"Piler Dirt" Survey for the Sampling and Detection of Potato Cyst Nematodes.

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.

Host Range and Genetic Characterization of Ditylenchus dipsaci Populations from Eastern Canada.

The stem and bulb nematode, Ditylenchus dipsaci, is a plant-parasitic nematode affecting over 500 plant species worldwide. Since 2012, garlic producers from Ontario and Quebec have been particularly affected with economic losses caused by this pest. Reproduction of D. dipsaci on a particular host depends on its biological race, and races are unknown for these populations from eastern Canada. As a polyphagous pest, D. dipsaci can possibly be a threat and have negative impact on many crops grown in Quebec, such as field and vegetable crops (e.g., onion). In this study, the host range of four populations of D. dipsaci from Quebec and Ontario was determined in a greenhouse experiment using 11 crops. Garlic, onion, and green onion showed high susceptibility to the nematode, whereas reproduction on potato was poor. No reproduction was observed on corn, soybean, barley, alfalfa, mustard, carrot, and lettuce. These crops could therefore be used as rotational crops in a control program. Thirty-two populations of D. dipsaci were also genetically characterized using genotyping-by-sequencing. The comparison of allele frequencies at 481 loci showed that most of the populations had a genotype similar to a reference population from northern Ontario. However, a sample from eastern Quebec exhibited a distinct genotype and will require further phenotyping in a greenhouse to preclude the possibility of a different race.

Analysis of Globodera rostochiensis effectors reveals conserved functions of SPRYSEC proteins in suppressing and eliciting plant immune responses.

Potato cyst nematodes (PCNs), including Globodera rostochiensis (Woll.), are important pests of potato. Plant parasitic nematodes produce multiple effector proteins, secreted from their stylets, to successfully infect their hosts. These include proteins delivered to the apoplast and to the host cytoplasm. A number of effectors from G. rostochiensis predicted to be delivered to the host cytoplasm have been identified, including several belonging to the secreted SPRY domain (SPRYSEC) family. SPRYSEC proteins are unique to members of the genus Globodera and have been implicated in both the induction and the repression of host defense responses. We have tested the properties of six different G. rostochiensis SPRYSEC proteins by expressing them in Nicotiana benthamiana and N. tabacum. We have found that all SPRYSEC proteins tested are able to suppress defense responses induced by NB-LRR proteins as well as cell death induced by elicitors, suggesting that defense repression is a common characteristic of members of this effector protein family. At the same time, GrSPRYSEC-15 elicited a defense responses in N. tabacum, which was found to be resistant to a virus expressing GrSPRYSEC-15. These results suggest that SPRYSEC proteins may possess characteristics that allow them to be recognized by the plant immune system.

A new method for studying population genetics of cyst nematodes based on Pool-Seq and genomewide allele frequency analysis.

Cyst nematodes are important agricultural pests responsible for billions of dollars of losses each year. Plant resistance is the most effective management tool, but it requires a close monitoring of population genetics. Current technologies for pathotyping and genotyping cyst nematodes are time-consuming, expensive and imprecise. In this study, we capitalized on the reproduction mode of cyst nematodes to develop a simple population genetic analysis pipeline based on genotyping-by-sequencing and Pool-Seq. This method yielded thousands of SNPs and allowed us to study the relationships between populations of different origins or pathotypes. Validation of the method on well-characterized populations also demonstrated that it was a powerful and accurate tool for population genetics. The genomewide allele frequencies of 23 populations of golden nematode, from nine countries and representing the five known pathotypes, were compared. A clear separation of the pathotypes and fine genetic relationships between and among global populations were obtained using this method. In addition to being powerful, this tool has proven to be very time- and cost-efficient and could be applied to other cyst nematode species.

Analysis of putative apoplastic effectors from the nematode, Globodera rostochiensis, and identification of an expansin-like protein that can induce and suppress host defenses.

The potato cyst nematode, Globodera rostochiensis, is an important pest of potato. Like other pathogens, plant parasitic nematodes are presumed to employ effector proteins, secreted into the apoplast as well as the host cytoplasm, to alter plant cellular functions and successfully infect their hosts. We have generated a library of ORFs encoding putative G. rostochiensis putative apoplastic effectors in vectors for expression in planta. These clones were assessed for morphological and developmental effects on plants as well as their ability to induce or suppress plant defenses. Several CLAVATA3/ESR-like proteins induced developmental phenotypes, whereas predicted cell wall-modifying proteins induced necrosis and chlorosis, consistent with roles in cell fate alteration and tissue invasion, respectively. When directed to the apoplast with a signal peptide, two effectors, an ubiquitin extension protein (GrUBCEP12) and an expansin-like protein (GrEXPB2), suppressed defense responses including NB-LRR signaling induced in the cytoplasm. GrEXPB2 also elicited defense response in species- and sequence-specific manner. Our results are consistent with the scenario whereby potato cyst nematodes secrete effectors that modulate host cell fate and metabolism as well as modifying host cell walls. Furthermore, we show a novel role for an apoplastic expansin-like protein in suppressing intra-cellular defense responses.

Life Cycle of the Golden Cyst Nematode, Globodera rostochiensis, in Quebec, Canada.

In 2006, the golden cyst nematode, Globodera rostochiensis, was discovered in the province of Quebec, Canada. We report here the life cycle of G. rostochiensis under the climatic conditions of southwestern Quebec. Only one full generation was completed per year under these latitudes. On susceptible potato cv. Snowden, G. rostochiensis needed a minimum of 579 growing degree units (GDU) (base 5.9°C) to complete its life cycle and the first mature cysts were observed 42 to 63 days after planting (DAP). In soil, second-stage juveniles (J2) were first observed 14 to 21 DAP, whereas both white females on roots and males in soil appeared synchronously after 35 to 42 days. The duration of the life cycle was affected by temperature but not by soil type. A second wave of hatching systematically occurred later in the season and a second generation of males was observed during the 2011 growth season. No complete second cycle was observed before plant senescence. Climate change and later maturing cultivars/crops could allow the development of a full second generation in the future.

Genetic diversity of the golden potato cyst nematode Globodera rostochiensis and determination of the origin of populations in Quebec, Canada.

The golden cyst nematode (Globodera rostochiensis), native to South America, has been introduced in many parts of the world, including Europe and North America. Recently, it was found for the first time in the province of Quebec, Canada in the locality of St. Amable near Montreal. To date, very few studies have examined the population genetics of this pest. Consequently, there is a lack of knowledge about the genetic structure and evolution of this nematode. In this study, twelve new microsatellite markers were developed in order to explore these questions. These markers were used to genotype fifteen populations originating from different regions of the world, including five from Canada. Within populations, the highest genetic diversity was consistently observed in the populations from Bolivia, the postulated region of origin of the golden nematode, and the lowest in populations from British Columbia (Canada) and New York (USA). The two Quebec populations were very similar to each other and to the population found in Newfoundland, but surprisingly, they were significantly different from three other North American populations including those from New York and British Columbia. Based on our results, we conclude that the golden cyst nematode has been introduced in North America at least twice from distinct regions of the world.

Pearl Millet for the Management of Pratylenchus penetrans in Flue-Cured Tobacco in Quebec.

Two 1-year rotation experiments were conducted from 1998 to 2000 to assess the impact of forage and grain pearl millet (Pennisetum glaucum) on Pratylenchus penetrans populations in a tobacco field (Nicotiana tabacum cv. Delgold) in Quebec. Performance of these crops was compared with rye, the standard rotation crop. Rye plots were doubled to include a fumigated standard (application of metham sodium at 67.4 liters/ha on a band). Forage pearl millet increased tobacco yields by an average of 103% compared with nonfumigated rye. In 2000, leaf dry weights of tobacco following grain pearl millet CGPM H5 and CGPM H6 was increased by 70 and 73%, respectively, when compared with nonfumigated rye and were not significantly different from the fumigated plots following rye. Forage and grain pearl millet should be considered as a viable alternative to fumigation for controlling P. penetrans infestation in flue-cured tobacco production in Quebec.