【作者】 王戈；

【导师】 杨焕文；

【作者基本信息】 云南农业大学 ， 作物遗传育种， 2012， 博士

【摘要】 本研究以4个不同烤烟品种（云85、K326、净叶黄和红花大金元）为试验材料，通过溶液培养等方法研究了不同烤烟品种根系分泌物酚酸和有机酸组分差异及其与品种抗性的相关性，通过模拟连作条件研究了根系分泌物和酚酸对幼苗生长的影响，并通过盆栽等方法研究了不同烤烟品种根际微生物数量及多样性差异。旨在丰富烤烟黑胫病抗性机制研究，明确病害发生机制，同时为今后开展生物防治和抗性栽培提供必要的理论依据。主要研究结果如下：1.采用反相色谱法以30%甲醇-0.2%乙酸体系和磷酸盐体系为分析条件适用于烤烟根系分泌物中酚酸和有机酸的定性和定量分析。两种方法操作简单、准确度高、重现性好。2.不同烤烟品种不同生育时期根系分泌物酚酸类物质共检出4种酚酸，其中以对羟基苯甲酸和丁香酸为主，可检出酚酸种类和含量因生育时期和品种抗性存在差异，抗病品种根系分泌物中较少或不检出对羟基苯甲酸，整个生育期酚酸检出总量与病情指数显著正相关。人工接种黑胫病菌促进了酚酸分泌量，各品种根系分泌物对羟基苯甲酸和丁香酸含量均呈上升趋势，且感病品种分泌量大于抗病品种，不同品种对羟基苯甲酸、丁香酸和酚酸总量与病情指数显著或极显著正相关。添加不同酚酸对黑胫病菌落生长和孢子囊产生量具有促进或抑制作用，作用方式和强度取决于作用浓度，各浓度下阿魏酸均抑制了孢子囊的产生，香草酸、香豆酸和阿魏酸在高浓度时完全抑制，相同浓度下羟基苯甲酸、丁香酸和香豆酸促进作用较大，香草酸和阿魏酸抑制作用较大；添加不同酚酸基本上促进了游动孢子释放，其作用强度随浓度升高而增大，当作用浓度大于0.05g/L和0.10g/L促进作用减小。3.不同烤烟品种不同生育时期根系分泌物有机酸类物质共检出7种，其中以草酸、乳酸和马来酸为主，可检出有机酸种类和含量因生育时期和品种抗性存在差异，抗病品种主要检出马来酸，而感病品种主要检出乳酸，感病品种有机酸检出总量大于抗病品种。人工接种黑胫病菌促进了有机酸的分泌，且分泌量随接种时间延长而增加，除抗病品种酒石酸检出量大于感病品种外其余有机酸检出量均为感病品种大于抗病品种，接种后整个时期感病品种有机酸检出总量均大于抗病品种。添加有机酸基本抑制了黑胫病菌菌落生长，且抑制作用随浓度增加而增大，添加苹果酸、乳酸和富马酸对孢子囊产生和游动孢子释放具有促进或抑制作用，作用方式和强度取决于作用浓度，草酸、酒石酸和丁二酸在各作用浓度下均表现为抑制孢子囊产生和游动孢子释放，且随作用浓度加大抑制加强，乙酸基本完全抑制孢子囊产生和游动孢子释放。4.添加不同烤烟品种根系分泌物对黑胫病菌生长的影响存在差异。添加抗病品种根系分泌物一定程度上抑制了黑胫病菌丝生长，而感病品种根系分泌物在各个生育期均促进了菌丝生长；除旺长期外，添加感病品种根系分泌物均明显促进了孢子囊的产生和游动孢子的释放，抗病品种根系分泌物对孢子囊产生量促进作用较小，且在旺长期表现出对游动孢子释放的抑制作用。5.在不同浓度根系分泌物作用下，自身种子萌发和幼苗生长明显受到抑制，幼苗叶绿素总量和根系活力下降，叶片和根系相对电导率增大，叶片和根际pH值降低，根系分泌物对烤烟幼苗的不利影响随作用浓度的增加而增大；随根系分泌物作用增加，除幼苗叶片POD活性保持较高活性呈一直上升趋势外，其它SOD、POD和CAT活性均呈先增加后降低趋势，幼苗叶片和根系MDA生成量增加。6.添加外源酚酸抑制了烤烟种子发芽率和幼苗株高，酚酸对地上部和地下部鲜重的影响因酚酸种类及作用浓度差异表现为抑制或促进；添加酚酸基本抑制了叶绿素总量（SPAD）和根系活力，并提高和促进了叶片pH值、根系相对电导率和根系丙二醛含量；添加酚酸对根系POD活性的影响均表现为“低促高抑”，香草酸和丁香酸处理根系SOD活性分别呈持续上升和下降趋势，其余处理表现为先增后减趋势，香草酸处理根系CAT活性低浓度变化不大，之后迅速升高且保持较高活性，其他酚酸处理CAT活性呈先增后减趋势。7.不同烤烟品种根际细菌数量随生育进程先增加后减少，真菌刚好相反，且细菌和真菌趋势拐点均出现在现蕾期，放线菌变化不大；团棵期和旺长期细菌和放线菌数量与品种抗性正相关，真菌数量与品种抗性呈负相关。人工接种黑胫病菌条件下，抗病品种根际环境刺激了细菌生长，而相对抑制感病品种细菌生长，不同品种均促进了根际真菌和放线菌生长，且真菌数量与品种抗性负相关。Biolog功能多样性分析表明，不同品种碳源总体利用能力及功能多样性存在一定差异，团棵期和旺长期抗病品种AWCD值、Shannon指数和Mclntosh指数大于感病品种；接种降低了感病品种碳源总体利用能力及功能多样性。更多还原

【Abstract】 Four different varieties of flue-cured tobacco (Nicotiana tobacum L.) including Yun85(highresistance, HR), K326(middle resistance, MR), Jingyehuang(middle susceptibility, MS) andHonghuadajingyuan(high susceptibility, HS) were selected as experiment materials. Compositiondifference of phenolic acids and organic acids of root exudates of different flue-cured tobacco varietiesand correlation between the difference and varieties resistance were studied with solution culture,difference of rhizosphere microorganism in difference varieties were studied with pot culture, and effectof root exudates and phenolic acids on seedling growth were studied with imitating condition ofcontinuous cropping. And main results were as follows:1. Analysis condition of30%methanol-0.2%acetic acid system and phosphate system with reversedphase chromatography were suitable for qualitative and quantitative detection of phenolic acids andorganic acids of root exudates. These methods were convenient, high accuracy and goodreproducibility.2. Four phenolic acids were detected in different growth stage of different flue-cured tobacco varieties,and P-hydrobenzoic and syringic acid were main compounds. And there were difference in speciesand content for growth stage and vareties resistance. P-hydrobenzoic acid was less or none detectedin root exudates of resistance varieties and there was significant positive correlation between totalphenolic acids during whole stage and disease index. In addition, secretions of phenolic acids werepromoted after inoculation, and content of P-hydrobenzoic and syringic acid showed an increasingtendency in root exudates of difference varieties. Moreover, secretion of susceptible varieties wasgreater than resistant varieties, and there was significant or very significant positive correlationbetween P-hydrobenzoic, syringic acid and total phenolic acids and disease index. However, addingdifference phenolic acids had promotive or inhibitory effect on colony growth and sporangium yieldof phytophora parasitica var. nicotiana, and mode of action and strength depend on actionconcentration. And sporangium yield were all restrained by ferulic acid in each concentration andwere completely restrained by vanillic acid, coumaric acid and ferulic acid in high-concentration.Promotive effect of P-hydrobenzoic, syringic acid and coumaric acid and inhibitory effect ofvanillic and ferulic acid were greatly in same concentration. Zoopore release was mainly promotedby adding different phenolic acids, and the higher concentration, the action strength increase.3. Seven organic acids were detected in different growth stage of different flue-cured tobacco varieties,and oxalic, lactic and maleic acid were main compounds. And there were difference in species andcontent for growth stage and vareties resistance. Maleic acid of resistance varieties and lactic acidof susceptible varieties was mainly detected, and total organic acids of susceptible varieties werebigger than resistance varieties. In addition, secretions of organic acids were promoted afterinoculation and the inoculation time extension, the secretion increase. Detectable amount of organicacids of susceptible varieties were bgger than resistance varieties except tartaric acid. Total organic acids of susceptible varieties were greater than resistance varieties. Colony growths of phytophoraparasitica var. nicotiana were restrained by adding organic acids, and the higher concentration, theinhibitory increase. Sporangium yield and zoopore release were promoted or restained by addingmaleic acid, lactic and fumaric acid, and mode and strength of action depend on actionconcentration. While sporangium yield and zoopore release were restrained by oxalic acid, tartaricand succinic acid in each concentration, and were completed restrained by acetic acid.4. Mycelium growth of phytophora parasitica var.nicotiana were inhibited as well as mycelium growthand biomass liveweight by resistant cultivar root exudates, but mycelium growth and biomassliveweight were stimulated by susceptible cultivar root exudates in all stage. Meanwhile, exceptrapid growing stage, sporangium yield and release zoospore rate of other stage were stimulated bysusceptible cultivar root exudates, but sporangium yield were relatively small stimulated byresistant cultivar root exudates and release zoospore rate were inhibited in rapid growing stage. Sothe whole results show that different resistant cultivar root exudates are related to cultivar resistance.And stimulation of susceptible and inhibition of resistant cultivars focus on rapid growing stage andflower budding stage.5. Seed germination and seeding growth was obvious inhibited, chlorophyll content and root activityreduced, relative conductivity of leaves and root increased and pH value of root and rhizospheredecreased. And the negative influence was enhanced with concentration increasing. In addition,SOD, POD and CAT activity all presented trend of first increased and then decreased exceptkeeping hot activity and presenting increase trend all the time in leaves POD activity. Meanwhile,MDA content of leaves and root was increased.6. Germination percentage and seeding height were restrained by phenolic acid. And impact ofphenolic acid on above-ground FW and under-ground FW were difference for species andconcentration of phenolic acid. At the same time, total chlorophyll and root activity were inhibitedand pH, relative conductivity of root and MDA content were increased by treatment of phenolicacid. The effect of treatment on enzyme activity was difference. POD activity of root all presentedincrease at low concentration and then decrease at high concentration in different treatment ofphenolic acid. And SOD activity of treatment of vanilic acid and syringic acid respectivelypresented continue rise and down trend, other treatment all presented trend of fist increase and thendecrease. CAT activity were changed little at low concentration and then rapid promoted andmaintained high activity, and CAT activity of others presented trend of fist increase and thendecrease.7. Bacterial numbers of different cultivars are first increased and then decreased with the growingprocess, while fungal numbers are reverse. Meanwhile, the trend inflexions of bacteria and fungiare all happened at the flower budding stage, and that of actinomyces is relatively stable. There arepositive correlations between rhizosphere microorganism counts of bacteria and actinomyces and cultivar resistance, and a negative correlation between fungal number and cultivar resistance at therosette stage and the rapid growing stage. In addition, growth of bacteria is stimulated inrhizosphere environment of the resistance cultivars, while the growth is relatively restrained in thesusceptible cultivars. And growths of fungi and actinomyces are promoted in all experimentmaterials. The analysis results of the Biolog method show that there are some differences in totalutilization ability of carbon source and functional diversity in different growth stages of differentcultivars, and the average well color development (AWCD), Shannon index and Mclntosh index ofthe resistance cultivars are more than that of the susceptible cultivars in different growth stages ofdifferent cultivars. At the same time, AWCD values and functional diversity of the susceptiblecultivars are decreased after inoculating phytophora parasitica var. nicotiana.更多还原