苏云金芽孢杆菌菌剂对棉花根际土壤细菌数量及多样性的影响

采用稀释平板培养法与PCR-DGGE技术, 以阿维菌素为阳性对照, 水为阴性对照, 研究了大田喷施推荐剂量(0.1 kg·hm^-2)和高剂量(10 kg·hm^-2)苏云金芽孢杆菌(Bt)菌剂对棉花根际土壤细菌种群数量及结构的影响。结果表明, 喷雾处理后1~3 d, 不同处理组土壤细菌数量间无显著性差异, 在3 d时细菌数量均值达到最大, 之后开始下降, 12 d后清水对照、推荐剂量Bt菌剂、高剂量Bt菌剂处理组土壤细菌数量均值维持在4.0×107 CFU·g^-1左右; 推荐剂量Bt菌剂处理样品土壤细菌数量在6 d时显著高于清水对照, 其余时间与清水对照间无显著性差异; 高剂量Bt菌剂处理与清水对照在整个试验期间均无显著性差异; 阿维菌素处理组土壤细菌数量在0~6 d内与清水对照无显著性差异, 而在12~45 d内显著低于其他3个处理组。DGGE图谱显示, Bt菌剂处理对棉花根际土壤17种细菌均无显著抑制作用。聚类分析结果表明, Bt菌剂对土壤细菌群落结构的扰动在12 d后得到恢复。与阴性对照组相比, Bt菌剂对土壤细菌多样性指数无显著影响, 而阳性对照阿维菌素对土壤细菌种群消长和多样性指数有较强的影响。对DGGE图谱中17条电泳条带的序列分析, 证明棉花根际土壤中存在起固氮作用的慢生根瘤菌属细菌和具有污染修复与净化活性的鞘脂菌属、鞘氨醇单胞菌属和红球菌属细菌。Bt菌剂与阿维菌素处理均对这些土壤有益菌群无明显不利影响。总体结果表明, Bt菌剂无论是在正常推荐剂量下还是在较高剂量(推荐剂量的100倍)下使用, 对棉花根际土壤微生态环境产生的冲击都较小, 是一种生态安全性较高的生物农药。     

水分管理对水稻籽粒硒积累及根际土壤细菌群落多样性的影响

采用盆栽试验方法,研究不同水分管理方式对水稻根际土壤硒组分、籽粒硒积累以及根际土壤细菌群落多样性的影响。结果表明：在水稻的各生育期,好氧和干湿交替较淹水灌溉一定程度上提高了土壤pH和氧化还原电位（Eh）,土壤可溶性和可交换态硒含量增加,从而提高了土壤硒的有效性。水稻成熟后,不同部位的含硒量由高到低依次为根（1.38~2.22 mg·kg^-1）、叶（0.55~0.85 mg·kg^-1）、茎（0.53~0.61 mg·kg^-1）和籽粒（0.15~0.53 mg·kg^-1）。水稻籽粒含硒量以干湿交替灌溉最高,淹水灌溉最低,二者含硒量差异达显著水平。干湿交替灌溉的水稻产量显著高于常规淹水灌溉,且较淹水灌溉提高了7.83%,较好氧灌溉提高13.51%。水稻根际土壤优势菌为变形菌门、绿弯菌门、拟杆菌门、酸杆菌门、Patescibacteria和芽单胞菌门,变形菌门是不同水分管理方式下水稻根际土壤中丰度最高的细菌,水分管理措施显著影响其丰富度,干湿交替和好氧灌溉中变形菌门的丰富度明显高于淹水灌溉。从纲水平看,Gammaproteobacteria的丰度与土壤有效硒含量呈正相关,故Gammaproteobacteria丰度的增加可能是土壤硒生物有效性增加的另一个重要原因。综上,干湿交替灌溉不但能提高水稻产量和稻米硒含量,且较正常淹水管理节约用水,在水稻生产中,是一种优先推荐的水分管理方法。     

水稻根际芽孢杆菌抗砷胁迫作用及其微生物机制

砷甲基化过程作为微生物的砷抗性机制改变砷的毒性和移动性，对土壤砷污染控制有重要意义。砷抗性根际促生菌对砷胁迫下水稻生长产生积极影响，然而水稻根际菌的砷甲基化效率及其影响水稻砷胁迫的机制研究还较为缺乏。从砷污染稻田根际土中筛选出一株砷甲基化功能芽孢杆菌Bacillus sp. LH14，探究该菌株的砷甲基化效率、砷抗性和促生相关特性，和菌株接种对土壤砷形态、水稻生长和根际微生物相互作用的影响。结果表明，菌株LH14具有砷甲基化和挥发能力，34 h内将三价无机砷转化为甲基砷的效率为54.9%，主要形态为二甲基砷和三甲基砷。LH14接种显著提高了土壤中砷甲基转移基因(arsM)丰度，增加土壤溶液甲基砷浓度，表明LH14参与了土壤砷形态转化。LH14能在砷胁迫下产生吲哚-3-乙酸(IAA)，菌株浸染显著增加高砷条件下种子萌发率、根和芽长及生物量。接种LH14对砷污染土壤中水稻植株生长有促进作用，可能与根际有益菌(例如Burkholderiaceae和Gemmatimonadaceae)相对丰度增加有关。所以，水稻根际存在砷甲基化功能植物促生菌，接种该菌改变水稻根际砷形态，并能产生植物激素和富集根际有益菌从而直接和间接地促进水稻生长，有利于缓解水稻砷胁迫，为砷甲基化功能菌应用于砷污染土壤修复和缓解植物砷胁迫提供理论支撑。     

水稻根际土壤及根组织内外固氮微生物的遗传多样性分析

利用PCR—RFLP检测了水稻根际土壤及根组织内外固氮微生物的nifH基因。对54个阳性克隆的nifH 限制性内切酶Mnl I的RFLP图谱进行UPGMA聚类分析和最大似然法分析，结果表明了8对相似的带型(相似性> 50%)和2对同源性为100%的带型分别来源于水稻根际土壤及根组织内外，3种特有的带型均来源水稻根际土壤和3种特有带型来源于水稻根组织内外。证明了水稻根际土壤和水稻根组织的固氮微生物具有显著的多样性，也初步显示了土壤中某些固氮微生物能定植于水稻根内或根表。     

多粘类芽孢杆菌LRS-1对辣椒疫霉病害根际土壤细菌多样性的影响

【目的】研究多粘类芽孢杆菌LRS-1生防菌对患疫霉病的辣椒根际土壤细菌多样性的影响。【方法】通过对无病阴性对照(CK)、辣椒疫霉菌阳性处理(PC)和生防菌+辣椒疫霉菌处理(LRS1) 3个处理的盆栽根际土壤样品16S r RNA基因的V3-V4区进行高通量测序,并对下机数据予以相关生物信息学分析,获得了不同处理根际土壤细菌的OTU丰度、Alpha多样性、OTUs分布、群落组成差异以及PCA聚类分析等数据结果。【结果】各处理间的根际土壤细菌群落变化明显,疫霉菌处理明显降低了根际土壤细菌群落的丰富度和多样性,而接种多粘类芽孢杆菌LRS-1则可明显改善根际土壤细菌群落丰富度与多样性;接种LRS-1明显提高了辣椒根际土壤细菌种类组成的相似性,并改善疫霉菌胁迫下的细菌种类组成;各处理的优势物种类群均分布于变形菌门(Proteobacteria)、厚壁菌门(Firmicutes)、放线菌门(Actinobacteria),但疫霉胁迫条件下Sphingomonadaceae、Clostridiales物种丰度显著降低,而Burkholderiales、Micrococcales等物种丰度显著增加,接...     

水稻根际耐镉细菌的筛选与鉴定

为获得稻田土壤镉污染修复的微生物,采用培养基加镉平板法,从水稻根际土壤分离耐镉细菌。本研究分离得到3株耐镉菌株,经16S rDNA测序、比对,鉴定为假单胞菌属细菌。对菌株细胞形态的电镜观察、菌株生长曲线的测定结果表明,在镉处理浓度为100 mg L 1条件下,菌株生长未受明显抑制,但菌株形态发生了适应性的变化;在镉处理浓度超过300 mg L 1时,菌株生长受到明显抑制;在镉处理浓度700 mg L 1条件下,菌株仍能生长,但生长延滞期延长,对数期后的生长量亦低;对菌体镉含量分析表明,在100 mg L 1镉处理下,3株菌株菌体中的镉含量分别为9.04 mg g 1、4.96 mg g 1和28.58 mg g 1,富集系数依次为90.4、49.6和285.8。可见,分离得到的3株菌株具有较强镉耐性和镉吸收能力,将为稻田土壤重金属镉污染的修复提供重要微生物资源。     

有机肥对水稻根际土壤中微生物和酶活性的影响

利用根际箱在红壤上研究了有机肥对水稻根际有效磷、根际微生物和土壤酶活性的影响。试验结果表明,有机肥明显地提高水稻根际和非根际土壤真菌、放线菌和细菌的数量及土壤有效磷的含量。根际土壤各类微生物的数量大于非根际土壤,表现出明显的根际效应。施用有机肥使根际效应增加,其效应为细菌 放线菌 真菌。有机肥还明显地促进水稻根际无机磷溶解菌和有机磷分解数量以及磷酸酶和脲酶的活性,同时对水稻根际土壤磷转化速率有明显的提高,从而加速了土壤养分的转化。     

西瓜枯萎病对根际细菌多样性的影响

通过系统分析北京地区西瓜根际微生物组,显示了细菌群落结构分布的显著区域性,磷元素和钾元素是影响西瓜根际菌群组成最主要的因子;扩增子测序分析表明,健康植株比发病植株根际土壤群落结构更为稳定,节点微生物更多;网络分析发现了来自5 个菌门的19 个菌属与镰刀菌属直接相关,其中芽孢菌属与假单胞菌属与镰刀菌属丰度呈正相关。通过对比西瓜健康植株与镰刀菌侵染植株根际细菌组成差异,并构建相关性网络发现了潜在的枯萎病防治菌群,为北京西瓜枯萎病预警和防控奠定了基础。     

玉米根际土壤芽胞杆菌的多样性

为了解玉米(Zea mays)不同品种根际芽胞杆菌种群多样性信息,本研究采用稀释平板法,对10个玉米品种根际的芽胞杆菌进行了分离,并对其进行16S r RNA序列分析。结果表明,从10个玉米品种根际共分离到69个形态差异的芽胞杆菌菌株;16S r RNA序列分析表明,69个菌株鉴定为23个种,归属于3个属,芽胞杆菌属(Bacillus)、赖氨酸芽胞杆菌属(Lysinibacillus)和嗜冷芽胞杆菌属(Psychrobacillus),其中芽胞杆菌属的种类和数量最多。玉米不同品种根际的芽胞杆菌的种类数量不同,QB662×QB2219和QB1013×QB446根际分离到的芽胞杆菌种类最多,均为8种;J106×QB572的芽胞杆菌菌落含量最高。玉米根际的优势种群主要为嗜气芽胞杆菌(B.aerophilus)、阿氏芽胞杆菌(B.aryabhattai)、简单芽胞杆菌(B.simplex)和苏云金芽胞杆菌(B.thuringiensis),其他芽胞杆菌种类仅在一种或少数的玉米品种出现。QB662×QB2219根际的芽胞杆菌种类香农-威纳(Shannon-Wiener)指数(H)最大;QB948×QB48根际的芽胞杆菌种群香农-威纳多样性指数次之,但均匀度指数最高;J106×QB572的多样性指数和均匀度指数皆最低。菌株FJAT-17411、FJAT-17472、FJAT-17430和FJAT-17442与Gen Bank中已报道16S r RNA基因序列的相似性为97%~98%,可能为芽胞杆菌的新种。本研究结果表明,玉米根际具有较为丰富的芽胞杆菌种群多样性,并存在一些潜在的新的微生物菌种资源。本研究对芽胞杆菌资源的开发和利用提供良好的实验材料和理论基础。     

水稻根际促生细菌促生生理活性评价

为了考察水稻特异性PGPR菌株各生理活性对水稻幼苗初生根数、次生根数、茎干重和根干重的影响,筛选优良PGPR菌株,采用改良的CAS检测法、Salkowski比色法、钼蓝比色法和1-氨基环丙烷-1-羧酸(ACC)脱氨酶检测法分别测定菌株产铁载体、产吲哚乙酸(IAA)、溶磷能力和ACC脱氨酶活性,利用Hoagland半固体培养基和土壤盆栽法培养水稻幼苗。结果表明,IAA产量低的菌株对水稻幼苗初生根数、次生根数和茎干重的增长有明显的促进效果;根干重明显随铁载体产量增加而增加;溶磷活性高的菌株对水稻幼苗茎干重和根干重的增加均有明显的促进作用;低ACC脱氨酶活性的菌株促进水稻幼苗初生根的生长,活性高的菌株促进次生根数的增加。根据半固体试验结果,选择7株具有明显促生效果的菌株进行盆栽试验,结果证实,这些菌株均具有显著的促生效应。     

Influences of organic debris and rice root on the nitrogen fixation in the submerged soil

Abstract

A series of experiments has been conducted on the N2 fixation in the paddy soils by the authors (1–4). The amount of organic substrates for microorganisms and the degree of reduction of the soil are found to be two major factors affecting the N₂-fixing activity of the heterotrophic microorganisms in the submerged soil. Organic debris, rice root and their neighboring soils are identified to be the important micro-sites for the heterotrophic N₂ fixers. The organic debris and the rice root are considered to play dual roles by supplying the organic substances; (1) increase of the population of the heterotrophic N₂ fixers—the amount of nitrogenase, (2) preparation of the reduced conditions favorable for the nitrogenase activity.

However, it is not yet clearly known which of these two roles of the organic substrates is more essential to the N₂-fixing activity in the paddy soil. In addition, it is expected that there must be some differences between the organic debris and rice root in their contribution to the N₂ fixation in the paddy soil.

An experiment was carried out to clarify these problems. Moist soil sample was collected from the plough layer of the paddy field at Central Agric. Exp. Sta. in Konosu City, Saitama Pref., passed through a 5 mm sieve and placed in pots (3 kg moist soil/pot). Ammonium sulfate, calcium superphosphate, and potassium chloride at the rate of 0.4-0.4-0.4 (N-P₂O₅-K₂0) g/pot were incorporated into the soil 7 days before transplanting. Split application of ammonium sulfate at the rates of 0.2 and 0.4 g N/pot were also incorporated at 30 and 41 days after transplanting respectively. These pots were divided into three series; planted (P-series), non-planted (N-series), and non-planted and applied with organic manure (O-series). In case of O-series, 60 g of fairly rotted organic manure was applied to each pot. Each pot of P-series was planted with two 4O-day-old seedlings of rice plant at 7 days after submergence. The Nseries was regarded as a control. Each series was not replicated in this preliminary experiment.     

光合细菌和有机肥对土壤主要微生物类群的影响

将光合细菌菌液和发酵有机肥施入农田土壤，研究表层土壤中微生物区系变化。结果表明，光合细菌和有机肥显著增加土壤中放线菌、异养细菌、固氮菌、氨化细菌和硝化细菌数量，减少反硝化细菌数量，改善土壤微生物区系，光合细菌作用比有机肥更稳定和持久。光合细菌还能明显降低真菌数量，提高放线菌／真菌比值，促进土壤健康。     

Effect of soil microorganisms on the growth of roots in rice seedlings

Abstract

Root system of rice seedlings grown on nutrient solution inoculated with soil microorganisms were examined morphologically in comparison with those obtained under sterile condition. In the presence of soil microorganisms, primary roots increased in their number and decreased in the total length. Inoculated plants had more secondary roots equipped with tertiary roots. In addition, longer root hairs developed densely on primary and secondary roots of the inoculated seedlings.

Anatomical examination of the primary roots revealed that the number and width of cortical layers, as well as the length and width of the cortical cells, were increased by the effect of microorganisms. Microbial effect on outer morphology of rice roots, consequently, was estimated to have been induced from the alteration in histological and cytological activities including the activation of the periclinal divisions of the epidermal cells, the inactivation of the transverse divisions of the cortical cells and the activation of the elongation of cortical cells.     

Enhanced rice yields are related to pronounced shifts in soil resident bacterial community structures in response to Rhodopseudomonas palustris and Bacillus subtilis inoculation

Journal of Soils and Sediments - Microbial inoculation is a promising way to improve soil fertility in an eco-friendly manner. Despite the extensive studies of microbial inoculation used for soil...     

Effect of root exudates on growth of newly isolated nitrifying bacteria from barley rhizoplane

An ammonia-oxidizing bacterium, strain PJA1, and nitrite-oxidizing bacterium, strain PJN1, were newly isolated from the rhizoplane of barley. The cells of strain PJA1 were lobate, compartmentalized, and showed characteristics of the genus Nitrosolobus. The similarity of the 16S rRNA gene to Nitrosolobus multiformis ATCC25196 was 99.04%. The cells of strain PJN1 were rod-shaped. The similarity of the 16S rRNA gene to Nitrobacter agilis ATCC14123 was 98.57%. These newly isolated bacteria were identified as Nitrosolobus sp. PJA1 and Nitrobacter sp. PJN1, respectively. The exudates prepared from barley roots of both the pre-heading and post-heading stages promoted the growth of strain PJA1 (15–20% increase). The growth of the non-rhizoplane strains like Nitrosomonas europaea ATCC25978 was remarkably inhibited by the exudates. The growth of strain PJN1 was also remarkably promoted by the root exudates at the pre-heading stage (6 time increase), and by the root exudates at the post-heading stage (2.5 time increase). The root exudates did not effect the growth of Nitrobacter winogradskyi IFO14297, isolated from the non-rhizoplane.     

Fe2+对水稻生长及土壤微生物活性的影响

通过盆栽试验,模拟冷浸田土壤亚铁毒害,研究了土壤-水稻-亚铁-微生物相互作用的体系中,外加Fe2+ 不同处理水平 (0、 100、 200、 400、 800和1600 mg/kg) 对水稻苗期和分蘖期相关生理指标、 土壤微生物活性及其生态特征的影响。结果表明, 在含一定亚铁本底（207.77 mg/kg）的正常稻田土壤中,外源性Fe2+的加入将逐步抑制水稻生长、 降低土壤微生物活性。外源Fe2+浓度达100 mg/kg后,水稻的株高、 干物质积累量显著降低; 水稻叶片生理指标叶绿素含量（SPAD值）、 脯氨酸含量、 抗氧化酶系统活性则显著增加,表明外源Fe2+浓度100 mg/kg 是本研究条件下外源Fe2+ 对水稻生长产生显著毒害影响的临界点; 同时随外源Fe2+浓度的增加, 土壤微生物活性指标土壤微生物量碳、 微生物三大基础菌系总量（细菌、 真菌、 放线菌）、 功能菌系总量（氨化细菌、 固氮菌、 纤维分解菌）、 铁还原菌总量总体是先快速下降,后逐渐平稳降低。 半效应浓度EC50分析表明,外源Fe2+浓度100 mg/kg 为多数土壤微生物活性指标（微生物基础菌系总量、 功能菌系总量、 铁还原菌）EC50变化的临界值; 体系中土壤微生物活性指标和水稻生长指标的变化存在显著的相关性, 表明供试土壤亚铁对水稻生长的影响是亚铁对土壤-植物-土壤微生物系统同步影响的结果。综上结果可知,外源Fe2+浓度100 mg/kg为导致供试土壤中水稻生长及土壤微生物活性受到显著负效应的临界值,进而推知,本研究所用土壤对水稻生长和微生物活性的亚铁毒胁迫临界浓度约为300 mg/kg（含本底）, Fe2+含量超出该浓度时,需采取合理的农艺措施控制其负效应。     

Effects of lead upon the actions of sulfate-reducing bacteria in the rice rhizosphere

Microbe-mineral interactions play an important role in affecting geochemical transformations of heavy metals in the soil environment. The formation of metal sulfide, which is mediated by sulfate-reducing bacteria (SRB) through contributing to sulfate reduction is an important pathway for heavy metal stabilization in anoxic soil. In oxic rice rhizospheres, there are abundant sulfur oxidizing bacteria (SOB) which can enhance sulfur oxidation and hence the availability of heavy metals, resulting in the uptake of such metals by the plant and a potential risk to human health. In this study, the potential existence of SRB in oxic rice rhizospheres, their contribution to sulfate reduction, and potential to reduce the availability of heavy metal was investigated. PCR-DGGE fingerprinting and real-time PCR results showed increasing numbers of SRB with Pb addition, which corresponded with increases in soil pH and reduction in Eh, suggesting the enhancement of sulfur reduction and SRB activity. Sulfur K-edge XANES, which characterized sulfur speciation in situ, revealed reduced states of sulfur. The SRB mediated the sulfate reduction and contributed to the formation of reduced sulfur which interacted with Pb, leading to the formation of stable metal sulfide and reduction of Pb availability. In return, acclimated SRB populations developed in Pb-polluted conditions. Hence stabilization of reduced sulfur by Pb enhanced the activity of SRB and sulfate reduction in rice rhizosphere.     

半干旱农田生态系统作物根系和施肥对土壤微生物体氮的影响

盆栽和大田试验表明,作物根系显著影响土壤微生物体氮的含量。在田间试验条件下,根际土壤微生物体氮比非根际土壤平均高出N54.7μg/g;盆栽试验中,根际土壤微生物体氮平均含量为N77.1±13.6μg/g,而非根际土壤为N65.2±17.0μg/g,差异达显著水平,根际微生物体氮含量为非际根际土壤的1.10～2.04倍。施肥能明显增加土壤微生物体氮含量,但影响程度因肥料种类而不同。秸秆和富含有机物质的厩肥对土壤微生物体氮的影响远大于化学肥料,而且土壤微生物体氮含量随秸秆施用量增加而增加。在红油土上进行的20年长期田间定位试验结果表明,对不施肥和施氮磷处理,0—20cm土层的微生物体氮分别是N102.2和110.4μg/g;在施氮磷的基础上,每公顷配施新鲜玉米秸秆9375kg、18750kg、37500kg和厩肥37500kg时,相应土层微生物体氮分别是N147.5、163.2、286.4和265.3μg/g。培养条件下,当有效能源物质缺乏时,微生物对NH4+-N的同化固定能力远大于NO3--N,但在加入有效能源物质葡萄糖后,微生物对2种形态氮的固定量大幅度增加,且对2种形态氮的固定量趋于一致。     

Mechanisms of production of soil protease by proteolytic Bacillus subtilis in wetland rice soil

In sterile soil inoculated with proteolytic Bacillus subtilis, the correlation between variations in protease activity and cell numbers was investigated during incubation, and the results were compared with those obtained in different growth media. In sterile soil inoculated with B. subtilis, the extracted soil z-FLase activity (hydrolytic activity towards benzyloxycarbonyl-L-phenylalanyl-L-leucine extracted with 0.1 M phosphate buffer) was correlated with the extracted soil protein content (r=0.95; P<0.01) and the number of vegetative cells (r=0.88, P<0.05), while the extracted soil caseinase activity was well correlated with the number of spores (r=0.82, P<0.05) and not with the number of vegetative cells. Extracellular z-FLase activity in different growth media, inoculated with the proteolytic B. subtilis, was correlated with the extracellular protein concentration (r=0.73, P<0.01) and the number of cells (r=0.73, P<0.01), but extracellular caseinase activity was not correlated with either of these measurements. From these results, we concluded that soil z-FLase production might relate to vegetative growth of the proteolytic B. subtilis and soil caseinase might relate to B. subtilis sporulation.