【作者】 林茂兹；

【导师】 林文雄；

【作者基本信息】 福建农林大学 ， 生态学， 2010， 博士

【摘要】 以太子参连作障碍自毒效应为主线,采用大田和盆栽试验相结合的方法,研究了连作引起的太子参根、叶显微结构的变化、光合作用系统参数的变化、苗期叶片差异蛋白质表达的变化、产量和品质的变化。进一步用GC-MS鉴定了太子参根际土壤与对照土壤物质的差异,用微生物培养法和t-RFLP技术分析了太子参根际土壤与对照土壤微生物区系的变化,用差异蛋白质组学方法分析了太子参根际土壤与对照土壤差异蛋白质的表达。主要结果如下: (1)太子参根际土壤提纯物对莴苣的根和茎生长具有极显著(P<0.01)的抑制作用,且抑制程度随浓度升高也极显著(P<0.01)增大。说明太子参根际土壤提取纯化物有较强的化感作用潜力。太子参根际土壤提纯物处理正常长势的太子参,3周后太子参叶尖和叶缘发黄或枯萎,且发黄或枯萎的面积大小与受试的提取物浓度成正比。提纯物对太子参叶片光合作用系统起毒害作用,且毒害程度随受试提取纯化物浓度升高而趋向严重。盆栽模拟连作与水旱轮作结果也显示连作对太子参光合作用系统有毒害作用。盆栽生长45天的太子参苗期叶片和根的显微结构研究结果显示:连作导致太子参叶片和根部细胞排列更为疏松,单位面积内细胞密度降低,视野内有些细胞死亡而缺失。大田试验研究结果显示连作导致:太子参产量极显著(P<0.01)降低,约为水旱轮作的1/3;太子参分根数呈极显著(P<0.01)减少,约为水旱轮作条件下的一半;太子参根长显著(P<0.05)缩短,约比水旱轮作条件下的缩短8%;太子参品质下降极显著(P<0.01),其中多糖含量仅为轮作的88.08%,人参皂苷Rb1含量仅为轮作的44.33%。(2)太子参苗期叶片蛋白质差异表达分析结果显示:连作导致太子参叶片中与植物衰老或致病相关的5个蛋白表达量全部上调;与植物抗性相关的5个蛋白(磷脂氢谷胱苷肽过氧化物酶、植物性丝氨酸-苏氨酸蛋白激酶、钙依赖蛋白激酶、查尔酮合成酶和硫氧还相关蛋白)表达量上调,4个蛋白(细胞壁富含甘氨酸蛋白、SKP1同源蛋白、锌指蛋白和成熟酶K)表达量下调;与光合作用相关的2个蛋白表达量下调(Rubisco大亚基和细胞色素b6),2个蛋白表达量上调(核酮糖二磷酸碳酸酵素小链c和细胞色素b6/f复合体亚基VI);与能量代谢相关的蔗糖-UDP葡萄糖基转移酶和细胞分裂相关的NF-Y5表达量均下调,而蛋白酶1型β亚基、ATP合成酶β亚基、细胞分裂素组氨酸磷光体转移蛋白4和NAD依赖-3-磷酸甘油醛脱氢酶表达量均上调。(3)GC-MS鉴定结果显示:与对照土壤相比,太子参根际土壤中酚醌类、有机酸类、酯类和醇类相对含量较高,而醛酮类、胺类、苯类、烃类、杂环类含量较低。酚醌类、有机酸类、酯类和醇类化感作用物质。(4)稀释平板培养法测定结果显示:与正茬相比,太子参连作导致土壤中细菌数量减少,真菌和放线菌数量增多。特化培养基微生物培养方法测定结果显示:太子参连作导致土壤好气性自生固氮菌和好气性纤维素分解菌下降极显著(P<0.01),厌气性纤维素分解菌极显著(P<0.01)上升,硝化菌变化不显著,未检测出亚硝酸细菌。大田土壤的t-RFLP技术分析结果显示:对照土壤细菌涉及50个种(属),其中具有致病作用,或属于病原体的有19个种(属);太子参根际土壤细菌涉及45个种(属),其中具有致病作用,或属于病原体的有23个种(属);大田太子参根际土壤细菌物种多样性低于对照土壤的;太子参根际土壤真菌有28个种(属),除了腐生生物可能为植物致病菌或病原菌外,未发现其他直接对植物致病的菌;大田太子参根际土壤真菌物种多样性低于对照土壤的。盆栽土壤的t-RFLP技术分析结果显示:对照(CK)土壤共查询到84个种(属)片断,其中涉及病原菌或致病菌的种(属)片断为36个,轮作(Tr1)土壤共查询到122个种(属)片断,其中涉及病原菌或致病菌的种属片断为11个,连作(Tr2)土壤共查询到118个种(属)片断,其中涉及病原菌或致病菌的种(属)片断为23个,太子参盆栽连作土壤(Tr2)中检测到大量(28个片断)链霉菌属,盆栽模拟连作的土壤细菌多样性比轮作土壤的低。对照(CK)土壤真菌涉及24个种(属),轮作(Tr1)土壤真菌涉及34个种(属),连作(Tr2)土壤真菌涉及38个种(属),可见土壤真菌种(属)随太子参种植年限增加,有增多趋势。但是检索到的这些真菌中未发现有致病菌或病原菌。真菌多样性研究结果表明:随着太子参耕作年限的增加,土壤真菌多样性呈下降趋势。以盆栽土壤细菌MSPI酶切的细菌片断为例,采用主成份分析方法分析引起太子参连作障碍的主要土壤细菌种类。结果表明:引起太子参连作障碍的主要细菌种属有好酸性细菌等56个种(属)。引起太子参连作障碍的次要细菌种属有脂环酸芽孢杆菌等4个种(属)。(5)太子参根际与对照土壤差异蛋白质表达分析结果显示:太子参根际土壤中与氮素利用相关的天冬酰胺合酶和谷氨酸合酶表达量均下调;太子参根际土壤中与细胞分裂、蛋白质合成相关的4种蛋白质(脯氨酰-tRNA合酶、推测的RNA聚合酶δ-24亚基ECF家族蛋白、DNA聚合酶IIIα亚基和转录调节子)表达量均下调,而另两种蛋白质(DNA解链酶和延长因子Tu)表达量上调,这从总体上引起土壤中细菌数量减少,细菌物种多样性下降,与土壤微生物变化趋势研究结果相一致,是太子参连作障碍的一个重要原因;太子参根际土壤中含Zn的乙醇脱氢酶家族蛋白、多糖去乙酰酶、3,4-二羟基-2-丁酮-4-磷酸合酶、反应调节子和热激蛋白等与抗性相关的蛋白质表达量均上调,是太子参根际土壤中有毒有害物质积累后,土壤微生物产生应答反应的表现;太子参根际土壤中天冬氨酸转氨甲酰酶催化亚基和醋酸盐激酶表达量均上调,可能与太子参根际土壤有机酸类相对含量升高有关。更多还原

【Abstract】 In this study, field experiment and pot experiment were used to reveal the mechanism of continuous cropping obstacle of Pseudostellariae heterophylla. The yield and quality of P. heterophylla were surveyed by field experiment. The microscopic structure of the leaf and root of P. heterophylla were surveyed with microscope through paraffin slice method. The relative chlorophyll content of leaf (SPAD) of P. heterophylla was measured with the SPAD-502 (made in Japan), the net photosynthetic rate of leaf was measured with Li-6400 (made in USA), the fluorescence kinetics parameters (F0、Fm、Fv、Fp、QY_max、Ft_Lss、NPQ_Lss、F0_Lss、Fm_Lss、qP_Lss、QY_Lss) were measured with FluorCam (made in Czech republic). The differential proteomics method was used to analyze the differential expression of proteins in P. heterophylla leaves under the circumstances of P. heterophylla-Rice-P. heterophylla crop rotation or P. heterophylla continuous cropping. The extract from the rhizospheric soil of P. heterophylla was identified by GC-MS after dissolved by methanol. The non-rhizospheric soil extract was used for control. The rhizospheric soil microbial flora and their variation trends under continuous cropping circumstance were surveyed through dilution plate method, microbe culture method and terminal restriction fragment length polymorphism (t-RFLP) technique. The citrate and SDS buffers sequential extraction method were respectively used to extract the soil protein from the rhizospheric soil and non-rhizospheric soil of P. heterophylla. By means of 2-dimensional electrophoresis and contrast with the dimensional gel map of total soil proteins, the data of differential proteins were searched through bioinformatics researching.The results could be summarized as follows: (1) the allelochemicals extremely significantly inhibited the growth of root and shoot of lettuce (P<0.01), and the inhibition degree was extremely significantly (P<0.01) proportional to the concentrations of allelochemicals. It was indicated that the allelochemicals from rhizospheric soil of P. heterophylla has strong allelopathic potential.The allelochemicals, which were extracted from the rhizospheric soil of P. heterophylla, were used to treat the normal growth P. heterophylla. Three weeks after treatment, the leaf tip and the leaf rim of P. heterophlla was withered, the withered area of the leaf was proportional to the concentration of extract. The allelochemicals, had autotoxic effect on photosynthetic system of P. heterophylla, and the toxic effect degree been proportional to the concentrations of extract. The pot experiment was conducted to simulate continuous cropping and“P. heterophylla-Rice-P. heterophylla”rotation cropping was taken as the control. The results showed that continuous cropping led to autotoxic effect on photosynthetic system of P. heterophlla.After planted 45 days, the transverse sections of leaf of P. heterophylla showed that continuous cropping led to decreased cell density in the leaf, compared with control (The P. heterophylla was planted in pot with non-rhizospheric soil); cellulas were absence in some areas under microscope, compared with control. The transverse sections of root showed that continuous cropping also led to decreased cell density in the root, and cellulas were also absence in some areas under microscope. It suggested that continuous cropping resulted in some cell death occurred in the leaf and root of P. heterophylla. That is, the continuous cropping obstacle of P. heterophylla was occurred at cellular level.Continuous cropping led to extremely significantly (P<0.01) yield decreasing, the yield of the continuous cropping was only one third of the control. Compared the continuous cropping with the control experiment, the root numbers of P. heterophylla was extreme significantly (P<0.01) reduced, and the root length of P. heterophylla was significantly (P<0.05) shortened, the root numbers of the continuous cropping was about half of the control, the root length of the continuous cropping declined by 8% from that of the control. The polysaccharide content of the root from continuous cropping reduced to 88.08% of the control; the Ginseng saponins Rb1 of the root from continuous cropping reduced to 44.33% of the control. It was suggested that continuous cropping also decreased the medicinal quality of P. heterophylla.(2) The senescence-associated similar protein, the Ras-related protein, the senescence-related receptor-like kinase, the PR-protein and the Ras-related protein RHN1 were all upregulated, these proteins are related to plant senescence or disease; the phospholipid hydroperoxide glutathione peroxidase, the plant-type serine-threonine protein kinase, the calcium-dependent protein kinase, the chalcone synthase and the thioredoxin-related protein were all up-regulatively expressed, the cell wall glycine-rich protein, the homologue to SKP1, the zinc finger protein, and the maturase K were all down-regulated, these proteins are related to plant resistance; both the ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit and the cytochrome b6 were down-regulatively expressed, the ribulose bisphosphate carboxylase small chain c and the cytochrome b6/f complex subunit VI were up-regulatively expressed, these proteins are related to photosynthesis; the sucrose-UDP glucosyltransferase and the NF-YB5 were down -regulatively expressed, the proteasome subunit beta type 1, the synthase beta subunit, the cytokinin histidine phosphotransfer protein 4 and the NAD-dependent glyceraldehyde-3-phosphate dehydrogenase were all up-regulatively expressed, these proteins are related to plant metabolism of energy or cell division. It was suggested that the P. heterophylla replanting disease resulted from up-regulated expression of the proteins associated with senescence or disease, and resulted in disturbed expressions of the proteins, which are related to plant resistance or energy metabolism or cell division. It was also suggested that the continuous planting of P. heterophylla resulted in down-regulated expression of the important photosynthesis related protein; this is the molecular base of the declined photosynthesis rate in the replanted P. heterophylla.(3) The relative content of phenol quinines, organic acids, esters and alcohols in the rhizospheric soil were higher than that in non-rhizospheric soil, respectively. And the relative content of aldehyde ketones, amines, benzenes, hydrocarbons, and heterocycles in the rhizospheric soil were lower than that in non-rhizospheric soil, respectively. It was concluded that the phenol quinines, the organic acids, the esters and the alcohols were mainly autotoxins of the P. heterophylla.(4) The results based on the dilution plate method showed that the soil bacteria numbers of Tr2 (continuous cropping) were extremely significant more than that of Tr1 (rotation of P. heterophylla-Rice-P. heterophylla), the soil bacteria numbers of the CK (non-rhizospheric soil never planted P. heterophylla) were least, they were extremely significantly less than that of the Tr2, but non-significant (P>0.05) less than that of Tr1. As far as fungi and actinomycetes, their variation trends were both extremely significant (P<0.01) increased as the continuous cropping years. It was concluded that the continuous cropping of P. heterophylla led to the soil turning from bacterial types into fungal types.The results based on the specialized culture method showed that the nitrobacteria in the soil was not influenced by continuous cropping of P. heterophylla; the aerobic nitrogen-fixing bacteria numbers increased in the first year of cropping of P. heterophylla, abrupt decreased as the continuous cropping of P. heterophylla, less than the numbers of CK; the aerobic cellulose decomposition bacteria numbers also increased in the first year of cropping of P. heterophylla, and extremely significanty (P<0.01) decreased as the continuous cropping of P. heterophylla; the anaerobic cellulose decomposition bacteria numbers extremely significanty increased as the continuous cropping of P. heterophylla. It was concluded that continuous cropping of P. heterophylla would destroy the balance of rhizosphere soil microbial flora.The field survey results based on t-RFLP technique showed that there were 45 genera of bacteria in the rhizospheric soil of P. heterophylla, and 23 genera of them belonged to pathogens; there were 50 genera of bacteria in the non-rhizospheric soil, and 19 genera of them belonged to pathogens; there were 28 genera of fungi in the rhizospheric soil of P. heteropylla, none of them belonged to pathogens exempt for the Saprotroph.The pot experiment survey results based on t-RFLP technique showed that there were 118 genera of bacteria in the Tr2 (continuous cropping rhizospheric soil of P. heterophylla), and 23 genera of them belonged to pathogens; there were 122 genera of bacteria in the Tr1 (“P. heterophylla-Rice-P. heterophylla”rotation cropping rhizospheric soil of P. heterophylla), and 11 genera of them belonged to pathogens; there were 84 genera of bacteria in the CK (non-rhizospheric soil never planted P. hetrophylla), and 36 genera of them belonged to pathogens; there were 28 genera of Streptomyces in the Tr2 were detected. The pot experiment survey results based on t-RFLP technique also showed that there were 24 genera of fungi in the CK, 34 genera of fungi in the Tr1 and 38 genera of fungi in the Tr2, that is, the fungi numbers increased with the continuous cropping of P. heterophylla. Neither there were pathogens in rhizospheric soil, nor pathogens in non-rhizospheric soil of P. heterophylla. The diversity of soil bacteria analyzed results based on t-RFLP technique showed that the bacteria diversity of Tr2 was worse than that of Tr1, but the bacteria diversity of CK was the worst. The diversity of soil fungi analyzed results based on t-RFLP technique showed that the fungi diversity of Tr2 was the worst, and the fungi diversity of CK was the best. In light of t-RFLP technique survey results, it also indicated that continuous cropping of P. heterophylla would destroy the balance of rhizosphere soil microbial flora.Which kinds of microbes were mainly related to the toxic potential of rhizospheric soil of P. heterophylla was an issue in this research. The principal component analysis was used to reveal this issue based on t-FRLP profiles digested by MspI enzyme of the bacterial flora. The results showed that the first principal component (the first main microbes) related to the toxic potential of rhizospheric soil of P. heterophylla had 56 genera, e.g. Acidosphaera rubrifaciens str., the second principal component (the second main microbes) related to the toxic potential of rhizospheric soil of P. heterophylla had 4 genera, e.g. Alicyclobacillus sp. str..(5) The Asparagine synthase and Glutamate synthase content of rhizospheric soil were both lower than that of non-rhizospheric soil, these enzymes are related to nitrogen cycle; the prolyl-tRNA synthetase, putative RNA polymerase sigma-24 subunit ECF subfamily, DNA polymerase III subunit alpha and transcriptional regulator content of rhizospheric soil were all lower than that of non-rhizospheric soil, DNA helicase II and elongation factor Tu content of rhizospheric soil were higher than that of non-rhizospheric soil, these proteins are related to cell division or protein synthesis. The results of these cell division or protein synthesis related protein might result in the decrease of bacteria numbers and its biodiversity. The results also showed that the zinc-containing alcohol dehydrogenase superfamily protein, polysaccharide deacetylase, 3,4-dihydroxy-2-butanone 4-phosphate synthase, response regulator and heat shock protein HtpG content of rhizospheric soil were all higher than that of non-rhizospheric soil, these proteins, which are related to resistance of organism, resulted from response of soil microbe to the accumulation of allelochemicals in rhizospheric soil of P. heterophylla. The results finally showed that the aspartate carbamoyltransferase catalytic subunit and acetate kinase content of rhizospheric soil are both higher than that of non-rhizospheric soil; it might be relative to the higher of organic acid in the rhizospheric soil.更多还原