【作者】 王明元；

【导师】 夏仁学；

【作者基本信息】 华中农业大学 ， 果树学， 2008， 博士

【摘要】 丛枝菌根真菌是地球上广谱分布的一类土壤微生物,能够与寄主植物形成互惠共生体,帮助寄主植物从土壤中吸收更多的矿质元素和水分,因而对植物的生长和发育尤其重要。在栽培条件下柑橘根系根毛很少且短,甚至无,需要依赖丛枝菌根的吸收作用维持其正常生长,从而对丛枝菌根的依赖性强。柑橘是一类缺铁敏感的果树,生产上常常因土壤中高钙或高pH等因子影响出现缺铁现象,严重引起产量降低、品质下降。本研究以柑橘常用砧木枳和红橘为试材、以丛枝菌根真菌球囊霉属（Glomus）中的3个菌种为菌剂,探讨其对柑橘铁吸收的影响及其作用机理,为阐明菌根真菌提高矿质营养吸收提供理论依据,进而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）丛枝菌根真菌对柑橘及其微域环境影响的研究。以G.mosseae、G.versiforme和G.diaphanum为供试菌种,接种于枳（Poncirus trifoliata（L）Raf.）实生苗上进行4种基质盆栽试验,从菌根发育、植株生长和土壤微生物等方面比较上述3种菌种的接种效果。结果表明,丛枝菌根真菌对柑橘的作用存在种间差异,4种基质中,接种G.versiforme的枳实生苗菌根发育最好（如菌根侵染率和菌丝密度最高）,其株高、茎粗和根系体积明显高于接种G.mosseae和G.diaphanum的,且根围土壤中细菌、真菌和放线菌的微生物量最大,分别达到了155.66×10⁶ cfu/g、71.25×10⁶ cfu/g和36.61×10⁵ cfu/g,其它两种真菌效应比较没有一定规律。因此,G.versiforme是这3种丛枝菌根真菌中最适合柑橘接种的。（2）不同pH下丛枝菌根真菌对柑橘铁吸收影响。以G.versiforme为供试菌种,接种于枳（Poncirus trifoliata（L.）Raf.）和红橘（Citrus reticulata Blanco）实生苗上进行盆栽砂培试验,营养液pH值分别设置4个水平,枳的为pH 5.0、6.0、7.0和pH 8.0,红橘的设置为pH 5.2、6.2、7.2和pH 8.2。从植株生长量、叶绿素和叶片铁含量、根系质子分泌等方面衡量菌种G.versiforme对柑橘铁吸收的影响。结果显示,丛枝菌根真菌提高了枳和红橘实生苗株高、干重、叶绿素、叶片活性铁和全铁含量,最大增幅分别为33.34%、26.83%、32.72%、24.37%和13.75%。在高pH处理下,未接种的枳和红橘根系分泌大量的质子,表现出强烈的缺铁信号,而接种处理的质子分泌量明显较少,这说明丛枝菌根真菌提高了柑橘体内铁营养。（3）缺铁和重碳酸盐处理下丛枝菌根真菌对柑橘铁吸收的作用。以G.versiforme为供试菌种,接种于枳和红橘实生苗上进行盆栽砂培试验,缺铁和重碳酸盐处理分别设置4个水平,枳的为CK（pH 6.0）、-Fe（pH 6.0）、CaCO₃（pH 7.0）和CaCO₃ {pH 8.0),红橘的设置为CK（pH 6.2）、-Fe（pH 6.2）、CaCO₃（pH 7.2）和CaCO₃（pH 8.2）。从植株生长量、叶绿素和叶片活性含量、叶片组织结构以及叶片矿质元素的比值及根系FCR活性、总酚等方面研究接种G.versiforme对柑橘铁吸收的作用。结果显示,缺铁条件下,柑橘叶片变薄,栅栏组织和海绵组织二者界限模糊,细胞排列紧密;接种G.versiforme促进了叶片活性铁的积累和叶绿素的合成,提高了叶片Fe/Mn和K/Ca的比值,增强了叶片POD、CAT以及根系FCR活性和总酚含量,最大增幅分别为66.67%、22.46%、54.04%、50.17%、12.87%和12.70%;降低了P/Fe、50（10P+K）/Fe,最大降幅分别达到了52.33%和35.58%。（4）丛枝菌根真菌与柑橘不同根围土壤中各形态铁的关系。以G.versiforme为供试菌种,接种于枳和红橘实生苗上进行盆栽砂培试验,利用根袋技术将根围土壤分为0-2 cm、2-4 cm和4-8 cm 3个土层,研究丛枝菌根真菌对柑橘不同根围土壤中交换态铁,氧化锰结合态铁,碳酸盐结合态铁,有机质结合态铁,无定型氧化铁结合态铁,晶形氧化铁结合态铁、残渣态铁和有效铁及土壤球囊霉素年动态变化的影响。结果表明,残渣态铁、有机结合态铁以及交换态铁是土壤中有效性铁的组成部分,而碳酸盐结合态铁和无定形氧化铁结合态铁则抑制了土壤中铁的有效性。丛枝菌根真菌基本上降低了不同根围土壤中残渣态铁、有机结合态铁以及交换态铁的含量,最大降幅分别达到55.27%、42.15%和59.10%,促进有效铁的吸收。此外,土壤中球囊霉素能够螯合铁、锰、铜和锌等金属,具有缓解土壤铁紧张的作用。（5）柑橘根系FR01基因克隆以及丛枝菌根真菌对FR01表达分析。提取枳和红橘根系RNA,利用同源序列法克隆柑橘根系FR01基因,并利用Real time QT-PCR技术分析接种G.versiforme对柑橘根系FR01表达的影响。结果显示,同源序列法克隆了枳和红橘根系FR01基因,片段长度同为350 bp,与豌豆、苜蓿中三价铁螯合物还原酶基因同源性达到80%以上。接种丛枝菌根真菌提高了不同时期柑橘根系FR01表达量,且缺铁条件下,其表达量要高于正常铁处理的。更多还原

【Abstract】 Arbuscular mycorrhizal（AM） fungus is a kind of microbe widely present in the earth, which could form mutualistic symbioses with host plants.AM fungi play very important role in plant growth due to it’s improvement of mineral elements and water.Citrus has no or,if any short root hairs in field and is fairly dependent on AM fungi that are most of Glomus species.Citrus is very susceptible to iron chlorosis that is the most frequent nutritional problem in this type of fruit due to high calcium or high pH in soil,which cause decreased yield and neglectly affected of fruit quality.This study deals with two citrus rootstoks（Poncirus trifoliata（L.） Raf.and Citrus reticulata Blanco） and three arbuscular mycorrhizal fungi of Glomus in order to evaluate the effect and mechanism of AM fungi on iron uptake in citrus,so as to provide basic theory for improvement of mineral elements.The main contents and results in the present study are as follows:（1）Study on effects of AM fungi on citrus and microenvironmentExperiment was carried out in pot culture and effects of three Glomus species（Glomus mosseae,G.versiforme and G.diaphanum） on mycorrhizal development,plant growth and soil microbes in trifoliate orange（Poncirus trifoliata（L.） Raf.） were studied. Different AM fungi showed the difference of inter-species in citrus seedlings.Inoculation with G.versiforme showed the greatest effects compared with G mosseae and G. diaphanum in trifoliate orange seedlings which had highest AM colonization,hyphal density,plant height,stem diameter,root volume and density of bacteria（×10⁶cfu/g）, fungus（×10⁶cfu/g） and actonimycetes（×10⁵ cfu/g） in rhizospheric soil.It suggested that G. versiforme was the best in the three AM fungus in citrus.（2）Effects of AM fungus on iron uptake in citrus under different pH conditionsThe effect of the arbuscular mycorrhizal（AM） fungus（Glomus versiforme） on iron contents in two citrus rootstocks[trifoliate orange（Poncirus trifoliata L.Raf） and red tangerine（Citrus reticulata Blanco）]was studied in sand culture under different pH conditions.The experiment was carried out at four pH levels by applying nutrient solution at pH 5.0,6.0,7.0 or 8.0 to P.trifoliata and pH 5.2,6.2,7.2 or 8.2 to C.reticulata.Plant biomass,chlorophyll,leaf active iron and H⁺ excreted were used to analyze the effects of AM fungi on iron uptake.The results indicated that colonization by G.versiforme led to higher plant height,dry weights,chlorophyll content,active iron and total iron of leaves compared with non-mycorrhizal treatments and the maximum extent was 33.34%, 26.83%,32.72%,24.37%and 13.75%.In heigh pH treatments,amounts of H⁺ excreted from the two rootstock roots without AM fungi inoculum showed strong signal of iron deficient.However,H⁺ excreted less in AM treatments,suggesting that.AM fungi improved the iron nutrition in citrus.（3）Function of AM fungi on iron uptake in citrus under iron deficiency and heavy bicarbonate stressEffects of arbuscular mycorrhizal fungus（Glomus versiforme） on iron nutriton of trifoliate orange and red tangerine were investigated with a sand culture under iron deficiency and heavy bicarbonate stress.The experiment was carried out at four pH levels by applying nutrient solution at CK（pH 6.0）,-Fe（pH 6.0）,CaCO₃（pH 7.0） or CaCO₃ （pH 8.0） to trifoliate orange and CK（pH 6.2）,-Fe（pH 6.2）,CaCO₃（pH 7.2） or CaCO₃ （pH 8.2） to red tangerine.The plant biomass,chlorophyll,active iron,tissue structure and ratios of mineral elements in leaves,root ferric-chelate reductase（FCR） activities and total phenolics were measured.Results showed that the frontier between palisade and spongy tissue got vague and cells were arranged tightly.The colonization of G versiforme significantly increased active iron accumulation and the sythesis of chlorophyll,improved Fe/Mn and K/Ca,and enhanced activities of POD,CAT and root FCR and the contents of total phenolics in citrus roots,and the maximum extent was 66.67%,22.46%,54.04%, 50.17%,12.87%and 12.70%respectively;decreased P/Fe,50（10P+K）/Fe,and the depth downrange was 52.33%and 35.58%respectively.（4）Relation ship between AM fungi and each iron species in different rhizospherie soil of citrusSand culture was carried out in trifoliate orange and red tangerine inoculded with G versiforme.Seasonal variation of soil iron species such as Exch-Fe,MnOX-Fe,Carb-Fe, OM-Fe,AOFe-Fe,COFe-Fe,RES-Fe,available Fe and glomalin in different soil range （0-2 cm,2-4 cm,4-8 cm） apart from taproot by root confinement were studied in normal management.The results indicated that RES-Fe,OM-Fe and Exch-Fe made up of soil available Fe,and Carb-Fe and AOFe-Fe showed the negative effects.Colonization of G versiforme decreased RES-Fe,OM-Fe and Exch-Fe,and the depth downrange was 55.27%,42.15%and 59.10%respectively,improved available Fe uptake.Furthermore,Fe, Mn,Cu and Zn could be sequestered by soil glomalin,showing the buffer action of iron deficient.（5）FRO1（FCR gene） gene clone in citrus root and effects of AM fungi on FRO1 expressionTotal RNA was extracted from trifoliate orange and red tangerine roots and cDNA fragment encoding FRO1 gene was obtained by RT-PCR.Effects of AM fungi on FRO1 expression was studied via Real time PCR.Results showed that the partial cDNA length of FRO1 gene in trifoliate orange and red tangerine was 350 bp in size,and above 80% homology was found with Pisum sativum and Medicago truncatula.Inoculated with AM fungi enhanced the gene expression during different stage and higher in iron dificient treatment than normal iron conditions.更多还原