粪产碱菌对稻根氧化还原特性及水稻多元酚和内根际激素水平的影响

联合固氮粪产碱菌结合于水稻表时能增强水稻根部还原力和稻根超氧化物歧化酶活性,实验室和田间试验证明粪产碱菌能提高水稻幼苗对高、低温不良环境的抗逆性,经浸种处理的水稻幼菌植株内多元酚含量增加１２．５％。粪产碱菌对接种水稻多元酚抽提物有强烈的趋化性,而该抽提物对粪产碱菌的固氮活性有明显的刺激作用,多元酚抽提物经双向纸层析和薄层层析表明接种诱导了至少一个特征组分含量提高,采用粪产碱菌野生型Ａ１５０１和固氮     

粪产碱菌对水稻根质子分泌作用及根际微生态的影响

用粪产碱菌浸种或沾根均能增强水稻根的质子分泌能力,促使介质酸化,pH值下降约2.2,浸种的效应更为明显。接种粪产碱菌能刺激水稻在缺铁胁迫条件下的质子分泌作用,提高根际溶液和根内ATP含量,同时增强根际土壤中铁、磷元素的有效性,促进植物根系对磷的吸收利用。     

稻根联合固氮细菌的研究I．菌种的分离和鉴定

从水稻根内分离到两株固氮能力较强的细菌,A-15和 E-26。经鉴定,A-15的表现性状与粪产碱菌的性状相符,但其DNA中GC含量为62．95—63 93克分子％,略高于文献报道的58．9克分子％。 由于缺乏粪产碱菌的模式株,暂将A—15归属于粪产碱菌(Alcaligenesfaecalis)。E-26的表观性状与阴沟肠杆菌属相符,因此将E一26归属于阴沟肠杆菌(Entcroba-cter cloacae)。据报道,在玉米根际曾分离到固氮的阴沟肠杆菌,却未见到有分离出固氮的粪产碱菌者。     

盐胁迫下粪产碱菌在水稻根表的定殖和异源固氮基因的表达

异源nif LacZ融合基因在粪产碱菌A15 6 1中的表达活性随盐浓度增加而升高 ,然后逐渐降低 ,nifH LacZ融合基因可以正常表达的盐浓度在 0 .1%～0 .5 %之间 ,盐浓度为 0 .0 5 %时活性最高。A15 6 1在盐浓度为 0 .0 6 %时趋化能力最强 ,随着盐浓度的提高逐渐下降 ,当盐浓度为 3 .0 %时完全丧失趋化能力。一定的盐浓度 (0 .5 % )对固氮粪产碱菌的根表定殖有促进作用 ,该条件下根表定殖的菌体数远大于对照。 3种nif LacZ融合基因在根内的表达部位有显著差异。nifH的表达部位主要分布于根的皮层薄壁组织细胞间隙 ,在条件适宜 (无铵和微量氧 )的部位或某些特殊位置如侧根伸出部位高水平表达。盐胁迫下水稻 耐盐粪产碱菌A15 6 1的联合固氮效率明显高于A15 6 1纯培养物     

水稻内生联合固氮细菌的筛选,鉴定及其分布特性

利用乙炔还原法和固定１５Ｎ２ 活性测定法对分离自水稻（ Ｏｒｙｚａ ｓａｔｉｖａ Ｌ．）“越富”种子、根、茎和叶的内生细菌进行了筛选,获得２９ 株具有体外固氮能力的水稻内生联合固氮细菌。鉴定结果表明它们分属于根癌土壤杆菌（ Ａｇｒｏｂａｃｔｅｒｉｕｍ ｔｕｍｅｆａｃｉｅｎｓ （Ｓｍｉｔｈ ｅｔ Ｔｏｗｎｓｅｎｄ） Ｃｏｎｎ） ,放射土壤杆菌（ Ａ． ｒａｄｉｏｂａｃｔｅｒ （Ｂｅｉｊｅｒｉｎｃｋ ｅｔ ｖａｎ Ｄｅｌｄｅｎ） Ｃｏｎｎ） ;阴沟肠杆菌（ Ｅｎｔｅｒｏｂａｃｔｅｒ ｃｌｏａｃａｅ （Ｊｏｒｄａｎ） Ｈｏｒｍａｅｃｈｅ ｅｔ Ｅｄｗａｒｄｓ） ,成团肠杆菌（ Ｅ． ａｇｇｌｏｍｅｒａｎｓ （Ｂｅｉｊｅｒｉｎｃｋ） Ｅｗｉｎｇ ｅｔ Ｆｉｆｅ） ,坂崎肠杆菌（ Ｅ． ｓａｋａｚａｋｉｉ Ｆａｍｅｒ ｅｔ ａｌ．） ;皮氏产碱菌（ Ａｌｃａｌｉｇｅｎｅｓ ｐｉｅｃｈａｕｄｉｉ Ｋｉｒｅｄｊｉａｎ ｅｔ ａｌ．） ,反硝化产碱菌（ Ａ． ｄｅｎｉｔｒｉｆｉｃａｎｓ （Ｌｅｉｆｓｏｎ ｅｔ Ｈｕｇｈ） Ｒｕｇｅｒ ｅｔ Ｔａｎ） ;类产碱假单胞菌（ Ｐｓｅｕｄｏｍｏｎａｓ ｐｓｅｕｄｏａｌｃａｌｉｇｅｎｅｓ Ｓｔａｎｉｅｒ） ,产碱假单胞菌（ Ｐ． ａｌｃａｌ     

水稻根际联合固氮细菌的研究

从我国南方水稻根部分离到3株氧化型革兰氏阴性细菌,编号为A1601,A1701和A1702。经15N示踪实验证明,它们均有较高的固氮能力。在无氮培养基中加入少量稻根浸出液进行培养后,可使固氮酶活性明显提高。根据菌株的形态和生理生化等特征鉴定,3株菌均为产孽菌属的细菌,分别为争论产碱菌(Alcaligenes paradoxus A1601),反硝化产碱蔺木糖氧化亚种(Alcaligenes denitrificans subsp.xylosoxydons A1701)和反硝化产碱菌反硝化亚种(Alcaligenes denitridicans subsp.denitrificans A1702)。这是继粪产碱菌(Alcaligenesfaecalis)之后,发现的产碱菌属中另外3株未见报道的固氮细菌。     

水稻幼苗根质膜氧化还原系统活性与抗冷性的关系

本试验以水稻幼苗为材料,研究冷胁迫和钙浸种、低温锻炼、低温锻炼结合钙浸种预处理分别对幼苗根质膜Ｆｅ（ＣＮ）３－６还原活性的影响。实验结果表明：冷胁迫降低了质膜Ｆｅ（ＣＮ）３－６还原活性;钙浸种、低温锻炼、低温锻炼结合钙浸种预处理均提高了质膜Ｆｅ（ＣＮ）３－６还源活性,尤其是削减了冷胁迫降低质膜Ｆｅ（ＣＮ）３－６还原活性的作用。根质膜Ｆｅ（ＣＮ）３－６还原活性与水稻幼苗抗冷力密切相关。     

三种蓝莓根际细菌的分离及其对蓝莓苗生长发育的影响

【目的】从3种蓝莓根际土壤中分离细菌,探究蓝莓根际土壤细菌多样性,并筛选具有产酸、促生长、抑菌性能的菌株,为蓝莓专用微生物肥料的研究提供优质菌株资源和理论基础。【方法】选用5种培养基分离3种蓝莓根际土壤细菌,并进行16S rRNA基因测序和系统发育分析。筛选产酸、产吲哚-3-乙酸（indole-3-acetic acid,IAA）和铁载体、固氮、溶磷和抑制灰葡萄孢生长的菌株,挑选最适菌株制备菌剂进行蓝莓苗盆栽实验验证促生能力,并检测菌剂对蓝莓元素吸收和根际土壤肥力的影响。【结果】从3种蓝莓根际土壤分离得到124株细菌,挑选70株代表性菌株进行16S rRNA基因测序,分布于3个门21个属,其中芽孢杆菌属（Bacillus）、假单胞菌属（Pseudomonas）、链霉菌属（Streptomyces）和红球菌属（Rhodococcus）为优势分离菌群。代表性菌株中,21.4%的菌株能产酸,21.4%的菌株产吲哚-3-乙酸,47.1%的菌株具有固氮潜力,65.7%的菌株具有解磷能力,14.3%的菌株能产铁载体。少量菌株同时具有产酸、产IAA、固氮、解磷和抑菌等能力。选取具有产酸和多种促生特征的菌株绿针假单胞菌CSM-70和双鱼假单胞菌CSM-129进行盆栽蓝莓苗处理,发现2株菌均能显著促进蓝莓苗的生长发育并调控根际土壤pH,其中菌株CSM-70处理还显著促进了蓝莓叶片氮、磷元素的吸收,提升了土壤速效钾、碱解氮的含量。【结论】蓝莓根际细菌多样性高且蕴藏着丰富的促生长菌株,绿针假单胞菌CSM-70和双鱼假单胞菌CSM-129能够促进蓝莓苗生长、调控根际土壤pH和肥力,并促进植株养分吸收,具有蓝莓专用微生物菌剂研制与应用的潜力。     

粪产碱菌(Alcaligenes faecalis)A-15与水稻根的结合作用

近年来,联合共生固氮作用已逐渐为人们所重视,与共生、自生固氮生物不同,这些细菌多聚集在植物根表,在根部的粘胶层中,部分细菌可进入根表层内。粪产碱茵A—15是从水稻根分离到的固氮细菌,     

盐胁迫下烘产碱菌在水稻根表的定殖和异源固氮基因的表达

异源ｎｉｆ－ＬａｎＺ融合基因在粪产碱菌Ａ１５６１中的表达活性随盐浓度增加而升高,然而逐渐降。ｎｉｆＨ－ＬａｅＺ融合基因可以正常表达的盐浓度在０．１％～０．５％之间,盐浓度与０．０５％时活必同Ａ１５６１在盐浓度为０．０６％时趋化能力最强,随着盐浓度的提高逐渐下降,当盐浓度为３．０％时完全人趋化能力。一定的盐浓度（０．５％）对固氮立碱菌的数远大于对照。３种ｎｉｆ－ＬａｃＺ融合基因在根内的表达部位有显著     

Effect of associative bacteria on element composition of barley seedlings grown in solution culture at toxic cadmium concentrations

The response of barley seedlings to inoculation with associative rhizobacteria Azospirillum lipoferum 137, Arthrobacter mysorens 7, Agrobacterium radiobacter 10 and Flavobacterium sp. L30 was studied in hydroponic and quartz sand cultures in the presence of 50 microM CdCl2. Cadmium caused severe inhibition in the growth and uptake of nutrient elements by the plants. Inoculation with the bacteria slightly stimulated root length and biomass of hydroponically grown Cd-treated seedlings. The bacteria increased the content of nutrients such as P, Mg, Ca, Fe, Mn and Na in roots and or shoots of the plants grown in the absence of Cd. Positive changes in the element composition caused by the bacteria were less pronounced in Cd-treated plants, whereas the total amount of nutrients taken by the inoculated plants was generally increased significantly. The content of Cd in the inoculated plants was unchanged, except increased in roots upon addition of A. lipoferum 137. Inoculation did not affect the activity of peroxidase, alpha-mannosidase, phosphodiesterae, alpha-galactosidase, and concentration of sulfhydryl compounds used as biochemical markers of stress in plant roots. The results showed that associative bacteria were capable of decreasing partially the toxicity of Cd for the barley plants through the improvement in uptake of nutrient elements.     

Intercellular colonization and growth promoting effects of Methylobacterium sp. with plant-growth regulators on rice (Oryza sativa L. Cv CO-43)

The Methylobacterium sp. strain NPFM-SB3, isolated from Sesbania rostrata stem nodules possessed nitrogenase activity and nodA genes. Pure culture of NPFM-SB3 strain produced indole-3-acetic acid, cytokinins and on inoculation to rice plants resulted in numerous lateral roots. Inoculation of synthetic auxins 2,4-dichlorophenoxy acetic acid, naphthalene acetic acid or flavonoids naringenin and dihydroxy-4-methoxyisoflavone individually or to bacterial inoculated rice seedlings improved the plant growth and lateral root formation under hydroponic condition. The formation of nodule-like structure and nitrogenase activity which is purely auxin dependent was observed in 2,4-dichlorophenoxy acetic acid treatments to Methylobacterium sp. NPFM-SB3 inoculated rice plants. The rhizobia entered through fissures formed due to lateral root emergence and spread intercellularly in the nodular structures concluded that the effect of 2,4-dichlorophenoxy acetic acid treatment for rice seedlings grown under gnotobiotic conditions is to create a niche in which these bacteria can grow.     

Early defence reactions in Norway spruce seedlings inoculated with the mycorrhizal fungus <Emphasis Type="Italic">Pisolithus tinctorius</Emphasis> (Persoon) Coker &; Couch and the pathogen <Emphasis Type="Italic">Heterobasidion annosum</Emphasis> (Fr.) Bref.

The activities of the enzymes responsible for cell-wall strengthening and salicylic acid (SA) content in Norway spruce seedlings were investigated after inoculation with the ectomycorrhizal fungus Pisolithus tinctorius or the pathogen Heterobasidion annosum, and after treatment with elicitors from both of these fungi. Inoculation with both fungi increased guaiacol peroxidase (POD) activity in the roots of the pathogen-inoculated seedlings during the earliest phases of colonisation, and induced the activities of several POD isoforms. Two of these were only seen in pathogen-inoculated seedlings and corresponded with increased POD activity against ferulic acid. Colonisation with H. annosum triggered an increase in phenylalanine ammonia lyase (PAL) activity in the roots of the spruce seedlings, which was followed by an accumulation of free SA. One month after inoculation levels of free SA were increased also in the shoots of H. annosum-inoculated seedlings. In contrast increase in free SA content in the roots of P. tinctorius-inoculated seedlings was only transient. Similarly to inoculation, treatment with elicitors of H. annosum increased the PAL and POD activity, as well as SA content in the roots of spruce seedlings. A positive correlation between PAL activity and SA content in the H. annosum-inoculated seedlings and accumulation of SA precursors in the phenylpropanoid pathway indicate that the plant defence mechanisms, during which SA is synthesised through the PAL pathway, are exploited by H. annosum for facilitation of colonisation.     

野外模拟酸雨胁迫下接种外生菌根真菌对马尾松幼苗的缓解作用

外生菌根真菌能够提高宿主植物对外界环境胁迫的抵抗力。主要探讨野外条件下外生菌根真菌对酸雨胁迫下马尾松(Pinus massoniana)幼苗生长、养分元素以及表层土壤的影响,以期为酸雨严重区马尾松林恢复提供科学依据。以2年生马尾松幼苗为材料,采用原位试验,共设置6个处理:p H5.6(对照)处理未接种、对照处理接种、p H4.5酸雨处理未接种、p H4.5酸雨处理接种、p H3.5酸雨处理未接种、p H3.5酸雨处理接种。研究表明:(1)酸雨处理与对照处理相比显著降低了非菌根苗总生物量及各部位生物量(根、茎、叶),对株高无显著影响,接种外生菌根真菌可以缓解酸雨对马尾松幼苗生长的不利影响;(2)与对照处理相比,酸雨处理的非菌根苗的针叶中N、P、Ca含量升高,Mg含量降低,根系中N、P、Ca含量降低,Mg含量随p H的降低先升高后降低。接种外生菌根真菌显著提高了p H3.5酸雨处理的马尾松幼苗根系中N、P、Ca、Mg含量,而对针叶中N、P、Ca、Mg含量无显著影响。(3)在非菌根土壤中,p H3.5酸雨处理与对照处理相比显著降低了土壤中有机质、速效磷、速效钾、可溶性碳、可溶性氮、铵态氮、硝态氮含量,而接种外生菌根真菌显著提高了上述指标。酸雨对土壤阳离子交换量无显著影响。总而言之,接种外生菌根真菌促进了酸雨处理的马尾松幼苗生长、缓解了酸雨对马尾松幼苗养分元素和表层土壤的不利影响,由此可见接种外生菌根真菌是减轻酸雨对马尾松危害的一个重要途径。     

固氮粪产碱菌ntrC-lacZ融合基因的构建及其在水稻根部的表达

将粪产碱菌（Alcaligenesfaecalis）A1501ntrC基因和lacZ基因正向克隆于广泛性转移载体pLA2917上,获得多拷贝ntrC质粒pLAC1和含ntrC－lacZ融合基因的重组质粒pLAC2,采用双亲结合方法,将上述两个重组质粒导入A1501中,获得含ntrC－lacZ融合基因的结合子A15C2和ntrC多拷贝结合子A15C1。采用X－gal原位显色技术、显微切片、扫描电镜观察及ntrC部分缺失突变株研究粪产碱菌ntrC在根部的表达及功能,结果表明粪产碱菌A1501在水稻根部有较强的定殖能力,并能侵进入水稻根内定殖。在水稻主侧根伸长区及根内皮层薄壁细胞及侧根分生区ntrC－lacZ融合基因表达活性明显高于别的部位。高铵条件下多拷贝ntrC结合子根表定殖能力大于野生型,而ntrC突变株则低于野生型。表明ntrC基因参与固氮菌根表结合的过程。     

Artificial induction of the apple replant problem byPenicillium claviforme inoculation

Inoculation of apple seedling roots withPenicillium claviforme reduced plant growth and resulted in morphological changes of roots resembling those found in seedlings growing in ‘apple replant problem’ soil (apple-sick soil). The introduced fungus persisted in the rhizosphere throughout the 30-month test period. The numbers of colony-forming units (CFU) ofPenicillium claviforme, as well as the ‘total’ number of CFU of micromycetes, were higher in the rhizoplane of the inoculated seedling in comparison with uninoculated plants. The numbers of CFU of phytotoxic micromycetes in the rhizosphere soil of inoculated seedlings were also proportionally higher. Apple tree growth was also reduced when seedlings were inoculated with the flucrescent bacteriumPseudomonas putida; however, no morphological changes were observed in the roots. Both micro-organisms introduced into the apple seedling rhizosphere caused changes in the microbial community. Inoculation withPenicillium claviforme andPseudomonas putida caused a decrease in the number of mycolytic bacteria in the rhizoplane of apple seedlings.     

Reactive oxygen metabolism in mycorrhizal and non-mycorrhizal citrus (Poncirus trifoliata) seedlings subjected to water stress

The effect of the arbuscular mycorrhizal (AM) fungus, Glomus versiforme, on growth and reactive oxygen metabolism of trifoliate orange (Poncirus trifoliata) seedlings was studied in potted plants under well-watered (WW) and water stressed (WS) conditions. Water stress significantly decreased root colonization. Shoot dry weight, plant height and stem diameter were higher in AM than in non-AM seedlings regardless of the water status. Inoculation with G. versiforme increased root dry weight and leaf number per plant of WW seedlings. There was less malondialdehyde (MDA) concentration in leaves and roots of AM seedlings, as well as lower hydrogen peroxide (H(2)O(2)) and superoxide anion radical (O(2)(-)) concentrations in AM roots under WW and WS conditions. AM inoculation did not affect the H(2)O(2) and O(2)(-) concentrations of WW leaves. Whether WS or not, AM symbiosis notably increased the guaiacol peroxidase (G-POD) activity of leaves, glutathione reductase (GR) activity of leaves and ascorbate peroxidase (APX) activity of roots. AM infection also markedly increased the APX activity of WS leaves. Soluble proteins and glutathione (GSH) in leaves and roots and ascorbate (ASC) in leaves were higher in WW AM than in WW non-AM seedlings. AM infection also enhanced the ASC and GSH contents of leaves and roots in WS seedlings. Cross-tolerance might occur in AM plants and be enhanced by AM symbiosis. Our results suggest that the increased concentrations of antioxidant enzymes and non-enzymatic antioxidants found in AM plants may serve to protect the organism against oxidative damage, enhancing drought tolerance.     

Glutathione-Mediated Alleviation of Chromium Toxicity in Rice Plants

A hydroponic experiment was conducted to determine the possible effect of exogenous glutathione (GSH) in alleviating chromium (Cr) stress through examining plant growth, chlorophyll contents, antioxidant enzyme activity, and lipid peroxidation in rice seedlings exposed to Cr toxicity. The results showed that plant growth and chlorophyll content were dramatically reduced when rice plants were exposed to 100 μM Cr. Addition of GSH in the culture solution obviously alleviated the reduction of plant growth and chlorophyll content. The activities of some antioxidant enzymes, including superoxide dismutase, catalase (CAT) and glutathione reductase in leaves, and CAT and glutathione peroxidase in roots showed obvious increase under Cr stress. Addition of GSH reduced malondialdehyde accumulation and increased the activities of these antioxidant enzymes in both leaves and roots, suggesting that GSH may enhance antioxidant capacity in Cr-stressed plants. Furthermore, exogenous GSH caused significant decrease of Cr uptake and root-to-shoot transport in the Cr-stressed rice plants. It can be assumed that GSH is involved in Cr compartmentalization in root cells.     

Endophytic colonization of rice by a diazotrophic strain of Serratia marcescens

Six closely related N2-fixing bacterial strains were isolated from surface-sterilized roots and stems of four different rice varieties. The strains were identified as Serratia marcescens by 16S rRNA gene analysis. One strain, IRBG500, chosen for further analysis showed acetylene reduction activity (ARA) only when inoculated into media containing low levels of fixed nitrogen (yeast extract). Diazotrophy of IRBG500 was confirmed by measurement of 15N2 incorporation and by sequence analysis of the PCR-amplified fragment of nifH. To examine its interaction with rice, strain IRBG500 was marked with gusA fused to a constitutive promoter, and the marked strain was inoculated onto rice seedlings under axenic conditions. At 3 days after inoculation, the roots showed blue staining, which was most intense at the points of lateral root emergence and at the root tip. At 6 days, the blue precipitate also appeared in the leaves and stems. More detailed studies using light and transmission electron microscopy combined with immunogold labeling confirmed that IRBG500 was endophytically established within roots, stems, and leaves. Large numbers of bacteria were observed within intercellular spaces, senescing root cortical cells, aerenchyma, and xylem vessels. They were not observed within intact host cells. Inoculation of IRBG500 resulted in a significant increase in root length and root dry weight but not in total N content of rice variety IR72. The inoculated plants showed ARA, but only when external carbon (e.g., malate, succinate, or sucrose) was added to the rooting medium.