Monitoring physiological status of GFP-tagged Pseudomonas fluorescens SBW25 under different nutrient conditions and in soil by flow cytometry

Pseudomonas fluorescens SBW25, a plant growth promoting bacterium, has been widely studied due to its potential as an inoculum for improving crop yields. Environmental inoculants are usually applied on seeds or directly to soil and to effectively promote plant growth they need to be viable and active. However, it is difficult to study the physiological status of specific microorganisms in complex environments, such as soil. In this study, our aim was to use molecular tools to specifically monitor the physiological status of P. fluorescens SBW25 in soil and in pure cultures incubated under different nutritional conditions. The cells were previously tagged with marker genes (encoding green fluorescent protein and bacterial luciferase) to specifically track the cells in environmental samples. The physiological status of the cells was determined using the viability stains 5-cyano-2,3-ditolyl-tetrazolium chloride (CTC) and propidium iodide (PI), which stain active and dead cells, respectively. Luciferase activity was used to monitor the metabolic activity of the population. Most of the cells died after incubation for nine days in nutrient rich medium. By contrast when incubated under starvation conditions, most of the population was not stained with CTC or PI (i.e. intact but inactive cells), indicating that most of the cells were presumably dormant. In soil, a large fraction of the SBW25 cell population became inactive and died, as determined by a decline in luciferase activity and CTC-stained cells, an increase in PI-stained cells, and an inability of the cells to be cultured on agar medium. However, approximately 60% of the population was unstained, presumably indicating that the cells entered a state of dormancy in soil similar to that observed under starvation conditions in pure cultures. These results demonstrate the applicability of this approach for monitoring the physiological status of specific cells under stress conditions, such as those experienced by environmental inoculants in soil.     

Monitoring Population Size,Activity, and Distribution of gfp-luxAB-Tagged Pseudomonas fluorescens SBW25 during Colonization of Wheat

Increasingly, focus has been directed towards the use of microorganisms as biological control agents to combat fungal disease, as an alternative to chemical fungicides. Pseudomonas fluorescens SBW25 is one bacterial strain that has been demonstrated to promote plant growth by biocontrol of pathogenic fungi. To understand the mode of action of this bacterium, information regarding its localization and metabolic activity on plants is important. In this study, a gfp/luxAB-tagged derivative of P. fluorescens SBW25, expressing the green fluorescent protein (GFP) and bacterial luciferase, was monitored during colonization of wheat starting from seed inoculation. Since bacterial luciferase is dependent on cellular energy reserves for phenotypic expression, metabolically active cells were detected using this marker. In contrast, the stable GFP fluorescence phenotype was used to detect the cells independently of their metabolic status. The combination of these two markers enabled P. fluorescens SBW25 cells to be monitored on wheat plants to determine their specific location and metabolic activity. Studies on homogenized wheat plant parts demonstrated that the seed was the preferred location of P. fluorescens SBW25 during the 65-day time period studied, but the leaves and roots were also colonized. Interestingly, the bacteria were also found to be metabolically active on all plant parts examined. In situ localization of P. fluorescens SBW25 using a combination of different microscopic techniques confirmed the preference for the cells to colonize specific regions of the seed. We speculate that the colonization pattern of P. fluorescens SBW25 can be linked to the mechanism of protection of plants from fungal infection.     

Simultaneous Monitoring of Cell Number and Metabolic Activity of Specific Bacterial Populations with a Dual gfp-luxAB Marker System

A dual marker system was developed for simultaneous quantification of bacterial cell numbers and their activity with the luxAB and gfp genes, encoding bacterial luciferase and green fluorescent protein (GFP), respectively. The bioluminescence phenotype of the luxAB biomarker is dependent on cellular energy status. Since cellular metabolism requires energy, bioluminescence output is directly related to the metabolic activity of the cells. By contrast, GFP fluorescence has no energy requirement. Therefore, by combining these two biomarkers, total cell number and metabolic activity of a specific marked cell population could be monitored simultaneously. Two different bacterial strains, Escherichia coli DH5α and Pseudomonas fluorescens SBW25, were chromosomally tagged with the dual marker cassette, and the cells were monitored under different conditions by flow cytometry, plate counting, and luminometry. During log-phase growth, the luciferase activity was proportional to the number of GFP-fluorescent cells and culturable cells. Upon entrance into stationary phase or during starvation, luciferase activity decreased due to a decrease in cellular metabolic activity of the population, but the number of GFP-fluorescing cells and culturable cells remained relatively stable. In addition, we optimized a procedure for extraction of bacterial cells from soil, allowing GFP-tagged bacteria in soil samples to be quantitated by flow cytometry. After 30 days of incubation of P. fluorescens SBW25::gfp/lux in soil, the cells were still maintained at high population densities, as determined by GFP fluorescence, but there was a slow decline in luciferase activity, implicating nutrient limitation. In conclusion, the dual marker system allowed simultaneous monitoring of the metabolic activity and cell number of a specific bacterial population and is a promising tool for monitoring of specific bacteria in situ in environmental samples.     

Specific interactions between arbuscular mycorrhizal fungi and plant growth-promoting bacteria: as revealed by different combinations

The interactions between two plant growth-promoting rhizobacteria (PGPR, Pseudomonas fluorescens SBW25 and Paenibacillus brasilensis PB177), two arbuscular mycorrhizal (AM) fungi (Glomus mosseae and Glomus intraradices) and one pathogenic fungus (Microdochium nivale) were investigated on winter wheat (Triticum aestivum cultivar Tarso) in a greenhouse trial. PB177, but not SBW25, had strong inhibitory effects on M. nivale in dual culture plate assays. The results from the greenhouse experiment show very specific interactions; for example, the two AM fungi react differently when interacting with the same bacteria on plants. Glomus intraradices (single inoculation or together with SBW25) increased plant dry weight on M. nivale-infested plants, suggesting that the pathogenic fungus is counteracted by G. intraradices, but PB177 inhibited this positive effect. This is an example of two completely different reactions between the same AM fungus and two species of bacteria, previously known to enhance plant growth and inhibit pathogens. When searching for plant growth-promoting microorganisms, it is therefore important to test for the most suitable combination of plant, bacteria and fungi in order to achieve satisfactory plant growth benefits.     

Biocontrol of Pythium in the pea rhizosphere by antifungal metabolite producing and non-producing Pseudomonas strains

AIMS: Four well-described strains of Pseudomonas fluorescens were assessed for their effect on pea growth and their antagonistic activity against large Pythium ultimum inocula. Methods and RESULTS: The effect of Pseudomonas strains on the indigenous soil microflora, soil enzyme activities and plant growth in the presence and absence of Pythium was assessed. Pythium inoculation reduced the shoot and root weights, root length, and the number of lateral roots. The effect of Pythium was reduced by the Pseudomonas strains. Strains F113, SBW25 and CHAO increased shoot weights (by 20%, 22% and 35%, respectively); strains Q2-87, SBW25 and CHAO increased root weights (14%, 14% and 52%). Strains SBW25 and CHAO increased root lengths (19% and 69%) and increased the number of lateral roots (14% and 29%). All the Pseudomonas strains reduced the number of lesions and the root and soil Pythium populations, while SBW25 and CHAO increased the number of lateral roots. Pythium inoculation increased root and soil microbial populations but the magnitude of this effect was Pseudomonas strain-specific. Pythium increased the activity of C, N and P cycle enzymes, while the Pseudomonas strains reduced this effect, indicating reduced plant damage. CONCLUSION: Strains SBW25 and CHAO had the greatest beneficial characteristics, as these strains produced the greatest reductions in the side effects of Pythium infection (microbial populations and enzyme activities) and resulted in significantly improved plant growth. Strain SBW25 does not produce antifungal metabolites, and its biocontrol activity was related to a greater colonization ability in the rhizosphere. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first critical comparison of such important strains of Ps. fluorescens showing disease biocontrol potential.     

Genes encoding a cellulosic polymer contribute toward the ecological success of Pseudomonas fluorescens SBW25 on plant surfaces

Pseudomonas fluorescens SBW25 is a Gram-negative bacterium that grows in close association with plants. In common with a broad range of functionally similar bacteria it plays an important role in the turnover of organic matter and certain isolates can promote plant growth. Despite its environmental significance, the causes of its ecological success are poorly understood. Here we describe the development and application of a simple promoter trapping strategy (IVET) to identify P. fluorescens SBW25 genes showing elevated levels of expression in the sugar beet rhizosphere. A total of 25 rhizosphere-induced (rhi) fusions are reported with predicted roles in nutrient acquisition, stress responses, biosynthesis of phytohormones and antibiotics. One rhi fusion is to wss, an operon encoding an acetylated cellulose polymer. A mutant carrying a defective wss locus was competitively compromised (relative to the wild type) in the rhizosphere and in the phyllosphere, but not in bulk soil. The rhizosphere-induced wss locus therefore contributes to the ecological performance of SBW25 in the plant environment and supports our conjecture that genes inactive in the laboratory environment, but active in the wild, are likely to be determinants of fitness in natural environments.     

Risk assessment for engineered bacteria used in biocontrol of fungal disease in agricultural crops

A plant growth promoting rhizobacterium (PGPR)Pseudomonas fluorescens SBW25 (WT) protects a number of crop plant species from damping-off caused by Pythium ultimum. A genetically modified, phenazine-1-carboxylic acid (PCA) producing variant, 23.10, carries on its chromosome a single copy of phzABCDEFG, under the control of the P tac constitutive promoter. The genetically modified biological control agent (GM-BCA), 23.10, has improved biocontrol activity when compared to wild type SBW25, and can effectively suppress Pythium spp. present at up to 100 times normal field infestations. GM-BCA inocula establish high population densities which persist well in the phytosphere of several crop plants including pea, wheat and sugar beet, effectively suppressed infection and promoted increase in total plant biomass. It also has an improved spectrum of activity over other plant phytopathogens such as Fusarium spp. Gaeumannomyces graminis var. tritici, Phytophtora cinnamomi and Rhizoctonia solani. However in developing BCAs and in particular GMBCAs it is important to determine whether their use has any adverse effect in the environment. Any observed changes following inoculation with wild type BCA or GM BCA in microbial diversity (bacteria and fungi) were negligible when assessed by either quantitive selective plate count methods (CFU/g) or culture independent molecular assays (SSU rRNA based PCR-DGGE). Rhizosphere community diversity profiles (DGGE) in infected plants in the presence of inocula were highly similar to disease free systems. Histological assessment of the impact of inocula on established functional mycorrhizae associations were conducted on cores collected from an established field margin grassland pasture. No adverse impact on mycorrhizal colonization and root infection were recorded after addition of WT or GM-BCA bacterial inocula as a soil drench. This approach and the related culturable and culture independent methods have recorded only a minor, transient perturbation to microbial communities, but as far as we are aware this is the first direct demonstration that a functional, AFC producing GMM also has only a transient impact on mycorrhizal associations in established plant communities. In all instances studied the plant species, plant stage of development and disease, damping-off, had a greater impact on changes in rhizosphere diversity than the presence of an introduced GM bacterial inocula.     

Effect of Insertion Site and Metabolic Load on the Environmental Fitness of a Genetically Modified Pseudomonas fluorescens Isolate

An isolate of Pseudomonas fluorescens (SBW25) was modified with different marker genes (lacZY, aph-1, and xylE). These marker genes were inserted singly or in combination into two separate (1 Mbp apart) and presumably nonessential sites (-6- and Ee) on the chromosome of SBW25. This allowed the production of a range of genetically modified SBW25 variants that differed with respect to insertion site of the marker genes and metabolic burden. The environmental fitness of the different SBW25 variants was tested in soil, in the rhizosphere of wheat and pea, and on the phylloplane of wheat. Reduced environmental fitness of the different variants was mainly attributed to the extra metabolic burden of novel gene expression, whereas choice of insertion site was of little significance. Changes in environmental fitness were dependent on the environmental conditions; an environment, such as soil, with a low microbial carrying capacity had a negative effect on the environmental fitness of variants with a large metabolic load. In environments with a larger carrying capacity, such as the rhizosphere of pea, environmental fitness of variants with a large metabolic load was not significantly different from that of variants with a smaller metabolic burden.     

Impact of wild-type and genetically modified Pseudomonas fluorescens on soil enzyme activities and microbial population structure in the rhizosphere of pea

The aim of this study was to determine the impact of wild-type along with functionally and nonfunctionally modified Pseudomonas fluorescens strains in the rhizosphere. The wild-type F113 strain carried a gene encoding the production of the antibiotic 2,4-diacetylphloroglucinol (DAPG) useful in plant disease control, and was marked with a lacZY gene cassette. The first modified strain was a functional modification of strain F113 with repressed production of DAPG, creating the DAPG-negative strain F113 G22. The second paired comparison was a nonfunctional modification of wild-type (unmarked) strain SBW25, constructed to carry marker genes only, creating strain SBW25 EeZY-6KX. Significant perturbations were found in the indigenous bacterial population structure, with the F113 (DAPG+) strain causing a shift towards slower growing colonies (K strategists) compared with the nonantibiotic-producing derivative (F113 G22) and the SBW25 strains. The DAPG+ strain also significantly reduced, in comparison with the other inocula, the total Pseudomonas populations but did not affect the total microbial populations. The survival of F113 and F113 G22 were an order of magnitude lower than the SBW 25 strains. The DAPG+ strain caused a significant decrease in the shoot-to-root ratio in comparison to the control and other inoculants, indicating plant stress. F113 increased soil alkaline phosphatase, phosphodiesterase and aryl sulphatase activities compared to the other inocula, which themselves reduced the same enzyme activities compared to the control. In contrast to this, the β-glucosidase, β-galactosidase and N -acetyl glucosaminidase activities decreased with the inoculation of the DAPG+ strain. These results indicate that soil enzymes are sensitive to the impact of inoculation with genetically modified microorganisms (GMMs).     

Genome-based discovery, structure prediction and functional analysis of cyclic lipopeptide antibiotics in Pseudomonas species

Analysis of microbial genome sequences have revealed numerous genes involved in antibiotic biosynthesis. In Pseudomonads, several gene clusters encoding non-ribosomal peptide synthetases (NRPSs) were predicted to be involved in the synthesis of cyclic lipopeptide (CLP) antibiotics. Most of these predictions, however, are untested and the association between genome sequence and biological function of the predicted metabolite is lacking. Here we report the genome-based identification of previously unknown CLP gene clusters in plant pathogenic Pseudomonas syringae strains B728a and DC3000 and in plant beneficial Pseudomonas fluorescens Pf0-1 and SBW25. For P. fluorescens SBW25, a model strain in studying bacterial evolution and adaptation, the structure of the CLP with a predicted 9-amino acid peptide moiety was confirmed by chemical analyses. Mutagenesis confirmed that the three identified NRPS genes are essential for CLP synthesis in strain SBW25. CLP production was shown to play a key role in motility, biofilm formation and in activity of SBW25 against zoospores of Phytophthora infestans. This is the first time that an antimicrobial metabolite is identified from strain SBW25. The results indicate that genome mining may enable the discovery of unknown gene clusters and traits that are highly relevant in the lifestyle of plant beneficial and plant pathogenic bacteria.     

Effect of genetically modified Pseudomonas putida WCS358r on the fungal rhizosphere microflora of field-grown wheat

We released genetically modified Pseudomonas putida WCS358r into the rhizospheres of wheat plants. The two genetically modified derivatives, genetically modified microorganism (GMM) 2 and GMM 8, carried the phz biosynthetic gene locus of strain P. fluorescens 2-79 and constitutively produced the antifungal compound phenazine-1-carboxylic acid (PCA). In the springs of 1997 and 1998 we sowed wheat seeds treated with either GMM 2, GMM 8, or WCS358r (approximately 10(7) CFU per seed), and measured the numbers, composition, and activities of the rhizosphere microbial populations. During both growing seasons, all three bacterial strains decreased from 10(7) CFU per g of rhizosphere sample to below the limit of detection (10(2) CFU per g) 1 month after harvest of the wheat plants. The phz genes were stably maintained, and PCA was detected in rhizosphere extracts of GMM-treated plants. In 1997, but not in 1998, fungal numbers in the rhizosphere, quantified on 2% malt extract agar (total filamentous fungi) and on Komada's medium (mainly Fusarium spp.), were transiently suppressed in GMM 8-treated plants. We also analyzed the effects of the GMMs on the rhizosphere fungi by using amplified ribosomal DNA restriction analysis. Introduction of any of the three bacterial strains transiently changed the composition of the rhizosphere fungal microflora. However, in both 1997 and 1998, GMM-induced effects were distinct from those of WCS358r and lasted for 40 days in 1997 and for 89 days after sowing in 1998, whereas effects induced by WCS358r were detectable for 12 (1997) or 40 (1998) days. None of the strains affected the metabolic activity of the soil microbial population (substrate-induced respiration), soil nitrification potential, cellulose decomposition, plant height, or plant yield. The results indicate that application of GMMs engineered to have improved antifungal activity can exert nontarget effects on the natural fungal microflora.     

Nitrogen dynamics of crop and soil subjected to different water and nitrogen inputs, including daily irrigation and steady-state fertilization-measurements and modelling

Major carbon and nitrogen fluxes through crop and soil were studied in a series of field experiments. Barley, winter wheat, a grass mixture cut for hay and the energy crop reed canary-grass (Phalaris arundinacea) were studied.The treatments ranged from drought to daily irrigation/fertilization with high doses of water and nitrogen. Crop biomass and nitrogen dynamics above and below ground and incident light as well as soil temperature, moisture and mineral N content were monitored. Litter decomposition experiments were also performed in the field.The results were used to parameterize, validate and improve a set of soil/plant simulation models. Selected experimental results and experiences gained from the water, C and N budgeting and modelling work are presented.     

冬小麦叶片气孔导度模型水分响应函数的参数化

植物气孔导度模型的水分响应函数用来模拟水分胁迫对气孔导度的影响过程, 是模拟缺水环境下植物与大气间水、碳交换过程的关键算法。水分响应函数包括空气湿度响应函数和土壤湿度(或植物水势)响应函数, 该研究基于田间实验观测, 分析了冬小麦(Triticum aestivum)叶片气孔导度对不同空气饱和差和不同土壤体积含水量或叶水势的响应规律。一个土壤水分梯度的田间处理在中国科学院禹城综合试验站实施, 不同水分胁迫下的冬小麦叶片气体交换过程和气孔导度以及其他的温湿度数据被观测, 同时观测了土壤含水量和叶水势。实验数据表明, 冬小麦叶片气孔导度对空气饱和差的响应呈现双曲线规律, 变化趋势显示大约1 kPa空气饱和差是一个有用的阈值, 在小于1 kPa时, 冬小麦气孔导度对空气饱和差变化反应敏感, 而大于1 kPa后则反应缓慢; 分析土壤体积含水量与中午叶片气孔导度的关系发现, 中午叶片气孔导度随土壤含水量增加大致呈现线性增加趋势, 但在平均土壤体积含水量大于大约25%以后, 气孔导度不再明显增加, 而是维持在较高导度值上下波动; 冬小麦中午叶片水势与相应的气孔导度之间, 随着叶水势的增加, 气孔导度呈现增加趋势。根据冬小麦气孔导度对空气湿度、土壤湿度和叶水势的响应规律, 研究分别采用双曲线和幂指数形式拟合了水汽响应函数, 用三段线性方程拟合了土壤湿度响应函数和植物水势响应函数, 得到的参数可以为模型模拟冬小麦的各类水、热、碳交换过程采用。     

Analysis of Expression of a Phenazine Biosynthesis Locus of Pseudomonas aureofaciens PGS12 on Seeds with a Mutant Carrying a Phenazine Biosynthesis Locus-Ice Nucleation Reporter Gene Fusion

A derivative of Pseudomonas aureofaciens PGS12 expressing a promoterless ice nucleation gene under the control of a phenazine biosynthesis locus was used to study the expression of a phenazine antibiotic locus (Phz) during bacterial seed colonization. Seeds of various plants were inoculated with wild-type PGS12 and a PGS12 ice nucleation-active phz:inaZ marker exchange derivative and planted in soil, and the expression of the reporter gene was monitored at different intervals for 48 h during seed germination. phz gene expression was first detected 12 h after planting, and the expression increased during the next 36-h period. Significant differences in expression of bacterial populations on different seeds were measured at 48 h. The highest expression level was recorded for wheat seeds (one ice nucleus per 4,000 cells), and the lowest expression level was recorded for cotton seeds (one ice nucleus per 12,000,000 cells). These values indicate that a small proportion of bacteria in a seed population expressed phenazine biosynthesis. Reporter gene expression levels and populations on individual seeds in a sample were lognormally distributed. There was greater variability in reporter gene expression than in population size among individual seeds in a sample. Expression on sugar beet and radish seeds was not affected by different inoculum levels or soil matric potentials of -10 and -40 J/kg; only small differences in expression on wheat and sugar beet seeds were detected when the seeds were planted in various soils. It is suggested that the nutrient level in seed exudates is the primary reason for the differences observed among seeds. The lognormal distribution of phenazine expression on seeds and the timing and difference in expression of phenazine biosynthesis on seeds have implications for the potential efficacy of biocontrol microorganisms against plant pathogens.     

Fate and behaviour of a seed-applied Pseudomonas brassicacearum strain in a winter wheat field trial,as determined by analysis with SCAR markers

The fate and behaviour of the seed-applied biocontrol strain Pseudomonas brassicacearum MA250 in a field trial with winter wheat was determined using sequence-characterised amplified region (SCAR) markers. Samples of belowground plant parts from healthy and withered (due to snow mould infection) seedlings were collected approximately one and seven months after sowing, which was performed in early autumn. DNA was extracted from roots and remaining parts of seeds with adhering soil, and the abundance of the strain was determined in quantitative real-time PCR (qPCR) assays. The results show that the introduced strain persisted over the whole trial-period of seven months. On termination of the trial (after seven months) the belowground plant parts of each plant housed 10⁶–10⁷ cells, substantially less than the original approximately 10⁹ cells inoculated onto the seed. In healthy seedlings, there was a shift in cell numbers from seeds to roots between the samplings, suggesting colonisation of the roots during this time. The results show that with sufficient attention given to analytical control measures and the possibility of resident background populations, SCAR markers in combination with qPCR provide valuable information regarding the fate and behaviour of biocontrol micro-organisms under field conditions.     

The influence of season and stage of development of plant on Endogone mycorrhiza of field-grown wheat.

A quantitative and qualitative survey of the indigenous Endogone population in wheat field soil and vesicular-arbuscular mycorrhiza in wheat roots as influenced by season and by the stage of development of the wheat plant was made. The number of Endogone spores in wheat field soil remained relatively unchanged in winter until January during the period of maximum root growth. The extent of formation of arbuscules was influenced by the stage of development of the wheat plants.     

A COMPARATIVE STUDY OF THE GROWTH OF WILD OATS (AVENA FATUA L. AND A. LUDOVICIANA DUR.) AND OF CULTIVATED CEREALS WITH VARIED NITROGEN SUPPLY

Growth analysis of wild oats ( Avena fatua and A. ludoviciana ) grown in pots with different levels of nitrogen supply showed many similarities to spring barley, winter oats and winter wheat.
Small differences that could affect competition between wild oats and cereals occurred mainly in the seedlings. Wild oat seedlings were smaller than the corresponding cultivated cereals in total dry weight, total nitrogen content, leaf area and number of shoots. However, very young wild oat plants had higher net assimilation rates than the cultivated cereals and soon caught up and passed them. The difference in net assimilation rate did not persist, and in the later stages of growth differences in dry-matter production depended mainly on differences in leaf area. Another important difference between wild oats and cultivated cereals was that 98–100% of the wild oat seeds and none of the crop seeds were dormant 2 months after harvest.
Ear emergence in wild oats spread over a longer period, the range of ear heights was greater and the tallest ears were taller than in the corresponding cultivated cereals. Assimilation in the ear appeared to account for less of the total dry matter of the plants of wild and cultivated oats than of wheat. The wild oats produced more seeds per plant than the cultivated cereals, but the 1000-grain weight, and hence the total dry weight of seeds, was lower in the weeds than in the crop.
Addition of nitrogen to the soil affected the growth of the wild oats in the same ways as the cultivated cereals; they took up the same amount of nitrogen per plant as winter oats and winter wheat but more than spring barley.
It is concluded that wild oats are most susceptible in the seedling stage to competition from the crop and that nitrogenous fertilizer applied to an infested field is unlikely to alter the balance between the yields of crop and of wild oats.     

Growth enhancement and antitranspirant activity following seed treatment with a derivative of 5-hydroxybenzimidazole (Ambiol) in four drought-stressed agricultural species

The biostimulating action of seed treatment with the synthetic antioxidant, Ambiol (2-methyl-4-Edimethylaminomethyl-5-hydroxybenzimidazole dihydrochloride) on subsequent growth and transpiration of seedlings was studied. To study growth and transpiration responses, seeds of four agricultural species, soybean ( Glycine max L.), rapeseed ( Brassica napus L.), winter wheat ( Trilicum aestivum L.) and corn ( Zea mays L.), were soaked in Ambiol for 24 h, using the following concentrations: 0, 0.01, 0.1, 1, 10 and 100 mg 1¹. The subsequent seedlings were subject to simulated soil drought, using computer-automated root misting chambers. The influence of Ambiot on transpiration rate under simulated air drought was studied by growing plants under low humidity in a controlled humidity chamber. Response to Ambiol varied, depending on its concentration, the species used and the environment. Compared to untreated plants, 10 mg 1-⁻¹ Ambiol reduced the mid-day transpiration rate and total daily water usage of soybean by approximately 25%. Under simulated soil drought in the root misting chamber, 10 and 100 mg 1-⁻¹ Ambiol increased growth of rapeseed and soybean by 25–45%, relative to the 0 mg 1-⁻¹ treatment, yielding plants comparable in size to the fully-irrigated controls. However, Ambiol failed to promote growth of two drought-stressed monocotyledons (corn and winter wheat). At 100 mg P. Ambiol inhibited growth of both well-watered wheat and rapeseed, although this inhibition was mitigated by drought.     

Short photoperiod induces dormancy in Lotus (Nelumbo nucifera)

BACKGROUND AND AIMS: Lotus (Nelumbo nucifera) has been cultivated as an ornamental and food plant in Japan for more than 1000 years. As large areas are required for its cultivation (approximately 2 m2 per plant), physiological research, such as into the effect of environmental factors on dormancy, has not been well studied until recently. In this paper, seedlings were used to examine environmental factors affecting dormancy induction. METHODS: In a first experiment, seeds were sown from 6 April to 6 October at 2-month intervals, and cultivated for 2 months in an unheated greenhouse. In a second experiment, seeds were prepared for germination on 16 November and 16 May and the seedlings were grown at 25 or 30 degrees C under natural daylength in phytotron growth rooms. After 1 month, the seedlings were cultivated at 20, 25 or 30 degrees C for a further month. The number of leaves and rhizome branches on the main stem were counted, and growth of rhizomes on the main stem was calculated using a rhizome enlargement index (= maximum internode diameter/internode length) after 2 months of culture in both experiments. KEY RESULTS: Rhizomes elongated without enlargement when the seeds were sown in April and June. Sowing the seeds in August and October resulted in rhizome enlargement from the tenth and fifth internodes, respectively. Rhizomes enlarged in the November-sowing but elongated in the May-sowing irrespective of temperature treatments under natural daylength in the phytotron rooms. The seedlings cultivated from May at 25-30 degrees C for 2 months had more leaves, and more rhizome branches and nodes than those cultivated from November. CONCLUSIONS: Short days led to induced dormancy in lotus.     

Effect of Genetically Modified Pseudomonas putida WCS358r on the Fungal Rhizosphere Microflora of Field-Grown Wheat

We released genetically modified Pseudomonas putida WCS358r into the rhizospheres of wheat plants. The two genetically modified derivatives, genetically modified microorganism (GMM) 2 and GMM 8, carried the phz biosynthetic gene locus of strain P. fluorescens 2-79 and constitutively produced the antifungal compound phenazine-1-carboxylic acid (PCA). In the springs of 1997 and 1998 we sowed wheat seeds treated with either GMM 2, GMM 8, or WCS358r (approximately 10⁷ CFU per seed), and measured the numbers, composition, and activities of the rhizosphere microbial populations. During both growing seasons, all three bacterial strains decreased from 10⁷ CFU per g of rhizosphere sample to below the limit of detection (10² CFU per g) 1 month after harvest of the wheat plants. The phz genes were stably maintained, and PCA was detected in rhizosphere extracts of GMM-treated plants. In 1997, but not in 1998, fungal numbers in the rhizosphere, quantified on 2% malt extract agar (total filamentous fungi) and on Komada's medium (mainly Fusarium spp.), were transiently suppressed in GMM 8-treated plants. We also analyzed the effects of the GMMs on the rhizosphere fungi by using amplified ribosomal DNA restriction analysis. Introduction of any of the three bacterial strains transiently changed the composition of the rhizosphere fungal microflora. However, in both 1997 and 1998, GMM-induced effects were distinct from those of WCS358r and lasted for 40 days in 1997 and for 89 days after sowing in 1998, whereas effects induced by WCS358r were detectable for 12 (1997) or 40 (1998) days. None of the strains affected the metabolic activity of the soil microbial population (substrate-induced respiration), soil nitrification potential, cellulose decomposition, plant height, or plant yield. The results indicate that application of GMMs engineered to have improved antifungal activity can exert nontarget effects on the natural fungal microflora.