High throughput pH bioassay demonstrates pH adaptation of Rhizobium strains isolated from the nodules of Trifolium subterraneum and T. repens.

The purpose of developing this high throughput assay was to determine whether there was evidence of pH adaptation in strains of rhizobia which nodulate subterranean clover (SC) and white clover (WC), and whether this was related to the pH of the soil of origin. pH is a first-order factor influencing the niche preferences of soil microorganisms and has been convincingly shown to be a key driver of soil bacterial communities. Naturalised strains of Rhizobium spp. that are pH-adapted may have the potential to better compete and/or persist in acidic or alkaline soils compared with introduced commercial strains. Three pilot studies were conducted to design the optimised bioassay. This bioassay tested the effect of pH-amended yeast mannitol broth (seven pH values from pH 4.5-9.0), across three time points, on the in vitro growth of 299 Rhizobium strains isolated from the nodules of SC and WC. The media pH where strains demonstrated fastest growth was related to the pH of the soil that strains were isolated from. However, the correlation between media pH and soil pH was strongly influenced by the growth of strains from alkaline soils (alkaline adaptation), especially in strains isolated from SC nodules.

Non-host larvae negatively impact persistence of the entomopathogen Beauveria bassiana in soil.

A better understanding of the ecology of the insect pathogenic fungus, Beauveria bassiana, in soil is needed to identify reasons behind the variable efficacy often seen after field application. A transformed strain of a candidate commercial strain of B. bassiana (F418 gfp tr3), expressing the green fluorescent protein and the hygromycin B resistance gene, was used to assess the effects of the larvae of a host insect, Tenebrio molitor L. (Coleoptera: Tenebrionidae), a non-host, Costelytra zealandica (Coleoptera: Scarabaeidae) and the absence of larvae on the persistence of F418 gfp tr3 in pasteurised and non-sterile soil over 4 months. In the presence of a T. molitor larvae, F418 gfp tr3 populations increased significantly in pasteurised and non-sterile soil; however, populations increased less in non-sterile soil than in pasteurised soil. Lower populations of F418 gfp tr3 were recovered in pasteurised soil in the presence of C. zealandica larvae than in pasteurised soil without larvae. No difference was observed between F418 gfp tr3 populations in non-sterile soil with a non-host larvae or without larvae. Accompanying studies showed that F418 gfp tr3 conidia germinated and produced appressoria on live and excised cuticle of non-host (C. zealandica) larvae but infection did not occur, leading to a net loss of viable conidia in the soil. Conidia administrated orally to C. zealandica larvae were viable on recovery from faecal samples, suggesting that ingestion of the fungus by the larvae had little impact on the viable fungal population. Soil bacterial and fungal community patterns were analysed using Single-Strand Conformation Polymorphism (SSCP) and showed a correlation between changes in F418 gfp tr3 persistence in pasteurised and non-sterile soil and changes in soil communities in the presence of a host insect, non-host insect or in the absence of insect. In pasteurised soil, non-specific germination of F418 gfp tr3 conidia on the non-host larval cuticle and the presence of antagonistic bacteria introduced with the field-collected larvae are most likely responsible for the differences observed. The more complex microbial community structures in non-sterile soil could lead to fungistasis, preventing potentially antagonistic bacteria degrading conidia or inhibiting attachment and germination on the non-host larval cuticle, resulting in the observed lack of difference between non-host and no larval treatments.

Genome sequence of the clover symbiont Rhizobium leguminosarum bv. trifolii strain CC275e.

Rhizobium leguminosarum bv. trifolii strain CC275e is a highly effective, N2-fixing microsymbiont of white clover (Trifolium repens L.). The bacterium has been widely used in both Australia and New Zealand as a clover seed inoculant and, as such, has delivered the equivalent of millions of dollars of nitrogen into these pastoral systems. R. leguminosarum strain CC275e is a rod-shaped, motile, Gram-negative, non-spore forming bacterium. The genome was sequenced on an Illumina MiSeq instrument using a 2 × 150 bp paired end library and assembled into 29 scaffolds. The genome size is 7,077,367 nucleotides, with a GC content of 60.9 %. The final, high-quality draft genome contains 6693 protein coding genes, close to 85 % of which were assigned to COG categories. This Whole Genome Shotgun project has been deposited at DDBJ/EMBL/GenBank under the accession JRXL00000000. The sequencing of this genome will enable identification of genetic traits associated with host compatibility and high N2 fixation characteristics in Rhizobium leguminosarum. The sequence will also be useful for development of strain-specific markers to assess factors associated with environmental fitness, competiveness for host nodule occupancy, and survival on legume seeds (New Zealand Ministry of Business, Innovation and Employment program, 'Improving forage legume-rhizobia performance' contract C10X1308 and DairyNZ Ltd.).

Mapping a candidate gene (MdMYB10) for red flesh and foliage colour in apple.

BACKGROUND: Integrating plant genomics and classical breeding is a challenge for both plant breeders and molecular biologists. Marker-assisted selection (MAS) is a tool that can be used to accelerate the development of novel apple varieties such as cultivars that have fruit with anthocyanin through to the core. In addition, determining the inheritance of novel alleles, such as the one responsible for red flesh, adds to our understanding of allelic variation. Our goal was to map candidate anthocyanin biosynthetic and regulatory genes in a population segregating for the red flesh phenotypes. RESULTS: We have identified the Rni locus, a major genetic determinant of the red foliage and red colour in the core of apple fruit. In a population segregating for the red flesh and foliage phenotype we have determined the inheritance of the Rni locus and DNA polymorphisms of candidate anthocyanin biosynthetic and regulatory genes. Simple Sequence Repeats (SSRs) and Single Nucleotide Polymorphisms (SNPs) in the candidate genes were also located on an apple genetic map. We have shown that the MdMYB10 gene co-segregates with the Rni locus and is on Linkage Group (LG) 09 of the apple genome. CONCLUSION: We have performed candidate gene mapping in a fruit tree crop and have provided genetic evidence that red colouration in the fruit core as well as red foliage are both controlled by a single locus named Rni. We have shown that the transcription factor MdMYB10 may be the gene underlying Rni as there were no recombinants between the marker for this gene and the red phenotype in a population of 516 individuals. Associating markers derived from candidate genes with a desirable phenotypic trait has demonstrated the application of genomic tools in a breeding programme of a horticultural crop species.