芘降解菌Pseudomonas sp.PR3的植物促生特性及其对芘胁迫下水稻生长的影响北大核心CSCD

农田中不断积累的多环芳烃不仅严重影响作物生长,同时增加粮食安全风险。筛选兼具促进植物生长特性和降解污染物功能的微生物菌株是解决上述问题的一种有效手段。从油田附近生长的植物根表分离得到一株具有芘降解能力,同时还具有溶磷、产吲哚乙酸和铁载体等植物促生特性的菌株PR3,经16S rDNA序列同源性分析确定为假单胞菌(Pseudomonas sp.)。菌株PR3在无机盐培养液中生长14 d后,对芘(20 mg/L)的降解率可达94%,对萘(50 mg/L)、菲(50 mg/L)、苯并(a)芘(10 mg/L)的降解率也分别达到92%,84%和47%。同时,该菌株7 d内最大溶磷量为756.25 mg/L,2 d内IAA合成量可达14.46 mg/L,4 d内生成铁载体的活性单位可达58.53%。在不同芘污染浓度处理下的盆栽实验表明,接种PR3可有效促进水稻生长并提高根际土壤中芘的降解,去除率可达72.02%-86.22%,同时显著降低水稻根及地上部中的芘含量,分别为21.81%-53.01%和49.81%-57.17%。因此,菌株PR3有助于实现芘污染土壤的生态修复以及降低作物芘暴露的风险。     

锐劲特降解菌株R-2的分离、鉴定及降解特性

从长期受锐劲特污染的农药厂活性污泥中分离到一株锐劲特降解菌株R-2, 根据其生理生化特征和16S rRNA基因序列同源性分析, 将该菌株鉴定为Paracoccus sp.。菌株R-2能以锐劲特为唯一碳源生长, 在含有50 mg/L的锐劲特的基础盐培养基中, 3 d的降解率达到85%。菌株R-2降解锐劲特的最适温度为30 °C, 最适pH值为6.0?7.0, 其降解锐劲特的效率与锐劲特初始浓度呈负相关。添加0.1 mmol/L的Zn2+或Fe3+能够显著促进菌株对锐劲特的降解。灭菌与非灭菌土壤降解试验表明, 菌株R-2均可以在10 d内降解63.4%?71.2%的100 μg/g的锐劲特。     

烟嘧磺隆降解菌Chryseobacterium sp.LAM-M5的分离、鉴定及其降解机理研究

为获得更丰富的烟嘧磺隆降解菌资源,以合肥某烟嘧磺隆生产厂活性污泥为研究对象,从中分离并筛选得到一株能以葡萄糖为唯一碳源、烟嘧磺隆为唯一氮源生长的菌株,根据其表型特征、16S rRNA序列相似性、DNA-DNA杂交值(DDH)和平均核苷酸一致性(ANI)的分析结果,将其鉴定为Chryseobacterium lacus ...     

烤烟根际烟碱降解细菌的多样性及其促生特性分析

【背景】挖掘兼具烟碱降解和植物根际促生细菌(Plant Growth-Promoting Rhizobacteria,PGPR)功能的细菌资源,有助于保护土壤质量,实现绿色种植。【目的】分析烤烟根际细菌多样性,筛选可降解高浓度烟碱的PGPR。【方法】采用纯培养法在选择性培养基上分离烟碱降解细菌。通过BOXA1R-PCR分析技术、16SrRNA基因测序及系统发育树构建,对菌株的遗传多样性和分类学地位进行分析。进一步评价了菌株的吲哚乙酸(Indole-3-Acetic Acid,IAA)活性、溶磷能力、病原菌拮抗能力等PGPR指标,以筛选出高效PGPR,最后通过盆栽试验验证其促生效果。【结果】分离得到58株烟碱降解细菌,根据BOXA1R-PCR指纹图谱选取11株菌进行16S rRNA基因序列测定,结果表明,58株菌分别属于芽孢杆菌属(Bacillus)、假单胞菌属(Pseudomonas)、拉乌尔菌属(Raoultella)和短波单胞菌属(Brevundimonas)4个属,以芽孢杆菌属(Bacillus)为优势菌属。58株细菌中48.28%的菌株可产IAA,27.59%具备溶磷能力,37.93%具备纤维素降解能力,G2-13、G2-3及HT2-8因促生与抗病特性突出而被筛选为目标功能菌。盆栽试验结果表明,G2-13菌株对幼苗生长的促进作用明显,可使株高与地上部鲜重分别增加33.05%与53.32%。【结论】烤烟根际存在较为丰富多样的烟碱降解细菌,它们在种植业上具有潜在的应用价值。     

内生型解淀粉芽孢杆菌YTB1407对甘薯根系的促生作用及其调控机理

本实验室从西洋参根中分离筛选获得兼具生防和促生效果的内生型解淀粉芽孢杆菌YTB1407.为调查其应用潜力,以甘薯为材料,以无菌水灌根处理(CK)为对照,采用不同浓度YTB1407菌悬液灌根盆栽甘薯植株,通过对甘薯生长前期3个主要生育时间点YTB1407在植株内的内生定殖检测和根系表型效应比较,筛选菌悬液灌根的最适处理浓度.对根系内源生长素吲哚乙酸(IAA)、细胞分裂素玉米素核苷(ZR)、反式玉米素核糖核苷(t-ZR)的含量和吲哚乙酸氧化酶类中的吲哚乙酸氧化酶(IAAO)、过氧化物酶(POD)和多酚氧化酶(PPO)的活性进行分析.结果表明: YTB1407促进了甘薯生长前期根系统的定殖、不定根幼根的伸长生长及侧根的发生,提高了根系活力;在甘薯生长后期形成了更大的吸收根系生物量和更低的地上部和总根系生物量比.其中OD₆₀₀值为0.50的菌悬液处理(T_0.50)促生效应显著,在甘薯茎叶封垄期的单株块根鲜质量和有效薯块数量最高.YTB1407通过提高ZR、t-ZR含量,降低IAAO活性、提高PPO活性和IAA含量以及(t-ZR+ZR)/IAA,促进块根分化建成初期甘薯不定根幼根向块根的分化;通过降低IAAO、PPO活性和提高IAA含量,降低(t-ZR+ZR)/IAA,促进块根分化建成后期甘薯分化根向块根的建成.     

吲哚-3-甲醛对志贺菌感染的保护作用及其机制研究

【目的】探究吲哚-3-甲醛(indole-3-carboxaldehyde,I3A)对志贺菌感染性结肠炎的缓解作用及其机制研究。【方法】体外实验,通过微量肉汤稀释法测定I3A对志贺菌的最低抑菌浓度(minimum inhibitory concentration,MIC)和I3A的抗菌活性,运用酶标仪测定600 nm处不同浓度I3A作用的菌液吸光度(A₆₀₀),并通过平板菌落计数检测志贺菌量。体内实验,将18只小鼠随机分为对照(NC)组、SF301组和SF301+I3A组。然后SF301+I3A组进行连续8 d的200 µL I3A灌胃处理,同时NC组和SF301组用200 µL无菌水灌胃处理,SF301组和SF301+I3A组于灌药第4天制备志贺菌感染性结肠炎小鼠模型,体重和疾病活动指数(disease activity index,DAI)用于评价小鼠状况。上述实验结束后将所有小鼠处死,取中段结肠进行组织病理学检测观察炎症情况,下段结肠检测白细胞介素-6 (interleukin-6,IL-6)和肿瘤坏死因子-α (tumor necrosis factor-α,TNF-α)的表达水平,粪便、盲肠内容物检测载菌量。同时进行14 d的I3A处理小鼠监测I3A的体内应用安全性。【结果】体外实验：I3A的MIC为128 µg/mL,不同浓度的I3A涂板计数和A₆₀₀结果与对照组比较,均有统计学差异(均P<0.05)。体内实验：与SF301组相比,SF301+I3A组小鼠体重和DAI更接近NC组,肉眼观结肠组织无明显损伤,组织病理学检查未见明显炎症,载菌量和炎性因子均有统计学差异(均P<0.05);I3A对小鼠体重变化无显著影响(P>0.05)。【结论】本研究证明I3A在志贺菌感染性结肠炎中对肠道具有保护作用,I3A可通过抑制志贺菌载菌量、降低结肠损伤和炎症水平来缓解志贺菌感染性结肠炎,并且I3A在体内没有显示毒性。     

荧光假单胞菌对食用菌的促生作用及其机理

从小麦根际分离到1株对食用菌有较强促生作用的荧光假单胞菌,命名为假单胞菌P2-10(Pseudomonas sp.P2-10)。该菌株与平菇天达300(Pleurotus ostreatus Td 300)、杏鲍菇杏6(Pleurotus eryngii X6)或鸡腿菇瑞7(Coprinus comatus R7)混合培养,可显著提高这些食用菌菌丝生长速度及诱导并促进子实体的形成和发育。假单胞菌P2-10对食用菌的促生作用来自其代谢产物,可能是通过其1-氨基环丙烷-1-羧酸(ACC)脱氨酶降解食用菌产生的ACC、减少食用菌乙烯的合成及乙烯对菌丝生长和子实体发育的抑制作用。     

阿特拉津降解菌Acinetobacter sp. DNS32对无机氮源的响应

【目的】研究Acinetobacter sp.DNS32的生长、阿特拉津降解能力和降解基因转录水平的表达对无机氮素的响应关系,为菌株的工程应用提供指导与理论基础。【方法】以Acinetobactersp.DNS32为对象,采用摇瓶法研究菌株在阿特拉津培养基中菌株生长情况及降解能力对外加硝态氮与铵态氮的响应关系,利用荧光定量PCR技术检测DNS32降解基因表达量对外加无机氮源的响应关系。【结果】外加无机氮源可以促进DNS32菌株的生长,提高阿特拉津降解能力,无机氮源对DNS32菌株的trzN、atzB和atzC 3种降解基因表达均有促进作用,加入无机氮源的试验处理中DNS32菌株trzN基因的表达量最高可达对照的11.252±2.408倍,推断DNS32菌株的这3种降解基因所编码的酶是稳定表达的组成酶。【结论】DNS32降解阿特拉津不受"氮饥饿"诱导机制调控,且无机氮源的存在对菌株的生长与降解有促进作用,因此菌株在土壤修复实践中具有广阔的应用前景。     

一株苯胺降解菌的分离及其苯胺降解特性的研究

目的:筛选高效苯胺降解菌并研究其降解特性,为利用微生物进行苯胺环境污染物修复奠定基础.方法:利用含苯胺的A15培养基分离筛选苯胺降解菌,探讨苯胺降解最佳条件、降解代谢途径,利用16S rDNA基因扩增测序法对株菌进行分子鉴定.结果:获得了一株以苯胺为惟一碳源、氮源生长的高效苯胺降解菌AN6-4.该菌降解苯胺的最高浓度为2500mg/L,降解苯胺的最适温度和pH值分别为30℃、7.0;该菌在60h内可以将1500mg/L浓度的苯胺完全降解;重金属离子对该菌株降解苯胺有不同程度的抑制作用;代谢机制研究表明,该菌株可以诱导合成邻苯二酚-2,3-双加氧酶并分泌到胞外降解苯胺;16S rDNA基因序列同源性比较结果表明该菌属芽孢杆菌的一种.结论:所获得的苯胺降解菌对于研究苯胺降解机制和苯胺环境污染物的生物修复具有重要的理论和潜在应用价值.     

3-苯氧基苯甲酸降解菌Sphingomonas sp. SC-1降解苯酚环境条件及其降解中间产物的研究

【目的】探究高效降解3-苯氧基苯甲酸(3-Phenoxybenzoic acid,3-PBA)的鞘氨醇单胞菌(Sphingomonas sp.) SC-1对苯酚的降解特性。【方法】采用HPLC测定微生物降解体系中苯酚残留量,考察环境条件对菌株SC-1降解苯酚的影响;分析不同培养时间苯酚降解体系混合样品的HPLC谱图,确定其降解中间产物。【结果】菌株SC-1能在基础盐培养基中以苯酚为唯一碳源和能源生长,在初始pH 7.0、30 °C条件下,24 h可完全降解100 mg/L苯酚;Cu2+、Ba2+、Mn2+等对其降解苯酚有不同程度的抑制作用;HPLC谱图分析,初步确定邻苯二酚是菌株SC-1降解苯酚的中间产物,且该菌株可在48 h内完全降解100 mg/L邻苯二酚。【结论】菌株SC-1对苯酚及邻苯二酚均有较强的降解能力,为完善3-PBA的降解途径及污染3-PBA或含酚废水或含酚农药残留的降解提供了数据参考。     

An <Emphasis Type="Italic">ipdC</Emphasis> gene knock-out of <Emphasis Type="Italic">Azospirillum brasilense</Emphasis> strain SM and its implications on indole-3-acetic acid biosynthesis and plant growth promotion

The indole-3-pyruvate decarboxylase gene (ipdC), coding for a key enzyme of the indole-3-pyruvic acid pathway of IAA biosynthesis in Azospirillum brasilense SM was functionally disrupted in a site-specific manner. This disruption was brought about by group II intron-based Targetron gene knock-out system as other conventional methods were unsuccessful in generating an IAA-attenuated mutant. Intron insertion was targeted to position 568 on the sense strand of ipdC, resulting in the knock-out strain, SMIT568s10 which showed a significant (∼50%) decrease in the levels of indole-3-acetic acid, indole-3-acetaldehyde and tryptophol compared to the wild type strain SM. In addition, a significant decrease in indole-3-pyruvate decarboxylase enzyme activity by ∼50% was identified confirming a functional knock-out. Consequently, a reduction in the plant growth promoting response of strain SMIT568s10 was observed in terms of root length and lateral root proliferation as well as the total dry weight of the treated plants. Residual indole-3-pyruvate decarboxylase enzyme activity, and indole-3-acetic acid, tryptophol and indole-3-acetaldehyde formed along with the plant growth promoting response by strain SMIT568s10 in comparison with an untreated set suggest the presence of more than one copy of ipdC in the A. brasilense SM genome.     

Pseudomonas induces salinity tolerance in cotton (Gossypium hirsutum) and resistance to Fusarium root rot through the modulation of indole-3-acetic acid

Abiotic stresses cause changes in the balance of phytohormones in plants and result in inhibited root growth and an increase in the susceptibility of plants to root rot disease. The aim of this work was to ascertain whether microbial indole-3-acetic acid (IAA) plays a role in the regulation of root growth and microbially mediated control of root rot of cotton caused by Fusarium solani. Seed germination and seedling growth were improved by both NaCl and Mg₂SO₄ (100 mM) solutions when treated with root-associated bacterial strains Pseudomonas putida R4 and Pseudomonas chlororaphis R5, which are able to produce IAA. These bacterial strains were also able to reduce the infection rate of cotton root rot (from 70 to 39%) caused by F. solani under gnotobiotic conditions. The application of a low concentration of IAA (0.01 and 0.001 μg/ml) stimulated plant growth and reduced disease incidence caused by F. solani (from 70 to 41–56%, respectively). Shoot and root growth and dry matter increased significantly and disease incidence was reduced by bacterial inoculants in natural saline soil. These results suggest that bacterial IAA plays a major role in salt stress tolerance and may be involved in induced resistance against root rot disease of cotton.     

Auxin activity and molecular structure of 2-alkylindole-3-acetic acids

2-Methylindole-3-acetic acid (2-Me-IAA) is a known auxin, but its 2-ethyl homologue has been considered inactive. Here we show that the compound previously bioassayed as 2-ethylindole-3-acetic acid (2-Et-IAA) was, in fact, 3-(3-methylindol-2-yl)propionic acid. The proper 2-Et-IAA and its 2-(n-propyl) homologue (2-Pr-IAA) are prepared, unambiguously characterized, and their auxin activity is demonstrated in the Avena coleoptile-section straight-growth test. Their half-optimal concentrations are approximately the same as for 2-Me-IAA (2 × 10^–5mol L^–1), and hence about ten times larger than for unsubstituted indole-3-acetic acid (IAA) and its derivatives alkylated in positions 4, 5, 6 or 7. The optimal response elicited by 2-Et-IAA and 2-Pr-IAA is about half that observed for 2-Me-IAA. These characteristics place the three 2-alkyl-IAAs along the borderline between the classes of strong and weak auxins, thus corroborating the results of interaction similarity analysis, a mathematical approach based on the capability of auxin molecules to participate in non-bonding interactions with a generalized receptor protein. X-ray diffraction analysis shows no explicit structural features to be blamed for the decrease in auxin activity caused by attaching a 2-alkyl substituent to the IAA molecule; sterical interference of the 3-CH₂COOH group and the 2-alkyl moiety is barely recognizable in the crystalline state. Quantum-chemical calculations and molecular dynamics simulations suggest that 2-alkyl-IAAs, in the absence of crystal-packing restraints, prefer conformations with the CH₂-COOH bond tilted to the heterocyclic ring system. Substantially higher conformational energy (and hence lower abundance) is predicted for planar conformers which were previously shown to prevail for IAA and many of its derivatives substituted in the benzene moiety of the indole nucleus. This shift in the rotational preferences of the -CH₂COOH moiety may be one of the reasons for the reduced plant-growth promoting activity of 2-alkyl-IAAs.     

Physiological and genetic analysis of root responsiveness to auxin-producing plant growth-promoting bacteria in common bean (Phaseolus vulgaris L.)

Plant root development can be largely affected through the association of roots with plant growth-promoting rhizobacteria (PGPR). However, little is known about the identity of plant genes enabling such PGPR-plant root associations. Differences in the responsiveness to PGPR among cultivars suggest genetic variation for this trait within germplasm. In this study, two genotypes of common bean (Phaseolus vulgaris L.), BAT477 and DOR364, were identified showing contrasting responsiveness in root development to inoculation with the PGPR Azospirillum brasilense Sp245. Inoculation with an A. brasilense Sp245 mutant strain strongly reduced in auxin biosynthesis or addition of increasing concentrations of exogenous auxin to the plant growth medium, indicated that the differential response to A. brasilense Sp245 among the bean genotypes is related to a differential response to the bacterial produced auxin. To further assess the role of the plant host in root responsiveness, a population of Recombinant Inbred Lines (RILs) of the DOR364×BAT477 cross was used to evaluate the efficacy of exogenous auxin on root development. We detected significant phenotypic variation among the RILs for basal root formation during germination upon addition of auxin to the growth medium. Genetic analysis revealed two quantitative trait loci (QTLs) associated with basal root responsiveness to auxin of which one explained 36% of the phenotypic variation among the RILs. This latter QTL mapped to the same location as a QTL for root tip formation at low P, suggesting that the host effect on root responsiveness to IAA interacts with specific root development. Also, significant correlations between basal root responsiveness to auxin and growth, root tips and root dry weight at low P were identified. To our knowledge, this is the first report on QTL detection for root responsiveness to auxin.     

IAA Producing Enterobacter sp. I-3 as a Potent Bio-herbicide Candidate for Weed Control: A Special Reference with Lettuce Growth Inhibition

Development of bio-herbicides is an emerging method to weed management in agricultural field. Very few studies were conducted on identification of microbial bio-herbicides to weed control. The present study was aimed to isolate and identify the effective bio-herbicide potential bacterium from soil and assess their role on plant growth inhibition. Three-hundred and one rhizobacteria were isolated from agriculture field soil samples collected from various parts of Republic of Korea. Two bacterial strains, I-4-5 and I-3 were significantly reduced the seedling growth of radish when compared to their controls. The highest rate of seedling growth inhibition was observed in I-3 bacterial isolate treatment in lettuce and radish. The mechanism of an effective bio-herbicide I-3 to plant growth inhibition was determined by analyzing IAA in their culture medium. IAA biosynthesis pathway of Enterobacter sp. I-3 was identified as tryptophan-dependent pathway and its production was increased due to addition of tryptophan in culture medium as quantified by using GC–MS SIM. In an in vitro study revealed that I-3 bacterial culture exudate combined with tryptophan significantly decreased leaf length, leaf width, root length and increased the number of lateral roots of lettuce. Indeed, the genomic DNA of I-3 bacterium was isolated and 16S rDNA was sequenced to find out the name of the bacterium. Based on phylogenetic analysis, I-3 isolate was identified and named into Enterobacter sp. I-3. The results of this study suggest that the utilization of Enterobacter sp. I-3 to crop field can be act as a potential bio-herbicide against weed growth.

Electronic supplementary material

The online version of this article (doi:10.1007/s12088-015-0515-y) contains supplementary material, which is available to authorized users.     

Optimization of indole acetic acid production by isolated bacteria from Stevia rebaudiana rhizosphere and its effects on plant growth

The ability to synthesize Indole-3-acetic acid (IAA) is widely associated with the plant growth promoting rhizobacteria (PGPR). The present work deals with isolation and characterization of such bacteria from the rhizosphere of medicinal plant Stevia rebaudiana and optimization of IAA production from its isolates. The optimization of IAA production was carried out at different pH and temperature with varied carbon and nitrogen sources of culture media. Out of different isolates obtained, three of them were screened as efficient PGPRs on the basis of different plant growth promoting attributes. Isolates CA1001 and CA2004 showed better production of IAA at pH 9 (91.7?µg?ml^?1) and at temperature 37?°C (81.7?µg?ml^?1). Dextrose (1%) was found to be the best carbon source for isolate CA1001 with 104?µg?ml^?1 IAA production. Isolate CA 2004 showed best production of IAA 36?µg?ml^?1 and 34?µg?ml^?1 at 1.5% and 1% Beef extract as nitrogen source respectively. Isolate CA 1001 showed 32?µg?ml^?1 IAA production at 0.5% nicotinic acid concentration. From the current study, CA1001 and CA2004 emerged as noble alternatives for IAA production further which also resulted in root and shoot biomass generation in crop plants, hence can be further used as bio-inoculants for plant growth promotion.     

The effect of kinetin on the level of an indoleacetylaspartic acid like substance in germinating seeds and young plants of Phaseolus vulgaris

Kinetin treatment increased the level of an extractable indoleacetyl conjugate, apparently indoleacetylaspartic acid (IAAsp) in germinating seeds and young plants of Phaseolus vulgaris L. cv. Alabaster. The level of this substance in extracts of treated seeds was always higher than that from water treated seeds irrespective of whether the seeds had been extracted 24, 48 or 72 h after the beginning of the treatment. In all parts of young bean plants treated with kinetin there was more of the substance than in the corresponding parts of water treated plants.     

Indole compounds released by the ectendomycorrhizal fungal strain MrgX isolated from a pine nursery

The production and metabolism of indole compounds in pure cultures of the ectendomycorrhizal strain MrgX, a common symbiont of Scots pine in forest nurseries, were investigated. Different indole compounds produced by this fungus were purified and identified by thin-layer chromatography, high-performance liquid chromatography and mass spectrometry. Indole-3-acetic acid (IAA) and indole-3-carboxylic acid were the most abundant. Although MrgX is able to synthesize IAA when cultivated on a medium without tryptophan, much higher IAA production was obtained when 1 mM tryptophan was added. Buffering of the medium at pH 5.8 was shown to be essential for IAA accumulation in the culture filtrate. In vitro IAA-synthesizing activity of the enzymes extracted from the mycelia of MrgX was also maximal when mycelia were grown in a buffered, tryptophan-supplemented medium. The hydrogen ion concentration strongly affected in vivo activity of IAA-synthesizing enzymes. This activity was rather weak at acid pH and was stimulated by increase in pH up to 8.5. These results and their possible significance for ectendo-mycorrhizal symbiosis are discussed with reference to the hormonal metabolism of ectomycorrhizal fungi and ectomycorrhizae.     

The influence of kinetin on the endogenous content of indoleacetic acid in swelling seeds of Phaseolus, Zea and Pinus and young plants of Phaseolus

Treatment of different plant materials, seeds of Phaseolus vulgaris, Zea mays and Pinus silvestris and young plants of Phaseolus, with kinetin increased the level of extractable IAA. For seeds this increase was most pronounced in bean seeds, which contained the lowest amount of endogenous IAA and cytokinins, and lower in maize seeds with high endogenous content of IAA and cytokinins. – For young bean plants the kinetin treatment significantly increased the extractable amounts of IAA from all parts of the plant, hypocotyls, cotyledons, epicotyls and primary leaves, when the cut plants were placed for 24 h in kinetin solution. For plants sprayed with kinetin solution only the primary leaves showed a significantly higher level of extractable IAA, which could be explained by the fact that the plants were growing very close together, so that the primary leaves received most of the kinetin during spraying.