Bacterial differentiation within Moraxella bovis colonies growing at the interface of the agar medium with the Petri dish.

Moraxella bovis was found to colonize the interface between agar and the polystyrene Petri dish, producing circular colonies when the inoculum was stabbed at a single point. The bacteria occurred in a thin layer of nearly uniform thickness, and colonial expansion occurred in at least two temporal phases. In the first phase, the radial colonial expansion was slow and non-linear. In the second phase, the radial expansion was linear. The interfacial colonies possessed three characteristic concentric growth zones. At the periphery was a narrow ring zone that enclosed another wider ring zone, which, in turn, surrounded a central circular zone. Different bacterial phase variants were recovered from these zones. The two outer ring zones yielded bacteria that formed agar surface colonies of spreading-corroding morphology, while cells from the innermost zone always yielded colonies with a different morphology. The uniform thickness of the colonies implied that replication was restricted to the outermost ring, and that the bacteria within the inner ring and inner circle had entered a quiescent state. The inner ring appeared to represent the lag in time needed for the replicative form to differentiate into the quiescent form. A different kind of variant was associated with wedge-shaped sectors within the colonies. The greatest number of these clonal variants appeared shortly after inoculation and their frequency decreased after the onset of linear growth. The period of slowest colonization coincided with highest frequency of clonal variant expression. It is proposed that the proliferative rate of the parental bacterial population exerted selective pressure on the expression of new clonal variants.     

Cell surface expansion in polarly growing root hairs of Medicago truncatula

Fluorescent microspheres were used as material markers to investigate the relative rates of cell surface expansion at the growing tips of Medicago truncatula root hairs. From the analysis of tip shape and microsphere movements, we propose three characteristic zones of expansion in growing root hairs. The center of the apical dome is an area of 1- to 2- microm diameter with relatively constant curvature and high growth rate. Distal to the apex is a more rapidly expanding region 1 to 2 microm in width exhibiting constant surges of off-axis growth. This middle region forms an annulus of maximum growth rate and is visible as an area of accentuated curvature in the tip profile. The remainder of the apical dome is characterized by strong radial expansion anisotropy where the meridional rate of expansion falls below the radial expansion rate. Data also suggest possible meridional contraction at the juncture between the apical dome and the cell body. The cell cylinder distal to the tip expands slightly over time, but only around the circumference. These data for surface expansion in the legume root hair provide new insight into the mechanism of tip growth and the morphogenesis of the root hair.     

Osmotic effects on radial growth rate and specific growth rate of three soil fungi

The radial growth rate on osmotically adjusted agar medium and the relative specific growth rate in osmotically adjusted liquid medium were determined for Rhizoctonia solani, Pythium ultimum, and Verticillium dahliae. On basal medium, an isolate of P. ultimum and R. solani had similar radial growth rates of 0.52 and 0.47 mm/h, respectively, whereas V. dahliae grew at a rate of 0.08 mm/h. Radial growth rate was reduced 50% at osmotic potentials of -16, -27, and -32 bars for P. ultimum, R. solani, and V. dahliae, respectively. No growth occurred at -32 bars for P. ultimum, -56.2 bars for R. solani, and -100 bars for V. dahlia. Specific growth rates in liquid culture were 0.011 h-1 for P. ultimum, 0.008 h-1 for V. dahliae, and 0.026 h-1 for R. solani. Ratios of radial growth rate (Kr) to specific growth rate (alphas) were computed for each fungus growing at different osmotic potentials. There was not a constant relationship between Kr on agar and alphas in liquid medium, e.g., Kr/alphas ratios varied from 8-41% from a mean ratio for a particular species. The results indicated that radial growth rate on osmotic agar was not useful as a measure of relative specific growth rate of a fungus in osmotically adjusted liquid medium.     

Effects of growth medium, inoculum size, and incubation time on culturability and isolation of soil bacteria

Soils are inhabited by many bacteria from phylogenetic groups that are poorly studied because representatives are rarely isolated in cultivation studies. Part of the reason for the failure to cultivate these bacteria is the low frequency with which bacterial cells in soil form visible colonies when inoculated onto standard microbiological media, resulting in low viable counts. We investigated the effects of three factors on viable counts, assessed as numbers of CFU on solid media, and on the phylogenetic groups to which the isolated colony-forming bacteria belong. These factors were inoculum size, growth medium, and incubation time. Decreasing the inoculum size resulted in significant increases in the viable count but did not appear to affect colony formation by members of rarely isolated groups. Some media that are traditionally used for soil microbiological studies returned low viable counts and did not result in the isolation of members of rarely isolated groups. Newly developed media, in contrast, resulted in high viable counts and in the isolation of many members of rarely isolated groups, regardless of the inoculum size. Increased incubation times of up to 3 months allowed the development of visible colonies of members of rarely isolated groups in conjunction with the use of appropriate media. Once isolated, pure cultures of members of rarely isolated groups took longer to form visible colonies than did members of commonly isolated groups. Using these new media and extended incubation times, we were able to isolate many members of the phyla Acidobacteria (subdivisions 1, 2, 3, and 4), Gemmatimonadetes, Chloroflexi, and Planctomycetes (including representatives of the previously uncultured WPS-1 lineage) as well as members of the subclasses Rubrobacteridae and Acidimicrobidae of the phylum Actinobacteria.     

Growth characteristics of the slow-growing actinobacterium Frankia sp. strain CcI3 on solid media

Frankia sp. strains are slow-growing Actinobacteria that form N₂-fixing root nodules on certain angiosperm trees and shrubs. These organisms are generally cultured in liquid media as routine growth on solid media is impractical because of lengthy incubation periods. Liquid media is problematic for filamentous organisms because unicellular spores are difficult to isolate, and mutants are difficult to separate. The goal of this study was to optimize Frankia CcI3 growth on solid media. Starting with an appropriate basal medium containing pyruvate as a carbon source, various peptones were individually tested to determine if the time needed for colonies to appear could be reduced. Bacto™ Proteose Peptone #3 at 1.6 mg ml⁻¹ final concentration promoted the best growth with colonies visible after 3 days, compared with the 7- to 10-day incubation required for control unamended media. Gellan gum gave a more rapid growth response as compared with agar. Increased calcium concentrations were required to promote solidification of gellan gum. Sporulation occurred within 2 weeks of plating CcI3 with colonies changing color from cream to reddish-brown. Scanning and transmission electron micrographs revealed the architecture of colonial development. Colonies developed in a lens-shaped manner mainly by penetration into the gel. Older colonies had numerous empty hyphae with sporangia developing near or at the surface and with crystalline material, presumably pigment, interspersed among the hyphae. Occasional vesicles were seen mixed within the colonies. Colonial development of Frankia CcI3 is reminiscent of that described for other members of the filamentous Suborder Frankineae.     

A high-throughput ultrasonic spraying inoculation method promotes colony cultivation of rare microbial species

Current method for obtaining microbial colonies still relies on traditional dilution and spreading plate (DSP) procedures, which is labor-intensive, skill-dependent, low-throughput and inevitably causing dilution-to-extinction of rare microorganisms. Herein, we proposed a novel ultrasonic spraying inoculation (USI) method that disperses microbial suspensions into millions of aerosols containing single cells, which lately be deposited freely on a gel plate to achieve high-throughput culturing of colonies. Compared with DSP, USI significantly increased both distributing uniformity and throughput of the colonies on agar plates, improving the minimal colony-forming abundance of rare Escherichia coli mixed in a lake sample from 1% to 0.01%. Applying this novel USI to a lake sample, 16 cellulose-degrading colonies were screened out among 4766 colonies on an enlarged 150-mm-diameter LB plate. Meanwhile, they could only be occasionally observed when using commonly used DSP procedures. 16S rRNA sequencing further showed that USI increased colony-forming species from 11 (by DSP) to 23, including seven completely undetectable microorganisms in DSP-reared communities. In addition to avoidance of dilution-to-extinction, operation-friendly USI efficiently inoculated microbial samples on the agar plate in a high-throughput and single-cell form, which eliminated masking or out-competition from other species in associated groups, thereby improving rare species cultivability.     

Media for the detection of Bacillus larvae spores in honey

Bacillus larvae, the causative agent of American foulbrood in honey bees completes its life cycle of germination, outgrowth and sporulation in young honey bee larvae by killing them and often bringing about the destruction of the entire hive. While B. larvae germinates and outgrows on complex organic media in vitro, the literature suggests, for reasons that are not at all clear, that a relatively large number of spores of B. larvae are required to yield each visible colony (colony forming units, CFU) on media. Various researchers have reported that from 16 to 3,000 or more spores of B. larvae are required to yield a single colony on an agar plate. HANSEN in Denmark designed a useful method of spreading approximately 80 mg of honey directly on the surface of a PETRI plate containing “J” agar medium to determine if B. larvae spores are present in the honey. In the present study, selected media were tested for the ability to recover B. larvae spores in honeys in the form of visible colonies (CFU) using HANSEN's strek method. A modification of a medium (TMYGP) developed by DINGMAN and STAHLY, (T-HCL-YGP agar), recovered considerably more viable B. larvae spores in the form of visible colonies (CFU) than HANSEN's “J” medium. When “J” medium was fortified with 0.1% sodium pyruvate, it was comparable to modified T-HCL-YGP medium in its recovery of B. larvae spores. Brain heart infusion agar (BHIA) with the addition of thiamine recovered more spores in the form of viable colonies than did “J” medium but it was not as efficient as T-HCL-YGP medium. Serial dilution from 100 to 10,000 times of weighed samples of honey with deionized water led to higher spore counts (CFU per g honey) than that indicated by undiluted honeys plated at 80 mg levels directly onto the surface of media by the HANSEN procedure.     

Life form and life history explain variation in population processes in a grassland community invaded by exotic plants and mammals

The existence of general characteristics of plant invasiveness is still debated. One reason we may not have found these characteristics is because we do not yet understand how processes underlying population dynamics contribute to community composition in invaded communities. Here I modify Ricker stock-recruitment models to parameterize processes important to community dynamics in an invaded grassland community: immigration, maximum intrinsic growth rate, self-regulation, and limitation by other species. I then used the parameterized models in a multi-species stochastic simulation to determine how processes affected long-term community dynamics. By parameterizing the models using the frequency of the 18 most common species in the grassland, I determined that life history and life form are stronger predictors of underlying processes than is native status. Immigration maintains exotic annual grasses and the dominant native perennial grass in the community. Growth rate maintains other perennial species. While the model mirrors the frequency of native species well, exotic species have lower observed than parameterized frequencies, suggesting that they are not reaching their potential frequency. These results, combined with results from past research, suggest that disturbance may be key to maintaining exotic species in the community. Here I showed that a continuous modified Ricker model fit discrete grassland frequency data well. This allowed me to model the dominant species in the community simultaneously and gain insight into the processes that determine community composition.     

Microbial communities associated with skeletal tumors on Porites compressa

Coral tumors are atypical skeletal forms found on coral reefs worldwide. Here we present an analysis of the microbial communities associated with skeletal tumors on the coral Porites compressa. Microbial growth rates on both healthy and tumorous P. compressa were decoupled from the surrounding water column. Microbial communities associated with tumorous colonies had a significantly faster growth rate than those associated with healthy P. compressa. The microbial community associated with the tumors contained more culturable Vibrio spp. and could utilize more carbon sources than the microbes associated with healthy colonies. Presence of tumors affected the composition and dynamics of the microbial population associated with the entire colony.     

Storage of a mixed microbial consortium capable of growth on biphenyl

A mixed microbial consortium, BSEN-2, was isolated from a polychlorinated biphenyl-contaminated soil using biphenyl as the sole source of carbon and energy. The community was found to contain nine members based on colony morphology on plate count agar with biphenyl. BSEN-2 was stored on glycerol (15% v/v) at - 80°C and was regenerated 3, 30, 100 and 250 d after freezing. No loss of community members or significant change in growth kinetics were observed. This method provides a simple, convenient and reliable method of producing mixed microbial inocula.     

Age and radial growth pattern of four tree species in a subtropical forest of China

Subtropical forests are usually composed of many tree species. Knowledge of the age and radial growth variation of the dominant tree species is useful for understanding forest dynamics and community structure and function. The aims of this study are to explore whether there are identifiable annual growth rings in the main tree species and to examine the growth characteristics within and among the species in Mount Gutian subtropical forest of China. The results showed that four out of eight tree species from which samples were collected had visible and cross-datable rings. There were no stable relationships between the age and diameter for these subtropical trees. Significant differences existed in radial growth rate within and among the four species, suggesting a high spatial heterogeneity in the mixed-species subtropical forest. The common pattern in age distribution of multiple species suggests a stand-wide disturbance occurring around the 1960s. It is interesting to note that the growth rate at the same age intervals was different for trees younger than 40 years of age and older than 40 years of age, suggesting a change in climate or forest structure in the two time periods. The results obtained from this study help understand the growth dynamics in other subtropical forests having these tree species.     

中国东南部不同海拔亚热带森林中马尾松径向生长对气候的响应

为了解我国东南部亚热带森林不同海拔树木生长对气候响应的差异,建立了福建省武夷山脉东麓2个样点的4个马尾松(Pinus massoniana)轮宽年表,对树木径向生长与气候因子进行了bootstrapped相关分析和线性混合模型(LME)拟合。结果表明,在高海拔地区马尾松径向生长对气候因子年际波动敏感性较强,主要表现为与生长季前冬季光温条件以及生长季内7月降水的正相关,生长-气候关系在不同样点间表现出较强的一致性。线性混合模型可以较好地拟合高海拔树木生长变化,当使用前1年12月平均日最高温、当年1月日照时长和当年7月降雨量3个气候变量进行拟合时,模型解释量达到0.5,其中前1年12月最高温和当年1月日照时数在模型中起到主导作用,累积相对贡献率约占80%,说明生长季前冬季的光热条件是限制高海拔马尾松径向生长最主要的气候因子。因此,我国亚热带地区高海拔的树木径向生长可能对未来气候变化有更强的敏感性,相关森林管理政策的制定需要将此纳入考虑;同时我国亚热带地区高海拔森林中的树木有被用于树轮气候重建的潜力。     

Functional traits explain phytoplankton community structure and seasonal dynamics in a marine ecosystem

A fundamental yet elusive goal of ecology is to predict the structure of communities from the environmental conditions they experience. Trait‐based approaches to terrestrial plant communities have shown that functional traits can help reveal the mechanisms underlying community assembly, but such approaches have not been tested on the microbes that dominate ecosystem processes in the ocean. Here, we test whether functional traits can explain community responses to seasonal environmental fluctuation, using a time series of the phytoplankton of the English Channel. We show that interspecific variation in response to major limiting resources, light and nitrate, can be well‐predicted by lab‐measured traits characterising light utilisation, nitrate utilisation and maximum growth rate. As these relationships were predicted a priori, using independently measured traits, our results show that functional traits provide a strong mechanistic foundation for understanding the structure and dynamics of ecological communities.     

Studies on Plover Cove Reservoir, Hong Kong

The four periphytic communities established in Plover Cove Reservoir (the epiphyton, epihthon, epipelon and epipsammon) were investigated between November, 1970 and December, 1971. A rich flora of diatoms was found to dominate all four peri-phytic communities, and, for this reason, assessment of diatom numbers was used to monitor each community during this survey. The standing crop of each community, measured by cell counts and by chlorophyll a determinations, showed distinct seasonal patterns. Except for the epipsammon, each community displayed a maximum growth in winter, a moderate spring and autumn growth period and a low summer growth. The episammic flora stood apart from the other attached floras in that it exhibited a prolonged maximum growth period in summer. Relatively small numbers of species were found to form the main bulk of all four communities (the so-called ‘constant forms’) and these belonged predominantly to the pennate genera Achnanthes, Fragilaria, Nitzschia and Synedra.     

Measuring Functional Diversity in Plant Communities with MixedLife Forms: A Problem of Hardand Soft Attributes

This paper examines the functional relationships among species in an Australian rangeland community with mixed life forms. It follows a previous study (Walker and others 1999) that explored the role of dominant and minor species in maintaining functional diversity and resilience in a rangeland ecosystem. Unlike our previous results, which were based on estimates of five plant functional attributes, the dominant species in this second community apparently are functionally no more dissimilar to each other than to all other species. We suggest that the lack of clear results in mixed life form communities represents a confounding of the relationships between the “hard” attributes that actually govern how a plant performs in an ecosystem and the “soft” attributes that we use as surrogates. There are very few data on the hard functional attributes of plant species and consequently little information on precisely how the (soft) measurable traits are related to their imputed functions. What evidence there is shows that although the relationships are strong within life forms, they differ between life forms. This poses a problem for the development of research relating plant biodiversity to ecosystem function. Until such a database is developed, it will be very difficult to advance our understanding and measurement of functional diversity in mixed life form communities. Received 30 April 2001; Accepted 23 January 2002.     

Growth of Chlorella vulgaris and associated bacteria in photobioreactors

The aim of this study was to test three flat plate photobioreactor configurations for growth of Chlorella vulgaris under non‐axenic conditions and to characterize and quantify associated bacterial communities. The photobioreactor cultivations were conducted using tap water‐based media to introduce background bacterial population. Growth of algae was monitored over time with three independent methods. Additionally, the quantity and quality of eukaryotes and bacteria were analysed using culture‐independent molecular tools based on denaturing gradient gel electrophoresis (PCR‐DGGE) and quantitative polymerase chain reaction (QPCR). Static mixers used in the flat plate photobioreactors did not generally enhance the growth at the low light intensities used. The maximum biomass concentration and maximum specific growth rate were 1.0 g l^?1 and 2.0 day^?1 respectively. Bacterial growth as determined by QPCR was associated with the growth of C. vulgaris. Based on PCR‐DGGE, bacteria in the cultures mainly originated from the tap water. Bacterial community profiles were diverse but reproducible in all flat plate cultures. Most prominent bacteria in the C. vulgaris cultures belonged to the class Alphaproteobacteria and especially to the genus Sphingomonas. Analysis of the diversity of non‐photosynthetic microorganisms in algal mass cultures can provide useful information on the public health aspects and unravel community interactions.     

Automated image analysis of bacterial colony growth as a tool to study individual lag time distributions of immobilized cells

A method to determine the individual lag time (lag) distributions of immobilized bacteria was presented. The method was based on the image analysis of the bacterial colony growth. The lag distributions were retrieved from the distributions of the detection times (Td) required to form macroscopically visible colonies. Using this method, the lag distributions on agar for Listeria monocytogenes cells previously subjected to two situations reproducing conditions encountered during the contamination of cheese, were determined. The results were presented and compared with lag distributions obtained with an established method based on the time to detection of turbidity in broth. An original method to retrieve lag in broth and agar without any knowledge of the growth rate was also proposed. In order not to bias the distributions of lag on agar the impact of spatial separation between colonies on colony growth rates was quantified. Means and standard deviations of lag distributions for the two different stresses were found to be similar in broth and on agar. Extreme Value type II distributions fitted the best the different datasets of lag distributions.