藤黄轮丝链霉菌T0907-107抑菌活性研究

从海产虾壳上分离筛选拮抗放线菌、研究其抑菌活性具有重要的实践意义。藤黄轮丝链霉菌(Streptomyces luteoverticillatus)T0907-107是分离自海产虾壳上并经初筛和鉴定的一株链霉菌,为了进一步开发和利用该菌,本研究采用极性不同的5种溶剂(丙酮、乙酸乙酯、正丁醇、乙醇和甲醇),对该菌株的发酵液菌体活性物质进行了提取,对粗提物的理化性质和抑菌活性进行了初步研究。结果表明,其发酵液活性物质大部分存在于菌体中,采用甲醇和丙酮浸提的效果好,其抑菌圈直径分别为26.20和22.50 mm;对粗提物进行耐热性实验,结果表明,粗提物是一种能够耐60℃高温和一定酸碱的物质;粗提物对蛋白酶K稳定;菌体及粗提物对多种植物病原菌物如烟草炭疽病菌(Colletotrichum nicotianae Sacc.)、烟草赤星病菌(Alternaria alternata(Fries)Keissler)、烟草立枯丝核病菌(Rhizoctonia solani Kühn)、枇杷炭疽菌(C.gloeosporioides(Penz.)Sacc.)和白菜炭疽病菌(C.higginsianum Sacc.)有较强抑制作用。研究结果表明,甲醇和丙酮是较合适的萃取溶剂,该菌发酵液菌体粗提物极性强、稳定性好、有较广的抑菌谱,显示出好的开发前景。本研究为进一步鉴定该活性物质及开发和利用该菌提供了基础资料。     

链霉菌表达系统的研究进展

链霉菌是广泛应用于工业生产的革兰氏阳性细菌，链霉菌作为继大肠杆菌、枯草芽孢杆菌之后又一个有价值的原核表达宿主近年来已有较为深入的研究。本文主要综述如下几个内容：链霉菌作为外源基因表达宿主的优点和缺点；链霉菌启动子－特别是严紧控制型启动子的研究进展；链霉菌基因转录的终止和翻译的起始；链霉菌中蛋白质分泌的信号；链霉菌分泌的蛋白酶及影响外源基因表达的其它因素。     

不同菌落形态的链霉菌对产谷氨酰胺转胺酶的影响

通过对链霉菌STK-203进行自然选育,挑选出5种不同菌落形态的单菌落。摇瓶发酵培养48h后检测发酵液中的谷氨酰胺转胺酶的活性、残余氨基氮含量、残余甘油含量、菌种生物量等4种生理指标。结果表明,编号为Ⅰ即馒头型的单菌落的酶活最高,高达5．84U／mL,其残余甘油含量为0．071g／L,残余氨基氮含量为1．48g／L,菌体浓度为36．01％。根据菌落形态和产酶的相关性,为产谷氨酰胺转胺酶的链霉菌STK-203菌株的选育和生产提供了一种简便有效的方法。     

变铅青链霉菌66的多效性突变子

变铅青链霉菌ＤＮＡ存在一种异常的位点特异性修饰，使其在含有Ｆｅ＾２＋的电泳缓冲液中电泳时遭到双链切割。用ＮＴＧ诱变变铅青链霉菌ＪＴ４６，获得了一株修饰缺陷突变株，命名为ＺＸ１。该突变菌株除了丧失修饰其ＤＮＡ的能力以外（亦即其ＤＮＡ在含有Ｆｅ＾２＋的电泳缓冲液中电泳时不被降解），在某些基因［如黑色素基因（ｍｅｌ）和某些抗生素抗性基因］的表达方面也发生了变化，同时还丧失了产孢能力和对唾噬体ΦＨＡＵ３的     

番茄内生链霉菌S5的分离及其除草活性

采用植物内生放线菌分离方法从健康番茄（Lycopersicon esculentum）根中分离纯化出58株内生放线菌，从中挑选部分代表性菌株进行代谢产物除草活性检测，发现编号为S5的菌株的代谢产物对小麦（Triticum aesfivum L．）和绿豆（Phaseolus radiatus L．）种子的发芽有强烈的抑制作用，但对发芽后的幼苗生长无明显影响。以百喜草（Paspalum notatum）和狗牙根（Cynodon dactylon）为实验对象，证明S5菌株的代谢产物的确能抑制草籽的发芽，该活性具有潜在的除草效能。经初步鉴定，S5菌株为淡紫灰链霉菌淡青变种（Streptomyces lavendulaevar．glaucescens）。发酵条件实验结果表明，S5菌株在1％葡萄糖和0．3％牛肉膏的S培养基中，以2％接种量在pH7．0和25℃摇床培养，可得到最强的抑制种子发芽的生物活性。     

绿色产色链霉菌E-219菌落的形态分化

菌株E-219由原始菌株CGMCC4.1119经60Coγ诱变及原生质体融合等手段改造后所得,本文通过分子荧光探针、荧光显微镜和电镜对绿色产色链霉菌E-219在固体基质表面的发育分化周期、斜面培养时间对阿维拉霉素产量的影响进行了研究,以为工业发酵选择适当的斜面种子提供理论依据。试验表明该菌株菌落发育过程伴有间断性菌丝凋亡,分生孢子的产生是活性菌丝分化的结果。与原始菌株相比该菌落形态呈现显著不同,其孢子萌发形成杂色初生菌丝,基内菌丝出现二次死亡(48h)及其中活性菌丝片段的二次快速生长,分生孢子形成数量多。原始菌株呈螺旋状孢子丝,孢子表面带刺,而E-219为直线型孢子丝,分生孢子表面光滑无刺突。摇瓶试验表明,菌种斜面种龄以106h左右为宜,此时分生孢子活性高,阿维拉霉素发酵单位可达1200mg/L。     

天蓝色链霉菌中新型双组分系统DraR-K对抗生素生物合成的差异调控

双组分调控系统(two-component system, TCS)是原核生物中最主要的信号转导系统,通常由组氨酸激酶和效应调节蛋白两类在结构上相对保守的蛋白组成。双组分调控系统在链霉菌抗生素合成复杂的代谢网络调控中起着重要的作用。近期中国中科院上海生命科学研究院植物生理生态研究所的研究人员发现天蓝色链霉菌(Streptomyces coelicolor)中存在一个新型双组分调控系统由 DraR 和 DraK 两个蛋白组成,被命名为 DraR-K。他们的研究发现 DraR 和 DraK 任何一个基因缺失都能导致分别添加不同氮源的基础培养基上生长的天蓝色链霉菌中放线紫红素的合成显著降低,但是十一烷基灵菌红素能够显著增加。同时发现这些突变体在添加谷氨酸盐的基础培养基上能够过量表达 I 型聚酮类黄色素。     

橄榄绿链霉菌A1所产43kD木聚糖酶XYNA的纯化及其酶学性质

橄榄绿链霉菌A1(Streptomyces olivaceoviridisA1)所产木聚糖酶XYNA经阴离子交换层析和分子筛层析分离,得到纯化的XYNA.XYNA的分子量约为43 kD.其最适温度为60℃,最适pH5.6.XYNA在55℃下以4-O-Me-D-glucurono-D-xylan的Km值和Vmax分别是332 g/kg和15.72μmol/(mL@min).SDS、EDTA对XYNA有轻微的抑制作用.XYNA无纤维素酶活性.胃蛋白酶、胰蛋白酶处理30 min,对酶活性无影响.XYNA成熟蛋白N端10个氨基酸序列为Ala-G1u-Ser-Thr-Leu-Gly-Ala-A1a-Ala-Ala.     

链霉菌Snea253菌株的变异分析及对根结线虫活性影响

为明确委内瑞拉链霉菌(Streptomyces venezuelae)自发突变对南方根结线虫活性的影响。本研究利用继代培养法选育Snea253突变株,对形态分化及相关基因进行分析,并测其代谢产物的生物活性。获得6株菌落形态差异大的自发突变菌株,其中不产色素的3株突变菌株Snea253-G、Snea253-B和Snea253-F杀线虫活性分别为61.0%、45.5%和35.0%;产色素的3株突变株Snea253-A、Snea253-H和Snea253-J杀线虫活性分别为48.7%、37.2%和37.9%。ssgA、bldC、bldD、whiB、whiE和whiI基因克隆测序结果显示各突变株基因序列没有差异,可能此6个基因没有直接参与控制Snea253菌株的自发突变。链霉菌自发突变不但使其形态发生变异,而且也影响了其代谢产物的生物活性,研究为从形态上筛选优良菌株提供了相关理论依据。     

农抗5102产生菌与泾阳链霉菌5406融合的初步研究

本文报道农抗５１０２产生菌衍生菌株９０－１１和ＷＨ－１与泾阳链霉菌５４０６之间融合研究的前期结果。研究表明，二级生长培养基中蔗糖浓度增加至１７％及匀浆打散菌丝团，均能提高５４０６菌株的原生质体释放量。农抗５１０２产生菌与泾阳链霉菌原生质体制备的适宜条件差异较大。     

秸秆降解过程中拮抗链霉菌对禾谷镰刀菌消长动态的影响

由禾谷镰刀菌(Fusarium graminearum)引起的小麦赤霉病是导致小麦产量和品质降低的主要因素，其在田间生长的基质主要为秸秆。基于各种拮抗菌的生物防治是控制小麦赤霉病的主要措施之一，但秸秆降解过程中拮抗菌的拮抗效应是否与平板对峙获取的拮抗效应一致尚不清楚。本研究首先通过平板对峙试验，筛选出对禾谷镰刀菌CGMCC3.4598有潜在拮抗作用的3株菌：吸水链霉菌ACCC41648 (Streptomyces hygroscopicus)、吸水链霉菌井冈变种ACCC40051 (S. hygroscopicus var. jingganggensis)和娄彻氏链霉菌ACCC41594(S.rochei)，其抑菌带平均宽度依次为2.67、1.67和1.12mm。基于室内70d培育试验，发现秸秆降解7、42和70 d后，吸水链霉菌ACCC41648及其井冈变种ACCC40051未表现抑制秸秆中禾谷镰刀菌生长的效应，而娄彻氏链霉菌ACCC41594在秸秆降解7 d和42 d后显著降低禾谷镰刀菌丰度分别达30.91%和69.28%。与对照相比，娄彻氏链霉菌ACCC41594显著促进秸秆降解...     

链霉菌JD211对水稻幼苗促生作用及土壤细菌多样性的影响

以春光1号水稻为供试种子,链霉菌JD211为供试菌,通过浅盘试验研究链霉菌JD211对水稻生物量和土壤细菌多样性的影响。结果表明:菌剂用量10 g kg-1对水稻幼苗的生长有极显著的促进作用,幼苗总干重、地上部分干重、地下部分干重与对照相比分别提高52.15%、44.56%、65.55%,植株全氮、全磷分别提高90.10%、58.51%。与CK土壤相比,菌剂用量10 g kg-1的土壤速效氮、有效磷均有显著提高,分别提高了37.49%、40.62%。细菌多样性的末端限制性片段长度多样性(T-RFLP)分析表明链霉菌JD211能促进一些稀有或生态势较弱的细菌生长,使参与土壤营养循环、改善土壤质地及防治植物病害的功能菌成为优势菌群。土壤功能微生物类群的变化,加速了N、P等土壤养分循环,增强了水稻对N、P等矿质养分的吸收,从而促进水稻生长。     

土壤因子对链霉菌S506定殖和促生功能的影响

链霉菌S506是从植物根际分离获得的具有促生、防病和降解根系毒素功能的根际微生物,为了解土壤环境条件对其在根际定殖和功能表达的影响,以链霉菌S506为试材,研究了土壤质地、环境温度和土壤湿度对S506在番茄根际土壤定殖及番茄植株生长的影响.结果表明,适宜链霉菌S506在番茄根际定殖和促生作用发挥的土壤质地为壤土,其次为砂土和黏土;试验所设温度梯度中,利于S506在根际定殖的环境温度为30℃,其次为22.5℃和15℃,而利于目的菌株促生功能表达的环境温度则依次为22.5℃、30℃和15℃;适宜于目的菌株在根际定殖和促生功能发挥的土壤相对湿度为20%、25%,其次为15%和30%.     

Screening of Streptomyces strains helping arbuscular mycorrhizal symbiosis against pepper (Capsicum annuum L.) Phytophthora blight

Mycorrhiza helper bacteria (MHB) can promote the formation and functioning of arbuscular mycorrhizal (AM) symbiosis, but their role and application potential in coping with soil-borne diseases are still unclear. A 14-week greenhouse pot experiment was conducted to obtain several actinomycete strains helping AM symbiosis in suppressing the Phytophthora blight of pepper (Capsicum annuum L.), using a soil inoculated with Phytophthora capsici after sterilization. Five Streptomyces strains, including S. pseudogriseolus, S. albogriseolus, S. griseoaurantiacus, S. tricolor, and S. tendae, as well as the AM fungus (Funneliformis caledonium) were tested. The Phytophthora blight severity reached 66% at full productive stage in the uninoculated control, and inoculation of F. caledonium, S. griseoaurantiacus, and S. tricolor alone significantly decreased (P ＜ 0.05) it to 47%, 40%, and 35%, respectively. Compared to F. caledonium alone, additional inoculation of S. tricolor or S. tendae, which were isolated from the rhizosphere of a healthy individual in an infected field, significantly elevated (P ＜ 0.05) root mycorrhizal colonization, root biomass, fruit yield, and total K acquisitions of pepper and further significantly decreased (P ＜ 0.05) blight severity. According to the feature of enhancing disease-suppression by AM symbiosis, both S. tricolor and S. tendae were confirmed as MHB strains here. Specifically, S. tendae had a stronger performance in directly accelerating mycorrhization, while S. tricolor antagonist to the pathogenic P. capsici. Furthermore, S. griseoaurantiacus with the independent disease-suppression function was not an MHB strain here. The redundancy analyses demonstrated that when AM fungus was present, root mycorrhizal colonization replaced soil pH becoming the main factor affecting pepper Phytophthora blight. Thus, S. tricolor and S. tendae seemed to have the value of preparation and application in the future to help AM symbiosis against pepper Phytophthora blight.     

利用微生物防除根寄生杂草列当

根寄生杂草列当(Orobanche spp.)已经严重制约全球许多地区的农业发展,寻找有效防除措施迫在眉睫。由于列当具有特殊生活史且与寄主关系密切,常规防除杂草措施难以达到理想防效。目前,尚无既能有效防除列当又不对寄主造成危害且便于大规模推广应用的列当防除措施。在众多列当防除措施中,微生物防除越来越引起关注和重视。本文对微生物防除列当的国内外研究进展及防除机理进行了综述。目前,列当生防微生物的研究主要集中在镰刀菌(Fusarium spp.)等列当病原菌和根瘤菌(Rhizobium spp.)等列当寄主植物共生菌上。微生物防除列当的机制主要包括两方面:一是通过产生代谢产物直接影响列当的萌发和生长,或通过降解列当种子萌发诱导物质间接影响列当的萌发;二是通过提高寄主植物自身对列当的抗性间接影响列当的寄生和生长。此外,本文还重点介绍了植物土传病害的土壤拮抗微生物防除列当杂草的可行性及研究进展。植物土传病害病原菌和列当均首先通过在地下侵染作物的根系进而危害作物正常生长,而作物抗土传病害的机理也与抗列当的机理类似。因此,存在于土壤中具有防治植物土传病害能力的微生物可能也具有防除根寄生杂草列当的功能。本团队前期试验从植物土传病害的土壤拮抗微生物中筛选到在盆栽试验中能够有效防除向日葵列当(O.cumana Wallr.)和瓜列当(O.aegyptiaca Pers.)的放线菌各1株,分别为淡紫褐链霉菌(Streptomyces enissocaesilis Sveshnikova)和密旋链霉菌(Streptomyces pactum Bhuyan B.K)。其中,密旋链霉菌的菌剂在田间试验中既降低了瓜列当的出土数量又增加了番茄的产量。总之,微生物是防除根寄生杂草列当的一条有效途径。     

苋菜对钾素吸收和利用上的基因型差异

The kinetics of K^ absorption and the utilization of both applied K and K in a calcareous alluvisol with low available K by different genotypes of grain amaranth(Amaranthus spp.),an ancient pseudo-cereal that produces a large biomass and a rich source of health nutrients and mineral elements,were studied by means of ion depletion technique and pot experiments.Grain amaranth had a high affinity for K^ absorption comparison with corn and cotton.However,there were differences among Amaranthus spp.in Kabsorption and utilization.Some cultivars such as R104,CX-4,Du001,Hy015 and Hr029 which had a higher rate of K absorption and stronger affinity for K^ were typical varieties with high K-use efficiency(KUE).The results showed that high KUE cultivars grew quickly,possessed stronger ability to take up soil slowly available K and Mineral K,and did not respond to K fertilization in the soil with low available K.Correspondingly,grain amaranth cultivars CX-77,Cr024,Vd001,Re003 and Sn003 were relatively low in KUE,Compared with high KUE cultivars,they took up more soil available K and both of their dry matter accumulation and K uptake responded to K applied significalty.     

梭梭和柽柳土壤微生物多样性初步分析

直接从沙漠土壤中提取混合微生物DNA,利用Illuminamiseq测序平台,对16S r DNA进行测序和分析。结果表明:1荒漠植被土壤微生物数量很少,生物活性极弱,DNA提取难度大;2沙漠植被土壤细菌多样性丰富,所有测得细菌分属到23个门和316个属,其中还存在一定数量的微生物新种,部分测得代表新属和种的序列提交Gen Bank,获得序列号(KT984242~KT984249);3不同沙漠土壤样品微生物群落有相似性,但也有较明显的差异,其差异与土壤理化因子等因素有关:如土壤含水量与酸杆菌和变形菌的分布相关;相对于空地,梭梭和柽柳群落土壤独有的迷踪菌门(Elusimicrobia)与固氮菌密切相关;梭梭的土壤环境p H明显高于柽柳土壤环境这决定了它们不同的优势种;土壤微生物生物量碳的大小不能反映微生物量的种类多少,但可以反映微生物数量的多少。     

百脉根根瘤菌自体诱导物合成酶基因mrlI2的初步研究

许多细菌均能产生一类叫做自体诱导物的信号分子,用于感知自身密度的变化,从而调控其生理行为,这种现象称为群体感应[1]。越来越多的研究表明,在根瘤菌中普遍存在着群体感应调控系统[2],并且它可能在根瘤菌与豆科植物共生结瘤固氮过程中起着重要作用。目前,在豌豆根瘤菌中共发现有4种群体感应系统(rai,rhi,cin和tra),其中cin系统位于上游,起总调控作用,tra系统控制着质粒的转移,rhi系统与结瘤数量有关,rai系统的功能还不清楚,它们共同组成了一个复杂的群体感应调控系统[3]。在苜蓿中华根瘤菌中,也发现群体感应系统与根瘤菌的生长和结瘤效率有关[4]。对中慢生型华癸根瘤菌和天山根瘤菌的研究     

Quantitative real-time PCR effectively detects and quantifies colonization of sclerotia of Sclerotinia sclerotiorum by Trichoderma spp.

Some members of the fungal genus Trichoderma are able to colonize and destroy sclerotia, the thick-walled resting structures of the soilborne plant pathogenic fungus Sclerotinia sclerotiorum, thus providing a potential means of biological disease control. However, current methods to detect and quantify colonization of sclerotia are labor-intensive, and generally qualitative rather than quantitative in nature. Our objective was to develop quantitative real-time PCR (polymerase chain reaction) methods to detect and measure colonization of sclerotia by Trichoderma spp. Specific PCR primer/probe sets were developed for Trichoderma spp. and for S. sclerotiorum. A total of 180 ITS1 (internal transcribed spacer) and ITS2 sequences from different species in the genus Trichoderma were aligned, and consensus sequences were determined. Six candidate primer sets were based on conserved regions of the consensus sequence, and the specificity of each nucleotide sequence was examined using BLAST (Basic Local Alignment Search Tool; NCBI) software. Each candidate primer set was tested on genomic DNA of T. harzianum strain ThzID1-M3, as well as six different Trichoderma isolates from soil, and on genomic DNA of S. sclerotiorum. The optimum primer/probe set selected, TGP4, successfully amplified genomic DNA of all Trichoderma isolates tested, and showed high precision and reproducibility over a linear range of eight orders of magnitude, from 85 ng to 8.5 fg of T. harzianum genomic DNA. TGP4 did not amplify S. sclerotiorum DNA. A specific PCR primer/probe set (TMSCL2) was developed for S. sclerotiorum, based on the calmodulin gene sequence. TMSCL2 did not amplify Trichoderma DNA. Quantitative real-time PCR with the primers then was evaluated in experiments to test differential effects of two soil moisture levels (−50 kPa, −1000 kPa matric potential) on biocontrol of S. sclerotiorum by indigenous Trichoderma spp. Periodically over 40 days, Trichoderma and S. sclerotiorum DNA in sclerotia were quantified by PCR with appropriate primers. Over 90% of the sclerotia were colonized by indigenous Trichoderma spp. at −1000 kPa, over the 40-day period, compared to only 60% at −50 kPa. In addition to determining incidence of colonization, the PCR method allowed measurement of the extent of sclerotial colonization, which also was significantly greater in the drier soil. Quantitative real-time PCR with the TGP4 primer/probe set provides a sensitive detection and measurement tool to evaluate colonization of sclerotia by Trichoderma spp.     

硫杆菌以及好养异养型硫氧化菌对元素硫的氧化

The prediction of the oxidation rate of elemental sulfur (S⁰) is a critical step in sulfur (S) fertilizer strategy to supply plant-available sulfur. An incubation experiment was conducted to determine the rate and amount of S⁰ oxidation in relation to the contribution of Thiobacillus spp. and aerobic heterotrophic S-oxidizing bacteria. After 84 days, 16.3% and 22.4% of the total S⁰ applied to the soil were oxidized at 20 and 30 ℃, respectively. The oxidation of S⁰ proved to be a two-step process with a rapid oxidation during the first 28 days and a slow oxidation from then on. The highest oxidation rate of 12.8 μg S cm^-2 d^-1 was measured during the first two weeks at 30 ℃. At 20 ℃ the highest oxidation rate of 10.2 μg S cm^-2 d^-1 was obtained from two to four weeks after start of the experiment. On an average the soil pH declined by 3.6 and 4.0 units after two weeks of experiment. At the same time the electric conductivity increased nine times. With the oxidation of S⁰ the population of Thiobacillus spp. and aerobic heterotrophic S-oxidizing bacteria increased. The corresponding values for Thiobacillus spp. and aerobic heterotrophic S-oxidizing bacteria increased from 2.9 × 10⁵ and 1.4 × 10⁵ g^-1 soil at the start of the experiment to 4 × 10⁸ and 5.6 × 10⁸ g^-1 soil 14 days after S⁰ application, respectively. No Thiobacillus spp. was present eight weeks after S⁰ application. The results suggested that oxidation of residual S⁰ completely relied on aerobic heterotrophic S-oxidizing bacteria.