Interactions between Pasteuria Penetrans and Plant Parastic Nematodes

Pasturia penetrans is a naturally occurring obligate bacterial hyperparasite of nematodes. It has been developed as a biocontrol agent for use in integrated pest management programs and is influential in establishing and maintaining nematode suppressive soils. The recent development of mass production systems for culturing P. penetrans by Pasteuria Bioscience, Inc. has led to the commercial production of products for Sting, Lance, Reniform and Soybean nematode control. This biocontrol approach is particularly appropriate where plant resistance to the nematode parasite is not available and where environmental restrictions preclude the use of chemical control products. This approach is also valuable in low-input and small-scale production systems. The development of other Pasteuria products to control other economically important plant parasitic nematodes is in progress.
The specificity between the attachment and penetration of P. penetrans and root knot nematodes (Meloidogyne spp.) has been found to be complex due to the presence of bacterial biotypes and the diversity of RKN isolates. Host preference and compatibility is determined in host-parasite attachment tests and the presence or absence of endospores on the cuticle is assayed by microscopic observation. Following attachment and penetration of the cuticle, which requires several days, endospore infection is assayed using a plant infection assay. Both the visual examination and the plant bioassays are time consuming and throughput is constrained by the resources required to conduct these assays.
This project aims to investigate the basis of differences between attachment, penetration and infection of P. penetrans stains and isolates of globally important parasites, Meloidogyne spp. and the potato cyst nematodes Globodera rostochiensis and G. pallida. Pools of nematodes showing high and low levels of attachment and high and low levels of infection will be prepared. These pools will be subjected to gene expression profiling in order to assess the molecular differences between the groups. The availability of genome information from G. pallida, M. incognita and Pasteuria will allow these experiments to be undertaken using either an RNAseq or microarray based approach. This will allow identification of genes from both nematode and bacteria whose expression profiles differ in the pools showing differential attachment and/or infection rates. A mutagenesis approach will be used to develop a pool of bacterial strains deficient in genes identified in the expression analysis for testing attachment, penetration and infection behaviour. Similarly, it will be possible to knock out nematode genes identified in this analysis by RNAi.
This project also proposes to use large particle flow cytometry to obtain rapid quantitative information about attachment of Pasteuria to nematodes. Access, for example, to the Union Biometra COPAS (Complex Object Parametric Analyzer and Sorter) instrument, specifically developed for high throughput manipulation of C. elegans in drug screening assays, which can sort and dispense live nematodes based on size (time of flight), optical density and the presence and intensity of fluorescent markers would provide a novel approach for investigating nematode/P. penetrans interactions relevant to the commercial development of P. penetrans for use in biocontrol.
This project will combine expertise in nematology, bacteriology and bioinformatics at the James Hutton Institute (Dr Vivian Blok, Prof John Jones, Dr Peter Cock, Prof Ian Toth), University of Hertfordshire (Dr Keith Davies) and Syngenta (Dr Tom Hewlett, Dr Anthony Flemming). The outputs of this work will include 1) the development of assays to improve the throughput and sensitivity of investigations of interactions between P. pentrans and nematode hosts 2) identification of the genetic basis and markers associated with differences in attachment, penetration and infection of P. pentrans and nematode hosts 3) a mutant panel of P. penetrans for screening for variants in attachment, penetration and infection.