芽孢杆菌绿色荧光蛋白标记及其在小麦体表定殖的初探

 将来自质粒pAD4412的启动子和绿色荧光蛋白基因gfpmut3a插入大肠杆菌-枯草芽孢杆菌穿梭载体pBE2,构建成芽孢杆菌表达载体pGF P4412,用其转化野生型生防芽孢杆菌83-6和A-47等8个菌株,均得到良好的发光表型。质粒稳定性实验表明重组质粒pG FP4412稳定性为92%。借助荧光显微镜对gfp标记的菌株A-47-gfp在小麦体表的定殖进行初步的研究。结果表明:A-47-gfp能够在小麦根际及小麦体表定殖(包括根表和茎叶表面);相对于在茎叶表面定殖的A-47-gf p在根表定殖的菌体与根的结合更为牢固;从根基到根尖A-47-gfp的定殖量有明显的减少趋势。     

Endophytic-Host Selectivity of Discula umbrinella on Quercus alba and Quercus rubra Characterized by Infection, Pathogenicity and Mycelial Compatibility

The fungal endophytic–host relationships of Discula umbrinella and two oak species, Quercus alba and Quercus rubra, were characterized on the basis of endophytic infection, pathogenicity, and mycelial compatibility. Isolates of D. umbrinella were cultured from leaves of Q. alba and Q. rubra collected from a hardwood forest located in Patuxent Wildlife Research Center in Laurel, Maryland, USA. Endophytic infection was observed on sterile leaf discs and living 2-month-old seedlings of Q. alba and Q. rubra. Fungal-host reciprocal inoculations revealed the presence of conidiomata on both hosts but conidiomata production was more abundant on Q. alba. Isolates from Q. rubra produced milder disease symptoms on both oak species. Mycelial compatibility studies identified seven different MCG groups. MCG groups 1–3 contained isolates from both oak species whereas MCG groups 4–7 contained host specific isolates. Field studies monitored the seasonal appearance of the sexual fruiting structures, perithecia, as a possible source of new genetic variation that might alter host specificity/pathogenicity of the D. umbrinella isolates on Q. alba and Q. rubra hosts. Only 1–2% of the leaves contained perithecia throughout the sampling period (April–September). Isolates collected from Q. alba differed from those collected from Q. rubra in endophytic infection, pathogenic response, and perithecia production. The results of this study suggest that the endophyte–host relationship is one of host selective preference for Q. alba, but that the endophyte has the ability to maintain the endophytic/pathogenic life cycle on the less preferred host species, Q. rubra.     

Contributions of ammonia-oxidizing archaea and bacteria to nitrification in Oregon forest soils

Ammonia oxidation, the first step of nitrification, is mediated by both ammonia-oxidizing archaea (AOA) and bacteria (AOB); however, the relative contributions of AOA and AOB to soil nitrification are not well understood. In this study we used 1-octyne to discriminate between AOA- and AOB-supported nitrification determined both in soil-water slurries and in unsaturated whole soil at field moisture. Soils were collected from stands of red alder (Alnus rubra Bong.) and Douglas-fir (Pseudotsuga menziesii Mirb. Franco) at three sites (Cascade Head, the H.J. Andrews, and McDonald Forest) on acidic soils (pH 3.9–5.7) in Oregon, USA. The abundances of AOA and AOB were measured using quantitative PCR by targeting the amoA gene, which encodes subunit A of ammonia monooxygenase. Total and AOA-specific (octyne-resistant) nitrification activities in soil slurries were significantly higher at Cascade Head (the most acidic soils, pH < 5) than at either the H.J. Andrews or McDonald Forest, and greater in red alder compared with Douglas-fir soils. The fraction of octyne-resistant nitrification varied among sites (21–74%) and was highest at Cascade Head than at the other two locations. Net nitrification rates of whole soil without NH₄⁺ amendment ranged from 0.4 to 3.3 mg N kg⁻¹ soil d⁻¹. Overall, net nitrification rates of whole soil were stimulated 2- to 8-fold by addition of 140 mg NH₄⁺-N kg⁻¹ soil; this was significant for red alder at Cascade Head and the H.J. Andrews. Red alder at Cascade Head was unique in that the majority of NH₄⁺-stimulated nitrifying activity was octyne-resistant (73%). At all other sites, NH₄⁺-stimulated nitrification was octyne-sensitive (68–90%). The octyne-sensitive activity—presumably AOB—was affected more by soil pH whereas the octyne-resistant (AOA) activity was more strongly related to N availability.     

Fungal and bacterial N2O production regulated by soil amendments of simple and complex substrates

Fungal N₂O production results from a respiratory denitrification that reduces NO₃⁻/NO₂⁻ in response to the oxidation of an electron donor, often organic C. Despite similar heterotrophic nature, fungal denitrifiers may differ from bacterial ones in exploiting diverse resources. We hypothesized that complex C compounds and substances could favor the growth of fungi over bacteria, and thereby leading to fungal dominance for soil N₂O emissions. Effects of substrate quality on fungal and bacterial N₂O production were, therefore, examined in a 44-d incubation after soils were amended with four different substrates, i.e., glucose, cellulose, winter pea, and switchgrass at 2 mg C g⁻¹ soil. During periodic measurements of soil N₂O fluxes at 80% soil water-filled pore space and with the supply of KNO₃, substrate treatments were further subjected to four antibiotic treatments, i.e., no antibiotics or soil addition of streptomycin, cycloheximide or both so that fungal and bacterial N₂O production could be separated. Up to d 8 when antibiotic inhibition on substrate-induced microbial activity and/or growth was still detectable, bacterial N₂O production was generally greater in glucose- than in cellulose-amended soils and also in winter pea- than in switchgrass-amended soils. In contrast, fungal N₂O production was more enhanced in soils amended with cellulose than with glucose. Therefore, fungal-to-bacterial contribution ratios were greater in complex than in simple C substrates. These ratios were positively correlated with fungal-to-bacterial activity ratios, i.e., CO₂ production ratios, suggesting that substrate-associated fungal or bacterial preferential activity and/or growth might be the cause. Considering substrate depletion over time and thereby becoming limited for microbial N₂O production, measurements of soil N₂O fluxes were also carried out with additional supply of glucose, irrespective of different substrate treatments. This measurement condition might lead to potentially high rates of fungal and bacterial N₂O production. As expected, bacterial N₂O production was greater with added glucose than with added cellulose on d 4 and d 8. However, this pattern was broken on d 28, with bacterial N₂O production lower with added glucose than with added cellulose. In contrast, plant residue impacts on soil N₂O fluxes were consistent over 44-d, with greater bacterial contribution, lower fungal contribution, and thus lower fungal-to-bacterial contribution ratios in winter pea- than in switchgrass-amended soils. Real-time PCR analysis also demonstrated that the ratios of 16S rDNA to ITS and the copy numbers of bacterial denitrifying genes were greater in winter pea- than in switchgrass-amended soils. Despite some inconsistency found on the impacts of cellulose versus glucose on fungal and bacterial leading roles for N₂O production, the results generally supported the working hypothesis that complex substrates promoted fungal dominance for soil N₂O emissions.     

Fractionation of the reference inoculum of epizootic rabbit enteropathy in discontinuous sucrose gradient identifies aetiological agents in high density fractions

Epizootic rabbit enteropathy (ERE) is a major cause of economic loss in intensive rabbit production. Since its first recognition in 1997, much work has been done to determine the pathogenic mechanisms of the disease and to identify the aetiological agent(s). Unfortunately, the quest for aetiology has only met with limited success despite the ability to reproduce the syndrome by inoculation of intestinal contents from field cases. These intestinal inocula contain a huge number of microorganisms which could all be involved in the aetiology of ERE. To decrease the number of putative agents, the French reference inoculum TEC3 was fractionated on a discontinuous sucrose gradient so that seven fractions (supernatant, 10%, 20%, 30%, 40%, 50% and pellet) were obtained. Specific-pathogen-free rabbits were inoculated with three out of these seven fractions (supernatant, 30%, and pellet). The objectives were: (1) to characterise the seven fractions by bacteriological examination; (2) to verify whether the aetiological agent was present in the fractions by inoculation of rabbits; (3) to assign the aetiological agent of ERE to a morphological group of pathogens; (4) to identify a fraction which could replace the reference inoculum TEC3 in applications such as cell cultures or egg inoculation. The results strongly suggest that ERE is a bacterial disease and does not have a viral or parasitic aetiology.     

2株重组根瘤菌在4个大豆品种根圈中定殖动态的比较研究

在灭菌和未灭菌土盆栽缩影条件下，比较研究了重组大豆根瘤菌HN01DL和TA113QD在4个不同品种大豆根圈中的定殖动态与水平，并初步考察了其与共生效应的关系。结果表明：HN01DL在渝豆1号和宁镇1号大豆根圈表现出较强的适应性和竞争性，而TA113QD则在Williams大豆根圈的适应性较强，在宁镇1号大豆根圈的适应性较差，且供试菌在4种大豆根圈定殖动态与水平的差异在共生效应上也有一定程度的反映，但未达到显著水平。     

毛乌素沙地3种豆科植物根际AM真菌生态分布研究

通过对毛乌素沙地草木樨(Melilotus suaveolens)、胡枝子(Leapedeza bicolor)和沙打旺(Astragalus adsurgens)等3种豆科植物根际土壤样品和根样的采集和分析,研究了3种植物根际AM真菌的群落组成和生态分布.试验结果表明,在已分离出的3属28种AM真菌中,球囊霉属(Glomus)种类占绝对优势;3种豆科植物都有较高的AM真菌定殖率和孢子密度,但不同种之间或同一种植物在不同样地之间的AM真菌种类和分布差异显著.AM真菌泡囊定殖率和总定殖率与土壤速效N有显著负相关,其定殖强度可能主要取决于宿主植物和AM真菌的相互选择性.     

Effect of Arbuscular Mycorrhizal Fungi and Phosphate-Solubilizing Bacteria Consortia Associated with Phospho-Compost on Phosphorus Solubilization and Growth of Tomato Seedlings (Solanum lycopersicum L.)

ABSTRACT

The exploitation of phosphate mines generates an important quantity of phosphate sludge that remains accumulated and not valorized. In this context, composting with organic matter and rhizospheric microorganisms offers an interesting alternative and that is more sustainable for agriculture. This work aims to investigate the synergetic effect of arbuscular mycorrhizal fungi (AMF), phosphate-solubilizing bacteria (PSB) and phospho-compost (PC), produced from phosphate-laundered sludge and organic wastes, and their combination on plant growth, phosphorus solubilization and phosphatase activities (alkaline and acid). Inoculated mycorrhizae and bacteria strains used in this study were selected from plant rhizosphere grown on phosphate-laundered sludge. Significant (p < .05) increases in plant growth was observed when inoculated with both consortia and PC (PC+ PSB+ AMF) similar to those recorded in plants amended with chemical fertilizer. Tripartite inoculated tomato had a significantly (p < .05) higher shoot height; shoot and root dry weight, root colonization and available P content, than the control. Co-inoculation with PC and AMF greatly increased alkaline phosphatase activity and the rate of mycorrhizal intensity. We conclude that PC and endophytic AMF and PSB consortia contribute to a tripartite inoculation in tomato seedlings and are coordinately involved in plant growth and phosphorus solubilization. These results open up promising prospects for using formulate phospho-compost enriched with phosphorus-solubilizing microorganisms (PSM) in crop cultivation as biofertilizers to solve problems of phosphate-laundered sludge accumulation.     

短小芽孢杆菌BX-4抗生素标记及定殖效果研究

为研究短小芽孢杆菌BX-4在作物根际的定殖及防病效果,通过浓度梯度法用氨苄青霉素对其进行了抗性标记,并通过番茄盆栽试验研究了其在根际土壤中的定殖规律。结果表明,筛选出的突变体菌株BX-4＇能够耐受浓度为200 μg·mL^-1的氨苄青霉素,并且具有耐药和遗传双重稳定性;应用试验显示该突变体菌株能成功在番茄根际定殖,接种20 d后根际土壤中存活数量达到最高值1.34×10^8 cfu·g^-1干土,以后逐渐下降,到50 d时趋于稳定;筛选的突变体菌株对番茄青枯病具有明显的防治效果,防效达37.9%-50.9%。短小芽孢杆菌BX-4在作物根部的定殖规律为揭示其生防机理及应用该菌提供了科学根据。     

植烟土壤提取物质对烟株生长及根际土壤细菌多样性的影响

对盆栽烟草外源添加不同浓度植烟土壤提取物质(T1:40μg·mL-1;T2:120μg·mL-1;CK:蒸馏水对照),探讨植烟土壤提取物质对烟草生长及土壤细菌多样性的影响。结果表明,植烟土壤提取物处理使烟株生长受抑制,且随处理浓度的增加受抑制程度显著提高,具体表现为烟株变矮,叶面积变小,光合作用能力降低,且烟草的保护酶系统受到破坏,丙二醛含量随处理浓度加大而增加,T2处理的丙二醛含量是对照的3.44倍。对外源添加物质处理后烟草根际土壤微生物T-RFs分析发现,在对照检测到17个门24个纲,T1处理有14个门21个纲,T2有10个门17个纲;丰富度指数的变化也和门纲的变化一致,随着处理浓度的增加而显著降低。可见外源添加物质处理后,根际土壤细菌群落减少,多样性水平下降。对各处理的根际土壤微生物T-RFs变化与烟株生长变化进行相关性分析表明,在外源添加物质处理的土壤中存在较多的负相关T-RFs片段,且这些片段中较多为病原菌;而正相关的T-RFs片段主要存在于对照土壤中,其中有较多与土壤营养元素循环相关的微生物。本研究结果显示,在外源添加植烟土壤提取物质处理下,烟草的生长受抑制,烟草根际土壤的微生态受到破坏,且随浓度的提升而加重。因此,连作土壤中自毒物质的富集是造成烟草连作障碍的主要原因。关键词烟草连作障碍根际细菌自毒作用T-RFLP