Nitrification inhibitors effectively target N2 O-producing Nitrosospira spp. in tropical soil.

The nitrification inhibitors (NIs) 3,4-dimethylpyrazole (DMPP) and dicyandiamide (DCD) can effectively reduce N2 O emissions; however, which species are targeted and the effect of these NIs on the microbial nitrifier community is still unclear. Here, we identified the ammonia oxidizing bacteria (AOB) species linked to N2 O emissions and evaluated the effects of urea and urea with DCD and DMPP on the nitrifying community in a 258 day field experiment under sugarcane. Using an amoA AOB amplicon sequencing approach and mining a previous dataset of 16S rRNA sequences, we characterized the most likely N2 O-producing AOB as a Nitrosospira spp. and identified Nitrosospira (AOB), Nitrososphaera (archaeal ammonia oxidizer) and Nitrospira (nitrite-oxidizer) as the most abundant, present nitrifiers. The fertilizer treatments had no effect on the alpha and beta diversities of the AOB communities. Interestingly, we found three clusters of co-varying variables with nitrifier operational taxonomic units (OTUs): the N2 O-producing AOB Nitrosospira with N2 O, NO3 - , NH4 + , water-filled pore space (WFPS) and pH; AOA Nitrososphaera with NO3 - , NH4 + and pH; and AOA Nitrososphaera and NOB Nitrospira with NH4 + , which suggests different drivers. These results support the co-occurrence of non-N2 O-producing Nitrososphaera and Nitrospira in the unfertilized soils and the promotion of N2 O-producing Nitrosospira under urea fertilization. Further, we suggest that DMPP is a more effective NI than DCD in tropical soil under sugarcane.

High-Level Abundances of Methanobacteriales and Syntrophobacterales May Help To Prevent Corrosion of Metal Sheet Piles.

Iron sheet piles are widely used in flood protection, dike construction, and river bank reinforcement. Their corrosion leads to gradual deterioration and often makes replacement necessary. Natural deposit layers on these sheet piles can prevent degradation and significantly increase their life span. However, little is known about the mechanisms of natural protective layer formation. Here, we studied the microbially diverse populations of corrosion-protective deposit layers on iron sheet piles at the Gouderak pumping station in Zuid-Holland, the Netherlands. Deposit layers, surrounding sediment and top sediment samples were analyzed for soil physicochemical parameters, microbially diverse populations, and metabolic potential. Methanogens appeared to be enriched 18-fold in the deposit layers. After sequencing, metagenome assembly and binning, we obtained four nearly complete draft genomes of microorganisms (Methanobacteriales, two Coriobacteriales, and Syntrophobacterales) that were highly enriched in the deposit layers, strongly indicating a potential role in corrosion protection. Coriobacteriales and Syntrophobacterales could be part of a microbial food web degrading organic matter to supply methanogenic substrates. Methane-producing Methanobacteriales could metabolize iron, which may initially lead to mild corrosion but potentially stimulates the formation of a carbonate-rich protective deposit layer in the long term. In addition, Methanobacteriales and Coriobacteriales have the potential to interact with metal surfaces via direct interspecies or extracellular electron transfer. In conclusion, our study provides valuable insights into microbial populations involved in iron corrosion protection and potentially enables the development of novel strategies for in situ screening of iron sheet piles in order to reduce risks and develop more sustainable replacement practices.IMPORTANCE Iron sheet piles are widely used to reinforce dikes and river banks. Damage due to iron corrosion poses a significant safety risk and has significant economic impact. Different groups of microorganisms are known to either stimulate or inhibit the corrosion process. Recently, natural corrosion-protective deposit layers were found on sheet piles. Analyses of the microbial composition indicated a potential role for methane-producing archaea. However, the full metabolic potential of the microbial communities within these protective layers has not been determined. The significance of this work lies in the reconstruction of the microbial food web of natural corrosion-protective layers isolated from noncorroding metal sheet piles. With this work, we provide insights into the microbiological mechanisms that potentially promote corrosion protection in freshwater ecosystems. Our findings could support the development of screening protocols to assess the integrity of iron sheet piles to decide whether replacement is required.

Moisture Is More Important than Temperature for Assembly of Both Potentially Active and Whole Prokaryotic Communities in Subtropical Grassland.

Moisture and temperature play important roles in the assembly and functioning of prokaryotic communities in soil. However, how moisture and temperature regulate the function of niche- versus neutral-based processes during the assembly of these communities has not been examined considering both the total microbial community and the sole active portion with potential for growth in native subtropical grassland. We set up a well-controlled microcosm-based experiment to investigate the individual and combined effects of moisture and temperature on soil prokaryotic communities by simulating subtropical seasons in grassland. The prokaryotic populations with potential for growth and the total prokaryotic community were assessed by 16S rRNA transcript and 16S rRNA gene analyses, respectively. Moisture was the major factor influencing community diversity and structure, with a considerable effect of this factor on the total community. The prokaryotic populations with potential for growth and the total communities were influenced by the same assembly rules, with the niche-based mechanism being more influential in communities under dry condition. Our results provide new information regarding moisture and temperature in microbial communities of soil and elucidate how coexisting prokaryotic populations, under different physiological statuses, are shaped in native subtropical grassland soil.

Co-Variation of Bacterial and Fungal Communities in Different Sorghum Cultivars and Growth Stages is Soil Dependent.

Rhizosphere microbial community composition can be influenced by different biotic and abiotic factors. We investigated the composition and co-variation of rhizosphere bacterial and fungal communities from two sorghum genotypes (BRS330 and SRN-39) in three different plant growth stages (emergence of the second leaf, (day10), vegetative to reproductive differentiation point (day 35), and at the last visible emerged leaf (day 50)) in two different soil types, Clue field (CF) and Vredepeel (VD). We observed that either bacterial or fungal community had its composition stronger influenced by soil followed by plant growth stage and cultivar. However, the influence of plant growth stage was higher on fungal community composition than on the bacterial community composition. Furthermore, we showed that sorghum rhizosphere bacterial and fungal communities can affect each other's composition and structure. The decrease in relative abundance of the fungus genus Gibberella over plant growth stages was followed by decrease of the bacterial families Oxalobacteracea and Sphingobacteriacea. Although cultivar effect was not the major responsible for bacterial and fungal community composition, cultivar SRN-39 showed to promote a stronger co-variance between bacterial and fungal communities.

Genomic comparison of chitinolytic enzyme systems from terrestrial and aquatic bacteria.

Chitin degradation ability is known for many aquatic and terrestrial bacterial species. However, differences in the composition of chitin resources between aquatic (mainly exoskeletons of crustaceans) and terrestrial (mainly fungal cell walls) habitats may have resulted in adaptation of chitinolytic enzyme systems to the prevalent resources. We screened publicly available terrestrial and aquatic chitinase-containing bacterial genomes for possible differences in the composition of their chitinolytic enzyme systems. The results show significant differences between terrestrial and aquatic bacterial genomes in the modular composition of chitinases (i.e. presence of different types of carbohydrate binding modules). Terrestrial Actinobacteria appear to be best adapted to use a wide variety of chitin resources as they have the highest number of chitinase genes, the highest diversity of associated carbohydrate-binding modules and the highest number of CBM33-type lytic polysaccharide monooxygenases. Actinobacteria do also have the highest fraction of genomes containing ß-1, 3-glucanases, enzymes that may reinforce the potential for degrading fungal cell walls. The fraction of bacterial chitinase-containing genomes encoding polyketide synthases was much higher for terrestrial bacteria than for aquatic ones supporting the idea that the combined production of antibiotics and cell-wall degrading chitinases can be an important strategy in antagonistic interactions with fungi.

Exploring the genomic traits of fungus-feeding bacterial genus Collimonas.

BACKGROUND: Collimonas is a genus belonging to the class of Betaproteobacteria and consists mostly of soil bacteria with the ability to exploit living fungi as food source (mycophagy). Collimonas strains differ in a range of activities, including swimming motility, quorum sensing, extracellular protease activity, siderophore production, and antimicrobial activities. RESULTS: In order to reveal ecological traits possibly related to Collimonas lifestyle and secondary metabolites production, we performed a comparative genomics analysis based on whole-genome sequencing of six strains representing 3 recognized species. The analysis revealed that the core genome represents 43.1 to 52.7% of the genomes of the six individual strains. These include genes coding for extracellular enzymes (chitinase, peptidase, phospholipase), iron acquisition and type II secretion systems. In the variable genome, differences were found in genes coding for secondary metabolites (e.g. tripropeptin A and volatile terpenes), several unknown orphan polyketide synthase-nonribosomal peptide synthetase (PKS-NRPS), nonribosomal peptide synthetase (NRPS) gene clusters, a new lipopeptide and type III and type VI secretion systems. Potential roles of the latter genes in the interaction with other organisms were investigated. Mutation of a gene involved in tripropeptin A biosynthesis strongly reduced the antibacterial activity against Staphylococcus aureus, while disruption of a gene involved in the biosynthesis of the new lipopeptide had a large effect on the antifungal/oomycetal activities. CONCLUSIONS: Overall our results indicated that Collimonas genomes harbour many genes encoding for novel enzymes and secondary metabolites (including terpenes) important for interactions with other organisms and revealed genomic plasticity, which reflect the behaviour, antimicrobial activity and lifestylesof Collimonas spp.

Revisiting the dilution procedure used to manipulate microbial biodiversity in terrestrial systems.

It is hard to assess experimentally the importance of microbial diversity in soil for the functioning of terrestrial ecosystems. An approach that is often used to make such assessment is the so-called dilution method. This method is based on the assumption that the biodiversity of the microbial community is reduced after dilution of a soil suspension and that the reduced diversity persists after incubation of more or less diluted inocula in soil. However, little is known about how the communities develop in soil after inoculation. In this study, serial dilutions of a soil suspension were made and reinoculated into the original soil previously sterilized by gamma irradiation. We determined the structure of the microbial communities in the suspensions and in the inoculated soils using 454-pyrosequencing of 16S rRNA genes. Upon dilution, several diversity indices showed that, indeed, the diversity of the bacterial communities in the suspensions decreased dramatically, with Proteobacteria as the dominant phylum of bacteria detected in all dilutions. The structure of the microbial community was changed considerably in soil, with Proteobacteria, Bacteroidetes, and Verrucomicrobia as the dominant groups in most diluted samples, indicating the importance of soil-related mechanisms operating in the assembly of the communities. We found unique operational taxonomic units (OTUs) even in the highest dilution in both the suspensions and the incubated soil samples. We conclude that the dilution approach reduces the diversity of microbial communities in soil samples but that it does not allow accurate predictions of the community assemblage during incubation of (diluted) suspensions in soil.

Soil microbiome responses to the short-term effects of Amazonian deforestation.

Slash-and-burn clearing of forest typically results in increase in soil nutrient availability. However, the impact of these nutrients on the soil microbiome is not known. Using next generation sequencing of 16S rRNA gene and shotgun metagenomic DNA, we compared the structure and the potential functions of bacterial community in forest soils to deforested soils in the Amazon region and related the differences to soil chemical factors. Deforestation decreased soil organic matter content and factors linked to soil acidity and raised soil pH, base saturation and exchangeable bases. Concomitant to expected changes in soil chemical factors, we observed an increase in the alpha diversity of the bacterial microbiota and relative abundances of putative copiotrophic bacteria such as Actinomycetales and a decrease in the relative abundances of bacterial taxa such as Chlamydiae, Planctomycetes and Verrucomicrobia in the deforested soils. We did not observe an increase in genes related to microbial nutrient metabolism in deforested soils. However, we did observe changes in community functions such as increases in DNA repair, protein processing, modification, degradation and folding functions, and these functions might reflect adaptation to changes in soil characteristics due to forest clear-cutting and burning. In addition, there were changes in the composition of the bacterial groups associated with metabolism-related functions. Co-occurrence microbial network analysis identified distinct phylogenetic patterns for forest and deforested soils and suggested relationships between Planctomycetes and aluminium content, and Actinobacteria and nitrogen sources in Amazon soils. The results support taxonomic and functional adaptations in the soil bacterial community following deforestation. We hypothesize that these microbial adaptations may serve as a buffer to drastic changes in soil fertility after slash-and-burning deforestation in the Amazon region.

Soil-borne microbiome: linking diversity to function.

Soil microorganisms are sensitive to environment disturbances, and such alterations have consequences on microbial diversity and functions. Our hypothesis is that alpha diversity of microbial communities and functional diversity decrease from undisturbed to disturbed soils, with consequences for functional redundancy in the soil ecosystem. To test this hypothesis, we used soil DNA shotgun metagenomics approach to assess the soil microbiome in a chronosequence of land-use from a native tropical forest, followed by deforestation and cultivation of soybean croplands and pasture in different seasons. Agriculture and pasture soils were among the most diverse and presented higher functional redundancy, which is important to maintain the ecosystem functioning after the forest conversion. On the other hand, the ecosystem equilibrium in forest is maintained based on a lower alpha diversity but higher abundance of microorganisms. Our results indicate that land-use change alters the structure and composition of microbial communities; however, ecosystem functionality is overcome by different strategies based on the abundance and diversity of the communities.

Biosynthetic genes and activity spectrum of antifungal polyynes from Collimonas fungivorans†Ter331.

The antifungal activity of bacteria from the genus Collimonas has been well documented, but the chemistry and gene functions that underlie this phenotype are still poorly understood. Screening of a random plasposon insertion library of Collimonas fungivorans Ter331 for loss-of-function mutants revealed the importance of gene cluster K, which is annotated to code for the biosynthesis of a secondary metabolite and which features genes for fatty acid desaturases and polyketide synthases. Mutants in gene cluster K had lost the ability to inhibit hyphal growth of the fungus Aspergillus niger and were no longer able to produce and secrete several metabolites that after extraction and partial purification from wildtype strain Ter331 were shown to share a putative ene-triyne moiety. Some but not all of these metabolites were able to inhibit growth of A. niger, indicating functional variation within this group of Collimonas-produced polyyne-like 'collimomycins'. Polymerase chain reaction analysis of isolates representing different Collimonas species indicated that the possession of cluster K genes correlated positively with antifungal ability, further strengthening the notion that this cluster is involved in collimomycin production. We discuss our findings in the context of other bacterially produced polyynes and the potential use of collimomycins for the control of harmful fungi.

Soil-borne microbial functional structure across different land uses.

Land use change alters the structure and composition of microbial communities. However, the links between environmental factors and microbial functions are not well understood. Here we interrogated the functional structure of soil microbial communities across different land uses. In a multivariate regression tree analysis of soil physicochemical properties and genes detected by functional microarrays, the main factor that explained the different microbial community functional structures was C : N ratio. C : N ratio showed a significant positive correlation with clay and soil pH. Fields with low C : N ratio had an overrepresentation of genes for carbon degradation, carbon fixation, metal reductase, and organic remediation categories, while fields with high C : N ratio had an overrepresentation of genes encoding dissimilatory sulfate reductase, methane oxidation, nitrification, and nitrogen fixation. The most abundant genes related to carbon degradation comprised bacterial and fungal cellulases; bacterial and fungal chitinases; fungal laccases; and bacterial, fungal, and oomycete polygalacturonases. The high number of genes related to organic remediation was probably driven by high phosphate content, while the high number of genes for nitrification was probably explained by high total nitrogen content. The functional gene diversity found in different soils did not group the sites accordingly to land management. Rather, the soil factors, C : N ratio, phosphate, and total N, were the main factors driving the differences in functional genes across the fields examined.

Shifting carbon flow from roots into associated microbial communities in response to elevated atmospheric CO2.

Rising atmospheric CO(2) levels are predicted to have major consequences on carbon cycling and the functioning of terrestrial ecosystems. Increased photosynthetic activity is expected, especially for C-3 plants, thereby influencing vegetation dynamics; however, little is known about the path of fixed carbon into soil-borne communities and resulting feedbacks on ecosystem function. Here, we examine how arbuscular mycorrhizal fungi (AMF) act as a major conduit in the transfer of carbon between plants and soil and how elevated atmospheric CO(2) modulates the belowground translocation pathway of plant-fixed carbon. Shifts in active AMF species under elevated atmospheric CO(2) conditions are coupled to changes within active rhizosphere bacterial and fungal communities. Thus, as opposed to simply increasing the activity of soil-borne microbes through enhanced rhizodeposition, elevated atmospheric CO(2) clearly evokes the emergence of distinct opportunistic plant-associated microbial communities. Analyses involving RNA-based stable isotope probing, neutral/phosphate lipid fatty acids stable isotope probing, community fingerprinting, and real-time PCR allowed us to trace plant-fixed carbon to the affected soil-borne microorganisms. Based on our data, we present a conceptual model in which plant-assimilated carbon is rapidly transferred to AMF, followed by a slower release from AMF to the bacterial and fungal populations well-adapted to the prevailing (myco-)rhizosphere conditions. This model provides a general framework for reappraising carbon-flow paths in soils, facilitating predictions of future interactions between rising atmospheric CO(2) concentrations and terrestrial ecosystems.

A review on the impact of domestication of the rhizosphere of grain crops and a perspective on the potential role of the rhizosphere microbial community for sustainable rice crop production.

The rhizosphere-associated microbiome impacts plant performance and tolerance to abiotic and biotic stresses. Despite increasing recognition of the enormous functional role of the rhizomicrobiome on the survival of wild plant species growing under harsh environmental conditions, such as nutrient, water, temperature, and pathogen stresses, the utilization of the rhizosphere microbial community in domesticated rice production systems has been limited. Better insight into how this role of the rhizomicrobiome for the performance and survival of wild plants has been changed during domestication and development of present domesticated crops, may help to assess the potential of the rhizomicrobial community to improve the sustainable production of these crops. Here, we review the current knowledge of the effect of domestication on the microbial rhizosphere community of rice and other crops by comparing its diversity, structure, and function in wild versus domesticated species. We also examine the existing information on the impact of the plant on their physico-chemical environment. We propose that a holobiont approach should be explored in future studies by combining detailed analysis of the dynamics of the physicochemical microenvironment surrounding roots to systematically investigate the microenvironment-plant-rhizomicrobe interactions during rice domestication, and suggest focusing on the use of beneficial microbes (arbuscular mycorrhizal fungi and Nitrogen fixers), denitrifiers and methane consumers to improve the sustainable production of rice.

Comparison of methane metabolism in the rhizomicrobiomes of wild and related cultivated rice accessions reveals a strong impact of crop domestication.

Microbial communities from rhizosphere (rhizomicrobiomes) have been significantly impacted by domestication as evidenced by a comparison of the rhizomicrobiomes of wild and related cultivated rice accessions. While there have been many published studies focusing on the structure of the rhizomicrobiome, studies comparing the functional traits of the microbial communities in the rhizospheres of wild rice and cultivated rice accessions are not yet available. In this study, we used metagenomic data from experimental rice plots to analyze the potential functional traits of the microbial communities in the rhizospheres of wild rice accessions originated from Africa and Asia in comparison with their related cultivated rice accessions. The functional potential of rhizosphere microbial communities involved in alanine, aspartate and glutamate metabolism, methane metabolism, carbon fixation pathways, citrate cycle (TCA cycle), pyruvate metabolism and lipopolysaccharide biosynthesis pathways were found to be enriched in the rhizomicrobiomes of wild rice accessions. Notably, methane metabolism in the rhizomicrobiomes of wild and cultivated rice accessions clearly differed. Key enzymes involved in methane production and utilization were overrepresented in the rhizomicrobiome samples obtained from wild rice accessions, suggesting that the rhizomicrobiomes of wild rice maintain a different ecological balance for methane production and utilization compared with those of the related cultivated rice accessions. A novel assessment of the impact of rice domestication on the primary metabolic pathways associated with microbial taxa in the rhizomicrobiomes was performed. Results indicated a strong impact of rice domestication on methane metabolism; a process that represents a critical function of the rhizosphere microbial community of rice. The findings of this study provide important information for future breeding of rice varieties with reduced methane emission during cultivation for sustainable agriculture.

Defensive properties of pyrrolizidine alkaloids against microorganisms.

The understanding of the selection factors that drive chemical diversification of secondary metabolites of constitutive defence systems in plants, such as pyrrolizidine alkaloids (PAs), is still incomplete. Historically, plants always have been confronted with microorganisms. Long before herbivores existed on this planet, plants had to cope with microbial pathogens. Therefore, plant pathogenic microorganisms may have played an important role in the early evolution of the secondary metabolite diversity. In this review, we discuss the impact that plant-produced PAs have on plant-associated microorganisms. The objective of the review is to present the current knowledge on PAs with respect to anti-microbial activities, adaptation and detoxification by microorganisms, pathogenic fungi, root protection and PA induction. Many in vitro experiments showed effects of PAs on microorganisms. These results point to the potential of microorganisms to be important for the evolution of PAs. However, only a few in vivo studies have been published and support the results of the in vitro studies. In conclusion, the topics pointed out in this review need further exploration by carrying out ecological experiments and field studies.

Facilitation in the soil microbiome does not necessarily lead to niche expansion.

BACKGROUND: The soil microbiome drives soil ecosystem function, and soil microbial functionality is directly linked to interactions between microbes and the soil environment. However, the context-dependent interactions in the soil microbiome remain largely unknown. RESULTS: Using latent variable models (LVMs), we disentangle the biotic and abiotic interactions of soil bacteria, fungi and environmental factors using the Qinghai-Tibetan Plateau soil ecosystem as a model. Our results show that soil bacteria and fungi not only interact with each other but also shift from competition to facilitation or vice versa depending on environmental variation; that is, the nature of their interactions is context-dependent. CONCLUSIONS: Overall, elevation is the environmental gradient that most promotes facilitative interactions among microbes but is not a major driver of soil microbial community composition, as evidenced by variance partitioning. The larger the tolerance of a microbe to a specific environmental gradient, the lesser likely it is to interact with other soil microbes, which suggests that facilitation does not necessarily lead to niche expansion.

Reduction of rare soil microbes modifies plant-herbivore interactions.

Rare species are assumed to have little impact on community interactions and ecosystem processes. However, very few studies have actually attempted to quantify the role of rare species in ecosystems. Here we compare effects of soil community assemblages on plant-herbivore interactions and show that reduction of rare soil microbes increases both plant biomass and plant nutritional quality. Two crop plant species growing in soil where rare microbes were reduced, had tissues of higher nutritional quality, which theoretically makes them more susceptible to pest organisms such as shoot-feeding aphids and root-feeding nematodes. Reduction of rare microbes increased aphid body size in the absence of nematodes; nematodes always reduced aphid body size independent of the soil microbial community. This study is the first to show that rare soil microbes are not redundant but may play a role in crop protection by enhancing aboveground and belowground plant defence. It remains to be tested whether these are direct effects of rare soil microbes on plants and herbivores, or indirect effects via shifts in the microbial soil community assemblages.

Comparative analysis of acidobacterial genomic fragments from terrestrial and aquatic metagenomic libraries, with emphasis on acidobacteria subdivision 6.

The bacterial phylum Acidobacteria has a widespread distribution and is one of the most common and diverse phyla in soil habitats. However, members of this phylum have often been recalcitrant to cultivation methods, hampering the study of this presumably important bacterial group. In this study, we used a cultivation-independent metagenomic approach to recover genomic information from soilborne members of this phylum. A soil metagenomic fosmid library was screened by PCR targeting acidobacterial 16S rRNA genes, facilitating the recovery of 17 positive clones. Recovered inserts appeared to originate from a range of Acidobacteria subdivisions, with dominance of subdivision 6 (10 clones). Upon full-length insert sequencing, gene annotation identified a total of 350 open reading frames (ORFs), representing a broad range of functions. Remarkably, six inserts from subdivision 6 contained a region of gene synteny, containing genes involved in purine de novo biosynthesis and encoding tRNA synthetase and conserved hypothetical proteins. Similar genomic regions had previously been observed in several environmental clones recovered from soil and marine sediments, facilitating comparisons with respect to gene organization and evolution. Comparative analyses revealed a general dichotomy between marine and terrestrial genes in both phylogeny and G+C content. Although the significance of this homologous gene cluster across subdivision 6 members is not known, it appears to be a common feature within a large percentage of all acidobacterial genomic fragments recovered from both of these environments.

Downward vascular translocation of a green fluorescent protein-tagged strain of Dickeya sp. (Biovar 3) from stem and leaf inoculation sites on potato.

Translocation of a green fluorescent protein (GFP)-tagged Dickeya sp. from stems or from leaves to underground parts of potato plants was studied in greenhouse experiments. Thirty days after stem inoculation, 90% of plants expressed symptoms at the stem base and 95% of plants showed browning of internal stem tissue. The GFP-tagged Dickeya sp. was detected by dilution plating in extracts of the stem interiors (100%), stem bases (90%), roots (80%), stolons (55%), and progeny tubers (24%). In roots, the GFP-tagged Dickeya sp. was found inside and between parenchyma cells whereas, in stems and stolons, the GFP-tagged Dickeya sp. was found in the xylem vessels and protoxylem cells. In progeny tubers, this strain was detected in the stolon end. Thirty days after leaf inoculation, the GFP-tagged Dickeya sp. was detected in extracts of 75% of the leaves, 88% of the petioles, 63% of the axils, and inside 25% of the stems taken 15 cm above the ground level. UV microscopy confirmed the presence of the GFP-tagged Dickeya sp. inside petioles and in the main leaf veins. No blackleg or aerial stem rot and no translocation of the GFP-tagged Dickeya sp. to underground plant parts was observed. The implications for contamination of progeny tubers are discussed.

The analysis of pyrrolizidine alkaloids in Jacobaea vulgaris; a comparison of extraction and detection methods.

INTRODUCTION: Pyrrolizidine alkaloids (PAs) serve an important function in plant defence.Objective - To compare different extraction methods and detection techniques, namely gas chromatography with nitrogen phosphorus detection (GC-NPD) and liquid chromatography tandem mass spectrometry (LC-MS/MS) with quadrupole analysers for analysing PAs in Jacobaea vulgaris. METHODOLOGY: Both formic acid and sulfuric acid were tested for PA extraction from dry plant material. For GC-NPD, reduction is required to transform PA N-oxides into tertiary amines. Zinc and sodium metabisulfite were compared as reducing agents. RESULTS: The lowest PA concentration measured with GC-NPD was approximately 0.03 mg/g and with LC-MS/MS 0.002 mg/g. The detection of major PAs by both techniques was comparable but a number of minor PAs were not detected by GC-NPD. With the LC-MS/MS procedure higher concentrations were found in plant extracts, indicating that losses may have occurred during the sample preparation for the GC-NPD method. Zinc proved a more effective reducing agent than sodium metabisulfite. The sample preparation for LC-MS/MS analysis using formic acid extraction without any reduction and purification steps is far less complex and less time consuming compared to GC-NPD analysis with sulfuric acid extraction and PA N-oxide reduction with zinc and purification. CONCLUSIONS: In terms of sensitivity and discrimination, formic acid extraction in combination with LC-MS/MS detection is the method of choice for analysing PAs (both free and N-oxides forms) in plant material.