Comparison of two fatty acid analysis protocols to characterize and differentiate Fusarium oxysporum f. sp. lycopersici and F. oxysporum f. sp. radicis-lycopersici

The utility of fatty acid methyl ester (FAME) profiles for characterization and differentiation of isolates of Fusarium oxysporum f. sp. lycopersici and F. oxysporum f. sp. radicis-lycopersici was investigated. Two fatty acid analysis protocols of the normal (MIDI) and a modified MIDI method were used for their utility. Only the modified MIDI method allowed a clear differentiation between F. oxysporum f. sp. lycopersici and F. oxysporum f. sp. radicislycopersici. FAME profiles using the modified MIDI method gave the most consistent and reproducible analyzed fatty acid data. Evaluation of the FAME profiles based on cluster analysis and principal-component analysis revealed that FAME profiles from tested isolates were correlated with the same vegetative compatibility groups (VCGs) compared to the same races in F. oxysporum f. sp. lycopersici. Results indicated that FAME profiles could be an additional tool useful for characterizing isolates and forma species of F. oxysporum obtained from tomato.     

Diversity among bacteria isolated from the deep subsurface

Culturable bacteria from the deep subsurface (179 m) at Cerro Negro, New Mexico were isolated and characterized. The average number of viable aerobic bacteria was estimated to be 5×10⁵g^–1 of sediment, but only about 0.1% of these could be recovered on agar medium when incubated under aerobic conditions. Of 158 strains isolated from this depth, 92 were characterized by cellular fatty acid profiles (FAME), 36 by analysis of partial 16S rDNA sequences, and 44 by rep-PCR genome fingerprint analysis using three different sets of oligonucleotide primers (REP, BOX, or ERIC). These analyses showed the majority of isolates (67%) were Gram-positive bacteria and primarily members of genera with a high %G+C DNA. The remaining isolates were -subdivisionProteobacteria (19%) and members of the flavobacteria group (14%). The diversity indices based on these different methods of characterization were very high suggesting this subsurface habitat harbors a highly diverse microbial community.     

Diversity within a Colony Morphotype: Implications for Ecological Research

Sets of bacterial isolates with the same colony morphologies were selected from spread plates of bacteria from deep subsurface rock samples; each set had a unique morphology. API-rapid-NFT analysis revealed that isolates within a set were the same. Fatty acid methyl ester analysis of one set of isolates clustered organisms within the same species, defining variation between isolates at the biotype (subspecies) and strain levels. Metal resistances consistently tracked with colony morphology, while antibiotic resistances were less reliable.     

Changes in Bacteria Recoverable from Subsurface Volcanic Rock Samples during Storage at 4°C

The abundance of viable microorganisms recovered from deep subsurface volcanic rock samples increased after rock perturbation and storage for 1 week at 4°C, while the diversity and evenness of recoverable heterotrophic bacterial communities generally decreased. One sample of each morphologically distinct colony type, recovered both before and after storage of U12n rock samples, was purified and characterized by fatty acid methyl ester (MIDI) and API rapid NFT strips. As determined by MIDI cluster analysis, the composition of the recoverable microbial communities changed with storage of rock samples; some groups of organisms were recovered only before, only after, or at both sample times. In general, the isolates recovered only after storage of rock samples had a greater ability to utilize the carbohydrates included in API test strips and had faster generation times than isolates recovered only on initial plating. The nutritional versatility and faster growth rates of organisms recovered in higher proportions after sample storage provide evidence that some microbial community changes may be due to the proliferation of a few bacterial types. However, because some new genera are recovered only after storage, the possibility also exists that dormant bacterial types are resuscitated during sample perturbation and storage.     

Capnocytophaga: New genus of Gram-negative gliding bacteria. II. Morphology and ultrastructure

Gram-negative, anaerobic gliding bacteria were isolated from normal supragingival plaque and from periodontal lesions. Isolates could be divided into two size classes: small 2.4–4.2 m×0.38–0.5 m and large 4.8–5.8 m×0.42–0.6 m cells. The outer membrane was either loose-fitting and wavy, or taut, and of variable thickness. An electron-dense fuzz was discernible on several of the isolates. The periplasmic region was of variable electron-density. The genus Capnocytophaga has been proposed for these organisms based on morphological and cultural characteristics.     

Changes in Bacteria Recoverable from Subsurface Volcanic Rock Samples during Storage at 4 degrees C

The abundance of viable microorganisms recovered from deep subsurface volcanic rock samples increased after rock perturbation and storage for 1 week at 4 degrees C, while the diversity and evenness of recoverable heterotrophic bacterial communities generally decreased. One sample of each morphologically distinct colony type, recovered both before and after storage of U12n rock samples, was purified and characterized by fatty acid methyl ester (MIDI) and API rapid NFT strips. As determined by MIDI cluster analysis, the composition of the recoverable microbial communities changed with storage of rock samples; some groups of organisms were recovered only before, only after, or at both sample times. In general, the isolates recovered only after storage of rock samples had a greater ability to utilize the carbohydrates included in API test strips and had faster generation times than isolates recovered only on initial plating. The nutritional versatility and faster growth rates of organisms recovered in higher proportions after sample storage provide evidence that some microbial community changes may be due to the proliferation of a few bacterial types. However, because some new genera are recovered only after storage, the possibility also exists that dormant bacterial types are resuscitated during sample perturbation and storage.     

Evaluation of fatty acid methyl ester profile and amplified fragment length polymorphism analyses to distinguish five species of Phytophthora associated with ornamental plants

Fatty acid methyl ester (FAME) profiles and amplified fragment length polymorphisms (AFLPs) were evaluated as tools for identifying species of Phytophthora. Five isolates of each of Phytophthora cactorum, Phytophthora citrophthora, Phytophthora cinnamomi, Phytophthora nicotianae and Phytophthora cryptogea were subjected to both analyses to examine variation among and within species. In FAME analysis, isolates of P. cactorum, P. cinnamomi and P.nicotianae were clustered by species, but isolates of P. citrophthora and P.cryptogea were divided into multiple clusters based on greater variations within these two species. The AFLP analysis differentiated all five species of Phytophthora. The five isolates of each species were grouped in a separate terminal cluster, but diversity within a species cluster varied considerably with variation greater in P. cryptogea and P. citrophthora. Comparing the dendrograms based on FAME and AFLP analyses, the overall patterns of both were similar. The P. cactorum cluster was distinct from clusters of the other four species, which formed one large cluster. The higher values of percentages of polymorphic loci and gene diversity in AFLP analysis substantiated diversity observed among isolates of P. citrophthora and P. cryptogea in FAME and AFLP dendrograms. Both FAME and AFLP appear to be useful tools for identifying species of Phytophthora, but only AFLP analysis has potential to study genetic and phylogenetic relationships within and among species in this genus.     

A study of the vascular arrangement in the rat adrenal gland using non-radioactive microspheres

Summary The intra-glandular vascular arrangement in the adrenal has been studied using non-radioactive microspheres injected by three different routes: in-vivo injection into the left ventricle under pentobarbital anesthesia, postmortem orthograde, and postmortem retrograde injection. The doses of microspheres were 10⁵ (average size 24.7 m), 10⁶ (15.8 m) and 10⁷ (9.9 m). The entrapment rate of microspheres by the medulla as compared with the whole gland was measured in the serially sectioned tissue (section thickness 60 m).The entrapment rates of 25-m microspheres differed between the orthograde and retrograde injections, while the entrapment rates for 15-m microspheres were essentially similar irrespective of the route of injection.Our results support the conclusion from previous microangiographic studies that the adrenal cortex and medulla are supplied by different arteries but have a common venous outflow, and that direct communication between cortical and medullary sinusoids is not likely. The medullary blood flow per gram tissue weight is estimated to be larger than cortical blood flow.     

Plant response to variations in nitrogen availability in a desert shrubland community

Spatial variations in nitrogen availability were studied in a desert community codominated byLarrea tridentata (DC.) Cov. andProsopis glandulosa Torr. Measurements of natural ¹⁵N values in tissues suggested thatProsopis obtains approximately half of its nitrogen through direct symbiotic fixation. Soils were collected under 1)Prosopis shrubs, 2)Larrea shrubs 2 m fromProsopis (LP), and 3)Larrea 2 m from otherLarrea but> 5 m from the nearestProsopis (LL).Prosopis soils showed significantly higher rates of nitrogen mineralization than LL soils in both A and B horizons. Rates of mineralization in LP soils were significantly higher than rates in LL soils only in the B horizon and were not significantly different from rates inProsopis soils. Leaf nitrogen concentrations were significantly higher in LP shrubs (2.06%) than in LL shrubs (1.78%), although ¹⁵N values did not differ between the two shrub types. Nitrogen concentrations inPerezia nana Gray, a perennial herb, were greater in plants underProsopis shrubs (2.09%) than under LP shrubs (1.93%) or LL shrubs (1.67%). Despite apparent differences in nitrogen availability, biomass ofLarrea and density ofPerezia did not differ significantly among these sites.     

Stable oxygen isotopes in<Emphasis Type="Italic"> Porites</Emphasis> corals monitor weekly temperature variations in the northern Gulf of Aqaba,Red Sea

In order to assess the ability of Porites corals to accurately record environmental variations, high-resolution (weekly/biweekly) coral ¹⁸O records were obtained from four coral colonies from the northern Gulf of Aqaba, which grew at depths of 7, 19, 29, and 42 m along one transect. Adjacent to each colony, hourly temperatures, biweekly salinities, and monthly ¹⁸O of seawater were continuously recorded over a period of 14 months (April 1999 to June 2000). Contrary to water temperature, which shows a regular and strong seasonal variation and change with depth, seawater ¹⁸O exhibits a weak seasonality and little change with depth. Positive correlations between seawater ¹⁸O and salinity were observed. The two parameters were related to each other by the equation ¹⁸O _Seawater (, VSMOW) = 0.281 × Salinity – 9.14. The high-resolution coral ¹⁸O records from this study show a regular pattern of seasonality and are able to capture fine details of the weekly average temperature records. They resolve more than 95% of the weekly average temperature range. On the other hand, attenuation and amplification of coral seasonal amplitudes were recorded in deep, slow-growing corals, which were not related to environmental effects (temperature and/or seawater ¹⁸O) or sampling resolution. We propose that these result from a combined effect of subannual variations in extension rate and variable rates of spine thickening of skeletal structures within the tissue layer. However, no smoothing or distortion of the isotopic signals was observed due to calcification within the tissue layer in shallow-water, fast-growing corals. The calculations from coral ¹⁸O calibrations against the in situ measurements show that temperature (T) is related to coral ¹⁸O ( _c ) and seawater ¹⁸O ( _w ) by the equation T (°C) = –5.38 ( _c – _w ) –1.08. Our results demonstrate that coral ¹⁸O from the northern Gulf of Aqaba is a reliable recorder of temperature variations, and that there is a minor contribution of seawater ¹⁸O to this proxy, which could be ignored.     

Survival and growth of early-instar nymphs of Ephemerella subvaria fed various diets

In two laboratory tests at 15 °C early-instar nymphs of the mayfly, Ephemerella subvaria were kept on different diets and their survival and growth recorded. The diets were either conditioned maple leaf discs; maple leaf disc remnants + Gammarus faecal material; maple leaf disc remnants + Hesperophylax faecal material; a clump of Cladophora strands or periphytic growth on small stones. A control group received no food. In both the tests, nymphs kept on Cladophora showed significantly (P 0.05 or P 0.01) higher percentage survival than for those on any other diet and the growth of these nymphs was second only to nymphs given periphyton as a diet. Although the nymphs on a diet of periphyton showed significantly (P 0.01) higher growth as compared to those on other food sources, the survival percentage of these nymphs was the poorest, presumably because of food limitation. As expected, a diet of maple leaf discs resulted in significantly higher survival and growth of nymphs as compared to the nymphs not given any food. The results clearly demonstrated that autochthonous sources of organic matter can serve as excellent food for stream invertebrates.     

Effects of feeding and starvation on growth and swimming activity in an obligatory air-breathing fish

Reared in (tubular) aquaria containing different depths of water, Ophiocephalus striatus (0.7 g, 4.5 cm body length), an obligatory air-breathing tropical fish, swam long or short distances to enable themselves to exchange atmospheric air. In each tested depth (2.5, 5.0, 15.5, 31.0 and 40.0 cm) series, one group was starved, while the other was fed ad libitum twice a day on fish muscle. In the shallowest water (2.5 cm depth), the feeding group surfaced 1,294 times, travelling 64.7 m at an energy cost of 20.4 mg dry fish substance/g live fish/day, against those exposed to the deepest water (40 cm depth), which expended 35.8 mg/g/day, swimming 1,503.4 m on 1,879 visits to the surface. The starving group surfaced only 482 times, travelling 24.1 m at an expense of 5.8 mg/g/day in the shallowest water, while those at 40 cm depth surfaced 504 times, swimming 403.2 m at an energy cost of 7.4 mg/g/day. Owing to the sustained swimming activity and the consequent fatigue, the test individuals belonging to both groups in all the tested series hang to the surface for a definite interval, repaying the O₂ debt. Observations were also made to assess the duration of hanging to precisely estimate the distance travelled. Irrespective of changes in depths of water, the duration of hanging to surface was only 3.0 hr/day for the feeding groups, while it was as much as 15.5 hr/day for the starving groups. The maximum sustained metabolic level of O.striatus reared in 40 cm depth was equivalent to 1.23 ml O₂/g/hr, which is about 2 times higher than the value reported for the active metabolism of swimming Oncorhynchus nerka at 15°C in Brett's (1964) respirometer. O.striatus reared in 2.5 cm depth fed 32.0 mg and converted 6.7 mg dry food/g live fish/day, while those exposed to the deepest water fed 49.1 mg, but converted only 5.5 mg/g/day. Culturing obligatory air-breathing fishes in shallow waters will be advantageous.     

Isolation of zoosporogenous actinomycetes from desert soils

We have undertaken a study to estimate the species diversity of zoosporogenous actinomycetes that can be isolated from an arid environment. The study site encompassed an area of approximately 22 000 square kilometers of the Mojave Desert along the California-Nevada border. A series of 29 soil samples was collected along two intersecting transects of approximately 190 and 240 km which traversed a number of distinct ecosystems. A₀ horizon soils were collected from the rhizosphere of the predominant vegetation at each sampling site and screened for the target genera using selective isolation techniques: chemoattraction (xylose and -collidine) and baiting with hair. Following incubation of primary isolation plates for 28 days at 28°C, all colonies that exhibited filamentous growth, presence of sporangia and/or motile spores upon direct microscopic observation (450 and 1000×) were further characterized by fatty acid analysis (FAME). Most of the isolates fell into three broad clusters that roughly correlated with presumptive genus assignments. Individual isolates could be assigned to 226 FAME biotypes based on chromatographic similarity (85%). The dominant species (514/826 isolates) belong to a previously undescribed taxon that morphologically resemblesGeodermatophilus but possesses unique FAME profiles that include at least three novel lipids. The remainder of the isolates were species ofActinoplanes, indeterminate species or vagrant isolates ofStreptomyces.     

RNA polymerase mutants of Escherichia coli. Streptolydigin resistance and its relation to rifampicin resistance

Summary Several streptolydigin-resistant mutants of Escherichia coli were shown to produce RNA polymerase with increased drug resistance due to a recessive mutation (stl) located between argH and thiA. With one mutant studied, enzyme reconstitution experiments directly demonstrated that the altered subunit is responsible for its drug resistance. It was also found that some mutations (rif or stv) conferring resistance to rifampicin (or streptovaricin) lead to a simultaneous change in resistance to streptolydigin, suggesting certain functional relationship between the polymerase structure affected by rif (or stv) and stl mutations. This inference was further supported by the results of cistron analysis and of extensive transductional mapping involving two stl and a number of rif mutations. Thus it was found that all the mutational sites affecting sensitivity of RNA polymerase to streptolydigin and to rifampicin are closely localized, with partial overlap to each other, within a short segment of the cistron which determines the structure of subunit. These results led us to propose that the subunit is directly responsible for catalyzing both initiation and chain elongation steps of RNA synthesis. The possible bearing of the present findings on the structure-function relationship of the polymerase is discussed.     

Phenotypic and genotypic characterization of antagonistic bacteria associated with roots of transgenic and non-transgenic potato plants

Rhizobacteria obtained during a risk assessment study from parental and transgenic T4 lysozyme-expressing potato plants were investigated to determine whether or not the strains could be grouped based on the source of isolation, transgenic or non-transgenic plants, respectively. A total of 68 representative bacterial strains of the group of enterics and pseudomonads were investigated by phenotypic profiling (the antagonistic activity towards bacterial and fungal plant pathogens, the production of the plant growth hormone indole-3-acetic acid [auxin], and the sensitivity to T4 lysozyme in vitro) and genotypic profiling by PCR fingerprints using BOX primers. All isolates were identified by fatty acid methyl ester (FAME) analysis. Computer-based analysis of the phenotypic characteristics showed that both, enterics and Pseudomonas strains clustered into six to seven groups at an Euclidian distance of 10. According to their BOX-PCR-generated fingerprints the Pseudomonas strains clustered into seven groups and the enterobacteria into two groups at the same genetic distance level of 10. The majority of groups were heterogeneous and contained isolates from all plant lines. In conclusion, cluster analysis of the phenotypic and genotypic features did not reveal correlations between bacterial isolates and transgenic character of plants.     

Bacterial and Fungal Diversity of Quaternary Cave Sediment Deposits

The bacterial and fungal assemblages of clastic sediments collected from two caves located in north-western Romania were investigated by assessing ITS and 16S rRNA gene diversity. Bacterial members belonging to Chloroflexi, Nitrospirae, Proteobacteria, Firmicutes, Acidobacteria, Gemmatimonadetes, and fungal members of Ascomycota were identified. Except for Bacillus sp., all bacteria were related to uncultured or unknown species and the majority (86%) of the bacterial sequences from one of the caves had no close GenBank relatives. The bacterial sequences obtained clustered with species found in extreme environments. Half of the bacterial operational taxonomic units were clustered with clones isolated from deep subsurface sediments of a radioactively contaminated site in the USA. The present study represents the first attempt to identify microorganisms in Quaternary cave sediments.

Supplemental materials are available for this article. Go to the publisher's online edition of Geomicrobiology Journal to view the supplemental file.     

Lack of convexity of threshold curves for compound grating: implications for modelling visual pattern detection

Threshold contrasts were determined for complex gratings made from pairs of sine-wave gratings with various spatial frequencies and contrast ratios. It was found that the plots representing the relationship between the contrast of two-component gratings with frequencies f ₁=2.0 deg^–1 and f ₂=kf ₁, (1.03 k 3.0), when the compound was at threshold (the so-called contrast threshold curves), tend to have either an elliptical (for k 1.5) or a rectangular (for k=3) form. Nevertheless, the elliptical fits as the matched-channel model predicted, and the roundedly rectangular fits, as the probability-summation models predicted, were very poor. Furthermore, statistical analysis shows that two of the six contrast threshold curves exhibiting a significant local disturbance fail to be convex.Theoretical treatment in terms of convex analysis shows that such a convexity violation of the contrast threshold curves rules out the possibility of detection being produced by a single linear channel as well as a parallel array of linear channels, either with or without probability summation, provided that the detection principle of the most sensitive channel is adopted. The adaptive matched-channel model originally proposed by Hauske cannot also account for the results obtained; however, it can be modified to be in line with them. We hypothesise that the detection in our experiment is likely to occur by means of several (no more than seven or eight) adaptive partially matched channels.     

Peptide des β-Alanins als Ersatz für die freie Aminosäure bei pantothensäurebedürftigen Hefezellen und ihr Verhalten gegenüber dem β-Alanin-Antagonisten Asparagin

Zusammenfassung Pantothensäurebedürftige Hefezellen können ihren Bedarf an diesem Vitamin nicht allein aus -Alanin decken, sondern auch aus Benzoyl--Alanin, -Alanyl-d,l-Norleucin und -Alanyl-l-Histidin. Der Antagonist Asparagin hemmt die Verwertung dieser Peptide genauso wie diejenige der freien Aminosäure. Durch höhere Konzentrationen an -Alanin oder -Alanyl-d,l-Norleucin läßt sich die Hemmwirkung nicht allein kompensieren, es kommt sogar zu einer Förderung des Hefewachstums. Der Antagonist wird dann zum Synergisten.

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Summary The -alanine containing peptides benzoyl--alanine, -alanyl-d,l-norleucine and -alanyl-l-histidine can substitute for the amino acid -alanine in a pantothenic acid requiring yeast. Asparagine, an antagonist of -alanine, affects these peptides in a similar manner. In combination with an overdose of -alanine or -alanyl-d,l-norleucine, asparagine is no longer an antagonist but becomes a synergist.

     

Spatial variability of soil total C and N and their stable isotopes in an upland Scottish grassland

As preparation for a below ground food web study, the spatial variability of three soil properties (total N, total C and pH) and two stable isotopes (¹³C and ¹⁵N of whole soil) were quantified using geostatistical approaches in upland pastures under contrasting management regimes (grazed, fertilised and ungrazed, unfertilised) in Scotland. This is the first such study of upland, north maritime grasslands. The resulting patterns of variability suggest that to obtain statistically independent samples in this system, a sampling distance of 13.5 m is required. Additionally, temporal change (a decline of 1) was observed in whole soil ¹⁵N for the grazed, fertilised plot. This may have been caused by new inputs of symbiotically-fixed atmospheric N₂.     

Use of cluster analysis with monoclonal antibodies for taxonomic differentiation of phytopathogenic fungi and for screening and clustering antibodies

Cluster analysis of ELISA (A405) values resulting from tests using all possible combinations of 43 monoclonal antibodies (raised againstPhytophthora megasperma f.sp.glycinea) and 14 phytopathogenic fungi separated these fungi into taxonomically appropriate clusters. Two distance measurements (Pearson's correlation coefficient and normalized Euclidean distance) and five linkage methods (single, complete, median, centroid, and average) were evaluated. The combination of Pearson's correlation coefficient and complete linkage produced the most natural clusters. A modification of the data matrix allowed clustering of the monoclonal antibodies by specificity. Numerically coded ELISA values clustered identically with the orginal data. The taxonomic and evolutionary implications of using monoclonal antibodies specific to carbohydrates are discussed.