【作者】 王中华；

【导师】 徐阳春；

【作者基本信息】 南京农业大学 ， 植物营养学， 2023， 博士

【摘要】 梨是我国重要果树作物,其总产量占全球产量的80%以上。生物有机肥含有促进植物生长的特定功能微生物,能活化土壤养分并改善根际环境,有效提高果实产量和品质。梨树作为多年生木本植物,生物有机肥的施用对提高果实产量和品质的效应及其与根际微生物群落的关系亟待研究。本研究采用田间试验和室内分析相结合的方法,在等氮钾养分条件下,以纯化肥（CF）为对照,设置普通有机肥（OF）和生物有机肥（BF）处理,连续2年（2018-2020）开展施肥对梨树根系和地上部生长的影响研究,利用微生物宏基因组、果实代谢组和果实转录组联合解析生物有机肥促进根系生长、提升梨果产量和品质的组学基础,探讨生物有机肥提升产量和品质的机制。主要结果如下:1.施用生物有机肥能够提高梨果产量,改善果实品质。连续施肥2年后,BF和OF处理的梨果产量比CF分别提高10.63%和8.43%。BF处理成熟期梨果实总糖和蔗糖含量分别比CF处理提高11.15%和131.48%,果糖和葡萄糖含量则分别下降了29.40%和41.95%。相比CF,BF处理的总酸含量降低,而OF处理总酸提高。BF处理果实糖酸比分别比CF和OF提高26.34%和15.02%。果实总游离氨基酸含量CF处理分别比BF和OF高82.34%、54.41%,Asn、Glu、Gln和Asp等4种氨基酸总量分别是BF和OF的2倍和1.69倍。BF显著促进了B、Ca、K和Mo等矿质营养元素在果实中的积累,而CF显著增加了果实中N的积累,相关性分析表明果实矿质元素积累差异是不同施肥处理导致果实品质差异的重要因素。2.与CF处理相比,OF和BF处理均显著促进梨树一年生枝条数量、粗度和长度,且BF促进枝条长度的效果优于OF。比叶重大小顺序为OF>BF>CF（P<0.05）。与CF相比,BF和OF处理下梨树叶片净光合速率以及光、水分和CO₂的利用效率更高。OF和BF处理总根长分别是CF的8.49倍和4.04倍,根表面积分别是CF的5.74倍和3.14倍,根体积分别是CF的3.92倍和2.49倍,根尖数是分别CF的12.49倍和5.64倍。相关性分析表明,总根长、根表面积和根尖数是根系响应地上部的关键因子,BF对梨果产量和品质的提升与其促进根系生长和叶片的光合生理生态有关。3.施肥影响根际土壤p H、有机质含量和土壤养分有效性。CF处理下根际土壤p H、有机质、总氮和碱解氮含量显著低于OF和BF处理。生物有机肥处理施肥区梨树根际土壤p H（6.4）,显著高于化肥（5.4）和有机肥（6.0）处理,说明生物有机肥能够提高弱酸性土壤p H,对产量的形成有利。宏基因组分析结果显示,与CF和OF处理相比,芽孢杆菌属、分枝杆菌属和鞘氨醇菌属在BF处理中最为丰富,且与产量呈正相关。冗余分析表明,超过60%的根际微生物分类变异与土壤p H、总氮和EC有关。BF处理中有更多功能基因参与能量生产和转化、碳水化合物运输和代谢、辅酶运输和代谢,主导这些功能的基因簇M6的累积丰度与产量正相关。SEM模型进一步证实,p H、EC和生态模块对梨产量有很强的促进作用,BF通过降低生态模块M10和M15细菌群落组成的独特性和增加特定细菌类群的相对丰度提高了产量和品质。总之,与CF相比,施用BF能够提高土壤p H、有机质和土壤有效养分含量,并通过调控根际土壤特定的细菌生态模块和功能基因簇影响了产量和品质。4.应用广泛靶向代谢组学技术对梨幼果期和成熟期果实初生代谢物进行了分析。发现氨基酸及其衍生物、糖醇、有机酸、酚酸、维生素等37个差异代谢物随着梨果实发育而逐渐积累,且OF和BF处理更利于它们的积累。无论是在幼果期还是成熟期,OF和BF处理都会导致1,5-酐-D-山梨糖醇、松二糖、琥珀酸酐、甲基丙二酸、琥珀酸、氨基丙二酸等6个代谢物积累量上升。KEGG分析发现,碳代谢、TCA循环和谷胱甘肽代谢是重要的分配通路。可见,与CF处理相比,BF和OF在果实发育早期即启动与果实品质相关的代谢通路,且调控了碳代谢和TCA循环、谷胱甘肽代谢等代谢通路,为果实生长和品质形成提供了更多的物质和能量支持,最终影响了可溶性糖和有机酸的积累。5.为进一步揭示BF促进果实品质形成的分子机理,分析了不同施肥处理对果实转录组学的影响。结果表明,BF和OF处理显著影响梨果实成熟期离子运输、糖代谢和转运、有机酸合成与转运、氨基酸代谢过程相关基因的表达。通过构建相关性网络,筛选得到84个与品质相关的节点基因,其中生长素响应蛋白基因占11.9%;BF显著影响GH3、SAUR和Aux/IAA家族基因表达,推测BF处理较CF处理单果重显著提高的原因可能与生长素响应蛋白基因的表达调控有关。果实矿质元素中N、B和Mo与品质形成关键基因最为密切,其中B和Mo对果实生长发育和品质形成起到积极作用,而N不利于品质形成。CF显著促进了果实硝酸还原酶和离子通道蛋白基因CNGCs的表达,而BF处理则显著促进离子通道蛋白基因VDAC2、GLRs的表达,说明不同施肥处理对果实产量和品质的影响与离子转运、代谢通道蛋白以及信号转导关键酶基因密切相关。综上所述,施用生物有机肥能够通过改变梨树根际土壤理化特性和特定细菌生态模块和功能基因簇,促进根系和地上部枝叶生长,增强叶片光合作用,调节果实离子和糖酸代谢、转运关键基因表达,影响碳氮代谢和能量循环,最终提升了果实产量和品质。更多还原

【Abstract】 Pear is an important fruit crop in China,with the total yield accounting for more than 80%of global production.Bio-organic fertilizer（BF）,containing specific functional microorganisms that promote plant growth,can activate soil nutrients and improve the rhizosphere environment,thereby effectively enhancing fruit yield and quality.The effects of BF applications on pear,a perennial woody plant,to improve fruit yield and quality,and their relationships with the rhizosphere microbial community,need to be investigated.In this study,field experiments and indoor analyses were combined to investigate the effects of fertilization on the growth of pear tree roots and shoots for two consecutive years.A pure chemical fertilizer（CF）was used as the control and ordinary organic fertilizer（OF）and BF treatments were applied under equal nitrogen and potassium nutrient conditions.Metagenomics,fruit metabolomics and the fruit transcriptome were used to analyze the omics-based ways BFs promoted root growth and improved pear yield and quality,including the mechanisms BF apply to enhance yield and quality.The main findings were as follows.1.The application of a BF increased pear fruit yield and improved fruit quality.After 2 years of continuous fertilization,the yields of pear trees treated independently with BF and OF were 10.63%and 8.43%higher,respectively,than those treated with CF.The total sugar and sucrose contents of pear fruits treated with BF were 11.15%and 131.48%higher,respectively than those treated with CF,whereas the fructose and glucose contents decreased by 29.40%and 41.95%,respectively.Compared with those treated with CF,the total acid contents of those treated with BF decreased,whereas the total acid contents of those treated with OF increased.The sugar acid ratios of fruits treated with BF were 26.34%and 15.02%higher than that of those treated with CF and OF,respectively.The total free amino acid contents of fruits treated with CF were 82.34%and 54.41%higher than that treated with BF and OF,respectively,and the total contents of Asn,Glu,Gln and Asp were 2 times and 1.69 times of those treated with BF and OF,respectively.BF significantly promoted the accumulation of B,Ca,K and Mo in the fruit,and CF significantly increased the accumulation of N in the fruit.A correlation analysis revealed that the difference in the mineral element accumulation in the fruit was an important factor in the fruit quality differences that resulted from the various fertilization treatments.2.Both OF and BF treatments significantly promoted the numbers,thicknesses and lengths of annual branches of pear trees compared with CF treatment,and the BF treatment was superior to the OF treatment in promoting branch length.For specific leaf weight,the order of increase was as follows:those treated with OF>BF>CF（P<0.05）.The net photosynthetic rate and the utilization efficiencies of light,water and CO₂of pear leaves under BF and OF treatments were greater than those under the CF treatment.For those treated with OF and BF,the total root lengths were 8.49times and 4.04 times those treated with CF,and the root surface areas were 5.74 times and 3.14times,respectively,those treated with CF,the root volumes were 3.92 times and 2.49 times,respectively,those treated with CF,and the numbers of root tips were 12.49 times and 5.64 times,respectively,those treated with CF.According to the correlation analysis,total root length,root surface area and root tip number were the key factors for root responses to above-ground shoots.The improvements in the yield and quality of pear fruit by BF were related to its promotion of root growth and the photosynthetic physiological ecology of the leaves.3.Fertilization affected rhizosphere soil p H,organic matter content and soil nutrient availability.The rhizosphere soil p H,organic matter content,total nitrogen and alkali-hydrolyzable nitrogen content after the CF treatment were significantly lower than after the OF and BF treatments.The soil p H after the CF treatment decreased from 5.5 before fertilization to 5.2,and that after BF treatment increased to 6.4,indicating that CF alone led to soil acidification,and CF inhibited the decrease of soil p H,which was beneficial to the yield and quality.A metagenomics analysis suggested that Bacillus,Mycobacterium and Sphingomonas were the most abundant in soil after the BF treatment compared with those after the CF and OF treatments,and they were positively correlated with yield.Moreover,the redundancy analysis revealed that more than 60%of the rhizosphere microbial taxonomic variation was associated with soil p H,total nitrogen and EC.After the BF treatment,more functional genes were involved in energy production and transformation,carbohydrate transport and metabolism,and coenzyme transport and metabolism.The cumulative abundance of gene cluster M6,which dominated these functions,was positively correlated with yield.The structural equation modeling（SEM）model further demonstrated that p H,EC and ecological modules strongly promoted the pear yield,and BF enhanced yield and quality by reducing the uniqueness of bacterial community composition in ecological modules M10 and M15and by increasing the relative abundance of specific bacterial groups.In summary,compared with CF,the BF application was able to increase soil p H,organic matter content and effective soil nutrient content,and it influenced yield and quality by regulating specific bacterial ecological modules and functional gene clusters in rhizosphere soil.4.Extensive targeted metabolomics were analyzed to determine primary metabolites of pear fruit at the young and ripening stages.In total,37 differential metabolites,such as amino acids and their derivatives,sugar alcohols,organic acids,phenolic acids and vitamins,gradually accumulated along with pear fruit development,and the OF and BF treatments were more favorable for their accumulation.The OF and BF treatments increased the accumulation of six metabolites,1,5-anhydride-D-sorbitol,turanose,succinic anhydride,methylmalonic acid,succinic acid and aminomalonic acid,both at the young fruit and ripening stages.According to the Kyoto Encyclopedia of Genes and Genomes（KEGG）analysis,carbon metabolism,tricarboxylic acid（TCA）cycle and glutathione metabolism were important distribution pathways.Notably,compared with the CDF treatment,the BF and OF treatments initiated metabolic pathways related to fruit quality at the early stage of fruit development and regulated carbon metabolism and metabolic pathways,such as TCA cycle and glutathione metabolism,which provided more material and energy support for fruit growth and quality formation,and this ultimately affected the accumulations of soluble sugars and organic acids.5.The effects of different fertilization treatments on fruit transcriptome were analyzed to further reveal the molecular mechanism of BF to promote fruit quality.The BF and OF treatments significantly affected the expression levels of genes related to ion transport,sugar metabolism and transport,organic acid synthesis and transport,and amino acid metabolism during pear fruit ripening.A total of 84 node genes related to quality were screened by constructing a correlation network,with auxin response protein genes accounting for 11.9%.The BF treatment significantly affected the expression levels of GH3,SAUR and Aux/IAA family genes,and the significantly higher single-fruit weight after the BF treatment compared with after the CF treatment may be related to the regulation of growth hormone responsive protein gene expression.Among the mineral elements,N,B and Mo were the most closely related to the key genes of quality formation.In addition,B and Mo played positive roles in fruit growth and quality formation,and N was detrimental to quality formation.The CF treatment significantly promoted the expression of nitrate reductase and the ion channel protein genes CNGCs,whereas the BF treatment significantly promoted the expression of ion channel protein genes VDAC2 and GLRs,indicating that the effects of different fertilization treatments on fruit yield and quality were closely related to ion transport,metabolic channel proteins and signal transduction key enzyme genes.In conclusion,the BF application promoted the growth of roots and shoots,enhanced leaf photosynthesis,regulated fruit ion and sugar acid metabolism,transported key gene expression,and affected carbon and nitrogen metabolism and the energy cycle by changing the physical and chemical properties of pear rhizosphere soil,specific bacterial ecological modules and functional gene clusters,which ultimately improved fruit yield and quality.更多还原