Distribution and multiplication of Ralstonia solanacearum strain race 1 biovar 4 in vegetable sweet potato cuttings

Ralstonia solanacearum (RS) race 1 biovar 4 (R1bv4), causal agent of bacterial wilt in vegetable sweet potato (VSP), is often latent in VSP vines and is important in introduction of the pathogen to newly planted fields. In this study, the effects of biological and environmental factors on the distribution and multiplication of R1bv4 in VSP tissues were examined. Based on stem-injection inoculation, the R1bv4strain of NC01 could cause 49.0% and 33.0% wilting on VSP cultivars TN71 and WS, respectively. The populations of NC01 in diseased TN71 and WS were 10⁸–10⁹ cfu/g tissue at 28th day after inoculation. On the other hand, the R1bv4 could not cause symptom in cultivars of TN57 and VSPSL-1 vine and the NC01 was confined to near the injection sites. Temperature tests indicated that NC01 could cause 28.0% and 14.0% wilting on cultivar TN71 at 28 and 20°C, respectively. Moreover, the populations of NC01in diseased plants were 1.6 × 10⁹ and 7.9 × 10⁸ cfu/g tissue at 28 and 20°C, respectively. The distribution of NC01 in VSP stem indicated that the isolation frequency of NC01 was lower than 31.0% in terminal shoots or erect stems and 45.0 to 100.0% in creeping stem after 8 wks planted in infested soil (10⁶ cfu/g soil). The results demonstrated that terminal shoots or erect stems were not common carrier for transmitting R1bv4. Furthermore, two R1bv4 strains, NC01 and HsinT01, were examined the ability for latent infection on cv. TN71. The results revealed that NC01 and HsinT01 showed different ability of latent infection on cultivar TN71. NC01 had lower percentage (46.8% and 45.1%) than HsinT01 (93.4% and 75.3%) at 20 and 28°C.     

青枯菌hrp基因的研究进展

青枯菌的hrp基因可诱发植物的超敏反应.对其基因组全序列测定表明:hrp基因簇位于基因组的大质粒上,共有20多个基因组成.从青枯菌中分离得到的可直接诱发植物超敏反应的效应蛋白主要为pop基因编码,它由hrp基因编码的类型Ⅲ蛋白分泌通道释放.目前的研究表明:(1)在hrp基因簇中,hrpY、hrpX及hrpV与分泌通道的一种纤毛的组装有关;(2)hrpB是整个类型Ⅲ蛋白分泌通道基因的转录激活子并作用于基因组中的其它效应基因;(3)hrpG是植物信号对hrp,基因的表达进行级联调控的组分之一.     

Ring-cleaving cyanuric acid amidohydrolase activity in the atrazine-mineralizing Ralstonia basilensis M91-3

The purpose of this study was to characterize the cyanuric acid amidohydrolase reaction in Ralstonia basilensis M91-3, an atrazine-mineralizing soil bacterium. This ring fission reaction is the last aromatic step in the degradative pathway of atrazine and other s-triazines. The products and molar stoichiometry of the cyanuric acid amidohydrolase reaction were one mol biuret (H₂N·CO·NH·CO·NH₂) and one mol CO₂ per mol cyanuric acid hydrolyzed, as confirmed by ¹³C-NMR and gas chromatography. The optimum pH and temperature, substrate specificity, and kinetic parameters were also characterized for the purified enzyme. The native enzyme had two forms of different sizes, 204 kDa and 160 kDa. Each was a tetramer or pentamer of 44 kDa and 33 kDa, respectively.     

Characteristics of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) production byRalstonia eutropha NCIMB 11599 and ATCC 17699

Ralstonia eutropha NCIMB 11599 and ATCC 17699 were grown, and their productions of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB)] compared. In flask cultures ofR. eutropha NCIMB 11599, cell concentration, P(3HB-co-4HB) concentration and polymer content decreased considerably with increases in the γ-butyrolactone concentration, and the 4HB fraction was also very low (maximum 1.74 mol%). In fed-batch cultures ofR. eutropha NCIMB 11599, glucose and γ-butyrolactone were fed as the carbon sources, under a phosphate limitation strategy. When glucose was fed as the sole carbon source, with its concentration controlled using an on-line glucose analyzer, 86% of the P(3HB) homopolymer was obtained from 201 g/L of cells. In a two-stage fed-batch culture, where the cell concentration was increased to 104 g/L, with glucose fed in the first step and constant feeding of γ-butyrolactone, at 6 g/h, in the second, final cell concentration at 67 h was 106 g/L, with a polymer content of 82%, while the 4HB fraction was only 0.7 mol%. When the same feeding strategy was applied to the fedbatch culture ofR. eutropha ATCC 17699, where the cell concentration was increased to 42 g/L, by feeding fructose in the first step and γ-butyrolactone (1.5 g/h) in the second, the final cell concentration, polymer content and 4HB fraction at 74 h were 51 g/L, 35% and 32 mol%, respectively. In summary,R. eutropha ATCC 17699 was better thanR. eutropha NCIMB 11599 in terms of P(3HB-co-4HB) production with various 4HB fractions.     

基于EMA-qPCR的茄科青枯菌活体检测技术的建立

【目的】利用特异性核酸染料叠氮溴乙锭(Ethidium monoazide bromide, EMA)与实时荧光定量PCR技术相结合, 建立一种能有效区分青枯菌死活细胞的检测方法。【方法】样品DNA制备前经EMA渗透预处理, 再进行实时荧光定量PCR特异扩增菌体DNA。【结果】终浓度为2.0 mg/L的EMA能有效排除1.0×107 CFU/mL灭活青枯菌细胞DNA的扩增, 对活细胞和不可培养状态(Viable but non-culturable, VBNC)活菌的DNA扩增均没有影响。当每个定量PCR反应体系中的活细胞在5.0×100?5.0×104 CFU范围内时, 扩增Ct值与定量PCR反应体系中活细胞CFU对数值呈良好的负相关性(R2=0.992 5)。比较EMA-qPCR法和平板计数法对经过不同温度短期保存的青枯菌检测结果发现, 待检样品可在24 °C与4 °C冷藏条件下短期保存。【结论】本研究建立的EMA-qPCR方法能有效检测青枯菌VBNC细胞和有效区分死活菌, 避免或减少青枯菌PCR检测的假阳性和假阴性。     

Interactions of Ralstonia solanacearum and Pectobacterium carotovorum with Meloidogyne incognita on potato

Effect of interactions of Meloidogyne incognita with Ralstonia solanacearum and interaction of M. incognita with Pectobacterium carotovorum were studied in sequential and simultaneous inoculations on potato (Solanum tuberosum). Inoculation of M. incognita caused a lesser reduction in plant growth than caused by R. solanacearum. Inoculation of M. incognita plus R. solanacearum caused a greater reduction in plant growth than the damage caused by either pathogen. Inoculation of M. incognita prior to R. solanacearum resulted in a greater reduction in plant growth than R. solanacearum was inoculated prior to M. incognita. However, inoculation of M. incognita or P. carotovorum caused similar reduction in plant growth. Inoculation of P. carotovorum prior to M. incognita caused lesser reduction in plant growth than simultaneous inoculation of both pathogens. Inoculation of M. incognita caused galling in potato roots but the size of galls was small. Inoculation of P. carotovorum or R. solanacearum with M. incognita had adverse effect on galling and nematode multiplication. Wilting or soft rot index was 3 when R. solanacearum or P. carotovorum was inoculated alone. In other treatments, where R. solanacearum or P. carotovorum was inoculated with M. incognita, wilting or soft rot indices were 5.     

Thymol and acibenzolar-s-methyl reduce incidence and severity of bacterial wilt of tomato caused by race I biovar III (R1B3) strain of Ralstonia solanacearum in Nigeria

Abstract

In Nigeria, most strains of Ralstonia solanacearum, the causative agent of tomato bacterial wilt disease; belong to race 1 biovar III (RIB3). Control strategies to assuage its destructive effect are highly necessary. A randomised complete-block design (RCBD) was used for the experiment. Thymol (0.7%) and Acibenzolar-s-methyl (ASM, 30 and 15?µg/ml) were used. Results indicated that the combination of thymol and ASM recorded the highest numbers of days for fruiting in Beske which were 74 and 75 while 59 and 60?days were recorded for UC82-B in both early and late seasons, respectively. When thymol and/or ASM were applied, bacterial wilt disease incidence and disease severity were significantly reduced and this was translated to a significant yield increase when compared with the untreated control plots. The results suggested that the combined application of thymol and ASM could be advantageous to tomato-growing farmers where R. solanacearum is prevalent.     

Biochemical characterization of an alcohol dehydrogenase from Ralstonia sp.

Stereoselective reduction towards pharmaceutically potent products with multi‐chiral centers is an ongoing hot topic, but up to now catalysts for reductions of bulky aromatic substrates are rare. The NADPH‐dependent alcohol dehydrogenase from Ralstonia sp. (RADH) is an exception as it prefers sterically demanding substrates. Recent studies with this enzyme indicated outstanding potential for the reduction of various alpha‐hydroxy ketones, but were performed with crude cell extract, which hampered its detailed characterization. We have established a procedure for the purification and storage of RADH and found a significantly stabilizing effect by addition of CaCl₂. Detailed analysis of the pH‐dependent activity and stability yielded a broad pH‐optimum (pH 6–9.5) for the reduction reaction and a sharp optimum of pH 10–11.5 for the oxidation reaction. The enzyme exhibits highest stability at pH 5.5–8 and 8–15°C; nevertheless, biotransformations can also be carried out at 25°C (half‐life 80 h). Under optimized reaction parameters a thorough study of the substrate range of RADH including the reduction of different aldehydes and ketones and the oxidation of a broad range of alcohols was conducted. In contrast to most other known alcohol dehydrogenases, RADH clearly prefers aromatic and cyclic aliphatic compounds, which makes this enzyme unique for conversion of space demanding substrates. Further, reductions are catalyzed with extremely high stereoselectivity (>99% enantio‐ and diastereomeric excess). In order to identify appropriate substrate and cofactor concentrations for biotransformations, kinetic parameters were determined for NADP(H) and selected substrates. Among these, we studied the reduction of both enantiomers of 2‐hydroxypropiophenone in more detail. Biotechnol. Bioeng. 2013; 110: 1838–1848. © 2013 Wiley Periodicals, Inc.     

麻楝果实化学成分及其抗烟草青枯病菌活性的研究

为研究麻楝(Chukrasia tabularis)的化学成分,采用色谱法从麻楝果实乙醇提取物中分离得到15个化合物,利用波谱学方法鉴定其结构分别为:没食子酸甲酯(1)、没食子酸乙酯(2)、没食子酸(3)、ozoroalide(4)、stigmast-4-en-6β-ol-3-one(5)、黄柏呈(6)、chukranin A(7)、chisopanin M(8)、21α,24α-methylmelianodiol(9)、toonaciliatin K(10)、21α,25-dimethylmelianodiol(11)、odoratone(12)、bourjotinolone A(13)、hispidone(14)和phragmalin di-isobutyrate(15)。化合物4~14为首次从麻楝属植物中分离得到。采用滤纸片琼脂扩散法对单体化合物进行抗烟草青枯病菌(Ralstonia solanacearum)的活性研究,结果表明化合物1、2和3具有中等拮抗活性。     

茄科劳尔氏菌单克隆抗体的制备及其特性鉴定

茄科劳尔氏菌(Ralstonia solanacearum,RS)是番茄、茄子、辣椒、马铃薯等茄科蔬菜青枯病害的致病菌。为实现对RS的快速高效检测,以茄科劳尔氏菌株1.76免疫BALB/c小鼠,经细胞融合后利用间接酶联免疫吸附分析(Enzyme-linked Immunosorbent Assay,ELISA)筛选出3株能稳定分泌抗茄科劳尔氏菌株1.76的单克隆杂交瘤细胞株1C1、1B3和9D7。然后利用小鼠体内诱生腹水,1C1、1B3和9D7效价分别为1:1 024 000、1:64 000、1:256 000。采用饱和硫酸铵沉淀及Protein-G亲和层析法纯化腹水,经SDS-PAGE鉴定显示纯化后的单克隆抗体(Monoclonal Antibodies,mAb)纯度较高。纯化后单克隆抗体(2 mg/mL)效价分别为1:17 529、1:35 819、1:50 000,抗体亚型均为IgG1。对3株抗体进行特异性检测结果显示,1C1和9D7均不能与RS-5结合,1B3不能结合1.74和RS-5。此外,检测结果还表明3株单克隆抗体与桑肠杆菌JX-6、苏云金芽胞杆菌SYJ及实验室现有11株燕麦嗜酸菌卡特莱兰亚种、燕麦嗜酸菌西瓜亚种、玉米细菌性条斑菌、嗜酸菌魔芋亚种,梨火疫病菌QB0809、 XL-4,玉米细菌性枯萎病菌QB0241、QB0242,水稻细菌性谷枯病菌QB0017、QB0753、QB0755均无交叉情况。此次茄科劳尔氏菌抗体的制备,为后期青枯病菌的快速检测提供参考。