Ecology of uncultivated oscillospira species in the rumen of cattle, sheep, and reindeer as assessed by microscopy and molecular approaches

The ecology of the uncultured, but large and morphologically conspicuous, rumen bacterium Oscillospira spp. was studied. Oscillospira-specific 16S rRNA gene sequences were detected in North American domestic cattle, sheep from Australia and Japan, and Norwegian reindeer. Phylogenetic analysis of the sequences obtained allowed definition of three operational taxonomic units within the Oscillospira clade. Consistent with this genetic diversity, we observed atypical smaller morphotypes by using an Oscillospira-specific fluorescence in situ hybridization probe. Despite the visual disappearance of typical large Oscillospira morphotypes, the presence of Oscillospira spp. was still detected by Oscillospira-specific PCR in the rumen of cattle and sheep. These observations suggest the broad presence of Oscillospira species in various rumen ecosystems with the level, and most likely the morphological form, dependent on diet. An ecological analysis based on enumeration of the morphologically conspicuous, large-septate form confirms that the highest counts are associated with the feeding of fresh forage diets to cattle and sheep and in two different subspecies of reindeer investigated.

Molecular tools for studying puberty in the grey mullet, Mugil cephalus: cloning of cDNAs that regulate reproductive function

The cDNAs coding for the brain GnRHs (AY373449-51), pituitary GH, SL and PRL, and liver IGFs (AY427954-5) were isolated. Partial cDNA sequences of the brain (Cyp19b) and gonadal (Cyp19a) aromatases have also been obtained. These tools would be utilized to study the endocrine regulation of puberty in the grey mullet.

Sustaining productivity of a Vertosol at Warra, Queensland, with fertilisers, no-tillage or legumes. 7. Yield, nitrogen and disease-break benefits from lucerne in a two-year lucerne–wheat rotation

Continuous cultivation and cereal cropping of southern Queensland soils previously supporting native vegetation have resulted in reduced soil nitrogen supply, and consequently decreased cereal grain yields and low grain protein. To enhance yields and protein concentrations of wheat, management practices involving N fertiliser application, with no-tillage and stubble retention, grain legumes, and legume leys were evaluated from 1987 to 1998 on a fertility-depleted Vertosol at Warra, southern Queensland. The objective of this study was to examine the effect of lucerne in a 2-year lucerne–wheat rotation for its nitrogen and disease-break benefits to subsequent grain yield and protein content of wheat as compared with continuous wheat cropping. Dry matter production and nitrogen yields of lucerne were closely correlated with the total rainfall for October–September as well as March–September rainfall. Each 100 mm of total rainfall resulted in 0.97 t/ha of dry matter and 26 kg/ha of nitrogen yield. For the March–September rainfall, the corresponding values were 1.26 t/ha of dry matter and 36 kg/ha of nitrogen yield. The latter values were 10% lower than those produced by annual medics during a similar period. Compared with wheat–wheat cropping, significant increases in total soil nitrogen were observed only in 1990, 1992 and 1994 but increases in soil mineralisable nitrogen were observed in most years following lucerne. Similarly, pre-plant nitrate nitrogen in the soil profile following lucerne was higher by 74 kg/ha (9–167 kg N/ha) than that of wheat–wheat without N fertiliser in all years except 1996. Consequently, higher wheat grain protein (7 out of 9 seasons) and grain yield (4 out of 9 seasons) were produced compared with continuous wheat. There was significant depression in grain yield in 2 (1993 and 1995) out of 9 seasons attributed to soil moisture depletion and/or low growing season rainfall. Consequently, the overall responses in yield were lower than those of 50 kg/ha of fertiliser nitrogen applied to wheat–wheat crops, 2-year medic–wheat or chickpea–wheat rotation, although grain protein concentrations were higher following lucerne. The incidence and severity of the soilborne disease, common root rot of wheat caused by Bipolaris sorokiniana, was generally higher in lucerne–wheat than in continuous wheat with no nitrogen fertiliser applications, since its severity was significantly correlated with plant available water at sowing. No significant incidence of crown rot or root lesion nematode was observed. Thus, productivity, which was mainly due to nitrogen accretion in this experiment, can be maintained where short duration lucerne leys are grown in rotations with wheat.

Functionally associated molecular genetic marker map construction in perennial ryegrass (Lolium perenne L.)

A molecular marker-based map of perennial ryegrass (Lolium perenne L.) has been constructed through the use of polymorphisms associated with expressed sequence tags (ESTs). A pair-cross between genotypes from a North African ecotype and the cultivar Aurora was used to generate a two-way pseudo-testcross population. A selection of 157 cDNAs assigned to eight different functional categories associated with agronomically important biological processes was used to detect polymorphic EST–RFLP loci in the F1(NA6 × AU6) population. A comprehensive set of EST–SSR markers was developed from the analysis of 14,767 unigenes, with 310 primer pairs showing efficient amplification and detecting 113 polymorphic loci. Two parental genetic maps were produced: the NA6 genetic map contains 88 EST–RFLP and 71 EST–SSR loci with a total map length of 963 cM, while the AU6 genetic map contains 67 EST–RFLP and 58 EST–SSR loci with a total map length of 757 cM. Bridging loci permitted the alignment of homologous chromosomes between the parental maps, and a sub-set of genomic DNA-derived SSRs was used to relate linkage groups to the perennial ryegrass reference map. Regions of segregation distortion were identified, in some instances in common with other perennial ryegrass maps. The EST-derived marker-based map provides the basis for in silico comparative genetic mapping, as well as the evaluation of co-location between QTLs and functionally associated genetic loci.

Molecular characterization and pathogenicity of Pythium species associated with damping-off in greenhouse cucumber (Cucumis sativus) in Oman

A study was undertaken in 2004 and 2005 to characterize pathogens associated with damping-off of greenhouse-grown cucumber seedlings in 13 districts in Oman. Identification of Pythium to the species level was based on sequences of the internal transcribed spacer (ITS) of the ribosomal DNA. Of the 98 Pythium isolates collected during the survey, Pythium aphanidermatum, P. spinosum, P. splendens and P. oligandrum accounted for 76%, 22%, 1% and 1%, respectively. Pythium aphanidermatum was isolated from all of the districts, while P. spinosum was isolated from seven districts. Pathogenicity tests showed inter- and intraspecific variation in aggressiveness between Pythium species. Pythium aphanidermatum, P. spinosum and P. splendens were found to be highly aggressive at 25°C. However, the aggressiveness of P. spinosum decreased when the temperature was raised to 30°C, which was found to correspond to the lower frequency of isolation of P. spinosum in the warmer seasons, compared to the cooler time of the year. Pythium aphanidermatum exhibited limited intraspecific variation in the sequences of the ITS region of the rDNA and showed 100% similarity to the corresponding P. aphanidermatum sequences from GenBank. The ITS sequence data, as well as morphological characteristics of P. spinosum isolates, showed a high level of similarity within and between P. spinosum and P. kunmingense, and suggested that the two species were synonymous. This study represents the first report of P. spinosum, P. splendens and P. oligandrum in Oman.

Molecular discrimination of Perna (Mollusca: Bivalvia) species using the polymerase chain reaction and species-specific mitochondrial primers

This work was prompted by the need to be able to identify the invasive mussel species, Perna viridis, in tropical Australian seas using techniques that do not rely solely on morphology. DNA-based molecular methods utilizing a polymerase chain reaction (PCR) approach were developed to distinguish unambiguously between the three species in the genus Perna. Target regions were portions of two mitochondrial genes, cox1 and nad4, and the intergenic spacer between these that occurs in at least two Perna species. Based on interspecific sequence comparisons of the nad4 gene, a conserved primer has been designed that can act as a forward primer in PCRs for any Perna species. Four reverse primers have also been designed, based on nad4 and intergenic spacer sequences, which yield species-specific products of different lengths when paired with the conserved forward primer. A further pair of primers has been designed that will amplify part of the cox1 gene of any Perna species, and possibly other molluscs, as a positive control to demonstrate that the PCR is working.

Recent advances in the molecular biology of Leifsonia xyli subsp. xyli, causal organism of ratoon stunting disease

Twelve years ago our understanding of ratoon stunting disease (RSD) was confined almost exclusively to diagnosis of the disease and control via farm hygiene, with little understanding of the biology of the interaction between the causal agent (Leifsonia xyli subsp. xyli) and the host plant sugarcane (Saccharum spp. hybrids). Since then, research has focused on developing the molecular tools to dissect L. xyli subsp. xyli, so that better control strategies can be developed to prevent losses from RSD. Within this review, we give a brief overview of the progression in research on L. xyli subsp. xyli and highlight future challenges. After a brief historical background on RSD, we discuss the development of molecular tools such as transformation and transposon mutagenesis and discuss the apparent lack of genetic diversity within the L. xyli subsp. xyli world population. We go on to discuss the sequencing of the genome of L. xyli subsp. xyli, describe the key findings and suggest some future research based on known deficiencies that will capitalise on this tremendous knowledge base to which we now have access.

Assessing the relationship between patch type and soil mites: A molecular approach

An urgent need exists for indicators of soil health and patch functionality in extensive rangelands that can be measured efficiently and at low cost. Soil mites are candidate indicators, but their identification and handling is so specialised and time-consuming that their inclusion in routine monitoring is unlikely. The aim of this study was to measure the relationship between patch type and mite assemblages using a conventional approach. An additional aim was to determine if a molecular approach traditionally used for soil microbes could be adapted for soil mites to overcome some of the bottlenecks associated with soil fauna diversity assessment. Soil mite species abundance and diversity were measured using conventional ecological methods in soil from patches with perennial grass and litter cover (PGL), and compared to soil from bare patches with annual grasses and/or litter cover (BAL). Soil mite assemblages were also assessed using a molecular method called terminal-restriction fragment length polymorphism (T-RFLP) analysis. The conventional data showed a relationship between patch type and mite assemblage. The Prostigmata and Oribatida were well represented in the PGL sites, particularly the Aphelacaridae (Oribatida). For T-RFLP analysis, the mite community was represented by a series of DNA fragment lengths that reflected mite sequence diversity. The T-RFLP data showed a distinct difference in the mite assemblage between the patch types. Where possible, T-RFLP peaks were matched to mite families using a reference 18S rDNA database, and the Aphelacaridae prevalent in the conventional samples at PGL sites were identified, as were prostigmatids and oribatids. We identified limits to the T-RFLP approach and this included an inability to distinguish some species whose DNA sequences were similar. Despite these limitations, the data still showed a clear difference between sites, and the molecular taxonomic inferences also compared well with the conventional ecological data. The results from this study indicated that the T-RFLP approach was effective in measuring mite assemblages in this system. The power of this technique lies in the fact that species diversity and abundance data can be obtained quickly because of the time taken to process hundreds of samples, from soil DNA extraction to data output on the gene analyser, can be as little as 4 days.

Rice molecular breeding laboratories in the genomics era: Current status and future considerations

Using DNA markers in plant breeding with marker-assisted selection (MAS) could greatly improve the precision and efficiency of selection, leading to the accelerated development of new crop varieties. The numerous examples of MAS in rice have prompted many breeding institutes to establish molecular breeding labs. The last decade has produced an enormous amount of genomics research in rice, including the identification of thousands of QTLs for agronomically important traits, the generation of large amounts of gene expression data, and cloning and characterization of new genes, including the detection of single nucleotide polymorphisms. The pinnacle of genomics research has been the completion and annotation of genome sequences for indica and japonica rice. This information-coupled with the development of new genotyping methodologies and platforms, and the development of bioinformatics databases and software tools-provides even more exciting opportunities for rice molecular breeding in the 21st century. However, the great challenge for molecular breeders is to apply genomics data in actual breeding programs. Here, we review the current status of MAS in rice, current genomics projects and promising new genotyping methodologies, and evaluate the probable impact of genomics research. We also identify critical research areas to "bridge the application gap" between QTL identification and applied breeding that need to be addressed to realize the full potential of MAS, and propose ideas and guidelines for establishing rice molecular breeding labs in the postgenome sequence era to integrate molecular breeding within the context of overall rice breeding and research programs.

Concordant molecular and phenotypic data delineate new taxonomy and conservation priorities for the endangered mountain yellow-legged frog

The mountain yellow-legged frog Rana muscosa sensu lato, once abundant in the Sierra Nevada of California and Nevada, and the disjunct Transverse Ranges of southern California, has declined precipitously throughout its range, even though most of its habitat is protected. The species is now extinct in Nevada and reduced to tiny remnants in southern California, where as a distinct population segment, it is classified as Endangered. Introduced predators (trout), air pollution and an infectious disease (chytridiomycosis) threaten remaining populations. A Bayesian analysis of 1901 base pairs of mitochondrial DNA confirms the presence of two deeply divergent clades that come into near contact in the Sierra Nevada. Morphological studies of museum specimens and analysis of acoustic data show that the two major mtDNA clades are readily differentiated phenotypically. Accordingly, we recognize two species, Rana sierrae, in the northern and central Sierra Nevada, and R. muscosa, in the southern Sierra Nevada and southern California. Existing data indicate no range overlap. These results have important implications for the conservation of these two species as they illuminate a profound mismatch between the current delineation of the distinct population segments (southern California vs. Sierra Nevada) and actual species boundaries. For example, our study finds that remnant populations of R. muscosa exist in both the southern Sierra Nevada and the mountains of southern California, which may broaden options for management. In addition, despite the fact that only the southern California populations are listed as Endangered, surveys conducted since 1995 at 225 historic (1899-1994) localities from museum collections show that 93.3% (n=146) of R. sierrae populations and 95.2% (n=79) of R. muscosa populations are extinct. Evidence presented here underscores the need for revision of protected population status to include both species throughout their ranges.

Sustaining productivity of a Vertosol at Warra, Queensland, with fertilisers, no-tillage or legumes. 9. Production and nitrogen benefits from mixed grass and legume pastures in rotation with wheat

Reduced supplies of nitrogen (N) in many soils of southern Queensland that were cropped exhaustively with cereals over many decades have been the focus of much research to avoid declines in profitability and sustainability of farming systems. A 45-month period of mixed grass (purple pigeon grass, Setaria incrassata Stapf; Rhodes grass, Chloris gayana Kunth.) and legume (lucerne, Medicago sativa L.; annual medics, M. scutellata L. Mill. and M. truncatula Gaertn.) pasture was one of several options that were compared at a fertility-depleted Vertosol at Warra, southern Queensland, to improve grain yields or increase grain protein concentration of subsequent wheat crops. Objectives of the study were to measure the productivity of a mixed grass and legume pasture grown over 45 months (cut and removed over 36 months) and its effects on yield and protein concentrations of the following wheat crops. Pasture production (DM t/ha) and aboveground plant N yield (kg/ha) for grass, legume (including a small amount of weeds) and total components of pasture responded linearly to total rainfall over the duration of each of 3 pastures sown in 1986, 1987 and 1988. Averaged over the 3 pastures, each 100 mm of rainfall resulted in 0.52 t/ha of grass, 0.44 t/ha of legume and 0.97 t/ha of total pasture DM, there being little variation between the 3 pastures. Aboveground plant N yield of the 3 pastures ranged from 17.2 to 20.5 kg/ha per 100 mm rainfall. Aboveground legume N in response to total rainfall was similar (10.6 - 13.2 kg/ha. 100 mm rainfall) across the 3 pastures in spite of very different populations of legumes and grasses at establishment. Aboveground grass N yield was 5.2 - 7.0 kg/ha per 100mm rainfall. In most wheat crops following pasture, wheat yields were similar to that of unfertilised wheat except in 1990 and 1994, when grain yields were significantly higher but similar to that for continuous wheat fertilised with 75 kg N/ha. In contrast, grain protein concentrations of most wheat crops following pasture responded positively, being substantially higher than unfertilised wheat but similar to that of wheat fertilised with 75 kg N/ha. Grain protein averaged over all years of assay was increased by 25 - 40% compared with that of unfertilised wheat. Stored water supplies after pasture were < 134mm (< 55% of plant available water capacity); for most assay crops water storages were 67 - 110 mm, an equivalent wet soil depth of only 0.3 - 0.45 m. Thus, the crop assays of pasture benefits were limited by low water supply to wheat crops. Moreover, the severity of common root rot in wheat crop was not reduced by pasture - wheat rotation.

Molecular and serological detection of A. centrale- and A. marginale-infected cattle grazing within an endemic area

A reverse line blot hybridization (RLB) one-stage nested PCR (nPCR) for Anaplasma centrale and a nested PCR for Anaplasma marginale were used to detect infected cattle grazing within an endemic region in Israel. A novel set of PCR primers and oligonucleotide probes based on a 16S ribosomal RNA gene was designed for RLB detection of both Anaplasma species, and the performance of the molecular assays compared. The immunofluorescent antibody test (IFA) was used to detect antibodies to both Anaplasma species, whereas, a highly sensitive and specific competitive enzyme-linked immunosorbent assay (cELISA) was used to detect antibodies in A. centrale-vaccinated cattle. The RLB and the nested PCR procedures showed bacteremia with sensitivity of 50 infected erythrocytes per milliliter. Up to 93% of the A. centrale vaccinates carried specific antibodies that were detected by cELISA, and up to 71% of the vaccinated cattle were found to be naturally infected with A. marginale according to the PCR and the RLB assays. Nevertheless, no severe outbreaks of A. marginale infection occurred among vaccinated herds in this endemic region. It appears that both, molecular tools and serology are useful for evaluation of the vaccine efficacy. In the light of wide natural field infection with A. marginale, strong recommendations to continue the A. centrale vaccination program regime will continue until a new generation of non-blood-based vaccine will be developed.

Molecular characterisation, pathogenesis and fungicide sensitivity of Pythium spp. from table beet (Beta vulgaris var. vulgaris) grown in the Lockyer Valley, Queensland

Table beet production in the Lockyer Valley of south-eastern Queensland is known to be adversely affected by soilborne root disease from infection by Pythium spp. However, little is known regarding the species or genotypes that are the causal agents of both pre- and post-emergence damping off. Based on RFLP analysis with HhaI, HinfI and MboI of the PCR amplified ITS region DNA from soil and diseased plant samples, the majority of 130 Pythium isolates could be grouped into three genotypes, designated LVP A, LVP B and LVP C. These groups comprised 43, 41 and 7% of all isolates, respectively. Deoxyribonucleic acid sequence analysis of the ITS region indicated that LVP A was a strain of Pythium aphanidermatum, with greater than 99% similarity to the corresponding P. aphanidermatum sequences from the publicly accessible databases. The DNA sequences from LVP B and LVP C were most closely related to P. ultimum and P. dissotocum, respectively. Lower frequencies of other distinct isolates with unique RFLP patterns were also obtained with high levels of similarity (>97%) to P. heterothallicum, P. periplocum and genotypes of P. ultimum other than LVP B. Inoculation trials of 1- and 4-week-old beet seedlings indicated that compared with isolates of the LVP B genotype, a higher frequency of LVP A isolates caused disease. Isolates with the LVP A, LVP B and LVP C genotypes were highly sensitive to the fungicide Ridomil MZ, which suppressed radial growth on V8 agar between approximately four and thirty fold at 5 μg/mL metalaxyl and 40 μg/mL mancozeb, a concentration far lower than the recommended field application rate.

A new semi-dwarfing gene identified by molecular mapping of quantitative trait loci in barley.

Semi-dwarfing genes have been widely used in spring barley (Hordeum vulgare L.) breeding programs in many parts of the world, but the success in developing barley cultivars with semi-dwarfing genes has been limited in North America. Exploiting new semi-dwarfing genes may help in solving this dilemma. A recombinant inbred line population was developed by crossing ZAU 7, a semi-dwarf cultivar from China, to ND16092, a tall breeding line from North Dakota. To identify quantitative trait loci (QTL) controlling plant height, a linkage map comprised of 111 molecular markers was constructed. Simple interval mapping was performed for each of the eight environments. A consistent QTL for plant height was found on chromosome 7HL. This QTL is not associated with maturity and rachis internode length. We suggest the provisional name Qph-7H for this QTL. Qph-7H from ZAU 7 reduced plant height to about 3/4 of normal; thus, Qph-7H is considered a semi-dwarfing gene. Other QTLs for plant height were found, but their expression was variable across the eight environments tested.

Interactions between rotation breaks, tillage and N management on sugarcane grown at Bundaberg and Ingham.

The impact of cropping histories (sugarcane, maize and soybean), tillage practices (conventional tillage and direct drill) and fertiliser N in the plant and 1st ratoon (1R) crops of sugarcane were examined in field trials at Bundaberg and Ingham. Average yields at Ingham (Q200) and Bundaberg (Q151) were quite similar in both the plant crop (83 t/ha and 80 t/ha, respectively) and the 1R (89 t/ha v 94 t/ha, respectively), with only minor treatment effects on CCS at each site. Cane yield responses to tillage, break history and N fertiliser varied significantly between sites. There was a 27% yield increase in the plant crop from the soybean fallow at Ingham, with soybeans producing a yield advantage over continuous cane, but there were no clear break effects at Bundaberg - possibly due to a complex of pathogenic nematodes that responded differently to soybeans and maize breaks. There was no carryover benefit of the soybean break into the 1R crop at Ingham, while at Bundaberg the maize break produced a 15% yield advantage over soybeans and continuous cane. The Ingham site recorded positive responses to N fertiliser addition in both the plant (20% yield increase) and 1R (34% yield increase) crops, but there was negligible carryover benefit from plant crop N in the 1R crop, or of a reduced N response after a soybean rotation. By contrast, the Bundaberg site showed no N response in any history in the plant crop, and only a small (5%) yield increase with N applied in the 1R crop. There was again no evidence of a reduced N response in the 1R crop after a soybean fallow. There were no significant effects of tillage on cane yields at either site, although there were some minor interactions between tillage, breaks and N management in the 1R crop at both sites. Crop N contents at Bundaberg were more than 3 times those recorded at Ingham in both the plant and 1R crops, with N concentrations in millable stalk at Ingham suggesting N deficiencies in all treatments. There was negligible additional N recovered in crop biomass from N fertiliser application or soybean residues at the Ingham site. There was additional N recovered in crop biomass in response to N fertiliser and soybean breaks at Bundaberg, but effects were small and fertiliser use efficiencies poor. Loss pathways could not be quantified, but denitrification or losses in runoff were the likely causes at Ingham while leaching predominated at Bundaberg. Results highlight the complexity involved in developing sustainable farming systems for contrasting soil types and climatic conditions. A better understanding of key sugarcane pathogens and their host range, as well as improved capacity to predict in-crop N mineralisation, will be key factors in future improvements to sugarcane farming systems.