Gary Keane

Densovirus induces winged morphs in asexual clones of the rosy apple aphid, Dysaphis plantaginea.

Winged morphs of aphids are essential for their dispersal and survival. We discovered that the production of the winged morph in asexual clones of the rosy apple aphid, Dysaphis plantaginea, is dependent on their infection with a DNA virus, Dysaphis plantaginea densovirus (DplDNV). Virus-free clones of the rosy apple aphid, or clones infected singly with an RNA virus, rosy apple aphid virus (RAAV), did not produce the winged morph in response to crowding and poor plant quality. DplDNV infection results in a significant reduction in aphid reproduction rate, but such aphids can produce the winged morph, even at low insect density, which can fly and colonize neighboring plants. Aphids infected with DplDNV produce a proportion of virus-free aphids, which enables production of virus-free clonal lines after colonization of a new plant. Our data suggest that a mutualistic relationship exists between the rosy apple aphid and its viruses. Despite the negative impact of DplDNV on rosy apple aphid reproduction, this virus contributes to their survival by inducing wing development and promoting dispersal.

Impact of Shortened Crop Rotation of Oilseed Rape on Soil and Rhizosphere Microbial Diversity in Relation to Yield Decline

Oilseed rape (OSR) grown in monoculture shows a decline in yield relative to virgin OSR of up to 25%, but the mechanisms responsible are unknown. A long term field experiment of OSR grown in a range of rotations with wheat was used to determine whether shifts in fungal and bacterial populations of the rhizosphere and bulk soil were associated with the development of OSR yield decline. The communities of fungi and bacteria in the rhizosphere and bulk soil from the field experiment were profiled using terminal restriction fragment length polymorphism (TRFLP) and sequencing of cloned internal transcribed spacer regions and 16S rRNA genes, respectively. OSR cropping frequency had no effect on rhizosphere bacterial communities. However, the rhizosphere fungal communities from continuously grown OSR were significantly different to those from other rotations. This was due primarily to an increase in abundance of two fungi which showed 100% and 95% DNA identity to the plant pathogens Olpidium brassicae and Pyrenochaeta lycopersici, respectively. Real-time PCR confirmed that there was significantly more of these fungi in the continuously grown OSR than the other rotations. These two fungi were isolated from the field and used to inoculate OSR and Brassica oleracea grown under controlled conditions in a glasshouse to determine their effect on yield. At high doses, Olpidium brassicae reduced top growth and root biomass in seedlings and reduced branching and subsequent pod and seed production. Pyrenochaeta sp. formed lesions on the roots of seedlings, and at high doses delayed flowering and had a negative impact on seed quantity and quality.

A bacmid approach to the genetic manipulation of granuloviruses

A Cydia pomonella granulovirus (CpGV) bacmid has been constructed, which allows rapid and efficient production of recombinant baculoviruses in Escherichia coli. An 8.6kbp bacterial DNA cassette derived from the AcMNPV Bac-to-Bac system was ligated into a unique PacI restriction site within an intergenic region flanking the DNA ligase gene of the CpGV genome. The CpGV bacmids produced in E. coli were transfected into a CpGV-permissive C. pomonella cell line and the transfected cells fed to larvae to amplify the virus. The enhanced green fluorescent protein (EGFP) gene under the constitutive Drosophila heat-shock promoter was transposed into the mini-attTn7 transposition site, using a modified pFASTBAC donor plasmid, to generate a recombinant CpGV bacmid which caused infected larvae to glow under UV light. Targeted homologous recombination was also achieved in a recombinant proficient E. coli strain (BJ5183). A chloramphenicol acetyl transferase (CAT) gene replaced the cathepsin (v-cath) gene in the bacmid to produce a v-cath-deletion mutant. This is the first published report of a granulovirus bacmid, which will allow easy manipulation of the CpGV genome, enabling future studies on granulovirus genes and biology.

Monitoring Infection Risk for Air and Soil Borne Fungal Plant Pathogens using Antibody and DNA Techniques and Mathematical Models describing Environmental Parameters

Economic losses resulting from disease development can be reduced by accurate and early detection of plant pathogens. Early detection can provide the grower with useful information on optimal crop rotation patterns, varietal selections, appropriate control measures, harvest date and post harvest handling. Classical methods for the isolation of pathogens are commonly used only after disease symptoms. This frequently results in a delay in application of control measures at potentially important periods in crop production. This paper describes the application of both antibody and DNA based systems to monitor infection risk of air and soil borne fungal pathogens and the use of this information with mathematical models describing risk of disease associated with environmental parameters.

Field Evaluation of a Competitive Lateral-flow Assay forDetection of Alternaria brassicae in Vegetable Brassica Crops

On-site detection of inoculum of polycyclic plant pathogens could potentially contribute to management of disease outbreaks. A 6-min, in-field competitive immunochromatographic lateral flow device (CLFD) assay was developed for detection of Alternaria brassicae (the cause of dark leaf spot in brassica crops) in air sampled above the crop canopy. Visual recording of the test result by eye provides a detection threshold of approximately 50 dark leaf spot conidia. Assessment using a portable reader improved test sensitivity. In combination with a weather-driven infection model, CLFD assays were evaluated as part of an in-field risk assessment to identify periods when brassica crops were at risk from A. brassicae infection. The weather-driven model overpredicted A. brassicae infection. An automated 7-day multivial cyclone air sampler combined with a daily in-field CLFD assay detected A. brassicae conidia air samples from above the crops. Integration of information from an in-field detection system (CLFD) with weather-driven mathematical models predicting pathogen infection have the potential for use within disease management systems.