"Piler Dirt" Survey for the Sampling and Detection of Potato Cyst Nematodes
Benjamin Mimee (Agriculture and Agri-Food Canada); Nathalie Dauphinais (Agriculture and Agri-Food Canada); Guy Bélair (Agriculture and Agri-Food Canada)
Potato cyst nematodes are a significant threat to potato production worldwide and have important economic impacts due to yield losses but also because of the expenses associated with regulation procedures. In order to reduce the sampling labor, an alternative strategy named the “Piler Dirt” that collects the soil carried with potato tubers during their transfer to storage was proposed. The method showed a better sensitivity than the reference method to detect fields infested with G. rostochiensis. The quantification of the number of cysts per kilogram of soil was proportional between the two methods at low and moderate population densities (R2 = 0.885) but no correlations were found at high density. However, the quantity of soil generated by the method was exceedingly large to be treated by diagnostic labs. It was shown that subsampling six aliquots, each equivalent to 5,000 cm3/ha, from the total quantity of soil generated by the Piler Dirt method, resulted in a probability of 97% to detect infested fields, 95% of the time in our dataset. Overall, Piler Dirt appears as a good compromise to reduce labor time and cost without significantly affecting sensitivity. However, it will be challenging to implement because it needs to be done simultaneously with harvest and will require the participation of farmers during a busy period.
Mimee, B., N. Dauphinais, and G.Bélair. 2019. "Piler Dirt" Survey for the Sampling and Detection of Potato Cyst Nematodes. Plant Dis 2019 https://doi.org/10.1094/PDIS-12-18-2188-RE
What determines host specificity in hyperspecialized plant parasitic nematodes?
Michael Sabeh (Agriculture and Agri-Food Canada); Etienne Lord (Agriculture and Agri-Food Canada); Marc St-Arnaud (University of Montreal); Benjamin Mimee (Agriculture and Agri-Food Canada)
In hyperspecialized parasites, the ability to grow on a particular host relies on specific virulence factors called effectors. These excreted proteins are involved in the molecular mechanisms of parasitism and distinguish virulent pathogens from non-virulent related species. The potato cyst nematodes (PCN) Globodera rostochiensis and G. pallida are major plant-parasitic nematodes developing on numerous solanaceous species including potato. Their close relatives, G. tabacum and G. mexicana are stimulated by potato root diffusate but unable to establish a feeding site on this plant host.
RNA sequencing was used to characterize transcriptomic differences among these four Globodera species and to identify genes associated with host specificity. We identified seven transcripts that were unique to PCN species, including a protein involved in ubiquitination. We also found 545 genes that were differentially expressed between PCN and non-PCN species, including 78 genes coding for effector proteins, which represent more than a 6-fold enrichment compared to the whole transcriptome. Gene polymorphism analysis identified 359 homozygous non-synonymous variants showing a strong evidence for selection in PCN species.
Overall, we demonstrated that the determinant of host specificity resides in the regulation of essential effector gene expression that could be under the control of a single or of very few regulatory genes. Such genes are therefore promising targets for the development of novel and more sustainable resistances against potato cyst nematodes.
Sabeh, M., E. Lord, É. Grenier, M, St-Arnaud, and B. Mimee. 2019. What determines host specificity in hyperspecialized plant parasitic nematodes? BMC Genomics 20:457 https://doi.org/10.1186/s12864-019-5853-4
Transcriptome-wide selection of a reliable set of reference genes for gene expression studies in potato cyst nematodes (Globodera spp.).
Relative gene expression analyses by qRT-PCR (quantitative reverse transcription PCR) require an internal control to normalize the expression data of genes of interest and eliminate the unwanted variation introduced by sample preparation. A perfect reference gene should have a constant expression level under all the experimental conditions. However, the same few housekeeping genes selected from the literature or successfully used in previous unrelated experiments are often routinely used in new conditions without proper validation of their stability across treatments. The advent of RNA-Seq and the availability of public datasets for numerous organisms are opening the way to finding better reference genes for expression studies. Globodera rostochiensis is a plant-parasitic nematode that is particularly yield-limiting for potato. The aim of our study was to identify a reliable set of reference genes to study G. rostochiensis gene expression. Gene expression levels from an RNA-Seq database were used to identify putative reference genes and were validated with qRT-PCR analysis. Three genes, GR, PMP-3, and aaRS, were found to be very stable within the experimental conditions of this study and are proposed as reference genes for future work.
Sabeh, M., Duceppe, M. O., St-Arnaud, M., & Mimee, B. (2018). Transcriptome-wide selection of a reliable set of reference genes for gene expression studies in potato cyst nematodes (Globodera spp.). PloS one, 13(3), e0193840. DOI:10.137/journal.pone.0193840.
An Evaluation of two H1-Linked Markers and their Suitability for Selecting Globodera rostochiensis Resistant Potatoes in the New York Breeding Program
Jaebum Park; Huijun Yang; Walter S. De Jong; Xiaohong Wang
The golden cyst nematode (Globodera rostochiensis) is a serious pest that can dramatically reduce potato crop yield. Pathotype Ro1 of G. rostochiensis was first detected in the United States in 1941 and is still present on several farms in New York State. The H1 gene confers high levels of resistance to pathotype Ro1 but screening for it with a bioassay is time consuming and expensive. In this study two known molecular markers, 57R and TG689, were evaluated for their ability to identify resistant clones among 38 global cultivars and 350 New York breeding clones. The ability of either marker to predict resistance was high – 99.7% and 98.3% for 57R and TG689, respectively – but the ability to predict susceptibility was much lower, 47% and 41%, respectively. As resistance is the trait of interest, either of these markers is sufficient to make selection decisions in a practical breeding program. Cases exhibiting discordance between presence/absence of diagnostic markers and bioassay results were investigated further. Recombination, inflow of other resistance genes, and occasional failure of marker- and/or bio-assays are discussed as potential causes.
Park, J., H. Yang, W. S. De Jong, X. Wang. 2018. An Evaluation of two H1-Linked Markers and their Suitability for Selecting Globodera rostochiensis Resistant Potatoes in the New York Breeding Program. American Journal of Potato Research. https://link.springer.com/article/10.1007/s12230-017-9623-z
A qRT-PCR method to evaluate viability of potato cyst nematode (Globodera spp.)
Benjamin Mimee; Brahim Soufiane; Nathalie Dauphinais; Guy Bélair
One of the main challenges to PCN management is the ability of PCN to remain dormant in the soil for several decades. For that reason, many countries have strict quarantine regulations for PCN. These regulations, although expensive and restrictive for growers, are necessary to prevent further spread of PCN but should be lifted when no more viable cysts are found. Here, we report a promising qRT-PCR method for the quantification of viable eggs and propose that this method be included in routine testing. The method was successful for quantifying G. ellingtonae, G. rostochiensis and G. pallida and was found to be very sensitive with the systematic detection of a single larva.
Citation: Mimee, B., B. Soufiane, N. Dauphinais, and G. Bélair. 2017. A qRT-PCR method to evaluate viability of potato cyst nematode (Globodera spp.). Canadian Journal of Plant Pathology. http://dx.doi.org/10.1080/07060661.2017.1382574
Analysis of survival and hatching transcriptomes from potato cyst nematodes, Globodera rostochiensis and G. pallida
Marc-Olivier Duceppe ; Joël Lafond-Lapalme; Juan Emilio Palomares-Rius; Michaël Sabeh; Vivian Blok; Peter Moffet; Benjamin Mimee
Citation: Duceppe, M.-O., J. Lafond-Lapalme, J. E. Palomares-Rius, M. Sabeh, V. Blok, P. Moffett, and B. Mimee. 2017. Analysis of survival and hatching transcriptomes from potato cyst nematodes, Globodera rostochiensis and G. pallida. Scientific Reports 7, Article number: 3882. doi:10.1038/s41598-017-03871-x
The Draft Genome of Globodera ellingtonae
Wendy S. Phillips; Danna K. Howe; Amanda M.V. Brown; Sebastian Eves-Van Den Akker; Levi Dettwyler; Amy B. Peetz; Dee R. Denver; and Inga A. Zasada
Globodera ellingtonae is a newly described potato cyst nematode (PCN) found in Idaho, Oregon, and Argentina. Here, we present a genome assembly for G. ellingtonae, a relative of the quarantine nematodes G. pallida and G. rostochiensis, produced using data from Illumina and Pacific Biosciences DNA sequencing technologies.
Phillips, W. S., Howe, D. K., Brown, A. M. V., Eves-van den Akker, S., Dettwyler, L., Peetz, A. B., Denver, R.E., Zasada, I. A. (2017). The Draft Genome of Globodera ellingtonae. Journal of Nematology, 49(2), 127–128. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5507130/
A new method for decontamination of de novo transcriptomes using a hierarchical clustering algorithm
Joël Lafond-Lapalme; Marc-Olivier Duceppe; Shengrui Wang; Peter Moffett; Benjamin Mimee
Citation: Lafond-Lapalme, J., M.-O. Duceppe, S. Wang, P. Moffett and B. Mimee. 2016. A new method for decontamination of de novo transcriptome using a hierarchical clustering algorithm. Bioinformatics. doi: 10.1093/bioinformatics/btw793
The genome of the yellow potato cyst nematode, Globodera rostochiensis, reveals insights into the genomic and transcriptomic bases of pathogenicity and virulence.
Eves-van den Akker, S., D.R. Laetsch, P. Thorpe, C.J. Lilley, E.G.J. Danchin, M. Da Rocha, C. Rancurel, N.E. Holroyd, J.A. Cotton, E. Grenier, J. Montarry, B. Mimee, M.-O. Duceppe, I. Boyes, J. Lapalme, M. Esquibet, M. Sabeh, M. Rott, H. Overmars, A. Tomczak, G. Smant, G. Koutsovoulos, V. Blok, S. Mantelin, P.J.A. Cock, W. Phillips, J. Marvin, L.M. Jones, H.B. Yusup, P.E. Urwin, M. Blaxter and J.T. Jones. 2016. The genome of the yellow potato cyst nematode, Globodera rostochiensis, reveals insights into the genomic and transcriptomic bases of pathogenicity and virulence. Genome Biology 17:124. DOI: 10.1186/s13059-016-0985-1.