转防治软腐病基因拟南芥的获得及其抗病性研究

AHLs是细菌群体感应中的关键信号分子,参与包括导致作物软腐病的胡萝卜软腐欧文氏菌在内的许多植物病原菌致病因子的表达。AHL内酯酶能降解AHLs使其达不到临界浓度,从而阻断了病原菌的致病过程。将克隆到的AHL内酯酶基因SS10置于CaMV 35S启动子下构建双元载体,通过根癌农杆菌介导转化拟南芥。通过抗性筛选和分离比获得纯合转基因植物种子,进行了PCR及RT-PCR鉴定,证明目的基因在转基因拟南芥中整合并表达。初步抗病性实验表明,部分转基因植株具有显著的抗软腐病能力。     

Rapid detection and identification of bacterial strains by Fourier transform near-infrared spectroscopy

The use of Fourier transform near-infrared (FT-NIR) spectroscopy and multivariate pattern recognition techniques for the rapid detection and identification of bacterial contamination in liquids was evaluated. The complex biochemical composition of bacteria yields FT-NIR vibrational transitions (overtone and combination bands) that can be used for classification and identification. Bacterial suspensions (Escherichia coli HB101, E. coli ATCC 43888, E. coli 1224, Bacillus amyloliquifaciens, Pseudomonas aeruginosa, Bacillus cereus, and Listeria innocua) were filtered to harvest the cells and eliminate the matrix, which has a strong NIR signal. FT-NIR measurements were done using a diffuse reflection-integrating sphere. Principal component analysis showed tight clustering of the bacterial strains at the information-rich spectral region of 6000-4000 cm(-1). The method reproducibly distinguished between different E. coli isolates and conclusively identified the relationship between a new isolate and one of the test species. This methodology may allow for the rapid assessment of potential bacterial contamination in liquids with minimal sample preparation.     

Selection for root colonising bacteria stimulating wheat growth in saline soils

High salinity of soils in arid and semi-arid regions results in desertification and decreased crop yield. One possibility to circumvent this problem is to use root colonising salt tolerant bacterial inoculants which can alleviate salt stress in plants. In the present work, the best five enhanced wheat root tip coloniser bacteria were selected from the rhizosphere of wheat grown in saline soil and were identified by the 16S rRNA gene sequence as Pseudomonas putida, Pseudomonas extremorientalis, Pseudomonas chlororaphis and Pseudomonas aurantiaca. The isolates tolerated salt of 5% NaCl and produced indole acetic acid under saline conditions. Four isolates proved to be very efficient in promoting a significant increase in the shoot, root and dry matter of wheat and were able to survive in saline soil. Four of the isolated strains appeared to be better competitive colonisers than reference strains and probably outcompeted with indigenous microorganisms of the rhizosphere. These results are promising for the application of selected environmentally save microbes in saline agricultural soils.     

酰基高丝氨酸内酯酶在蜡状芽胞杆菌中可增强双效菌素抗软腐病

双效菌素(zwittermicin A,ZwA)是一种广谱、新型的抗生素,对很多植物病原菌具有抑制作用;酰基高丝氨酸内酯酶(AHL)通过降解植物病原菌群体感应(quorum-sensing)系统的信号分子,减弱致病基因的表达从而达到抗病的效果。结合这两种物质的不同抗病机理,将来自苏云金芽胞杆菌(Bacillus thuringiensis)中的AHL基因导入到蜡状芽胞杆菌(B.cereus)中,得到相应的重组菌株。实验表明,AHL基因在重组菌中可正常表达,并且不影响受体菌ZwA的表达和产量;感病实验证明,同时表达ZwA和AHL的重组菌对于胡萝卜软腐欧文氏菌(Erwinia carotovora)感染马铃薯(Solanum tuberosum)所引起的病害的抗病效果比单独含产ZwA或AHL的蜡状芽胞杆菌的抗病效果有明显增强。说明结合ZwA和AHL的不同抗病机理来提高抗病效果是可行的。     

不同栽培基质对6种侧耳属食用菌风味成分的影响

为探究不同栽培基质对侧耳属食用菌风味成分的影响以及不同品种食用菌间的风味成分差异,本研究采用顶空固相微萃取结合气相色谱-质谱联用(HS-SPME-GC-MS)技术对桑枝、杂木屑和棉籽壳基质上栽培的6种侧耳属食用菌风味成分进行了分析。结果表明,18个样品中共检出47种挥发性化合物,包括13种醛类化合物、9种醇类化合物、9种酮类化合物、2种呋喃类化合物、2种酯类化合物等。18个样品中的共有成分有17种,包括正己醛、壬醛、1-辛烯-3-醇、3-辛酮、1-辛烯-3-酮、2-正戊基呋喃等。18个样品中挥发性化合物的种类和相对含量差异较大,但棉籽壳基质上栽培的6个菌株子实体中均出现了较高含量的酯类化合物。所有样品中的关键风味成分(ROAV≥1)均包括1-辛烯-3-酮和1-辛烯-3-醇,桑枝基质和杂木屑基质栽培的部分菌株中关键风味成分还包括正己醛、壬醛和2-壬烯醛等。18个样品中总5'-核苷酸含量和总游离氨基酸含量分别为7.86~43.86 mg·g^-1 DW和8.54~25.34 mg·g^-1 DW, 且杂木屑基质栽培的所有菌株中总游离氨基酸含量均高于桑枝基质和棉籽壳基质。综上所述,棉籽壳基质有利于侧耳属食用菌中酯类化合物的生成,而杂木屑基质有利于游离氨基酸的生成。本研究结果为侧耳属食用菌栽培及风味成分的开发利用提供了科学依据。     

AHL内酯酶在蜡状芽胞杆菌中可增强ZwA抗软腐病作用

双效菌素(Zwittermicin A, ZwA)是一种广谱、新型的抗生素，对很多植物病原菌具有抑制作用；酰基高丝氨酸内酯酶(AHL内酯酶)通过降解植物病原菌群体感应（Quorum-Sensing）系统的信号分子，减弱致病基因的表达从而达到抗病的效果。结合这两种物质的不同抗病机理，本研究将来自苏云金芽胞杆菌中的AHL内酯酶基因导入到ZwA产量不同的蜡状芽胞杆菌中，得到相应的重组菌株。实验表明，AHL内酯酶基因在重组菌中可正常表达，并且不影响受体菌ZwA的表达和产量；感病实验证明，同时表达ZwA和AHL内酯酶的重组菌对于胡萝卜软腐欧文氏菌感染马铃薯所引起的病害的抗病效果比单独含产ZwA或AHL内酯酶的蜡状芽胞杆菌的抗病效果有明显增强。说明结合ZwA和AHL内酯酶的不同抗病机理来提高抗病效果是可行的。     

西北半干旱荒漠草原与耕地土壤可培养微生物多样性及分布特征比较

采用稀释涂布平板、分离培养和16S r DNA序列分析法对我国甘肃白银地区半干旱荒漠草原土壤可培养细菌、放线菌、真菌数量及群落分布特征进行了分析,比较了荒漠草原和耕地土壤微生物多样性。发现荒漠草原土壤可培养细菌、放线菌、真菌数量分别为1.23×106、0.19×106、0.18×106cfu·g-1,耕地三类微生物数量分别是3.03×106、0.53×106、0.05×106cfu·g-1。荒漠草原可培养细菌、放线菌数量明显低于耕地,而真菌数量高于耕地。从荒漠草原分离出14株细菌,分别属于γ-变形菌纲(γ-Proteobacteria)噬冷杆菌属(Psychrobacter),放线菌门(Actinobacteria)皮球菌属(Kytococcus),厚壁菌门(Firmicutes)芽孢杆菌属(Bacillus)、亮氨酸芽孢杆菌属(Lysinibacillus)、土壤芽孢杆菌属(Solibacillus)、气球菌属(Aerococcus),优势菌为芽孢杆菌属和噬冷杆菌属。耕地分离出可培养细菌19株,分别属于ɑ-变形菌纲(ɑ-Proteobacteri)根瘤菌属(Rhizobium)、中华根瘤菌属(Sinorhizobium),γ-变形菌纲(γ-Proteobacteria)假单胞菌属(Pseudomonas),厚壁菌门(Firmicutes)芽孢杆菌属(Bacillus),放线菌门(Actinobacteria)微杆菌属(Microbacterium)、节杆菌属(Arthrobacter)、微球菌属(Micrococcus)、考克氏菌属(Kocuria),以放线菌门细菌为主(占57.9%)。从荒漠草原分离放线菌共8株,分别属于链霉菌属(Atreptomyces)、小单孢菌属(Micromonspora)、间孢囊菌属(Intrasporangium),而耕地主要为链霉菌属(Atreptomyces)、小单孢菌属(Micromonspora)。荒漠草原真菌主要是交链孢霉属(Alternaria)、芽枝霉属(Cladosporium),耕地土壤真菌包括青霉属(Penicillium)、交链孢霉属(Alternaria)、曲霉属(Aspergillus)、毛霉属(Mucor)、链孢霉属(Coniothecium)。试验结果表明,荒漠草原与耕地土壤微生物都具有较丰富的多样性,但微生物群落结构存在一定差异,同一区域不同深度土壤中微生物数量和种类也存在差异,耕地土壤微生物多样性明显高于荒漠草原。     

紫外线对杏鲍菇原生质体的诱变作用

对杏鲍菇（Pleurotus eryngii）菌株PL7的原生质体进行了紫外线诱变的研究。结果发现,用20W紫外灯（245nm）,在垂直距灯管30cm处照射90s,PL7菌株的原生质体致死率达74%。获得再生菌株234株,其中20株与亲本菌株PL7有明显的拮抗反应,6个菌株菌落生长速度提高11.0%～17.8%,液体培...     

Biochemistry of TBT-Degrading Marine Pseudomonads Isolated from Indian Coastal Waters

Tributyltin (TBT) is a very effective biocide and an active ingredient in antifouling paints. Screening along the Indian coast yielded 49 bacterial isolates capable of TBT assimilation. The screening was done based on the ability of bacteria to grow in mineral salt medium (MSM) containing TBT as the sole source of carbon. All the isolates produced exopolysaccharides (biosurfactants) in the medium which aid in emulsification and thus ease bioavailability of TBT. Five isolates were identified as potent TBT degraders (namely, Pseudomonas pseudoalcaligenes, Pseudomonas stutzeri, Pseudomonas mendocina, Pseudomonas putida, and Pseudomonas balearica) based on their biomass production in MSM containing TBT as the sole source of carbon. In addition to evaluating the potential of individual bacterial strains, the study also focused on using a consortium of bacteria to explore their synergistic effect when grown on TBT. Further tests like growth profile, rhamnolipid secretion profile, extracellular protein secretion profile, and detection of siderophores were performed on these isolates when grown in MSM supplemented with 2 mM TBT concentration. Emulsification activity of the crude extracellular polysaccharides against kerosene was evaluated. It can be therefore inferred that TBT degradation by these marine pseudomonads is a two-step process: (a) dispersion of TBT in the aqueous phase and (b) tin–carbon bond cleavage by siderophores affecting debutylation of TBT. The consortium of bacteria may be effective in the treatment of TBT-contaminated waste water in dry docks.     

SCAR标记技术鉴别榆黄蘑品种

为了建立一套基于DNA分子标记技术快速鉴定榆黄蘑菌株的有效方法,本研究通过对生产上常用的16个榆黄蘑菌株进行SRAP多态性分析,从榆黄蘑2号菌株中扩增获得了一个片段长为537bp的特异片段,将其克隆、测序并设计引物,成功转化为稳定的SCAR标记。试验结果表明,利用该特异SCAR标记,能在1d时间内准确鉴定出榆黄蘑2号菌株。由此可见,SCAR分子标记是一种快速、稳定、准确鉴定榆黄蘑菌株的新方法。     

Antibacterial activity of Cuminum cyminum L. and Carum carvi L. essential oils

Essential oils extracted by hydrodistillation from fruits of Cuminum cyminum L. and Carum carvi L. were analyzed by gas chromatography (GC) and GC-mass spectrometry (MS). The main components of C. cyminum oil were p-mentha-1,4-dien-7-al, cumin aldehyde, gamma-terpinene, and beta-pinene, while those of the C. carvi oil were carvone, limonene, germacrene D, and trans-dihydrocarvone. Antibacterial activity, determined with the agar diffusion method, was observed against Gram-positive and Gram-negative bacterial species in this study. The activity was particularly high against the genera Clavibacter, Curtobacterium, Rhodococcus, Erwinia, Xanthomonas, Ralstonia, and Agrobacterium, which are responsible for plant or cultivated mushroom diseases worldwide. In general, a lower activity was observed against bacteria belonging to the genus Pseudomonas. These results suggest the potential use of the above essential oils for the control of bacterial diseases.     

盐碱土耐盐碱细菌筛选及其植物促生能力研究

  目的  为寻找耐盐碱促生细菌资源。  方法  采用传统定向筛选培养基培养、摇瓶培养和16S rDNA鉴定的方法,自盐碱土筛选细菌并对其促生特性进行测定。  结果  筛选得到具有促生特性的细菌14株;其中,不动杆菌属6株,肠杆菌属3株,假单胞菌属5株。在筛选出的14株细菌,均具有解钾能力,培养液速效钾含量与对照12.86 ± 0.30 mg L?相比增加16.24 ~ 36.21 mg L?1;12株细菌具有溶磷能力,培养液磷含量对照组为0.8 ± 0.1 mg L?1,接种细菌菌株的为59.1 ~ 233.2 mg L?1,培养基pH值由6.86 ± 0.34下降至4.06 ~ 5.69,线性相关分析表明溶磷培养液磷含量与培养液pH值呈负相关;7株细菌具有产植物生长激素吲哚乙酸（IAA）能力,在底物色氨酸（L-Trp）浓度为500 mg L?1时产IAA能力为5.57 ~ 21.11 mg L?1·OD₆₀₀;9株细菌具有产铁载体能力,能力最强的Su值达到95%。14株细菌均能在pH值为8、NaCl浓度为0.5 mol L?1的培养液中生长;有4株能在NaCl浓度为1.5 mol L?1培养基中生长,且B1421生长良好;有5株细菌可在pH为10的培养基中生长,为B2222生长最为良好。  结论  松嫩平原盐碱土中存在具有不同促生能力的细菌,能够耐受高pH值和较高NaCl含量的胁迫,是可供以耐盐碱植物利用改良盐碱土的菌株资源。     

Antibacterial activity of Coriandrum sativum L. and Foeniculum vulgare Miller Var. vulgare (Miller) essential oils

Essential oils were extracted from the fruits of Coriandrum sativum L. and Foeniculum vulgare Miller var. vulgare (Miller) and assayed in vitro for antibacterial activity to Escherichia coli and Bacillus megaterium, bacteria routinely used for comparison in the antimicrobial assays, and 27 phytopathogenic bacterial species and two mycopathogenic ones responsible for cultivated mushroom diseases. A significant antibacterial activity, as determined with the agar diffusion method, was shown by C. sativum essential oil whereas a much reduced effect was observed for F. vulgare var. vulgare oil. C. sativum and F. vulgare var. vulgare essential oils may be useful natural bactericides for the control of bacterial diseases of plants and for seed treatment, in particular, in organic agriculture. The significant antibacterial activity of essential oils to the bacterial pathogens of mushrooms appears promising.     

细菌生物学特性对其生物膜形成的影响

Biofilm-associated microorganisms play crucial roles in terrestrial and aquatic nutrient cycling and in the biodegradation of environmental pollutants.Biofilm formation was determined for a total of 18 bacterial isolates obtained from the biofilms of wastewater treatment systems and of little carpolite in soil.Among these isolates,seven showed strong biofilm-forming capacity.The phylogenetic affiliation of the isolates showing high biofilm formation capacity was determined through 16S rDNA sequencing and the isolates were grouped into 7 bacterial species including Pseudomonas sp.,Pseudomonas putida,Aeromonas caviae,Bacillus cereus,Pseudomonas plecoglossicida,Aeromonas hydrophila,and Comamonas testosteroni.The biofilm-forming capacity was closely related with flagella,exopolysaccharide,and extracel-lular protein.According to the coefficient of determination,the relative importance of the five biological characteristics to biofilm formation was,in order from greatest to least,exopolysaccharide > flagella > N-acyl-homoserine lactones (AHLs) signaling molecules > extracellular protein > swarming motility.     

A quantitative evaluation and phylogenetic characterization of oligotrophic denitrifying bacteria harbored in subsurface upland soil using improved culturability

This is the first investigation to show that oligotrophic denitrifying bacteria are dominant denitrifiers in subsurface upland soil. We examined the vertical distribution of denitrifying bacterial populations in upland soil using two kinds of enumeration media. The number of denitrifying bacteria, enumerated in subsurface soil layers by a 100-fold diluted nutrient broth (DNB) medium with NO₃ ⁻, was two to three orders of magnitude greater than those enumerated by a conventional nutrient broth medium with NO₃ ⁻, suggesting the dominance of oligotrophic denitrifying bacteria. Seventy-four percent of the total denitrifying bacterial isolates were DNB organisms of the oligotrophic type, which did not show appreciable growth on a conventional nutrient broth medium. The isolates were heterogeneous and were categorized as alpha (35 strains) and beta (19 strains) subdivisions of proteobacteria and high G+C gram-positive bacteria (7 strains) by 16S rRNA gene sequence analysis. The 35-alpha subdivision of proteobacterial isolates was of oligotrophic type and widely distributed from the surface to subsurface soil layers. Phylogenetic analysis indicated that some isolates belonged to groups with few or no cultivated representatives, and that one isolate may be a member of a new genus. This isolation procedure, using diluted media, is valuable in detecting diverse and novel denitrifying bacteria in the subsurface soil.     

Activity of bacteria strains originaited from sewage sludge against some soil fungi

Abstract

The potential biological activity of three bacterial strains against the four phytopathogens was examined. The bacterial strains were originated from sewage sludge water extract and from the humic acid solution obtained from the sewage sludge. The fresh bacteria cultures and filtered culture broth were used. The noncoated sugar beet seeds were coated by soaking for 15 min in the liquid culture of bacteria, then they were sown in the sterilized soil inoculated by one of the following types of soil fungus: Aphanomyces cochlioides L3, Phoma betas PH8, Pythium irregulare PIR4, Rhizoctonia solani Z10-2. The test proved that Bacillus amytoliquefaciens O1 / O3 and Flaviomonas oryzihabitans K1 / K2 showed antifungal activity on the artificial medium, but they were not able to effectively suppress fungal pathogens in the soil. The bacteria did not injure sugar beet roots, so they can be regarded as microorganisms that are not harmful to plants. It may be concluded that these bacteria have the potential to support antifungal activity of the humis acid and the sewage sludge extracts.     

Hormonal interactions in the rhizosphere of maize (Zea mays L.) and their effects on plant development

The phytohormones indole acetic acid (IAA), abscisic acid (ABA), isopentenyladenosine (iPA), dihydrozeatin riboside (DHZR), and zeatinriboside (ZR) were determined quantitatively using monoclonal antibodies by an enzyme immunoassay in the following samples: a) culture filtrates of known bacterial species, growing naturally in close, loose or without contact with higher plants; b) culture filtrates of heterogeneous populations of microorganisms, isolated from the rhizoplane and rhizosphere of maize as well as from root-free soil; c) sterile and nonsterile maize root exudates; d) in the rhizosphere of field-grown maize plants and in soil fractions distant from the roots (bulk soil). ABA was not detected in the culture filtrates of bacteria and ZR was not found in bulk soil and rhizosphere soil of field-grown maize. All phytohormones were present in the other samples analyzed. Bacterial cultures with cell concentrations roughly equal to those in the rhizosphere of field-grown maize may produce under optimal laboratory conditions phytohormone concentrations comparable to those measured in the rhizosphere of field-grown maize. During the whole vegetation period there was a steep phytohormone gradient in the first centimeter of soil around a maize root in the field. Inoculation of maize seedlings growing in nutrient solution with rhizosphere bacteria resulted in a synergistic increase in phytohormone concentration in comparison to the sum of hormone production by sterile plant roots and by bacteria cultures. Using regression analysis, the relationship between phytohormone concentration changes in the rhizosphere and different morphological characteristics of the maize plant were shown to be highly significant.     

Characterization of phosphate solubilizing fluorescent pseudomonads from the rhizosphere of Aloe vera (L.)

Soil phosphorous (P) deficiency is a major constraint to plant production which is overcome by adding inorganic-phosphate as chemical fertilizers. Fluorescent pseudomonads are the diverse group of bacteria able to mobilize sparingly soluble phosphate form. Total three hundred seven fluorescent Pseudomonas isolates were obtained from the Aloe barbadensis (Miller) rhizosphere. These Pseudomonas strains were further evaluated in vitro for their ability to solubilize phosphate and to produce indole acetic acid (IAA), hydrogen cyanide (HCN), siderophore and 1-aminocyclopropane 1- carboxylate (ACC) deaminase. Fifty three (36.8%) isolates produced IAA and 52 (36.1%) isolates produced siderophores whereas 36 (25.0%) and 31 (21.5%) isolates produced HCN and ACC deaminase, respectively. A positive correlation existed between siderophore and ACC deaminase producers. Cluster analysis showed rhizosphere as the major factor influencing the ecological distribution and physiological characterization of phosphate solubilizing bacteria (PSB). Based on partial 16S rRNA gene sequencing PSB were identified as Pseudomonas putida, Pseudomonas sp. and Pseudomonas plecoglossicida with highest phosphate solubilization ability. In conclusion, these phosphate solubilizing fluorescent pseudomonads would help in understanding their role in phosphorus solubilization and identification of potent phosphorus solubilizers from the rhizosphere of commercially grown A. barbadensis.     

Extracts from Marine Macroalgae and Opuntia ficus-indica Cladodes Enhance Halotolerance and Enzymatic Potential of Diazotrophic Rhizobacteria and Their Impact on Wheat Germination Under Salt Stress

Soil salinity, which affects more than 6% of the earth’s land surface and more than 20% of its irrigated areas, is a major threat to agriculture. Diazotrophic bacteria are among the functional groups of soil microbiota that are threatened by this abiotic stress, as their activity is mostly inhibited by salt stress. Seventy bacterial strains with distinct characteristics were isolated from soils by using N-free Jensen’s selective medium. Based on their ability to produce metabolites of agricultural interest, four strains were selected and identified as Flavobacterium johnsoniae, Pseudomonas putida, Achromobacter xylosoxidans, and Azotobacter chroococcum. The selected strains were grown at different NaCl concentrations (0–600 mmol L~(-1) in N-free broth and 0–2 000 mmol L~(-1) in Luria-Bertani medium) in the presence and absence of glycine betaine (GB), aqueous and hydro-alcoholic extracts from marine macroalgae, Ulva lactuca and Enteromorpha intestinalis, and Opuntia ficus-indica cladodes. The selected bacterial strains, GB, and the aforementioned extracts were tested for their ability to promote the germination of wheat (Triticum durum) seeds at 0–300 mmol L~(-1) NaCl. Compared with the results obtained with the synthetic osmoprotectant GB, the extracts from O. ficus-indica, U. lactuca, and E. intestinalis significantly promoted bacterial growth and seed germination under salt stress.     

Seed priming with Pseudomonas putida isolated from rhizosphere triggers innate resistance against Fusarium wilt in tomato through pathogenesis-related protein activation and phenylpropanoid pathway

Pathogenesis-related (PR) proteins are one of the major and preliminary proteins accumulated as a defense against biotic stress. This defense response can be induced by using beneficial rhizobacteria, which has been studied in various host-pathogen interactions. In the present study, eleven Pseudomonas isolates were assessed for their potential to ferment sorbitol, reduce nitrate, and produce mycolytic enzymes, 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase, phenazine antibiotics, and N-acyl homoserine lactones (AHLs). All isolates were tested against the host-specific pathogen Fusarium oxysporum MTCC1755 in tomato under greenhouse conditions, and shortlisted isolates were tested for their rhizosphere competence. In-vitro test results showed that the isolates were able to produce mycolytic enzymes, including protease, lipase, chitinase, cellulase, and amylase, and the antibiotic phenazine and were negative for pyoluteorin. All the isolates except two were positive for ACC deaminase production. Greenhouse results showed that the isolates M80, M96, and T109 significantly reduced symptoms of Fusarium wilt. Extended greenhouse tests under autoclaved and unautoclaved soil conditions showed that M80, M96, and T109 were excellent rhizosphere competitors and were identified as Pseudomonas putida. In brief, the defense-specific biochemical variations in the host could describe the improved defense against Fusarium wilt occurring in the primed plants. These three Pseudomonas strains could be used as potential biocontrol agents, along with their rhizosphere competence.