荧光假单胞杆菌RB-42 RB-89的趋化性与烟草根部定殖研究

烟草PGPR(Plant Growth-Promoting Rhizobacteria)菌株RB-89、RB-42对烟草根部收集液有较强的趋化性。在稀释浓度为10^-5时，RB-89、RB-42的趋化值依次为1．79、1．83。RB-42和RB-89对氨基酸趋化性差异显著，对酪氨酸、脯氨酸、甘氨酸、笨丙氨酸表现出较强的趋化性，对组氨酸、苏氨酸没有表现出趋化性；RB-42和RB-89菌株对有机酸的趋化性没有明显区别，只是RB-42对乳酸的趋化性表现得比对其它有机酸的强些；菌株对糖类物质的趋化性最弱。菌株RB-89、RB一42在烟株根表的定殖受灭菌、趋化剂影响较大，土壤灭菌、趋化剂处理可增加菌株在根表的定殖量。     

荧光假单胞杆菌RB-42、RB-89促烟草生长机理初探

盆栽试验结果表明,接种Pseudomonas fluorescens RB-42和RB-89菌剂,能促进烟草幼苗对P、K的吸收,其中对P吸收的促进作用比对K明显;并促进了P、K元素在植株中的积累转化。用P. fluorescens RB-42和RB-89菌液浸根处理,烟草幼苗,幼苗叶鲜重、根重和烟叶中的P、K元素含量均高于不接菌处理(CK1)。RB-42、RB-89菌悬液处理烟草幼苗的叶鲜重分别比CK1增加8.42%和37.18%,比接种R4菌株处理(CK2)增加2.34%和29.44%;叶干重分别比CK1增加23.81%和40.47%,比CK2增加1.69%和15.68%;株高比CK1增长26.32%和64.29%,RB-89比CK2增高15%;叶面积比CK1增加32.48%和67.21%,比CK2增加11.65%和40.92%。用RB-42、RB-89菌悬液处理烟株,根际的质子分泌作用增强,RB-89处理引起根际的pH值下降1.5个单位。RB-42和RB-89能够引起根际微环境中Mn、Zn、P、K、Ca、Mg离子含量的增加,其中根际分泌物Zn元素的浓度含量变化最大。     

恶臭假单胞菌P861(Gus)在油菜根部定殖的生态研究

本研究采用Gus 基因标记技术和常规方法跟踪考察了恶臭假单胞菌P861(Gus) 在缩影系统油菜根圈的定殖情况,以及缩影系统内土壤类型、土壤含水量对根部定殖的影响。土壤含水量分别为60% FC和75% FC时,P861(Gus) 在砂姜黑土中的定殖水平高于50% FC的,不但能散布至种子下8cm 以内的根段部位,且定殖水平分别为7.5×10²和2.8×10³cfu·g^-1。在灰潮土缩影中,P861(Gus) 在油菜根圈的定殖动态表现为在油菜播种后3 ～6 天,定殖密度可达最高水平(5.5×10⁶cfu·g^-1) ,然后急速下降,最后保持在一个相对稳定的较低水平(7.6×10²cfu·g^-1) 。P861(Gus) 在不同根段部位的定殖密度并无从上到下逐渐递减的规律。     

恶臭假单胞菌P861(Gus)在油菜根部定殖的生态研究

本研究采用Gus 基因标记技术和常规方法跟踪考察了恶臭假单胞菌P861(Gus) 在缩影系统油菜根圈的定殖情况,以及缩影系统内土壤类型、土壤含水量对根部定殖的影响。土壤含水量分别为60% FC和75% FC时,P861(Gus) 在砂姜黑土中的定殖水平高于50% FC的,不但能散布至种子下8cm 以内的根段部位,且定殖水平分别为7.5102和2.8103cfug-1。在灰潮土缩影中,P861(Gus) 在油菜根圈的定殖动态表现为在油菜播种后3 ～6 天,定殖密度可达最高水平(5.5106cfug-1) ,然后急速下降,最后保持在一个相对稳定的较低水平(7.6102cfug-1) 。P861(Gus) 在不同根段部位的定殖密度并无从上到下逐渐递减的规律。     

巨大芽孢杆菌在油菜根部定殖和促生作用的研究

采用基因标记技术和常规方法跟踪巨大芽孢杆菌A6 (gusA)在缩影系统油菜根际的定殖情况。A6 (gusA)菌在油菜不同根段部位的定殖密度表现从上到下逐渐递减的现象。随着接种后时间的延长而逐渐下降。在根段 8cm以外的根区几乎检测不到接种菌。在油菜播种后 3d ,定殖密度可达最高水平 (8 7×10 5cfug-1根 ) ,然后急速下降 ,30d后保持相对稳定的较低水平 (2 2× 10 2 cfug-1根 )。在促生试验中 ,表现在不同程度上增加植株干重、全氮、全磷和全钾的含量     

用接合转移方法构建杀虫防病荧光假单胞菌

以经过改造的含有转座子Ｔｎ５的自杀质粒ｐＳＵＰ２０２１为载体，通过接合转移将苏云金杆菌杀虫蛋白基因ｃｒｙ Ｉ Ａ（ｃ）片段插入生防细菌荧光假单胞菌Ｐ３０３菌株染色体组。Ｓｏｕｔｈｅｒｎ和Ｗｅｓｔｅｒｎ印迹分析分别证实杀虫基因的导入和杀虫蛋白的表达。室内生物测定结果表明新构建的ＰＴ２１０、ＰＴ２１２等荧光假单胞菌工程菌菌株不仅保持了野生型自然菌株对小麦全蚀病良好的抑菌活性，而且表现出对小菜蛾和玉米暝     

内生菌YN201728的定殖能力及其防治烟草白粉病的效果研究

为研究生防菌YN201728在烟草体内的定殖规律及防病机制,本研究利用其绿色荧光标记菌株YN28-P43GFPmut3a实时动态观测标记菌的定殖部位及密度,并探究其对温室烟草白粉病的盆栽防效与定殖的关系。结果表明,YN28-P43GFPmut3a发酵液处理烟草种子和幼苗后,种子内的标记菌含量可达2.18×10⁶ CFU·g^-1,在幼苗的根表土、根际土、根、茎、叶等组织中均能检测到标记菌,且其定殖密度表现为根表土>根际土>根>茎>叶。激光共聚焦显微镜观察显示,标记菌主要聚集在烟草根表皮、木质部导管、茎表皮、韧皮部及维管束组织、叶片表面和叶肉细胞间隙以及种皮与胚等部位。盆栽防效试验结果表明,YN201728野生型和标记菌株对烟草白粉病均有较好的保护和治疗效果,持效期长达21 d。此外,烟草叶片中内生菌的定殖量与其防效呈正相关。本研究结果表明,内生菌YN201728在烟草体内有良好的定殖和防治白粉病效果,具很好的开发潜力,为烟草病害的生物防治提供了一定的理论基础。     

内生枯草芽孢杆菌B-001菌株内生定殖研究及生物学特性

用浸种、浸根、淋根、伤茎、伤叶和喷雾等接种方法测试枯草芽孢杆菌B-001菌株入侵烟草植株的途径,发现该菌可通过自然孔口和伤口入侵寄主植物;取接种植株的茎部组织切片在电镜下观察,发现该菌株在烟草的微管束和细胞内定殖。生长特性研究结果表明该菌株最适生长pH为7.5,最适生长温度为30℃,48～96h内处于稳定生长期。     

巨大芽胞杆菌WY4的GFP标记及其在小白菜上的定殖

解磷菌(phosphate solubilizing bacteria,PSB)可以通过提高土壤有效磷含量而增加作物产量,目前已有许多解磷菌被分离并应用于农业生产中,但关于解磷菌在植物根际中的定殖情况仍缺乏系统性的研究.WY4为本实验室前期从小白菜(Brassica chinensis)根际分离得到的一株高效解磷菌,本研究利用绿色荧光蛋白(green fluorescent protein,GFP)标记技术研究了WY4在小白菜根际及土壤中的定殖规律.与原菌株相比,GFP标记对菌株WY4-GFP生长及解磷活性具有较小影响,同时在促进小白菜生长上WY4-GFP与WY4无显著性差异;WY4-GFP具有持久的定殖能力(接种21d的自然土及30 d的小白菜根际土中,WY4-GFP的定殖数量分别为105和104 CFU/g左右),同时随时间的增加WY4-GFP在土壤中定殖数量逐渐减少;WY4-GFP在小白菜根冠及分生区大量定殖,在伸长区及侧根根毛处数量较少,同时表皮细胞间隙上也有较多的标记菌株.研究结果表明,WY4-GFP在小白菜根际及土壤中具有良好的定殖能力,这为后期深入研究解磷菌与植物间的关系提供了重要参考.     

发光酶基因(luxAB)标记的荧光假单胞菌CP1108的定殖能力研究

利用三亲本杂交方法,将luxAB发光酶基因标记至荧光假单胞菌(Pseudomonas fluorescens)CP1108上,研究标记菌株CP1108L的个体生态学特征及其在棉花根圈的定殖动态.结果表明,标记菌株连续传代20次均未发生质粒丢失现象,标记质粒在受体菌株中较为稳定,CP1108L菌株的生长及部分生理生化特性...     

枯草芽孢杆菌Tu-100对油菜根系分泌物所含氨基酸的趋化性研究

应用水培的方法采集油菜根系分泌物;采用高效液相色谱测定出油菜根系分泌物中所含的16种氨基酸;使用烧杯法和平板趋化法进行趋化性试验,分别测定枯草芽孢杆菌Tu-100对16种氨基酸、油菜根系和油菜菌核的趋化性。结果表明Tu-100仅对组氨酸、脯氨酸、精氨酸、天门冬氨酸和谷氨酸等5种氨基酸有趋化性,其中组氨酸的趋化性最强;Tu-100对油菜根系和油菜菌核也有趋化性,对油菜根系的趋化性要大于油菜菌核。     

巴西大西洋森林中巴西松地面根部显示丛枝菌根真菌高定殖率和多样性

Almost 30 different arbuscularmycorrhizal fungi (AMF)species, distributed in different genera such as Glomus, Acaulospora,Scutellospora,Entrophospora,Ambispora,Kuklospora,Gigaspora,and Archeospora, have been identified in the root zone of Araucaria angustifolia, known as Brazil Pine. During our AMF survey in this ecosystem, our attention was called to the presence of many superficially growing Araucaria roots. Our hypothesis was that these roots were colonized with AMF because of the presence of AMF spores in organic material aboveground. Samples of these superficial roots and the organic substrate they were growing on were evaluated for their mycorrhizal status. DNA was extracted from the AMF colonized superficial roots and submitted to polymerase chain reaction (PCR) amplification using the NS31-AM1 primer pair, followed by cloning and sequencing. We found that the root colonization percentages were between 31% and 52%, and the number of AMF spores in the substrate ranged from 27 to 164 spores per 50 g dry substrate.The phylogenetic analyses and tree construction using maximum parsimony (MP) and neighbor-joining (NJ) methods identified 13 different species of the phylum Glomeromycota belonging to the genera Glomus, Funneliformis, Rhizophagus, Gigaspora, Acaulospora,and Archaeospora, and five isolates were identified only at the genus level. To our knowledge, this is the first report on Araucaria angustifolia with roots growing aboveground, producing runner roots that develop on dead tree trunks and organic material. The higher colonization of the aboveground roots than those commonly found in belowground Araucaria roots suggests that they may present active metabolic uptakeof nutrients.     

烤烟根系分泌物对烤烟幼苗生长和养分吸收的影响

连作是烟草栽培中的普遍现象,已引起生长抑制、产量下降和品质恶化等问题。本试验采用溶液培养的方法,在培养液中分别加入未分组和分组后的烤烟根系分泌物,研究根系分泌物对烤烟幼苗生长和养分吸收的影响。结果表明,加入未分组的烤烟根系分泌物显著抑制幼苗的生长,降低根系活力,并随加入量的增加抑制作用增强;加入酸溶性、碱溶性、中性组分的根系分泌物,均降低幼苗根系活力,以中性组分的抑制作用较强。三种不同组分的根系分泌物均显著降低根系对NO3-、PO43-、K+离子的吸收,其中中性组分对NO3-吸收的影响最大,而酸溶性组分对K+的吸收抑制作用较强。推测在烤烟根系分泌物中,可能存在多种抑制烤烟生长和养分吸收的化学物质。     

Effects of root exudates of Rj2Rj3- and Rj4-genotype soybeans on growth and chemotaxis of Bradyrhizobium japonicum

Almost all of the soybean cultivars (Glycine max L. Merr.) form nodules on their roots by infection with Bradyrhizobium japonicum. However, it has been observed that the soybean cultivars harboring nodulation conditioning genes, for instance Rj ₂, Rj ₃, and Rj ₄, do not form effective nodules with some strains of B. japonicum. Ishizuka et al. (1991b) classified the rhizobia isolated from field-grown soybeans into nodulation-types A, B, and C based on the compatibility with these Rj-soybean cultivars. Nodulation-type B is incompatible with Rj ₂ Rj ₃-cultivars and type C is incompatible with Rj ₄-cultivars. Nodulation-type A rhizobia are compatible with both Rj ₂ Rj ₃ - and Rj ₄-cultivars. Furthermore, the Rj-cultivars were found to prefer appropriate nodulation-type rhizobia for nodulation, that is, non-Rj-, Rj ₂ Rj ₃ -, and Rj ₄-cultivars prefer nodulation-types A, C, and B rhizobia, respectively (Ishizuka et al. 1991a). In the present study, in order to examine the mechanisms of preference of Rj-cultivars for rhizobial strains for nodulation, root exudates from Rj-cultivars were examined for their effects on the growth rate and chemotaxis of various types of B. japonicum strains.     

Observation of microbial colonization on the surface of rice roots along with their development and degradation

Microbial colonization on the surface of rice roots along with their development and degradation was examined throughout the growth period of rice plants with the naked eye, by light microscopy, and scanning electron microscopy. Four stages were recognized in the development and degradation processes of rice roots and microbial colonization pattern. At stage I, both crown and lateral roots were undergoing development, white, and covered with a mucigel layer. Microbial colonization was rarely observed. At stage II, lateral roots developed further, and they stained brownish due to the deposition of iron oxides/hydroxides on the root surface. Microbial colonies commonly developed along the grooves between epidermal cells. At stage III, lateral roots ceased to develop, and the root surfaces were widely covered with deposits of iron oxides/hydroxides. Microbial colonies were found inside epidermal and exodermal cells as well as on epidermal cells. At stage IV, the roots became dark brown or transparent. Most of the epidermal and exodermal cells disappeared from the root surface. Roots went through stage I to stage IV as they aged, and respective degradation stages were observed from the nodal roots from higher nodes to the nodal roots from lower nodes. There were fewer microorganisms on the surface of lateral roots than on crown roots. About 50% of the roots were at stages I and II on June 20 and July 20, which corresponded to the early to middle growth stages, while most of the roots were at stages III or IV from August 10.     

Survival of,and root colonization by,alginate-encapsulated Pseudomonas fluorescens cells following introduction into soil

Summary The survival of Pseudomonas fluorescens cells encapsulated in alginate beads and colonization of wheat roots was studied in soil microcosms inoculated with the cells in alginate beads of varying composition. Cells encapsulated in beads and introduced into a non-sterile loamy sand survived better than cells added directly to the same soil. A recovery/growth step for the bead-encapsulated cells was added before they were introduced into the soil, in an attempt to obtain optimal population levels in the soil. Further, bacterial populations that grew to the highest density in the beads subsequently showed the highest survival levels in soil. The addition of 3% skim milk, or 3% skim milk and 3% bentonite clay to all bead types consistently resulted in the highest survival of the encapsulated cells in soil. Root colonization by P. fluorescens was generally not impaired by the encapsulation in alginate. One week after inoculation into the soil, encapsulated cells in the various bead types were able to colonize the wheat rhizoplane at high population levels, similar to or exceeding those found when free cells were inoculated. In a second root colonization experiment the wheat rhizoplane was also efficiently colonized 7 weeks after the inoculant cells had been introduced into the soil in different bead types. In both assays, the cells encapsulated in beads amended with skim milk plus bentonite clay showed the highest root colonization rates. It is clear, therefore, that alginate-mediated establishment of inoculants can improve inoculant effectiveness.     

Production of B-group vitamins by plant growth-promoting Pseudomonas fluorescens strain 267 and the importance of vitamins in the colonization and nodulation of red clover

 We have estimated the production of water-soluble B vitamins by plant growth-promoting rhizobacterium Pseudomonas fluorescens strain 267 in a minimal medium with different C sources and at different pH values. In the minimal medium, strain 267 produced large amounts of niacin (0.92 μg ml^–1) and pantothenic acid (0.75 μg ml^–1), but also other vitamins such as biotin, thiamine, cobalamine and pyridoxine. The production of B vitamins was dependent on the C source and pH of the growth medium. By random Tn5 mutagenesis, thiamine and niacin auxotrophs were isolated from P. fluorescens strain 267 and mutants were used to evaluate the vitamin production on colonization of clover roots under controlled conditions. Red clover root colonization decreased by about 1 order of magnitude in the case of the niacin auxotroph. The vitamin auxotrophs of P. fluorescens in a mixed inoculation of clover with R. leguminosarum bv. trifolii strain 24.1 showed no plant growth-promotion activity. Received: 23 May 2000     

Ectomycorrhizal‐fungal colonization induces physio‐morphological changes in Quercus serrata leaves and roots

To determine how colonization by different ectomycorrhizal (ECM)‐fungal species affects the physiology and morphology of Quercus serrata seedlings, we assessed the net photosynthetic rate, the respiration rate of the lateral roots, leaf and root nitrogen (N) concentrations, specific leaf area, and specific root length in 9‐month‐old Q. serrata seedlings inoculated with Pisolithus tinctorius, Scleroderma citrinum, Laccaria amethystea, and Astraeus hygrometricus. While uninoculated control plants showed no colonization, the percentage of ECM colonization of root tips attained 35% with P. tinctorius and about 86% with the other three ECM species. Similar to ECM root colonization, the photosynthetic as well as the root‐respiration rates were higher in seedlings with S. citrinum, L. amethystea, and A. hygrometricus than that in the control and those with P. tinctorius. Both the photosynthetic and root‐respiration rates were positively correlated with ECM‐fungal colonization. Similar trends were observed for the N concentration, specific leaf area, and specific root length, which differed significantly among ECM‐fungal species and were related with ECM‐fungal colonization. The results suggest that both physiological and morphological traits are specific to ECM‐fungal species. As Q. serrata seedlings with high colonization can exhibit better resource‐acquisition ability, the identification of strongly colonizing ECM‐fungal species is essential. Comparisons with high‐ and low‐colonizing ECM‐fungal species improve our understanding of source–sink relationships in carbon allocation of forest tree species.     

The flavonoid naringenin enhances intercellular colonization of rice roots by<Emphasis Type="Italic"> Azorhizobium caulinodans</Emphasis>

The intercellular colonization of rice roots by Azorhizobium caulinodans and other diazotrophic bacteria has been studied using strains marked with the lacZ reporter gene. A. caulinodans were able to enter the roots of rice at emerging lateral roots (lateral root cracks) by crack entry and this was observed by light microscopy. After colonization of lateral roots, bacteria moved into intercellular space within the cortical cell layer of roots. Naringenin at 1×10^-5 and 5×10^-5 M concentration significantly enhanced root colonization. The role of nodABC and regulatory nodD genes was also studied; lateral root crack (LRC) colonization of rice was shown to be Nod factor and NodD independent. Lateral root crack colonization of rice was also observed with similar frequency following inoculation with Azospirillum brasilense and the colonization by A. brasilense was stimulated by naringenin and other flavonoid molecules.