干旱胁迫下灰色链霉菌对桔梗幼苗根际土壤酶活性、养分及微生物的影响

以不同浓度的灰色链霉菌发酵液处理干旱胁迫的桔梗根际土壤为研究对象,将不同浓度的灰色链霉菌发酵液(原液,原液稀释10倍,原液稀释50倍,原液稀释100倍(菌数1×10~8cfu·mL~(-1)),原液稀释150倍)200m L分别与花盆0~20 cm的土搅拌,无菌水作为对照(CK),土壤的持水量控制在45%左右,待桔梗生长至四叶期时,分别于7、14、21、28、35 d取其根际土壤(0~20 cm)。采用比色法和高锰酸钾滴定法测定桔梗根际土壤蔗糖酶、脲酶、过氧化氢酶、纤维素酶和蛋白酶活性的变化,稀释平板计数法测定微生物数量。结果表明,随着灰色链霉菌发酵液稀释倍数增加,桔梗根际土壤蔗糖酶、脲酶、过氧化氢酶、纤维素酶和蛋白酶活性均逐渐升高,发酵液稀释到100倍时,土壤蔗糖酶、脲酶、过氧化氢酶、纤维素酶和蛋白酶活性均达到峰值,比CK的峰值分别提高了68%、12.63%、11.53%、128.24%、43.51%。在第35天时,用灰色链霉菌发酵液稀释100倍处理干旱胁迫的桔梗根际土壤有机质、全氮、有效磷、速效钾含量比CK分别升高了280.13%、99.15%、60%和60.12%;灰色链霉菌发酵液处理的土壤中细菌不断增加,在第35天时比CK增加了355.43%,而土壤中放线菌和真菌先增加后降低,但高于CK。土壤酶活性、土壤养分因子和土壤微生物数量的相关性分析表明,脲酶与有机质、全氮、有效磷、速效钾和细菌呈极显著正相关(P0.01),纤维素酶与有机质、全氮、有效磷、速效钾、细菌和放线菌呈极显著或显著正相关,蛋白酶与有效磷、速效钾、细菌、放线菌和真菌呈极显著或显著正相关,蔗糖酶和过氧化物酶与有机质、全氮、有效磷、速效钾和细菌呈负相关且不显著,与放线菌、真菌正相关且不显著。     

纳塔尔链霉菌产纳他霉素发酵条件优化

纳他霉素(Natamycin)广泛应用在医药、食品和保健等领域,具有良好的应用前景。作为一种高效的广谱抗真菌剂,它能有效的抑制真菌及真菌孢子的生长。为了提高纳他霉素的发酵水平,通过摇瓶分批发酵试验的方法应用纳塔尔链霉菌(Streptomyces natalensis)生产纳他霉素,重点研究了种子质量、发酵培养基成分和培养条件的优化等单因素条件。结果表明：通过改变单因素变量确定了最佳发酵培养基：葡萄糖40g/L,大豆蛋白胨20g/L,酵母粉5g/L,初始pH7.0;最佳发酵条件：培养至48h的种子液以6%的接种量接种于装液量为25mL发酵培养基的250mL三角瓶中;28℃,220r/min进行发酵。纳他霉素的产量在此最优工艺下可达1.472g/L,比优化前提高了150%,为相关的试验研究提供了可靠的基础依据。     

放线菌JS2对石榴枯萎病菌及南方根结线虫的毒力研究

石榴枯萎病是一种比较难防治的土传病害,石榴根结线虫病的广泛发生加重了石榴枯萎病发生程度及病害的防控难度,所以探索兼具拮抗石榴枯萎病及根结线虫病的生防菌,才能有效环保地控制石榴枯萎病。首次报道了链霉菌JS2兼有拮抗石榴枯萎病菌及抑杀南方根结线虫的活性。本研究从云南蒙自、建水石榴园采集的土样中分离筛选得到对石榴枯萎病菌有较强拮抗活性的放线菌,其中菌株JS2对石榴枯萎病菌的室内抑菌直径为3.5 mm。研究结果表明菌株JS2对南方根结线虫有较好的抑杀作用,50%JS2菌悬上清液处理南方根结线虫卵6 d的孵化率与对照相比降低了53.33%,50%JS2菌悬上清液处理南方根结线虫二龄幼虫72 h后,校正死亡率增加了83.88%。菌株JS2兼有拮抗石榴枯萎病菌及抑杀南方根结线虫的活性,具有潜在生防价值。结合菌株JS2在各鉴定培养基上的培养特征、形态学观察及16S rD NA序列分析,将JS2初步鉴定为公牛链霉菌(Streptomyces tauricus)。     

硫藤黄链霉菌基因组表达文库的构建及筛选

为了探讨硫藤黄链霉菌中可能存在的次级代谢产物合成及调控机制,以链霉菌整合型质粒pJTU2554为载体,构建了硫藤黄链霉菌的基因组表达文库。以模式菌株变铅青链霉菌为异源表达宿主,通过生物活性筛选获得9株具有抑菌活性的异源表达突变菌株,同时通过PCR筛选获得多个含有聚酮合酶和非核糖体多肽合成酶基因的克隆子。TLC及HPLC-MS分析发现6个携带有aureothin生物合成基因簇的cosmid,通过异源表达在变铅青链霉菌中成功合成聚酮类抗生素aureothin,证实文库异源表达及筛选的有效性。     

Potential for Biological Control of Botrytis cinerea in Pinus sylvestris Seedlings

Grey mould (Botrytis cinerea Pers.: Fr.) is the most common economically important fungal disease in Swedish forest nurseries. In tests in a growth room, foliage of predisposed (preinoculation incubation at 35°C for 4 days) Scots pine (Pinus sylvestris L.) seedlings was sprayed with suspensions containing Mycostop®, Binab® TF.WP or GlioMix® at concentrations of 0.5, 1 or 0.5 g l^?1, respectively, and/or conidia of B. cinerea (10⁶ spores ml^?1). Binab and GlioMix reduced grey mould in needles by 94 and 92%, respectively, and were as effective as the fungicide Euparen® M 50 WG, while Mycostop reduced disease by 51%. In one trial in a forest nursery, Mycostop, Binab and GlioMix, each applied two and four times during the growing season, suppressed spontaneous B. cinerea infections in needles of first year container-grown P. sylvestris seedlings by 16–57%, and were as effective as recommended fungicidal sprays. It was concluded that biological control has potential to effectively suppress grey mould in seedlings in forest nurseries.     

链霉菌769接合转移体系的建立

本研究确定了质粒pSET152从大肠杆菌(Escherichia coli)ET12567(pUZ8002)转移到链霉菌769中的接合转移的条件。在MS培养基上得到了最高的接合转移效率;孢子在50℃下热激10 min,供体菌和受体菌的比例为1:10,16～18 h后进行抗生素覆盖的情况下结合转移效率为1.03×10-6～1.23×10-5。经过连续5代的选择压力和PCR验证,表明质粒pSET152已经整合到受体菌株的染色体中。本研究首次建立了链霉菌769接合转移系统。     

密旋链霉菌Act12遗传转化系统的建立与优化

密旋链霉菌(Streptomyces pactum)Act12是分离自黄土高原的1株多效放线菌,具有良好的生防应用价值,为了深入研究与利用,需建立该菌株的高效遗传转化系统。本试验以整合型质粒pSET152为出发质粒,大肠杆菌S17-1为供体,Act12的孢子为受体时,在改良2CMY培养基上进行接合转移,孢子预萌发条件为50℃热激10min,阿普霉素质量浓度为10.0mg/L,覆盖时间为18h,转化效率达到2.079×10~(-5)。以抗阿普霉素基因为目的基因,通过PCR验证,成功地将质粒pSET152转入到Act12中。同时,利用该转化系统,成功敲出Act12基因组中一可能的负调控转录因子spa0520,并获得了次级代谢图谱明显变化的缺失突变株Δspa0520。所构建的Act12遗传转化系统,为后续对其生防活性机理的研究以及深入开发利用奠定了基础。另外,通过培养基的改良发现,Act12在氮源含有NO-3时产孢丰富,为后续研究提供了思路。     

Insights into the Variation in Bioactivities of Closely Related Streptomyces Strains from Marine Sediments of the Visayan Sea against ESKAPE and Ovarian Cancer

Marine sediments host diverse actinomycetes that serve as a source of new natural products to combat infectious diseases and cancer. Here, we report the biodiversity, bioactivities against ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.) and ovarian cancer, and metabolites variation among culturable actinomycetes isolated from the marine sediments of Visayan Sea, Philippines. We identified 15 Streptomyces species based on a 16S rRNA gene sequence analysis. The crude extracts of 10 Streptomyces species have inhibited the growth of ESKAPE pathogens with minimum inhibitory concentration (MIC) values ranging from 0.312 mg/mL to 20 mg/mL depending on the strain and pathogens targeted. Additionally, ten crude extracts have antiproliferative activity against A2780 human ovarian carcinoma at 2 mg/mL. To highlight, we observed that four phylogenetically identical Streptomyces albogriseolus strains demonstrated variation in antibiotic and anticancer activities. These strains harbored type I and II polyketide synthase (PKS) and non-ribosomal synthetase (NRPS) genes in their genomes, implying that their bioactivity is independent of the polymerase chain reaction (PCR)-detected bio-synthetic gene clusters (BGCs) in this study. Metabolite profiling revealed that the taxonomically identical strains produced core and strain-specific metabolites. Thus, the chemical diversity among these strains influences the variation observed in their biological activities. This study expanded our knowledge on the potential of marine-derived Streptomyces residing from the unexplored regions of the Visayan Sea as a source of small molecules against ESKAPE pathogens and cancer. It also highlights that Streptomyces species strains produce unique strain-specific secondary metabolites; thus, offering new chemical space for natural product discovery.     

提高耐热链霉菌转化泰乐菌素能力的分子改造与育种

BIB0830是将泰乐菌素转化为乙酰异戊酰泰乐菌素(AIV)的耐热链霉菌(Streptomyces thermotolerans)工业菌株。以pIJ8600为载体分别构建了acyB1-B2基因整合型表达载体pBIB425和带红霉素突变启动子ermEp*的红霉素抗性ermE基因整合型表达载体pBIB304。通过大肠杆菌(Escherichia coli)-链霉菌属间接合转移分别将pBIB425、pBIB304和pBIB308(耐热链霉菌的nsdA基因中断载体)导入工业菌株BIB0830,抗性筛选分别得到接合子BIB425、BIB304和BIB308。经PCR验证均为阳性克隆。摇瓶发酵结果表明,与出发菌株BIB0830相比,BIB425和BIB308泰乐菌素转化率分别提高了14%和22%,而BIB304则无明显影响。     

井冈霉素高产菌株的复合诱变筛选

吸水链霉菌井冈变种(Streptomyces hygroscopicus var. jinggangensis Yen)KN-16经紫外线、60Coγ射线、亚硝酸-紫外线复合诱变处理,摇瓶发酵筛选得到一株井冈霉素A高产菌株F-101,该菌株产井冈霉素A含量较出发菌株提高了33.5%.