Targeted transcriptomics reveals signatures of large-scale independent origins and concerted regulation of effector genes in Radopholus similis.

The burrowing nematode, Radopholus similis, is an economically important plant-parasitic nematode that inflicts damage and yield loss to a wide range of crops. This migratory endoparasite is widely distributed in warmer regions and causes extensive destruction to the root systems of important food crops (e.g., citrus, banana). Despite the economic importance of this nematode, little is known about the repertoire of effectors owned by this species. Here we combined spatially and temporally resolved next-generation sequencing datasets of R. similis to select a list of candidates for the identification of effector genes for this species. We confirmed spatial expression of transcripts of 30 new candidate effectors within the esophageal glands of R. similis by in situ hybridization, revealing a large number of pioneer genes specific to this nematode. We identify a gland promoter motif specifically associated with the subventral glands (named Rs-SUG box), a putative hallmark of spatial and concerted regulation of these effectors. Nematode transcriptome analyses confirmed the expression of these effectors during the interaction with the host, with a large number of pioneer genes being especially abundant. Our data revealed that R. similis holds a diverse and emergent repertoire of effectors, which has been shaped by various evolutionary events, including neofunctionalization, horizontal gene transfer, and possibly by de novo gene birth. In addition, we also report the first GH62 gene so far discovered for any metazoan and putatively acquired by lateral gene transfer from a bacterial donor. Considering the economic damage caused by R. similis, this information provides valuable data to elucidate the mode of parasitism of this nematode.

What is the Optimal Way to Assess Meloidogyne Spp. Reproduction in Greenhouse Pot Experiments?

Often research efforts that address both the practical concerns of managing Meloidogyne spp. and understanding their basic biology involve greenhouse reproduction assays. However, there is little consensus in regards to what parameters should be used to conduct greenhouse assays. The goal of this research was to evaluate how pot size, Meloidogyne spp. inoculation life stage, inoculation density, and time of assay impacted final reproduction factor (RF = initial nematode density/final nematode density) values. In experiments with M. incognita, the factor of the pot size mattered, with higher RF values in pots containing 500 g soil vs. pots with 100 g soil; larger pots containing 3,000 g soil did not have RF values different from the aforementioned sizes. Inoculating with M. incognita J2 resulted in RF values on average of >2 fold higher then when inoculating with eggs at comparable densities. Inoculation density of M. incognita did not have an impact on final M. incognita RF values. In experiments that considered time of assay, three species were evaluated: M. incognita, M. chitwoodi, and M. hapla. There was no difference in M. incognita RF values when assays were conducted for 5 wk, 6 wk, 7 wk, and 8 wk. However, a longer assay time resulted in higher RF values for M. hapla and M. chitwoodi, with at least a 7 week assay required. In conclusion, a moderate pot size (500 g of soil) inoculated with M. incognita J2 resulted in maximum RF values. The length of the assay required will depend on the Meloidogyne spp. in question, with longer duration assays required for M. hapla and M. chitwoodi than for M. incognita.

Genome Announcement: The Draft Genome of the Carrot Cyst Nematode Heterodera Carotae.

Heterodera carotae, the carrot cyst nematode, is a significant pest affecting carrot globally. Here we present the draft genome of H. carotae, which was generated from short read libraries from Illumina HiSeq technology, and the corresponding genome annotation.

Differential Response of Meloidogyne, Pratylenchus, Globodera, and Xiphinema Species to the Nematicide Fluazaindolizine.

This research focused on the effects of fluazaindolizine on a diversity of plant-parasitic nematodes. In microwell assays, 24-h dose-response curves were generated for several species and populations of Meloidogyne, Pratylenchus neglectus, P. penetrans, Globodera ellingtonae, and Xiphinema americanum. In a greenhouse study, the impact of fluazaindolizine on fecundity of M. incognita, M. hapla, and M. chitwoodi was tested by exposing nematodes for 24 h in solution and inoculating on tomato. The average 24-h ED50s (dose that resulted in the immobility of 50% of exposed nematodes) for M. hapla, M. chitwoodi, and M. incognita were 325.7, 223.4, and 100.7 ppm, respectively. M. hapla had the most variation among populations, with 24-h ED50s ranging from 72 to 788 ppm. G. ellingtonae had the lowest 24-h ED50 at 30 ppm. Pratylenchus spp. were unaffected by fluazaindolizine. X. americanum was the only species where effects of fluazaindolizine were reversible, but had a 24-h ED50 that fell in the range of the Meloidogyne spp. In the greenhouse study, M. chitwoodi was the least sensitive with reproduction reaching 62% of the untreated control after a pre-exposure to 47 ppm, whereas M. incognita and M. hapla at the same exposure dose had reproduction rates of 27 and 36% of the untreated control, respectively. Despite varying in in vitro responses to fluazaindolizine, reproduction of all Meloidogyne spp. was suppressed after only 24 h of exposure. This study expanded our understanding of how G. ellingtonae, P. thornei, P. penetrans, and X. americanum respond to fluazaindolizine.

New reduced-risk agricultural nematicides - rationale and review.

The last decade has seen a sharp increase in nematicide research in the agricultural industry. As a result, several new synthetic nematicides have become available to growers, and several more are expected in the near future. This new interest in nematicides is directly related to the growing demand for safer and more selective products, and the increasing regulatory pressure on many of the traditional nematicides. This has led to a ban of several widely used fumigant (e.g. methyl bromide) and non-fumigant (e.g. aldicarb) nematicides. The loss of traditional nematicides, combined with a lack of replacement products and awareness of the damage that nematodes can cause, has not only raised concern among growers, but has also created new opportunities for the crop protection industry. Nematicides have become a priority, and many companies are now allocating significant research dollars to discover new nematicides. The new nematicides are very different from previous products: (i) they are more selective, often only targeting nematodes, and (ii) they are less toxic, and safer to use. This review article describes these new developments by discussing the challenges that are associated with finding new nematicides, reviewing the nature, characteristics, and efficacy of new nematicides, and discussing the impact they could have on future nematode management.

Short-Term Effects of Sublethal Doses of Nematicides on Meloidogyne incognita.

Meloidogyne species are one of the most important groups of plant-parasitic nematodes globally because of their ability to damage most cultivated plants. Although they are widespread and economically important, there are limited control measures to combat these nematodes. New nonfumigant nematicides have been discovered that have the potential to be widely utilized for the management of plant-parasitic nematodes. Because of the longer half-lives in soil and lower toxicity of new nematicides compared with traditional fumigant and nonfumigant nematicides, understanding how nematodes respond to sublethal doses of nematicides is imperative to understanding whether nematicide resistance has the potential to develop. Characterizing responses of nematodes to sublethal doses will provide the foundation for future work, such as gene expression studies. In this study, the nematicides oxamyl (Vydate), fluazaindolizine (Salibro), fluensulfone (Nimitz), and fluopyram (Velum), were evaluated to understand how sublethal doses affect the fecundity and mobility of Meloidogyne incognita second-stage juveniles (J2). Using a microwell assay system, dose-response curves for each nematicide were established for M. incognita J2. Fluopyram was the most toxic nematicide, with effective doses up to 230 times lower than that of other nematicides. The other nematicides had predicted ED50 values (effective doses that resulted in 50% of the population becoming inactive) of 89.4, 131.7, and 180.6 ppm for oxamyl, fluensulfone, and fluazaindolizine, respectively. The 24-h ED50 of each nematicide was then used in both motility and infectivity assays. The motility and activity of M. incognita J2 exposed to ED50 doses of fluazaindolizine and fluensulfone was significantly reduced, with nematodes initially being motile but eventually becoming inactive. However, the motility of M. incognita J2 exposed to ED50 doses of fluopyram and oxamyl was not different from a water control. In a pot assay, M. incognita J2 exposed to ED50 doses of fluazaindolizine, oxamyl, and fluensulfone were unable to reproduce on tomato, with reproduction factors (RF = final population density/initial population density) of 0 to 0.03. Fluopyram did not reduce reproduction of M. incognita, with a mean RF of 38.7 ± 4.5, which was similar to the RF of 46.3 ± 4.6 for the water control. This study is the first comprehensive evaluation of M. incognita activity, motility, and fecundity after exposure to the traditional nematicide, oxamyl, as well as three new nematicides, fluazaindolizine, fluopyram, and fluensulfone.

Genome Announcement: The Draft Genomes of Two Radopholus similis populations from Costa Rica.

Radopholus similis is an economically important pest of both banana and citrus in tropical regions. Here we present draft genomes from two populations of R. similis from Costa Rica that were created and assembled using short read libraries from Illumina HiSeq technology.

Transcriptional response of Meloidogyne incognita to non-fumigant nematicides.

There is limited research about the impacts of new nematicides, including fluazaindolizine, fluopyram, and fluensulfone, on the plant-parasitic nematode Meloidogyne incognita, despite it being a pervasive agricultural pest. In this study, M. incognita second-stage juveniles were exposed for 24-h to fluensulfone, fluazaindolizine, fluopyram, and oxamyl and total RNA was extracted and sequenced using next-generation sequencing to determine gene expression. The effects of nematicide exposure on cellular detoxification pathways, common differentially expressed (DE) genes, and fatty acid and retinol-binding genes were examined. Fluopyram and oxamyl had the smallest impacts on the M. incognita transcriptome with 48 and 151 genes that were DE, respectively. These compounds also elicited a weak response in the cellular detoxification pathway and fatty acid and retinol-binding (FAR) genes. Fluensulfone and fluazaindolizine produced robust transcriptional responses with 1208 and 2611 DE genes, respectively. These compounds had strong impacts on cellular detoxification, causing differential regulation of transcription factors and genes in the detox pathway. These compounds strongly down-regulated FAR genes between 52-85%. Having a greater understanding of how these compounds function at a molecular level will help to promote proper stewardship, aid with nematicide discovery, and help to stay a step ahead of nematicide resistance.

Transcriptional changes of biochemical pathways in Meloidogyne incognita in response to non-fumigant nematicides.

Meloidogyne incognita is a destructive and economically important agricultural pest. Similar to other plant-parasitic nematodes, management of M. incognita relies heavily on chemical controls. As old, broad spectrum, and toxic nematicides leave the market, replacements have entered including fluensulfone, fluazaindolizine, and fluopyram that are plant-parasitic nematode specific in target and less toxic to applicators. However, there is limited research into their modes-of-action and other off-target cellular effects caused by these nematicides in plant-parasitic nematodes. This study aimed to broaden the knowledge about these new nematicides by examining the transcriptional changes in M. incognita second-stage juveniles (J2) after 24-h exposure to fluensulfone, fluazaindolizine, and fluopyram as well as oxamyl, an older non-fumigant nematicide. Total RNA was extracted and sequenced using Illumina HiSeq to investigate transcriptional changes in the citric acid cycle, the glyoxylate pathway, [Formula: see text]-fatty acid oxidation pathway, oxidative phosphorylation, and acetylcholine neuron components. Observed transcriptional changes in M. incognita exposed to fluopyram and oxamyl corresponded to their respective modes-of-action. Potential targets for fluensulfone and fluazaindolizine were identified in the [Formula: see text]-fatty acid oxidation pathway and 2-oxoglutarate dehydrogenase of the citric acid cycle, respectively. This study provides a foundation for understanding how potential nematicide resistance could develop, identifies cellular pathways as potential nematicide targets, and determines targets for confirming unknown modes-of-action.

The plant-parasitic cyst nematode effector GLAND4 is a DNA-binding protein.

Cyst nematodes are plant pathogens that infect a wide range of economically important crops. One parasitic mechanism employed by cyst nematodes is the production and in planta delivery of effector proteins to modify plant cells and suppress defences to favour parasitism. This study focuses on GLAND4, an effector of Heterodera glycines and H. schachtii, the soybean and sugar beet cyst nematodes, respectively. We show that GLAND4 is recognized by the plant cellular machinery and is transported to the plant nucleus, an organelle for which little is known about plant nematode effector functions. We show that GLAND4 has DNA-binding ability and represses reporter gene expression in a plant transcriptional assay. One DNA fragment that binds to GLAND4 is localized in an Arabidopsis chromosomal region associated with the promoters of two lipid transfer protein genes (LTP). These LTPs have known defence functions and are down-regulated in the nematode feeding site. When expressed in Arabidopsis, the presence of GLAND4 causes the down-regulation of the two LTP genes in question, which is also associated with increased susceptibility to the plant-pathogenic bacterium Pseudomonas syringae. Furthermore, overexpression of one of the LTP genes reduces plant susceptibility to H. schachtii and P. syringae, confirming that LTP repression probably suppresses plant defences. This study makes GLAND4 one of a small subset of characterized plant nematode nuclear effectors and identifies GLAND4 as the first DNA-binding, plant-parasitic nematode effector.