Comparative genomics among cyst nematodes reveals distinct evolutionary histories among effector families and an irregular distribution of effector-associated promoter motifs.

Potato cyst nematodes (PCNs), an umbrella term used for two species, Globodera pallida and G. rostochiensis, belong worldwide to the most harmful pathogens of potato. Pathotype-specific host plant resistances are essential for PCN control. However, the poor delineation of G. pallida pathotypes has hampered the efficient use of available host plant resistances. Long-read sequencing technology allowed us to generate a new reference genome of G. pallida population D383 and, as compared to the current reference, the new genome assembly is 42 times less fragmented. For comparison of diversification patterns of six effector families between G. pallida and G. rostochiensis, an additional reference genome was generated for an outgroup, the beet cyst nematode Heterodera schachtii (IRS population). Large evolutionary contrasts in effector family topologies were observed. While VAPs (venom allergen-like proteins) diversified before the split between the three cyst nematode species, the families GLAND5 and GLAND13 only expanded in PCNs after their separation from the genus Heterodera. Although DNA motifs in the promoter regions thought to be involved in the orchestration of effector expression ("DOG boxes") were present in all three cyst nematode species, their presence is not a necessity for dorsal gland-produced effectors. Notably, DOG box dosage was only loosely correlated with the expression level of individual effector variants. Comparison of the G. pallida genome with those of two other cyst nematodes underlined the fundamental differences in evolutionary history between effector families. Resequencing of PCN populations with different virulence characteristics will allow for the linking of these characteristics to the composition of the effector repertoire as well as for the mapping of PCN diversification patterns resulting from extreme anthropogenic range expansion.

Genomic Reconstruction of the Introduction and Diversification of Golden Potato Cyst Nematode Populations in Indonesia.

Potato cyst nematodes (PCNs), the umbrella term for Globodera rostochiensis and G. pallida, coevolved with their Solanaceous hosts in the Andean Mountain region. From there, PCN proliferated worldwide to virtually all potato production areas. PCN is a major factor limiting the potato production in Indonesia. In our survey, only G. rostochiensis was found. Fourteen field populations were collected on Java and Sumatra, and unique variants were called by mapping resequencing data on a G. rostochiensis reference genome. A phylogenetic tree based on 1.4 million unique variants showed a genotypic separation between the outgroup, a Scottish Ro1 population, and all Indonesian populations. This separation was comparable in size with the genotypic distinction between the Javanese and the Sumatran PCN populations. Next, variants within PCN effector gene families SPRYSEC, 1106, 4D06, and venom allergen-like protein (VAL) that all interfere with the host innate immune system were compared. Distinct selective pressures acted on these effector families; while SPRYSECs (4,341 single-nucleotide polymorphisms [SNPs]/insertions or deletions of bases [indels]) behaved like neutral genes, the phylogenetic trees of 1106, 4D06, and VAL proteins (235, 790, and 150 SNPs/indels, respectively) showed deviating topologies. Our data suggest that PCN was introduced on Java not too long after the introduction of potato in the middle of the eighteenth century. Soon thereafter, the pathogen established on Sumatra and started to diversify independently. This scenario was corroborated by diversification patterns of the effector families 1106, 4D06, and VAL. Our data demonstrate how genome resequencing data from a nonindigenous pathogen can be used to reconstruct the introduction and diversification process.

Comparative genomics of two inbred lines of the potato cyst nematode Globodera rostochiensis reveals disparate effector family-specific diversification patterns.

BACKGROUND: Potato cyst nematodes belong to the most harmful pathogens in potato, and durable management of these parasites largely depends on host-plant resistances. These resistances are pathotype specific. The current Globodera rostochiensis pathotype scheme that defines five pathotypes (Ro1 - Ro5) is both fundamentally and practically of limited value. Hence, resistant potato varieties are used worldwide in a poorly informed manner. RESULTS: We generated two novel reference genomes of G. rostochiensis inbred lines derived from a Ro1 and a Ro5 population. These genome sequences comprise 173 and 189 scaffolds respectively, marking a ≈ 24-fold reduction in fragmentation as compared to the current reference genome. We provide copy number variations for 19 effector families. Four dorsal gland effector families were investigated in more detail. SPRYSECs, known to be implicated in plant defence suppression, constitute by far the most diversified family studied herein with 60 and 99 variants in Ro1 and Ro5 distributed over 18 and 26 scaffolds. In contrast, CLEs, effectors involved in feeding site induction, show strong physical clustering. The 10 and 16 variants cluster on respectively 2 and 1 scaffolds. Given that pathotypes are defined by their effectoromes, we pinpoint the disparate nature of the contributing effector families in terms of sequence diversification and loss and gain of variants. CONCLUSIONS: Two novel reference genomes allow for nearly complete inventories of effector diversification and physical organisation within and between pathotypes. Combined with insights we provide on effector family-specific diversification patterns, this constitutes a basis for an effectorome-based virulence scheme for this notorious pathogen.

Characterization of the Habitat- and Season-Independent Increase in Fungal Biomass Induced by the Invasive Giant Goldenrod and Its Impact on the Fungivorous Nematode Community.

Outside its native range, the invasive plant species giant goldenrod (Solidago gigantea) has been shown to increase belowground fungal biomass. This non-obvious effect is poorly characterized; we don't know whether it is plant developmental stage-dependent, which fractions of the fungal community are affected, and whether it is reflected in the next trophic level. To address these questions, fungal assemblages in soil samples collected from invaded and uninvaded plots in two soil types were compared. Although using ergosterol as a marker for fungal biomass demonstrated a significant increase in fungal biomass, specific quantitative PCR (qPCR) assays did not point at a quantitative shift. MiSeq-based characterization of the belowground effects of giant goldenrod revealed a local increase of mainly Cladosporiaceae and Glomeraceae. This asymmetric boost in the fungal community was reflected in a specific shift in the fungivorous nematode community. Our findings provide insight into the potential impact of invasive plants on local fungal communities.

Organic amendment strengthens interkingdom associations in the soil and rhizosphere of barley (Hordeum vulgare).

Anthropogenic modification of soil systems has diverse impacts on food web interactions and ecosystem functioning. To understand the positive, neutral or adverse effects of agricultural practices on the associations of community members of soil microbes and microfaunal biomes, we characterized the effects of different fertilization types (organic, inorganic and a combination of organic and inorganic) on the food web active communities in the bulk soil and rhizosphere compartments in field conditions. We examined the influence of fertilization on (i) individual groups (bacteria, protozoa and fungi as microbe representatives and metazoans as microfauna representatives) and (ii) inter-kingdom interactions (focusing on the interactions between bacteria and eukaryotic groups) both neglecting and considering environmental factors in our analysis in combination with the microbial compositional data. Our results revealed different patterns of biota communities under organic versus inorganic fertilization, which shaped food web associations in both the bulk and rhizosphere compartments. Overall, organic fertilization increased the complexity of microbial-microfaunal ecological associations with inter- and intra- connections among categories of primary decomposers (bacteria and fungi) and predators (protozoa and microfauna) and differences in potential function in the soil food web in both the bulk and rhizosphere compartments. Furthermore, the inter-connections between primary decomposers and predators in bulk soil were more pronounced when environmental factors were considered. We suggest that organic fertilization selects bacterial orders with different potential ecological functions and interactions as survival, predation and cooperation due to more complex environment than those of inorganic or combined fertilization. Our findings support the importance of a comprehensive understanding of trophic food web patterns for soil management systems.

Conventional and organic soil management as divergent drivers of resident and active fractions of major soil food web constituents.

Conventional agricultural production systems, typified by large inputs of mineral fertilizers and pesticides, reduce soil biodiversity and may negatively affect ecosystem services such as carbon fixation, nutrient cycling and disease suppressiveness. Organic soil management is thought to contribute to a more diverse and stable soil food web, but data detailing this effect are sparse and fragmented. We set out to map both the resident (rDNA) and the active (rRNA) fractions of bacterial, fungal, protozoan and metazoan communities under various soil management regimes in two distinct soil types with barley as the main crop. Contrasts between resident and active communities explained 22%, 14%, 21% and 25% of the variance within the bacterial, fungal, protozoan, and metazoan communities. As the active fractions of organismal groups define the actual ecological functioning of soils, our findings underline the relevance of characterizing both resident and active pools. All four major organismal groups were affected by soil management (p < 0.01), and most taxa showed both an increased presence and an enlarged activity under the organic regime. Hence, a prolonged organic soil management not only impacts the primary decomposers, bacteria and fungi, but also major representatives of the next trophic level, protists and metazoa.

Shifts in the Active Rhizobiome Paralleling Low Meloidogyne chitwoodi Densities in Fields Under Prolonged Organic Soil Management.

Plants manipulate their rhizosphere community in a species and even a plant life stage-dependent manner. In essence plants select, promote and (de)activate directly the local bacterial and fungal community, and indirectly representatives of the next trophic level, protists and nematodes. By doing so, plants enlarge the pool of bioavailable nutrients and maximize local disease suppressiveness within the boundaries set by the nature of the local microbial community. MiSeq sequencing of specific variable regions of the 16S or 18S ribosomal DNA (rDNA) is widely used to map microbial shifts. As current RNA extraction procedures are time-consuming and expensive, the rRNA-based characterization of the active microbial community is taken along less frequently. Recently, we developed a relatively fast and affordable protocol for the simultaneous extraction of rDNA and rRNA from soil. Here, we investigated the long-term impact of three type of soil management, two conventional and an organic regime, on soil biota in fields naturally infested with the Columbian root-knot nematode Meloidogyne chitwoodi with pea (Pisum sativum) as the main crop. For all soil samples, large differences were observed between resident (rDNA) and active (rRNA) microbial communities. Among the four organismal group under investigation, the bacterial community was most affected by the main crop, and unweighted and weighted UniFrac analyses (explaining respectively 16.4% and 51.3% of the observed variation) pointed at a quantitative rather than a qualitative shift. LEfSe analyses were employed for each of the four organismal groups to taxonomically pinpoint the effects of soil management. Concentrating on the bacterial community in the pea rhizosphere, organic soil management resulted in a remarkable activation of members of the Burkholderiaceae, Enterobacteriaceae, and Pseudomonadaceae. Prolonged organic soil management was also accompanied by significantly higher densities of bacterivorous nematodes, whereas levels of M. chitwoodi had dropped drastically. Though present and active in the fields under investigation Orbiliaceae, a family harboring numerous nematophagous fungi, was not associated with the M. chitwoodi decline. A closer look revealed that a local accumulation and activation of Pseudomonas, a genus that includes a number of nematode-suppressive species, paralleled the lower M. chitwoodi densities. This study underlines the relevance of taking along both resident and active fractions of multiple organismal groups while mapping the impact of e.g. crops and soil management regimes.

Disparate gain and loss of parasitic abilities among nematode lineages.

Plant parasitism has arisen time and again in multiple phyla, including bacteria, fungi, insects and nematodes. In most of these organismal groups, the overwhelming diversity hampers a robust reconstruction of the origins and diversification patterns of this trophic lifestyle. Being a moderately diversified phylum with ≈ 4,100 plant parasites (15% of total biodiversity) subdivided over four independent lineages, nematodes constitute a major organismal group for which the genesis of plant parasitism could be mapped. Since substantial crop losses worldwide have been attributed to less than 1% of these plant parasites, research efforts are severely biased towards this minority. With the first molecular characterisation of numerous basal and supposedly harmless plant parasites as well as their non-parasitic relatives, we were able to generate a comprehensive molecular framework that allows for the reconstruction of trophic diversification for a complete phylum. In each lineage plant parasites reside in a single taxonomic grouping (family or order), and by taking the coverage of the next lower taxonomic level as a measure for representation, 50, 67, 100 and 85% of the known diversity was included. We revealed distinct gain and loss patterns with regard to plant parasitism per se as well as host exploitation strategies between these lineages. Our map of parasitic nematode biodiversity also revealed an unanticipated time reversal in which the two most ancient lineages showed the lowest level of ecological diversification and vice versa.

Rather than by direct acquisition via lateral gene transfer, GHF5 cellulases were passed on from early Pratylenchidae to root-knot and cyst nematodes.

BACKGROUND: Plant parasitic nematodes are unusual Metazoans as they are equipped with genes that allow for symbiont-independent degradation of plant cell walls. Among the cell wall-degrading enzymes, glycoside hydrolase family 5 (GHF5) cellulases are relatively well characterized, especially for high impact parasites such as root-knot and cyst nematodes. Interestingly, ancestors of extant nematodes most likely acquired these GHF5 cellulases from a prokaryote donor by one or multiple lateral gene transfer events. To obtain insight into the origin of GHF5 cellulases among evolutionary advanced members of the order Tylenchida, cellulase biodiversity data from less distal family members were collected and analyzed. RESULTS: Single nematodes were used to obtain (partial) genomic sequences of cellulases from representatives of the genera Meloidogyne, Pratylenchus, Hirschmanniella and Globodera. Combined Bayesian analysis of ≈ 100 cellulase sequences revealed three types of catalytic domains (A, B, and C). Represented by 84 sequences, type B is numerically dominant, and the overall topology of the catalytic domain type shows remarkable resemblance with trees based on neutral (= pathogenicity-unrelated) small subunit ribosomal DNA sequences. Bayesian analysis further suggested a sister relationship between the lesion nematode Pratylenchus thornei and all type B cellulases from root-knot nematodes. Yet, the relationship between the three catalytic domain types remained unclear. Superposition of intron data onto the cellulase tree suggests that types B and C are related, and together distinct from type A that is characterized by two unique introns. CONCLUSIONS: All Tylenchida members investigated here harbored one or multiple GHF5 cellulases. Three types of catalytic domains are distinguished, and the presence of at least two types is relatively common among plant parasitic Tylenchida. Analysis of coding sequences of cellulases suggests that root-knot and cyst nematodes did not acquire this gene directly by lateral genes transfer. More likely, these genes were passed on by ancestors of a family nowadays known as the Pratylenchidae.

SSU ribosomal DNA-based monitoring of nematode assemblages reveals distinct seasonal fluctuations within evolutionary heterogeneous feeding guilds.

Soils are among the most complex, diverse and competitive habitats on Earth and soil biota are responsible for ecosystem services such as nutrient cycling, carbon sequestration and remediation of freshwater. The extreme biodiversity prohibits the making of a full inventory of soil life. Hence, an appropriate indicator group should be selected to determine the biological condition of soil systems. Due to their ubiquity and the diverse responses to abiotic and biotic changes, nematodes are suitable indicators for environmental monitoring. However, the time-consuming microscopic analysis of nematode communities has limited the scale at which this indicator group is used. In an attempt to circumvent this problem, a quantitative PCR-based tool for the detection of a consistent part of the soil nematofauna was developed based on a phylum-wide molecular framework consisting of 2,400 full-length SSU rDNA sequences. Taxon-specific primers were designed and tested for specificity. Furthermore, relationships were determined between the quantitative PCR output and numbers of target nematodes. As a first field test for this DNA sequence signature-based approach, seasonal fluctuations of nematode assemblages under open canopy (one field) and closed canopy (one forest) were monitored. Fifteen taxa from four feeding guilds (covering ∼ 65% of the free-living nematode biodiversity at higher taxonomical level) were detected at two trophic levels. These four feeding guilds are composed of taxa that developed independently by parallel evolution and we detected ecologically interpretable patterns for free-living nematodes belonging to the lower trophic level of soil food webs. Our results show temporal fluctuations, which can be even opposite within taxa belonging to the same guild. This research on nematode assemblages revealed ecological information about the soil food web that had been partly overlooked.

Small subunit ribosomal DNA-based phylogenetic analysis of foliar nematodes (Aphelenchoides spp.) and their quantitative detection in complex DNA backgrounds.

Foliar nematodes, plant-parasitic representatives of the genus Aphelenchoides, constitute a minority in a group dominated by fungivorous species. Distinction between (mostly harmless) fungal feeding Aphelenchoides species and high impact plant parasites such as A. besseyi, A. fragariae, A. ritzemabosi, and A. subtenuis is severely hampered by the scarcity of informative morphological characters, some of which are only observable in specific developmental stages. Poor description of a number of non-plant-parasitic Aphelenchoides species further complicates identification. Based on (nearly) full-length small subunit ribosomal DNA (SSU rDNA) sequences (≈1,700 bp), a phylogenetic tree was generated, and the four target species appeared as distinct, well-supported groups. Notably, this genus does not constitute a monophyletic group: A. besseyi and A. ritzemabosi cluster together and they are phylogenetically isolated from A. fragariae, A. subtenuis, and most other fungivorous species. A phylum-wide SSU rDNA framework was used to identify species-specific DNA motifs. For the molecular detection of four plant-parasitic Aphelenchoides species, polymerase chain reaction primers were developed with high, identical annealing temperatures (63°C). Within the molecular framework presented here, these primers can be used for the rapid screening of plant material and soil for the presence of one or multiple foliar nematode species.

A rapid, sensitive, and cost-efficient assay to estimate viability of potato cyst nematodes.

Potato cyst nematodes (PCNs) are quarantine organisms, and they belong to the economically most relevant pathogens of potato worldwide. Methodologies to assess the viability of their cysts, which can contain 200 to 500 eggs protected by the hardened cuticle of a dead female, are either time and labor intensive or lack robustness. We present a robust and cost-efficient viability assay based on loss of membrane integrity upon death. This assay uses trehalose, a disaccharide present at a high concentration in the perivitelline fluid of PCN eggs, as a viability marker. Although this assay can detect a single viable egg, the limit of detection for regular field samples was higher, ≈10 viable eggs, due to background signals produced by other soil components. On the basis of 30 nonviable PCN samples from The Netherlands, a threshold level was defined (ΔA(trehalose) = 0.0094) below which the presence of >10 viable eggs is highly unlikely (true for ≈99.7% of the observations). This assay can easily be combined with a subsequent DNA-based species determination. The presence of trehalose is a general phenomenon among cyst nematodes; therefore, this method can probably be used for (for example) soybean, sugar beet, and cereal cyst nematodes as well.

A ribosomal DNA-based framework for the detection and quantification of stress-sensitive nematode families in terrestrial habitats.

Indigenous communities of soil-resident nematodes have a high potential for soil health assessment as nematodes are diverse, abundant, trophically heterogeneous and easily extractable from soil. The conserved morphology of nematodes is the main operational reason for their under-exploitation as soil health indicators, and a user-friendly biosensor system should preferably be based on nonmorphological traits. More than 80% of the most environmental stress-sensitive nematode families belong to the orders Mononchida and Dorylaimida. The phylogenetic resolution offered by full-length small subunit ribosomal DNA (SSU rDNA) sequences within these two orders is highly different. Notwithstanding several discrepancies between morphology and SSU rDNA-based systematics, Mononchida families (indicated here as M1-M5) are relatively well-supported and, consequently, family-specific DNA sequences signatures could be defined. Apart from Nygolaimidae and Longidoridae, the resolution among Dorylaimida families was poor. Therefore, a part of the more variable large subunit rDNA (≈ 1000 bp from the 5'-end) was sequenced for 72 Dorylaimida species. Sequence analysis revealed a subclade division among Dorylaimida (here defined as D1-D9, PP1-PP3) that shows only distant similarity with 'classical' Dorylaimid systematics. Most subclades were trophically homogeneous, and - in most cases - specific morphological characteristics could be pinpointed that support the proposed division. To illustrate the practicability of the proposed molecular framework, we designed primers for the detection of individual subclades within the order Mononchida in a complex DNA background (viz. in terrestrial or freshwater nematode communities) and tested them in quantitative assays (real-time polymerase chain reaction). Our results constitute proof-of-principle for the concept of DNA sequence signatures-based monitoring of stress sensitive nematode families in environmental samples.

Phylum-wide analysis of SSU rDNA reveals deep phylogenetic relationships among nematodes and accelerated evolution toward crown Clades.

Inference of evolutionary relationships between nematodes is severely hampered by their conserved morphology, the high frequency of homoplasy, and the scarcity of phylum-wide molecular data. To study the origin of nematode radiation and to unravel the phylogenetic relationships between distantly related species, 339 nearly full-length small-subunit rDNA sequences were analyzed from a diverse range of nematodes. Bayesian inference revealed a backbone comprising 12 consecutive dichotomies that subdivided the phylum Nematoda into 12 clades. The most basal clade is dominated by the subclass Enoplia, and members of the order Triplonchida occupy positions most close to the common ancestor of the nematodes. Crown Clades 8-12, a group formerly indicated as "Secernentea" that includes Caenorhabditis elegans and virtually all major plant and animal parasites, show significantly higher nucleotide substitution rates than the more basal Clades 1-7. Accelerated substitution rates are associated with parasitic lifestyles (Clades 8 and 12) or short generation times (Clades 9-11). The relatively high substitution rates in the distal clades resulted in numerous autapomorphies that allow in most cases DNA barcode-based species identification. Teratocephalus, a genus comprising terrestrial bacterivores, was shown to be most close to the starting point of Secernentean radiation. Notably, fungal feeding nematodes were exclusively found basal to or as sister taxon next to the 3 groups of plant parasitic nematodes, namely, Trichodoridae, Longidoridae, and Tylenchomorpha. The exclusive common presence of fungivorous and plant parasitic nematodes supports a long-standing hypothesis that states that plant parasitic nematodes arose from fungivorous ancestors.