Maureen O’Callaghan

Microbial inoculation of seed for improved crop performance: issues and opportunities

There is increasing interest in the use of beneficial microorganisms as alternatives to chemical pesticides and synthetic fertilisers in agricultural production. Application of beneficial microorganisms to seeds is an efficient mechanism for placement of microbial inocula into soil where they will be well positioned to colonise seedling roots and protect against soil-borne diseases and pests. However, despite the long history of inoculation of legume seeds with Rhizobia spp. and clear laboratory demonstration of the ability of a wide range of other beneficial microorganisms to improve crop performance, there are still very few commercially available microbial seed inoculants. Seed inoculation techniques used for research purposes are often not feasible at a commercial scale and there are significant technical challenges in maintaining viable microbial inocula on seed throughout commercial seed treatment processes and storage. Further research is needed before the benefits of a wide range of environmentally sensitive potential seed inoculants can be captured for use in agriculture, ecosystem restoration and bioremediation. There is no single solution to the challenge of improving the ability of seed inoculants to establish and function consistently in the field. Development of novel formulations that maintain the viability of both inoculant and seed during storage will result from multidisciplinary research in microbial and seed physiology and adjuvant chemistry.

Bacterial and fungal communities in the rhizosphere of field-grown genetically modified pine trees (Pinus radiata D.)

This study assessed the impact of Pinus radiata (D. Don) genetically modified (GM) by biolistic insertion of the LEAFY and nptII genes on rhizosphere microbial communities of field grown trees. Rhizosphere soil was sampled quarterly for two consecutive years. A culture-independent approach was used to characterise the microbial communities based on PCR and denaturing gradient gel electrophoresis (DGGE) of 16S/18S rDNA gene fragments, and internal transcribed spacer (ITS) fragments amplified from total rhizosphere DNA. Trees from two independent transformation events were sampled, together with non-modified control trees of the same parental genotype. DGGE profiles of rhizosphere general Bacteria did not differ between GM and control trees with one exception (summer 2006 sample). For Alphaproteo- and Actinobacteria, significant differences between treatments were detected in one out of eight samplings. Small seasonal shifts could be detected in all bacterial communities. General fungal and ectomycorrhizal communities did not differ significantly between GM and control trees with the exception of summer 2006, when ectomycorrhizal communities associated with GM trees from one transformation event differed from those associated with control trees. Small seasonal shifts of general fungal and ectomycorrhizal communities were seen over the two-year sampling period. More detailed analysis of microbial communities at one sampling date (using amplified rDNA restriction analysis (ARDRA) and 16S/18S rDNA sequencing) revealed significant differences in four ARDRA groups between one GM treatment and the control (bacteria), and significant differences in one ARDRA group between the two GM treatments (fungi). When data from all sampling dates are considered together, the low incidence of statistical differences in the microbial communities associated with the genetically modified and control trees suggests that there was no significant impact of this genetic modification on rhizosphere microbial communities.

Nucleotide sequence of the Serratia entomophila plasmid pADAP and the Serratia proteamaculans pU143 plasmid virulence associated region

Some strains of Serratia entomophila and S. proteamaculans cause amber disease of the New Zealand grass grub Costelytra zealandica (Coleoptera: Scarabaeidae), an important pasture pest in New Zealand. The disease determinants of S. entomophila, are encoded on a 153,404-bp plasmid, termed pADAP for amber disease associated plasmid. The S. proteamaculans strain 143 (Sp143) exhibits an unusual pathotype, where only 60-70% of C. zealandica larvae infected with the bacterium succumb to disease. DNA sequence analysis of the Sp143 pU143 virulence associated region identified high DNA similarity to the pADAP sep virulence associated region, with DNA sequence variation in the sepA gene and the variable region of the sepC component. No pADAP anti-feeding prophage orthologue was detected in the Sp143 genome. The region of pADAP replication was cloned and found to replicate in S. entomophila but not in Escherichia coli. DNA sequence analysis of the plasmid pSG348 repA gene from the French isolate of Serratia grimesii, identified 93% DNA identity to the pADAP repA gene. A comparison of the pU143 virulence associated region with the completed pADAP nucleotide sequence is given.

Peripheral sequences of the Serratia entomophila pADAP virulence-associated region.

Some strains of the Enterobacteriaceae Serratia entomophila and Serratia proteamaculans cause amber disease in the grass grub, Costelytra zealandica (Coleoptera: Scarabaeidae), an important pasture pest in New Zealand. The genes responsible for this disease reside on a large, 155-kb plasmid designated amber disease-associated plasmid (pADAP). Herein, we report the DNA sequencing of approximately 50 kb upstream and 10 kb downstream of the virulence-encoding region. Based on similarity with proteins in the current databases, and potential ribosome-binding sites, 63 potential ORFs were determined. Eleven of these ORFs belong to a type IV pilus cluster (pilL-V) and a further eight have similarities to the translated products of the plasmid transfer traH-N genes of the plasmid R64. In addition, a degenerate 785-nt direct repeat flanks a 44.7-kb region with the potential to encode three Bacillus subtilis Yee-type proteins, a fimbrial gene cluster, the sep virulence-associated genes and several remnant IS elements.

Environmental Impacts of Bacterial Biopesticides

Bacteria have been used in the biological control of insect pests since the early 20th century, but very few entomopathogenic bacteria have been developed into commercially available biopesticides. Bacillus thuringiensis Berliner (Bt) is currently used in over 90% of all biopesticides sold worldwide. Bt has been developed into over 100 products (Glare and O’Callaghan 2000) which are used, collectively, against at least 1000 pest species. The species Bt is comprised of numerous strains and subspecies (Lecadet et al. 1999), that can produce a wide variety of invertebrate-specific toxins.

Isolation of root-associated Pseudomonas and Burkholderia spp. with biocontrol and plant growth-promoting traits

ABSTRACT Novel, root-associated Pseudomonas and Burkholderia strains with biological control and plant growth-promoting (PGP) traits are being sought for biotechnological application in agriculture. We present a new isolation approach for recovery of rhizoplane and/or endophytic Pseudomonas and Burkholderia spp. with desirable biocontrol and PGP phenotypes. The method may enable better targeted biodiscovery of these two important genera.

The Role of Bacteria and Archaea in Nitrification, Nitrate Leaching and Nitrous Oxide Emissions in Nitrogen-Rich Grassland Soils

Nitrate (\( {\rm NO}_{3}{}^{-} \)) leaching into fresh water and nitrous oxide (N2O) greenhouse gas emissions are two serious environmental impacts that occur from intensively grazed grassland soils. The oxidation of ammonia (NH3) to \( {\rm NO}_{3}{}^{-} \) is a key process in the nitrogen (N) cycle which has implications both in influencing nitrous oxide emissions and \( {\rm NO}_{3}{}^{-} \) leaching. We investigated the relationships between nitrification rate, \( {\rm NO}_{3}{}^{-} \) leaching and N2O emissions with ammonia oxidizing bacteria (AOB) and ammonia oxidizing archaea (AOA) in nitrogen rich grassland soils. Both AOA and AOB were detected in large numbers in these grassland soils. The AOB abundance grew by 3.2–10.4 fold and activity increased by 177 fold in response to the addition of urine-N, and the AOB growth was significantly inhibited by a nitrification inhibitor, dicyandiamide (DCD). However, neither the AOA abundance, nor activity, increased with the application of urine-N substrate. AOB prefer to grow under high nitrogen environments whereas AOA prefer to grow under low nitrogen environments. DCD decreased \( {\rm NO}_{3}{}^{-} \) leaching by 59% and decreased N2O emissions by 64% from animal urine patches. Significant quantitative relationships were found between the AOB abundance and the nitrification rate, \( {\rm NO}_{3}{}^{-} \)-N leaching losses, and N2O emissions, whereas no such relationships were found with AOA. These findings suggest that nitrification, \( {\rm NO}_{3}{}^{-} \) leaching and N2O emissions are driven by bacteria rather than archaea in these nitrogen rich grassland soils.

Bioassay of bacterial entomopathogens against insect larvae

Bacillus thuringiensis (Bt) is the leading biopesticide used around the world to combat lepidopteran agricultural pests. Identification of Bt subsp. tenebrionis strains active against coleopteran pests and Bt subsp. israelensis and serovars of Bacillus sphaericus active against Diptera has led to development of products for control of species that vector human disease. The selection and subsequent development of bacterial entomopathogens as biopesticides depend on the ability to accurately assay their activity against the target insect. This chapter outlines methods for the bioassay of Bt against key insect pests, ranging from the use of relatively simple diet- and foliar-based assays to more complex experimental procedures required for assay of aquatic species. Use of specialized equipment for the testing of environmental constraints, such as UV radiation, on the activity of the bacterial pathogens is outlined. Other bacterial genera, previously unrecognized as insect pathogens, have recently been discovered and methods for testing some of these non-sporeforming bacteria are briefly described.

Using existing data to predict and quantify the risks of GM forage to a population of a non-target invertebrate species: A New Zealand case study

Determining the effects of genetically modified (GM) crops on non-target organisms is essential as many non-target species provide important ecological functions. However, it is simply not possible to collect field data on more than a few potential non-target species present in the receiving environment of a GM crop. While risk assessment must be rigorous, new approaches are necessary to improve the efficiency of the process. Utilisation of published information and existing data on the phenology and population dynamics of test species in the field can be combined with limited amounts of experimental biosafety data to predict possible outcomes on species persistence. This paper presents an example of an approach where data from laboratory experiments and field studies on phenology are combined using predictive modelling. Using the New Zealand native weevil species Nicaeana cervina as a case study, we could predict that oviposition rates of the weevil feeding on a GM ryegrass could be reduced by up to 30% without threat to populations of the weevil in pastoral ecosystems. In addition, an experimentally established correlation between feeding level and oviposition led to the prediction that a consistent reduction in feeding of 50% or higher indicated a significant risk to the species and could potentially lead to local extinctions. This approach to biosafety risk assessment, maximising the use of pre-existing field and laboratory data on non-target species, can make an important contribution to informed decision-making by regulatory authorities and developers of new technologies.

Draft Genome Sequence of a Kale (Brassica oleracea L.) Root Endophyte, Pseudomonas sp. Strain C9

ABSTRACT Pseudomonas sp. strain C9 is a plant growth–promoting bacterium isolated from the root tissue of Brassica oleracea L. grown in soil from Marlborough, New Zealand. Its draft genome of 6,350,161 bp contains genes associated with plant growth promotion and biological control.