生防放线菌ZZ-9耐药菌株的微波诱变选育

为了获得耐甲基硫菌灵的菌株,采用微波诱变法对放线菌ZZ-9菌株进行0～75 s的处理,筛选出在甲基硫菌灵致死浓度下仍能较好生长的突变菌株,并测定其对苹果树腐烂病菌拮抗性和传代稳定性的影响,选育出性能优良的耐药菌株,与甲基硫菌灵协同作用进行防治苹果树腐烂病试验。结果表明,在微波辐照15～60 s条件下,获得4株耐70μg·mL^(-1)甲基硫菌灵的突变菌株ZZ-9A、ZZ-9B、ZZ-9C和ZZ-9D。通过对4株突变菌株进行抑菌率和传代稳定性测定,筛选出一株抑菌率较好且传代稳定的突变菌株ZZ-9B。菌株ZZ-9B与70μg·mL^(-1)甲基硫菌灵协同作用测定表明,当菌株ZZ-9B与甲基硫菌灵配比为8∶2时,在离体枝条上对苹果树腐烂病的协同防效为89.42%,明显高于单独使用突变菌株和甲基硫菌灵的防效。因此,在使用菌株ZZ-9B来防治苹果树腐烂病时,添加少量的甲基硫菌灵,可显著提高防效。

链霉菌702菌株所产抗真菌单体组分DZP-8和DZP-9的分离纯化与制备

用乙醇浸提链霉菌702菌株发酵液,滤液真空浓缩成油状物,再用乙酸乙酯萃取,萃取液真空浓缩成浸膏,浸膏通过硅胶柱层析,以氯仿与甲醇不同配比进行梯度洗脱,收集含抗真菌活性物质馏分洗脱液,进行真空浓缩至干燥为抗真菌活性物质粗品。粗品通过制备型HPLC仪进一步分离纯化,用乙腈∶水∶TFA不同浓度进行梯度洗脱,按单组分保留时间收集不同馏分洗脱液,将洗脱液冻干成抗真菌活性单体组分样品。抗菌活性和单组分样品纯度检测分别用管碟法检测对黑曲霉的抑菌活性和分析型HPLC。结果表明,含抗真菌活性单位10 000 mg/L发酵液200 L经乙醇浸提液浓缩后得到含抗真菌活性单位22 500 mg/L的油状物80 L,浸提率为90%;80 L油状物经乙酸乙酯萃取浓缩得含抗真菌活性单位120 000 mg/L的12 L浸膏,萃提率为80%。500 g浸膏经硅胶柱层析,获得含抗真菌活性单位达750 mg/g的粗品48.0802 g。粗品经制备型HPLC仪获得纯度分别达到99.47%和90.28%的抗真菌单组分DZP-8和DZP-9。

Multiplex genome editing using a dCas9-cytidine deaminase fusion in Streptomyces

CRISPR/Cas-mediated genome editing has greatly facilitated the study of gene function in Streptomyces. However, it could not be efficiently employed in streptomycetes with low homologous recombination(HR) ability. Here, a deaminase-assisted base editor d Cas9-CDA-ULstr was developed in Streptomyces, which comprises the nuclease-deficient Cas9(dCas9), the cytidine deaminase from Petromyzon marinus(PmCDA1), the uracil DNA glycosylase inhibitor(UGI) and the protein degradation tag(LVA tag). Using d Cas9-CDA-ULstr , we achieved single-, double-and triple-point mutations(cytosine-to-thymine substitutions)at target sites in Streptomyces coelicolor with efficiency up to 100%, 60% and 20%, respectively. This base editor was also demonstrated to be highly efficient for base editing in the industrial strain, Streptomyces rapamycinicus, which produces the immunosuppressive agent rapamycin. Compared with base editors derived from the cytidine deaminase rAPOBEC1, the PmCDA1-assisted base editor dCas9-CDA-ULstr could edit cytosines preceded by guanosines with high efficiency, which is a great advantage for editing Streptomyces genomes(with high GC content). Collectively, the base editor dCas9-CDA-ULstr could be employed for efficient multiplex genome editing in Streptomyces. Since the d Cas9-CDA-ULstr -based genome editing is independent of HR-mediated DNA repair, we believe this technology will greatly facilitate functional genome research and metabolic engineering in Streptomyces strains with weak HR ability.

Biosorption of mercury and lead by aqueous Streptomyces VITSVK9 sp. isolated from marine sediments from the bay of Bengal, India

Toxic heavy metals are increasingly accumu- lating in the environment worldwide and are considered to be life threatening contaminants. The biosorption of mercury and lead by marine actinomycetes isolated from marine sediment collected from the Bay of Bengal coast of Puducherry, India, was evaluated. The maximum tolerance concentration （MTC） of Streptomyces sp. was determined by a well diffusion method and a broth dilution method. The effects of the initial metal ion concentration, the pH and the biomass dosage on the biosorption of mercury and lead ions were investigated. The MTC of the isolate to metals was 200 mg. L 1 for mercury and 1800 mg. L-1 for lead. At neutral pH, the isolate had a maximum biosorption of metal ions of 200 mg.L 1 and 150 mg.L~ for mercury and lead respectively. Fourier transform infrared （FTIR） absorption spectra showed the chemical interactions between the functional groups in the biomass such as hydroxyl （- OH）, amine （-NH2）, earboxyl （- COOH） and the metal ions. The isolate was further characterized by molecular taxonomy and identified as a member of the genus Streptomyces. Based on the phenotypic and phylogenetic analysis, the strain was classified as a new species of the genus Streptomyces and designated as Streptomyces VITSVK9 sp. （HM137310）. A blast search of the 16S rDNA sequence of the strain showed the most similarity （95%） with Streptomyces sp. A515 Ydz-FQ （EU384279）. Based on the results, it can be concluded that this marine Streptomyces could be used as a biosorbent for the removal of heavy metal ions from aqueous environments.

放线菌ZZ-9发酵液与腐植酸钠混施对小麦的促生作用

为探讨放线菌ZZ-9发酵液与腐植酸钠混施对小麦生长的影响,将ZZ-9发酵液与腐植酸钠按照不同质量比混合,探究放线菌ZZ-9发酵液与腐植酸钠混施后对小麦的生长指标和生理生化指标的影响。结果表明,ZZ-9发酵液与腐植酸钠质量比为16∶4时,对小麦的各生理指标的影响较明显,与空白对照相比较,生长30 d后,小麦株高和根长分别增加了32.7%和30.6%;PAL和PPO活性增加了129.7%、68.0%;叶绿素含量增加了57.8%;MDA含量减少了69.4%。与单施发酵液相比较,小麦株高和根长分别增加了12.16%、7.57%;PAL和PPO活性分别增加了85.10%、96.30%;叶绿素含量增加了37.92%;MDA含量减少了56.65%。证明放线菌ZZ-9发酵液与腐植酸钠按16:4比例混施后处理小麦,对小麦具有更好的促生效果。

2种生防菌对苹果树3种病害病原菌拮抗作用比较

以生防放线菌长枝木霉T6和放线菌ZZ-9为试材,采用圆盘滤膜法和对口培养法比较T6、ZZ-9所产生的代谢物对苹果树腐烂病病菌、苹果树轮纹病病菌和苹果树早期落叶病病菌的拮抗作用,结果表明:长枝木霉T6的非挥发性物质对这3种病原菌抑制效果明显,抑制率分别达到97.50%、83.69%、68.35%。放线菌ZZ-9的非挥发性物质对苹果树腐烂病菌和苹果树轮纹病菌的抑制率达到了96.25%和72.42%%,对早期落叶病菌的拮抗作用较弱,抑制率小于50%。长枝木霉T6和放线菌ZZ-9的挥发性物质对这3种病原菌的拮抗作用不明显,抑制率均小于50%。长枝木霉T6和放线菌ZZ-9对苹果树三种病害的拮抗作用主要由二者所产生的非挥发性物质起作用。