巨大芽孢杆菌ZS-3溶无机磷机制及其对樟树的促生作用

旨在探究巨大芽孢杆菌ZS-3菌株溶无机磷特性和相应的溶磷机制,为利用和提高其改善土壤中难溶磷资源的利用率提供理论依据和技术途径。通过NBRIP培养基、钼锑抗比色法、稀释涂布法、酸碱滴定法等方法测定菌株的溶磷量、菌落生长情况和可滴定酸度,通过扫描电镜(SEM)观察Ca3(PO4)2表面形态特征,通过高效液相色谱法和对硝基苯磷酸二钠法测定发酵液中有机酸种类、含量和磷酸酶活性,选择2种不同类型的土壤,通过盆栽实验测定菌株ZS-3对樟树生长及土壤理化性质的影响。结果显示,菌株ZS-3在连续培养168 h期间最大溶磷量出现在培养96 h时,为188μg/mL;菌体数量最大达2.34×109 CFU/mL,出现在培养72 h时;培养初期24 h内pH下降速度最快,从初始8.06降至5.14,可滴定酸度呈缓慢上升趋势。SEM实验表明菌株ZS-3与Ca3(PO4)2作用后能使其表面变得凸凹不平。ZS-3的溶磷过程中酸性磷酸酶和碱性磷酸酶活性较低。ZS-3在培养过程中会产生大量有机酸,以外泌乙酸和甲酸为主,含量分别为484.12 ng/μL和255.1 ng/μL;施菌处理对2种不同类型土壤栽种的樟树都具有促生作用,土壤速效磷含量均提高且低磷土壤速效磷含量增长量更显著。菌株ZS-3能够高效溶解无机磷,促进樟树生长,有望为溶磷微生物肥料的开发提供物质资源。     

一株凝结芽孢杆菌的分离筛选及产孢条件优化

【背景】凝结芽孢杆菌除了具有一般乳酸菌的益生功能外,还具较强的耐酸、耐胆盐、耐高温、易贮存等生物特性。【目的】从泡菜中筛选一株性能优良的凝结芽孢杆菌用于微生态制剂的制备,并对其产孢率进行优化,为该菌株的进一步工业化生产提供参考依据。【方法】采用选择性培养基通过特定的培养条件,筛选到一株抑菌效果良好的产酸芽孢杆菌,并对其进行特异性引物的鉴定、16S rRNA基因序列分析及生理生化实验。通过单因素及正交试验对菌株的产芽孢条件进行优化。【结果】筛选得到一株凝结芽孢杆菌BC01,该菌株对大肠杆菌(Escherichia coli CVCC 1527)、鼠伤寒沙门氏菌(Salmonella typhimurium CVCC 2228)、产气荚膜梭菌(Clostridium perfringens CVCC 46)、猪霍乱沙门氏菌(Salmonella choleraesuis CVCC 503)等均有较强的抑制作用;模拟胃液处理120 min存活率达到94%;0.3%的胆盐存活率达到84.3%。单因素及正交试验优化后的最适培养基配方:糖蜜10.0 g/L,酵母浸出粉20.0 g/L,NaCl 5.0 g/L,K_2HPO_4 5.0 g/L,MnSO_4 10.0 mg/L;最适培养条件:接种量4%,温度45°C,初始pH 7.0,转速200 r/min,培养时间36 h。在该优化条件下,其活菌数最高达到6.7×10~9 CFU/mL,产孢率达到89.2%。【结论】筛选得到一株可用于微生态制剂的菌株——凝结芽孢杆菌BC01,对其产孢率进行了优化,为工业化生产奠定了基础。     

一株芽孢杆菌的分离和鉴定

从中国农业科学院北京畜牧兽医研究所鸡舍附近土壤中分离到一株芽孢杆菌P-25,并进行了分子鉴定。通过形态鉴定、革兰氏染色、生理生化测定、16SrRNA序列分析和系统发育树构建,确定该菌株为蜡状芽孢杆菌(Bacillus cereus),其16SrRNAGenBank登录号为GU271135。     

一株高效溶磷伯克霍尔德菌的筛选鉴定及对马尾松幼苗的促生作用

采用标准稀释平板法从马尾松根际土中分离溶磷细菌,利用钼锑抗比色法测定溶磷细菌的溶磷特性;通过分析溶磷菌的溶磷能力与发酵液pH的关系,以及液相色谱-质谱 (HPLC-MS)联用对发酵液中有机酸的测定,探究其溶磷机制;通过对接种溶磷菌马尾松盆栽苗生长、生理、土壤养分和土壤酶活性的测定,明确溶磷菌对马尾松生长和生理的影响。结果表明: 由马尾松根际土壤中共筛选到溶磷细菌16株,其中菌株WJ27溶磷效果最优,液体培养5 d时的溶磷量达411.98 mg·L^-1。经过表型观察、生理生化鉴定和系统发育树分析,发现菌株WJ27属于伯克霍尔德菌属;其对不同磷源的溶磷特性存在差异,溶磷能力依次为: Ca₃(PO₄)₂(220.85 mg·L^-1)＞AlPO₄(182.33 mg·L^-1)＞FePO₄·2H₂O(129.19 mg·L^-1)＞CaHPO₄·2H₂O (115.23 mg·L^-1)。胞外有机酸测定结果表明,该菌株通过分泌柠檬酸、丙二酸等有机酸降低发酵液中pH,进而发挥溶磷作用;盆栽试验结果表明,接种菌株WJ27对马尾松幼苗生长、生理、土壤养分和土壤酶活性有积极作用。与对照相比,接种WJ27的马尾松的苗高、主根长、侧根数量、地上部(茎、枝、叶)鲜重、干重和根系鲜重、干重分别增加了14.3%、36.9%、56.1%、44.7%、60.0%、158.3%和100.0%;叶绿素b、总叶绿素、地上部可溶性蛋白和可溶性糖、根系活力和根系可溶性蛋白分别增加了145.8%、45.2%、206.3%、59.4%、80.5%和260.0%;根系超氧化物歧化酶、过氧化物酶和地上部过氧化氢酶的活性分别提高了71.2%、197.5%和36.6%;根际土壤中速效氮、速效钾、速效磷含量和土壤脲酶、过氧化氢酶、磷酸酶活性分别较对照土壤显著增加18.1%、17.0%、11.9%和34.3%、45.5%、62.6%。说明接种WJ27可以改善土壤养分和土壤酶活性,进而促进马尾松幼苗的生长。     

一株产纤溶酶凝结芽孢杆菌的筛选及其生物学特性

为了筛选性能良好的产纤溶酶菌株,本研究通过脱脂乳平板及纤维蛋白平板双层筛选从传统发酵食品豆豉中,分离到1株具有溶解纤维蛋白能力的凝结芽孢杆菌(Bacillus coagulans) HQ-1。分析其发酵上清液中纤溶酶的纤溶能力、组成特征和作用方式,并研究菌株HQ-1生物学特性。结果表明:菌株HQ-1发酵上清液中的纤溶酶活力为383.1 U/mL;Native-PAGE实验表明HQ-1发酵上清液中能溶解纤维蛋白的纤溶酶组分仅有一种,并且通过激活纤溶酶原实现HQ-1降解纤维蛋白。HQ-1生物学特性实验表明:菌株HQ-1对阿莫西林等11种常见抗生素敏感;对沙门氏菌、大肠杆菌和金黄色葡萄球菌的抑制作用显著。菌株HQ-1产纤溶酶活性及稳定性较好,具有潜在开发价值,本研究结果为产纤溶酶微生物资源的开发提供理论依据。     

2株耐盐碱芽孢杆菌对盐胁迫下葡萄的促生作用研究

为评价耐盐碱微生物对盐胁迫下植物的促生效应,本文以1年生“阳光玫瑰”葡萄为试验材料,利用盆栽试验评估2株耐盐碱芽胞杆菌菌株(HMB38720和HMB38667)对葡萄生长及生理特性的影响.研究结果表明,与对照相比,在盐胁迫末期(44 d, 6次灌施后10 d),灌根使用2株耐盐碱芽孢杆菌菌株后,葡萄新梢相对生长幅度显著提高了77.37%和198.69%,叶面积显著提高了50.61%和38.35%,葡萄叶片SPAD值显著提高了22.40%和16.60%,而叶片相对电导率显著降低了30.00%和23.50%,施用菌株HMB38720后叶片丙二醛(MDA)含量显著降低18.10%;菌株HMB38720处理对育苗基质电导率和pH值无影响,而菌株HMB38667处理后这2种指标显著下降了19.17%和0.32.同时芽孢杆菌菌株均能明显或显著增加葡萄叶片中防御酶(SOD, POD, PAL, PPO)的活性.冗余分析表明,植物生长表型(叶片面积和叶片SPAD值、新梢相对生长幅度)与叶片的4种防御酶活性呈正相关.研究结果表明,2株耐盐碱芽孢杆菌菌株均能够缓解葡萄植株遭受盐胁迫的损害,具有生产应用潜力...     

3株芽孢杆菌对番茄的促生作用及对番茄根域微生物的影响

通过种子萌发和盆栽促生试验研究3株芽孢杆菌Bs10、Ba12和Bl10对番茄的促生作用及其对番茄根域微生物区系的调节作用.结果表明: 3株芽孢杆菌对番茄种子的胚轴、胚根和番茄植株的生长有明显的促进作用,处理后番茄根系的总长度、总表面积和总体积均显著增加;处理后土壤中细菌数量和比例显著增加,真菌数量和比例明显减少.与对照相比,土壤微生物区系优势菌数量发生改变:优势甲基营养型芽孢杆菌在番茄根区、根表土壤中和根内的数量大幅提高;病原真菌腐皮镰刀菌和尖孢镰刀菌在根区和根表土壤中的数量显著减少.推知芽孢杆菌对根系微生物区系的调节作用是其发挥防病促生作用的重要机制之一.     

一株产絮凝剂芽孢杆菌的分离及成份分析

从超高压灭菌的哈密瓜汁中筛选出一株具有高絮凝活性的芽孢杆菌。经形态及生理生化指标测试,初步确定该菌株为Bacillussp.B53。对此菌产生的絮凝活性成分纯化后经高效液相色谱、薄层层析分析表明该絮凝活性物质是聚γ谷氨酸,产量达到12.48g/L,该物质仅由谷氨酸组成,在212nm处有最大吸收峰,电泳结果表明Bacillussp.B53所产聚γ谷氨酸是分子量集中在440～669kD之间的多分子量聚集体,该絮凝剂有效地絮凝各种无机和有机的介质,高岭土和Ca(OH)2效果明显。     

一株类芽孢杆菌的分离鉴定及其抗革兰氏阴性菌活性测定

【背景】随着碳青霉烯类和多粘菌素类可转移耐药基因的发现及扩散,多重耐药革兰氏阴性细菌感染更加难以治疗。【目的】筛选有效拮抗革兰氏阴性菌的菌株,为新型抗生素的发掘奠定基础。【方法】利用胰蛋白胨大豆琼脂培养基筛选土壤源细菌,通过16S rRNA基因序列鉴定其种属;通过全基因组测序,antiSMASH比对分析菌株产抗生素潜能,双层琼脂平板法验证其抗菌活性;通过甲醇萃取其次级代谢产物,高效液相色谱串联质谱(HPLC-MS/MS)进行次级代谢产物分析。【结果】从北京周边土壤样品中分离到一株类芽孢杆菌CAU136 (Paenibacillus pabuli CAU136),经过生物信息学分析和antiSMASH比对,表明该菌株有较强合成次级代谢产物的潜能,双层琼脂平板法验证其能抑制多株革兰氏阴性菌生长,HPLC-MS/MS检测结果显示其可能分泌多粘菌素E。【结论】类芽孢杆菌CAU136可能分泌多粘菌素E,能有效拮抗革兰氏阴性菌。     

一株具有防病促生功能的贝莱斯芽孢杆菌SF327

【目的】筛选植物根际促生贝莱斯芽孢杆菌,分析菌株的生防潜力和全基因组特征。【方法】通过温室小青菜促生试验以及植物益生表型的分析,明确具有促生功能的菌株SF327。用滤纸片法测定菌株SF327对5种植物病原真菌以及4种植物病原细菌的拮抗活性。通过大田喷雾接种的方式评价菌株SF327对水稻白叶枯病的防治潜力。利用antiSMASH分析预测菌株SF327产生的二次代谢产物。通过比较基因组分析SF327与2株植物根际益生贝莱斯芽孢杆菌的代表性菌株FZB42和SQR9的亲缘关系、核心基因以及二次代谢产物合成基因簇。【结果】菌株SF327能够产生生长素吲哚-3-乙酸,是一株有益的根围促生菌;对稻瘟病菌、黄瓜枯萎病菌、辣椒疫霉菌、橡胶树胶孢炭疽菌、尖孢炭疽病菌都具有明显的拮抗作用;也具有防治水稻白叶枯病的生防潜力。菌株SF327基因组全长4.08 Mb,GC含量为46.49%,共编码4 033个基因,含有13个潜在的次生代谢产物编码基因簇,不含有质粒。SF327与FZB42和SQR9具有较近的亲缘关系,有87%以上的核心基因相同,但与SQR9的亲缘关系较近。【结论】B. velezensis SF327是一株具有宽广拮抗谱的多功能菌株,具有较好的生防应用潜力。     

Isolation of a Paenibacillus sp. strain and structural elucidation of its broad-spectrum lipopeptide antibiotic

This research was initiated to search for novel antimicrobial compounds produced by food or environmental microorganisms. A new bacterial strain, designated OSY-SE, which produces a unique and potent antimicrobial agent was isolated from soil. The isolate was identified as a Paenibacillus sp. through cultural, biochemical, and genetic analyses. An antimicrobial compound was extracted from Paenibacillus OSY-SE with acetonitrile and purified using liquid chromatography. After analyses by mass spectrometry (MS) and nuclear magnetic resonance (NMR), the antimicrobial compound was determined to be a cyclic lipopeptide consisting of a C(15) fatty acyl (FA) chain and 13 amino acids. The deduced sequence is FA-Orn-Val-Thr-Orn-Ser-Val-Lys-Ser-Ile-Pro-Val-Lys-Ile. The carboxyl-terminal Ile is connected to Thr by ester linkage. The new compound, designated paenibacterin, showed antagonistic activities against most Gram-positive and Gram-negative bacteria tested, including Listeria monocytogenes, methicillin-resistant Staphylococcus aureus, Escherichia coli O157:H7, and Salmonella enterica serovar Typhimurium. Paenibacterin is resistant to trypsin, lipase, α-glucosidase, and lysozyme. Its antimicrobial activity was lost after digestion by pronase and polymyxin acylase. Paenibacterin is readily soluble in water and fairly stable to exposure to heat and a wide range of pH values. The new isolate and its antimicrobial agent are being investigated for usefulness in food and medical applications.     

Isolation of Paenibacillus sp. and Assessment of its Potential for Enhancing Mineral Weathering

One mineral-solubilizing bacterial strain designated KT was isolated from a soil in Henan Province, China. The full-length 16S rRNA gene sequence showed 95.2 to 96.7% similarity to the bacteria of the genus Paenibacillus, indicating that the strain KT belonged to the genus Paenibacillus. The potential of this strain to release potassium from silicate minerals was investigated using a potassium-bearing rock as the sole source of potassium to support its growth. After inoculation for 7 days, the concentrations of water-soluble Al, Ca and Fe released from the potassium-bearing rock in active bacterial culture were higher than those from the control with autoclaved inoculum, but the concentration of water-soluble K in active bacterial culture was similar to that in the control. The concentrations of HNO₃-extractable Al, Ca, Fe and K from the bacterial culture were also higher than those in the control. These results showed strain KT was able to release potassium from potassium-bearing rock. These results have important implications for extraction of potassium from rocks to support plant growth.     

生淀粉酶生产菌株Paenibacillus sp.的筛选和酶的纯化及酶学性质

分离得到1株产生淀粉酶的菌株,通过扩增和测定16S rDNA序列并进行比对,发现是Paenibacillus属的细菌。液体摇瓶发酵结束后,其产生的生淀粉酶比酶活达108.5U/mL。通过饱和(NH4)2SO4沉淀、Sephacryl S-300层析的方法对其所产的生淀粉酶进行分离纯化,得到纯化的酶蛋白比酶活为5112.04U/mg,纯化倍数为14.1,相对分子质量约为1.0×105。该酶以木薯生淀粉为底物时,最适pH5.6,最适温度50℃。金属离子Ca2+、Mg2+对该酶具有激活作用,Zn2+、Cu2+、Fe2+、Ni2+、Mn2+、Co2+和EDTA2-对该酶均具有抑制作用。     

溶磷真菌ATMT转化条件优化及其对矮牵牛的促生效果评价

[目的]建立和优化一株溶磷真菌咖啡果小蠹青霉菌Penicillium brocae的转化体系,并利用分子标记观察其在根部的定殖;检测接种咖啡果小蠹青霉菌对植物的促生作用,为菌肥的研发奠定基础.[方法]利用农杆菌介导的转化体系(Agrobacterium tumefaciens-mediated transformati...     

假单胞菌BP16的分离鉴定及其植物促生性状和效应

【背景】Pseudomonas最突出的植物促生性状是可抑制植物病原微生物,但其它植物促生性状报道相对较少。【目的】明确Pseudomonassp.BP16的植物促生性状,揭示其对小麦幼苗和马铃薯的促生效应,以期为该菌株的田间应用提供理论依据。【方法】基于无机磷培养基,以涂布划线技术进行分离纯化;通过多相分类对BP16进行分类鉴定;以选择性培养基或比色法等方法对促生性状进行定性或定量分析;设置3种处理pBP16、VK、CK,分别代表施加含BP16的颗粒微生物肥料、施加不含菌的空载体颗粒、颗粒微生物肥料和空载体颗粒都不施加的空白处理,采用盆栽试验分析不同处理下的小麦和马铃薯生长性状及其根围土理化性质的变化。【结果】共分离得到41株菌;1株编号为BP16菌株的16Sr RNA基因序列与P.brassicacearumsubsp.neoaurantiaca的相似性最高(99.38%),但一些生理生化特征如明胶液化存在一定的差异;BP16菌株的解磷酸钙-磷能力最强,其发酵液中水溶性磷含量可达67.15μg/mL菌液,而且该菌具有固氮能力、产IAA (Indole-3-acetic acid)能力(8.93 mg/L)、产铁载体能力(D/d为2.46)、ACC脱氨酶活性,同时能够抑制植物病原菌马铃薯晚疫病病原菌、亚麻枯萎病病原菌和向日葵菌核病病原菌,其抑菌率分别为76.19%、32.00%和22.22%;pBP16相比VK和CK处理,显著提高了小麦的株高、根长、叶绿素含量以及干重(提高约5.29%-90.90%),根围土速效磷和速效氮含量也显著提高,提高约8.63%-83.81%;同时,p BP16较VK和CK处理的单株产量以及根围土速效磷都有极显著的提高,而速效氮水平高于CK。【结论】菌株BP16为P. brassicacearum的一个新亚种,具有多种植物促生性状,对植物促生效应良好,可作为植物促生菌剂用菌种、具有良好的应用前景。     

Isolation and characterization of a novel polychlorinated biphenyl-degrading bacterium, Paenibacillus sp. KBC101

The biphenyl-utilizing bacterial strain KBC101 has been newly isolated from soil. Biphenyl-grown cells of KBC101 efficiently degraded di- to nonachlorobiphenyls. The isolate was identified as Paenibacillus sp. with respect to its 16S rDNA sequence and fatty acid profiles, as well as various biological and physiological characteristics. In the case of highly chlorinated biphenyl (polychlorinated biphenyl; PCB) congeners, the degradation activities of this strain were superior to those of the previously reported strong PCB degrader, Rhodococcus sp. RHA1. Recalcitrant coplanar PCBs, such as 3,4,3,4-CB, were also efficiently degraded by strain KBC101 cells. This is the first report of a representative of the genus Paenibacillus capable of degrading PCBs. In addition to growth on biphenyl, strain KBC101 could grow on dibenzofuran, xanthene, benzophenone, anthrone, phenanthrene, naphthalene, fluorene, fluoranthene, and chrysene as sole sources of carbon and energy. Paenibacillus sp. strain KBC101 presented heterogeneous degradation profiles toward various aromatic compounds.     

一株耐盐甲基杆菌Methylobacterium sp.W-1的分离及促生潜能研究

[背景] 多种甲基杆菌属细菌对寄主植物有促生作用,其分布区域较广。筛选具有耐盐与促生特性的甲基杆菌属菌株可为微生物菌肥的开发提供依据。[目的] 从新疆乌尔禾地区盐渍土壤中筛选耐盐促生菌,对其培养基成分进行优化及促生能力进行研究,为微生物菌肥的开发提供依据。[方法] 采用阿须贝无氮培养基筛选耐盐菌株,对菌株进行基因序列分析及生理生化测定,采用平板试验法初步研究该菌对拟南芥的生长影响。[结果] 筛选出中度耐盐菌株W-1,经鉴定为甲基杆菌属（Methylobacteriumsp.）。菌株生长最佳无机盐为NaCl,最适浓度为1%–3%,最高耐受浓度达7%。最佳氮源为酸水解酪蛋白,产生长素最高达33.53 mg/L。溶磷能力达28.71 mg/L。菌株W-1接种拟南芥幼苗后叶绿素a和叶绿素b含量均高于对照组,同时对其根系发育有显著的促进作用。[结论] 菌株W-1促生性能显著,可为生物肥料制备提供菌种资源。     

The growth-promoting effect of Beijerinckia and Clostridium sp. cultures on some agricultural crops

New strains of Beijerinckia mobilis and Clostridium sp. isolated from the pea rhizosphere were studied with respect to their promoting effect on the growth and development of some agricultural crops. Seed soaking in bacterial suspensions followed by the soil application of the suspensions or their application by means of foliar spraying was found to be the most efficient method of bacterization. The application of B. mobilis and Clostridium sp. cultures in combination with mineral fertilizers increased the crop production by 1.5-2.5 times. The study of the population dynamics of B. mobilis by the method of genetic marking showed that this bacterium quickly colonized the rhizoplane of plants and, therefore, had characteristics of an r-strategist. At the same time, Clostridium sp. was closer to K-strategists, since this bacterium slowly colonized the econiches studied. The introduction of the bacteria into soil did not affect the indigenous soil bacterial complex. The presence of Clostridium sp. slowed down the colonization of roots by the fungal mycelium. The possible mechanisms of the plant growth-promoting activity of B. mobilis and Clostridium sp. are discussed.