Histologial Studies on the Root-Knot Nematodes Nonresistant and Resistant Root Apex of Humulus lupulus L.

The nematodes (Meloidogy incognita Chitwood.) invade near the root apex of the hop (Humulus lupulus L.), and usually lie near the meristematic zone. Fallowing their entrance, there are the formation of the giant cells and the consequent continuous divisions and activities of the flat meristematic cells, which result in formation of the nematode galls. Their sizes are also related to the number of nematodes. String beads nematode galls are often formed at the nematode-non resistant root part of the "Comet". The process of nematodes entering and later changing of the cells and tissue is almost the same as the formation of the nematode gall in other plants. Fewer nematodes can also enter the root tip of the highly resistant "Qingdao Dahua", but the subsequent special differentiation of the cells and tissues in the root have greatly restrained the activities of nematodes and ultimately to their death.     

The Effect of Endophytic Fungi on Nematode Populations in Summer-dormant and Summer-active Tall Fescue

Summer-active (continental) and summer-dormant (Mediterranean) tall fescue morphotypes are each adapted to different environmental conditions. Endophyte presence provides plant parasitic nematode resistance, but not with all endophyte strains and cultivar combinations. This study sought to compare effects of four nematode genera on continental and Mediterranean cultivars infected with common toxic or novel endophyte strains. A 6-mon greenhouse study was conducted with continental cultivars, Kentucky 31 (common toxic) and Texoma MaxQ II (novel endophyte) and the Mediterranean cultivar Flecha MaxQ (novel endophyte). Endophyte-free plants of each cultivar were controls. Each cultivar × endophyte combination was randomly assigned to a control, low or high inoculation rate of a mixed nematode culture containing stunt nematodes (Tylenchorhynchus spp.), ring nematodes (Criconemella spp.), spiral nematodes (Helicotylenchus spp.), and lesion nematodes (Pratylenchus spp.). Endophyte infection had no effect on nematode population densities. The cultivar × endophyte interaction was significant. Population densities of stunt nematode, spiral nematode, and ring nematodes were higher for Flecha MaxQ than other cultivar × endophyte combinations. Novel endophyte infection enhances suitability of Flecha MaxQ as a nematode host.     

The ultrastructure of the microfilaria of Brugia, Nematoda: Filarioidea

The microfilaria of Brugia pahangi is a differentiated nematode larva. The basic nematode body plan is present showing cuticle, hypodermis, dorsal, ventral, and lateral cords, muscle cells, longitudinal nerves, papillary nerves, amphids and phasmids. Secretory granules are present in ganglionic cells and in axons in the nerve ring. There is no differentiated pseudocoelom. There is only a single row of muscle cells between each pair of cords. The excretory cell complex is similar in structure to the hypodermal gland cells of other nematodes. The alimentary canal of the microfilaria is very much modified. The pharyngeal cells are attached to the pharyngeal thread which is circular in cross section and there is no pharyngeal musculature. The intestine is represented by the solid mass of the inner body within paired intestinal cells. The intestine is separated from the rectum. The three rectal cells form a syncytium of villi in the anal vesicle. The structure in Brugia is related to the ultrastructure of other microfilariae and it is concluded that the evolution of the modifications of the basic larval structure is due to the small size of these nematodes as a consequence of their adaptation to a parasitic mode of life in the capillaries of the vertebrate host with transmission through an intermediate arthropod vector.     

Heterotrimeric G-protein and signal transduction in the nematode-trapping fungus Arthrobotrys dactyloides

The fungus Arthrobotrys dactyloides produces specialized constricting rings to trap and then consume nematodes. The signal transduction pathway involved in the nematode-trapping process was examined. Mastoparan, an activator of G-protein, had a stimulatory effect on the inflation of ring cells, whereas a G-protein inhibitor, pertussis toxin, prevented ring-cell expansion. The 40-kDa G_α of heterotrimeric G-proteins was specifically ADP-ribosylated by pertussis toxin. Using an antibody specific to the 35-kDa subunit G_β, we showed that immunogold-labeled G_β was more concentrated in ring cells than in the hyphae. In the absence of nematodes, the rings could be inflated by either pressurizing the culture in a syringe, raising intracellular Ca²⁺ concentrations, or adding warm water. We used these methods to reveal differences in responses to antagonists. The results support a model in which the pressure exerted by a nematode on the ring activates G-proteins in the ring cells. The activation leads to an increase in cytoplasmic Ca²⁺, activation of calmodulin, and finally the opening of water channels. The ring cells expand to constrict the ring and thus immobilize the nematode. Received: 13 April 2000 / Accepted: 22 June 2000     

Plant-nematode interactions

Root-knot nematodes and cyst nematodes are obligate, biotrophic pathogens of numerous plant species. These organisms cause dramatic changes in the morphology and physiology of their hosts. The molecular characterization of induced plant genes has provided insight into the plant processes that are usurped by nematodes as they establish their specialized feeding cells. Recently, several gene products have been identified that are secreted by the nematode during parasitism. The corresponding genes have strong similarity to microbial genes or to genes that are found in nematodes that parasitize animals. New information on host resistance genes and nematode virulence genes provides additional insight into this complex interaction.     

A conserved metalloprotease mediates ecdysis in Caenorhabditis elegans

Molting is required for progression between larval stages in the life cycle of nematodes. We have identified four mutant alleles of a Caenorhabditis elegans metalloprotease gene, nas-37, that cause incomplete ecdysis. At each molt the cuticle fails to open sufficiently at the anterior end and the partially shed cuticle is dragged behind the animal. The gene is expressed in hypodermal cells 4 hours before ecdysis during all larval stages. The NAS-37 protein accumulates in the anterior cuticle and is shed in the cuticle after ecdysis. This pattern of protein accumulation places NAS-37 in the right place and at the right time to degrade the cuticle to facilitate ecdysis. The nas-37 gene has orthologs in other nematode species, including parasitic nematodes, and they undergo a similar shedding process. For example, Haemonchus contortus molts by digesting a ring of cuticle at the tip of the nose. Incubating Haemonchus larvae in extracted exsheathing fluids causes a refractile ring of digested cuticle to form at the tip of the nose. When Haemonchus cuticles are incubated with purified NAS-37, a similar refractile ring forms. NAS-37 degradation of the Haemonchus cuticle suggests that the metalloproteases and the cuticle substrates involved in exsheathment of parasitic nematodes are conserved in free-living nematodes.     

Effect of neonicotinoid synergists on entomopathogenic nematode fitness

In previous greenhouse and field studies, the neonicotinoid insecticide imidacloprid interacted synergistically with five entomopathogenic nematode species against five scarab species. Two other neonicotinoids, thiamethoxam and acetamiprid, showed a weaker interaction with nematodes in scarab larvae. Entomopathogenic nematodes have the potential to recycle in hosts after inundative applications, thereby increasing the persistence of nematodes and insect control. Thus we investigated the effect of neonicotinoids on nematode fitness after tank mixing and after combined applications. Tank mixing only had a negative effect on nematode survival and infectivity in a few nematode–insecticide combinations and only if both insecticide concentration and exposure time were several times higher than typical for field applications. Combined application of nematodes with imidacloprid generally had no negative effect on the percentage of scarab cadavers producing progeny or the number of nematode progeny emerging per cadaver. In experiments with a synergistic increase in scarab mortality, the total number of progeny in combination treatments was up to four times higher than in nematodes only treatments. Similarly, nematode populations in soil from combination treatments were 13.2 times greater than for nematodes only treatments at 28 days after treatment. Combined imidacloprid–nematode applications did not affect the pathogenicity or infectivity of the nematode progeny. Combining thiamethoxam with nematodes had no negative effects on nematode reproduction in the majority of treatments. However, due to the weaker interaction of thiamethoxam and nematodes on scarab mortality, the total number of nematode progeny per treatment generally did not increase compared with nematodes only treatments. The demonstrated tank mix compatibility of imidacloprid and nematodes improves the feasibility of combining these agents for curative white grub control. The positive effect of imidacloprid on nematode reproduction after combined application may increase the likelihood of infection of white grubs by subsequent generations of nematodes, thereby improving their field persistence and biological control potential.     

Identification of genes involved in Meloidogyne incognita-induced gall formation processes in Arabidopsis thaliana

Root-knot nematodes (RKN; Meloidogyne incognita) are phytoparasitic nematodes that cause significant damage to crop plants worldwide. Recent studies have revealed that RKNs disrupt various physiological processes in host plant cells to induce gall formation. However, little is known about the molecular mechanisms of gall formation induced by nematodes. We have previously found that RNA expression levels of some of genes related to micro-RNA, cell division, membrane traffic, vascular formation, and meristem maintenance system were modified by nematode infection. Here we evaluated these genes importance during nematode infection by using Arabidopsis mutants and/or β-glucronidase (GUS) marker genes, particularly after inoculation with nematodes, to identify the genes involved in successful nematode infection. Our results provide new insights not only for the basic biology of plant–nematode interactions but also to improve nematode control in an agricultural setting.     

Evolution of Parasitism in Insect-transmitted Plant Nematodes

Nematode-insect associations have evolved many times in the phylum Nematoda, but these lineages involve plant parasitism only in the Secernentean orders Aphelenchida and Tylenchida. In the Aphelenchida (Aphelenchoidoidea), Bursaphelenchus xylophilus (Pine wood nematode), B. cocophilus (Red ring or Coconut palm nematode) (Parasitaphelenchidae), and the many potential host-specific species of Schistonchus (fig nematodes) (Aphelenchoididae) nematode-insect interactions probably evolved independently from dauer-forming, mycophagous ancestors that were phoretically transmitted to breeding sites of their insect hosts in plants. Mycophagy probably gave rise to facultative or obligate plant-parasitism because of opportunities due to insect host switches or peculiarities in host behavior. In the Tylenchida, there is one significant radiation of insect-associated plant parasites involving Fergusobia nematodes (Fergusobiinae: Neotylenchidae) and Fergusonina (Fergusoninidae) flies as mutualists that gall myrtaceous plant buds or leaves. These dicyclic nematodes have different phases that are parasitic in either the insect or the plant hosts. The evolutionary origin of this association is unclear.     

The bacterial community of entomophilic nematodes and host beetles

Insects form the most species‐rich lineage of Eukaryotes and each is a potential host for organisms from multiple phyla, including fungi, protozoa, mites, bacteria and nematodes. In particular, beetles are known to be associated with distinct bacterial communities and entomophilic nematodes. While entomopathogenic nematodes require symbiotic bacteria to kill and reproduce inside their insect hosts, the microbial ecology that facilitates other types of nematode–insect associations is largely unknown. To illuminate detailed patterns of the tritrophic beetle–nematode–bacteria relationship, we surveyed the nematode infestation profiles of scarab beetles in the greater Los Angeles area over a five‐year period and found distinct nematode infestation patterns for certain beetle hosts. Over a single season, we characterized the bacterial communities of beetles and their associated nematodes using high‐throughput sequencing of the 16S rRNA gene. We found significant differences in bacterial community composition among the five prevalent beetle host species, independent of geographical origin. Anaerobes Synergistaceae and sulphate‐reducing Desulfovibrionaceae were most abundant in Amblonoxia beetles, while Enterobacteriaceae and Lachnospiraceae were common in Cyclocephala beetles. Unlike entomopathogenic nematodes that carry bacterial symbionts, insect‐associated nematodes do not alter the beetles' native bacterial communities, nor do their microbiomes differ according to nematode or beetle host species. The conservation of Diplogastrid nematodes associations with Melolonthinae beetles and sulphate‐reducing bacteria suggests a possible link between beetle–bacterial communities and their associated nematodes. Our results establish a starting point towards understanding the dynamic interactions between soil macroinvertebrates and their microbiota in a highly accessible urban environment.     

Trichostrongylus colubriformis larvae induce necrosis and release of IL33 from intestinal epithelial cells in vitro: implications for gastrointestinal nematode vaccine design

Gastrointestinal nematodes represent a major production problem for ruminant livestock. Enhancing immunity to gastrointestinal nematodes through vaccination is desirable but mechanistic understanding of initial host responses that facilitate gastrointestinal nematode protective immunity is limited. We hypothesise that gastrointestinal nematode invasion induces mucosal epithelium damage and alarmin (e.g. IL33) release, thereby contributing to initiation of protective gastrointestinal nematode immunity. To test this, an in vitro air-liquid interface human HT-29 epithelial cell-Trichostrongylus colubriformis co-culture system was developed. Exsheathed L3 T. colubriformis exhibited both sinusoidal and burrowing motions in the co-culture system. Burrowing parasites, but not ivermectin-paralysed larvae, induced necrotic death of epithelial cells (annexin V(+)/propidium iodide(+)/caspase 3/7(-)). Microscopy confirmed that larvae consumed labelled necrotic epithelial cell contents. Trichostrongylus colubriformis larvae and their post-exsheathment antigens (excretory/secretory products) significantly induced IL33 mRNA expression in the epithelial cells. Immunoblot confirmed that IL33 was released from epithelial cells due to the damage caused by motile larvae. Exposure of HT-29 cells to alum or Sigma proprietary adjuvants induced significant epithelial cell IL33 mRNA expression without inducing cellular necrosis. Hence, the intracellular contents were not released externally where they might exert alarmin activity and this may limit their ability to trigger a protective anti-gastrointestinal nematode response. We conclude that T. colubriformis motion at the infection site induces intestinal epithelial cell necrosis which facilitates the release of intracellular contents, including IL33, and may be fundamental to the initiation of an appropriate host response to gastrointestinal nematodes. Our co-culture model is useful for studying initial epithelial cell-parasite interactions without conducting expensive animal trials.     

In vitro assessment of plant lectins with anti-pinwood nematode activity

Two lectin proteins were purified from the corms of Pinellia ternata and Lycoris radiata. Both P. ternata agglutinin (PTA) protein and L. radiata agglutinin (LRA) protein formed polymers and coagulated both rabbit red blood cells and yeast cells. The two proteins were each diluted to different concentration and then mixed with pinewood nematodes, and nematode survival was measured. Results showed that the two lectin proteins showed significant levels of resistance against nematodes and the nematode population was significantly reduced, compared to PBS buffer without protein control group. The mean number of nematodes of two lectin proteins group was significantly lower than that of control group constantly throughout the assay period with differences being very significant at P < 0.01 after 24 h. After 96 h, when 500 μg/ml proteins were used, nematode number significantly declined to an average of 26 (approximately 43% of the controls) and 32.2 (approximately 53.3% of the controls) nematodes at LRA and PTA protein, respectively, compared to the control group. Results also indicated that higher concentrations of protein were more toxic to the pinewood nematode. Even when the concentration was as low as 30 μg/ml, the toxic proteins retained their anti-nematode activity. Furthermore, pinewood nematode was exposed to the proteins for longer, more pinewood nematodes were killed. Our results indicated the two lectin proteins both apparently have a toxic effect on the pinewood nematode that affects its survival in vitro.     

Loss of susceptibility as an alternative for nematode resistance

Among plant pathogens, sedentary endoparasitic nematodes are one of the most damaging pests in global agriculture. These obligate parasites interact with their hosts in a quite unique and intriguing way. They induce the redifferentiation of root cells into specialized feeding cells essential for nematode growth and reproduction; thus, nematodes have evolved the ability to exploit plant genes and hijack host functions for their own requirements. Various approaches to engineer plants with resistance to parasitic nematodes have been pursued, most focusing on the introduction of resistance genes. An alternative strategy to achieve resistance is to exploit the susceptibility of plant disease. Better knowledge of the plant response during the compatible interaction should allow the identification of targets to engineer resistance to parasitic nematodes in crop species.     

昆虫病原线虫共生细菌共生性的分子生物学研究进展

嗜线虫致病杆菌属Xenorhabdus和发光杆菌属Photorhabdus细菌隶属肠杆菌科Enterobacteriaceae,对多种害虫致病能力强,分别与斯氏属Steinernema和异小杆属Heterorhabditis昆虫病原线虫互惠共生。该两属共生细菌既存在对昆虫寄主的病原性,又存在与线虫寄主的共生性。共生细菌与其线虫寄主的共生性主要表现以下4方面：（1）细菌产生食物信号诱导滞育不取食的感染期线虫恢复;（2）细菌为线虫生长与繁殖提供营养;（3）细菌能于感染期线虫的肠道定殖与生长;（4）细菌产生杀线虫毒素杀死非共生线虫。本文综述了共生菌以上4方面的共生性及其相关的分子机制。     

An insight into critical endocycle genes for plant-parasitic nematode feeding sites establishment

Root-knot and cyst nematodes are biotrophic parasites that invade the root apex of host plants and migrate toward the vascular cylinder where they cause the differentiation of root cells into galls (or root-knots) containing hypertrophied multinucleated giant-feeding cells, or syncytia, respectively. The precise molecular mechanisms that drive the formation of such unique nematode feeding sites are still far-off from being completely understood. The diverse gene expression changes occurring within the host cells suggest that both types of plant-parasitic nematodes modulate a variety of plant processes. Induction and repression of genes belonging to the host cell cycle control machinery have shown to be essential to drive the formation of such specialized nematode feeding cells. We demonstrate that nematodes usurp key components regulating the endocycle in their favor. This is illustrated by the involvement of anaphase-promoting complex (APC) genes (CCS52A and CCS52B), the endocycle repressor DP-E2F-like (E2F/DEL1) gene and the ROOT HAIRLESS 1 PROTEIN (RHL1), which is part of a multiprotein complex of the toposiomerase VI, in the proper formation of nematode feeding sites. Altering the expression of these genes in Arabidopsis plants by down- or overexpressing strategies strongly influences the extent of endoreduplication in both types of nematode feeding site leading to a disturbance of the nematode’s life cycle and reproduction.     

Enhanced levels of plant cell cycle inhibitors hamper root-knot nematode-induced feeding site development

Root-knot nematodes (RKN) are highly specialized, obligatory plant parasites. These animals reprogram root cells to form large, multinucleate, and metabolically active feeding cells (giant cells) that provide a continuous nutrient supply during 3–6 weeks of the nematode’s life. The establishment and maintenance of physiologically fully functional giant cells are necessary for the survival of these nematodes. As such, giant cells may be useful targets for applying strategies to reduce damage caused by these nematodes, aiming the reduction of their reproduction. We have recently reported the involvement of cell cycle inhibitors of Arabidopsis, named Kip-Related Proteins (KRPs), on nematode feeding site ontogeny. Our results have demonstrated that this family of cell cycle inhibitors can be envisaged to efficiently disrupt giant cell development, based on previous reports which showed that alterations in KRP concentration levels can induce cell cycle transitions. Herein, we demonstrated that by overexpressing KRP genes, giant cells development is severely compromised as well as nematode reproduction. Thus, control of root-knot nematodes by modulating cell cycle-directed pathways through the enhancement of KRP protein levels may serve as an attractive strategy to limit damage caused by these plant parasites.