A Comparison of the Use of In Vitro-Transcribed and Native rRNA for the Quantification of Microorganisms in the Environment

Abstract Nearly full-length, small subunit (SSU) rRNA was transcribed in vitro from clones of SSU rDNA genes. Comparing the use of in vitro-transcribed and native rRNA indicated that, when in vitro-transcribed rRNA was used as a standard for quantitative hybridizations with oligonucleotide probes, the population was consistently underestimated. The population abundance was expressed as a percentage of specific target SSU rRNA (determined with a specific oligonucleotide probe), relative to the total SSU rRNA (measured with a universal probe). Differences in hybridization signals could be related to specific probe target locations and rRNA denaturation conditions, suggesting that higher order structure is important in quantitative membrane hybridizations. Therefore, in vitro-transcribed rRNA cannot always be used for the absolute quantification of microbial populations, but can be employed as a standard to quantify shifts in population abundance over time, and to compare community structure in various environments.     

Fertility table and rate of population growth of the predator Supputius cincticeps (Heteroptera: Pentatomidae) on one plant of Eucalyptus cloeziana in the field

The reproductive potential and population growth (r_m and λ) of the predator Supputius cincticeps (Heteroptera: Pentatomidae) were evaluated using a life and fertility table. S. cincticeps was reared on one plant of Eucalyptus cloeziana in the field and fed with Tenebrio molitor (Coleoptera: Tenebrionidae) pupae. Females of this predator had a net reproductive rate (R₀) of 21.02 females/female; an intrinsic rate of population increase (r_m) of 0.041 and finite rate of increase (λ) of 1.042. This resulted in population growth of S. cincticeps in the eucalyptus plant with a doubling time of 17.01 days. This natural enemy can be reared under field conditions with alternative prey for use in biological control. Such individuals of S. cinticeps will be better adapted to field conditions when they are liberated.     

Mitochondrial DNA sequence variation in the spruce budworm species complex (Choristoneura: Lepidoptera)

A combination of polymerase-chain-reaction amplification and automated DNA sequencing was used to survey variation in a species complex of pest insects, the spruce budworms (Choristoneura fumiferana species group), and an outgroup species, C. rosaceana. We sequenced an mtDNA region of 1,573 bp that extends from the middle of cytochrome oxidase subunit I (COI) through tRNA leucine (UUR) to the end of cytochrome oxidase subunit II. In addition, we examined levels of intraspecific variation within a 470-bp region of the COI gene. Choristoneura fumiferana clearly represented the oldest lineage within its species group, with 2.7%-2.9% sequence divergence from the other species. In contrast, the four remaining species (C. pinus, C. biennis, C. occidentalis, and C. orae) had closely related or identical mtDNA, with < 1% divergence among most of their haplotypes. Despite its older lineage and widespread geographic distribution, C. fumiferana showed significantly lower intraspecific genetic diversity than did C. occidentalis. Choristoneura orae shared haplotypes with C. occidentalis and C. biennis, and species-level separation of these three species was not supported. Two divergent, uncommon haplotypes were also found in C. occidentalis and C. biennis. The divergent haplotype in C. biennis had an unusually high number of inferred amino acid replacements, suggesting selective differences between mitochondrial DNA haplotypes. Transition:transversion ratios in Choristoneura paralleled those found in Drosophila; transition:transversion ratios were highest in closely related sequences but decreased with increasing sequence divergence. Nucleotide composition showed an A+T bias that was near the high end of the range known for insects. This work illustrates the potential utility of direct DNA sequencing in assessing population structures, species limits, and phylogenetic relationships among organisms that have not previously been subjected to DNA analysis.      

A Synthetic Medium for Comparative Nutritional Studies of Lactobacilli

The composition of a synthetic medium supporting the growth of lactobacilli is given (Table 1). The medium, containing glucose, amino acids, vitamins, mineral salts, purines and pyrimidines, allows the study of nutritional requirements of different strains of lactobacilli under identical environmental conditions. It was found that all the strains tested needed L-glutamic acid, L-valine and L-leucine, and a group of them also required L-arginine, L-tyrosine and L-tryptophan. Some strains required vitamins, e.g. L. bulgaricus (pantothenic acid), L. fermenti (pantothenic acid and niacin). These results are compared with those found by others employing different media.     

Monocaryotization of Cultures of Lenzites trabea (Pers.) Fr. and other Wood-destroying Basidiomycetes

Cultures of Lenzites trabea and some other basidiomycetes grownon nutrient agar containing high concentrations of sodium arsenateconsistently reverted from the dicaryotic to the monocaryoticcondition. This effect could also be induced by a wide varietyof other toxicants, including copper sulphate, zinc sulphate,sodium dichromate, sodium pentachlorophenate, creosote, crystalviolet, boric acid, and sodium taurocholate, but sodium arsenatewas the most reliable monocaryotizing agent, being effectivewith eight of the fourteen basidio-mycete species tested withit. The neohaplonts formed by sodium arsenate and other toxicantsmate normally with each other and with compatible monosporeisolates. The value of chemical monocaryotization as a techniquein fungal genetics and experimental taxonomy is discussed. Byusing the mating factor alleles as genetic markers, it was demonstratedthat when L. trabea is monocaryo-tized by sodium arsenate orcopper sulphate, only one of the two nuclei in the dicaryoncan be recovered in the neohaplonts, the direction of this completenuclear selection depending on the toxicant and the dicaryonconcerned. It was shown to occur in treated wood blocks duringroutine soil-block decay tests of inorganic wood preservativesand it is probably a frequent, though hitherto unrecognized,occurrence in the laboratory testing of wood preservatives andother fungicides against basidiomycetes.     

Phosphorus benefits of different legume crops to subsequent wheat grown in different soils of Western Australia

Certain legume crops, including white lupin (Lupinus albus L.), mobilise soil-bound phosphorus (P) through root exudates. The changes in the rhizosphere enhance P availability to these crops, and possibly to subsequent crops growing in the same soil. We conducted a pot experiment to compare phosphorus acquisition of three legume species with that of wheat, and to determine whether the legume crops influence growth and P uptake of a subsequent wheat crop. Field pea (Pisum sativum L.), faba bean (Vicia faba L.), white lupin (Lupinus albus L.) and wheat (Triticum aestivum L.) were grown in three different soils to which we added no or 20 mg P kg^–1 soil (P0, P20). Growth, P content and rhizosphere carboxylates varied significantly amongst crops, soils and P levels. Total P content of the plants was increased with applied phosphorus. Phosphorus content of faba bean was 3.9 and 8.8 mg/pot, at P0 and P20, respectively, which was about double that of all other species at the respective P levels. Field pea and white lupin had large amounts of rhizosphere carboxylates, whereas wheat and faba bean had negligible amounts in all three soils at both P levels. Wheat grew better after legumes than after wheat in all three soils. The effect of the previous plant species was greater when these previous species had received P fertiliser. All the legumes increased plant biomass of subsequent wheat significantly over the unplanted pots in all the soils. Faba bean was unparalleled in promoting subsequent wheat growth on all fertilised soils. This experiment clearly demonstrated a residual benefit of the legume crops on the growth of the subsequent wheat crop due to enhanced P uptake. Faba bean appeared to be a suitable P-mobilising legume crop plant for use in rotations with wheat.