Multiple-strand displacement and identification of single nucleotide polymorphisms as markers of genotypic variation of Pasteuria penetrans biotypes infecting root-knot nematodes

Pasteuria species are endospore-forming obligate bacterial parasites of soil-inhabiting nematodes and water-inhabiting cladocerans, e.g. water fleas, and are closely related to Bacillus spp. by 16S rRNA gene sequence. As naturally occurring bacteria, biotypes of Pasteuria penetrans are attractive candidates for the biocontrol of various Meloidogyne spp. (root-knot nematodes). Failure to culture these bacteria outside their hosts has prevented isolation of genomic DNA in quantities sufficient for identification of genes associated with host recognition and virulence. We have applied multiple-strand displacement amplification (MDA) to generate DNA for comparative genomics of biotypes exhibiting different host preferences. Using the genome of Bacillus subtilis as a paradigm, MDA allowed quantitative detection and sequencing of 12 marker genes from 2000 cells. Meloidogyne spp. infected with P. penetrans P20 or B4 contained single nucleotide polymorphisms (SNPs) in the spoIIAB gene that did not change the amino acid sequence, or that substituted amino acids with similar chemical properties. Individual nematodes infected with P. penetrans P20 or B4 contained SNPs in the spoIIAB gene sequenced in MDA-generated products. Detection of SNPs in the spoIIAB gene in a nematode indicates infection by more than one genotype, supporting the need to sequence genomes of Pasteuria spp. derived from single spore isolates.     

Phylogenetic analysis of Pasteuria penetrans by use of multiple genetic loci

Pasteuria penetrans is a gram-positive, endospore-forming eubacterium that apparently is a member of the Bacillus-Clostridium clade. It is an obligate parasite of root knot nematodes (Meloidogyne spp.) and preferentially grows on the developing ovaries, inhibiting reproduction. Root knot nematodes are devastating root pests of economically important crop plants and are difficult to control. Consequently, P. penetrans has long been recognized as a potential biocontrol agent for root knot nematodes, but the fastidious life cycle and the obligate nature of parasitism have inhibited progress on mass culture and deployment. We are currently sequencing the genome of the Pasteuria bacterium and have performed amino acid level analyses of 33 bacterial species (including P. penetrans) using concatenation of 40 housekeeping genes, with and without insertions/deletions (indels) removed, and using each gene individually. By application of maximum-likelihood, maximum-parsimony, and Bayesian methods to the resulting data sets, P. penetrans was found to cluster tightly, with a high level of confidence, in the Bacillus class of the gram-positive, low-G+C-content eubacteria. Strikingly, our analyses identified P. penetrans as ancestral to Bacillus spp. Additionally, all analyses revealed that P. penetrans is surprisingly more closely related to the saprophytic extremophile Bacillus haladurans and Bacillus subtilis than to the pathogenic species Bacillus anthracis and Bacillus cereus. Collectively, these findings strongly imply that P. penetrans is an ancient member of the Bacillus group. We suggest that P. penetrans may have evolved from an ancient symbiotic bacterial associate of nematodes, possibly as the root knot nematode evolved to be a highly specialized parasite of plants.     

Interactions between bacteria and plant-parasitic nematodes: now and then

Based on genome-to-genome analyses of gene sequences obtained from plant-parasitic, root-knot nematodes (Meloidogyne spp.), it seems likely that certain genes have been derived from bacteria by horizontal gene transfer. Strikingly, a common theme underpinning the function of these genes is their apparent direct relationship to the nematodes' parasitic lifestyle. Phylogenetic analyses implicate rhizobacteria as the predominant group of 'gene donor' bacteria. Root-knot nematodes and rhizobia occupy similar niches in the soil and in roots, and thus the opportunity for genetic exchange may be omnipresent. Further, both organisms establish intimate developmental interactions with host plants, and mounting evidence suggests that the mechanisms for these interactions are shared too. We propose that the origin of parasitism in Meloidogyne may have been facilitated by acquisition of genetic material from soil bacteria through horizontal transfer, and that such events represented key steps in speciation of plant-parasitic nematodes. To further understand the mechanisms of horizontal gene transfer, and also to provide experimental tools to manipulate this promising bio-control agent, we have initiated a genomic sequence of the bacterial hyper-parasite of plant parasitic nematodes, Pasteuria penetrans. Initial data have established that P. penetrans is closely related to Bacillus spp., to the extent that considerable genome synteny is apparent. Hence, Bacillus serves as a model for Pasteuria, and vice versa.     

Integrated management of root-knot nematode, Meloidogyne incognita infestation in tomato and grapevine

An integrated approach with the obligate bacterial parasite, Pasteuria penetrans and nematicides was assessed for the management of the root-knot nematode, Meloidogyne incognita infestation in tomato and grapevine. Seedlings of tomato cv. Co3 were transplanted into pots filled with sterilized soil and inoculated with nematodes (5000 juveniles/pot). The root powder of P. penetrans at 10 mg/pot was applied alone and in combination with carbofuran at 6 mg/pot. Application of P. penetrans along with carbofuran recorded lowest nematode infestation (107 nematodes/200 g soil) compared to control (325 nematodes/200 g soil). The rate of parasitization was 83.1% in the carbofuran and P. penetrans combination treatment as against 61.0% in the P. penetrans treatment only. The plant growth was also higher in the combination treatment compared to all other treatments. A field trial was carried out to assess the efficacy of P. penetrans and nematicides viz., carbofuran and phorate in the management of root-knot nematode, M. incognita infestation of grapevine cv. Muscat Hamburg. A nematode and P. penetrans infested grapevine field was selected and treatments either with carbofuran or phorate at 1 g a.i/vine was given. The observations were recorded at monthly interval. The results showed that the soil nematode population was reduced in nematicide treated plots. Suppression of nematodes was higher under phorate (117 nematodes/200 g soil) than under carbofuran (126.7 nematodes/200 g soil) treatment. The number of juveniles parasitized was also influenced by nematicides and spore load carried/juvenile with phorate being superior and the increase being 17.0 and 29.0% respectively over the control. The results of these experiment confirmed the compatibility of P. penetrans with nematicides and its biological control potential against the root-knot nematode.     

Identification of new single nucleotide polymorphism-based markers for inter- and intraspecies discrimination of obligate bacterial parasites (Pasteuria spp.) of invertebrates

Protein-encoding and 16S rRNA genes of Pasteuria penetrans populations from a wide range of geographic locations were examined. Most interpopulation single nucleotide polymorphisms (SNPs) were detected in the 16S rRNA gene. However, in order to fully resolve all populations, these were supplemented with SNPs from protein-encoding genes in a multilocus SNP typing approach. Examination of individual 16S rRNA gene sequences revealed the occurrence of "cryptic" SNPs which were not present in the consensus sequences of any P. penetrans population. Additionally, hierarchical cluster analysis separated P. penetrans 16S rRNA gene clones into four groups, and one of which contained sequences from the most highly passaged population, demonstrating that it is possible to manipulate the population structure of this fastidious bacterium. The other groups were made from representatives of the other populations in various proportions. Comparison of sequences among three Pasteuria species, namely, P. penetrans, P. hartismeri, and P. ramosa, showed that the protein-encoding genes provided greater discrimination than the 16S rRNA gene. From these findings, we have developed a toolbox for the discrimination of Pasteuria at both the inter- and intraspecies levels. We also provide a model to monitor genetic variation in other obligate hyperparasites and difficult-to-culture microorganisms.     

Attachment tests of Pasteuria penetrans to the cuticle of plant and animal parasitic nematodes, free living nematodes and srf mutants of Caenorhabditis elegans.

Populations of Pasteuria penetrans isolated from root-knot nematodes (Meloidogyne spp.) and cyst nematodes (Heterodera spp.) were tested for their ability to adhere to a limited selection of sheathed and ex-sheathed animal parasitic nematodes, free living nematodes, including Caenorhabditis elegans wild type and several srf mutants, and plant parasitic nematodes. The attachment of spores of Pasteuria was restricted and no spores were observed adhering to any of the animal parasitic nematodes either with or without their sheath or to any of the free living nematodes including C. elegans and the srf mutants. All spore attachment was restricted to plant parasitic nematodes; however, spores isolated from cyst nematodes showed the ability to adhere to other genera of plant parasitic nematodes which was not the case with spores isolated from root-knot nematodes. The results are discussed in relationship to cuticular heterogeneity.     

线虫寄生菌巴斯德杆菌遗传多样性分析

应用16S rDNA-PCR技术快速检测不同根系样品中的巴斯德杆菌(Pasteuria spp.),并采用PCR-RFLP和PCR-SSCP法初步分析这些样品中巴斯德杆菌群体的遗传多样性,结果表明,来自福建、广东的30份感染根结线虫病的根系中,有9份样品含有巴斯德杆菌;PCR-RFLP分析表明,克隆子的EcoH I酶切带型分为5类,其中2类占相对优势,PCR-SSCP带型分类的情况与PCR-RFLP的基本一致,但表现出更大的差异性.选取12个克隆子进行测序分析,结果显示,克隆子的16S rDNA序列与穿刺巴斯德杆菌(Pasteuria penetrans)的相应序列具有较高的同源性(97.8%～99.7%);系统进化关系分析进一步表明,不同根系样品中的巴斯德杆菌(Pasteuria spp.)16S rDNA序列与GenBank已收录的穿刺巴斯德杆菌(P.penetrans)序列形成1个主要分支和7个独立分支,具有一定的遗传差异.     

Biological control potential of the obligate parasite Pasteuria penetransagainst the root-knot nematode, Meloidogyne incognita infestation in Brinjal

The efficacy of the obligate bacterial parasite, Pasteuria penetrans against the rootknot nematode, Meloidogyne incognita infestation was assessed in brinjal. The seedling pans with sterilized soil were inoculated with nematodes and root powder of P. penetrans were applied at different dosages viz., 0 x 10(6), 0.5 x 10(6) spores and 1 x 10(6) spores/pan. Seeds of brinjal cv Co2 were sown in the pans and seedlings were allowed to grow. The seedlings were transplanted to microplots containing sterilized soil. Observations on nematode infestation and plant growth were recorded at seedling, flowering, and fruiting stages. Nematode infestation was significantly reduced by P. penetrans treatment. There was 22, 75 and 86% reduction in nematode population of soil over control at seedling, flowering and fruiting stages, respectively, at higher spore density (1 x 10(6)). Egg mass production was decreased by 63, 78 and 89% over control at 35 (seedling), 100 (flowering) and 160 (fruiting) days after sowing respectively, at 1 x 10(6) spores treated soil. The parasitizing ability of P. penetrans increased with the age of the crop. At higher spore density the percentage of parasitization was increased from 52.0 (35 days after sowing) to 90.0 (160 days after sowing) %. At these stages of the crop, the spore load per juvenile also increased at the higher dose. The P. penetrans application enhanced the plant growth. The weight of the shoot was increased by 17.6% whereas root weight by 41.0% over the control at fruiting stage. The experimental results revealed the potential use of P. penetrans as biological control agent of M. incognita. Application of P. penetrans spores in the nursery is a good strategy since the mass multiplication is quite difficult.     

Molecular and Morphological Characterization and Biological Control Capabilities of a Pasteuria ssp. Parasitizing Rotylenchulus reniformis, the Reniform Nematode

Rotylenchulus reniformis is one of 10 described species of reniform nematodes and is considered the most economically significant pest within the genus, parasitizing a variety of important agricultural crops. Rotylenchulus reniformis collected from cotton fields in the Southeastern US were observed to have the nematode parasitic bacterium Pasteuria attached to their cuticles. Challenge with a Pasteuria-specific monoclonal antibody in live immuno-fluorescent assay (IFA) confirmed the discovery of Pasteuria infecting R. reniformis. Scanning and transmission electron microscopy were employed to observe endospore ultrastructure and sporogenesis within the host. Pasteuria were observed to infect and complete their life-cycle in juvenile, male and female R. reniformis. Molecular analysis using Pasteuria species-specific and degenerate primers for 16s rRNA and spoII, and subsequent phylogenetic assessment, placed the Pasteuria associated with R. reniformis in a distinct clade within established assemblages for the Pasteuria infecting phytopathogenic nematodes. A global phylogenetic assessment of Pasteuria 16s rDNA using the Neighbor-Joining method resulted in a clear branch with 100% boot-strap support that effectively partitioned the Pasteuria infecting phytopathogenic nematodes from the Pasteuria associated with bacterivorous nematodes. Phylogenetic analysis of the R. reniformis Pasteuria and Pasteuria spp. parasitizing a number of economically important plant parasitic nematodes revealed that Pasteuria with different host specificities are closely related and likely constitute biotypes of the same species. This suggests host preference, and thus effective differentiation and classification are most likely predicated by an influential virulence determinant(s) that has yet to be elucidated. Pasteuria Pr3 endospores produced by in vitro fermentation demonstrated efficacy as a commercial bionematicide to control R. reniformis on cotton in pot tests, when applied as a seed treatment and in a granular formulation. Population control was comparable to a seed-applied nematicide/insecticide (thiodicarb/imidacloprid) at a seed coating application rate of 1.0 x 10(8) spores/seed.     

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).     

The electrostatic nature of the spore of Pasteuria penetrans, the bacterial parasite of root-knot nematodes

The response of spores of Pasteuria penetrans , the Gram-positive obligate nematode hyperparasite, was studied in a direct current electric field and monitored using a microscope attached to a video recorder apparatus. Fluorescence measurements were performed on the spore's surface using HEXCO as a fluorescent probe. The mobilities of the spores and fluorescence measurements were performed in different salt concentrations and at different pH values. The results showed a significant electronegative potential at the spore surface which was dependent on the pH, salt concentration and valency of the cation present in the electrolyte medium. The results of the fluorescence experiments using HEXCO correlated well with the results obtained from the electrophoretic mobility experiments. A polyclonal antiserum raised to spores of P. penetrans affected the surface charge density and the data presented suggest that electrostatic interactions may be important in the binding of spores to the nematode cuticle. The binding of HEXCO to hydrophobic sites on the spores' surfaces suggests the possibility of other attractive forces also being important in the binding process.     

Population Dynamics of Meloidogyne arenaria and Pasteuria penetrans in a Long-Term Crop Rotation Study

The endospore-forming bacterium Pasteuria penetrans is an obligate parasite of root-knot nematodes (Meloidogyne spp.). The primary objective of this study was to determine the effect of crop sequence on abundance of P. penetrans. The experiment was conducted from 2000 to 2008 at a field site naturally infested with both the bacterium and its host Meloidogyne arenaria and included the following crop sequences: continuous peanut (Arachis hypogaea) (P-P-P) and peanut rotated with either 2 years of corn (Zea mays) (C-C-P), 1 year each of cotton (Gossypium hirsutum) and corn (Ct-C-P), or 1 year each of corn and a vegetable (V-C-P). The vegetable was a double crop of sweet corn and eggplant (Solanum melongena). A bioassay with second-stage juveniles (J2) of M. arenaria from a greenhouse (GH) population was used to estimate endospore abundance under the different crop sequences. A greater numerical increase in endospore densities was expected in the P-P-P and V-C-P sequences than in the other sequences because both peanut and eggplant are good hosts for M. arenaria. However, endospore densities, as determined by bioassay, did not substantially increase in any of the sequences during the 9-year experiment. To determine whether the nematode population had developed resistance to the resident P. penetrans, five single egg-mass (SEM) lines from the field population of M. arenaria were tested alongside the GH population for acquisition of endospores from the field soil. Four of the five SEM lines acquired 9 to 14 spores/J2 whereas the GH population and one of the SEM lines acquired 3.5 and 1.8 spores/J2, respectively. Endospore densities estimated with the four receptive SEM lines were highest in the P-P-P plots (14-20 spores/J2), intermediate in the V-C-P plots (6-7 spores/J2), and lowest in the Ct-C-P plots (< 1 spore/J2). These results indicate that the field population of M. arenaria is heterogeneous for attachment of P. penetrans endospores. Moreover, spore densities increased under intensive cropping of hosts for M. arenaria, but the GH population of the nematode was not receptive to spore attachment. However, previously, the GH population was very receptive to spore acquisition from this field site. One explanation for this inconsistency is that the M. arenaria population in the field became resistant to the dominant subpopulation of P. penetrans that had been present, and this led to the selection of a different subpopulation of the bacterium that is incompatible with the GH population.     

Temporal regulation of the Bacillus subtilis early sporulation gene spo0F

The initiation of sporulation in Bacillus subtilis depends on seven genes of the spo0 class. One of these, spo0F, codes for a protein of 14,000 daltons. We studied the regulation of spo0F by using spo0F-lacZ translational fusions and also measured Spo0F protein levels by immunoassays. spo0F-lacZ and Spo0F levels increased as the cells entered the stationary phase, and this effect was repressed by glucose and glutamine. Decoyinine, which lowers GTP levels and allows sporulation in the presence of normally repressing levels of glucose, induced spo0F-lacZ expression and raised Spo0F levels. The expression of spo0F-lacZ was dependent on spo0A, -0B, -0E, -0F, and -0H genes, a spo0H deletion causing the strongest effect. In most respects, the spo0F gene was regulated in a manner similar to that of spoVG. However, the presence of an abrB mutation did not relieve the dependence of spo0F gene expression on spo0A, as it does with spoVG (P. Zuber and R. Losick, J. Bacteriol. 169:2223-2230, 1987).     

Occurrence, hosts, morphology, and molecular characterisation of Pasteuria bacteria parasitic in nematodes of the family Plectidae

Parasitic bacteria of the genus Pasteuria are reported for three Anaplectus and four identified and several unidentified Plectus species found in eight countries in various habitats. The pasteurias from plectids agree in essential morphological characters of sporangia and endospores as well as in developmental cycle with those of the Pasteuria species and strains described from tylenchid nematodes, but appear to be mainly distinguished from these by absence of a distinct perisporium in the spores and the endospores obviously not being cup- or saucer-shaped. The wide range of measurements and morphological peculiarities of sporangia and endospores suggest that probably several Pasteuria species have to be distinguished as parasites in Plectidae. From an infected juvenile of an unidentified plectid species the 16S rRNA gene sequence of Pasteuria sp. was obtained. Substantial sequence divergence from described Pasteuria species and its phylogenetic position on molecular trees indicate that this Pasteuria sp. could be considered as a new species. Preliminary results of the analysis of DNA phylogeny of Pasteuria spp. and their nematode hosts provide evidence for incongruence of their phylogenetic history and of host switching events during evolution of the bacterial parasites.     

Effects of the nematicide imicyafos on soil nematode community structure and damage to radish caused by Pratylenchus penetrans

The effects of the non-fumigant nematicide imicyafos on soil nematode community structure and damage to radish caused by Pratylenchus penetrans were evaluated in two field experiments in consecutive years (2007 and 2008). Nematode densities in soil at 0 - 10 cm (the depth of nematicide incorporation) and 10 - 30 cm were measured. The application of imicyafos had a significant impact on the density of P. penetrans at 0 - 10 cm but had no effect on free-living nematode density. PCR-DGGE analysis conducted using extracted nematodes showed that the nematode community structure 12 d after application in 2007 was altered by the application of imicyafos at the 0 - 10 cm depth, but not at 10 - 30 cm. No significant differences were observed in the diversity of the nematode community at harvest (89 and 91 d after application) between the control and imicyafos treatments in both depths and both years. In both years, the damage to radish caused by P. penetrans was markedly suppressed by the nematicide. Overall, the nematicide imicyafos decreased populations of P. penetrans in soil and thereby decreased damage to radish, while having little impact on the soil nematode community.