【作者】 吴强盛；

【导师】 夏仁学；

【作者基本信息】 华中农业大学 ， 果树学， 2006， 博士

【摘要】 丛枝菌根是土壤中的丛枝菌根真菌与寄主植物根系形成的一种互惠共生体，能帮助寄主植物从土壤中吸收更多的水分和矿质元素，从而成为土壤与植物间物质的运输载体，对植物的生长和发育显得尤为重要。柑橘多栽培于丘陵山地，水分是其获得高产优质的主要限制因子之一。在栽培条件下柑橘根系根毛少且短，甚至无，需要依赖丛枝菌根的吸收作用维持其生长，从而对丛枝菌根的依赖性强。本研究从丛枝菌根真菌的角度，探讨其对柑橘抗旱性的作用及其机理，为阐明菌根抗旱提供理论依据；期望从球囊霉属（Glomus）中筛选出适合我国柑橘发展的真菌／菌种，为丛枝菌根真菌在柑橘上的应用提供参考。本研究主要内容和结果如下：（1）柑橘丛枝菌根真菌菌种抗旱比较研究。以G．mosseae、G．versiforme、G．diaphanum、G．geosporum和G．etunicatum为供试菌种，接种于正常水分或水分胁迫下的枳（Poncirus trifoliata（L．）Raf．）和红橘（Citrus tangerine Hort．ex Tanaka）实生苗上进行盆栽试验，从菌根发育、植株生长、碳水化合物以及抗氧化酶等方面比较上述5种菌种的抗旱效果。结果表明，水分胁迫明显抑制5种菌种对柑橘根系的侵染，但菌根的各部分结构联系更紧密。丛枝菌根真菌对柑橘抗旱性的作用存在种间差异，正常水分和水分胁迫下对枳实生苗高效的菌种分别是G．versiforme和G．mosseae，G．diaphanum是一个很弱的菌种，不具有抗旱潜力；正常水分和水分胁迫下G．mosseae和G．geosporum均对红橘实生苗具有很强的抗旱潜力，G．etunicatum在红橘实生苗上不具备抗旱潜力。（2）丛枝菌根真菌对柑橘水分和渗透调节的影响。以G．mosseae和G．versiforme为供试菌种，接种于不同水分处理的枳或红橘实生苗上进行盆栽试验。结果表明，在20％、16％和12％土壤含水量条件下接种G．mosseae的枳实生苗根系总吸收面积和活跃吸收面积均明显高于对应的未接种植株，有的达到显著水平；G．mosseae接种提高枳实生苗的水分利用效率达20.0％～39.9％。研究还表明，无论在正常水分还是水分胁迫下，G．versiforme接种明显提高枳或红橘实生苗叶片和根系的可溶性糖、可溶性淀粉和非结构性碳水化合物含量，降低脯氨酸含量；在2种水分条件下接种G．versiforme提高枳实生苗根系葡萄糖、果糖和蔗糖含量，对叶片则依水分状况而异。G．versiforme接种提高了2种水分状况下的枳或红橘实生苗叶片和根系的K⁺和Ca²⁺浓度，对Mg²⁺反应不一致。接种G．versiforme显著提高正常水分和水分胁迫下的红橘实生苗叶片蒸腾速率、光合速率和气孔导度。本研究首次观察到，无论正常水分还是水分胁迫下接种G．versiforme都显著降低红橘实生苗叶片温度（降温效果为0.80℃～1.82℃），从而减少叶片水分向外界的蒸散。较高的渗透调节物质提高了菌根化柑橘的渗透调节能力，这种提高与脯氨酸无关，与非结构性碳水化合物、葡萄糖、果糖、蔗糖、K⁺、Ca²⁺、Mg²⁺相关。（3）丛枝菌根真菌对柑橘活性氧代谢的影响。以G．versiforme为供试菌种接种于正常水分或水分胁迫的枳和红橘实生苗上进行盆栽试验，结果表明接种丛枝菌根真菌降低或显著降低柑橘叶片和根系的MDA、H₂O₂和O₂^·—含量，提高各种抗氧化酶（如SOD、G-POD、CAT、GR和APX）活性和各种抗氧化剂（如ASC、TASC、GSH、TASC）含量，使ASC-GSH循环更快，清除更多的ROS，从而减轻水分胁迫产生的氧化破坏，使菌根化柑橘在水分胁迫下处于更有利的地位。（4）磷和球囊霉素在丛枝菌根提高柑橘抗旱性的作用及磷与抗氧化的关系。以G．mosseae、G．versiforme和G．diaphanum为供试菌种，接种于正常水分或水分胁迫下的枳实生苗上进行盆栽试验。结果表明水分胁迫增加根际菌丝密度和球囊霉素含量，降低根系ALP和SDH活性。在根系中，菌根侵染率＞SDH＞ALP。接种丛枝菌根真菌提高根际酸性磷酸酶和总磷酸酶活性，使土壤中更多的有机P转化为无机P被植株吸收，导致植株叶片和根系的P含量升高，土壤有效P含量降低。相关性分析表明，菌根的结构、土壤磷酸酶和根内SDH共同促进植株吸收P。根系抗氧化的菌根贡献与菌根对P的贡献之间存在间接的相关关系，与菌根对植株生长和矿质元素（除Ca和Mg）的贡献间没有相关关系。研究首次发现，无论在正常水分还是水分胁迫下接种丛枝菌根真菌在枳实生苗根际都产生一种糖蛋白——球囊霉素，范围在1.63 mg／g～1.93 mg／g。由于球囊霉素的“超级胶水”功能，将小的团聚体粘合成大的团聚体，使2 cm、1 cm和大于0.25 cm的水稳性团聚体提高，0.25 cm的水稳性团聚体降低。据此菌根化土壤具有高度的空隙性和持水性，延迟水分向土壤毛细管流动，最终提高植株的抗旱性。（5）柑橘丛枝菌根发育特性研究。田间条件下观察到柑橘根系具有泡囊、丛枝、侵入点、根内菌丝、根外菌丝等结构，说明柑橘是丛枝菌根类植物。田间条件下国庆1号温州蜜柑（Citrus unshiu Marc Guoqing No．1）和国庆4号温州蜜柑（C．unshiu Marc Guoqing No．4）菌根侵染率和孢子密度年变化均呈“∧”形，有效磷和中性磷酸酶年变化呈“V”形。在不同的土壤深度以10 cm～20 cm的菌根侵染率和孢子密度最高。覆草有利于柑橘菌根发育，施N肥和翻耕抑制柑橘菌根发育。红橘+枳、红橘+粗柠檬、枳、枳橙和枳柚5种盆栽柑橘砧木的侵染率和孢子密度以红橘+枳最高。相关性表明，2种试材的菌根侵染率都与孢子密度呈极显著正相关，与有效磷呈极显著负相关，说明较高的孢子密度和较低的有效磷对菌根侵染率有促进作用；2柑橘品种的孢子密度均与有效磷极显著负相关，与中性磷酸酶和总磷酸酶极显著正相关，表明较高的中性磷酸酶和总磷酸酶对孢子密度有刺激作用，而较高的有效磷对其有抑制作用。柑橘园土壤有机磷矿化主要以中性磷酸酶的酶促为主。最后，结合前人的研究，作者初步提出丛枝菌根真菌提高寄主植物抗旱性的机理模式图，并探讨了菌根化柑橘存在交叉抗性的特性。更多还原

【Abstract】 Arbuscular mycorrhizas are mutualistic symbioses that form between arbuscular mycorrhizal （AM） fungi and the roots of host plants. The symbioses are characterized by bi-movement of nutrients where carbon flows to the symbiosis and inorganic nutrients or water move to the plants, thereby becoming a transported carrier between the roots of host plant and soil. Thus, AM symbioses are important for growth and development of host plants. Citrus is one of important fruit species in southern China. Most of citrus trees are cultivated in highland, and water is a restricted factor to acquire high yield and good quality of citrus. Citrus has rare and short root hairs in field and is fairly dependent on AM fungi that are most Glomus species. This study is to evaluate the effect and mechanism of AM fungi on drought resistance of citrus, and to select some effective fungi using the citrus. The main contents and results in the present study are as follows:（1） Selection of AM fungi for drought resistance of citrusExperiment was carried out in potted culture and effects of five Glomus species （Glomus mosseae, G. versiforme, G. diaphanum, G. geosporum and G. etunicatum） on mycorrhizal development, plant growth, carbohydrate and antioxidant enzymes in two citrus rootstocks species—trifoliate orange （Poncirus trifoliata （L.） Raf.） and tangerine （Citrus tangerine Hort. ex Tanaka）—under well-watered and water stress conditions were studied. Water stress significantly decreased the mycorrhizal infection of two citrus roots, whereas all structures of arbuscular mycorrhizae were more closely joined under water stress conditions than under well-watered conditions. Different AM fungi showed the difference of inter-species in drought resistance of citrus seedlings. Inoculation with G. versiforme showed the greatest effects in trifoliate orange seedlings under well-watered conditions and inoculated with G. mosseae the greatest effects under water stress conditions. G. diaphanum was a rather weak fungus for the drought resistance of trifoliate orange seedlings under well-watered and water stress conditions. Inoculation with G. mosseae or G. geosporum represented greater effects in tangerine seedlings whether water stressed or not, and inoculation with G. etunicatum did not represent effects in tangerine seedlings.（2） Effects of AM fungi on water and osmotic adjustment in citrusPotted experiments were conducted to research the effects of AM fungi on water status and osmotic adjustment of citrus seedlings under different water treatments conditions. G. mosseae-inoculation increased the total absorption and active absorption areas of roots in trifoliate orange seedlings under 20%, 16% and 12% water content of soil conditions. Trifoliate orange seedling colonized by G. mosseae had higher 20.0%～39.9% water use efficiency than non-mycorrhizal trifoliate orange seedlings, when the water content of soil was 20%, 16% and 12% respectively. The results also showed that G. versiforme- inoculation increased the soluble sugar contents, the soluble starch contents and the non-structural carbohydrate contents in leaves and roots of either trifoliate orange or tangerine seedlings, but G. versiforme-inoculation decreased the proline contents of two citrus rootstocks under well-watered and water stress conditions, respectively. There were greater the glucose, fructose and sucrose contents of roots of trifoliate orange seedlings, as well as greater the K⁺ and Ca²⁺ levels in leaves and roots of trifoliate orange or tangerine seedlings grown under well-watered and water stress conditions. However, the responses to inoculation with G. versiforme on Mg²⁺ levels of two rootstocks and the glucose, fructose and sucrose contents of leaves of trifoliate orange seedlings were varied. AM tangerine seedlings had higher leaf water potential, transpiration rates, photosynthetic rates, stomatal conductance, and lower leaf temperature （0.80℃～1.82℃） than corresponding non-AM control. AM colonization improved the osmotic adjustment originating not from proline but from non-structural carbohydrate, K⁺, Ca²⁺, Mg²⁺, glucose, fructose and sucrose, resulting in the enhancement of drought resistance.（3） Effect of G. versiforme inoculation on reactive oxygen metabolism in trifoliate orange and tangerine seedlings in potted culture under well-watered and water stress conditionsWhether water stressed or not, AM symbiosis decreased the concentrations of malondialdehyde, superoxide anion radical and hydrogen peroxide in leaves or roots of two citrus rootstocks. AM infection also increased the activities of antioxidant enzymes including superoxide dismustase, guaiacol peroxidase, catalase, glutathione reductase and ascorbate peroxide and enhanced the contents of antioxidants including reduced ascorbate, reduced glutathione, total ascorbate and total glutathione regardless of water status. The more antioxidant enzymes and antioxidants in AM seedlings would result in faster removal of hydrogen peroxide through ascorbate-glutathione cycle, helping to alleviate oxidative damage and enhancing drought resistance of host plants.（4） Effects of phosphorus and glomalin on drought resistance of trifoliate orange seedlings colonized by G. versiforme, G. mosseae and G. diaphanum in potted culture and relationship between phosphorus and anti-oxidation under well-watered and water stress conditionsWater stress enhanced fungal density and glomalin in rhizosphere of AM citrus seedlings and decreased alkaline phosphatase and succinate dehydrogenase activies of citrus roots. In AM roots, the ranking of AM colonization, alkaline phosphatase and succinate dehydrogenase was as follows: AM colonization＞succinate dehydrogenase＞alkaline phosphatase. The activities of acid phosphorylase and total phosphorylase were increased by AM colonization, resulting in more organic phosphorus translated into inorganic phosphorus for providing host plants. As a result, the contents of phosphorus in leaves and roots of AM citrus seedlings were increased by AM colonization, and the content of available phosphorus in rhizosphere was decreased. When analysed relativity, the results showed that mycorrhizal structures, soil phosphorylase and succinate dehydrogenase together affected the phosphorus absorption of plant. There was indirect correlation between contribute of AM to phosphorus and contribute of AM to antioxidant enzymes and antioxidants of roots, as well as no correlation between contribute of AM to plant growth or nutrient element （excluding Ca and Mg） and contribute of AM to both antixodant enzymes and antioxidants of roots.Whether water stressed or not, glomalin was firstly observed in rhizosphere of AM seedlings, and its levels were from 1.63 mg/g to 1.93 mg/g. As a result of "super glue" of glomalin, small aggregations were agglutinated big aggregation, resulting in the increments of 2 cm, 1 cm and＞0.25 cm water-stable aggregations and the decreases of 0.25 cm water-stable aggregation. Thus, mycorrhizal soils presented considerable characterists of interspace and holding water. Water would be delayed to flow to soil capillary, and drought resistance of AM host plants was enhanced.（5） Study on AM characterists of citrus in fieldModel AM structures including vesicles, arbuscles, entry points, internal hyphae and external hyphae were observed in roots of citrus in filed. This showed that Citrus belonged to AM plants. Both AM colonization and spore density showed "A" shape on Guoqing No.1 （Citrus unshiu Marc cv Guoqing No.1）/trifoliate orange （Poncirus trifoliata （L.） Raf.） and Guoqing No. 4 （C. unshiu Marc Guoqing No.4）/trifoliate orange, reaching lower status in Feb. and Dec., middle status in Apr. and Oct., and higher status in Jun. and Aug. "V" sharp was found in soil avail phosphorus or litmusless phosphatase. The highest AM colonization and spore density were within the soil depth of 10 cm～20 cm in Guoqing No.1 and Guoqing No.4 in field. Covered ruderals significantly enhanced AM colonization and spore density of two citrus species in field. However, AM colonization and spore density of two citrus in field were decreased by application of N fertilization and tillage. AM development of tangerine+trifoliate orange was highest in five citrus rootstocks （tangerine+trifoliate orange, trifoliate orange, troyer citrange, swingle citrumelo, tangerine+rough lemon） and was significantly higher than that of other rootstocks.The study indicated that AM colonization was significantly positive correlation with spore density （p＜0.01） and notably negative correlation with soil avail phosphorus （p＜0.01） on Guoqing No.1 and Guoqing No. 4. Thus, the higher spore density and the lower soil avail phosphorus could accelerate mycorrhizai infection to roots. Spore density was significantly negative correlation with soil avail phosphorus （p＜0.01） and markedly positive correlation with litmusless phosphatase （p＜0.01） or total phosphatase （p＜0.01） on Guoqing No.1 and Guoqing No. 4. Therefore, we deduced that higher soil avail phosphorus had a function of restraint, and both higher litmusless phosphatase and total phosphatase had function of stimulation to spore density. The mineralization of organic phosphorus gave priority to litmusless phosphatase in citrus trees in field.Finally, model figure of mechanisms about drought resistance of host plants by AM fungi was primarily established according to both this study and former results. The study also discussed the possibility of cross-tolerance in mycorrhizal plants.更多还原