Real-Time Duplex Applications of Loop-Mediated AMPlification (LAMP) by Assimilating Probes

Isothermal nucleic-acid amplification methods such as Loop-Mediated isothermal AMPlification (LAMP) are increasingly appealing alternatives to PCR for use in portable diagnostic system due to the low cost, weight, and power requirements of the instrumentation. As such, interest in developing new probes and other functionality based on the LAMP reaction has been intense. Here, we report on the development of duplexed LAMP assays for pathogen detection using spectrally unique Assimilating Probes. As proof of principle, we used a reaction for Salmonella enterica as a model coupled with a reaction for λ-phage DNA as an internal control, as well as a duplexed assay to sub-type specific quarantine strains of the bacterial wilt pathogen Ralstonia solanacearum. Detection limits for bacterial DNA analyzed in individual reactions was less than 100 genomic equivalents in all cases, and increased by one to two orders of magnitude when reactions were coupled in duplexed formats. Even so, due to the more robust activity of newly available strand-displacing polymerases, the duplexed assays reported here were more powerful than analogous individual reactions reported only a few years ago, and represent a significant advance for incorporation of internal controls to validate assay results in the field.     

Coupling System for Food Wastes Anaerobic Digestion and Polyhydroxyalkanoates Production with Ralstonia eutropha

A new technology was developed to couple the anaerobic digestion of food wastes with production of polyhydroxyalkanoates (PHAs). Acetic, propionic, butyric and lactic acids were produced during food wastes anaerobic digestion and their concentrations reached 5.5, 1.8, 27.4 and 32.7 g/L, respectively under appropriate digestion conditions. The fermentative acids were transferred through a dialysis membrane to an air-lift reactor for PHA synthesis by Ralstonia eutropha. Dry cell concentration and PHA content reached 22.7 g/L and 72.6%, respectively. The obtained PHA was a copolymer of b-hydroxybutyrate (HB) and b-hydroxyvalerate (HV) with 2.8% (mole ratio) of HV units in polymer.     

Leaf-to-Whole Plant Spread Bioassay for Pepper and Ralstonia solanacearum Interaction Determines Inheritance of Resistance to Bacterial Wilt for Further Breeding

Bacterial wilt (BW) disease from Ralstonia solanacearum is a serious disease and causes severe yield losses in chili peppers worldwide. Resistant cultivar breeding is the most effective in controlling BW. Thus, a simple and reliable evaluation method is required to assess disease severity and to investigate the inheritance of resistance for further breeding programs. Here, we developed a reliable leaf-to-whole plant spread bioassay for evaluating BW disease and then, using this, determined the inheritance of resistance to R. solanacearum in peppers. Capsicum annuum ‘MC4′ displayed a completely resistant response with fewer disease symptoms, a low level of bacterial cell growth, and significant up-regulations of defense genes in infected leaves compared to those in susceptible ‘Subicho’. We also observed the spreading of wilt symptoms from the leaves to the whole susceptible plant, which denotes the normal BW wilt symptoms, similar to the drenching method. Through this, we optimized the evaluation method of the resistance to BW. Additionally, we performed genetic analysis for resistance inheritance. The parents, F₁ and 90 F₂ progenies, were evaluated, and the two major complementary genes involved in the BW resistance trait were confirmed. These could provide an accurate evaluation to improve resistant pepper breeding efficiency against BW.     

烟草青枯病菌(Pseudomonas solanacearum)细菌素的生物学特性研究

本文研究了烟草青枯病菌(Pseudomonas solanacearum)无毒产细菌素菌株 A_(3-5)产生的细菌素的生物学特性,结果表明,用不同制备方法提取的 A_(3-5)的细菌素,Au 值均为8。失活温度为60℃,在30℃以下温度放置25天抑菌活性不变,35℃下放置20天,活性开始减弱,40℃放置10天,活性即减弱,25天对几乎完全失活。这说明 A_(3-5)的细菌素虽具有较强耐热能力,但高温不利于其存活。此外,0.5%的胰蛋白酶即刻使其完全失活,说明对胰蛋白酶敏感。经1万转/分钟和5万转/分钟连续离心后,细菌素仍存在于上清液中,说明为非沉淀性的。以上特性表明 A_(3-5)的细菌素可能是一种小分子物质。     

烟草青枯病拮抗内生菌的发酵条件

研究了发酵时间、发酵温度、菌龄、接种量、摇瓶转速、溶氧量对菌量的影响,以及烟草青枯病拮抗菌湘2-3发酵条件与菌量之间的关系,并运用正交试验确定了最佳发酵条件。结果表明:当发酵时间24h,发酵温度28℃,菌龄18h~22h,接种量4%,摇瓶转速210 r/min,溶氧量100mL/150mL时,湘2-3菌的发酵最好。     

我国主要烟草青枯病病圃青枯菌系统发育分析

为探究我国主要植烟区烟草青枯病鉴定病圃中青枯菌的系统发育,采用演化型分类框架下的序列变种分类方法,对从各烟株和土壤样品中分离得到的100株青枯菌进行系统发育分析。结果表明,所有菌株可归为4个分支,分别为序列变种15、17、34、54。其中,云南文山州广南县病圃中的菌株为序列变种17和54,福建泰宁县的菌株为序列变种34,福建三明市三元区的菌株为序列变种15和34,广东白云区的菌株为序列变种15和17,广东南雄市的菌株为序列变种15、17和34,安徽宣城市宣州区的菌株为序列变种34和54,贵州福泉的菌株为序列变种17,重庆彭水的菌株为序列变种17。本研究发现同一病圃中存在多个序列变种,某些病圃同一地块中烟株与土壤分离所得菌株序列变种不一致。     

Optimal biostimulation strategy for phenol degradation with indigenous rhizobium Ralstonia taiwanensis

This study provides a first attempt from a perspective of Gaden's classification of fermentation and phase-plane to put forward phenol degradation using various augmented nutrient media for biostimulation. It aimed to identify the most promising nutrient source(s) to attenuate synergistic interactions with phenol for optimal phenol degradation. Therefore, the growth association of phenol degradation using various nutrient media in place of combined toxic interactions was established via Gaden's classification scheme of fermentation and phase-plane analysis. In cultures grown on medium bearing dual carbon sources (glycerol and phenol) or phenol alone, phenol was found to be firstly biodegraded for microbial growth (i.e., growth-associated degradation). In contrast, when yeast extract or acetate was supplemented, a diauxic growth behavior was observed as the augmented nutrient was primarily utilized while phenol degradation was repressed. Moreover, using glycerol as the nutrient source, phenol degradation seemed to be enhanced simultaneously during the consumption of glycerol for cellular growth after ca. 2h response lag in growth. Although gluconic acid could enhance cell growth as well as phenol degradation, the phenol degradation performance was still not as good as that of glycerol. Thus, biostimulation with glycerol appeared to show the most favorable metabolic characteristics against phenol toxicity on Ralstonia taiwanensis, leading to better degradation efficiency of the toxic pollutant. Phase-plane trajectories also clearly confirmed that glycerol was the optimal biostimulating nutrient source for phenol degradation.     

白肋烟栽培土壤中青枯病菌的鉴定与生化型分析

为建立一套成本低廉、简单快速的分离检测土壤青枯病菌的方法,采用半选择性培养基(PCCG)和PCR技术相结合,从白肋烟栽培土壤中分离鉴定出12个菌株,并分析了这12个菌株的ITS序列 .结果表明:分离出的12个菌株ITS序列与青枯病菌同源性最高,达到92.6％,该方法适合于土壤中青枯病菌的分离鉴定.Hayward的青枯病菌生化型鉴定结果表明,这12个菌株均为生化型Ⅲ.