侧柏内生真菌的分离鉴定及抗菌活性筛选

采用组织分离法从侧柏的根、茎、叶中分离获得内生真菌110株,经形态观察将其中103株鉴定分类为4个目、6个科、15个属,未产孢7株;内生真菌以链格孢属为优势属,茎叶中内生真菌的株数、种类多于根中.通过与苹果腐烂病菌、葡萄黑痘病菌等4种植物病原真菌平板对峙培养,从分离的内生真菌中筛选出21株(占总分离株数的19.1%)有拮抗活性的菌株,其至少对1种植物病原真菌有生长抑制作用,活性菌株主要分布在链格孢属,其抗菌活性成分值得进一步研究.     

拮抗油菜菌核病的海洋细菌筛选及其活性物质的分子检测

以油菜核盘菌为病原指示菌,对1021株海洋细菌进行抗菌筛选,从中得到了144株具抗菌活性的菌株.在建立筛选新型特异性拮抗化合物资源库的同时,重点分析了筛选的302株海洋假单胞菌,对其中具有较强抗菌活性的19株假单胞菌进行了产活性物质合成基因的分子检测.结果表明,菌株1A06832和SH-46扩增获得了2,4-二乙酰基间...     

海洋细菌抗菌筛选及深海独岛枝芽胞杆菌A493活性物质分离鉴定

以水稻黄单胞菌等植物病原菌为指示菌,采用平板对峙法对411株海洋细菌进行了抗菌筛选,初筛获得具有抗菌活性的海洋菌株81株,复筛获得具有稳定抗菌活性的菌株7株,最后通过测定抗菌谱,得到1株抗菌谱特异并且稳定拮抗水稻黄单胞菌的深海独岛枝芽胞杆菌（Virgibacillus dokdonensis）A493.对水稻白叶枯病害的温室生防实验表明,经过A493无菌发酵上清波处理,水稻生长20 d后对水稻白叶枯病害的防治效果达到了66.7％,且对水稻的正常生长无不良影响.通过离子交换色谱法提取,再经2次薄层层析硅胶板回收分离,得到了纯化活性物质,经过ESI-MS＂分析,初步判断活性物质分子量为317 Da.Doskochilova系统纸层析结果显示活性物质很可能为新的氨基糖苷类物质.     

9种药用植物内生或附生真菌的分离及其杀虫、抑菌活性

从9种药用植物的根、茎、叶、花中分离得到358株内生或附生真菌,其中9株真菌的发酵液对白纹伊蚊3龄幼虫的毒杀活性较强,24 h死亡率均达到80%以上;15株真菌的发酵液对至少1种供试病原真菌的菌丝生长抑制率超过50%;4株真菌的发酵液对5种供试细菌均有明显的抑菌活性.     

放线菌S-159-05抑菌活性物质的初步研究

从陕西渭北旱原的土壤中分离筛选得到一株对多种植物病原真菌拮抗性较好且遗传稳定的放线菌新菌株,编号为S-159-05。生测结果表明,S-159-05菌株活性产物粗提物对水稻稻瘟病菌和小麦根腐病菌有很好的抑制活性,1mg/L药剂的抑制率都在60%以上。采用大孔树脂柱层析和反相制备高效液相色谱切割分离,从40%乙醇洗脱液中得到一个有抑菌活性的单组分化合物An-2。经波谱数据分析,An-2的结构为1-(4-氨基甲酰基-呋喃-3-基)-2-(3-胍基)葡萄苷,这是在氨基糖苷类化合物中发现的一种新抑菌活性物质,从而为新的氨基糖苷类农用抗生素的研发打下良好基础。     

苦参杀虫活性菌株的筛选

为筛选苦参内生菌中对水稻害虫有毒杀作用的菌株,本研究采用研磨法从健康苦参植物的根、茎、叶和种子中进行菌株分离。用玻片浸渍法和喷雾法筛选产杀虫活性物质菌株,获得细菌93株、放线菌15株、真菌5株。经初筛及复筛,发现RoF2和LeB8两株菌株的发酵液粗提物分别对稻飞虱Rice planthopper、稻纵卷叶螟Cnaphalocrocis medinalis Guenee致死率达85%以上。苦参内生菌株LeB8和RoF2的粗提代谢物对稻纵卷叶螟和稻飞虱有明显的毒杀作用,为开发新的生物源农药提供了可能的生物质。     

海洋放线菌BM-2菌株的抗菌特性

采用平板对峙法和打孔法测定海洋放线菌BM-2菌株及其无菌发酵液对12种植物病原真菌和5种细菌的抑制作用.结果表明:BM-2菌株及其无菌发酵液对立枯丝核菌等12种植物病原真菌和大肠杆菌、枯草芽孢杆菌、嗜水气单胞菌3种细菌具有较强的抗菌作用;发酵液能抑制细链格孢菌的分生孢子萌发和芽管伸长;该菌株的抑菌作用对pH值和蛋白酶比较稳定,紫外线、高温对发酵液的抑菌活性有一定的影响.     

生防芽孢杆菌的分离筛选与初步鉴定

从不同环境中采集7份土壤样品用于芽孢杆菌的分离纯化,通过多次平板划线分离纯化得到30个菌落形态不同的菌株。以黄瓜枯萎病菌为指示菌对得到的菌株进行抑菌活性筛选,其中的4个菌株具有明显的抑菌作用。采用对峙生长法,测定4株芽孢杆菌对其它植物病原真菌的活性,结果显示4个芽孢杆菌均具有广谱抑菌性。进一步试验表明,4株芽孢杆菌的发酵液也具有明显的抑菌作用,说明对病原真菌有抑制作用的物质水溶性较大,大部分释放到发酵液中,并且发酵液可使黄瓜枯萎等植物病原真菌菌丝膨大、褐变、末端钝圆,促进镰刀菌产生小孢子。通过菌落形态特征、系列生化测定对其进行初步鉴定,结果表明其中一株为枯草芽孢杆菌。     

两种红树植物抑菌活性内生真菌的研究

文章通过稀释涂布平板法分离广州南沙红树植物海桑和桐花树的内生真菌,并采用改良菌饼法对所有菌株进行抑菌活性筛选。结果显示,从两种红树植物中共分离得到38株内生真菌,均对至少一种指示菌有抑菌活性,其中有31株可抑制两种或以上指示菌,占总菌株的81.6%。     

放线菌BOS-009的分离、拮抗活性及鉴定

对从长白山不同海拔环境中分离筛选出可以产生抗菌活性物质的BOS-009放线菌菌株进行了研究,测定了该菌的抗菌谱,发现该菌株对多种植物病原性真菌及细菌有拮抗作用.通过形态特征、培养特征观察,生理生化特性及抑菌谱等系列比较.发现该菌株与链霉菌属的除虫链霉菌(Streptomyces avermitilis)的特征基本相符;通过16s rDAN序列比对,发现该菌株16s rDAN与除虫链霉菌的16s rDAN同源性达99%.根据多项分类原则和系统进化树的构建分析,将该菌株暂归入除虫链霉菌类.     

细菌抑制剂离体生物活性试验

[目的]为明确化学药剂对病原细菌的离体抑菌活性,进而为田间病害防治提供优秀的候选药剂。[方法]将黄瓜细菌性角斑病菌(Pseudomonas syringae pv.lachrymans)等常见病原细菌作为防治靶标,利用带菌平板法,对药剂的离体抑菌活性进行对比研究。[结果]不同的药剂对病原细菌的离体抑菌活性差异很大,链霉素、土霉素等药剂对多种病原细菌具有较高的离体抑菌活性。[结论]试验采用带菌平板法,能够对药剂的离体抑菌活性进行准确检测。     

海洋放线菌Y-0117农用活性代谢产物的研究

在农用抗生素筛选中发现代号为Y-0117的海洋放线茵菌株所产生的代谢产物对植物病原真菌具有良好的抑制作用。对Y-0117发酵液进行萃取、硅胶柱层析、制备薄层色谱、制备HPLC,分离纯化得到两个活性组分0117A和0117B。通过紫外光谱、质谱、核磁共振等手段,确定0117A的分子式为C35H58O8,分子量为604。结构与已知化合物Bafilomycin D相同;0117 B的分子式为C42H60O12．分子量为756,结构与近期发现具有抗癌活性的已知化合物Hygrobafilomycin相同。研究发现0117B对多种植物病原真菌具有良好的抑制活性,对稻瘟病菌的最小抑制浓度为15．63μg·mL^-1。     

石榴皮抑菌活性物质的初步研究

研究了石榴皮初提物对黄瓜枯萎病菌的室内生物活性.结果表明:在甲醇、氯仿及正己烷提取物中,甲醇提取物的抑菌活性最为显著.采用生长速率法和孢子萌发法测定了石榴皮溶剂萃取物对黄瓜枯萎病菌、番茄灰霉病菌、玉米小斑病菌和棉花立枯病菌等4种病原菌的生物活性.在甲醇提取物的石油醚、氯仿、乙酸乙酯及正丁醇萃取物中,以正丁醇萃取物的抑菌活性最强,在质量浓度为200 g/L时.对4种供试真菌的抑制率均大于70%,而对玉米小斑菌的抑制作用达90.67%,其EC50值为26.2 g/L.     

CAN SIMULTANEOUS INHIBITION OF SEEDLING GROWTH AND STIMULATION OF RHIZOSPHERE BACTERIAL POPULATIONS PROVIDE EVIDENCE FOR PHYTOTOXIN TRANSFER FROM PLANT RESIDUES IN THE BULK SOIL TO THE RHIZOSPHERE OF SENSITIVE SPECIES?

In order to demonstrate that allelopathic interactions are occurring, one must, among other things, demonstrate that putative phytotoxins move from plant residues on or in the soil, the source, through the bulk soil to the root surface, a sink, by way of the rhizosphere. We hypothesized that the incorporation of phytotoxic plant residues into the soil would result in a simultaneous inhibition of seedling growth and a stimulation of the rhizosphere bacterial community that could utilize the putative phytotoxins as a sole carbon source. If true and consistently expressed, such a relationship would provide a means of establishing the transfer of phytotoxins from residue in the soil to the rhizosphere of a sensitive species under field conditions. Presently, direct evidence for such transfer is lacking. To test this hypothesis, cucumber seedlings were grown in soil containing various concentrations of wheat or sunflower tissue. Both tissue types contain phenolic acids, which have been implicated as allelopathic phytotoxins. The level of phytotoxicity of the plant tissues was determined by the inhibition of pigweed seedling emergence and cucumber seedling leaf area expansion. The stimulation of cucumber seedling rhizosphere bacterial communities was determined by the plate dilution frequency technique using a medium containing phenolic acids as the sole carbon source. When sunflower tissue was incorporated into autoclaved (to reduce the initial microbial populations) soil, a simultaneous inhibition of cucumber seedling growth and stimulation of the community of phenolic acid utilizing rhizosphere bacteria occurred. Thus, it was possible to observe simultaneous inhibition of cucumber seedlings and stimulation of phenolic acid utilizing rhizosphere bacteria, and therefore provide indirect evidence of phenolic acid transfer from plant residues in the soil to the root surface. However, the simultaneous responses were not sufficiently consistent to be used as a field screening tool but were dependent upon the levels of phenolic acids and the bulk soil and rhizosphere microbial populations present in the soil. It is possible that this screening procedure may be useful for phytotoxins that are more unique than phenolic acids.     

Genetic Diversity and Phylogeny of Antagonistic Bacteria against Phytophthora nicotianae Isolated from Tobacco Rhizosphere

The genetic diversity of antagonistic bacteria from the tobacco rhizosphere was examined by BOXAIR-PCR, 16S-RFLP, 16S rRNA sequence homology and phylogenetic analysis methods. These studies revealed that 4.01% of the 6652 tested had some inhibitory activity against Phytophthora nicotianae. BOXAIR-PCR analysis revealed 35 distinct amplimers aligning at a 91% similarity level, reflecting a high degree of genotypic diversity among the antagonistic bacteria. A total of 25 16S-RFLP patterns were identified representing over 33 species from 17 different genera. Our results also found a significant amount of bacterial diversity among the antagonistic bacteria compared to other published reports. For the first time; Delftia tsuruhatensis, Stenotrophomonas maltophilia, Advenella incenata, Bacillus altitudinis, Kocuria palustris, Bacillus licheniformis, Agrobacterium tumefaciens and Myroides odoratimimus are reported to display antagonistic activity towards Phytophthora nicotianae. Furthermore, the majority (75%) of the isolates assayed for antagonistic activity were Gram-positives compared to only 25% that were Gram-negative bacteria.     

Modification of Biphenolic Anti-Bacterial to Achieve Broad-Spectrum Activity

The Gram-positive bacteria, methicillin-resistant Staphylococcus aureus (MRSA) and Gram-negative bacteria, Acinetobacter baumannii, are pathogens responsible for millions of nosocomial infections worldwide. Due to the threat of bacteria evolving resistance to antibiotics, scientists are constantly looking for new classes of compounds to treat infectious diseases. The biphenolic analogs of honokiol that were most potent against oral bacteria had similar bioactivity against MRSA. However, all the compounds proved ineffective against A. baumannii. The inability to inhibit A. baumannii is due to the difficult-to-penetrate lipopolysaccharide-coated outer membrane that makes it challenging for antibiotics to enter Gram-negative bacteria. The C 2 scaffold was optimized from the inhibition of Gram-positive bacteria to broad-spectrum antibacterial compounds that inhibit the dangerous Gram-negative pathogen A. baumannii.     

Induction and/or Selection of Phenolic Acid-Utilizing Bulk-Soil and Rhizosphere Bacteria and Their Influence on Phenolic Acid Phytotoxicity

Bulk-soil and rhizosphere bacteria are thought to exert considerable influence over the types and concentrations of phytotoxins, including phenolic acids, that reach a root surface. Induction and/or selection of phenolic acid-utilizing (PAU) bacteria within the bulk-soil and rhizosphere have been observed when soils are enriched with individual phenolic acids at concentrations 0.25 mol/g soil. However, since field soils frequently contain individual phenolic acids at concentrations well below 0.1 mol/g soil, the actual importance of such induction and/or selection remains uncertain. Common bacteriological techniques (e.g., isolation on selective media, and plate dilution frequency technique) were used to demonstrate in Cecil A_p soil systems: (1) that PAU bacterial communities in the bulk soil and the rhizosphere of cucumber seedlings were induced and/or selected by mixtures composed of individual phenolic acids at concentrations well below 0.25 mol/g soil; (2) that readily available carbon sources other than phenolic acids, such as glucose, did not modify induction and/or selection of PAU bacteria; (3) that the resulting bacterial communities readily utilize mixtures of phenolic acids as a carbon source; and (4) that depending on conditions (e.g., initial PAU bacterial populations, and phenolic acid concentration) there were significant inverse relationships between PAU bacteria in the rhizosphere of cucumber seedlings and absolute rates of leaf expansion and/or shoot biomass. The decline in seedling growth could not be attributed to resource competition (e.g., nitrogen) between the seedlings and the PAU bacteria in these studies. The induced and/or selected rhizosphere PAU bacteria, however, reduced the magnitude of growth inhibition by phenolic acid mixtures. For a 0.6 mol/g soil equimolar phenolic acid mixture composed of p-coumaric acid, ferulic acid, p-hydroxybenzoic acid, and vanillic acid, modeling indicated that an increase of 500% in rhizosphere PAU bacteria would lead to an approximate 5% decrease (e.g., 20–25%) in inhibition of absolute rates of leaf expansion. As far as we know, this is the first time that such a relationship has been quantified.     

A Degeneration Gradient of Poplar Trees Contributes to the Taxonomic,Functional, and Resistome Diversity of Bacterial Communities in Rhizosphere Soils

Bacterial communities associated with roots influence the health and nutrition of the host plant. However, the microbiome discrepancy are not well understood under different healthy conditions. Here, we tested the hypothesis that rhizosphere soil microbial diversity and function varies along a degeneration gradient of poplar, with a focus on plant growth promoting bacteria (PGPB) and antibiotic resistance genes. Comprehensive metagenomic analysis including taxonomic investigation, functional detection, and ARG (antibiotics resistance genes) annotation revealed that available potassium (AK) was correlated with microbial diversity and function. We proposed several microbes, Bradyrhizobium, Sphingomonas, Mesorhizobium, Nocardioides, Variovorax, Gemmatimonadetes, Rhizobacter, Pedosphaera, Candidatus Solibacter, Acidobacterium, and Phenylobacterium, as candidates to reflect the soil fertility and the plant health. The highest abundance of multidrug resistance genes and the four mainly microbial resistance mechanisms (antibiotic efflux, antibiotic target protection, antibiotic target alteration, and antibiotic target replacement) in healthy poplar rhizosphere, corroborated the relationship between soil fertility and microbial activity. This result suggested that healthy rhizosphere soil harbored microbes with a higher capacity and had more complex microbial interaction network to promote plant growing and reduce intracellular levels of antibiotics. Our findings suggested a correlation between the plant degeneration gradient and bacterial communities, and provided insight into the role of high-turnover microbial communities as well as potential PGPB as real-time indicators of forestry soil quality, and demonstrated the inner interaction contributed by the bacterial communities.     

Drug loaded electrospun polymer/ceramic composite nanofibrous coatings on titanium for implant related infections

Developing an effective antibacterial surface with the help of drugs that prevent bacterial adhesion, colonization, and proliferation into the surrounding tissues is of great demand. Rifampicin (Rf) is effective antibiotic drug proved has proved its potential in treating bacteria in biofilms, especially against the microbes causing bone infections. Hydroxyapatite (HA), a biocompatible osteoconductive ceramic, has been verified to be a significant material for bioactivity enhancement. Electrospinning is an effective inexpensive method for incorporating nanoparticles into nanofibers with uniform distribution for the drug delivery system for tissue engineering applications. In the current study, for improving bioactivity and antibacterial properties, novel functional polycaprolactone (PCL) composite nanofibers loaded HA and Rf was developed and coated on titanium (Ti). Different characterization techniques such as SEM, EDS, XRD, FITR were used to analyze these PCL/Rf/HA nanocomposites. The results showed that the bioactivity and tensile strength of the composite scaffold increased with the addition of HA nanoparticles. In vitro bioactivity demonstrated that the PCL/HA/Rf composite nanofibers possess enhanced calcium deposition when compared to the pure sample. Cellular interactive responses such as adhesive and proliferation were evaluated using hFOB human fetal osteoblast cell lines. After 6 days of culturing, the cellular properties on Ti sample coated with PCL/HA/Rf was significantly improved. Antibacterial evaluations on the substrates showed that Rf-loaded PCL/HA fibers displayed >3 log reduction against S.aureus MRSA, and S.epidermidis bacterial strain and >2 log reduction against P.aeruginosa bacteria. In vitro drug release study shows initial burst release of Rf, followed by sustained released of 62% at the end of 32 days. The cell viability, adhesion, and proliferation evaluation suggest that the PCL/HA/Rf coated substrate possess good cytocompatibility. Further incorporation of Rf enhanced the antibacterial property of this nanofibrous scaffold.     

Influence of Phenolic acids on microbial populations in the rhizosphere of cucumber

Experiments were conducted to determine whether changes in soil microbial populations that occur in response to additions of certain allelopathic phenolic acids to bulk soil also occur in the rhizosphere. Cucumber seedlings were transplanted into cups containing a nutrient-enriched mixture of Portsmouth B₁, soil and sand and were watered five times (once every 48 hr) with aqueous solutions of ferulic,p-coumaric, or vanillic acid (each at 0, 0.25, or 0.50ol/g soil material). Nutrient solution was applied on alternate days. Leaf growth was suppressed by up to 42% by phenolic acids, but changes in root growth varied with the compound and concentration in solution. Significant increases (over 600% relative to controls) in populations of fast-growing bacteria in the rhizosphere were detected after two but not after five treatments, and increases (400% relative to controls) in numbers of fungal propagules were detected after five treatments. Such increases suggested that chronic exposure to a phenolic acid might resuit in high populations of rhizosphere microorganisms that could metabolize the compounds and thus alter observable responses by the plant. To test this, plants were watered repeatedly with a low-concentration solution of ferulic acid (chronic treatments; 0.0 or 0.1mol/g soil material in one experiment, 0.000 or 0.025imol/g soil material in a second) and then once with a highconcentration solution (acute treatment; 0.0, 0.5, or 1.0mol/g soil material in the first experiment; 0.000, 0.125, or 0.250mol/g soil material in the second).Paper No. 12385 of the Journal Series of the North Carolina Agricultural Research Service, Raleigh, North Carolina. The use of trade names in this publication does not imply endorsement by the North Carolina Agricultural Research Service or the United States Department of Agriculture of the products named, nor criticism of similar ones not mentioned. Acute treatments and some chronic treatments suppressed leaf growth, but results were inconsistent for root growth. Acute treatments increased numbers of several types of bacteria in the rhizosphere but had inconsistent effects on fungi. Chronic treatments had no effect on numbers of bacteria or fungal propagules in the rhizosphere. Furthermore, chronic treatments did not alter responses of plants or microbial populations to the subsequent acute treatment. Results demonstrated that phenolic acids in soil, which must pass through the rhizosphere before interaction with plant roots can occur, alter the microbial ecology of the rhizosphere. However, microbially mediated acclimation of plants to relatively high concentrations of ferulic acid was not observed.