plant.However,only a few studies focused on the endophytic actinobacteria in phosphate solubilization,and the involved mechanism is rarely deciphered.Especially for C.oleifera,whose growth and yield are strictly limited by phosphorus deficiency,the studies of entophytes are still in the initial stage,and almost no attention has been paid to the regulation of phosphorus absorption and utilization in C.oleifera by endophytic actinobacteria.In this study,the endophytic actinobacteria Streptomyces sp.CoT10 and Streptomyces sp.CoH27 were isolated and identified from C.oleifera,and their mechanisms in activating insoluble phosphates in red soil and promoting phosphorus absorption and utilization by the host were systematically elucidated through physiological and biochemical,molecular biology,microbiomic,and transcriptomic approaches,which provides a theoretical basis for further development of biofertilizers for high-yield and high-quality cultivation of C.oleifera.The associated studies and results are as follows:（1）C.oleifera harbored a high diversity of endophytic actinobacteria:876 endophytic actinobacteria were isolated from four high-yield C.oleifera trees,and 156 endophytic actinobacteria pure isolates were obtained after purification.They were distributed in 17 genera,among which Streptomyces was the dominant genus,followed by Nocardia and other rare actinobacteria genera.Redundancy analysis showed that soil p H and organic matter played crucial roles in the composition of the endophytic community.Plant growth-promoting characteristics analysis of all isolates revealed that 86.54%of the isolates were able to produce IAA,16.03%had nitrogen fixation,21.15%had phosphate solubilization,and 35.26%produced siderophores.The above results indicated that endophytic actinobacteria of C.oleifera have the potential to be exploited as biofertilizers in C.oleifera woodlands.Through qualitative and quantitative determination of phosphate solubilization and siderophores production,two strains,CoT10 and CoH27,showed remarkable phosphate solubilization and high siderophores production ability and were considered as potential strains for further studies.（2）Endophytic actinobacteria CoT10 solubilized phosphates by producing siderophores to promote C.oleifera growth:In vitro tests showed that CoT10 could effectively solubilize different insoluble phosphate resources,among which the solubility of Fe PO₄ reached 72.49mg L^-1.Further studies confirmed that the Fe-P solubilization was directly mediated by its secreted siderophores.Soil experiments showed that CoT10 promoted the effective transformation between different phosphorus fractions in red soils,and exhibited significant phosphate solubilization effects on different red soils in C.oleifera cultivation areas from 9provinces of southern China.Under greenhouse conditions,CoT10 spore solution(10⁷ spores m L^-1)inoculation could effectively mobilize insoluble phosphates and iron in the rhizosphere soil of C.oleifera,which increased the available phosphorus content by 15.35%and significantly improved the absorption and utilization efficiency of phosphorus and iron by the host,showing obvious growth boost effects.Microbial diversity analysis showed that inoculation of CoT10 increased the rhizosphere microbial diversity,with the relative abundance of dominant phyla such as Actinobacteria,Proteobacteria,and Firmicutes increased by 21.32%,7.93%,and 38.95%,respectively.Redundancy analysis,co-occurrence network analysis,and functional prediction indicated that CoT10 inoculation improved the microbial interactions in C.oleifera rhizosphere soil and regulated the phosphorus-and iron-related functional roles.The above results suggested that CoT10 promoted the mobilization of iron-fixed phosphorus by secreting siderophores and regulated the dynamic transformation of phosphorus in red soil,which in turn promoted plant growth.（3）The endophytic actinobacteria CoH27 regulated efficient absorption and utilization of nitrogen and phosphorus,improved soil micro-ecology,and promoted plant growth of C.oleifera:The whole genome of CoH27 contains many growth promotion-related functional genes,and CoH27 exhibited a remarkable capacity to fix nitrogen and solubilize Al PO₄(64.42mg L^-1).Greenhouse experiments showed that inoculation of CoH27 spore solution at a concentration of 10⁷ spores m L^-1 increased the content of available phosphorus and nitrogen in the rhizosphere soil,and significantly promoted the growth,photosynthetic efficiency,and root development of C.oleifera grafted seedlings and cutting seedlings of different ages.In addition,CoH27 increased the absorption and utilization efficiency of nitrogen and phosphorus in C.oleifera by inducing the secretion and accumulation of organic acids such as formic acid,malic acid,and citric acid in C.oleifera root exudates,together with organic acid synthase and nitrogen assimilation-related enzymes activities activation in C.oleifera roots.Microbiomic analysis revealed that the introduction of CoH27 significantly increased the abundance and diversity of a variety of beneficial microorganisms in the rhizosphere soil,enhanced the interactions between rhizosphere microorganisms,and improved the soil micro-ecological environment by participating in the regulation of soil nitrogen and phosphorus nutrient cycling,therefore establishing a ground-underground interaction model for efficient nutrient absorption and utilization.In addition,CoH27 can be applied to other commercial crops such as Brassica napus L.and Capsicum annuum L.which also displayed plant growth-promoting performance with enhanced photosynthesis and fruit ripening,leading to crop yield increase.Thus,CoH27is suitable for developing broad-spectrum natural microbial fertilizers.（4）Transcriptomic analysis of C.oleifera elucidated the mechanism of endophytic actinobacteria CoH27 regulating host nitrogen and phosphorus absorption and utilization,improving plant growth and development:From C.oleifera transcriptome data,5 differentially expressed genes involved in phosphate absorption and transport,7 transcription factors involved in regulating phosphate absorption and transport,and 10 differentially expressed genes involved in nitrogen absorption and assimilation and 3 transcription factors involved in regulating nitrate assimilation were mined.By q RT-PCR verification,CoH27 induced the up-regulation of CoPHT1;4 in the root system and the down-regulation of CoPHO1;H1,CoPHO1;H3,CoPHR1,and CoPAP27,thus improving phosphorus absorption and utilization by the root system and alleviating the effects of low phosphorus stress on the growth of C.oleifera;CoH27 also induced up-regulation of CoNRT2.5 to promote NO₃^-absorption in roots,as well as the genes encoding nitrogen assimilation-related enzymes（CoNR,CoNi R,CoNADH-GOGAT,CoGS1,CoGDHA）to improve nitrogen uptake and utilization in C.oleifera.CoH27mediated the efficient uptake of nitrogen and phosphorus nutrients in C.oleifera by promoting photosynthesis,plant growth hormones syntheses such as gibberellin（Diterpenoid biosynthesis pathway）,cytokinin（Zeatin biosynthesis pathway）and abscisic acid（Carotenoid biosynthesis pathway）,and fatty acids,starch,and sucrose synthesis,which ultimately promoted the growth and development of C.oleifera.In summary,this study confirmed the rich diversity of endophytic actinobacterial communities in C.oleifera,screened out potential strains CoT10 and CoH27,and systematically analyzed the different mechanisms of their actions in regulating the efficient absorption and utilization of phosphorus in C.oleifera,which laid a theoretical foundation for the efficient mobilization of phosphorus in red soil regions of China and the rational application of endophytes in high-yielding and high-quality cultivation of C.oleifera.