On the abundance of marine naked amoebae on the surface of five species of macroalgae

Abstract Naked amoebae constituted a numerically significant component of the surface microbial community of five species of seaweeds. They were most abundant in the summer months on the brown macrophytes, Fucus and Laminaria , where numbers up to 23 cells cm⁻² of algal surface were recorded. This implies that littoral algal stands can support populations of up to 3.2 × 10⁶ amoebae m⁻². A total of 27 different species were recognized and, of these, six species were less than 10 μm in length, a size class overlooked in previous studies. Damaged tissue contained higher numbers of amoebae, up to 43 cells cm⁻², presumably due to higher bacterial densities and increased shelter at these sites. Some amoebae may have been utilizing algal carbon exudates directly, especially isolates of Trichosphaerium , which showed evidence of algal digestion. The numbers of amoebae found in this study suggest that these protozoa may play a significant, and previously overlooked, role in the cycling of estuarine carbon.     

Influence of PCBs on the predator-prey relation between bacteria and protozoa in soil

Abstract This work deals with the impact of a possible accidental pollutant, pyralene (Prodelec, France; PCBs in trichlorobenzene), intoduced into the soil. Its influence on the predator-prey relation between bacteria and amoebae was studied by comparing the population dynamics of (i) an inoculated bacterial population ( A. lipoferum ) chosen as a biological tracer, (ii) the indigenous bacterial microflora, (iii) the infigenous amoebae. In the absence of pyralene the inoculated bacterial population decreased from 10⁷ to 10⁴ bacteria g⁻¹ soil (dw), grazed by the infigenous amoebae whose numbers increased 3-fold. In contrast, in presence of 2500 ppm of pyralene the introduced bacteria survived at a higher level (3·10⁶ bacteria g⁻¹ soil (dw)) while the number of amoebae diminished slightly. No predation occurred with PCB contamination. The indigenous bacterial microflora was not affected quantitatively by pyralene. In pure liquid culture with 500 ppm of pyralene added, bacterial growth was inhibited and an amoebal strain isolated from an inoculated uncontaminated soil was killed. We conclude that the active form of the amoebae were killed, and encystement was inhibited by pyralene in the soil. Hence the protozoa were unable to regulate the introduced A. lipoferum strain as they did in the absence of the pollutant.     

Influence of woodlot floor topography on microbial decomposer distribution

The distribution of microbial populations that decomposed sugar, cellulose and lignin-related substrates was examined in a beech Fagus grandifolia Ehrh. and maple Acer saccharum Marsh. dominated woodlot developed on glacial till. The topography of the woodlot, characterized by rises, depressions and more extensive level areas about 1 m in diameter with a 0.5 m vertical maximum, produced a mosaic of decomposer habitats designated as high, level and low sites.
In general, populations of sugar, cellulose and lignin decomposing organisms (based on ten estimates made from April to October) were two to four times higher in litter and soil samples from low sites than those from high sites. Sugar decomposing bacteria in litter were most abundant at all topographic sites. 135 × 10⁶ g⁻¹ dry litter at high sites, 396 × 10⁶ g⁻¹ at level sites and 456 × 10⁶ g⁻¹ at low sites; lignolytic fungi were least abundant, 391 × 10² g⁻¹ dry litter at high sites. 700 × 10⁶ g⁻¹ at level sites and 954 × 10² g⁻¹ at low sites. Numbers of microbial decomposers in the topographic sites were correlated with organic matter content. Distribution of fungal genera did not appear to be related to topographic site. Most populations examined showed two numerical peaks, one in late May or June and one in late September or October. It is suspected that these peaks were influenced by the coincident timing of favourable physical conditions and priming by soluble nutrients leached from litter.     

Influence of soil pH on the nitrate-reducing microbial populations and their potential to reduce nitrate to NO and N2O

Abstract After the introduction of Rhizobium leguminosarum biovar trifolii into natural loamy sand and silt loam, bacterial numbers increased only directly after inoculation. Thereafter, bacterial numbers decreased until an equilibrium was reached. This decrease was exponential on a log scale and could be described by the function Y = A + B − R ', where Y is the log number of rhizobial cells at time: T ; A represents the lgo of the final population size; B is the difference between the log (initial number of bacteria) and A ; R is the daily reduction factor of Y−A and t is time in days after inoculation. The final population sizes increased with increasing inoculum densities (10⁴−10⁸ bacteria/g soil). In sterilized soil, however, the populations increased up to an equilibrium, which was not affected by the inoculum density.
The final population sizes were higher in silt loam than in loamy sand in natural, as well as in sterilized soil. The final population size was reached earlier in natural silt loam than in loamy sand. Also the growth rate in sterilized soil was higher in silt loam than in loamy sand. The growth rate of low inoculum densities in silt loam was exponential and approximately the same as in yeast extract mannitol broth. The growth rate in loamy sand could be improved by incresing the bulk density of the soil from 1.0 to 1.4 g/cm³.     

Survival of κ-carrageenan-encapsulated and unencapsulated Pseudomonas aeruginosa UG2Lr cells in forest soil monitored by polymerase chain reaction and spread plating

Abstract A method was developed for direct extraction, purification and amplification of DNA from forest soil. Eighty-two % of the DNA in Pseudomonas aeruginosa UG2Lr introduced into soil was recovered. The detection limit for the strain was approximately 800 cfu g⁻¹ of dry soil based on the polymerase chain reaction (PCR). Survival of κ-carrageenan-encapsulated and unencapsulated UG2Lr was monitored by antibiotic selective and bioluminescence-based nonselective plating and PCR-amplification of a tnsA fragment. After freeze-thaw treatment of soil samples, the unencapsulated UG2Lr declined from an initial population density of 1 × 10⁹ cfu g⁻¹ of dry soil to below the detection threshold of both selective (14 cfu g⁻¹ of dry soil) and nonselective (1 × 10³ cfu g⁻¹ of dry soil) plating. However, presence of nonculturable UG2Lr cells in the soil was revealed by PCR and resuscitation of the bacteria. Population density of the encapsulated UG2Lr increased from 2.7 × 10⁶ to 2.9 × 10⁸ cfu g⁻¹ of dry soil after a 3-week incubation at 22°C and declined to 6.3 × 10⁶ cfu g⁻¹ of dry soil after the freeze-thaw treatment.     

Protozoan predation of bacterial cells in soil aggregates

Abstract The development and survival of Aerobacter aerogenes IAM11022 in the inner and outer zones of soil aggregates (1–2 mm) was investigated in relation to a protozoan ( Colpoda sp.). With different dilutions of the bacterial cell suspension, a constant partition ratio of these cells was observed between outer and inner zones of the aggregates. Protozoa inoculated in the same manner were generally recovered only from the outer zone of the aggregates.
In the presence of protozoa, prey cell numbers of the outer zone were reduced from more than 10⁸ to approx. 10⁴ cells · g soil⁻¹ in 12 days. In contrast, 10⁸ cells · g soil⁻¹ remained in the inner zone of the aggregates, even after 12 days.
The increase in predator cell number was proportional to initial prey densities in the outer zone of the aggregates. At a constant initial prey density (1.8 × 10⁷ cells · g soil⁻¹), Colpoda sp. multiplied in proportion to the initial number of predators. When the initial density of the predator was low more prey cells survived in the outer zone.
Prey persistence was associated with 3 different types of protection: (1) small pore necks of the inner zone space of the aggregates; (2) the division of the outer zone space into compartments; and (3) the distribution of protozoan cells among soil aggregates. The latter two were closely related to the moisture condition of the soil.     

Survival of Ralstonia solanacearum in Soil under Irrigated Rice Culture and Aquatic Weeds

The survival of Ralstonia solanacearum in naturally infested sandy loam soil under irrigated rice culture was investigated in Sankhu village (1400 m above sea level) in central Nepal. The experimental plot had a previous history of bacterial wilt and a range of 1.5 × 10⁴–3 × 10⁴ colony-forming units (CFU) per g soil was present. The survival of R. solanacearum was monitored in roots of naturally growing aquatic weeds in the rice plot and in soil before and after rice harvest. The incidence of the bacterial infection in the weeds, Dopatrium sp. and Monochoria vaginalis , were 57.5 and 10%, respectively. The bacterial population detected in soil before rice harvest was 1.5 × 10⁴ CFU per g soil whereas a range of 7.5 × 10²–1.5 × 10³ CFU per g was detected after the rice harvest. Biovar typing of R. solanacearum isolated from potato plants, potato tubers, aquatic weeds, and the soil from the experimental plot yielded the diverse biovars 2 A, 3 and 4. This is the first report of the survival of these biovars in soil, which was under continuous flow of irrigation water for 3 months during rice culture.     

The effect of procaine on the partitioning of plasmid pSC101

Abstract An examination of samples obtained from a commercial fish smoker, using seawater agar with incubation at 4°, 15° and 37°C for up to 28 days, revealed the presence of large bacterial populations in smoked fish. However, initially only low bacterial numbers, i.e., 2 × 10³/g, were present in the muscle of fresh, whole haddock ( Melanogrammus aeglefinus ). With filleting, there was a sudden increase in numbers to 9.2 × 10⁵/g. Yet immediately after smoking, the bacterial populations decreased (5 × 10⁵/g), followed by a gradual increase with storage (e.g., 2 × 10⁶/g after 24 h). Representative colonies were presumptively identified as Acinetobacter, Alcaligenes , coryneforms, Pseudomonas and Vibrio spp.     

Post-processing microflora and the shelf stability of gari marketed in Port Harcourt

Gari was examined for its post-processing microbial content. Aerobic mesophilic bacteria and fungi were isolated from all samples. The total viable bacterial counts ranged from 2.0 × 10² to 8.0 × 10⁴ cfu/g. Fungal counts ranged from 1.0 × 10² to 1.5 × 10⁴ cfu/g. The total viable counts of fresh samples were much lower than those of market and packaged samples. Bacillus, Micrococcus and Proteus spp. were the bacteria isolated, Aspergillus niger, Aspergillus flavus and Penicillium spp. the fungi. Food borne parasites and pathogens such as Staph. aureus and Clostridium perfringens were not found. The gari samples were quite stable, having a shelf life of 3–6 months. The water activities of the samples ranged from 0.52 to 0.68. Based on the microbial counts of the samples, the critical upper limit for the safety of gari was set at 10⁴ cfu/g dry sample.     

Decreased endo-lysosomal acidification capacity in methylene diphosphonate-resistant mutants of Dictyostelium discoideum

Abstract The in vivo capacity for endo-lysosomal acidification has been monitored in Dictyostelium discoideum amoebae with acridine orange, a fluorescent weak base dye commonly used to probe transmembrane pH gradients. In the presence of aerobic amoebae, the initial rate of fluorescence quenching was found to be proportional to cell density between 5 × 10⁵ and 2.5 × 10⁶ cells ml⁻¹ and independent of acridine orange concentration in the 1.5 to 7.5 μM range. The dye response was sensitive to agents that perturb endo-lysosomal acidification such as NaN₃, nigericin or imidazole. Several mutant cell lines whose growth was resistant to methylene diphosphonate were found to be partially deficient in the acridine orange quenching test, suggesting that endo-lysosomal acidification was altered in these mutants.     

Selective grazing of methanotrophs by protozoa in a rice field soil

Biological methane oxidation is a key process in the methane cycle of wetland ecosystems. The methanotrophic biomass may be grazed by protozoa, thus linking the methane cycle to the soil microbial food web. In the present study, the edibility of different methanotrophs for soil protozoa was compared. The number of methanotroph-feeding protozoa in a rice field soil was estimated by determining the most-probable number (MPN) using methanotrophs as food bacteria; naked amoebae and flagellates were the dominant protozoa. Among ten methanotrophic strains examined as a food source, seven yielded a number of protozoa comparable with the yield with Escherichia coli [10⁴ MPN (g soil dry weight)⁻¹], and three out of four Methylocystis spp. yielded significantly fewer numbers [10²–10³ MPN (g soil dry weight)⁻¹]. The lower edibility of the Methylocystis spp. was not explained either by their growth phase or by harmful effects on protozoa. Incubation of the soil under methane resulted in a higher number of protozoa actively grazing on methanotrophs, especially on the less-edible group. Protozoa isolated from the soil demonstrated a grazing preference on the different methanotrophs consistent with the results of MPN counts. The results indicate that selective grazing by protozoa may be a biological factor affecting the methanotrophic community in a wetland soil.     

Effects of Beauveria bassiana and Metarhizium anisopliae on mortality, fecundity and egg fertility of Tetranychus evansi

Abstract:  The susceptibility of various developmental stages of Tetranychus evansi Baker & Pritchard (eggs, larvae, protonymphs, deutonymphs and adults) to the entomopathogenic fungi Metarhizium anisopliae (Metschnikoff) Sorokin and Beauveria bassiana (Balsamo) Vuillemin was evaluated under laboratory conditions. Three concentrations (3.0 × 10⁶, 1.0 × 10⁷ and 1.0 × 10⁸ conidia/ml) of both fungi were used for each stage. The effect of fungal infection on fecundity and egg fertility was also investigated using both fungal species. Deutonymphs that survived the infection and developed into adult females were allowed to oviposit. Adults and deutonymphs were more susceptible to fungal infection than larval and protonymphal stages at all the concentrations. Nevertheless, the concentration level influenced the mortality of the different mite stages. Eggs were also susceptible to fungal infection and mortality was dose-dependent. Fungus-treated female mites laid fewer eggs than the controls but there was no significant difference in egg hatchability between the treatments.     

Molecular Detection of Phytophthora cryptogea on Calendula officinalis and Gerbera jamesonii Artificially Inoculated with Zoospores

In this work, a protocol for zoospores production of Phytophthora cryptogea , an economically important plant pathogen was optimized. Five different concentrations of zoospores (5 × 10⁵, 5 × 10⁴, 5 × 10³, 5 × 10², 5 × 10¹ zoospores/ml) from four different isolates of P. cryptogea (Maria 1, Maria 2, S3 1-A, Amazzone) were used as inoculum on pot marigold ( Calendula officinalis ) and gerbera ( Gerbera jamesonii ) plants. Maria 1 was the most virulent isolate both on pot marigold and gerbera plants according to disease severity. A rapid and sensitive pathogen DNA extraction protocol suitable for large quantities of plant samples was adopted. Conventional polymerase chain reaction (PCR) was able to detect the pathogen in artificially inoculated symptomless pot marigold (collected day 12) and gerbera plants (day 8) after pathogen inoculation, with the suspension of 5 × 10⁵, 5 × 10⁴, 5 × 10³ P. cryptogea  zoospores/ml. Real-time PCR showed the possibility to detect the pathogen in artificially inoculated symptomless pot marigold (collected day 8) and gerbera plants (day 4) after pathogen inoculation, with the suspension of 5 × 10⁵, 5 × 10⁴ P. cryptogea  zoospores/ml. The first symptoms appeared on pot marigold plants 14 days after pathogen inoculation and on gerbera plants 10 days after inoculation. Real-time PCR showed the possibility to detect the pathogen 4 days before conventional PCR and 6 days before the appearance of disease symptoms both on pot marigold and gerbera plants.     

Bacterial microflora in the gastro-intestinal tract of Dover sole (Solea solea L.), with emphasis on the possible role of bacteria in the nutrition of the host

Abstract There was a progressive increase in the size of the aerobic heterotrophic bacterial populations along the gastro-intestinal tract of farmed Dover sole. Moreover, higher counts were recorded in juvenile than in adult animals. Thus, in juvenile fish, 5.2 × 10⁵, 8.0 × 10⁵ and 9.8 × 10⁶ aerobic heterotrophs/g were recovered from the stomach/foregut, midgut and hindgut/rectum, respectively. In adult fish, comparative samples revealed the presence of only 3.0 × 10⁴, 7.0 × 10⁴ and 2.3 × 10⁵ bacteria/g, respectively. There bacteria were equated with Acinetobacter, Alcaligenes , Enterobacteriaceae representatives, Flavobacterium, Micrococcus, Photobacterium, Staphylococcus and Vibrio . Of the compounds tested, many isolates, particularly those recovered from the hindgut/rectum, degraded p -nitrophenyl- β - N -acetylglucosaminide, chitin and collagen. Consequently, it is likely that such organisms may contribute to nutritional processes within Dover sole.     

Effects of Host Cell Density on Cell Infection Level in Antheraea eucalypti (Lepidoptera: Saturniidae) Cell Cultures Persistently Infected with Nosema bombycis (Microsporida: Nosematidae)

ABSTRACT. Spores of Nosema bombycis Y9101, isolated from the beet armyworm, Spodoptera exigua , were primed with an alkaline solution and inoculated into Antheraea eucalypti cell cultures. Infected cells were subcultured every five days at three cell densities (2.5 × 10³, 5.0 × 10³, and 1.0 × 10⁴ cells/cm²). A difference was observed in the spread of N. bombycis Y9101 infection between low-density and higher-density cultures of host cells. The host cell density did not affect the productivity of secondary infective forms of the parasite. The principal factor determining the rate of microsporidian infection in vitro was the number of host cells existing within the reach of extruded short-coiled polar tubes from spores germinated intracellularly.     

Growth Kinetics, Cell Shape, and the Cytoskeleton of Primary Astrocyte Cultures

Abstract: We examined correlations among growth kinetics, cell shape, and cytoskeletal protein content in rat astrocytes grown in primary culture. Cell suspensions from brains of newborn rats were seeded at densities from 0.2 to 3 × 10⁵/cm². At initial densities above 1 × 10⁵ the population increased to reach confluency by 10–12 days, after which cell number remained stable for many weeks. At low initial densities, 0.2–0.4 × 10⁵/cm², cells did not increase in number. Final density increased with increasing plating densities. High-density cells had small perikarya and several long cytoplasmic processes; low-density cells appeared flat and polygonal. All cultures were almost entirely astrocytic, as judged by immunofluorescent staining with antiserum against glial fibrillary acidic protein (GFAP). Cytoskeletal proteins were analyzed by gel electrophoresis after extraction from cells with nonionic detergent. Relative amounts of the proteins differed, in that low-density cells contained large amounts of cytoskeletal actin relative to the intermediate filament (IF) proteins vimentin and GFAP, whereas high-density cells contained relatively less actin and more IF proteins. Such differences in cytoskeletal proteins between the high- and low-density cultures were mirrored in the relative rates of synthesis of the cytoskeletal proteins. In the low-density cells amino acid incorporation into cytoskeletal-associated actin was more active than that into the IFs, whereas in the high-density cells higher rates of IF protein synthesis were observed.     

Monitoring the fate of genetically engineered bacteria sprayed on the phylloplane of bush beans and grass

Abstract The fate of a Bacillus amyloliquefaciens with the recombinant plasmid pSB20 sprayed on the phyllosphere of grass, and of a Tn 5 marked Pseudomonas syringae sprayed on the phyllosphere of bush beans was studied in planted soil microcosms. B. amyloliquefaciens showed a decline from 1.5×10⁸ to 3.1×10² cfu g⁻¹ on the phylloplane of grass in the course of the experiment. B. amyloliquefaciens was easy to follow by selective cultivation due to the complete absence of bacterial background growth. Southern blot hybridization of Hin dIII digested genomic DNA showed plasmid restriction patterns identical with pSB20 indicating high plasmid stability. In total DNA extracts from phyllosphere bacteria the recombinant plasmid was detectable by Southern blot hybridization up to 6×10⁴ cfu g⁻¹ (wet weight). Counts of hybridizing colonies showed that P. syringae established on the phyllosphere of bush beans at between 5×10³ and 4×10⁶ cfu g⁻¹ fresh weight. During senescence of the bean plants the strain was no longer detectable by selective cultivation and subsequent colony hybridization. In contrast, Tn5 marked DNA was detected after PCR amplification over the whole period of the experiment.     

Plasmid and chromosomally encoded luminescence marker systems for detection of Pseudomonas fluorescens in soil

Luminescent strains of Pseudomonas fluorescens 10586 were constructed in which luciferase production was constitutive by introduction of Vibrio fischeri luxABE genes on the chromosome and on a multicopy plasmid. Light production in liquid batch culture was directly proportional to biomass concentration during exponential growth and enabled detection by luminometry of 1.7 × 10³ and 8.9 × 10⁴ cells/ml for the plasmid and chromosomally marked strains, respectively. Luminescent colonies of both strains were detectable by eye, enabling viable cell enumeration on solid media against a background of non-luminescent strains. Following inoculation into sterile and non-sterile soil lower levels of detection were increased but detection of 8.1–59 × 10³and 2.2–30 × 10³ cells per g of soil was possible for plasmid and chromosomally marked strains. Maximum specific growth rate in liquid culture was unaffected by introduction of lux marker genes on the chromosome, but was reduced in the plasmid marked strain. The chromosomally encoded marker was stable in both liquid culture and in soil, but the plasmid was unstable during continuous subculturing in liquid medium and during growth in soil. The chromosomally encoded luminescence-marker system therefore provides a convenient, non-extractive technique for quantification of genetically modified soil microbial inocula.     

Colonization of Vibrio pelagius and Aeromonas caviae in early developing turbot (Scophthalmus maximus L.) larvae

Polyclonal antisera made in rabbits against whole washed cells of Vibrio pelagius and Aeromonas caviae were used for detection of these bacterial species in the rearing water and gastrointestinal tract of healthy turbot ( Scophthalmus maximus ) larvae exposed to V. pelagius and/or Aer. caviae . The results demonstrated that this method is suitable for detection of V. pelagius and Aer. caviae in water samples and larvae at population levels higher than 10³ ml⁻¹ and 10³ larva⁻¹. Populations of aerobic heterotrophic bacteria present in the gastrointestinal tract of turbot larvae, estimated using the dilution plate technique, increased from approximately 4 × 10² bacteria larva⁻¹ on day 3 post-hatching to approximately 10⁵ bacteria fish⁻¹ 16 days post-hatching. Sixteen days after hatching, Vibrio spp. accounted for approximately 3 × 10⁴ cfu larva⁻¹ exposed to V. pelagius on days 2, 5 and 8 post-hatching. However, only 10³ of the Vibrio spp. belonged to V. pelagius . When larvae were exposed to Aer. caviae on day 2 post-hatching, the gut microbiota of 5-day old larvae was mainly colonized by Aeromonas spp. (10⁴ larva⁻¹), of which 9 × 10³ belonged to Aer. caviae . Later in the experiment, at the time when high mortality occurred, 9 × 10⁵ Aer. caviae were detected. Introduction of V. pelagius to the rearing water seemed to improve larval survival compared with fish exposed to Aer. caviae and with the control group. It was therefore concluded that it is beneficial with regard to larval survival to introduce bacteria ( V. pelagius ) to the rearing water.     

Longevity of the freshwater anostracan Streptocephalus mackini (Crustacean: Anostraca) in relation to food (Chlorella vulgaris) concentration

SUMMARY 1. Temporary ponds are inhabited by a variety of invertebrates, of which anostracans are an important group. We studied the lifetables of male and female anostracan Streptocephalus mackini at 3 algal concentrations (0.5 × 10⁶, 1.0 × 10⁶ and 1.5 × 10⁶ cells mL⁻¹).
2. Regardless of sex, S. mackini showed better survivorship at lower food levels. The longest average lifespan observed was 85 ± 2 days for males fed Chlorella at 0.5 × 10⁶ cells mL⁻¹.
3. Both net reproductive rate and generation time decreased with increasing food level. The highest net reproductive rate was about 120 cysts per female. The longest generation time of about 40 days, observed at 0.5 × 10⁶ cells mL⁻¹, was more than three times that at 1.5 × 10⁶ cells mL⁻¹.
4. The rate of population increase ( r ) was nearly the same (0.31 ± 0.06) at high (1.5 × 10⁶ cells mL⁻¹) and intermediate (1.0 × 10⁶ cells mL⁻¹) food levels. The r -value at low food level (0.5 × 10⁶ cells mL⁻¹ of Chlorella ) was 0.20 ± 0.01 per day.