Morphological and molecular characteristics of a new species of Pasteuria parasitic on Meloidogyne ardenensis

A species of the hyper-parasitic bacterium Pasteuria was isolated from the root-knot nematode Meloidogyne ardenensis infecting the roots of ash (Fraxinus excelsior). It is morphologically different from some other Pasteuria pathogens of nematodes in that the spores lack a basal ring on the ventral side of the spore and have a unique clumping nature. Transmission electron microscopy (TEM) showed that the clumps of spores are not random aggregates but result from the disintegration of the suicide cells of the thalli. Sporulation within each vegetative mycelium was shown to be asynchronous. In addition to the novel morphological features 16S rRNA sequence analysis showed this to be a new species of Pasteuria which we have called P. hartismeri. Spores of P. hartismeri attach to juveniles of root-knot nematodes infecting a wide range of plants such as mint (Meloidogyne hapla), rye grass (unidentified Meloidogyne sp.) and potato (Meloidogyne fallax).     

Underlying effects of spatial aggregation (clumping) in relationships between plant diversity and resource uptake

In the debate on the influence of declining plant diversity on ecosystem functioning, spatial aggregation has received little attention, in spite of the fact that the local distribution of species determines interaction. Using a simple conceptual model, we visualize the effect that clumping has on below ground resource uptake (RU). Two general conclusions can be drawn from the model: (1) clumping results in a loss of RU, and does so to a larger extent when species differ more in rooting depth and when plant density increases; (2) the current view on complementarity may be flawed, as we find that neighborhood species richness only promotes spatial complementarity directly at the transition from monocultures to higher species richness levels, but not at transitions between higher richness levels. The number of species in a community does, however, affect the probability of the occurrence of clumping. In randomly assembled communities, clumps are statistically larger when the number of species is lower, leading to reduced overall below ground complementarity. Complementarity, and thus also RU, will therefore increase indirectly with increasing species richness. As a result of our findings, we propose that clumping could have a number of 'hidden' consequences, for example on invasibility of a community and on competition between species. Clumping could also prove to be a new and important explanatory factor in diversity studies, as it extends the definition of complementarity effects to include the effect of spatial heterogeneity of plant positions within the community.     

Metabolic Adaptations of Azospirillum brasilense to Oxygen Stress by Cell-to-Cell Clumping and Flocculation

The ability of bacteria to monitor their metabolism and adjust their behavior accordingly is critical to maintain competitiveness in the environment. The motile microaerophilic bacterium Azospirillum brasilense navigates oxygen gradients by aerotaxis in order to locate low oxygen concentrations that can support metabolism. When cells are exposed to elevated levels of oxygen in their surroundings, motile A. brasilense cells implement an alternative response to aerotaxis and form transient clumps by cell-to-cell interactions. Clumping was suggested to represent a behavior protecting motile cells from transiently elevated levels of aeration. Using the proteomics of wild-type and mutant strains affected in the extent of their clumping abilities, we show that cell-to-cell clumping represents a metabolic scavenging strategy that likely prepares the cells for further metabolic stresses. Analysis of mutants affected in carbon or nitrogen metabolism confirmed this assumption. The metabolic changes experienced as clumping progresses prime cells for flocculation, a morphological and metabolic shift of cells triggered under elevated-aeration conditions and nitrogen limitation. The analysis of various mutants during clumping and flocculation characterized an ordered set of changes in cell envelope properties accompanying the metabolic changes. These data also identify clumping and early flocculation to be behaviors compatible with the expression of nitrogen fixation genes, despite the elevated-aeration conditions. Cell-to-cell clumping may thus license diazotrophy to microaerophilic A. brasilense cells under elevated oxygen conditions and prime them for long-term survival via flocculation if metabolic stress persists.     

Expression of clumping and fibrinogen-binding activities of Staphylococcus aureus at various growth stages

Clumping and fibrinogen-binding activities of 4 Staphylococcus aureus strains (Cowan I, Newman D2C, Wood 46 and NCTC 5655) were assayed with a semiquantitative clumping test and an enzyme-linked immunosorbent assay (ELISA), respectively. Distinct positive clumping was detected with whole cells of the 3 strains except Wood 46. Amounts of fibrinogen required for a definite clumping depended greatly on strains as well as on their growth phases. On the other hand, fibrinogen-binding activities were detected both in culture supernatants and in cell lysates of all the 4 strains, and the levels were rather comparable with one another and relatively steady through their growth cycles. No significant correlation was thus found among expression behavior of clumping and fibrinogen-binding activities.     

Role of cyanide production by Pseudomonas fluorescens CHA0 in the suppression of root-knot nematode, Meloidogyne javanica in tomato

Summary Pseudomonas fluorescens strain CHA0 produces hydrogen cyanide (HCN), a secondary metabolite that accounts largely for the biocontrol ability of this strain. In this study, we examined the role of HCN production by CHA0 as an antagonistic factor that contributes to biocontrol of Meloidogyne javanica, the root-knot nematode, in situ. Culture filtrate of CHA0, resulting from 1/10-strength nutrient broth yeast extract medium amended with glycine, inhibited egg hatch and caused mortality of M. javanica juveniles in vitro. The bacterium cultured under high oxygen-tension conditions exhibited better inhibitory effects towards nematodes, compared to its cultivation under excess oxygen situation. Growth medium amended with 0.50 or 1.0 mM FeEDDHA further improved hatch inhibition and nematicidal activity of the strain CHA0. Strain CHA77, an HCN-negative mutant, failed to exert such toxic effects, and in this strain, antinematode activity was not influenced by culture conditions. Exogenous cyanide also inhibited egg hatch and caused mortality of M. javanica juveniles in vitro. Strains CHA0 or CHA77 applied in unsterilized sandy-loam soil as drench, caused marked suppression of root-knot disease development incited by M. javanica in tomato seedlings. However, efficacy of CHA77 was noticeably lower compared to its wild type counterpart CHA0. An increased bioavailability of iron following EDTA application in soil substantially improved nematode biocontrol potential of CHA0 but not that of CHA77. Soil infestation with M. javanica eggs resulted in significantly lower nematode population densities and root-knot disease compared to the juveniles used as root-knot disease-inducing agents. Strain CHA0 significantly suppressed nematode populations and inhibited galling in tomato roots grown in soil inoculated with eggs or juveniles and treated with or without EDTA. Strain CHA0 exhibited greater biocontrol potential in soil inoculated with eggs and treated with EDTA. To demonstrate that HCN synthesis by the strain CHA0 acts as the inducing agent of systemic resistance in tomato, efficacy of the strain CHA0 was compared with CHA77 in a split root trial. The split-root experiment, guaranteeing a spatial separation of the inducing agent and the challenging pathogen, showed that HCN production by CHA0 is not crucial in the induction of systemic resistance in tomato against M. javanica, because the HCN-negative-mutant CHA77 induced the same level of resistance as the wild type but exogenous cyanide in the form of KCN failed to trigger the resistance reaction. In the root section where both nematode and the bacterium were present, strain CHA0 reduced nematode penetration to a greater extent than CHA77, suggesting that for effective control of M. javanica, a direct contact between HCN-producing CHA0 and the nematode is essential.     

Host-plant selectivity of rhizobacteria in a crop/weed model system

Belowground microorganisms are known to influence plants' performance by altering the soil environment. Plant pathogens such as cyanide-producing strains of the rhizobacterium Pseudomonas may show strong host-plant selectivity. We analyzed interactions between different host plants and Pseudomonas strains and tested if these can be linked to the cyanide sensitivity of host plants, the cyanide production of bacterial strains or the plant identity from which strains had been isolated. Eight strains (four cyanide producing) were isolated from roots of four weed species and then re-inoculated on the four weed and two additional crop species. Bacterial strain composition varied strongly among the four weed species. Although all six plant species showed different reductions in root growth when cyanide was artificially applied to seedlings, they were generally not negatively affected by inoculation with cyanide-producing bacterial strains. We found a highly significant plant species x bacterial strain interaction. Partitioning this interaction into contrasts showed that it was entirely due to a strongly negative effect of a bacterial strain (Pseudomonas kilonensis/brassicacearum, isolated from Galium mollugo) on Echinochloa crus-galli. This exotic weed may not have become adapted to the bacterial strain isolated from a native weed. Our findings suggest that host-specific rhizobacteria hold some promise as biological weed-control agents.     

Tm1: a mutator/foldback transposable element family in root-knot nematodes

Three closely related parthenogenetic species of root-knot nematodes, collectively termed the Meloidogyne incognita-group, are economically significant pathogens of diverse crop species. Remarkably, these asexual root-knot nematodes are capable of acquiring heritable changes in virulence even though they lack sexual reproduction and meiotic recombination. Characterization of a near isogenic pair of M. javanica strains differing in response to tomato with the nematode resistance gene Mi-1 showed that the virulent strain carried a deletion spanning a gene called Cg-1. Herein, we present evidence that the Cg-1 gene lies within a member of a novel transposable element family (Tm1; Transposon in Meloidogyne-1). This element family is defined by composite terminal inverted repeats of variable lengths similar to those of Foldback (FB) transposable elements and by 9 bp target site duplications. In M. incognita, Tm1 elements can be classified into three general groups: 1) histone-hairpin motif elements; 2) MITE-like elements; 3) elements encoding a putative transposase. The predicted transposase shows highest similarity to gene products encoded by aphids and mosquitoes and resembles those of the Phantom subclass of the Mutator transposon superfamily. Interestingly, the meiotic, sexually-reproducing root-knot nematode species M. hapla has Tm1 elements with similar inverted repeat termini, but lacks elements with histone hairpin motifs and contains no elements encoding an intact transposase. These Tm1 elements may have impacts on root-knot nematode genomes and contribute to genetic diversity of the asexual species.     

Pollen clumping and wind dispersal in an invasive angiosperm

Pollen dispersal is a fundamental aspect of plant reproductive biology that maintains connectivity between spatially separated populations. Pollen clumping, a characteristic feature of insect-pollinated plants, is generally assumed to be a detriment to wind pollination because clumps disperse shorter distances than do solitary pollen grains. Yet pollen clumps have been observed in dispersion studies of some widely distributed wind-pollinated species. We used Ambrosia artemisiifolia (common ragweed; Asteraceae), a successful invasive angiosperm, to investigate the effect of clumping on wind dispersal of pollen under natural conditions in a large field. Results of simultaneous measurements of clump size both in pollen shedding from male flowers and airborne pollen being dispersed in the atmosphere are combined with a transport model to show that rather than being detrimental, clumps may actually be advantageous for wind pollination. Initial clumps can pollinate the parent population, while smaller clumps that arise from breakup of larger clumps can cross-pollinate distant populations.     

Surface characterization of two amoebae relative to cell adhesion

This is a comparative study of the surfaces of two Acanthamoeba castellanii strains, one of which clumps during exponential growth. Proteins from the isolated surface membranes of the strains were compared by disc electrophoresis. The number and similarity of the proteins between the two strains depended on the method of solubilization and electrophoresis. Periodic acid Schiff staining indicated that most of the proteins were glycoproteins. The molecular weights of the proteins ranged between 50 000–70 000 with those of the clumping strain being slightly larger. The non-clumping strain's surface membrane exhibited glucosyltransferase activity while the clumping strain did not. Galactosyltransferase activity was present in surface membranes of both strains but the specific activity of the clumping strain was greater. The non-clumping strain agglutinated in the presence of concanavalin A, soybean agglutinin and wheat germ agglutinin while the clumping strain was affected by soybean agglutinin only. Whole cell electrophoresis and enzymic treatments further indicated differences in the surfaces of the two strains. The data is discussed with reference to differences between the two amoebae strains and their possible role in cell-cell adhesion.     

Meiotic parthenogenesis in a root-knot nematode results in rapid genomic homozygosity

Many isolates of the plant-parasitic nematode Meloidogyne hapla reproduce by facultative meiotic parthenogenesis. Sexual crosses can occur, but, in the absence of males, the diploid state appears to be restored by reuniting sister chromosomes of a single meiosis. We have crossed inbred strains of M. hapla that differ in DNA markers and produced hybrids and F(2) lines. Here we show that heterozygous M. hapla females, upon parthenogenetic reproduction, produce progeny that segregate 1:1 for the presence or absence of dominant DNA markers, as would be expected if sister chromosomes are rejoined, rather than the 3:1 ratio typical of a Mendelian cross. Codominant markers also segregate 1:1 and heterozygotes are present at low frequency (<3%). Segregation patterns and recombinant analysis indicate that a homozygous condition is prevalent for markers flanking recombination events, suggesting that recombination occurs preferentially as four-strand exchanges at similar locations between both pairs of non-sister chromatids. With this mechanism, meiotic parthenogenesis would be expected to result in rapid genomic homozygosity. This type of high negative crossover interference coupled with positive chromatid interference has not been observed in fungal or other animal systems in which it is possible to examine the sister products of a single meiosis and may indicate that meiotic recombination in this nematode has novel features.     

Inter- and intra-specific cuticle variation between amphimictic and parthenogenetic species of root-knot nematode (Meloidogyne spp.) as revealed by a bacterial parasite (Pasteuria penetrans)

Specific host–parasite interactions exist between species and strains of plant parasitic root-knot nematodes and the Gram-positive bacterial hyperparasite Pasteuria penetrans. This bacterium produces endospores that adhere to the cuticle of migrating juveniles, germinate and colonise the developing female within roots. Endospore attachment of P. penetrans populations to second-stage juveniles of the root-knot nematode species Meloidogyne incognita and Meloidogyne hapla showed there were interactive differences between bacterial populations and nematode species. Infected females of M. incognita produced a few progeny which were used to establish two nematode lines from single infective juveniles encumbered with either three or 26 endospores. Single juvenile descent lines of each nematode species were produced to test whether cuticle variation was greater within M. hapla lines that reproduce by facultative meiotic parthenogenesis than within lines of M. incognita, which reproduces by obligate parthenogenesis. Assays revealed variability between broods of individual females derived from single second-stage juvenile descent lines of both M. incognita and M. hapla suggesting that progeny derived from a single individual can differ in spore adhesion in both sexual and asexual nematode species. These results suggest that special mechanisms that produced these functional differences in the cuticle surface may have evolved in both sexually and asexually reproducing nematodes as a strategy to circumvent infection by this specialised hyperparasite.     

Relationship between the successful infection by entomopathogenic nematodes and the host immune response

Reproduction of entomopathogenic nematodes requires that they escape recognition by a host's immune system or that they have mechanisms to escape encapsulation and melanization. We investigated the immune responses of larvae for the greater wax moth (Galleria mellonella), tobacco hornworm (Manduca sexta), Japanese beetle (Popillia japonica), northern masked chafer (Cyclocephala borealis), oriental beetle (Exomala orientalis) and adult house crickets (Acheta domesticus), challenged with infective juveniles from different species and strains of entomopathogenic nematodes. The in vivo immune responses of hosts were correlated with nematode specificity and survival found by infection assays. In P. japonica, 45% of injected infective juveniles from Steinernema glaseri NC strain survived; whereas the hemocytes from the beetle strongly encapsulated and melanized the Heterorhabditis bacteriophora HP88 strain, S. glaseri FL strain, Steinernema scarabaei and Steinernema feltiae. Overall, H. bacteriophora was intensively melanized in resistant insect species (E. orientalis, P. japonica and C. borealis) and had the least ability to escape the host immune response. Steinernema glaseri NC strain suppressed the immune responses in susceptible hosts (M. sexta, E. orientalis and P. japonica), whereas S. glaseri FL strain was less successful. Using an in vitro assay, we found that hemocytes from G. mellonella, P. japonica, M. sexta and A. domestica recognized both nematode species quickly. However, many S. glaseri in M. sexta and H. bacteriophora in G. mellonella escaped from hemocyte encapsulation by 24h. These data indicate that, while host recognition underlies some of the differences between resistant and susceptible host species, escape from encapsulation following recognition can also allow successful infection. Co-injected surface-coat proteins from S. glaseri did not protect H. bacteriophora in M. sexta but did protect H. bacteriophora in E. orientalis larva; therefore, surface coat proteins do not universally convey host susceptibility. Comparisons of surface coat proteins by native and SDS-PAGE demonstrated different protein compositions between H. bacteriophora and S. glaseri and between the two strains of S. glaseri.     

Nematode Population and Community Dynamics in Soybean-Wheat Cropping and Tillage Regimes

The nematode community structures of various soybean-wheat regimes and of a single-cropped, conventionally tilled soybean regime were studied at two sites in Tennessee. Each of the 100 nematode species identified in the study was placed in one of five trophic groups, the most diverse being plant parasites (31 species), followed by Dorylaimida (26 species), bacterivores (23 species), fungivores (15 species), and predators (5 species). No significant differences in overall diversity and dominance among treatments and trophic groups were found. Densities of Heterodera glycines Ichinohe infective juveniles were significantly higher in single-cropped, conventionally tilled soybeans in July. When data were subjected to ordination analysis, it was shown that plant-parasitic nematode communities produced an aggregation of conventionally tilled, single-cropped soybean plots when compared to all double-cropped treatments. Ordination of overall nematode communities yielded similar results.     

An Analysis of Clumping in the Soil Amoeba Mayorella palestinensis*

SYNOPSIS. Mayorella palestinensis, a small soil amoeba, clumped during active growth in axenic, fluid culture media. Clumping was not associated with starvation (as in the case of cellular slime molds) or with encystation Hartmannella rhysodes was used as a control amoeba which did not clump to the same extent, but otherwise resembled M. palestinensis. Mixtures of both amoebae formed mixed clumps, without subsequent segregation of amoebae in the clumps. Clumping by M. palestinensis was temperature dependent, localized to the surface of the amoeba and required surface conformation. The requirement for a living system could be separated from the surface specificity of M. palestinensis and satisfied with living amoebae that did not clump by themselves, i.e., H. rhysodes, but did clump with dead M. palestinensis. It is proposed that amoeba-to-amoeba adhesion is similar to foreign substrate adhesion, and that M. palestinensis adheres to itself because its surface is more suitable for adhesion than other surfaces present in the culture. A competition for adhesion exists between surfaces so that clumps can be dissociated by providing a foreign surface that is more suitable for adhesion than the surface of the amoeba.     

Grazer-induced changes in the desmid Staurastrum

In aquatic environments, predator kairomones have been shown to affect morphology of prey species. Past work on the interaction between zooplankton and phytoplankton was based mainly on the Daphnia–Scenedesmus model. Algae of the genus Staurastrum can produce mucilage, causing cell clumping and settling out of the water column. These clumps are too large to be eaten by daphniids. Thus, we hypothesised that this may be a grazer defence. We investigated whether Daphnia magna induces the formation of mucus globules in Staurastrum, how this occurs, and if the formation of clumps inhibits growth in juvenile Daphnia. Eight strains of Staurastrum were used to check whether mucus extrusion is induced by the presence of Daphniaor possibly by a chemical excreted by Daphnia magna. None of the strains reacted to the presence of Daphnia water alone, animals had to be present to induce clumping. Mechanical action (gentle stirring) caused the same strains to clump. The ecological relevance of clumping was then investigated. The different Staurastrum strains were used as food in a growth experiment with ecologically relevant densities of neonates of Daphnia hyalina. These small daphniids did not cause the same clumping observed for Daphnia magna when present in experiments at high densities. We observed that juvenile daphniids grew less well on strains with larger cell size.     

根结线虫放线菌及其生物防治活性研究

从感染植物根部的根结线虫卵和雌虫中,分离得到放线菌20株。形态、细胞壁氨基酸组分和16S rRNA基因序列分析表明:分离菌株分属于链霉菌属(Streptomyces)、诺卡氏菌属(Nocardia)和假诺卡氏菌属(Pseudonocardia),其中链霉菌占80.0%。分离菌株对根结线虫Meloidogynespp.卵的平均寄生率、卵的孵化率、幼虫死亡率分别为54.1%、40.4%和26.2%。根据体外测试的结果,选择具有高致病力的3株链霉菌(1-17,2-6,9-47)和1株诺卡氏菌(5-1)进行温室番茄防效测试,其生防效率分别为31.4%、37.7%、56.4%和42.4%。     

In vitro uptake of 140 kDa Bacillus thuringiensis nematicidal crystal proteins by the second stage juvenile of Meloidogyne hapla

Plant-parasitic nematodes (PPNs) are piercing/sucking pests, which cause severe damage to crops worldwide, and are difficult to control. The cyst and root-knot nematodes (RKN) are sedentary endoparasites that develop specialized multinucleate feeding structures from the plant cells called syncytia or giant cells respectively. Within these structures the nematodes produce feeding tubes, which act as molecular sieves with exclusion limits. For example, Heterodera schachtii is reportedly unable to ingest proteins larger than 28 kDa. However, it is unknown yet what is the molecular exclusion limit of the Meloidogyne hapla. Several types of Bacillus thuringiensis crystal proteins showed toxicity to M. hapla. To monitor the entry pathway of crystal proteins into M. hapla, second-stage juveniles (J2) were treated with NHS-rhodamine labeled nematicidal crystal proteins (Cry55Aa, Cry6Aa, and Cry5Ba). Confocal microscopic observation showed that these crystal proteins were initially detected in the stylet and esophageal lumen, and subsequently in the gut. Western blot analysis revealed that these crystal proteins were modified to different molecular sizes after being ingested. The uptake efficiency of the crystal proteins by the M. hapla J2 decreased with increasing of protein molecular mass, based on enzyme-linked immunosorbent assay analysis. Our discovery revealed 140 kDa nematicidal crystal proteins entered M. hapla J2 via the stylet, and it has important implications in designing a transgenic resistance approach to control RKN.     

Relationship between saprotrophic growth in soil of different biotypes of Pochonia chlamydosporia and the infection of nematode eggs

The ecology of Pochonia chlamydosporia in soil and its interaction with both plant and nematode hosts are important for the successful exploitation of the fungus as a biological control agent. Differences in saprotrophism and parasitism were assessed for biotypes of P. chlamydosporia, which had originated from the eggs of cyst or root‐knot nematodes. Colonisation in soils of different textures (compost, sandy loam and loamy sand) measured by the numbers of colony‐forming units, differed greatly. Most biotypes were more abundant in sterilised soil of the different textures compared with non‐sterilised soils. The proportion of nematode eggs parasitised in a baiting technique demonstrated that biotypes had host preferences. Those biotypes that originated from root‐knot nematodes (RKN‐biotypes) infected significantly more Meloidogyne hapla eggs than Globodera pallida eggs, whereas biotypes from cyst nematodes (CN‐biotypes) parasitised more G. pallida eggs than M. hapla eggs. Differences in virulence between biotypes in an in vitro assay in which the fungi were placed directly onto the egg masses of M. hapla and those differences observed in the baiting technique showed similar trends. There was a negative linear correlation between the growth of the eight biotypes in soil and the proportion of eggs they infected in compatible interactions (i.e. fungal biotype originated from the same nematode genus as the target eggs). Those biotypes that infected most nematode eggs colonised soil the least extensively, suggesting that virulence may have a fitness cost. However, the relationship between saprotrophic growth and virulence is complex. The relative abundance of the different biotypes in soil in Petri dish assays was similar to that under glasshouse conditions using potato but not tomato as the plant host. Chlamydospores of some biotypes applied to soil significantly reduced (>50%) the population densities of M. hapla on tomato and of G. pallida on potato plants. Some biotypes that were both effective and virulent are good candidates for biological control of specific nematode pests. Data presented here and elsewhere indicate that RKN‐biotypes have different host preferences to CN‐biotypes; the specific primers based on the vcp1 gene from P. chlamydosporia rapidly confirmed the host origin of seven of the eight biotypes.