Field Release Experiment Research Articles

Characterization of a microsatellite locus in the parasitoid waspAphelinus abdominalis(Hymenoptera: Aphelinidae)

AbstractPrimers for DNA amplification using the polymerase chain reaction (PCR) were synthesized for a microsatellite locus isolated from a partial genomic library of the aphid parasitoidAphelinus abdominalis(Dalman). Screening for genetic polymorphism at this locus in two laboratory strains of this wasp revealed the presence of two alleles different in the number of (GT) and (GGC) repeats. The relative frequencies of the two alleles were not significantly different between the two strains or between diploid females and haploid males. Heterozygosity at this microsatellite locus was estimated to be 0.40 which is within the range in other hymenopterous species. Given thatA. abdominalisis a good candidate for augmentative release programmes in greenhouses against aphids, we suggest that microsatellite markers may have application in discriminating among aphelinid sibling species and strains. The markers provide a means for studying the performance and impact of selected parasitoid lines on pest dynamics in field release experiments.

The acquisition of indigenous plasmids by a genetically marked pseudomonad population colonizing the sugar beet phytosphere is related to local environmental conditions

The transfer of naturally occurring conjugative plasmids from the indigenous microflora to a genetically modified population of bacteria colonizing the phytospheres of plants has been observed. The marked strain (Pseudomonas fluorescens SBW25EeZY6KX) was introduced as a seed dressing to sugar beets (Beta vulgaris var. Amethyst) as part of a field experiment to assess the ecology and genetic stability of deliberately released bacterial inocula. The sustained populations of the introduced strain, which colonized the phytosphere, were assessed throughout the growing season for the acquisition of plasmids conferring mercury resistance (Hg(supr)). Transconjugants were isolated only from root and leaf samples collected within a narrow temporal window coincident with the midseason maturation of the crop. Conjugal-transfer events were recorded during this defined period in two separate field release experiments conducted over consecutive years. On one occasion seven of nine individual plants sampled supported transconjugant P. fluorescens SBW25EeZY6KX, demonstrating that conjugative gene transfer between bacterial populations in the phytosphere may be a common event under specific environmental conditions. The plasmids acquired in situ by the colonizing inocula were identified as natural variants of restriction digest pattern group I, III, or IV plasmids from five genetically distinct groups of large, conjugative mercury resistance plasmids known to persist in the phytospheres of sugar beets at the field site. These data demonstrate not only that gene transfer may be a common event but also that the genetic and phenotypic stability of inocula released into the natural environment cannot be predicted.

The flagellin gene as a stable marker for detection of Pseudomonas fluorescens SBW25.

Flagellin gene central regions from 111 isolates of Pseudomonas fluorescens SBW25 obtained from soil during a field release experiment were analysed using a combined PCR/RFLP technique to look for variation. In addition, a 858 bp flagellin gene sequence from the original strain and the last isolate obtained from the release site were compared. There was no variation in flagellin gene sequences indicating that the gene was stable over the period of the release, and that the flagellin gene is a suitable marker for use in the detection of bacteria in release experiments. A comparison of Ps. fluorescens SBW25 flagellin with other sequenced flagellins revealed closest homology to the flagellin of Ps. putida PRS2000.

Comparative analysis of the genetic structure of a Rhizobium meliloti field population before and after environmental release of the highly competitive R. meliloti strain GR4

Rhizobium meliloti strain GR4, which exhibits a highly competitive ability for alfalfa root nodule occupancy, was used in a field release experiment in Granada, Spain. In order to analyze the ecological impact of the GR4 release, we characterized the R. meliloti indigenous population of the field site by ERIC-(enterobacterial repetitive intergenic consensus) PCR and IS (insertion sequence) fingerprinting. Both fingerprinting methods resulted in the same grouping of the isolates. Data obtained were compared with a previous analysis by plasmid based sequence-specific PCR. Isolates belonging to the major infective group, as defined by dominant plasmid types, were shown to have identical or nearly identical ERIC and IS fingerprint patterns. Hence, we conclude that all three typing methods are suited to characterize the genetic structure of the field population. The possible impact of the introduction of strain GR4 was examined two years after its release in its original environment. No effect on the genetic structure of the indigenous R. meliloti field population was observed.

Comparative analysis of the genetic structure of a Rhizobium meliloti field population before and after environmental release of the highly competitive R. meliloti strain GR4

Rhizobium meliloti strain GR4, which exhibits a highly competitive ability for alfalfa root nodule occupancy, was used in a field release experiment in Granada, Spain. In order to analyze the ecological impact of the GR4 release, we characterized the R. meliloti indigenous population of the field site by ERIC-(enterobacterial repetitive intergenic consensus) PCR and IS (insertion sequence) fingerprinting. Both fingerprinting methods resulted in the same grouping of the isolates. Data obtained were compared with a previous analysis by plasmid based sequence-specific PCR. Isolates belonging to the major infective group, as defined by dominant plasmid types, were shown to have identical or nearly identical ERIC and IS fingerprint patterns. Hence, we conclude that all three typing methods are suited to characterize the genetic structure of the field population. The possible impact of the introduction of strain GR4 was examined two years after its release in its original environment. No effect on the genetic structure of the indigenous R. meliloti field population was observed.

OECD's biosafety work on “large-scale” releases of transgenic plants

The OECD has recently published the work on safety of large-scale releases of crop plants modified by modern biotechnology. The work generated three major publications: (a) Historical Review of Traditional Crop Breeding Practices; (b) Analysis of Field Release Experiments of Transgenic Crop Plants; and (c) Safety Considerations for Biotechnology — Scale-up of Crop Plants. This paper outlines them.

Field Release of a Genetically Modified Pseudomonas fluorescens on Wheat: Establishment, Survival and Dissemination

The establishment, survival and dissemination of a strain of Pseudomonas fluorescens, that was originally isolated from the phylloplane of sugar beet and genetically modified with the marker genes lacZY and kanr-xylE was studied during a field release experiment on wheat. The genetically modified microorganism (GMM) was released into the environment onto the seed and as a foliar spray applied at tillering. Survival as well as dissemination of the GMM was greater in the field than could be predicted from experiments that were carried out in microcosm and pot experiments under contained conditions and in comparison with previous release studies with similar GMMs. The GMM used in our field release survived and multiplied especially well in the rhizosphere and phylloplane of wheat, and was found to disseminate up to 2 m away from the inoculated areas and to depths likely to be greater than the deepest sampling of 45 cm. GMM numbers in soil declined steadily over time, but numbers increased subsequently on volunteer wheat, weeds and resown wheat plants.

Impact of Field Release of Genetically Modified Pseudomonas fluorescens on Indigenous Microbial Populations of Wheat

In a field release experiment, an isolate of Pseudomonas fluorescens, which was chromosomally modified with two reporter gene cassettes (lacZY and Kan(supr)-xylE), was applied to spring wheat as a seed coating and subsequently as a foliar spray. The wild-type strain was isolated from the phylloplane of sugar beet but was found to be a common colonizer of both the rizosphere and phylloplane of wheat as well. The impact on the indigenous microbial populations resulting from release of this genetically modified microorganism (GMM) was compared with the impact of the unmodified, wild-type strain and a nontreated control until 1 month after harvest of the crop. The release of the P. fluorescens GMM and the unmodified, wild-type strain resulted in significant but transient perturbations of some of the culturable components of the indigenous microbial communities that inhabited the rhizosphere and phylloplane of wheat, but no significant perturbations of the indigenous culturable microbial populations in nonrhizosphere soil were found. Fast-growing organisms that did not produce resting structures (for example, fluorescent pseudomonads and yeasts) seemed to be most sensitive to perturbation. In terms of hazard and risk to the environment, the observed microbial perturbations that resulted from this GMM release may be considered minor for several reasons. First, the recombinant P. fluorescens strain caused changes that were, in general, not significantly different from those caused by the unmodified wild-type strain; second, perturbations resulting from bacterial inoculations were mainly small; and third, the release of bacteria had no obvious effects on plant growth and plant health.

Construction and characterization of a Rhizobium leguminosarum biovar viciae strain designed to assess horizontal gene transfer in the environment

An integration vector was developed which inserts cloned DNA in a non-essential site of the Rhizobium leguminosarum biovar viciae chromosome. The expression of integrated genes is under the control of the constitutive neomycin phosphotransferase II ( nptII) promotor of transposon Tn5. The design of the vector ensures that loss of vector sequences can be detected, enabling selection of progeny containing only the requisite DNA. The newly constructed vector was employed to insert the Escherichia coli gusA gene conferring GUS activity into R. leguminosarum bv. viciae strain LRS39401 which is cured of its symbiotic plasmid (pSym). One GUS-positive transconjugant, strain CT0370, was shown to have lost all vector sequences. Conjugal transfer of pSym2004 (a Tn5-tagged derivative of symbiotic plasmid pRL1JI, which specifies pea nodulation and symbiotic nitrogen fixation) to CT0370, restored the GUS-positive strain's symbiotic proficiency. Strain CT0370 is presently being used in a field release experiment in order to assess the extent of pSym transfer in a natural R. leguminosarum bv. viciae population under environmental conditions.

Construction and characterization of a Rhizobium leguminosarum biovar viciae strain designed to assess horizontal gene transfer in the environment.

An integration vector was developed which inserts cloned DNA in a non-essential site of the Rhizobium leguminosarum biovar viciae chromosome. The expression of integrated genes is under the control of the constitutive neomycin phosphotransferase II (nptII) promoter of transposon Tn5. The design of the vector ensures that loss of vector sequences can be detected, enabling selection of progeny containing only the requisite DNA. The newly constructed vector was employed to insert the Escherichia coli gusA gene conferring GUS activity into R. leguminosarum bv. viciae strain LRS39401 which is cured of its symbiotic plasmid (pSym). One GUS-positive transconjugant, strain CT0370, was shown to have lost all vector sequences. Conjugal transfer of pSym2004 (a Tn5-tagged derivative of symbiotic plasmid pRL1JI, which specifies pea nodulation and symbiotic nitrogen fixation) to CT0370, restored the GUS-positive strain's symbiotic proficiency. Strain CT0370 is presently being used in a field release experiment in order to assess the extent of pSym transfer in a natural R. leguminosarum bv. viciae population under environmental conditions.

Genotype-specific habitat selection for oviposition sites in the cactophilic species Drosophila buzzatii

Isofemale lines of the cactophilic species, Drosophila buzzatii, exhibit genetic variation for their oviposition response to cactus yeast species in the laboratory. In general, interactions between yeast species preclude the use of pairwise preferences as predictors of preferences in three-way choice experiments. Two isofemale lines with relatively high laboratory preference for ovipositing on the yeast Pichia cactophila (as opposed to Cryptococcus cereanus) and two isofemale lines with relatively low preference for P. cactophila were used in a series of field release experiments to determine if laboratory preferences were also realized under field conditions. The influence of yeast species on both settling behaviour (long-distance response) and oviposition preference (short-distance response) were tested. The four lines were identical in their settling behaviour, preferring P. cactophila. The analysis of the oviposition preference tests showed significant line effects which correlated with the laboratory results. Thus a genetic component for oviposition preference under laboratory and field conditions was demonstrated and this strengthens the evidence for genotype-specific habitat selection in D. buzzatii. One low line, however, did not differ significantly from the two high lines under field conditions. A laboratory retest of this low line showed that the laboratory preference had not changed. The reason for the difference in the two situations is unknown but undoubtedly is attributable to uncontrolled variables under the field situation. Settling behaviour and oviposition response, in general, appear to be proximately linked to differences in the volatiles produced by the different yeast species.

ETMOD: A New Environmental Tritium Model

The ETMOD (Environmental Tritium Model) computer code calculates the potential radiological impact of short duration releases of HT and HTO. The code is now able to predict key input parameters, such as the deposition velocity of HT to soil and the rate of HTO exchange between terrestrial surfaces and the atmosphere, on the basis of weather conditions, land use, season and time of day. In this paper, these features are illustrated by presenting model simulations of an HT field release experiment.