一株柠条内生解磷菌的分离鉴定及实时荧光定量PCR检测

结合形态观察与16S rDNA序列测定对柠条根系内分离筛选得到的解磷细菌C9进行鉴定,Blast比对结果表明C9为泛菌(Pantoea vagans).在无机磷液体培养基培养条件下,用钼锑抗比色法研究它的解磷能力.结果表明,随着时间的延长,培养液中速效磷含量逐渐增加到4.45mg/L,溶液pH可降至4.2.进一步利用实时荧光定量PCR检测柠条根系中、柠条根际土壤与柠条根围土壤中该细菌存在的相对基因拷贝数,结果发现该基因在3种样品中的数量为:柠条根系＞柠条根际土壤＞柠条根围土壤,表明泛菌解磷细菌能聚集生长在柠条根系内,随着与根系接触距离的增加而呈逐级递减趋势.     

高效溶磷菌的分离、筛选及在土壤中溶磷有效性的研究

从采自全国各地130个土样中.分离出1000余菌株,从中筛选出1株代号为Ap-2号的高效溶磷菌.经鉴定为黑曲霉Aspergillus niger.对该菌株的产酸特性以及接种土壤后的溶磷有效性进行了研究.试验表明,该菌株发酵过程产草酸、柠檬酸等多种有机酸,使土壤速效磷含量增加141.94%.其溶磷作用,与土壤pH含水量有密切关系,对温度要求不严,与接种量是一定正相关.并可与磷酸铵、三料、尿素、硫酸铵等化肥混合使用.     

类芦根际溶磷真菌的筛选、鉴定及其溶磷能力分析

为揭示类芦(Neyraudia reynaudiana)等水土保持植物的耐低磷机制,开发溶磷菌种质资源,提高赤红壤磷素利用率,从类芦根际土壤中筛选到一株溶磷能力较强的真菌FP1,经形态学和ITS序列分析,鉴定为黑曲霉(Aspergillus niger)。3种难溶性磷酸盐液体培养基接种菌株FP1后,其pH值和溶磷量的动态变化显示,培养液的pH值均呈显著下降趋势。溶磷量与培养时间有关,除磷酸三钙外,菌株FP1对其他难溶性磷酸盐的溶磷趋势均为先上升再下降并趋于稳定。菌株FP1对不同磷源的最大溶磷率顺序为:磷酸铝(92.02%)磷酸三钙(41.62%)3种磷酸盐的混合物(35.86%)磷酸铁(19.20%)。FP1对磷酸铝和磷酸铁都具有较强的溶磷能力,表明抗逆性强的水土保持植物类芦根际土壤蕴藏着高效的溶磷微生物资源。     

棉花根际解磷菌的解磷能力和分泌有机酸的初步测定

利用特殊培养基对盐碱地棉花根际解磷菌进行了分离以及pH值和分泌有机酸能力的初步测定。利用溶磷圈法筛选出10个解磷能力较高的菌株进行深入研究,其中液体培养条件下测定了菌株的溶磷能力,有效磷在4.04~185.63 mg/L,其中wpL2溶磷量达到185.63 mg/L;测定了培养液pH值,下降到5.12~6.67,但是pH与溶磷量之间没有线性关系,测定了培养液的有机酸含量,菌株溶磷量与有机酸总量没有线性相关性,其中所分离到的解无机磷菌株均可以分泌酒石酸,除此之外,wpc1还分泌乙酸,wpc2和wpL2还分泌柠檬酸;分离到的解有机磷菌株均可分泌乙酸,除此之外,ypL1和ypc2分泌酒石酸,ypL3分泌柠檬酸,ypL2和ypc3分泌柠檬酸和丁二酸,均不能产生苹果酸。     

一株高效解无机磷细菌BS06的鉴定及其解磷能力分析

【目的】对一株来源于广西甘蔗根际土壤的高效解无机磷细菌BS06的分类和解磷能力进行探讨,以期为解磷微生物在广西甘蔗生产上的开发和应用提供理论依据。【方法】通过形态观察、生理生化测定及16S rRNA基因序列同源性分析,进一步结合种特异的recA基因序列分析对解磷菌BS06进行分类鉴定;通过改变无机磷培养基中的碳源、氮源对菌株解磷能力的影响,分析菌株的解磷特性;通过盆栽试验了解菌株对甘蔗品种粤糖00236、桂糖28磷素吸收的影响。【结果】分类鉴定结果表明菌株BS06属于洋葱伯克霍尔德菌(Burkholderia cepacia);菌株在以乳糖为碳源条件下具有较强的解磷能力,其发酵液中水溶性磷含量为262.71 mg/L;在以硝酸钠为氮源条件下有较强解磷能力,其发酵液中水溶性磷含量达到305.85 mg/L;接种BS06菌株显著促进甘蔗组培苗的生长并提高甘蔗植株的含磷量。【结论】解磷细菌BS06具有较大的开发利用潜力。     

解磷黑曲霉L-1菌株原生质体诱变育种

以从龙泉山地区酸性红壤中分离的黑曲霉L-1(Aspergillus niger sp.L-1)为出发菌株,通过研究菌株L-1原生质体的形成和再生条件,发现培养30 h的菌丝体在以0.15 mol/L氯化钾为渗透压稳定剂的基本培养基上经过再次培养20 h后,所得菌丝体用0.3%纤维素酶和0.4%蜗牛酶在30 ℃条件下处理...     

纤维素酶产生菌和糖化酶产生菌液体混合发酵的研究

应用正交试验设计研究了糖化酶高产菌株黑曲霉UV-11(Aspergillus niger UV-11)和纤维素酶高产菌株黑曲霉F_(27)(A.niger F_27)液体混合发酵条件。结果表明,发酵液糖化酶活力为6500U/ml,CMC酶活力为99mg葡萄糖/ml·h。与UV-11单菌发酵相比,糖化酶活力提高16％,CMC酶活力提高266％。     

一株高效解磷真菌新菌株的筛选鉴定及解磷特性

从辽宁省辽中县多年耕种的日光温室番茄根际土壤中筛选出一株解磷真菌,通过菌落形态特征和ITS rDNA序列对比,鉴定该菌株为草酸青霉菌的一株新菌株,将其命名为PSF1.该菌株能利用葡萄糖、蔗糖、乳糖、半乳糖、可溶性淀粉等多种碳源和硫酸铵、氯化铵、硝酸铵、硝酸钾、尿素等多种氮源进行生长代谢并表现出较强的解磷能力,在C/N 10∶1~60∶1、初始pH 7~8的条件下生长情况较好且解磷能力较高.该菌株有很强的产酸能力,在培养过程中培养液pH由7.00~7.50下降至2.06~4.87;在4种磷源培养液中的最高解磷量分别为磷酸三钙(869.62 mg·L^-1)>磷矿粉(233.56 mg·L^-1)>磷酸铝(44.77 mg·L^-1)>磷酸铁(28.42 mg·L^-1).Pearson相关分析表明,菌株在磷酸三钙、磷矿粉和磷酸铁培养液中的解磷量与pH的变化之间呈极显著负相关关系,在磷酸铝培养液中无显著相关关系.菌株PSF1解磷能力强,生长条件广,推测其在土壤中有较强的解磷能力.     

产黄酮甘蔗叶内生真菌的分离与鉴定

采用显色反应、薄层层析色谱和紫外吸收光谱法,对甘蔗叶内16株内生真菌的代谢产物进行黄酮类化合物检测。结果共筛选到3株能够产黄酮类化合物的内生真菌(GZ-1、GZ-4和GZ-5)。依据真菌的形态特征及ITS序列分析,鉴定结果表明菌株GZ-1、GZ-5为棘孢曲霉(Aspergillus aculeatus),菌株GZ-4为黑曲霉(Aspergillus niger)。     

一株产赭曲霉毒素A黑曲霉的筛选与鉴定

【目的】筛选出可产生赭曲霉毒素A (OTA)的霉菌菌株。【方法】利用CYA和YES培养基从实验室32个霉菌样品中筛选目的菌株。利用高效液相色谱-荧光检测法对OTA产生菌进行初筛,利用高效液相色谱-质谱联用对OTA初筛菌株进行复筛。通过菌落形态、菌丝及分生孢子形态、ITS DNA序列、β-Tubulin基因序列及Calmodulin基因序列分析等鉴定目的菌株。【结果】得到一株OTA产生菌株1062,该菌株能在25 °C条件下,在CYA、YES和CA培养基中很好生长。结合形态学、对培养基的要求以及上述3个基因序列的进化树分析,该菌株属于黑曲霉(Aspergillus niger)。【结论】菌株1062具有OTA产生能力,是一株黑曲霉。     

一株耐盐日本曲霉的筛选及其溶磷促生作用

【目的】从内蒙古种植葵花的盐碱地中筛选高效溶磷真菌,为盐碱地增产节肥开发生物肥料提供溶磷菌种资源。【方法】利用ITS r DNA序列鉴定菌株、固体培养基测定耐盐性,液体摇床培养与盆栽试验结合分析菌株溶磷能力,盆栽和田间试验明确菌株M1促进作物生长和增产作用;LC-MS技术测定菌株M1在液体培养基中分泌有机酸和植物激素含量,明确菌株M1的溶磷和促生机理。【结果】溶磷菌株M1鉴定为日本曲霉(Aspergillus japonicus)。液体培养基接种菌株M1培养6 d,以Ca_3(PO_4)_2为磷源时上清液有效磷达1020.89 mg/L,溶解率为63.30%;以AlPO_4为磷源时有效磷达995.69 mg/L,溶解率为48.59%;以贵州开阳磷矿粉、江苏锦屏磷矿粉、云南晋宁磷矿粉、河北钒山磷矿粉和云南昆阳磷矿粉为磷源接种菌株M1,从晋宁磷矿粉释放的有效磷浓度最高,达到363.64 mg/L。菌株M1可耐受10%NaCl。将M1制备的菌剂分别接种于施用Ca_3(PO_4)_2、AlPO_4和开阳磷矿粉3种磷源的4种盆栽试验土壤包括北京石灰性潮土、安徽黏性潮土、安徽水稻土和山东沿海盐潮土。结果显示,菌株M1对玉米植株促生效果显著,玉米植株鲜重比对照提高2.14%–90.91%、干重增加22.15%–268.28%;土壤有效磷提高21.81–24.27 mg/kg。菌株M1与4种土壤的适配性均高于对照菌株DSM 821。田间小区花生产量结果显示,接种溶磷菌剂M1增产效果最好,花生果实产量达4.46 t/hm~2,比不接种菌剂的对照处理增加0.81 t/hm~2,增产22.19%。菌株M1在含有磷酸三钙、磷酸铝和开阳磷矿粉3种难溶磷培养液中经过6 d培养,均产生7种有机酸,其中草酸和柠檬酸含量最高,分别为616.16 mg/L和413.69 mg/L;培养液均能检测到吲哚乙酸(IAA)和玉米素,IAA含量为15.45–77.58 mg/L,玉米素浓度为0.06–0.11 mg/L。【结论】获得了一株高效溶解多种难溶磷的日本曲霉菌M1,它能显著增加土壤有效磷、促进玉米生长和花生增产,与4种典型土壤适配性好,具有良好的农业应用前景。     

Organic acid production in vitro and plant growth promotion in maize under controlled environment by phosphate-solubilizing fluorescent Pseudomonas

Background  

Phosphorus deficiency is a major constraint to crop production due to rapid binding of the applied phosphorus into fixed forms not available to the plants. Microbial solubilization of inorganic phosphates has been attributed mainly to the production of organic acids. Phosphate-solubilizing microorganisms enhance plant growth under conditions of poor phosphorus availability by solubilizing insoluble phosphates in the soil. This paper describes the production of organic acids during inorganic phosphate solubilization and influence on plant growth as a function of phosphate solubilization by fluorescent Pseudomonas.     

Thermotolerant phosphate solubilizing microorganisms and their interaction with mung bean

Several phosphate solubilizing microorganisms (PSM) were tested for their efficiency at 35°, 40° and 45°C. There was a marked variation in their ability to solubilise tricalcium phosphate and the effect was more pronounced at 45°C. Two bacterial and one fungal strain were found to be thermotolerant as they solubilised a large amount of tricalcium phosphate at the three tested temperatures. These thermotolerant strains were identified as Bacillus subtilis (TT₀), Bacillus circulans (TT₈) and Aspergillus niger (TT₁₀). Seed inoculation of mung bean showed a better establishment of temperature tolerant strains as revealed by the rhizosphere population. The inoculation improved nodulation, the available P₂O₅ content of the alluvial soil, root and shoot biomass, straw and grain yield and phosphorus and nitrogen uptake of the crop. Among the bacterial strains, the best effect on yield was obtained with B. subtilis. However, statistically it was equivalent to streptomycin resistant mutant (M-20) and Pseudomonas striata (27). A. niger was less effective than bacteria. Though superphosphate was found to be a better source of phosphate fertiliser, the use of rock phosphate (RP₄₀), coupled with phosphate solubilising bacteria (PSB), gave results comparable to superphosphate (SP₂₀) + PSB inoculants.     

Available phosphate content of an alluvial soil as influenced by inoculation of some isolated phosphate-solubilizing micro-organisms

Summary Among several phosphate-solibilizing micro-organisms isolated from an alluvial soil (Fluvaquent) in sucrose-Ca₃(PO₄)₂ agar plates, two fungal strains, ACF₂ (Aspergillus candidus) and ACF₁ (A. fumigatus) two bacterial strains, ACB₅ (Bacillus firmus B-7650) and ACB₆ (B. firmus B-7651) and one actinomycete strain, ACS₆ (Streptomyces sp.) were efficient solubilizers, solubilizing 297.0, 288.3, 49.0, 45.8 and 29.0 μg of P as free PO₄ ⁻³, respectively, containing 15 mg insoluble P from Ca₃(PO₄)₂ in broth. Solubilization was lesser from AlPO₄ and FePO₄. The isolates producing oxalic and tartaric acids without or with citric acid showed higher ability of solubilizing insoluble inorganic phosphates. All the above isolates possessed the ability of solubilizing rock phosphate in considerable amounts, ACF₁ (A. fumigatus) being the highest (31.5 μg), while ACB₆ (B. firmus B-7651) and both the aspergilli also possessed cellulose-decomposing ability in addition. Inoculation of the isolates, in a flask culture experiment, had no significant effect on the accumulation of available phosphorus in soil even when amended with rock phosphate (RP), farm yard manure (FYM), (FYM+RP), rice straw (RS) and (RS+RP). Nevertheless, the overall performance of ACF₂ (A. candidus) and ACB₆ (B. firmus B-7651) was better than that of the others, in this respect, while ACB₅ (B. firmus B-7650) and ACF₁ (A. fumigatus) intensified the enhancing effect of FYM and RS. Partial sterilization, by autoclaving, of the soil had no significant effect on available phosphorus content of the soil irrespective of any inoculation.     

The structure of Aspergillus niger phytase PhyA in complex with a phytate mimetic

Phytases hydrolyse the phosphomonoesters of phytate (myo-inositol-1,2,3,4,5,6-hexakis phosphate) and thus find uses in plant and animal production through the mobilisation of phosphorus from this source. The structure of partially deglycosylated Aspergillus niger PhyA is presented in apo form and in complex with the potent inhibitor myo-inositol-1,2,3,4,5,6-hexakis sulfate, which by analogy with phytate provides a snapshot of the Michaelis complex. The structure explains the enzyme’s preference for the 3′-phosphate of phytate. The apo-and inhibitor-bound forms are similar and no induced-fit mechanism operates. Furthermore the enzyme structure is apparently unaffected by the presence of glycosides on the surface. The new structures of A. niger PhyA are discussed in the context of protein engineering studies aimed at modulating pH preference and stability.     

Dephosphorization of LD slag by phosphorus solubilising bacteria

Phosphorus is one of the major nutrients, and microbial solubilisation of insoluble mineral phosphate in soil is an important process in natural ecosystem and in agricultural soil. Many soil microorganisms display the ability to solubilize many insoluble inorganic phosphates. They are generally referred as phosphorus solubilising microorganisms (PSM). In this study an attempt was made to look into the phosphorus solubilisation efficiency of some commonly available soil bacteria and their possible application in bio-beneficiation of metallurgical waste like LD Slag. Linz -Donawitz (LD) slag is produced in large quantities (200 kg LD slag per ton of hot metal) and poses a substantial disposal problem in the iron and steel making industry. LD slag contains around 29% Ca, 21% Fe, and 5% Mg. Its phosphorus content is about 1.5-6%. Due to presence of high amount of Ca, it can be used as flux in blast furnace, but presence of high amount of phosphorus in the LD slag makes them unsuitable for industrial application. Removal of phosphorus with the help of phosphorus solubilising microorganisms may be a great advantage in biotechnological applications. Two gram positive bacteria belonging to genus Bacillus and two gram negative bacteria belonging to genus Pseudomonas were selected in this study. Phosphorus solubilisation efficiency was studied initially with tricalcium phosphate as model insoluble phosphate compound at different sugar concentration, NaCl concentration and at different initial pH of the medium. About 35% of ‘P’ could be solubilized from LD slag by Pseudomonas aeruginosa at 2% solid content.     

Factors determining rock phosphate solubilization by microorganisms isolated from soil

Forty two soil isolates (31 bacteria and 11 fungi) were studied for their ability to solubilize rock phosphate and calcium phosphate in culture medium. Eight bacteria and 8 fungi possessed solubilizing ability. Pseudomonas cepacia and Penicillium purpurogenum showed the highest activity. There was a correlation between final pH value and titratable acidity (r=–0.29 to –0.87) and between titratable acidity and soluble phosphate (r=0.22 to 0.99). Correlation values were functions of insoluble phosphate and of the group of microorganisms considered. A high correlation was observed between final pH and soluble phosphate only for the rock phosphates inoculated with the highest concentration of solubilizing bacteria (r=–0.73 to –0.98).