Bacterial Predators of Micrococcus luteus in Soil

Micrococcus luteus cells died relatively rapidly when they were added to natural soil. Microscopic observation showed that the cells were being physically destroyed by bacterial predators in the soil. Two of these predators were responsible for the initial, main attack, and they were isolated. The isolates on laboratory media lysed M. luteus cells in a manner similar to the attacks that occurred in soil. Neither predator was obligate, however, nor were they nutritionally fastidious. One of these bacteria produced mycelium and conidia. Under nutritionally poor conditions it used slender filaments of mycelium to seek out host cells. It had at least some of the characteristics of a Streptoverticillium species. The other bacterium was a short, gram-negative rod that did not easily fit into any of the known groups of gram-negative bacteria. It attached to host cells, but its mechanism of lysing these cells is not known.     

Host Range and Ecology of Isolates of Pasteuria spp. from the Southeastern United States

Isolates of Pasteuria penetrans were evaluated for ecological characteristics that are important in determining their potential as biological control agents. Isolate P-20 survived without loss of its ability to attach to its host nematode in dry, moist, and wet soil and in soil wetted and dried repeatedly for 6 weeks. Some spores moved 6.4 cm (the maximum distance tested) downward in soil within 3 days with percolating water. The isolates varied greatly in their attachment to different nematode species and genera. Of five isolates tested in spore-infested soil, three (P-104, P-122, B-3) attached to two or more nematode species, whereas B-8 attached only to Meloidogyne hapla and B-I did not attach to any of the nematodes tested. In water suspensions, spores of isolate P-20 attached readily to M. arenaria but only a few spores attached to other Meloidogyne spp. Isolate P-104 attached to all Meloidogyne spp. tested but not to Pratylenchus scribneri. Isolate B-4 attached to all species of Meloidogyne and Pratylenchus tested, but the rate of attachment was relatively low. Isolate P-Z00 attached in high numbers to M. arenaria when spores were extracted from females of this nematode; when extracted from M. javanica females, fewer spores attached to M. arenaria than to M. javanica or M. incognita.     

The attachment of Enteromorpha zoospores to a bacterial biofilm assemblage

This study has investigated the relationship between bacterial biofilms and the attachment of zoospores of the green macroalga Enteromorpha. Zoospore attachment to glass slides was enhanced in the presence of a bacterial biofilm assemblage, and the number attaching increased with the number of bacteria present. Zoospores also attached to control surfaces, but at lower numbers; glass surfaces conditioned in autoclaved seawater had the same number of zoospores attached as new glass surfaces. The spatial relationship between bacterial cells and attached zoospores was quantified by image analysis. The hypothesis tested was that zoospores attached preferentially to, or in the very close vicinity of, bacterial cells. Spatial microscopic analysis showed that more bacteria were covered by zoospores than would be expected if zoospore attachment was a random process and zoospores appeared to attach to bacterial clusters. The most likely explanation is that zoospores are attracted to bacterial cells growing on surfaces and the presence of a bacterial biofilm enhances their settlement. The possibility is discussed that Enteromorpha zoospores respond to a chemical signal produced by bacteria, i.e. that there may be prokaryote‐eukaryote cell signalling.     

Effect of Bovine Manure on Fecal Coliform Attachment to Soil and Soil Particles of Different Sizes

Manure-borne bacteria can be transported in runoff as free cells, cells attached to soil particles, and cells attached to manure particles. The objectives of this work were to compare the attachment of fecal coliforms (FC) to different soils and soil fractions and to assess the effect of bovine manure on FC attachment to soil and soil fractions. Three sand fractions of different sizes, the silt fraction, and the clay fraction of loam and sandy clay loam soils were separated and used along with soil samples in batch attachment experiments with water-FC suspensions and water-manure-FC suspensions. In the absence of manure colloids, bacterial attachment to soil, silt, and clay particles was much higher than the attachment to sand particles having no organic coating. The attachment to the coated sand particles was similar to the attachment to silt and clay. Manure colloids in suspensions decreased bacterial attachment to soils, clay and silt fractions, and coated sand fractions, but did not decrease the attachment to sand fractions without the coating. The low attachment of bacteria to silt and clay particles in the presence of manure colloids may cause predominantly free-cell transport of manure-borne FC in runoff.     

Fate of Escherichia coli O157:H7 in Manure-Amended Soil

Escherichia coli O157:H7 cells survived for up to 77, >226, and 231 days in manure-amended autoclaved soil held at 5, 15, and 21°C, respectively. Pathogen populations declined more rapidly in manure-amended unautoclaved soil under the same conditions, likely due to antagonistic interactions with indigenous soil microorganisms. E. coli O157:H7 cells were inactivated more rapidly in both autoclaved and unautoclaved soils amended with manure at a ratio of 1 part manure to 10 parts soil at 15 and 21°C than in soil samples containing dilute amounts of manure. The manure-to-soil ratio, soil temperature, and indigenous microorganisms of the soil appear to be contributory factors to the pathogen's survival in manure-amended soil.     

Extracellular trehalose utilization by Saccharomyces cerevisiae

Two haploid strains of Saccharomyces cerevisiae viz. MATα and MATa were grown in glucose and trehalose medium and growth patterns were compared. Both strains show similar growth, except for an extended lag phase in trehalose grown cells. In both trehalose grown strains increase in activities of both extracellular trehalase activities and simultaneous decrease in extracellular trehalose level was seen. This coincided with a sharp increase in extracellular glucose level and beginning of log phase of growth. Alcohol production was also observed. Secreted trehalase activity was detected, in addition to periplasmic activity. It appeared that extracellular trehalose was hydrolyzed into glucose by extracellular trehalase activity. This glucose was utilized by the cells for growth. The alcohol formation was due to the fermentation of glucose. Addition of extracellular trehalase caused reduction in the lag phase when grown in trehalose medium, supporting our hypothesis of extracellular utilization of trehalose.     

Differentiating Intracellular from Extracellular Alkaline Phosphatase Activity in Soil by Sonication

Differentiating intracellular from extracellular enzyme activity is important in soil enzymology, but not easy. Here, we report on an adjusted sonication method for the separation of intracellular from extracellular phosphatase activity in soil. Under optimal sonication conditions [soil:water ratio  =  1/8 (w/v) and power density  =  15 watt ml^-1], the activity of alkaline phosphomonoesterase (phosphatase) in a Haplic Cambisol soil increased with sonication time in two distinct steps. A first plateau of enzyme activity was reached between 60 and 100 s, and a second higher plateau after 300 s. We also found that sonication for 100 s under optimal conditions activated most (about 80%) of the alkaline phosphatase that was added to an autoclaved soil, while total bacteria number was not affected. Sonication for 300 s reduced the total bacteria number by three orders of magnitude but had no further effects on enzyme activity. Our results indicate that the first plateau of alkaline phosphatase activity was derived from extracellular enzymes attached to soil particles, and the second plateau to the combination of extracellular and intracellular enzymes after cell lysis. We conclude that our adjusted sonication method may be an alternative to the currently used physiological and chloroform-fumigation methods for differentiating intracellular from extracellular phosphatase activity in soil. Further testing is needed to find out whether this holds for other soil types.     

Competitive Ability and Survival in Soil of Pseudomonas Strain 679-2, a Dominant, Nonobligate Bacterial Predator of Bacteria

A copper-resistant, nonobligate, bacterial predator of bacteria was isolated from soil. It was a Pseudomonas species, designated strain 679-2. It attacked most other nonobligate bacterial predators and hence could control their predatory and other activities in nature. It also inhibited various fungi. It attached to prey cells and produced a toxic, copper-related, growth initiation factor like that produced by Cupriavidus necator. In addition, it produced a second, novel compound that was both antibacterial and antifungal. Strain 679-2 appeared to have only a very limited natural occurrence. It was found only in the soil from one small area in one field. It was absent on the leaves of the plant species that were examined. Regardless of its rarity, however, it was highly competitive in soil. An inoculum consisting of only a few cells added to soil multiplied rapidly to become a major component of the soil microflora within 24 h. A small amount of glutamic acid could be added along with the cells to stimulate production of the toxic compounds noted above, but this was not necessary. After this multiplication, or when large numbers of cells were added to soil, the numbers decreased only slowly during the next several months. Cell survival also was good on plant leaves. The survival in soil and on plant leaves occurred in both laboratory and field experiments. Other than desiccation, the natural mechanism for controlling the numbers or activities of strain 679-2 in soil is not known. The various characteristics of this bacterium, as noted above, are of particular interest because they indicate a possible use of the cells or inhibitor compounds for controlling organisms in soil or on plant surfaces.     

Restoring Soil Ecosystems and Biomass Production of Arundo donax L. under Microbial Communities-Depleted Soil

In recent years, giant reed (Arundo donax L) has received considerable attention as a promising plant for energy production. Giant reed is able to grow in a range of environments, including wetlands and marginal soils, and has shown promise in phytoremediation efforts. A pot experiment was carried out to investigate the ability of giant reed to restore ecosystems of different soils, including bauxite-derived red mud-amended soil and pure red mud (red mud—a waste generated by the Bayer process in the aluminum industry—is strongly alkaline and has a high salt content and electrical conductivity (EC) dominated by sodium). Samples were exposed to high temperatures, which simulate the effects of bushfires. Selected soil properties that were measured included soil dehydrogenase, alkaline phosphatase, urease and catalase activities, soil organic carbon, soil pH, EC, available soil macronutrients NPK, and above- and below-ground plant biomass yield. The results showed that giant reed reduced EC in all autoclaved soils and red mud-contaminated soils by 24–82 %. Significantly, available N was increased, and a slight increase was recorded for available K. The presence of giant reed enhanced the soils’ enzyme activities to recover in all tested autoclaved soils and red mud-contaminated soils; specifically, dehydrogenase activity increased by 262 and 705 % in non-autoclaved and autoclaved soils, respectively, and urease and catalase activities increased by 591 and 385 % in autoclaved soils, respectively. Total bacterial and fungal counts were higher in autoclaved soils than non-autoclaved soils after cultivating giant reed for 12 weeks. Autoclaved soils enabled higher biomass production for giant reed than non-autoclaved soils. These results demonstrate that giant reed is not only able to survive on soil that has lost its microbial community as a result of heat, but can also yield significant amounts of biomass while assisting recovering soil ecosystems after bushfires.     

Methods for Detection of Anticarsia gemmatalis Nucleopolyhedrovirus DNA in Soil

Two methods, phenol-ether and magnetic capture-hybridization (MCH), were developed and compared with regard to their sensitivities and abilities to extract the DNA of the insect baculovirus Anticarsia gemmatalis nucleopolyhedrovirus (AgMNPV) from soil and to produce DNA amplifiable by PCR. Laboratory experiments were performed with 0.25 g of autoclaved soil inoculated with different viral concentrations to optimize both methods of baculovirus DNA extraction and to determine their sensitivities. Both procedures produced amplifiable DNA; however, the MCH method was 100-fold more sensitive than the phenol-ether procedure. The removal of PCR inhibitors from the soil appeared to be complete when MCH was used as the viral DNA isolation method, because undiluted aliquots of the DNA preparations could be amplified by PCR. The phenol-ether procedure probably did not completely remove PCR inhibitors from the soil, since PCR products were observed only when the AgMNPV DNA preparations were diluted 10- or 100-fold. AgMNPV DNA was detected in field-collected soil samples from 15 to 180 days after virus application when the MCH procedure to isolate DNA was coupled with PCR amplification of the polyhedrin region.     

Measurement of Glucose Utilization by Pseudomonas fluorescens That Are Free-Living and That Are Attached to Surfaces

The assimilation and respiration of glucose by attached and free-living Pseudomonas fluorescens were compared. The attachment surfaces were polyvinylidene fluoride, polyethylene, and glass. Specific uptake of [¹⁴C]glucose was determined after bacterial biomass was measured by (i) microscopic counts or (ii) prelabeling of cells by providing [³H]leucine as substrate, followed by dual-labeling scintillation counting. The glucose concentration was 1.4, 3.5, 5.5, 7.6, or 9.7 μM. Glucose assimilation by cells which became detached from the surfaces during incubation with glucose was also measured after the detached cells were collected by filtration. The composition of the substratum had no effect on the amount of glucose assimilated by attached cells. Glucose assimilation by attached cells exceeded that by free-living cells by a factor of between 2 and 5 or more, and respiration of glucose by surface-associated cells was greater than that by free-living bacteria. Glucose assimilation by detached cells was greater than that by attached bacteria. Measurements of biomass by microscopic counts gave more consistent results that those obtained with dual-labeling, but in general, results obtained by both methods were corroborative.     

In vitro attachment of phylloplane yeasts to Botrytis cinerea, Rhizoctonia solani, and Sclerotinia homoeocarpa

The ability of yeasts to attach to hyphae or conidia of phytopathogenic fungi has been speculated to contribute to biocontrol activity on plant surfaces. Attachment of phylloplane yeasts to Botrytis cinerea, Rhizoctonia solani, and Sclerotinia homoeocarpa was determined using in vitro attachment assays. Yeasts were incubated for 2 d on potato dextrose agar (PDA) prior to experimentation. A total of 292 yeasts cultured on PDA were screened for their ability to attach to conidia of B. cinerea; 260 isolates (89.1%) attached to conidia forming large aggregates of cells, and 22 isolates (7.5%) weakly attached to conidia with 1 or 2 yeast cells attached to a few conidia. Ten yeasts (3.4%), including 8 isolates of Cryptococcus laurentii, 1 isolate of Cryptococcus flavescens, and an unidentified species of Cryptococcus, failed to attach to conidia. All non-attaching yeasts produced copious extracellular polysaccharide (EPS) on PDA. Seventeen yeast isolates did not attach to hyphal fragments of B. cinerea, R. solani, and S. homoeocarpa after a 1 h incubation, but attachment was observed after 24 h. Culture medium, but not culture age, significantly affected the attachment of yeast cells to conidia of B. cinerea. The 10 yeast isolates that did not attach to conidia when grown on agar did attach to conidia (20%-57% of conidia with attached yeast cells) when cultured in liquid medium. Attachment of the biocontrol yeast Rhodotorula glutinis PM4 to conidia of B. cinerea was significantly greater at 1 x 10(7) yeast cells x mL(-1) than at lower concentrations of yeast cells. The ability of yeast cells to attach to fungal conidia or hyphae appears to be a common phenotype among phylloplane yeasts.     

Leishmania tropica: Biochemical aspects of promastigotes' attachment to macrophages in vitro

The in vitro attachment of promastigotes of Leishmania tropica to mouse peritoneal macrophages was studied under experimental conditions. Parasite binding by macrophages required an intact intracellular physiology as suggested by the action of several metabolic inhibitors. Parasite attachment was inhibited in the presence of azide, fluoride, iodoacetamide, and 2-deoxyglucose, but was affected very little by inhibitors of oxidative phosphorylation, by 2,4,-dinitrophenol and by cyanide. Parasite attachment was not prevented by cycloheximide, an inhibitor of protein synthesis. Successful parasite attachment required the simultaneous presence of magnesium, glucose, and a macromolecular component of fetal calf serum in the extracellular medium. Furthermore, glucose and serum supported parasite binding synergistically. The requirement for extracellular glucose could be replaced by mannose, suggesting that such a requirement is structural rather than metabolic. The active fraction of serum was nondialyzable, heat labile, and precipitable by ammonium sulfate. These various chemical ingredients of the culture medium were required mainly during the interaction of parasites with macrophages. The extracellular and metabolic requirements of successful parasite binding suggest that attachment of parasites to macrophages possess the characteristics of a biochemical reaction which is probably mediated by one or more enzymatic reactions.     

Interaction of Escherichia coli and Soil Particles in Runoff

A laboratory-scale model system was developed to investigate the transport mechanisms involved in the horizontal movement of bacteria in overland flow across saturated soils. A suspension of Escherichia coli and bromide tracer was added to the model system, and the bromide concentration and number of attached and unattached E. coli cells in the overland flow were measured over time. Analysis of the breakthrough curves indicated that the E. coli and bromide were transported together, presumably by the same mechanism. This implied that the E. coli was transported by advection with the flowing water. Overland-flow transport of E. coli could be significantly reduced if the cells were preattached to large soil particles (>45 μm). However, when unattached cells were inoculated into the system, the E. coli appeared to attach predominantly to small particles (<2 μm) and hence remained unattenuated during transport. These results imply that in runoff generated by saturation-excess conditions, bacteria are rapidly transported across the surface and have little opportunity to interact with the soil matrix.     

Prey Range Characterization, Ribotyping, and Diversity of Soil and Rhizosphere Bdellovibrio spp. Isolated on Phytopathogenic Bacteria

Thirty new Bdellovibrio strains were isolated from an agricultural soil and from the rhizosphere of plants grown in that soil. Using a combined molecular and culture-based approach, we found that the soil bdellovibrios included subpopulations of organisms that differed from rhizosphere bdellovibrios. Thirteen soil and seven common bean rhizosphere Bdellovibrio strains were isolated when Pseudomonas corrugata was used as prey; seven and two soil strains were isolated when Erwinia carotovora subsp. carotovora and Agrobacterium tumefaciens, respectively, were used as prey; and one tomato rhizosphere strain was isolated when A. tumefaciens was used as prey. In soil and in the rhizosphere, depending on the prey cells used, the concentrations of bdellovibrios were between 3 × 10² to 6 × 10³ and 2.8 × 10² to 2.3 × 10⁴ PFU g⁻¹. A prey range analysis of five soil and rhizosphere Bdellovibrio isolates performed with 22 substrate species, most of which were plant-pathogenic and plant growth-enhancing bacteria, revealed unique utilization patterns and differences between closely related prey cells. An approximately 830-bp fragment of the 16S rRNA genes of all of the Bdellovibrio strains used was obtained by PCR amplification by using a Bdellovibrio-specific primer combination. Soil and common bean rhizosphere strains produced two and one restriction patterns for this PCR product, respectively. The 16S rRNA genes of three soil isolates and three root-associated isolates were sequenced. One soil isolate belonged to the Bdellovibrio stolpii-Bdellovibrio starrii clade, while all of the other isolates clustered with Bdellovibrio bacteriovorus and formed two distantly related, heterogeneous groups.     

Protozoan Response to the Addition of Bacterial Predators and Other Bacteria to Soil

Representatives of several categories of bacteria were added to soil to determine which of them might elicit responses from the soil protozoa. The various categories were nonobligate bacterial predators of bacteria, prey bacteria for these predators, indigenous bacteria that are normally present in high numbers in soil, and non-native bacteria that often find their way in large numbers into soil. The soil was incubated and the responses of the indigenous protozoa were determined by most-probable-number estimations of total numbers of protozoa. Although each soil was incubated with only one species of added bacteria, the protozoan response for the soil was evaluated by using most-probable-number estimations of several species of bacteria. The protozoa did not respond to incubation of the soil with either Cupriavidus necator, a potent bacterial predator, or one of its prey species, Micrococcus luteus. C. necator also had no effect on the protozoa. Therefore, in this case, bacterial and protozoan predators did not interact, except for possible competition for bacterial prey cells. The soil protozoa did not respond to the addition of Arthrobacter globiformis or Bacillus thuringiensis. Therefore, the autochthonous state of Arthrobacter species in soil and the survival of B. thuringiensis were possibly enhanced by the resistance of these species to protozoa. The addition of Bacillus mycoides and Escherichia coli cells caused specific responses by soil protozoa. The protozoa that responded to E. coli did not respond to B. mycoides or any other bacteria, and vice versa. Therefore, addition to soil of a nonsoil bacterium, such as E. coli, did not cause a general increase in numbers of protozoa or in protozoan control of the activities of other bacteria in the soil.     

Functional Profiling and Distribution of the Forest Soil Bacterial Communities Along the Soil Mycorrhizosphere Continuum

An ectomycorrhiza is a multitrophic association between a tree root, an ectomycorrhizal fungus, free-living fungi and the associated bacterial communities. Enzymatic activities of ectomycorrhizal root tips are therefore result of the contribution from different partners of the symbiotic organ. However, the functional potential of the fungus-associated bacterial communities remains unknown. In this study, a collection of 80 bacterial strains randomly selected and isolated from a soil–ectomycorrhiza continuum (oak–Scleroderma citrinum ectomycorrhizas, the ectomycorrhizosphere and the surrounding bulk soil) were characterized. All the bacterial isolates were identified by partial 16S rRNA gene sequences as members of the genera Burkholderia, Collimonas, Dyella, Mesorhizobium, Pseudomonas, Rhizobium and Sphingomonas. The bacterial strains were then assayed for β-xylosidase, β-glucosidase, N-acetyl-hexosaminidase, β-glucuronidase, cellobiohydrolase, phosphomonoesterase, leucine-aminopeptidase and laccase activities, chitin solubilization and auxin production. Using these bioassays, we demonstrated significant differences in the functional distribution of the bacterial communities living in the different compartments of the soil–ectomycorrhiza continuum. The surrounding bulk soil was significantly enriched in bacterial isolates capable of hydrolysing cellobiose and N-acetylglucosamine. In contrast, the ectomycorrhizosphere appeared significantly enriched in bacterial isolates capable of hydrolysing glucopyranoside and chitin. Notably, chitinase and laccase activities were found only in bacterial isolates belonging to the Collimonas and Pseudomonas genera. Overall, the results suggest that the ectomycorrhizal fungi favour specific bacterial communities with contrasting functional characteristics from the surrounding soil.     

Evaluation of trichothecene and nontrichothecene mycotoxins produced byFusarium in soybeans

Each of 12 cultures ofFusarium, comprising four species, isolated from moldy soybeans suspected of being involved in illness of wild geese, were grown separately in autoclaved moist rice, in autoclaved moist soybeans, and in surface sterilized-disinfected soybeans, assayed for various mycotoxins, and fed to rats. Four additional cultures that produced known toxins on rice were also grown on soybeans as controls. All isolates, except one ofF moniliforme, grown in rice resulted in weight loss of rats, and that one resulted in weight gain; 12 of the isolates caused death. One isolate ofF poae grown in soybeans caused death when consumed by rats, but none of the other 15 resulted in weight loss or overt injury. Much larger amounts of zearalenone, deoxynivalenol (DON), T-2 toxin, neosolaniol, T-2 tetraol, wortmannin, and moniliformin were produced by the cultures on rice than on soybeans, but more HT-2 toxin was produced by one isolate ofF poae grown on soybeans than when grown on rice. Soybeans appear to be a poor substrate for elaboration of most of the toxins produced by the isolates tested.     

Isolation of Arthrobacter Bacteriophage from Soil

Soil was percolated with water and various nutrient solutions, and then the percolates were analyzed for bacteriophages which produced plaques on various Arthrobacter strains. The water percolates did not contain detectable phage. In contrast, phages for A. globiformis strains ATCC 8010 and 4336, and for several recent Arthrobacter species soil isolates, were easily detected in nutrient broth, soil extract, and cation-complete medium percolates. These percolates did not contain phage that produced plaques on A. oxydans and a recent Arthrobacter species soil isolate. Percolation with a selective nicotine-salts solution was required for demonstrating phage for these bacteria. None of the percolates contained phage for five additional named Arthrobacter species. In addition, phages were not detected for A. crystallopoietes in a 2-hydroxypyridine percolate of soil. Based on their lytic spectra, the phage isolates from this soil were relatively host specific.     

Adhesion of Cochliobolus heterostrophus Conidia and Germlings to Leaves and Artificial Surfaces

Braun, E. J., and Howard, R. J. 1994. Adhesion of Cochliobolus heterostrophus conidia and germlings to leaves and artificial surfaces. Experimental Mycology 18, 211-220. We have examined the nonspecific attachment of Cochliobolus heterostrophus germlings to a variety of surfaces (glass, cellophane, Mylar, polystyrene, Teflon, maize leaves) in an effort to more fully characterize this important stage of pathogenesis. Washing experiments showed that conidia began adhering to glass just prior to germ tube emergence, about 20 min after hydration and inoculation. By 50-60 min after inoculation, over 90% of the germinating conidia resisted washing and remained firmly attached. Similar results were obtained with the other surfaces. Both sodium azide and cycloheximide prevented attachment, indicating that metabolic activity was required for adhesion. Light microscopy and cryo scanning electron microscopy were used to document a temporal and spatial relationship between attachment, appearance of extracellular matrix materials, and germ tube emergence. Attachment of conidia to the substratum was correlated with the appearance of extracellular material exuded from the tips of conidia just prior to germination. The two-layered sheath of matrix materials associated with germ tubes also surrounded appressoria and appeared to aid in attachment of these structures to leaves and artificial surfaces. We conclude that extracellular matrix is produced and/or secreted within 20 min of hydration and serves in the nonspecific attachment of germlings to the substrate.