Effects of Klebsiella planticola SDF20 on soil biota and wheat growth in sandy soil

The potential for ecological effects to occur after the release of genetically engineered microorganisms is a global concern and the release of biotechnology products must be assessed on a case-by-case basis. In this research, a genetically engineered strain of Klebsiella planticola (SDF20) bacteria was added to microcosms containing sandy soil and wheat plants to assess the potential for effects on soil biota and plant growth. One half of the soil treatments in this study contained wheat plants to compare some effects on growing rhizosphere communities in the experimental system. When SDF20 was added to soil with plants, the numbers of bacterial and fungal feeding nematodes increased significantly, coinciding with death of the plants. In contrast, when the parental strain, SDF15 was added to soil with plants, only the number of bacterial feeding nematodes increased, but the plants did not die. The introduction of either SDF15 or SDF20 strains to soil without plants did not alter the nematode community. No effects were observed on the activity of native bacterial and fungal communities by either SDF15 or SDF20. This study is evidence that SDF20 can persist under conditions found in some soil ecosystems and for long enough periods of time to stimulate change in soil biota that could affect nutrient cycling processes. Further investigation is needed to determine the extent these observations may occur in situ but this study using soil microcosms was the first step in assessing potential for the release of genetically engineered microorganisms to result in ecological effects.     

Qualitative and quantitative detection of agricultural microorganisms expressing iturin and mop cyclase in soils

The environmental release of genetically engineered microorganisms (GEMs) to improve agriculture or remediate environmental hazards has raised concern over the fate of the organisms and their engineered genes. To detect the microorganisms released into the environment at the molecular level, Bacillus subtilis KB producing iturin and Pseudomonas fluorescens MX1 carrying the moc (mannityl opine catabolism) region from the Agrobacterium tumefaciens were employed as model microorganisms. Using specific fusion primers and the TaqMan probes, qualitative and quantitative detections of the model organisms by PCR and real-time PCR were conducted employing a small-scale soil-core device and pots during the six month period. The data indicate that the model bacteria can be easily detected by qualitative and quantitative methods in the test systems employed, and they do not give significant impacts on the other bacteria in soils on the Southern blotting analysis, although long-term observation may be needed.     

Strategies for bacterial tagging and gene expression in plant-host colonization studies

Bacteria are extraordinarily diverse microorganisms with a huge potential to benefit environmental and agricultural systems. Comprehensive studies in complex habitats such as soils and plants have led to the development of genetic tools to evaluate gene expression and bacterial colonization under controlled or environmental conditions and to obtain genetically engineered organisms for environmental release. In addition, current advances in genomic and metagenomic research will add to the number of genes with potential for biotechnological applications, which will require the development of appropriate genetic systems to fulfill their potential for both industrial and agricultural applications. The aim of the present review is to assess the approaches and recent progress in vector design and genetic tools to study and manipulate plant-bacterial interactions, as well as strategies to construct genetically modified strains for environmental release.     

农业微生物基因工程研究与展望

摘要：介绍了我国农业微生物基因工程研究现状，特别是饲料、肥料和农药用重组微生物制剂的研究成果。为争取5～10年内使我国农业微生物成为新兴的高技术支柱产业，建议进一步加强基础研究，重视资源开发利用，适时开展功能基因组学研究，从高起点切入，以加快源头创新。另建议将微生物农药、饲料和食品加工用酶制剂列为近期研究开发重点。同时也要不断革新生产工艺，改进加工剂型，调整研发结构，促进产业发展。     

Novel method for the study of the population dynamics of a genetically modified microorganism in the gut of the earthworm Lumbricus terrestris

Summary A laboratory microcosm study was used to investigate the survival and population dynamics of genetically modified microorganisms (GMM) in the gut of Lumbricus terrestris. Three methods of axenic earthworm production were investigated. An antibiotic mixture of streptomycin and cycloheximide was introduced either passively, mixed with sterile soil or cellulose, or actively, by teflon catheter. Worms treated by all methods lost weight but this was least for the catheter method which was also the only method to produce axenic earthworms. Axenic earthworms were used to determine the effect of competition with indigenous gut bacteria on ingested GMM. The GMM used was Pseudomonas fluorescens, strain 10586/FAC510, with chromosomally inserted Lux genes for bioluminescence, and chromosomal resistance to rifampicin. The bacteria were grown up to the mid-exponential phase before inoculation into earthworms. Bacteria in faecal material were enumerated by dilution plate counting using selective agar. The GMM were re-isolated from the casts of both antibiotic-treated and untreated earthworms. Lower concentrations of GMM and higher concentrations of indigenous bacteria in the casts of untreated compared to antibiotic-treated earthworms suggested that competition is a fundamental control on population dynamics of the introduced bacterial inocula ingested by earthworms.The catheter method, developed in this study, is proposed as a technique to contribute to the risk assessment of environmental release of GMM.     

Investigating the influence of transgenic tobacco plants codifying a protease inhibitor on soil microbial community

Serine protease inhibitors (PIs) are involved in several physiological processes, such as regulation of endogenous proteinases and defence against phytophageous insects. Transgenic modifications have enhanced protease inhibitor expression to develop insect resistant cultivars in several important crops. The fate of protease inhibitors released from genetically engineered plants is an important issue because of possible inhibition of soil proteases and effects of the insecticidal protein and its codifying sequence on soil microorganisms. The persistence of transgenic sequence mustard trypsin inhibitor-2 in soil and its hypothetical acquisition by soil microorganisms by horizontal gene transfer and the effect of transgenic plant material on soil microbial community structure and soil protease activity were investigated. With the aim to simulate the effects of plant litter on soil microorganisms, a microcosm experimental model was used. Despite the persistence of transgenic DNA sequences, no recombination event was detected between plant DNA and soil bacteria; molecular analysis of bacterial community also showed no significant influence on the dominant members of the bacterial community and soil protease activity was not inhibited by the release of constitutively over-expressed protease inhibitor.     

Effect of Azospirillum inoculants on arbuscular mycorrhiza establishment in wheat and maize plants

Plant growth-promoting rhizobacteria and arbuscular mycorrhizal (AM) fungi represent two main groups of beneficial microorganisms of the rhizosphere. The role of different strains of Azospirillum on AM fungi development was evaluated by measuring the percentage of AM colonisation of the root system in durum wheat and maize plants, grown under both greenhouse and field conditions. The effect of wild-type Azospirillum brasilense strain Sp245 and genetically modified (GM) derivatives overproducing indole-3-acetic acid was assessed at greenhouse level in (1) three different cultivars of durum wheat, in the presence of indigenous AM fungi and (2) maize plants artificially inoculated with Glomus mosseae and Glomus macrocarpum. In addition, the establishment of natural AM fungal symbiosis was evaluated using Azospirillum lipoferum CRT1 in maize plants at field level. Despite the stimulatory effect of the different Azospirillum inocula on root growth, no significant differences in AM colonisation were found, independently of the AM fungus involved, either in wheat or in maize plants. Similarly, GM A. brasilense, which strongly stimulates root development, did not affect AM formation. Although these results were obtained in conditions in which the mycorrhization rate was moderate (15–30%), overall considered they indicate that the use of wild-type or GM Azospirillum phytostimulators does not alter mycorrhization.     

Symbiosis with bacteria enhances the use of chitin by the springtail,Folsomia candida (Collembola)

Summary The relationship between Folsomia candida and chitin-degrading microorganisms was studied. On chitin agar, 10¹⁰ bacteria were isolated per g faeces, and 3.8×10¹¹ bacteria per g gut contents, 1/3 of them showing a clear (chitin-free) zone around the colony. The most abundant chitin-degrading bacteria were Xanthomonas maltophilia and Curtobacterium sp. To determine the bacterial contribution in the use of chitin by F. candida, a feeding experiment was carried out. F. candida were fed with chitin, either amended with or without tetracycline as an inhibitor of bacteria. When tetracycline was omitted the biomass of F. candida was increased compared to those fed chitin with tetracycline. However, this result was observed only when the food replacement intervals were long enough to allow bacterial colonization before ingestion of the food. In a food-selection experiment, a preference for chitin colonized with microorganisms as opposed to sterile chitin was found. The results indicate that a mutualistic symbiosis of F. candida with chitinolytic microorganisms is likely to enhance chitin degradation. This relationship is not only intra-intestinal but also involves an extra-intestinal phase.Dedicated to the late Prof. Dr. W. Kühnelt     

Transgenic cotton could safely be grown since CpTI toxin rapidly degrades in the rhizosphere soil

Abstract

Cultivation of transgenic plants is debated worldwide. Potential environmental risks have to be considered, before acceptance of expanding cultivation, despite the advantages of the use of fewer pesticides. Here, the potential effects on soil ecosystems of transgenic plants have been studied. As a model, genetically engineered cotton producing cowpea trypsin inhibitor (CpTI) has been used. The degradation of CpTI in the rhizosphere of the transgenic CpTI+Bt (Bacillus thuringiensis) cotton cultivar SGK321 was assessed. During plant development, concentrations of CpTI toxin in the rhizosphere were measured using an enzyme-linked immunosorbent assay (ELISA). As the plants developed, the residue of CpTI in the rhizosphere increased, and reached a peak at topping stage (100 days after planting). After this stage, the residue began to decrease, and was nil the following year (258 days after planting). The conclusion is that genetically engineered cotton can safely be cultivated since no accumulation of substances released from the transgenic plants was persistent in the soil.     

Vertical transport of a field-released genetically engineered microorganism through soil

The vertical movement of a genetically-engineered bacterium released into a sub-surface soil contained within a lysimeter was investigated. The strain used was Pseudomonas fluorescens HK44, a lux-based microbe capable of bioremediating specific polyaromatic hydrocarbons (PAHs). Strain HK44 was inoculated into a 4 m deep by 2.5 m diameter soil lysimeter outfitted with a hydraulic assembly to control groundwater levels. After 230 days, the lysimeter was subjected to a water table manipulation that resulted in significant transport of HK44 cells to depths 60 cm below the original inoculation zone within 20 days. A lysimeter not exposed to water level fluctuations exhibited no significant vertical transport. These results demonstrate the potential distribution of genetically engineered microorganisms in subsurface soil due to groundwater effects.     

Impact of heavy metal amended sewage sludge on forest soils as assessed by bacterial and fungal biosensors

Bacterial and fungal bioluminescence-based biosensors were used as indicators of potential heavy metal toxicity to microorganisms in the needle litter of a mature Pinus radiata forest under heavy metal contaminated sewage sludge. Sewage sludge was amended with increasing concentrations of Cu, Ni and Zn and applied to the surface of a mature P. radiata forest. The response of the bacterial and fungal biosensors to soluble Cu, Ni and Zn in needle litter extracts was investigated. The bioluminescence response of the bacterial biosensor Escherichia coli HB101 pUCD607 declined as water-soluble Zn concentrations increased. The effective concentrations that gave a 50% reduction in bioluminescence (EC₅₀ values) for water-soluble Zn and total litter Zn were 1.3 mg l⁻¹ and 3700 mg kg⁻¹, respectively. The bioluminescence response of the fungal biosensor Armillaria mellea declined as soluble Cu concentrations increased. The EC₅₀ values for water-soluble Cu and total litter Cu were 0.12 mg l⁻¹ and 540 mg kg⁻¹, respectively. No decline in bioluminescence was noted for either the bacterial or fungal biosensor on exposure to increasing concentrations of water-soluble Ni. The use of a combination of bacterial and fungal biosensors offers a rapid and sensitive tool for assessing toxicity of heavy metals to microorganisms and, thus, elucidating the environmental impact of contaminants in sewage sludge on litter dwelling microorganisms.     

Phosphate solubilizing microorganisms isolated from rhizosphere of maize cultivated in an oxisol of the Brazilian Cerrado Biome

Many soil microorganisms are able to transform insoluble forms of phosphorus to an accessible soluble form, contributing to plant nutrition as plant growth-promoting microorganisms (PGPM). The objective of this work was to isolate, screen and evaluate the phosphate solubilization activity of microorganisms in maize rhizosphere soil to manage soil microbial communities and to select potential microbial inoculants. Forty-five of the best isolates from 371 colonies were isolated from rhizosphere soil of maize grown in an oxisol of the Cerrado Biome with P deficiency. These microorganisms were selected based on the solubilization efficiency of inorganic and organic phosphate sources in a modified Pikovskaya's liquid medium culture containing sodium phytate (phytic acid), soybean lecithin, aluminum phosphate (AlPO₄), and tricalcium phosphate (Ca₃(PO₄)₂). The isolates were identified based on nucleotide sequence data from the 16S ribosomal DNA (rDNA) for bacteria and actinobacteria and internal transcribed spacer (ITS) rDNA for fungi. Bacteria produced the greatest solubilization in medium containing tricalcium phosphate. Strains B17 and B5, identified as Bacillus sp. and Burkholderia sp., respectively, were the most effective, mobilizing 67% and 58.5% of the total P (Ca₃(PO₄)₂) after 10 days, and were isolated from the rhizosphere of the P efficient L3 maize genotype, under P stress. The fungal population was the most effective in solubilizing P sources of aluminum, phytate, and lecithin. A greater diversity of P-solubilizing microorganisms was observed in the rhizosphere of the P efficient maize genotypes suggesting that the P efficiency in these cultivars may be related to the potential to enhance microbial interactions of P-solubilizing microorganisms.     

Changes in active microbial biomass by earthworms and grass amendments in agricultural soil

The influence of the earthworm Aporrectodea caliginosa on the biomass and the proportion of active and dormant soil microorganisms after the addition of cut perennial ryegrass (Lolium perenne) to upper soil from agricultural field was studied in a microcosm experiment. During a 2-month period, soil samples were taken 1, 8, 22, 36, 50, and 64 days after cut grass addition. A substrate-induced respiration (SIR) method was used to analyse the samples for total microbial biomass and the distribution of active and dormant microbial biomass. It was found that the addition of grass increased the microbial biomass (SIR) because of an increase in the active microbial biomass. After the initially high values, the active microbial biomass decreased slowly, and at day 64, it was still higher in the grass-amended soils than in the control treatment without grass addition. After 1 day, the active microbial biomass was higher in the soil with A. caliginosa than without the earthworm. At the subsequent samplings, there were no differences in microbial biomass or the proportion of dormant vs active microorganisms between the grass-amended soils. The average from all sampling occasions of SIR was higher in earthworm-treated soil.     

Root growth,and microorganisms associated with the rhizoplane and root zone soil of a native C4 grass on burned and unburned sand prairies

We investigated the root growth of native Schizachyrium scoparium, little bluestem grass, and the seasonal abundance of rhizoplane and root zone soil microorganisms on burned and unburned sand prairies. Root growth and abundances of rhizoplane and root zone microorganisms were greater in burned than unburned sites. Microbe populations were nearly always higher on the rhizoplane than in the root zone soils, although they were not always significantly different. The seasonal dynamics of total bacteria, total fungi, fluorescent pseudomonads, and microorganisms that decompose chitin, cellulose, and protein varied between burned and unburned sites. Some microbial populations showed significant, though weak, relationships with root growth. Populations of most microorganisms were usually highest from June through August, when roots were being shed following the peak standing crop of root mass in May. The production of fine roots on burned sites early in the growing season and the consequent shedding of fine roots probably have an important effect on microbe population dynamics.     

Isozyme variation in species of the section Comopyrum of Aegilops

Genetic variation at 21 enzyme loci was studied in Aegilops comosa and Ae. uniaristata, the two species belonging to section Comopyrum of Aegilops. In Ae. comosa, the mean number of alleles per locus was 2.00 and the proportion of polymorphic loci was 0.667; in Ae. uniaristata they were 1.19 and 0.143, respectively. The two species were genetically distant from each other (I=0.561) supporting the previously assigned different genome symbols, M and N.     

转CryIAb水稻对稻田杂草群落组成及多样性的影响

尽管基因工程技术可以增加作物产量, 但转基因作物是否对农田生态产生影响受到广泛关注。本研究通过田间定位试验, 应用群落生态学方法研究了转CryIAb基因抗虫水稻"Mfb"连续2年在传统栽培和半野生条件下对稻田杂草群落组成及多样性的影响。调查结果显示: 转CryIAb基因稻"Mfb"与非转基因稻"明恢86"田间杂草种类没有显著差异。稻田杂草的频度和密度与栽培方式有关, 半野生稻田杂草的频度和密度显著高于传统稻田, 但相同栽培条件下, 转基因稻"Mfb"与非转基因稻"明恢86"田间杂草频度和密度在整个生长期内均无显著差异。半野生稻田物种丰富度(Sr)指数明显大于传统稻田; 相同栽培条件下, 相同生长时期抗虫转基因水稻"Mfb"与其非转基因对照"明恢86"对稻田杂草群落丰富度的影响差异不显著。稻田杂草群落优势度(D)、均匀度(J)以及多样性(H)各处理、各生长时期内转基因稻与非转基因稻相比均没有显著差异。稻田杂草Shannon-Wiener多样性指数变化无明显规律, 相同栽培方式相同生长期的抗虫转基因水稻"Mfb"与其非转基因对照"明恢86"的Shannon-wiener指数差异不显著。综合上述分析, 转CryIAb基因抗虫稻对稻田杂草群落的组成及多样性没有显著影响。     

Inhibition of nitrification in soil by gaseous hydrocarbons

Summary Recent work has shown that gaseous hydrocarbons such as methane, ethane, and ethylene are competitive inhibitors of the monooxygenase enzyme responsible for oxidation of ammonia by chemoautotrophic nitrifying microorganisms such as Nitrosomonas europaea. Because methane, ethane, and ethylene are produced by microbial activity in soil, we studied the possibility that they may inhibit oxidation of ammonia by the nitrifying soil microorganisms. We found that all three of these gaseous hydrocarbons inhibited nitrification in soil and that their ability to inhibit nitrification decreased in the order: ethylene > ethane > methane. Ethylene was much more effective than ethane or methane for inhibiting nitrification of ammonium in soil, but it was much less effective than acetylene, and it seems unlikely that the amounts of ethylene produced in soils will be sufficient to cause significant inhibition of nitrification by soil microorganisms.     

Nitrogenase activity associated with leaf,root, and rhizosphere soils in an Indian tropical forest

Summary N₂ fixation (acetylene reduction assay) by phylloplane microorganisms was measured in dominant and co-dominant plant species growing in a tropical rain forest. No significant acetylene reduction was recorded with intact leaf samples. Azotobacter sp., Beijerinckia sp., Derxia sp., and Klebsiella pneumoniae were isolated as phylloplane N₂-fixing bacteria. Azospirillum lipoferum was only isolated from soil samples containing the roots of Poaceae. Nitrogenase activity was recorded in culture derived from the roots and rhizosphere soil samples, although low acetylene reduction activity indicates that these associations did not provide large amounts of N to the systems studied.     

Development of an algorithm identifying maximally diverse core collections

The development of a core collection, one which represents the genetic diversity of a crop with minimal redundancy and increases utility of the collection as a whole, is especially important as the funding for germplasm collections decreases. With limited resources, it is difficult to manage large germplasm collections and disperse genetically diverse germplasm to plant breeders. An algorithm was developed to assist in selection of core collections based on estimates of genetic distance. The criteria for selection of the maximum genetically diverse set were based on rankings of genetic distance between an accession with respect to all other accessions. Depending on the size core which a user wished, a zone around each selected accession was determined and no other accession within these limits was selected. The premise for the algorithm was that the genetic variability represented in the core must be representative of the distribution of genetic distances within the population of interest. In the present study, the algorithm was used with RAPD-marker-based estimates of genetic distance for 270 Theobroma cacao L. accessions and 134 Capsicum accessions that chose a set representing 18.5% of the population and representing the breadth of RAPD-based variation.     

发光酶基因标记荧光假单胞菌X16L2的发光研究

通过光光光度仪和ＡＭＰＮ法定量测定荧光假单胞菌Ｘ１６Ｌ２,在液培和土壤中的发光情况及其数量,发现Ｘ１６Ｌ２在液培和土才壤中的发光动力学曲线相似,达到稳定发光强度的时间均在加入反应底物癸醛后约７分钟。Ｘ１６Ｌ２在液培中的发光强度与生物量吴正相关。土壤中Ｘ１６Ｌ２的发光量既与活菌数有关,也与土壤条件有关,土著微生物对Ｘ１６Ｌ２的光量,土壤中Ｘ１６Ｌ２的发光量既与活菌数有关,也与土壤条件有关。土著微生物