【作者】 李忠；

【导师】 江立庚；

【作者基本信息】 广西大学 ， 作物栽培学与耕作学， 2016， 博士

【摘要】 连作障碍可导致花生产量显著下降,品质降低,土壤理化性状恶化,土壤肥力下降,病害加重。加强花生连作障碍机理及防控措施的研究,对提高花生产量和质量,促进花生产业可持续发展都具有十分重要的意义。通过相关的生理生化指标的检测分析,研究花生耐连作的生理生化机理,筛选鉴定出花生耐连作种质资源以及应用其选育出花生耐连作新品种,是目前防治花生连作障碍最为经济有效的途径。因此本研究以花生野生种A. correntina(Arachis.correntina)和栽培种桂花22、桂花17为材料,通过测定花生栽培种和野生种连作土壤理化和生物学性状,磷、钾素利用效率,水培根系分泌物和根际土壤物质的化感效应以及磷、钾转运蛋白相关基因的克隆与分析,研究花生栽培种和野生种耐连作差异的生理生化机制,为鉴定花生野生种A. correntina的耐连作能力提供科学依据,为其应用于花生耐连作的遗传改良提供理论基础和基因来源。主要研究结果如下：1、连作条件下,花生野生种A. correntina的土壤理化和生物学性状的变化幅度更小与正茬相比,花生野生种A. correntina和栽培种桂花17随着连作年限的增加,土壤细菌、放线菌数量、土壤有效养分含量和干物质产量均降低,A. correntina相比桂花17下降幅度小；两个花生品种随连作年限的增加,根际土壤的真菌数量均增加,A. correntina相比桂花17增加幅度小。桂花17各个连作年限处理的土壤脲酶、转化酶和酸性磷酸酶活性随连作年限的增加有下降的趋势A. correntina各生育期各连作年限处理间的土壤脲酶、转化酶、酸性磷酸酶和土壤过氧化氢酶活性的差异不大,变幅小。从连作条件下土壤理化与生物学性状变化的初步试验研究表明,A. correntina耐连作能力比栽培种桂花17更强。两个花生品种的不同连作年限的土壤过氧化氢酶活性的变化无规律性。相关性分析表明两个花生品种的土壤脲酶、转化酶和酸性磷酸酶活性与土壤细菌,放线菌数量及主要土壤养分含量间存在显著正相关,与土壤真菌存在负相关关系,这些酶可作为评价土壤肥力的指标。2、花生野生种A. correntina的光合生产受磷、钾营养水平的影响相对较小,磷、钾素利用效率高于栽培种桂花22。施磷、钾均可明显提高桂花22的叶片光合速率、叶片叶绿素含量、叶片蒸腾速率、叶片气孔导度和干物质产量,降低桂花22叶片胞间C02浓度。相比桂花22,A. correntina 5个施磷、钾处理间的叶片光合速率、叶片叶绿素含量、叶片蒸腾速率、叶片气孔导度、叶片胞间C02浓度和干物质产量的总体差异小,受施磷、钾量影响小。A. correntina各施磷、钾处理在栽培后96d、120d叶片叶绿素含量和净光合速率下降幅度小,保持较高含量。相比桂花22, A. correntina的植株含磷、钾量和磷、钾素利用效率在不同施磷、钾处理间变化幅度比桂花22的小,受施磷、钾量影响小。试验表明A. correntina各施磷、钾处理的磷、钾素利用效率普遍高于桂花22,受施磷、钾量影响小。本试验结果为A. correntina应用于花生的磷、钾高效遗传改良提供了基础依据。3、在受磷饥饿的诱导磷相关转运蛋白基因相对表达量野生种A. correntina低于栽培种桂花17。试验获得4条磷转运蛋白相关基因序列(P31、P32、P51和P52)长度在600bp左右,2条钾转运蛋白相关基因序列(K21和K22)长度为483bp,由此推导出的磷、钾转运蛋白相关的氨基酸序列与已知的其它作物的磷、钾转运蛋白同源性较高,推导出的钾转运蛋白相关的氨基酸序列与已知的其它作物的生长素抑制蛋白同源性较高。试验所得的磷、钾转运蛋白相关的氨基酸序列目前还较少有报道。运用试验所得的磷、钾转运蛋白基因的保守序列设计特异引物进行实时荧光定量PCR分析,实时荧光定量PCR分析表明,在低磷胁迫下,试验扩增所得的磷(P71、P72、P81和P82)、钾(K91和K92)转运蛋白同基因在A. correntina和桂花17的相对表达量总体表现出胁迫处理前期升高,胁迫处理后期逐渐降低的趋势。同时在两个花生品种4条磷转运蛋白相关基因序列(P71、P72、P81和P82)之间的相对表达量也存在明显差异,桂花17受低磷胁迫诱导的磷转运蛋白相关基因各时期相对表达水平明显高于4. correntina,推测桂花17受低磷胁迫的影响可能要大于A. correntina。在受钾饥饿的诱导钾相关转运蛋白基因相对表达量A. correntin和桂花17无明显差异。试验表明本试验所获得的花生磷、钾转运蛋白基因可能主要参与了花生的根从土壤中吸收磷、钾的过程,在分子水平上A. correntina受低磷胁迫的影响要小于桂花17。4、野生种A. correntina和栽培种桂花22根系分泌物及其化感作用存在差异,野生种植株生长受到化感物质的抑制作用小于栽培种。野生种和栽培种根系分泌物及其化感作用存在差异。连作条件下,分离鉴定出的桂花22和A. correntina根分泌物包括有机酸类、醇类、酯类、酮类、醛类、苯类、烃类等,主要有机化合物有：邻苯二甲酸、苯甲醛、草酸、对乙基苯甲酸、丙酰胺、丙氨酸、戊酸等。用花生根系分泌物对花生幼苗进行生物检测,从试验结果看,桂花22水培根系分泌物和土壤根际物质乙醚组分高浓度处理对桂花22的株高、根长、地上干重和地下部干重均表现出抑制作用,抑制作用随着组分浓度增大而增强,其他组分物质对花生幼苗的生长均影响不大,与对照相比差异未达到显著水平。而A. correntina各物质组分处理与对照相比未表现出显著抑制作用。桂花22D3组分株高、根长和地下部干重均显著低于对照,表现出抑制作用,而其他组分处理与对照的差异均未达显著水平。首次明确花生根分泌物中的苯甲醛及对乙基苯甲酸对花生具有化感作用。与对照比,苯甲醛,对乙基苯甲酸,阿魏酸的低浓度处理(1×10-5mo1/L)对两个花生品种的株高,根长,地上部干重和地下部干重有促进作用或抑制作用小,而苯甲醛,苯甲酸,阿魏酸的两个高浓度处理(1×10-3mol/L和1×10-4mol/L)都存在不同程度的抑制作用,并与对照存在显著性差异(P<0.05)。与对照相比,高浓度处理的苯甲醛,苯甲酸,阿魏酸的SOD酶活性,POD酶活性和MDA在两个花生品种中都表现出不同程度的上升趋势。试验表明根系物质的化感抑制作用差异可能是造成A. correntina和桂花22耐连作能力差异的主要原因之一综上所述,相比花生栽培种,花生野生种A. correntina连作条件下的土壤理化和生物学性状的变化幅度更小,植株光合生产受磷、钾营养水平的影响相对较小,磷、钾素利用效率高,在受磷、钾饥饿的诱导磷、钾相关转运蛋白基因相对表达量变幅低,植株生长受到化感物质的抑制作用小。以上试验结果表明,野生种A. correntina相比栽培种有较好的耐连作特性的生理生化机制,在花生耐连作新品种选育方面具有良好的应用前景。更多还原

【Abstract】 The continuous cropping obstacles can lead to significant reduction the peanut production and quality, deterioration of soil physical and chemical properties, soil fertility decline, disease worse. The study of mechanism of peanut continuous cropping obstacle, prevention and control measures improve the peanut yield and quality, and promote the sustainable development of the peanut industry,which has very important significance.Through the analysis of the test related to the physiological and biochemical indexes, screening out of peanut continuous cropping resistant germplasm resources and application of peanut continuous cropping resistant breeding, is one of the effective prevention and control of peanut continuous cropping obstacle.The objective of the experiment was to study the effect of peanut continuous cropping on soil microbial quantity, soil enzyme activity, soil nutrient content and dry matter yield, wich research on continuous cropping soil of peanut cultivars variety Guuihua17 and wild species A.correntina rhizosphere soil microbial species, quantity, the change of the soil enzyme activity and soil nutrient content. The paper studied the effects of different phosphorus and potassium application rate on photosynthetic characteristics and yield of peanut wild species A.correntina and cultivated variety Guihua22. The chlorophyll content, net photosynthetic rate and dry matter production of peanut function leaf were detected. The study was to analyze peanut root exudates and study the allelopathic effects of peanut root exudates. Putative phosphate and potassium transporter gene were cloned in A.correntina and Guihua17 with RT-PCR method in this study.The results were showed as follows:1 The soil for physical and chemical change, change of biological properties of peanut wild species is smaller than peanut cultivars variety under the condition of continuous cropping.The results showed that the quantities of fungi and bacteria significantly increased and actinomyces quantity significantly reduced in the two peanut varieties of rhizosphere soil along with the increasing length of continuous cropping,the decline in A.correntina. is smaller than Guihua 17.Compared with the control,two different peanut varieties of continuous cropping soil available nutrients content reduced along with the increasing length of continuous cropping. Compared with the control, the soil urease, invertase and acid phosphatase activity enzyme activity of Guihua 17 had dropped with the increasing length of continuous cropping.The soil urease, invertase and acid phosphatase activity enzyme activity of A.correntina had a fall and then rise tendency in various stages. The change of soil hydrogen peroxide enzyme activity was irregular in the two varieties.Correlation analysis showed that two peanut varieties of urease, invertase and acid phosphatase had markedly positive correlation with bacteria, actinomyces and soil nutrient content,it could be used as evaluation index of soil fertility.On the contrary, there was a negative correlation relationship with fungi.2 The photosynthetic production of peanut wild species affected by p, k application rate are relatively small, phosphorus and potassium use efficiency of peanut wild species is higher than peanut cultivars varietyThe results showed that the phosphorus and potassium treatments could significantly improve chlorophyll content, transpiration rate, Leaf stomatal conductance,dry matter weight and photosynthetic rate of Guihua22. Compared with Guihua22,and reduce the osmanthus leaf intercellular CO2 concentration.There’s no significant effect of different phosphorus and potassium application on photosynthetic characteristics and dry matter accumulation in A.correntin.Compared with Guihua22, there’s higher levels of chlorophyll and photosynthetic rate at 96 d and 120 d after planting in A.correntin. There’s small response to the change of plant phosphorus and potassium content and phosphorus and potassium use efficiency under different phosphorus and potassium treatments in A.correntin. A.correntina had a good photosynthetic physiological basis. The test results provided a theoretical basis for forther development and utilization of A.correntina.3 The relative expression levels of Phosphate transporter gene of peanut wild species was lower than peanut cultivars variety under low phosphorus stress in the roots.Phosphate and potassium transporter gene were obtained.Homology analysis shows that they shares high similarity with high affinity phosphate and potassium transporter proteins from other plants,which indicates that proteins may play a key role in phosphate and potassium transport of peanut.The methods of quantitative real- time PCR were performed for further research.The relative expression levels of P31 and P32, P51 and P52,K61 and K62 detected by quantitative real- time PCR exhibited up- regulation under low phosphorus stress and potassium stress in the roots, and expression patterns were shown differently between A.correntina and Guihua17.The expression of P31 and P32, P51 and P52 was up-regulated by phosphate starvation. The expression of K61 and K62 was not different between A.correntina and Guihual7 by potassium starvation.These transporter proteins may play a role during the process of transport phosphate and potassium. The relative expression levels of Phosphate and potassium transporter gene of peanut wild species was lower than peanut cultivars variety under low phosphorus stress and potassium stress in the roots.4 The root secretion and its allelopathy were shown differently between peanut wild species and peanut cultivars variety, plant growth of peanut wild species was inhibited by allelochemicals less than cultivated.Compounds extracted from peanut root exudates in water culture and soil were identified and allelopathy,wich were studied by GC-MS analysis and biologicall test. The peanutroot exudates included organicacide,alcohol,ester,acetone,aldehyde,pheyl, hydrocarbon and so on.Contrast with control,the influence of the peanut root exudates did not show significant allelopathyinhibition.Benzaldehyde,4-ethylbenzoic acid which was found to be the major component in peanut root exudates,was used to test the bioactivity. Treatments of H20,1×10-5mol/L,1×10-4mol/L,1×10-3mol/L concentration were applied to test their effects on early development of peanut.The results showed that 1×10-5mol/L concentration of benzaldehyde,4-ethylbenzoic acid and ferulic acid had less impact on stem length,root length, dry weight of root and shoot.Contrast with control, 1×10-3mol/L and 1×10-4mol/L concentrationof benzaldehyde,4-ethylbenzoic acid and ferulic acid reduced peanut seedling growth to varying degrees,wich existed significant differences (P<0.05);In addition to the low concentration treatment (1×10-5 mol/L) of benzaldehyde increase slightly the SOD activity, POD activity and MDA content in A.correntina and the SOD activity, POD activity in Guihua22,the SOD activity, POD activity and MDA content of different concentrations of benzaldehyde, 4-ethylbenzoic acid and ferulic acid showed rising trend to varying degrees in two peanut varieties.Tow peanut varieties of high concentrations of benzaldehyde treatment (1×10-3 mol/L) reached significant level (P<0.05) in addition to SOD activity, of A.correntina In conclusion, there were allelophathic substances in peanut root exudates, which was one of the causes of problems associated with allelopathy.To sum up, the long-term continuous cropping make the quantities of fungi and significantly increased, bacteria and actinomyces quantity significantly reduced in the two peanut varieties of rhizosphere soil along with the increasing length of continuous cropping,the decline in A.correntina. is smaller than Guihua 17.. Compared with Guihua22, there’s small response to the change of plant phosphorus and potassium content,phosphorus and potassium use efficiency under different phosphorus and potassium treatments in A.correntin. The relative expression levels of Phosphate transporter gene of peanut wild species was lower than peanut cultivars variety.Plant growth of peanut wild species was inhibited by allelochemicals less than cultivated.The test results provided a theoretical basis for forther development and utilization of A.correntina.更多还原