An overview of arbuscular mycorrhizal fungi–nematode interactions

Plant parasitic nematodes and arbuscular mycorrhizal fungi (AMF) share plant roots as a resource for food and space. The interest in AMF-nematode interactions lies in the possibility of enhanced resistance or tolerance of AMF-infected plants to nematodes, and the potential value of this for control of crop pests. Data collated from previous studies revealed a great diversity of AMF-nematode responses and we seek to generalise from these by evaluating and discussing interactions involving three groups of nematodes distinguished by their mode of parasitism: (i) ectoparasites; (ii) sedentary endoparasites; and (iii) migratory endoparasites. Based on proximity in tissue, we expected that the interactions between endoparasites and AMF would be stronger, i.e. more reciprocal effects of endoparasitic nematodes on AMF, than those between ectoparasites and AMF. Contrary to this hypothesis, we found that, relative to AMF-free plants, AMF-infected plants were damaged more by ectoparasites than by endoparasites. Of the sedentary endoparasites, numbers of root-knot nematodes were reduced more by mycorrhizal infection than were those of cyst nematodes. The reduction in nematode damage by AMF was not different for root-knot or cyst nematode infested plants. Migratory endoparasitic nematodes were the only group whose numbers were greater on AMF-infected plants. However, the experiments involving migratory nematodes were characterised by relatively high levels of AMF infection and little nematode damage compared to the other feeding types. The outcomes of the AMF-nematode interactions are determined by many factors during the interactions between organisms and their physical, physiological and temporal environments. Assessing effects by recording plant sizes and total nematode or AMF populations at the end of experiments gives very little information on the mechanisms of the interactions. It is time to stop doing studies of black boxes and time to start observing processes, directly by using microscopy and indirectly by application of molecular genetics.     

Cellular Signaling Pathways and Posttranslational Modifications Mediated by Nematode Effector Proteins

Plant-parasitic cyst and root-knot nematodes synthesize and secrete a suite of effector proteins into infected host cells and tissues. These effectors are the major virulence determinants mediating the transformation of normal root cells into specialized feeding structures. Compelling evidence indicates that these effectors directly hijack or manipulate refined host physiological processes to promote the successful parasitism of host plants. Here, we provide an update on recent progress in elucidating the molecular functions of nematode effectors. In particular, we emphasize how nematode effectors modify plant cell wall structure, mimic the activity of host proteins, alter auxin signaling, and subvert defense signaling and immune responses. In addition, we discuss the emerging evidence suggesting that nematode effectors target and recruit various components of host posttranslational machinery in order to perturb the host signaling networks required for immunity and to regulate their own activity and subcellular localization.The root-knot (Meloidogyne spp.) and cyst (Globodera and Heterodera spp.) nematodes are sedentary endoparasites of the root system in a wide range of plant species. These obligate parasites engage in intricate relationships with their host plants that result in the transformation of normal root cells into specialized feeding sites, which provide the nematodes with all the nutrients required for their development. The initiation and maintenance of functional feeding cells by root-knot nematodes (giant cells) and cyst nematodes (syncytia) seems to be a dynamic process involving active dialogue between the nematodes and their host plants. The nematodes use their stylet, a needle-like apparatus, to deliver effector proteins into the host cells (Williamson and Hussey, 1996; Davis et al., 2004). These effector proteins are mainly synthesized in the nematode esophageal glands, which consist of one dorsal cell and two subventral cells. The activity of these glands is developmentally regulated, with secretions from the two subventral glands being most dynamic during the early stage of infection, consisting of root penetration, migration, and feeding site initiation. Secretions from the single dorsal cell seem to be more active during the sedentary stage of nematode feeding (Hussey and Mims, 1990).Recent progress in the functional characterization of effector proteins from a number of phytonematodes has elucidated diverse mechanisms through which these effectors facilitate the nematode parasitism of host plants. One such mechanism involves depolymerization of the main structural polysaccharide constituents of the plant cell wall by using a diverse collection of extracellular effector proteins (Davis et al., 2011; Wieczorek, 2015). Another mechanism includes the molecular mimicry of host proteins in both form and function (Gheysen and Mitchum, 2011). This strategy could be highly successful when the nematode-secreted effectors imitate host functions to subvert cellular processes in favor of nematodes while escaping the regulation of host cellular processes. Another mechanism of effector action is the modulation of central components of auxin signaling to apparently generate unique patterns of auxin-responsive gene expression, leading to numerous physiological and developmental changes required for feeding site formation and development (Cabrera et al., 2015). In addition, cyst and root-knot nematodes have evolved to efficiently suppress defense responses during their prolonged period of sedentary biotrophic interaction with their hosts. Accordingly, a large number of nematode effectors are engaged in suppressing host immune responses and defense signaling (Hewezi and Baum, 2013; Goverse and Smant, 2014). Finally, there is accumulating evidence that nematode effector proteins target and exploit the host posttranslational machinery to the parasite’s advantage. Posttranslational modifications (PTMs) are tightly controlled and highly specific processes that enable rapid cellular responses to specific stimuli without the requirement of new protein synthesis (Kwon et al., 2006). Phosphorylation, ubiquitination, and histone modifications, among others, have recently been identified as fundamental cellular processes controlling immune signaling pathways (Stulemeijer and Joosten, 2008; Howden and Huitema, 2012; Marino et al., 2012; Salomon and Orth, 2013). This finding underscores the importance of targeting and coopting host posttranslational machinery by pathogen effectors to exert their virulence functions. Here, we review recent progress in the functional characterization of nematode effector proteins and the parasitic strategies that involve modifications of the plant cell wall, molecular mimicry of host factors, alteration of auxin signaling, subversion of defense signaling, and targeting and utilizing the host posttranslational machinery.     

A phylogenetic framework for root lesion nematodes of the genus Pratylenchus (Nematoda): Evidence from 18S and D2-D3 expansion segments of 28S ribosomal RNA genes and morphological characters

The root lesion nematodes of the genus Pratylenchus Filipjev, 1936 are migratory endoparasites of plant roots, considered among the most widespread and important nematode parasites in a variety of crops. We obtained gene sequences from the D2 and D3 expansion segments of 28S rRNA partial and 18S rRNA from 31 populations belonging to 11 valid and two unidentified species of root lesion nematodes and five outgroup taxa. These datasets were analyzed using maximum parsimony and Bayesian inference. The alignments were generated using the secondary structure models for these molecules and analyzed with Bayesian inference under the standard models and the complex model, considering helices under the doublet model and loops and bulges under the general time reversible model. The phylogenetic informativeness of morphological characters is tested by reconstruction of their histories on rRNA based trees using parallel parsimony and Bayesian approaches. Phylogenetic and sequence analyses of the 28S D2–D3 dataset with 145 accessions for 28 species and 18S dataset with 68 accessions for 15 species confirmed among large numbers of geographical diverse isolates that most classical morphospecies are monophyletic. Phylogenetic analyses revealed at least six distinct major clades of examined Pratylenchus species and these clades are generally congruent with those defined by characters derived from lip patterns, numbers of lip annules, and spermatheca shape. Morphological results suggest the need for sophisticated character discovery and analysis for morphology based phylogenetics in nematodes.     

Functional C‐TERMINALLY ENCODED PEPTIDE (CEP) plant hormone domains evolved de novo in the plant parasite Rotylenchulus reniformis

Sedentary plant‐parasitic nematodes (PPNs) induce and maintain an intimate relationship with their host, stimulating cells adjacent to root vascular tissue to re‐differentiate into unique and metabolically active ‘feeding sites’. The interaction between PPNs and their host is mediated by nematode effectors. We describe the discovery of a large and diverse family of effector genes, encoding C‐TERMINALLY ENCODED PEPTIDE (CEP) plant hormone mimics (RrCEPs), in the syncytia‐forming plant parasite Rotylenchulus reniformis. The particular attributes of RrCEPs distinguish them from all other CEPs, regardless of origin. Together with the distant phylogenetic relationship of R. reniformis to the only other CEP‐encoding nematode genus identified to date (Meloidogyne), this suggests that CEPs probably evolved de novo in R. reniformis. We have characterized the first member of this large gene family (RrCEP1), demonstrating its significant up‐regulation during the plant–nematode interaction and expression in the effector‐producing pharyngeal gland cell. All internal CEP domains of multi‐domain RrCEPs are followed by di‐basic residues, suggesting a mechanism for cleavage. A synthetic peptide corresponding to RrCEP1 domain 1 is biologically active and capable of up‐regulating plant nitrate transporter (AtNRT2.1) expression, whilst simultaneously reducing primary root elongation. When a non‐CEP‐containing, syncytia‐forming PPN species (Heterodera schachtii) infects Arabidopsis in a CEP‐rich environment, a smaller feeding site is produced. We hypothesize that CEPs of R. reniformis represent a two‐fold adaptation to sustained biotrophy in this species: (i) increasing host nitrate uptake, whilst (ii) limiting the size of the syncytial feeding site produced.     

Consequences of variation in species diversity in a community of root-feeding herbivores for nematode dynamics and host plant biomass

To date, no study has explicitly addressed effects of variation in species diversity of root‐feeding herbivores on host plant biomass. Root‐feeding nematodes typically occur in multi‐species communities. In a three‐year field experiment, we investigated how variation in species diversity of root‐feeding nematodes affected nematode dynamics and response of the dune grass Ammophila arenaria to root‐feeder activity. This plant species needs regular burial by fresh beach sand to remain vigorous, suggesting that A. arenaria benefits from a temporary escape from root‐feeding soil organisms and that root‐feeders are involved in plant degeneration in stabilized dunes. We created series of ceased and continued sand burial and added the endoparasitic nematodes Meloidogyne maritima, Heterodera arenaria and Pratylenchus penetrans alone or in combination to A. arenaria. We included treatments with and without the whole soil community, measured plant biomass and quantified numbers of nematodes. Addition of H. arenaria and P. penetrans decreased numbers of M. maritima juveniles and delayed the first appearance in time of both juveniles and females, while numbers of males only decreased when plants had been buried. Burial with sand and addition of the other two endoparasites affected numbers of H. arenaria juveniles, while numbers of P. penetrans were low and not affected. Shoot biomass of A. arenaria was lower when M. maritima had been added alone than when the three species had been added together. Addition of root zone soil decreased biomass of all plant parts. Burial with sand decreased aboveground shoot biomass, whereas it increased belowground shoot and root biomass. Our results point at idiosyncratic effects of nematode diversity on A. arenaria biomass. Heterodera arenaria and P. penetrans protected their host by reducing numbers and delaying activity of M. maritima to a later stage in the growth season, when root‐feeding activity was less harmful for plant biomass development.     

Proteins secreted by root-knot nematodes accumulate in the extracellular compartment during root infection

Root-knot nematodes are biotrophic parasites that invade the root apex of host plants and migrate towards the vascular cylinder where they induce the differentiation of root cells into hypertrophied multinucleated giant cells. Giant cells are part of the permanent feeding site required for nematode development into the adult stage. To date, a repertoire of candidate effectors potentially secreted by the nematode into the plant tissues to promote infection has been identified. However, the precise role of these candidate effectors during root invasion or during giant cell induction and maintenance remains largely unknown. Primarily, the identification of the destination of nematode effectors within plant cell compartment(s) is crucial to decipher their actual functions. We analyzed the fine localization in root tissues of five nematode effectors throughout the migratory and sedentary phases of parasitism using an adapted immunocytochemical method that preserves host and pathogen tissues. We showed that secretion of effectors from the amphids or the oesophageal glands is tightly regulated during the course of infection. The analyzed effectors accumulated in the root tissues along the nematode migratory path and along the cell wall of giant cells, showing the apoplasm as an important destination compartment for these effectors during migration and feeding cell formation.Key words: plant pathogen, effector, immunocytochemistry, root-knot nematode, secretion, plant apoplasm     

Effects of aldicarb and benomyl on nematodes, vesicular arbuscular mycorrhizas and the growth and yield of forage maize

The effects of aldicarb and benomyl on plant-parasitic nematodes, vesicular arbuscular mycorrhiza and the growth of forage maize were measured in 1980—1982 in two field experiments at Woburn, Bedfordshire and in a pot experiment using loamy sand soil from the field site. The most numerous migratory nematode, Tylenchorhynchus dubius increased three to four-fold during each season in untreated soil and was effectively controlled by aldicarb. Pratylenchus species were fewer but equally well controlled. The cereal cyst-nematode (Heterodera avenae), a serious maize pathogen in Northern France, was relatively scarce in untreated roots and was further decreased by aldicarb treatment; post-harvest H. avenae egg numbers were not affected by treatments; they declined equally because maize is such a poor host. Significant yield benefits (up to 37%) followed aldicarb treatment and were ascribed to nematode control in the absence of attribution to insect or other pests. Benomyl did not increase yields nor did it significantly affect the incidence of mycorrhiza. The results confirm that considerable losses of forage maize can be caused by nematodes in light soil and that aldicarb is an effective nematicide even at the rate of 1·7 kg a.i./ha.     

Activity profiling of barley vacuolar processing enzymes provides new insights into the plant and cyst nematode interaction

Vacuolar processing enzymes (VPEs) play an important role during regular growth and development and defence responses. Despite substantial attempts to understand the molecular basis of plant–cyst nematode interaction, the mechanism of VPEs functioning during this interaction remains unknown. The second-stage Heterodera filipjevi juvenile penetrates host roots and induces the formation of a permanent feeding site called a syncytium. To investigate whether infection with H. filipjevi alters plant host VPEs, the studies were performed in Hordeum vulgare roots and leaves on the day of inoculation and at 7, 14 and 21 days post-inoculation (dpi). Implementing molecular, biochemical and microscopic methods we identified reasons for modulation of barley VPE activity during interaction with H. filipjevi. Heterodera filipjevi parasitism caused a general decrease of VPE activity in infected roots, but live imaging of VPEs showed that their activity is up-regulated in syncytia at 7 and 14 dpi and down-regulated at 21 dpi. These findings were accompanied by tissue-specific VPE gene expression patterns. Expression of the barley cystatin HvCPI-4 gene was stimulated in leaves but diminished in roots upon infestation. External application of cyclotides that can be produced naturally by VPEs elicits in pre-parasitic juveniles vesiculation of their body, enhanced formation of granules, induction of exploratory behaviour (stylet thrusts and head movements), production of reactive oxygen species (ROS) and final death by methuosis. Taken together, down-regulation of VPE activity through nematode effectors promotes the nematode invasion rates and leads to avoidance of the induction of the plant proteolytic response and death of the invading juveniles.     

A Plant Health Care Program for Brambles in the Pacific Northwest

Pratylenchus and Xiphinema species have been associated with decline and mortality of brambles (Rubus species) in the Pacific Northwest of the United States. These nematodes cause direct feeding damage and (or) transmit viruses that result in poor fruit quality and plant decline. A nematode management program has been developed by the author to minimize chemical use and nematode-induced damage while optimizing fruit production. Nematode management is an integral part of a total plant health care program in which foliar and soil pests, plant stresses, and fertility are managed.     

Screening soybean cyst nematode effectors for their ability to suppress plant immunity

The soybean cyst nematode (SCN), Heterodera glycines, is one of the most destructive pathogens of soybeans. SCN is an obligate and sedentary parasite that transforms host plant root cells into an elaborate permanent feeding site, a syncytium. Formation and maintenance of a viable syncytium is an absolute requirement for nematode growth and reproduction. In turn, sensing pathogen attack, plants activate defence responses and may trigger programmed cell death at the sites of infection. For successful parasitism, H. glycines must suppress these host defence responses to establish and maintain viable syncytia. Similar to other pathogens, H. glycines engages in these molecular interactions with its host via effector proteins. The goal of this study was to conduct a comprehensive screen to identify H. glycines effectors that interfere with plant immune responses. We used Nicotiana benthamiana plants infected by Pseudomonas syringae and Pseudomonas fluorescens strains. Using these pathosystems, we screened 51 H. glycines effectors to identify candidates that could inhibit effector-triggered immunity (ETI) and/or pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI). We identified three effectors as ETI suppressors and seven effectors as PTI suppressors. We also assessed expression modulation of plant immune marker genes as a function of these suppressors.     

The effect of fungal parasitism and predation on the population dynamics of nematodes in the activated sludge process

Nematophagous fungi, both predators and endoparasites, were found to be common components of activated sludge. Although rotifers and ciliate protozoa (both potential prey) were also abundant, no fungi were parasitic on these organisms. Endoparasitic fungi, which were far more abundant than predators, were able to infect nematodes and complete their life cycles successfully. Neither of the predatory fungi observed were able to produce conidia: an unidentified net-forming species lived saprophytically and failed to capture any prey, although it played a minor role in the formation of microbial flocs, and a single conidium of Dactylella mammillata was observed to capture a nematode by spontaneous trap formation. Several endoparasites were recorded although single species dominated each sludge examined; these were Meria coniospora and Catenaria anguillulae. Both endoparasites were related to the population dynamics of the nematodes with 100% of the nematode population becoming infected at certain times. Clear predator (parasitetprey (host) associations were discernible and these are discussed.     

Effect of tillage,subsidiary crops and fertilisation on plant‐parasitic nematodes in a range of agro‐environmental conditions within Europe

The overall goal in nematode management is to develop sustainable systems where nematode populations are kept under the economic damage threshold. Conservation tillage and subsidiary crops, applied as cover crops and living mulches, generally improve soil health by increasing soil organic matter content and stimulating soil microbial activity. However, more permanent crop and weed cover associated with subsidiary crops and noninversion tillage, respectively, may benefit plant‐parasitic nematodes with broad host spectra such as Meloidogyne and Pratylenchus. These genera are major constraints to many field crops throughout Europe and there is a need to identify effective and reliable management options that can be applied to avoid excessive infestations. The dynamics of the indigenous fauna of plant‐parasitic nematodes were studied in eight coordinated multi‐environment field experiments (MEEs) under four agro‐environmental conditions in Europe (Continental, Nemoral, Atlantic North and Mediterranean North). The MEEs consisted of a 2‐year sequence of wheat combined with a living mulch or subsequent cover crops and second main crops maize, potatoes or tomatoes depending on site. Additionally, the effects of inversion tillage using the plough were compared with various forms of conservation tillage (no‐tillage, shallow and deep noninversion tillage). Overall, Helicotylenchus, Paratylenchus, Pratylenchus and Tylenchorhynchus were the most frequent genera across sites while Meloidogyne occurred only in Germany at very low densities. During the wheat–maize sequences in Switzerland, the populations of Pratylenchus increased from 63 to 146 nematodes per 100 mL soil and Helicotylenchus from 233 to 632 nematodes per 100 mL soil. The effects of tillage on plant‐parasitic nematodes were generally minor, although no tillage in Italy supported higher densities of Pratylenchus (184 nematodes per 100 mL soil) than inversion tillage (59 nematodes per 100 mL soil). Furthermore, Pratylenchus densities were 160 nematodes per 100 mL soil when leguminous subsidiary crops were grown, 122 nematodes per 100 mL soil in the green fallow and 84 nematodes per 100 mL soil after growing black oat (Avena strigosa) or oilseed radish (Raphanus sativus). The differences were greatest in Italy, in a sandy soil with low organic matter. Application of compost or nitrogen fertiliser had no consistent effects on plant‐parasitic nematodes. We conclude that crop rotations including specific subsidiary crops are prominent factors affecting the indigenous nematode community, while tillage and fertiliser are of lower importance.