The role of phytohormones ethylene and auxin in plant-nematode interactions

The phytohormones ethylene and auxin regulate many important processes in plants, including cell differentiation, cell expansion, and responses to abiotic stresses. These hormones also play important roles in many plant-pathogen interactions, including regulation of plant defense responses and symptom development. Sedentary plant-parasitic nematodes, which require the formation of a complex feeding site within the host root, are among the world’s most destructive plant pathogens. Nematode-induced feeding sites show dramatic changes in host cell morphology and gene expression. These changes are likely mediated, at least in part, by phytohormones. In the present review, current knowledge of the roles of ethylene and auxin will be explored in two main areas: the specific role of phytohormones in mediating feeding site development by plant-parasitic nematodes and the general role of phytohormones in affecting the ability of parasitic nematodes to cause disease. Published in Russian in Fiziologiya Rastenii, 2009, Vol. 56, No. 1, pp. 3–7. This article was presented in original.     

Naturally induced secretions of the potato cyst nematode co-stimulate the proliferation of both tobacco leaf protoplasts and human peripheral blood mononuclear cells.

Naturally induced secretions from infective juveniles of the potato cyst nematode Globodera rostochiensis co-stimulate the proliferation of tobacco leaf protoplasts in the presence of the synthetic phytohormones alpha-naphthaleneacetic acid (NAA) and 6-benzylaminopurine (BAP). With the use of a protoplast-based bioassay, a low-molecular-weight peptide(s) (< 3 kDa) was shown to be responsible for the observed effect. This mitogenic oligopeptide(s) is functionally dissimilar to auxin and cytokinin and, in addition, it does not change the sensitivity of the protoplasts toward these phytohormones. In combination with the mitogen phytohemagglutinin (PHA), cyst nematode secretions also co-stimulated mitogenesis in human peripheral blood mononuclear cells (PBMC). The stimulation of plant cells isolated from nontarget tissue--these nematodes normally invade the roots of potato plants--suggests the activation of a general signal transduction mechanism(s) by an oligopeptide(s) secreted by the nematode. Whether a similar oligopeptide-induced mechanism underlies human PBMC activation remains to be investigated. Reactivation of the cell cycle is a crucial event in feeding cell formation by cyst nematodes. The secretion of a mitogenic low-molecular-weight peptide(s) by infective juveniles of the potato cyst nematode could contribute to the redifferentiation of plant cells into such a feeding cell.     

Brief Review of the Associations of Xylobiont Nematodes with Bark Beetles (Coleoptera,Curculionidae: Scolytinae)

Nematode pathogens cause wilt diseases in conifers and deciduous trees. The longhorn beetles (Coleoptera: Cerambycidae) and bark beetles (Coleoptera: Curculionidae: Scolytinae) act as nematode vectors spreading the invasive juvenile stages during their maturation feeding or during oviposition on the plant hosts. There are numerous reviews of nematodes associated with bark beetles on conifers, while little attention has been paid to the nematodes of deciduous trees. The development of Dutch elm disease and ash dieback is mainly caused by fungal pathogens transmitted by bark beetles; the latter act as vectors of not only fungi but also nematodes enclosed in nematangia under their elytra, and also in the tracheae and Malpighian canals. Apart from phytopathogenic nematodes, bark beetles transmit mycophagous and bacterivorous nematodes and own parasites of bark beetles. The ecological groups of nematodes associated with Scolytinae are reviewed; the known records of associations of nematodes with bark beetles are listed for coniferous host plants of Russia and neighboring countries; the world-wide list of these associations for deciduous plant hosts is given.

     

Effects of genetic mutations and chemical exposures on Caenorhabditis elegans feeding: evaluation of a novel, high-throughput screening assay

Background

Government agencies have defined a need to reduce, refine or replace current mammalian-based bioassays with testing methods that use alternative species. Invertebrate species, such as Caenorhabditis elegans, provide an attractive option because of their short life cycles, inexpensive maintenance, and high degree of evolutionary conservation with higher eukaryotes. The C. elegans pharynx is a favorable model for studying neuromuscular function, and the effects of chemicals on neuromuscular activity, i.e., feeding. Current feeding methodologies, however, are labor intensive and only semi-quantitative.

Methodology/Principal Findings

Here a high-throughput assay is described that uses flow cytometry to measure C. elegans feeding by determining the size and intestinal fluorescence of hundreds of nematodes after exposure to fluorescent-labeled microspheres. This assay was validated by quantifying fluorescence in feeding-defective C. elegans (eat mutants), and by exposing wild-type nematodes to the neuroactive compounds, serotonin and arecoline. The eat mutations previously determined to cause slow pumping rates exhibited the lowest feeding levels with our assay. Concentration-dependent increases in feeding levels after serotonin exposures were dependent on food availability, while feeding levels decreased in arecoline-exposed nematodes regardless of the presence of food. The effects of the environmental contaminants, cadmium chloride and chlorpyrifos, on wild-type C. elegans feeding were then used to demonstrate an application of the feeding assay. Cadmium exposures above 200 µM led to a sharp drop in feeding levels. Feeding of chlorpyrifos-exposed nematodes decreased in a concentration-dependent fashion with an EC₅₀ of 2 µM.

Conclusions/Significance

The C. elegans fluorescence microsphere feeding assay is a rapid, reliable method for the assessment of neurotoxic effects of pharmaceutical drugs, industrial chemicals or environmental agents. This assay may also be applicable to large scale genetic or RNAi screens used to identify genes that are necessary for the development or function of the pharynx or other neuromuscular systems.     

Changes in community composition, carbon and nitrogen stable isotope signatures and feeding strategy in epilithic aquatic nematodes along a depth gradient

Periphyton is an important component within the littoral zones of lakes, but it is known to vary dramatically on small (cm–m) and large (km) spatial scales, showing differences in composition and abundance. Until relatively recently, changes in periphyton composition along depth gradients have not been studied sufficiently and the response of small meiobenthic invertebrates inhabiting the periphyton to vertically changing environmental conditions such as light are poorly understood. To investigate the changing community composition of epilithic (on stones) nematodes along a depth gradient, we conducted a field study at Lake Erken, Sweden, with the specific objective to investigate whether changes in periphyton composition (algae, bacteria) are reflected in changing nematode feeding types and what the consequences are for nematodes and their resource consumption. We analysed the abundance, species composition and feeding type distribution of epilithic nematodes along 11 depth levels, from 5 to 300?cm water depth. Our study resulted in the first measurements of carbon (¹³C) and nitrogen (¹⁵N) stable isotopes in free living nematodes in lakes. Nematode community composition and feeding type distribution exhibited dramatic changes along the depth gradient. Nematode feeding types changed from a dominance of algae-feeding species in the shallow littoral zone to one of bacteria-feeding species in the deep littoral zone. The ¹³C stable isotope signatures of nematodes and the small (<20?μm) periphyton fraction changed with increasing depth, with nematodes shown to feed on this small periphytic component. Nematodes were identified as primary consumers by means of trophic level calculations based on ¹⁵N stable isotopes.     

Cell cycle activation by plant parasitic nematodes

Sedentary nematodes are important pests of crop plants. They are biotrophic parasites that can induce the (re)differentiation of either differentiated or undifferentiated plant cells into specialized feeding cells. This (re)differentiation includes the reactivation of the cell cycle in specific plant cells finally resulting in a transfer cell-like feeding site. For growth and development the nematodes fully depend on these cells. The mechanisms underlying the ability of these nematodes to manipulate a plant for its own benefit are unknown. Nematode secretions are thought to play a key role both in plant penetration and feeding cell induction. Research on plant-nematode interactions is hampered by the minute size of cyst and root knot nematodes, their obligatory biotrophic nature and their relatively long life cycle. Recently, insights into cell cycle control in Arabidopsis thaliana in combination with reporter gene technologies showed the differential activation of cell cycle gene promoters upon infection with cyst or root knot nematodes. In this review, we integrate the current views of plant cell fate manipulation by these sedentary nematodes and made an inventory of possible links between cell cycle activation and local, nematode-induced changes in auxin levels.     

Assaying Predatory Feeding Behaviors in Pristionchus and Other Nematodes

This protocol provides multiple methods for the analysis and quantification of predatory feeding behaviors in nematodes. Many nematode species including Pristionchus pacificus display complex behaviors, the most striking of which is the predation of other nematode larvae. However, as these behaviors are absent in the model organism Caenorhabditis elegans, they have thus far only recently been described in detail along with the development of reliable behavioral assays ¹. These predatory behaviors are dependent upon phenotypically plastic but fixed mouth morphs making the correct identification and categorization of these animals essential. In P. pacificus there are two mouth types, the stenostomatous and eurystomatous morphs ², with only the wide mouthed eurystomatous containing an extra tooth and being capable of killing other nematode larvae. Through the isolation of an abundance of size matched prey larvae and subsequent exposure to predatory nematodes, assays including both "corpse assays" and "bite assays" on correctly identified mouth morph nematodes are possible. These assays provide a means to rapidly quantify predation success rates and provide a detailed behavioral analysis of individual nematodes engaged in predatory feeding activities. In addition, with the use of a high-speed camera, visualization of changes in pharyngeal activity including tooth and pumping dynamics are also possible.     

The effect of auxin on plant regeneration of wheat,barley and triticale

One of the basic components of a medium influencing somatic embryogenesis of cereals from immature embryos is the type of auxin. According to some researchers, phytohormones can also play an important role during Agrobacterium-mediated transformation. In this first part of research, the influence of three types of auxins used alone or in combination of two on somatic embryogenesis and plant regeneration in three cereal species has been tested. Eight cultivars of barley, five cultivars of wheat and three cultivars of triticale have been used. Efficiency of plant development on two regeneration media, with and without growth regulators has been compared. Efficiency of regeneration characterized by frequency of explants that form embryogenic callus ranged from 25% for wheat cultivar Torka to 100% for two barley cultivars. Mean number of plantlets regenerating per explant differed significantly (from 2 to 58) depending on the type of auxin in inducing media, the type of regenerating media as well as cultivar. The biggest differences in regeneration efficiency were observed between barley cultivars, however regeneration of plants occurred in all combinations tested. The best regeneration coefficients for most barley cultivars were obtained after culture on dicamba or dicamba with 2,4-D. However, in the case of highly regenerating cv Scarlett, the most effective culture media contained picloram or 2,4-D alone. The highest values of regeneration coefficients for two triticale cultivars (Wanad and Kargo) were obtained on picloram (26.1 and 21.4, respectively) and for `Gabo' on picloram with dicamba (12.6). The range of mean number of regenerated plantlets was from 12 to 30. Dicamba alone or lower concentrations of picloram with 2,4-D were the best media influencing embryogenic callus formation in five wheat cultivars. However, the highest values of regeneration coefficients ranging from 10.6 to 26.8 were obtained at lower concentrations of picloram with 2,4-D or picloram with dicamba. R2 regeneration medium containing growth regulators was significantly better for plantlet development in several combinations (cultivar and induction medium) than the one without growth regulators. Generally, regeneration coefficients for all tested cultivars of three cereal species on the best media were high, ranging from 5.5 for barley cultivar Rodion to 51.6 for another barley cultivar Scarlett. Plantlets developed normally, flowering and setting seed.     

Effects of native and exotic range‐expanding plant species on taxonomic and functional composition of nematodes in the soil food web

Due to climate warming, many plant species shift ranges towards higher latitudes. Plants can disperse faster than most soil biota, however, little is known about how range‐expanding plants in the new range will establish interactions with the resident soil food web. In this paper we examine how the soil nematode community from the new range responds to range‐expanding plant species compared to related natives. We focused on nematodes, because they are important components in various trophic levels of the soil food web, some feeding on plant roots, others on microbes or on invertebrates. We expected that range expanding plant species have fewer root‐feeding nematodes, as predicted by enemy release hypothesis. We therefore expected that range expanders affect the taxonomic and functional composition of the nematode community, but that these effects would diminish with increasing trophic position of nematodes in the soil food web. We exposed six range expanders (including three intercontinental exotics) and nine related native plant species to soil from the invaded range and show that range expanders on average had fewer root‐feeding nematodes per unit root biomass than related natives. The range expanders showed resistance against rather than tolerance for root‐feeding nematodes from the new range. On the other hand, the overall taxonomic and functional nematode community composition was influenced by plant species rather than by plant origin. The plant identity effects declined with trophic position of nematodes in the soil food web, as plant feeders were influenced more than other feeding guilds. We conclude that range‐expanding plant species can have fewer root‐feeding nematodes per unit root biomass than related natives, but that the taxonomic and functional nematode community composition is determined more by plant identity than by plant origin. Plant species identity effects decreased with trophic position of nematodes in the soil food web.     

Molecular phylogeny of the Tylenchina and evolution of the female gonoduct (Nematoda: Rhabditida)

Tylenchina are a morphologically and functionally diverse group of nematode species that range from free-living bacteriovores, over transitory grazing root-hair feeders to highly specialized plant-parasites with complex host associations. We performed phylogenetic analyses of small subunit rDNA sequences from 97 species including an analysis that account for the RNA secondary structure in the models of evolution. The present study confirms the sister relationship of the bacteriovore Cephalobidae with the predominantly plant-parasitic Tylenchomorpha. All analyses appoint the fungal-feeding Aphelenchidae and Aphelenchoididae as being polyphyletic but the morphology based hypothesis of their monophyly could not be significantly rejected. Within the Tylenchomorpha, the families that exclusively parasitize higher plants are joined in a single clade. However, only the monophyletic position of the (super)families Hoplolaimidae and Criconematoidea were supported; Anguinidae, Tylenchidae, Belonolaimidae and Pratylenchidae appeared to be paraphyletic or polyphyletic. Parsimony and likelihood ancestral state reconstruction revealed that burrowing endoparasitism and sedentary endoparasitism each evolved, respectively, at least six and at least three times independently, mostly from migratory ectoparasitic ancestors. Only root-knot nematodes have evolved from burrowing endoparasitic nematodes. Traditional classifications are partially misled by this convergent evolution of feeding type and associated morphology. Contrastingly, mapping attributes of the gonoduct cellular architecture, including newly obtained data of 18 species belonging to the Aphelenchoidea, Criconematoidea, Anguinidae and Panagrolaimidae, revealed a broad congruence of the gonoduct characters and the molecular phylogenetic hypothesis. Yet, the presence of an offset spermatheca and proliferation of uterus cells has evolved multiple times, the latter associated with derived endoparasitic feeding specialization and resulting reproduction mode. Ancestral state reconstruction further revealed that the gonoduct of the morphologically and ecologically dissimilar tylenchid and cephalobid nematodes evolved from a common ancestor.     

Nematodes surfing the waves: long‐distance dispersal of soil‐borne microfauna via sea swept rhizomes

Dispersal mechanisms of soil‐borne microfauna have hitherto received little attention. Understanding dispersal mechanisms of these species is important to unravel their basic life history traits, biogeography, exchange of individuals between populations, and local adaptation. Soil‐borne nematodes and root‐feeding nematodes in particular occupy a key position in soil‐food webs and can be determinants for plant growth and vegetation structure and succession. However, their dispersal abilities have been scarcely addressed, predominantly focusing on species of agricultural importance. Still, root‐feeding nematodes are usually considered as being extremely limited and bound to the rhizosphere of plants. We investigated a mechanism for long distance dispersal of root‐feeding nematodes associated to two widespread coastal dune grasses. The nematodes are known to be crucial for the functioning of these grasses. We experimentally tested the hypothesis that root‐feeding nematodes are able to move across long distances inside rhizome fragments that are dispersed by seawater. We also tested the survival capacities of the host plants in seawater. Our study demonstrates that root‐feeding nematodes and plants are able to survive immersion in seawater, providing a mechanism for long distance dispersal of root feeding nematodes together with their host plant. Drifting rhizome fragments enable the exchange of plant material and animals between dune systems. These results provide new insights to understand the ecology of dune vegetation, the interaction with soil‐borne organisms and more importantly, re‐set the scale of spatial dynamics of a group of organisms considered extremely constrained in its dispersal capacities.     

Nitric oxide synthase in invertebrates

Summary The gas nitric oxide is now recognized as an important signalling molecule that is synthesized froml-arginine by the enzyme nitric oxide synthase. This enzyme can be localized by different methods, including immunocytochemistry and the histochemical reaction for NADPH diaphorase. It has been demonstrated in various vertebrate cells and tissues, and recently several studies dealing with the production of nitric oxide in invertebrates have been published. Diploblastic animals, flatworms and nematodes seem to lack NADPH diaphorase activity but it has been found in the rest of the phyla studied. The most frequently reported sites for the production of nitric oxide are the central and peripheral nervous systems and, in primitive molluscs, the muscle cells. In insects, it has also been described in the Malpighian tubules. The roles of nitric oxide in invertebrates are closely related to the physiological actions described in vertebrates, namely, neurotransmission, defence, and salt and water balance. The recent cloning of the first nitric oxide synthase from an invertebrate source could open interesting avenues for further studies.     

The suitability of the free-living nematode Panagrellus redivivus as live food for first-feeding fish larvae

The free‐living nematode Panagrellus redivivus has been recommended as a suitable food source for first‐feeding fish. A new technology for mass production of P. redivivus enables fish hatchery operators to rely on an inexpensive, standardized and permanently available live food for first‐feeding fish larvae. The proximate composition, and the fatty acid and amino acid profiles of nematodes mass produced on oat‐based and purified ingredient media were determined. The quality of nematodes was significantly influenced by the culture medium used. The lipid content and fatty acid composition of nematodes could be modified by using lipid‐enriched media. Mass‐produced nematodes were tested on first‐feeding common carp (Cyprinus carpio L.) and whitefish (Coregonus lavaretus) larvae. Carp larvae, grown on nematodes cultured on oat medium enriched with sunflower oil, showed a higher survival rate (87.1%) than the control group fed frozen zooplankton (82.9%) at the end of the 1‐week feeding experiment. Differences in larval mass between the treatments disappeared after subsequent feeding of a dry diet for 2 weeks. Whitefish larvae can be reared exclusively on a dry diet; here, the initial feeding of nematodes had no effect on final biomass and survival of larvae.     

Feeding Activity and Survival of Slugs,Deroceras reticulatum,Exposed to the Rhabditid Nematode,Phasmarhabditis hermaphrodita: A Model of Dose Response

The slug, Deroceras reticulatum (Stylommatophora: Limacidae), was exposed to different concentrations of infective dauer juveniles of the rhabditid nematode Phasmarhabditis hermaphrodita, in a two-stage bioassay, at 10°C. Slugs were exposed in groups of 10 or 12 to nematodes in plastic boxes filled with soil aggregates for 3 or 5 days and then transferred individually to petri dishes each containing a disk of Chinese cabbage leaf as food. Subsequently, slug food consumption and survival were measured for 10 to 13 days. Models were developed to describe the way that exposure to the nematode caused inhibition of slug feeding followed by death. Both effects were related to nematode concentrations and time after exposure to the nematode. Following exposure to high concentrations (300,000 dauer juveniles per box), slugs were killed rapidly, within a few days after the end of the exposure period. Following exposure to low concentrations of nematodes (7000 or 15,000 per box), substantial numbers of slugs survived until the end of the bioassay, but feeding activity by these slugs was strongly inhibited. It is suggested that inhibition of slug feeding is important for the success of this nematode as a biocontrol agent.     

Cross habituation to feeding deterrents and acceptance of a marginal host plant byPieris rapae larvae

Sensitivity of the cabbage butterfly,Pieris rapae L. to feeding deterrents was compared for larvae reared on different food sources under laboratory conditions. Since cabbage-reared larvae normally reject nasturtium,Tropaeolum majus L., the effects of previous exposure to allelochemicals on larval acceptance or rejection of this plant were also examined. When compared with cabbage-reared larvae, nasturtium-reared larvae were less sensitive to feeding deterrents including cymarin, erysimoside and 2-O-β-d-glucosyl cucurbitacin E. Nasturtium-reared larvae were insensitive to chlorogenic acid, which was deterrent to cabbage-reared larvae. Feeding by larvae reared on a wheat germ diet was not deterred by these compounds. The results indicate that dietary experience can extensively affect larval sensitivity to feeding deterrents and that cross habituation of larvae to deterrents occurs in response to certain chemical constituents of nasturtium and wheat germ diet. Digitoxin, however, proved to be an exception. Larvae reared on either nasturtium or wheat germ diet were as sensitive to digitoxin as those reared on cabbage. Previous results have shown that rejectionof nasturtium by cabbage-reared larvae is due to the presence of strong feeding deterrents in this plant. However, more than 50% of 2nd instar larvae reared from neonate on cabbage leaves treated with strophanthidin, cymarin, erysimoside, digitoxigenin and digitoxin accepted nasturtium as a food source. 2-O-β-d-glucosyl cucurbitacin E, 2-O-β-d-glucosyl cucurbitacin I and rutin were also active in causing larvae to feed on nasturtium. Thus dietary exposure to unrelated plant chemicals can profoundly affect insect acceptance of a plant that contains feeding deterrents.     

Adaptive significance of gall formation for a gall-inducing aphids on Japanese elm trees

Insect galls are abnormal plant tissues induced by external stimuli from parasitizing insects. It has been suggested that the stimuli include phytohormones such as auxin and cytokinins produced by the insects. In our study on the role of hormones in gall induction by the aphid Tetraneura nigriabdominalis, it was found that feedback regulation related to auxin and cytokinin activity is absent in gall tissues, even though the aphids contain higher concentrations of those phytohormones than do plant tissues. Moreover, jasmonic acid signaling appears to be compromised in gall tissue, and consequently, the production of volatile organic compounds, which are a typical defense response of host plants to herbivory, is diminished. These findings suggest that these traits of the gall tissue benefit aphids, because the gall tissue is highly sensitive to auxin and cytokinin, which induce and maintain it. The induced defenses against aphid feeding are also compromised. The abnormal responsiveness to phytohormones is regarded as a new type of extended phenotype of gall-inducing insects.     

Sugar and phytohormone response pathways: navigating a signalling network

Many plant developmental, physiological and metabolic processes are regulated, at least in part, by nutrient availability. In particular, alterations in the availability of soluble sugars, such as glucose and sucrose, help regulate a diverse array of processes. Multiple lines of evidence indicate that many of these processes are also regulated in response to other signalling molecules, such as phytohormones. This review draws examples from a variety of plant systems, including bean, Arabidopsis, potato, and cereals. Five of the most interesting and best developed examples of processes regulated via 'interactions' or 'crosstalk' between sugars and phytohormones are described, including embryogenesis, seed germination, early seedling development, tuberization, and the regulation of alpha-amylase activity. The types of mechanisms by which different response pathways are known or postulated to interact are also described. These mechanisms include regulation of the metabolism and/or transport of a signalling molecule by a different response pathway. For example, sugars have been postulated to help regulate the synthesis, conjugation and/or transport of phytohormones, such as gibberellins and abscisic acid. Conversely, phytohormones, such as abscisic acid, gibberellins and cytokinins have been shown to help regulate sugar metabolism and/or transport. Similarly, sugars have been shown to regulate the expression of components of phytohormone-response pathways and phytohormones regulate the expression of some genes encoding possible components of sugar-response pathways. Examples of proteins and second messengers that appear to act in multiple response pathways are also described.