Transformation of Gaeumannomyces graminis with β‐Glucuronidase Reporter and Hygromycin Resistance Genes

Take‐all disease is caused by Gaeumannomyces graminis, (Sacc.) Arx & D. Olivier, a soil‐borne fungus, which colonizes the root and crown tissue of many members of the Poaceae plant family. This fungus is able to grow along the surface of roots as darkly pigmented runner hyphae, which has the ability to penetrate the root. Here, we describe a genetic transformation of G. graminis var. graminis by using polyethylene glycol (PEG)‐based protoplast transformation. Fungus cells were transformed with a plasmid, pHPG, containing the gusA reporter gene that codes for β‐glucuronidase (GUS) and the hph gene for hygromycin resistance as the selectable marker. A de novo transformant selection assay was developed to identify the putative transformants that were expressing the hph gene. In addition, the transformed cells maintained the ability to infect the plant tissues. The GUS‐expressing fungus can be used to study fungal infection processes including fungal penetration, colonization and the role(s) of melanin during pathogenesis. Thus, this study is the first report of G. graminis var. graminis transformed with a visibly detectable reporter gene that provides a useful tool to a better understanding of host–Gaeumannomyces interactions.     

蚕沙有机肥的养分特性及其肥效

将废弃蚕沙进行无害化处理和适度发酵开发出蚕沙有机肥,分析了其养分特点,并采用盆栽试验研究了蚕沙有机肥的肥效.结果表明:发酵蚕沙有机肥的全氮、全磷、全钾含量与堆肥前相比显著提高,分别比堆肥前提高了58.0%、84.4%和29.7%;添加微生物菌剂可有效缩短发酵时间,并能减少堆肥过程的碳、氮损失.施用发酵后蚕沙的小白菜和番茄种子的发芽指数均大于80%,对作物发芽没有抑制作用.施用发酵蚕沙有机肥不仅可提高小白菜产量、营养养分、Vc含量,减少硝酸盐积累量,还可提高土壤pH值,增加土壤速效养分和有机质含量,增强土壤酶活性,其效果优于发酵羊粪有机肥处理.     

黑麦草根系分泌物剂量对污染土壤芘降解和土壤微生物的影响

模拟根际根系分泌物梯度递减效应,研究了黑麦草根系分泌物剂量对污染土壤中芘降解特征和土壤微生物生态特征的影响.结果表明:污染土壤中芘残留量随根系分泌物添加剂量的增加呈现先下降后上升的非线性变化,达到最低芘残留量的添加剂量是总有机碳(TOC) 32.75 mg·kg-1,说明此浓度下根系分泌物显著促进了芘的降解;土壤微生物生物量碳和微生物熵的变化趋势与污染土壤中芘残留量变化趋势相反,表明土壤微生物与污染土壤 中芘残留量存在密切关系.芘污染土壤中微生物群落以细菌占主导地位,且细菌变化趋势与芘降解变化一致,表明芘以细菌降解为主,根系分泌物主要通过影响细菌数量,进而影响芘的降解.能催化有机物质脱氢反应的土壤微生物胞内酶——脱氢酶活性的变化与土壤微生物变化趋势一致,进一步证明微生物及其生物化学特性变化是污染土壤中芘残留量随根系分泌物添加剂量变化的生态机制.     

用人MHC转基因小鼠模型鉴定高致病禽流感病毒核蛋白CTL表位

目的:筛选高致病禽流感病毒核蛋白(NP)中可用于高致病禽流感病毒感染检测或疫苗设计的CTL表位,为评价疫苗接种效果和开发新型疫苗奠定基础。方法:根据NCBI公布的NP的核苷酸序列设计特异性引物,以2006年深圳株高致病禽流感H5N1病人分离的病毒cDNA为模板扩增NP全长基因(1500 bp)并测序。通过生物信息学方法,预测NP氨基酸序列中潜在的HLA-A觹0201限制性表位。构建重组pJW4303-NP核酸疫苗并肌肉免疫HLA-A2/DP4转基因小鼠,利用ELISPOT法筛选特异性CTL表位。结果:克隆了2006年深圳株高致病禽流感NP基因,构建的重组pJW4303-NP核酸疫苗能在体外COS-7细胞中表达,免疫小鼠后能引起小鼠产生特异性的体液免疫和细胞免疫。结论:生物信息学和转基因小鼠模型筛选相结合的方法,能用于高致病性禽流感核蛋白CTL表位的筛选。     

禽致病性大肠埃希菌强毒力岛（HPI）全岛缺失株的构建

目的构建大肠埃希菌强毒力岛（HPI）全岛缺失突变株,为进一步评价大肠埃希菌HPI的功能打下基础。方法根据已知大肠埃希菌HPI基因序列设计PCR敲除引物,引物5′端有50 bp的拟敲除基因的同源臂,3′端为扩增引物,以pKD3为模板,扩增两侧含FRT位点的氯霉素抗性基因,利用pKD46的λ重组系统替换E.coli ZL基因组上的毒力岛全岛基因,再利用表达Flp重组酶的质粒pCP20,可将FRT位点之间的氯霉素抗性基因删除,用鉴定引物进行鉴定并测序。结果构建的全岛缺失株与预期一致。结论成功构建了禽致病性大肠埃希菌强毒力岛（HPI）全岛缺失突变株。     

Probiotic (symbiotic) bacterial languages

Symbiotic gut microorganisms release of various soluble low molecular weight (LMW)molecules of different chemical nature (surface and exogenous proteins, nucleases, serpins, sirtuines, other enzymes, lectins, peptides, amines, bacteriocines, fatty and amino acids, lactones, furanons, miRNA, NO, etc). These LMW molecules are able to sense environment, interact with corresponding cell surface, membrane, cytoplasm and nucleic acid receptors, to reply quickly and coordinately by induction of special sets of genes, to support stability of host genome and microbiome, to modulate epigenomic regulation of gene phenotypic expression, to ensure the information exchange in numerous bacterial and bacteria-host systems playing an important role in the control for many genetic and physiological functions, biochemical and behaviour reactions, in supporting host health in general. Various symbiotic (probiotic) strains produce different spectrum of such LMW molecules. There is chemical and functional similarity between LMW molecules synthesized by host eukaryotic cells, indigenous and probiotic microorganisms and some micronutrients. It means many LMW compounds of different origin may be the universal regulators contributing to the transmission of information, quorum sensing effects, metagenome stability and epigenomic control for cell growth and development as well as phenotypic expression of different genes. Knowledge accumulated concerning molecular languages of symbiotic microorganisms allows us to better understand the mode of action of known probiotics and to design in principle novel probiotics (metabiotics) with increased health effectiveness. Now we are only at the beginning of a new era of molecular personal biotherapy and nutrition. Soon we can successfully manipulate both the host and its microbiota through interfering in their cross talk, stability and epigenomic regulation of expression of genes using various types of eukaryotic, prokaryotic and nutrition origin LMW molecules are capable to modulate genetic, metabolic and physiological activities.     

Resuscitation of Escherichia coli VBNC cells depends on a variety of environmental or chemical stimuli

Aims: To analyse viable but nonculturable (VBNC) state induction in Escherichia coli and resuscitation of VBNC suspensions in several conditions. Methods and Results: VBNC were induced in four media, two temperatures and six strains, but only cells produced at 4°C were able to resuscitate. Resuscitation of 14 VBNC suspensions obtained in several conditions occurred in the presence of supernatants of growing cells, in minimal medium supplemented with amino acids or after temperature change, depending on strain. A limited time period beyond no more resuscitation that could be observed was also confirmed. Conclusions: The supernatants positive effect is suggested to be because of a nonproteinaceous molecule, and a combination of methionine, glutamine, threonine, serine and asparagine could be used as primary mix for resuscitation experiments. Significance and Impact of the Study: Escherichia coli resuscitation was already attempted in several conditions, but it is the first time that a positive result was observed in minimal medium supplemented with amino acids or after temperature change. The role of amino acids in resuscitation is of special interest since was never reported for any species.     

Incidence and survival of non-O157 verocytotoxigenic Escherichia coli in soil

Aims: This study estimated the incidence of non‐O157 verocytotoxigenic Escherichia coli (VTEC) in farm pasture soils and investigated the survival of non‐O157 VTEC in clay and sandy loam soils. Methods and Results: Twenty farms were tested over a 12‐month period by sample enrichment in tryptone soya broth plus vancomycin, followed by PCR screening for the presence of vt1 and vt2 genes. Of the 600 soil samples, 162 (27%), across all farms, were found to contain vt1 and/or vt2 genes. The enrichment cultures from the 162 PCR‐positive samples were plated onto Chromocult tryptone bile X‐glucuronide agar (TBX), presumptive VTEC colonies recovered, confirmed as VTEC by PCR and serotyped. Samples of the two predominant soil types in Ireland (clay and sandy) were homogenized, characterized in terms of pH, boron, cobalt, copper, potassium, magnesium, manganese, phosphorus, zinc and organic matter content, inoculated with washed suspensions of eight non‐O157:H7 soil isolates and six bovine faecal isolates and stored at 10°C for up to 201 days. Inoculum survival rates were determined at regular intervals by recovering and plating soil samples on TBX. All inoculated non‐O157 serotypes had highest D‐values in the sandy loam soil with D‐values ranging from 50·26 to 75·60 days. The corresponding range in clay loam soils was 31·60–48·25 days. Conclusions: This study shows that non‐O157 VTEC occur widely and frequently in pasture soils and can persist in such environments for several months, with considerable opportunity for recycling through farm environments, and cattle, with clear potential for subsequent transmission into the human food chain. Significance and Impact of the Study: This is the first such study of non‐O157 VTEC in farm soils and found that these VTEC are frequent and persistent contaminants in farm soils. In light of recent epidemiological data, non‐O157 VTEC should be seen as an emerging risk to be controlled within the food chain.     

Inactivation of plant infecting fungal and viral pathogens to achieve biological containment in drainage water using UV treatment

Aim: To explore whether ultraviolet (UV) light treatment within a closed circulating and filtered water drainage system can kill plant pathogenic species. Methods and Results: Ultraviolet experiments at 254 nm were conducted to determine the inactivation coefficients for seven plant pathogenic species. At 200 mJ cm^?2, the individual species log reductions obtained for six Ascomycete fungi and a cereal virus were as follows: Leptosphaeria maculans (9·9‐log), Leptosphaeria biglobosa (7·1‐log), Barley stripe mosaic virus (BSMV) (4·1‐log), Mycosphaerella graminicola (2·9‐log), Fusarium culmorum (1·2‐log), Fusarium graminearum (0·6‐log) and Magnaporthe oryzae (0·3‐log). Dilution experiments showed that BSMV was rendered noninfectious when diluted to >1/512. Follow‐up large‐scale experiments using up to 400 l of microbiologically contaminated waste water revealed that the filtration of drainage water followed by UV treatment could successfully be used to inactivate several plant pathogens. Conclusions: By combining sedimentation, filtration and UV irradiation within a closed system, plant pathogens can be successfully removed from collected drainage water. Significance and Impact of the Study: Ultraviolet irradiation is a relatively low cost, energy efficient and labour nonintensive method to decontaminate water arising from a suite of higher biological containment level laboratories and plant growth rooms where genetically modified and/or quarantine fungal and viral plant pathogenic organisms are being used for research purposes.