Frequency, size, and localization of bacterial aggregates on bean leaf surfaces

Using epifluorescence microscopy and image analysis, we have quantitatively described the frequency, size, and spatial distribution of bacterial aggregates on leaf surfaces of greenhouse-grown bean plants inoculated with the plant-pathogenic bacterium Pseudomonas syringae pv. syringae strain B728a. Bacterial cells were not randomly distributed on the leaf surface but occurred in a wide range of cluster sizes, ranging from single cells to over 10(4) cells per aggregate. The average cluster size increased through time, and aggregates were more numerous and larger when plants were maintained under conditions of high relative humidity levels than under dry conditions. The large majority of aggregates observed were small (less than 100 cells), and aggregate sizes exhibited a strong right-hand-skewed frequency distribution. While large aggregates are not frequent on a given leaf, they often accounted for the majority of cells present. We observed that up to 50% of cells present on a leaf were located in aggregates containing 10(3) cells or more. Aggregates were associated with several different anatomical features of the leaf surface but not with stomates. Aggregates were preferentially associated with glandular trichomes and veins. The biological and ecological significance of aggregate formation by epiphytic bacteria is discussed.     

Effect of the Surfactant Tween 80 on the Detachment and Dispersal of Salmonella enterica Serovar Thompson Single Cells and Aggregates from Cilantro Leaves as Revealed by Image Analysis

Salmonella enterica has the ability to form biofilms and large aggregates on produce surfaces, including on cilantro leaves. Aggregates of S. enterica serovar Thompson that remained attached to cilantro leaves after rigorous washing and that were present free or bound to dislodged leaf tissue in the wash suspension were observed by confocal microscopy. Measurement of S. Thompson population sizes in the leaf washes by plate counts failed to show an effect of 0.05% Tween 80 on the removal of the pathogen from cilantro leaves 2 and 6 days after inoculation. On the contrary, digital image analysis of micrographs of single cells and aggregates of green fluorescent protein (GFP)-S. Thompson present in cilantro leaf washes revealed that single cells represented 13.7% of the cell assemblages in leaf washes containing Tween 80, versus 9.3% in those without the surfactant. Moreover, Tween 80 decreased the percentage of the total S. Thompson cell population located in aggregates equal to or larger than 64 cells from 9.8% to 4.4% (P < 0.05). Regression analysis of the frequency distribution of aggregate size in leaf washes with and without Tween 80 showed that the surfactant promoted the dispersal of cells from large aggregates into smaller ones and into single cells (P < 0.05). Our study underlines the importance of investigating bacterial behavior at the scale of single cells in order to uncover trends undetectable at the population level by bacterial plate counts. Such an approach may provide valuable information to devise strategies aimed at enhancing the efficacy of produce sanitization treatments.     

Changing cell aggregations and lignification in tobacco suspension cultures

The formation and dissociation of cell aggregates in tobaccocell suspensions were analyzed during 2,4-dichlorophenoxyaceticacid (2,4-D) (10^–6 M) stock culture and during 6-benzyladenine(BA) (10^–6 M) culture, for their effects on lignification. Aggregates were fractionated according to size by sieving them.Each fraction was cultured in both media and changes in thedistribution of cells in the cluster fractions were followedduring culture, after which the formation and dissociation ratesof cells in aggregates were estimated. There was a significant difference in the formation and dissociationof cell aggregates between the 2,4-D culture and the BA culture.In the 2,4-D culture, aggregates dissociated to smaller onesor to single cells in the early growth stage, and then grewinto large aggregates. Moreover, most cells had a uniform capacityfor aggregate formation. In the BA culture, aggregates grewrapidly to a large size in the early growth stage. Lignification and tracheary element formation in the BA culturewere especially stimulated in large aggregates derived fromthe large aggregates of the 2,4-D culture. Results of successivecultures of a particular cell aggregate fraction indicated thatmost cells had the potential to lignify or to differentiatetracheary elements. Thus, the switch-on of lignification ortracheid formation in the BA culture was determined only bycells in aggregate. (Received October 22, 1977; )     

Aggregate cell suspension cultures of Tripterygium wilfordii Hook. f. for triptolide, wilforgine, and wilforine production

The relationships between aggregate cell types, cell growth, and the triptolide, wilforgine, and wilforine content in aggregate cell suspension cultures of Tripterygium wilfordii Hook. f. were examined. Aggregate cells larger than 2?mm grew quickly and constituted the majority of the white aggregates. The accumulation of triptolide was strongly correlated with the size of the aggregates and the length of the culture period. The aggregates 0.5?C2?mm in diameter accumulated higher triptolide content than those with other sizes throughout the culture. However, the size of the aggregate cells did not significantly affect on the wilforgine and wilforine content. Two other kinds of aggregate cells, the brown and green aggregate cells, also formed in the suspension cultures. The smallest aggregates (0.1?C0.5?mm) had a lower biomass and growth rate and had more chloroplasts and higher alkaloid content. The results of this study can be used to improve the selection process for the mass production of triptolide, wilforgine, and wilforine from cell suspension cultures.     

Succession of Protists on Estuarine Aggregates

Abstract Colonization by and succession of bacteria and bacterivorous protists on laboratory-made aggregates were determined over a period of 14 days during winter and spring in 1997. Aggregates were generated from natural water from the limnetic zone of the Elbe Estuary using a tilting tube roller system. Within 1 h after the beginning of the experiments, macroaggregates started to form. Aggregates reached a maximum size of 1 mm with a tendency toward large sizes at the end of the experiment after the 10th day. On the first day, high bacterial densities of more than 10⁹ cells ml⁻¹ were detected within the aggregates. The abundances of flagellates and ciliates within aggregates were also two or three orders of magnitude higher than in the surrounding water. Densities of aggregate associated organisms are comparable to those occuring in sediments. The first protistan colonizers on the aggregates were small heterotrophic flagellates, such as choanoflagellates and small euglenids. Later, beginning on the 4th day, small sarcodines and ciliates became abundant. The most abundant ciliates associated with aggregates were small species of the Hypotrichia, Cyrtophorida, and Hymenostomata. After 9 days, large omnivorous and carnivorous ciliates, such as large members of the Hypotrichia and the Pleurostomatida, occurred. In spring, large heterotrophic flagellates and amebae also appeared at this time. These findings indicated the existence of a succession of protists on newly formed aggregates and a microbial food net within the aggregates based on bacterial production. Additionally, most of the species observed during this study were adapted for living on surfaces. In natural environments they are more common in benthic than in pelagic environments. For them, aggregates are havens in the water column comparable to sediment communities. Received: 7 January 2000; Accepted: 15 May 2000; Online Publication: 28 August 2000     

Influence of leaf size,orientation, and arrangement on temperature and transpiration in three high-elevation,large-leafed herbs

Summary The temperature and water relations of the largleafed, high-elevation species Frasera speciosa, Balsamorhiza sagittata, and Rumex densiflorus were evaluated in the Medicine Bow Mountains of southeast Wyoming (USA) to determine the influence of leaf size, orientation, and arrangement on transpiration. These species characteristically have low minimum stomatal resistances (<60 s m^-1) and high maximum transpiration rates (>260 mg m^-2s^-1 for F. speciosa). Field measurements of leaf and microclimatic parameters were incorporated into a computer simulation using standard energy balance equations which predicted leaf temperature (T _leaf) and transpiration for various leaf sizes. Whole-plant transpiration during a day was simulated using field measurements for plants with natural leaf sizes and compared to transpiration rates simulated for plants having identical, but hypothetically smaller (0.5 cm) leaves during a clear day and a typically cloudy day. Although clear-day transpiration for F. speciosa plants with natural size leaves was only 2.0% less per unit leaf area than that predicted for plants with much smaller leaves, daily transpiration of B. sagittata and R. densiflorus plants with natural leaf sizes was 16.1% and 21.1% less, respectively. The predicted influence of a larger leaf size on transpiration for the cloudy day was similar to clear-day results except that F. speciosa had much greater decreases in transpiration (12.7%). The different influences of leaf size on transpiration between the three species was primarily due to major differences in leaf absorptance to solar radiation, orientation, and arrangement which caused large differences in T _leaf. Also, simulated increases in leaf size above natural sizes measured in the field resulted in only small additional decreases in predicted transpiration, indicating a leaf size that was nearly optimal for reducing transpiration. These results are discussed in terms of the possible evolution of a larger leaf size in combination with specific leaf absorptances, orientations and arrangements which could act to reduce transpiration for species growing in short-season habitats where the requirement for rapid carbon fixation might necessitate low stomatal resistances.     

Emergence of seedlings of sugar beet (Beta vulgaris L.) as affected by the size,roughness and position of aggregates in the seedbed

Laboratory and field experiments were carried out to quantify the effects of the size and roughness of aggregates placed in the seedling path of sugar beet, in order to help in decision making for soil tillage and sowing operations. Graded aggregates (10, 15, 20, 30, 40, 50, 60 and 70 mm longest axis) were either laid on the soil surface or included in the soil over the seeds. The percent emergence decreased exponentially with aggregate size over 10 mm when the aggregates were included in the seedbed. The result was the same with aggregates laid on the soil surface, but for aggregates over 30 mm (mass>10 g). Aggregates on the soil surface could be lifted by the seedlings until their weight exceeded the seedling emergence force. Larger aggregates or aggregates in the soil layer could not be moved. Seedlings which did not emerge remained blocked in small cavities in the aggregate surface. No seedlings were blocked under smooth aggregates or glass beads. The experimental results fits with a model giving the probability to meet a hole according the distance covered and the diameter and density of holes. The results obtained under controlled conditions were similar to those obtained in field experiments for a wide range of aggregate sizes. These results will be incorporated into a computerised seedbed generator to simulate the effects of seedbed structure on seedling emergence.     

Effect of soil aggregate size on methanogenesis and archaeal community structure in anoxic rice field soil

In anoxically incubated slurries of Italian rice field soil, CH(4) production is initiated after a lag phase during which ferric iron and sulfate are reduced. The production of CH(4) was affected by the size of soil aggregates used for the preparation of the soil slurry. Rates of CH(4) production were lowest with small aggregates (<50 and 50-100 μm), were highest with aggregates of 200-2000 μm size and were intermediate with aggregates of 2000-15000 μm size. The different amounts of CH(4) accumulated were positively correlated to the concentrations of acetate, propionate and caproate that transiently accumulated in the slurries prepared from different aggregate sizes and also to the organic carbon content. The addition of organic debris that was collected from large-size aggregates to the aggregate size fractions <200 and <50 μm resulted in an increase of CH(4) production to amounts that were comparable to those measured in unamended aggregates of 200-2000 μm size, indicating that CH(4) production in the different aggregate size fractions was limited by substrate. The distribution of archaeal small-subunit rRNA genes in the different soil aggregate fractions was analyzed by terminal restriction fragment length polymorphism which allowed seven different archaeal ribotypes to be distinguished. Ribotype-182 (consisting of members of the Methanosarcinaceae and rice cluster VI), ribotype-389 (rice cluster I and II) and ribotype-820 (undigested DNA, rice cluster IV and members of the Methanosarcinaceae) accounted for >20, >30 and >10% of the total, respectively. The other ribotypes accounted for <10% of the total. The relative quantity of the individual ribotypes changed only slightly with incubation time and was almost the same among the different soil aggregate fractions. Ribotype-389, for example, slightly decreased with time, whereas ribotype-182 slightly increased. At the end of incubation, the relative quantity of ribotype-182 seemed to be slightly higher in soil fractions with larger than with smaller aggregates, whereas it was the opposite with ribotype-80 (Methanomicrobiaceae) and ribotype-88 (Methanobacteriaceae). Ribotype-280 (Methanosaetaceae and rice cluster V), ribotype-375 (rice cluster III), ribotype-389 and ribotype-820, on the other hand, were not much different among the different soil aggregate size fractions. However, the differences were not significant relative to the errors encountered during the extraction of polymerase chain reaction (PCR)-amplifiable DNA from soil. In conclusion, soil aggregate size and incubation time showed a strong effect on the function but only a small effect on the structure of the methanogenic microbial community.     

B. thuringiensis is a Poor Colonist of Leaf Surfaces

The ability of several Bacillus thuringiensis strains to colonize plant surfaces was assessed and compared with that of more common epiphytic bacteria. While all B. thuringiensis strains multiplied to some extent after inoculation on bean plants, their maximum epiphytic population sizes of 10⁶ cfu/g of leaf were always much less than that achieved by other resident epiphytic bacteria or an epiphytically fit Pseudomonas fluorescens strain, which attained population sizes of about 10⁷ cfu/g of leaf. However B. thuringiensis strains exhibited much less decline in culturable populations upon imposition of desiccation stress than did other resident bacteria or an inoculated P. fluorescens strain, and most cells were in a spore form soon after inoculation onto plants. B. thuringiensis strains produced commercially for insect control were not less epiphytically fit than strains recently isolated from leaf surfaces. The growth of B. thuringiensis was not affected by the presence of Pseudomonas syringae when co-inoculated, and vice versa. B. thuringiensis strains harboring a green fluorescent protein marker gene did not form large cell aggregates, were not associated with other epiphytic bacteria, and were not found associated with leaf structures, such as stomata, trichomes, or veins when directly observed on bean leaves by epifluorescent microscopy. Thus, B. thuringiensis appears unable to grow extensively on leaves and its common isolation from plants may reflect immigration from more abundant reservoirs elsewhere.     

Detrital aggregates in some Iowa lakes and reservoirs

Detrital aggregates in three eutrophic Iowa lakes and four eutrophic Iowa reservoirs were studied with light and scanning electron microscopy to determine if aggregate morphologies and concentrations were similar. Lake and reservoir aggregates were composed of organic and inorganic particles bound together in an organix matrix. Many of the inorganic particles were calcium carbonate. Obvious bacterial and fungal attachment to the aggregates was rare. Aggregate concentrations ranged from 4 to 274 million aggregates per liter. Aggregates smaller than 18 μm diameter dominated the hyperbolic size-frequency distribution of aggregates in all lakes. Reservoir and lake aggregate concentrations did not differ significantly, but mean aggregate concentrations were directly correlated to the mean chlorophyll a concentration of the lakes. These data strongly suggest that detrital aggregate concentrations are influenced by the trophic status of a lake. Journal Paper No. J-8705 of the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa. Project 2051. Journal Paper No. J-8705 of the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa. Project 2051.     

Caffeic acid-O-methyltransferase in a suspension of cell aggregates of tobacco

Aggregates of tobacco cells in suspension in 2,4-D (10^?6 M) and kinetin (10^?5 M) cultures were fractionated by size, then their O-methyltransferase (OMT) activities were assayed. Only the kinetin culture showed high OMT activity, which was higher in the larger than the smaller aggregates at all stages of cell growth. The contents of phenolic acids were also greater in the larger cell aggregates in the kinetin culture. However, when the kinetin cultured cells were transferred to a medium containing 10^?6 M of 2,4-D, the relationships between the cell size of the aggregates and OMT, lignin and the phenolic acids disappeared. The importance of kinetin and cell association for OMT and the subsequent lignification of the cells is discussed.     

Application of imaging tools to plant cell culture: Relationship between plant cell aggregation and flavonoid production

Summary Image analysis tools were developed to measure biomass concentration, aggregate size and distribution, and pigmentation from anthocyanin-producing cell suspension cultures of ohelo (Vaccinium pahalae). The ex situ imaging system could image cell aggregates from 30 μm to 2 mm in diameter. The image analysis algorithm was based on extracted geometric features and morphological methods for biomass volume estimates, and hue, saturation, and intensity color characteristics for pigmentation estimates. Detailed information available from sampled cell culture images was validated by comparison to standard destructive manual measurements. Image analysis measurements revealed that pigment accumulation was negatively correlated with aggregate size. Although a substantial proportion of small aggregates remained colorless, the highly-pigmented small aggregates, 18 to 238 μm in breadth, contributed over 70% of the culture anthocyanin production (mg L⁻¹), despite their minor contribution to the overall biomass. The relative frequency of pigmented aggregates was higher in large-size aggregate classes; however, the pigmented sectors were mostly confined to only the periphery of the aggregates. As a result, large aggregate classes had only a minor contribution to overall culture anthocyanin yield.     

Cultivation of mammalian cells as aggregates in bioreactors: effect of calcium concentration of spatial distribution of viability

Recombinant human kidney epithelial 293 cells were cultivated as aggregates in suspension. The concentration calcium ion, in the range of 100 muM to 1mM, affected the rate of aggregate formation. During the course of cultivation the size distribution of aggregates shifted and the fraction of larger aggregates increased. This effect was more profound in cultures with a high calcium concentration. Scanning and transmission microscopic examination of the aggregates revealed that cell packing was greater in the high calcium cultures and that ultrastructural integrity was retained in aggregates from both low and high calcium cultures. Confocal microscopy was applied to examine the viability of cells in the interior of the aggregates. High viability was observed in the aggregates obtained from exponentially growing cultures. Aggregates from the high calcium culture in the stationary phase exhibited lower viability in the interior. With its ease of retention in a perfusion bioreactor, aggregate cultures offer an alternative choice for large-scale operation. (c) 1993 John Wiley & Sons, Inc.     

Adhesive specificity in normal and transformed mouse fibroblasts

Adhesive specificity was studied in normal and transformed $\text{Balb}\text{c}$ mouse fibroblasts by comparing the number of labeled cells collected from a suspension of these cells by aggregates of various cell types. Aggregates of the two malignant cells examined collected either very many cells (aggregates of SV3T3 cells) or very few cells (aggregates of 3T12 cells). In addition, the relative adhesive behavior of these two aggregate types did not vary according to the cell suspension in which they were circulated. These data make it unnecessary to assume that malignancy is always accompanied by a decrease in intercellular adhesion.The adhesive behavior of normal 3T3 cell aggregates, compared to the aggregates composed of either malignant cell type, varied according to the type of cells in the suspension. Aggregates of 3T3 cells collected an appreciable number of SV3T3 cells but few 3T12 cells. Collection of 3T3 cells by 3T3 aggregates was also low if the 3T3 cells of the suspension were harvested from confluent cultures. However, collection of 3T3 cells by 3T3 aggregates increased significantly, as compared to collection by SV3T3 and 3T12 aggregates in the same cell suspension, if the 3T3 suspension was prepared from sparse cultures.Flat-revertants of SV3T3 cells were also studied. These cells behave like nonmalignant 3T3 cells rather than like the SV3T3 cells from which they were derived.We suggest that malignancy may not be caused by decreased intercellular adhesion as compared to normal cells but, perhaps, by decreased intercellular recognition.     

宁夏典型天然草地土壤团聚体稳定性及其有机碳分布特征

土壤团聚体作为土壤结构的基本单元和土壤有机碳存在的重要场所,对维系土壤质量和生态环境可持续发展具有重要作用。为探究宁夏天然草地土壤团聚体稳定性及其有机碳分布特征,以宁夏4种典型的天然草地（草甸草原、温性草原、草原化荒漠和荒漠草原）为研究对象,系统开展研究。结果表明：草甸草原、温性草原和草原化荒漠各粒级机械团聚体和水稳性团聚体含量均呈现随粒径减小而先减小后增大趋势,荒漠草原机械团聚体含量呈现随粒径减小而先减小后增大的趋势,水稳性团聚体含量呈现随粒径减小逐渐增大的趋势;且草甸草原和温性草原机械稳定性和水稳性团聚体在0-10 cm、10-20 cm和20-40 cm 3个土层均以>0.25 mm大团聚体为主,草原化荒漠和荒漠草原水稳性团聚体均以<0.25 mm的微团聚体为主。4种草地类型机械团聚体和水稳性团聚体MWD和GMD,以及各粒级土壤团聚体有机碳含量均表现为草甸草原和温性草原显著高于草原化荒漠和荒漠草原。草甸草原和温性草原在3个土层深度均以>0.25 mm的大团聚体对有机碳的贡献率最高,分别达到43.86%、59.26%、58.89%和58.02%、54.03%、57.15%;草原化荒漠和荒漠草原在3个土层深度,均以<0.25 mm的微团聚体贡献率高,分别达到了60.37%、55.86%、54.33%和75.61%、78.34%、78.74%。结果表明：草甸草原和温性草原较草原化荒漠和荒漠草原土壤团聚体稳定性更高,更有利于土壤有机碳累积。     

In vitro regeneration patterns of Platycerium bifurcatum leaf cell suspension culture

A simple suspension culture system of Platycerium bifurcatum was developed where sporophytes could be regenerated directly from leaf cells or indirectly through an aposporous gametophyte stage under the same culture conditions. Single cells and aggregates of up to 100 cells developed aposporous gametophytes which later gave rise to sporophytes. Such gametophytes started apogamy when they were mostly less than 0.7 mm in length, bearing only rhizoids. In most cases, only one sporophyte was regenerated from one gametophyte. Aggregates of 500–1000 or more cells, on the other hand, regenerated sporophytes directly. Intercellular interaction was considered to be the physiological cause, and the separation of leaf cells to a certain degree drove the cells to embark on different regeneration paths. It is suggested that the possible existence of a threshold size of cell aggregates separates the two regeneration patterns. Received: 3 March 1997 / Revision received: 11 April 1997 / Accepted: 3 June 1997     

Changes in animal cell natural aggregates in suspended batch cultures

Some anchorage-dependent animal cells can form natural aggregates in stirred tanks. Baby hamster kidney (BHK) natural aggregates are described and characterized. Total cell concentration and viability could be obtained after aggregate mechanical aissociation, with negligible cell lysis and no change in cell membrane permeability. During a normal batch run, aggregates were formed immediately after inoculation, a few spherical aggregates increasing size during the initial growth phase. At the end of the growth phase, an increase in aggregate concentration was observed, without a considerable increase in aggregate diameter. At the end of the batch run, 160 h after inoculation, aggregates disintegrated into smaller, non-spherical units, following a sharp viability decrease. Cell concentrations of 1. 2 · 10⁶ cells/ml were obtained, with 60% of the total cells being in aggregates; the cell concentration in aggregates achieved 5 · 10⁸ cells/ml, with a porosity of 55%. Viability was consistently in the range 85–90%, both for aggregate and suspended cells.     

Nonnative protein polymers: structure, morphology, and relation to nucleation and growth

Thermally induced aggregates of α-chymotrypsinogen A and bovine granulocyte-colony stimulating factor in acidic solutions were characterized by a combination of static and dynamic light scattering, spectroscopy, transmission electron microscopy, and monomer loss kinetics. The resulting soluble, high-molecular weight aggregates (∼10³-10⁵ kDa) are linear, semiflexible polymer chains that do not appreciably associate with one another under the conditions at which they were formed, with classic power-law scaling of the radius of gyration and hydrodynamic radius with weight-average molecular weight (M_w). Aggregates in both systems are composed of nonnative monomers with elevated levels of β-sheet secondary structure, and bind thioflavine T. In general, the aggregate size distributions showed low polydispersity by light scattering. Together with the inverse scaling of M_w with protein concentration, the results clearly indicate that aggregation proceeds via nucleated (chain) polymerization. For α-chymotrypsinogen A, the scaling behavior is combined with the kinetics of aggregation to deduce separate values for the characteristic timescales for nucleation (τ_n) and growth (τ_g), as well as the stoichiometry of the nucleus (x). The analysis illustrates a general procedure to noninvasively and quantitatively determine τ_n, τ_g, and x for soluble (chain polymer) aggregates, as well as the relationship between τ_n/τ_g and aggregate M_w.     

Botulinum hemagglutinin‐mediated in situ break‐up of human induced pluripotent stem cell aggregates for high‐density suspension culture

Large numbers of human induced pluripotent stem cells (hiPSCs) are required for making stable cell bank. Although suspension culture yields high cell numbers, there remain unresolved challenges for obtaining high‐density of hiPSCs because large size aggregates exhibit low growth rates. Here, we established a simple method for hiPSC aggregate break‐up using botulinum hemagglutinin (HA), which specifically bound with E‐cadherin and disrupted cell–cell connections in hiPSC aggregates. HA showed temporary activity for disrupting the E‐cadherin‐mediated cell–cell connections to facilitate the break‐up of aggregates into small sizes only 9 hr after HA addition. The transportation of HA into the aggregates was mediated by transcellular and paracellular way after HA addition to the culture medium. hiPSC aggregates broken up by HA showed a higher number of live cells, higher cell density, and higher expansion fold compared to those of aggregates dissociated with enzymatic digestion. Moreover, a maximum cell density of 4.5 ± 0.2 × 10⁶ cells ml^?1 was obtained by aggregate break‐up into small ones, which was three times higher than that with the conventional culture without aggregate break‐up. Therefore, the temporary activity of HA for disrupting E‐cadherin‐mediated cell–cell connection was key to establishing a simple in situ method for hiPSC aggregate break‐up in bioreactors, leading to high cell density in suspension culture.