Investigations on Microbes Attached to the Cuticle of Phytonematodes.

Nematodes form various associations with soil microbiome. Experimental studies on nematode-attached microbes can improve mechanistic understanding of these associations and lead to new discoveries relevant for the field of nematode biocontrol. Microbial attachment to the surface of phytonematodes is very specific and influenced by a multitude of factors, including the designation of nematodes and microbes, environmental and biological factors in soil, time of incubation, and the ratio and evolutionary trajectories between nematodes and microbes. Here, we describe how the classical nematological and microbiological techniques can be coupled with the advanced molecular tools to study the microbial attachment to phytonematodes in soil. We focus on the characterization of nematode-attached microbes using classical microbiological approaches and high-throughput amplicon sequencing and on the effects of nematode-attached microbes on plant defense responses.

Genetic Variation among Heterodera schachtii Populations Coincided with Differences in Invasion and Propagation in Roots of a Set of Cruciferous Plants.

Genes of host plants and parasitic nematodes govern the plant-nematode interaction. The biological receptors and parasitism effectors are variable among plant species and nematode populations, respectively. In the present study, hatch testing and bioassays on cabbage, oilseed radish, and mustard were conducted to compare the biological characteristics among six populations of the beet cyst nematode Heterodera schachtii. Genetic patterns of the vap1 gene for the studied populations were distinct as shown by denaturing the gradient gel electrophoresis of PCR-amplified gene fragments. Concurrently, significant differences in the hatching rates, number of penetrated J2 in roots, and eggs/cyst ratios among the six nematode populations for the three cruciferous species were observed. In conclusion, analyzing the population genetic structure of H. schachtii plays a pivotal role in illustrating the variability in the plant-nematode interaction among its populations and plant species, which in its role leads to developing nematode management depending on plant resistance.

Nematode-Microbe Complexes in Soils Replanted with Apple.

Apple replant disease is a severe problem in orchards and tree nurseries. Evidence for the involvement of a nematode-microbe disease complex was reported. To search for this complex, plots with a history of apple replanting, and control plots cultivated for the first time with apple were sampled in two fields in two years. Shoot weight drastically decreased with each replanting. Amplicon sequencing of the nematode community and co-extracted fungal and bacterial communities revealed significant differences between replanted and control plots. Free-living nematodes of the genera Aphelenchus and Cephalenchus and an unidentified Dorylaimida were associated with replanted plots, as indicated by linear discriminant analysis effect size. Among the co-extracted fungi and bacteria, Mortierella and Methylotenera were most indicative of replanting. Some genera, mostly Rhabditis, Streptomyces and a fungus belonging to the Chaetomiaceae indicated healthy control plots. Isolating and investigating the putative disease complexes will help to understand and alleviate stress-induced root damage of apple in replanted soil.

Responsiveness of Elite Cultivars vs. Ancestral Genotypes of Barley to Beneficial Rhizosphere Microbiome, Supporting Plant Defense Against Root-Lesion Nematodes.

Harnessing plant-microbe interactions to advance crop resistance to pathogens could be a keystone in sustainable agriculture. The breeding of crops to maximize yield in intensive agriculture might have led to the loss of traits that are necessary for beneficial plant-soil feedback. In this study, we tested whether the soil microbiome can induce a stronger plant defense against root-lesion nematodes in ancestral genotypes of barley than in elite cultivars. Plants were grown in a sterile substrate with or without the inoculation of rhizosphere microbiomes, and Pratylenchus neglectus was inoculated to the roots. Unexpectedly, elite cultivars profited significantly more from the microbiome than ancestral genotypes, by the reduction of nematodes in roots and the increased shoot weight relative to control plants. The elite cultivars had higher microbial densities in the rhizosphere, which were correlated with root weight. The structure of the bacterial and fungal community of elite and ancestral genotypes differed, as compared by 16S rDNA or internal transcribed spacer amplicon profiles in denaturing gradient gel electrophoresis. The elite cultivars differed in responsiveness to the microbiome. For the most responsive cultivars Beysehir and Jolgeh, the strong microbe-induced suppression of nematodes coincided with the strongest microbe-dependent increase in transcripts of salicylic acid-regulated defense genes after nematode invasion, while the jasmonate-regulated genes LOX2 and AOS were downregulated in roots with the inoculated microbiome. The microbe-triggered modulation of defense gene expression differed significantly between elite and ancestral genotypes of barley. Soil microbiomes conditioned by maize roots suppressed the nematodes in elite cultivars, while the corresponding bulk soil microbiome did not. In conclusion, cultivars Beysehir and Jolgeh harbor the genetic background for a positive plant-microbiome feedback. Exploiting these traits in breeding for responsiveness to beneficial soil microbiomes, accompanied by soil biome management for compatible plant-microbe interactions, will support low-input agriculture and sustainability.

Priming Soybean cv. Primus Leads to Successful Systemic Defense Against the Root-Lesion Nematode, Pratylenchus penetrans.

Root lesion nematodes, Pratylenchus penetrans, are major pests of legumes with little options for their control. We aimed to prime soybean cv. Primus seedlings to improve basic defense against these nematodes by root application of N-3-oxo-tetradecanoyl-L-homoserine lactone (oxo-C14-HSL). The invasion of soybean roots by P. penetrans was significantly reduced in plants that were pre-treated with the oxo-C14-HSL producing rhizobacterium Ensifer meliloti strain ExpR+, compared to non-inoculated plants or plants inoculated with the nearly isogenic strain E. meliloti AttM with plasmid-mediated oxo-C14-HSL degradation. The nematodes were more clustered in the root tissues of plants treated with the AttM strain or the control compared to roots treated with the ExpR+ strain. In split-root systems primed on one side with strain ExpR+, root invasion was reduced on the opposite side compared to non-primed plants indicating a systemic plant response to oxo-C14-HSL. No additional local effect was detected, when inoculating nematodes on the ExpR+ primed side. Removal of oxo-C14-HSL after root exposure resulted in reduced root invasion compared to non-primed plants when the nematodes were added 3, 7, or 15 days later. Thus, probably the plant memorized the priming stimulus. Similarly, the plants were primed by compounds released from the surface of the nematodes. HPLC analysis of the root extracts of oxo-C14-HSL treated and untreated plants revealed that priming resulted in enhanced phytoalexin synthesis upon P. penetrans challenge. Without root invading nematodes, the phytoalexin concentrations of primed and non-primed plants did not significantly differ, indicating that priming did not lead to a persistently increased stress level of the plants. Upon nematode invasion, the phytoalexins coumestrol, genistein, and glyceollin increased in concentration in the roots compared to control plants without nematodes. Glyceollin synthesis was significantly more triggered by nematodes in primed plants compared to non-primed plants. The results indicated that the priming of soybean plants led to a more rapid and strong defense induction upon root invasion of nematodes.

Plants Specifically Modulate the Microbiome of Root-Lesion Nematodes in the Rhizosphere, Affecting Their Fitness.

Plant-parasitic nematodes are a major constraint on agricultural production. They significantly impede crop yield. To complete their parasitism, they need to locate, disguise, and interact with plant signals exuded in the rhizosphere of the host plant. A specific subset of the soil microbiome can attach to the surface of nematodes in a specific manner. We hypothesized that host plants recruit species of microbes as helpers against attacking nematode species, and that these helpers differ among plant species. We investigated to what extend the attached microbial species are determined by plant species, their root exudates, and how these microbes affect nematodes. We conditioned the soil microbiome in the rhizosphere of different plant species, then employed culture-independent and culture-dependent methods to study microbial attachment to the cuticle of the phytonematode Pratylenchus penetrans. Community fingerprints of nematode-attached fungi and bacteria showed that the plant species govern the microbiome associated with the nematode cuticle. Bacteria isolated from the cuticle belonged to Actinobacteria, Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, Sphingobacteria, and Firmicutes. The isolates Microbacterium sp. i.14, Lysobacter capsici i.17, and Alcaligenes sp. i.37 showed the highest attachment rates to the cuticle. The isolates Bacillus cereus i.24 and L. capsici i.17 significantly antagonized P. penetrans after attachment. Significantly more bacteria attached to P. penetrans in microbiome suspensions from bulk soil or oat rhizosphere compared to Ethiopian mustard rhizosphere. However, the latter caused a better suppression of the nematode. Conditioning the cuticle of P. penetrans with root exudates significantly decreased the number of Microbacterium sp. i.14 attaching to the cuticle, suggesting induced changes of the cuticle structure. These findings will lead to a more knowledge-driven exploitation of microbial antagonists of plant-parasitic nematodes for plant protection.

Biological Suppression of Populations of Heterodera schachtii Adapted to Different Host Genotypes of Sugar Beet.

Productivity of sugar beet and brassica vegetable crops is constrained by the nematode Heterodera schachtii worldwide. In sugar beet cropping areas of Central Europe and North America, H. schachtii is managed by crop rotation, and cultivation of resistant brassica cover crops. The recently released nematode-tolerant sugar beet cultivars suffer less damage than susceptible cultivars at high initial population densities of H. schachtii. Many tolerant cultivars allow for less nematode reproduction than susceptible cultivars. Monoculture of susceptible hosts can facilitate the evolution of suppressive soil. Objectives of this study were to determine if susceptible hosts are required for this process, and if monoculture with sugar beet genotypes of different host status (susceptible, resistant, tolerant) impact this capacity. Additionally, we tested if amending soil with the cyst nematode pathogens Pasteuria nishizawae or Hyalorbilia sp. strain DoUCR50 favored the establishment of soil suppressiveness. In 4-year microplot studies with H. schachtii Schach0 or Schach1, one susceptible, one Schach0-resistant, and one tolerant sugar beet genotype were monocultured. In 2010, plots were amended with P. nishizawae or DoUCR50, the last being introduced into non-treated soil for Schach0, and into previously biocide-treated soil for Schach1. In 2011, respective Schach0 plots received a second amendment with DoUCR50. Nematode population densities and growth and yield parameters were determined annually. Effects of P. nishizawae and DoUCR50 on populations of H. schachtii were limited and not consistent. Starting in the second year of the monoculture, eggs of both H. schachtii pathotypes became diseased. Up to 90% of the total eggs were encumbered by the third cropping cycle, under the susceptible, resistant, and tolerant cultivar. In all years, the tolerant genotype produced the highest and most stable white sugar yields while yields of the other cultivars slowly improved during the monoculture. Results of this study suggested the presence of egg-infecting factors in this sugar beet monoculture that dramatically increased the proportions of diseased eggs. The tolerant cultivar allowed establishment of soil suppressiveness without the initial yield decline observed when susceptible sugar beet genotypes are grown in monoculture.

Microbes Attaching to Endoparasitic Phytonematodes in Soil Trigger Plant Defense Upon Root Penetration by the Nematode.

Root-knot nematodes (Meloidogyne spp.) are among the most aggressive phytonematodes. While moving through soil to reach the roots of their host, specific microbes attach to the cuticle of the infective second-stage juveniles (J2). Reportedly, the attached microorganisms affect nematodes and reduce their performance on the host plants. We have previously shown that some non-parasitic bacterial strains isolated from the cuticle of Meloidogyne hapla in different soils affected J2 mortality, motility, hatching, and root invasion. Here we tested whether cuticle-attached microbes trigger plant defenses upon penetration of J2. In in vitro assays, M. hapla J2-attached microbes from a suppressive soil induced pathogen-associated molecular pattern-triggered immunity (PTI) in tomato roots. All tested PTI-responsive defense genes were upregulated after root invasion of J2 with attached microbes, compared to surface-sterilized J2, particularly the jasmonic acid-mediated PTI marker genes TFT1 and GRAS4.1. The strain Microbacterium sp. K6, that was isolated from the cuticle, significantly reduced root invasion when attached to the J2. Attached K6 cells supported plant defense and counteracted suppression of plant basal defense in roots by invaded J2. The plant response to the J2-attached K6 cells was stronger in leaves than in roots, and it increased from 1 to 3 days post inoculation (dpi). At 1 dpi, the plant responded to J2-attached K6 cells by ameliorating the J2-triggered down-regulation of defense genes mostly in roots, while at 3 dpi this response was systemic and more pronounced in leaves. In a reactive oxygen species (ROS) assay, the compounds released from J2 with attached K6 cells triggered a stronger ROS burst in tomato roots than the compounds from nematodes without K6, or the metabolites released from strain K6 alone. Leaves showed a 100 times more sensitive response than roots, and the metabolites of K6 with or without J2 induced strong ROS bursts. In conclusion, our results suggest the importance of microorganisms that attach to M. hapla in suppressive soil, inducing early basal defenses in plants and suppressing nematode performance in roots.

Plants and Associated Soil Microbiota Cooperatively Suppress Plant-Parasitic Nematodes.

Disease suppressive soils with specific suppression of soil-borne pathogens and parasites have been long studied and are most often of microbiological origin. As for the plant-parasitic nematodes (PPN), which represent a huge threat to agricultural crops and which successfully defy many conventional control methods, soil progression from conducive to suppressive state is accompanied by the enrichment of specific antagonistic microbial consortia. However, a few microbial groups have come to the fore in diminishing PPN in disease suppressive soils using culture-dependent methods. Studies with cultured strains resulted in understanding the mechanisms by which nematodes are antagonized by microorganisms. Recent culture-independent studies on the microbiome associated with soil, plant roots, and PPN contributed to a better understanding of the functional potential of disease suppressive microbial cohort. Plant root exudation is an important pathway determining host-microbe communication and plays a key role in selection and enrichment of a specific set of microbial antagonists in the rhizosphere as first line of defense against crop pathogens or parasites. Root exudates comprising primary metabolites such as amino acids, sugars, organic acids, and secondary metabolites can also cause modifications in the nematode surface and subsequently affect microbial attachment. A positive interaction between hosts and their beneficial root microbiota is correlated with a low nematode performance on the host. In this review, we first summarized the historical records of nematode-suppressive soils and then focused on more recent studies in this aspect, emphasizing the advances in studying nematode-microbe interactions over time. We highlighted nematode biocontrol mechanisms, especially parasitism, induced systemic resistance, and volatile organic compounds using microbial consortia, or bacterial strains of the genera Pasteuria, Bacillus, Pseudomonas, Rhizobium, Streptomyces, Arthrobacter, and Variovorax, or fungal isolates of Pochonia, Dactylella, Nematophthora, Purpureocillium, Trichoderma, Hirsutella, Arthrobotrys, and Mortierella. We discussed the importance of root exudates in plant communication with PPN and soil microorganisms, emphasizing their role in microbial attachment to the nematode surface and subsequent events of nematode parasitism. Comprehensive understanding of the plant-beneficial microbial consortia and the mechanisms underlying disease suppression may help to develop synthetic microbial communities for biocontrol of PPN, thereby reducing nematicides and fertilizers inputs.

Symbiosis of soybean with nitrogen fixing bacteria affected by root lesion nematodes in a density-dependent manner.

Early maturing varieties of soybean have a high yield potential in Europe, where the main biotic threat to soybean cultivation are root lesion nematodes (Pratylenchus spp.). Nitrogen fixation in root nodules by highly efficient inoculants of Bradyrhizobium japonicum is an incentive to grow soybean in low-input rotation systems. We investigated density-dependent effects of Pratylenchus penetrans on nitrogen fixation by co-inoculated B. japonicum. Less than 130 inoculated nematodes affected the number and weight of nodules, the density of viable bacteroids in nodules, and nitrogen fixation measured as concentration of ureides in leaves. With more inoculated nematodes, the percentage that invaded the roots increased, and adverse effects on the symbiosis accelerated, leading to non-functional nodules at 4,000 and more nematodes. When P. penetrans invaded roots that had fully established nodules, growth of nodules, density of bacteroids, and nitrogen fixation were affected but not the number of nodules. In contrast, nodulation of already infested roots resulted in a high number of small nodules with decreased densities of bacteroids and nitrogen fixation. P. penetrans invaded and damaged the nodules locally, but they also significantly affected the nodule symbiosis by a plant-mediated mechanism, as shown in an experiment with split-root systems.

Bacteria isolated from the cuticle of plant-parasitic nematodes attached to and antagonized the root-knot nematode Meloidogyne hapla.

Plant-parasitic nematodes are associated with specifically attached soil bacteria. To investigate these bacteria, we employed culture-dependent methods to isolate a representative set of strains from the cuticle of the infective stage (J2) of the root-knot nematode Meloidogyne hapla in different soils. The bacteria with the highest affinity to attach to J2 belonged to the genera Microbacterium, Sphingopyxis, Brevundimonas, Acinetobacter, and Micrococcus as revealed by 16S rRNA gene sequencing. Dynamics of the attachment of two strains showed fast adhesion in less than two hours, and interspecific competition for attachment sites. Isolates from the cuticle of M. hapla J2 attached to the lesion nematode Pratylenchus penetrans, and vice versa, suggesting similar attachment sites on both species. Removal of the surface coat by treatment of J2 with the cationic detergent CTAB reduced bacterial attachment, but did not prevent it. Some of the best attaching bacteria impaired M. hapla performance in vitro by significantly affecting J2 mortality, J2 motility and egg hatch. Most of the tested bacterial attachers significantly reduced the invasion of J2 into tomato roots, suggesting their beneficial role in soil suppressiveness against M. hapla.

Plant-Nematode Interactions Assisted by Microbes in the Rhizosphere.

Plant health is strongly influenced by the interactions between parasites/pathogens and beneficial microorganisms. In this chapter we will summarize the up-to date knowledge on soil suppressiveness as a biological tool against phytonematodes and explore the nature of monoculture versus crop rotation in this regard. Since nematodes are successfully antagonized by different microbiological agents, we highlighted this phenomenon with respect to the most important antagonists, and a nature of these interactions. The focus is on the hyperparasitic microbes of phytonematodes such as Pasteuria sp. and egg parasites. Furthermore, we comprised the studies on the defence system expressions in plants triggered by nematode-associated microbes. The attachment of bacteria and fungi to phytonematodes and putative effects of the attachment on the induced systemic resistance in plants are discussed. Finally, our chapter is rounded up with the importance of incorporating the knowledge on plant-nematode-microbe interactions in the integrated pest management.

Free-Living Nematodes Together With Associated Microbes Play an Essential Role in Apple Replant Disease.

Apple replant disease (ARD) is a severe problem in apple production worldwide. It is caused by a complex of soil biota, leading to small discolorated roots, as well as increased biosynthesis of phytoalexins, total phenolic compounds and antioxidants. We sampled soil from randomized field plots with either apple trees affected by ARD, which were five times replanted every second year, or with healthy trees growing in plots, which had a grass cover during this period. We investigated the contribution of nematodes to ARD by dissecting the soil biota from plots infested with ARD and non-infested control plots into a nematode and a microbe fraction. Nematode communities significantly differed between ARD and control soil as revealed by high-throughput sequencing of 18S rRNA genes. Plant-parasitic nematodes were too low in abundance to explain root damage, and did not significantly differ between ARD and control soil. Their separate and synergistic effect on ARD symptoms of susceptible M26 apple rootstocks was analyzed 4 and 8 weeks after inoculation in three greenhouse experiments. Inoculants were either nematodes from ARD plots (NARD), NARD plus microbes from ARD plots (MARD), NARD plus microbes from control plots (MCon), nematodes from control plots NCon plus MARD, NCon plus MCon, MARD, or MCon, or non-inoculated control. In all three experiments, the combination NARD plus MARD had the strongest adverse effect on the plants, with respect to growth parameters of shoots and roots, total phenolic compounds and phytoalexins in roots, and antioxidants in leaves. NARD also induced ARD but less than NARD plus MARD. NARD plus MCon had delayed effects on the plants compared to NARD plus MARD, suggesting that detrimental nematode-microbe interactions built up with time. Effects of MARD or NCon plus MARD were minor or not distinguishable from those of MCon or non-inoculated control. Overall, the source of the inoculated nematodes -ARD or control soil- and the interaction between ARD nematodes and microbes were highly significant factors determining ARD. In conclusion, exploring the associations of nematodes and microbes in ARD soils will give the chance to unravel the etiology of ARD.

Rhizosphere Microbiomes Modulated by Pre-crops Assisted Plants in Defense Against Plant-Parasitic Nematodes.

Plant-parasitic nematodes cause considerable damage to crop plants. The rhizosphere microbiome can affect invasion and reproductive success of plant-parasitic nematodes, thus affecting plant damage. In this study, we investigated how the transplanted rhizosphere microbiome from different crops affect plant-parasitic nematodes on soybean or tomato, and whether the plant's own microbiome from the rhizosphere protects it better than the microbiome from fallow soil. Soybean plants growing in sterilized substrate were inoculated with the microbiome extracted from the rhizosphere of soybean, maize, or tomato. Controls were inoculated with extracts from bulk soil, or not inoculated. After the microbiome was established, the root lesion nematode Pratylenchus penetrans was added. Root invasion of P. penetrans was significantly reduced on soybean plants inoculated with the microbiome from maize or soybean compared to tomato or bulk soil, or the uninoculated control. In the analogous experiment with tomato plants inoculated with either P. penetrans or the root knot nematode Meloidogyne incognita, the rhizosphere microbiomes of maize and tomato reduced root invasion by P. penetrans and M. incognita compared to microbiomes from soybean or bulk soil. Reproduction of M. incognita on tomato followed the same trend, and it was best suppressed by the tomato rhizosphere microbiome. In split-root experiments with soybean and tomato plants, a systemic effect of the inoculated rhizosphere microbiomes on root invasion of P. penetrans was shown. Furthermore, some transplanted microbiomes slightly enhanced plant growth compared to uninoculated plants. The microbiomes from maize rhizosphere and bulk soil increased the fresh weights of roots and shoots of soybean plants, and microbiomes from soybean rhizosphere and bulk soil increased the fresh weights of roots and shoots of tomato plants. Nematode invasion did not affect plant growth in these short-term experiments. In conclusion, this study highlights the importance of the rhizosphere microbiome in protecting crops against plant-parasitic nematodes. An effect of pre-crops on the rhizosphere microbiome might be harnessed to enhance the resistance of crops towards plant-parasitic nematodes. However, nematode-suppressive effects of a particular microbiome may not necessarily coincide with improvement of plant growth in the absence of plant-parasitic nematodes.

Effector gene vap1 based DGGE fingerprinting to assess variation within and among Heterodera schachtii populations.

Populations of beet cyst nematodes Heterodera schachtii vary in aggressiveness and virulence toward sugar beet varieties, but also in traits like host range, or decline rate in the field. Diversity of their essential pathogenicity gene vap1 is shaped by diversifying selection and gene flow. The authors developed a technique to study inter-population variation and intra-population diversity and dynamics of H. schachtii based on the gene vap1. Degenerate primers were designed to amplify, clone, and sequence this gene from diverse species and populations of cyst nematodes. This resulted in a high diversity of sequences for H. schachtii, and allowed to design non-degenerated primers to amplify a fragment suitable for sequence dependent separation of gene variants in denaturing gradient gel electrophoresis (DGGE). The developed primers span a highly variable intron and part of a slightly variable exon. A marker comprised of the 14 mostly detected gene variants was established for gel-to-gel comparisons. For individual juveniles up to six gene variants were resolved and substantial variation within and among cysts was observed. A fast and easy DNA extraction procedure for 20 pooled cysts was established, which provided DGGE patterns with high similarity among replicate samples from field populations. Permutation tests on pairwise similarities within and among populations showed significant differences among vap1 patterns of field populations of H. schachtii. Similarly, gene diversity as expressed by the Shannon index was statistically different among field populations. In conclusion, the DGGE technique is a fast and - compared to sequencing approaches - inexpensive tool to compare populations of H. schachtii and link observed biological characteristics to genetic pattern.Populations of beet cyst nematodes Heterodera schachtii vary in aggressiveness and virulence toward sugar beet varieties, but also in traits like host range, or decline rate in the field. Diversity of their essential pathogenicity gene vap1 is shaped by diversifying selection and gene flow. The authors developed a technique to study inter-population variation and intra-population diversity and dynamics of H. schachtii based on the gene vap1. Degenerate primers were designed to amplify, clone, and sequence this gene from diverse species and populations of cyst nematodes. This resulted in a high diversity of sequences for H. schachtii, and allowed to design non-degenerated primers to amplify a fragment suitable for sequence dependent separation of gene variants in denaturing gradient gel electrophoresis (DGGE). The developed primers span a highly variable intron and part of a slightly variable exon. A marker comprised of the 14 mostly detected gene variants was established for gel-to-gel comparisons. For individual juveniles up to six gene variants were resolved and substantial variation within and among cysts was observed. A fast and easy DNA extraction procedure for 20 pooled cysts was established, which provided DGGE patterns with high similarity among replicate samples from field populations. Permutation tests on pairwise similarities within and among populations showed significant differences among vap1 patterns of field populations of H. schachtii. Similarly, gene diversity as expressed by the Shannon index was statistically different among field populations. In conclusion, the DGGE technique is a fast and ­ compared to sequencing approaches ­ inexpensive tool to compare populations of H. schachtii and link observed biological characteristics to genetic pattern.

Response of the bacterial community in an on-farm biopurification system, to which diverse pesticides are introduced over an agricultural season.

A biopurification system (BPS) is used on-farm to clean pesticide-contaminated wastewater. Due to high pesticide loads, a BPS represents a hot spot for the proliferation and selection as well as the genetic adaptation of discrete pesticide degrading microorganisms. However, while considerable knowledge exists on the biodegradation of specific pesticides in BPSs, the bacterial community composition of these systems has hardly been explored. In this work, the Shannon diversity, the richness and the composition of the bacterial community within an operational BPS receiving wastewater contaminated with various pesticides was, for the first time, elucidated over the course of an agricultural season, using DGGE profiling and pyrosequencing of 16S rRNA gene fragments amplified from total community DNA. During the agricultural season, an increase in the concentration of pesticides in the BPS was observed along with the detection of significant community changes including a decrease in microbial diversity. Additionally, a significant increase in the relative abundance of Proteobacteria, mainly the Gammaproteobacteria, was found, and OTUs (operational taxonomic units) affiliated to Pseudomonas responded positively during the course of the season. Furthermore, a banding-pattern analysis of 16S rRNA gene-based DGGE fingerprinting, targeting the Alpha- and Betaproteobacteria as well as the Actinobacteria, indicated that the Betaproteobacteria might play an important role. Interestingly, a decrease of Firmicutes and Bacteroidetes was observed, indicating their selective disadvantage in a BPS, to which pesticides have been introduced.

Microbiomes associated with infective stages of root-knot and lesion nematodes in soil.

Endoparasitic root-knot (Meloidogyne spp.) and lesion (Pratylenchus spp.) nematodes cause considerable damage in agriculture. Before they invade roots to complete their life cycle, soil microbes can attach to their cuticle or surface coat and antagonize the nematode directly or by induction of host plant defenses. We investigated whether the nematode-associated microbiome in soil differs between infective stages of Meloidogyne incognita and Pratylenchus penetrans, and whether it is affected by variation in the composition of microbial communities among soils. Nematodes were incubated in suspensions of five organically and two integrated horticultural production soils, recovered by sieving and analyzed for attached bacteria and fungi after washing off loosely adhering microbes. Significant effects of the soil type and nematode species on nematode-associated fungi and bacteria were revealed as analyzed by community profiling using denaturing gradient gel electrophoresis. Attached microbes represented a small specific subset of the soil microbiome. Two organic soils had very similar bacterial and fungal community profiles, but one of them was strongly suppressive towards root-knot nematodes. They were selected for deep amplicon sequencing of bacterial 16S rRNA genes and fungal ITS. Significant differences among the microbiomes associated with the two species in both soils suggested specific surface epitopes. Among the 28 detected bacterial classes, Betaproteobacteria, Bacilli and Actinobacteria were the most abundant. The most frequently detected fungal genera were Malassezia, Aspergillus and Cladosporium. Attached microbiomes did not statistically differ between these two soils. However, Malassezia globosa and four fungal species of the family Plectosphaerellaceae, and the bacterium Neorhizobium galegae were strongly enriched on M. incognita in the suppressive soil. In conclusion, the highly specific attachment of microbes to infective stages of phytonematodes in soil suggested an ecological role of this association and might be involved in soil suppressiveness towards them.

Statistical test for tolerability of effects of an antifungal biocontrol strain on fungal communities in three arable soils.

A statistical method was developed to test for equivalence of microbial communities analysed by next-generation sequencing of amplicons. The test uses Bray-Curtis distances between the microbial community structures and is based on a two-sample jackknife procedure. This approach was applied to investigate putative effects of the antifungal biocontrol strain RU47 on fungal communities in three arable soils which were analysed by high-throughput ITS amplicon sequencing. Two contrasting workflows to produce abundance tables of operational taxonomic units from sequence data were applied. For both, the developed test indicated highly significant equivalence of the fungal communities with or without previous exposure to RU47 for all soil types, with reference to fungal community differences in conjunction with field site or cropping history. However, minor effects of RU47 on fungal communities were statistically significant using highly sensitive multivariate tests. Nearly all fungal taxa responding to RU47 increased in relative abundance indicating the absence of ecotoxicological effects. Use of the developed equivalence test is not restricted to evaluate effects on soil microbial communities by inoculants for biocontrol, bioremediation or other purposes, but could also be applied for biosafety assessment of compounds like pesticides, or genetically engineered plants.

Do drying and rewetting cycles modulate effects of sulfadiazine spiked manure in soil?

Naturally occurring drying-rewetting events in soil have been shown to affect the dissipation of veterinary antibiotics entering soil by manure fertilization. However, knowledge of effects on the soil microbial community structure and resistome is scarce. Here, consequences of drying-rewetting cycles on effects of sulfadiazine (SDZ) in soil planted with Dactylis glomerata L. were investigated in microcosms. Manure containing SDZ or not was applied to the pregrown grass and incubated for 56 days in a climate chamber. Water was either added daily or reduced during two drying events of 7 days, each followed by a recovery phase. Total community DNA was analyzed to reveal the effects on the bacterial community structure and on the abundance of sul1, sul2, intI1 ,intI2, qacE+qacEΔ1, traN and korB genes relative to 16S rRNA genes. 16S rRNA gene-based DGGE fingerprints indicated that drying-rewetting cycles modulated the effects of SDZ on the bacterial community structure in the soil. Furthermore, the SDZ treatment increased the relative abundance of sulfonamide resistance and integrase genes compared to the control. However, this increase was not different between moisture regimes, indicating that drying-rewetting had only a negligible effect on the selection of the resistome by SDZ in the manured soil.

Characterization of tet(Y)-carrying LowGC plasmids exogenously captured from cow manure at a conventional dairy farm.

Manure from dairy farms has been shown to contain diverse tetracycline resistance genes that are transferable to soil. Here, we focus on conjugative plasmids that may spread tetracycline resistance at a conventional dairy farm. We performed exogenous plasmid isolation from cattle feces using chlortetracycline for transconjugant selection. The transconjugants obtained harbored LowGC-type plasmids and tet(Y). A representative plasmid (pFK2-7) was fully sequenced and this was compared with previously described LowGC plasmids from piggery manure-treated soil and a GenBank record from Acinetobacter nosocomialis that we also identified as a LowGC plasmid. The pFK2-7 plasmid had the conservative backbone typical of LowGC plasmids, though this region was interrupted with an insert containing the tet(Y)-tet(R) tetracycline resistance genes and the strA-strB streptomycin resistance genes. Despite Acinetobacter populations being considered natural hosts of LowGC plasmids, these plasmids were not found in three Acinetobacter isolates from the study farm. The isolates harbored tet(Y)-tet(R) genes in identical genetic surroundings as pFK2-7, however, suggesting genetic exchange between Acinetobacter and LowGC plasmids. Abundance of LowGC plasmids and tet(Y) was correlated in manure and soil samples from the farm, indicating that LowGC plasmids may be involved in the spread of tet(Y) in the environment.