A fluorescence‐activated cell sorting‐based strategy for rapid isolation of high‐lipid Chlamydomonas mutants

There is significant interest in farming algae for the direct production of biofuels and valuable lipids. Chlamydomonas reinhardtii is the leading model system for studying lipid metabolism in green algae, but current methods for isolating mutants of this organism with a perturbed lipid content are slow and tedious. Here, we present the Chlamydomonas high‐lipid sorting (CHiLiS) strategy, which enables enrichment of high‐lipid mutants by fluorescence‐activated cell sorting (FACS) of pooled mutants stained with the lipid‐sensitive dye Nile Red. This method only takes 5 weeks from mutagenesis to mutant isolation. We developed a staining protocol that allows quantification of lipid content while preserving cell viability. We improved separation of high‐lipid mutants from the wild type by using each cell's chlorophyll fluorescence as an internal control. We initially demonstrated 20‐fold enrichment of the known high‐lipid mutant sta1 from a mixture of sta1 and wild‐type cells. We then applied CHiLiS to sort thousands of high‐lipid cells from a pool of about 60 000 mutants. Flow cytometry analysis of 24 individual mutants isolated by this approach revealed that about 50% showed a reproducible high‐lipid phenotype. We further characterized nine of the mutants with the highest lipid content by flame ionization detection and mass spectrometry lipidomics. All mutants analyzed had a higher triacylglycerol content and perturbed whole‐cell fatty acid composition. One arbitrarily chosen mutant was evaluated by microscopy, revealing larger lipid droplets than the wild type. The unprecedented throughput of CHiLiS opens the door to a systems‐level understanding of green algal lipid biology by enabling genome‐saturating isolation of mutants in key genes.     

Structural analysis of chloroplast tail‐anchored membrane protein recognition by ArsA1

In mammals and yeast, tail‐anchored (TA) membrane proteins destined for the post‐translational pathway are safely delivered to the endoplasmic reticulum (ER) membrane by a well‐known targeting factor, TRC40/Get3. In contrast, the underlying mechanism for translocation of TA proteins in plants remains obscure. How this unique eukaryotic membrane‐trafficking system correctly distinguishes different subsets of TA proteins destined for various organelles, including mitochondria, chloroplasts and the ER, is a key question of long standing. Here, we present crystal structures of algal ArsA1 (the Get3 homolog) in a distinct nucleotide‐free open state and bound to adenylyl‐imidodiphosphate. This approximately 80‐kDa protein possesses a monomeric architecture, with two ATPase domains in a single polypeptide chain. It is capable of binding chloroplast (TOC34 and TOC159) and mitochondrial (TOM7) TA proteins based on features of its transmembrane domain as well as the regions immediately before and after the transmembrane domain. Several helices located above the TA‐binding groove comprise the interlocking hook‐like motif implicated by mutational analyses in TA substrate recognition. Our data provide insights into the molecular basis of the highly specific selectivity of interactions of algal ArsA1 with the correct sets of TA substrates before membrane targeting in plant cells.     

Sun‐induced fluorescence – a new probe of photosynthesis: First maps from the imaging spectrometer HyPlant

Variations in photosynthesis still cause substantial uncertainties in predicting photosynthetic CO₂ uptake rates and monitoring plant stress. Changes in actual photosynthesis that are not related to greenness of vegetation are difficult to measure by reflectance based optical remote sensing techniques. Several activities are underway to evaluate the sun‐induced fluorescence signal on the ground and on a coarse spatial scale using space‐borne imaging spectrometers. Intermediate‐scale observations using airborne‐based imaging spectroscopy, which are critical to bridge the existing gap between small‐scale field studies and global observations, are still insufficient. Here we present the first validated maps of sun‐induced fluorescence in that critical, intermediate spatial resolution, employing the novel airborne imaging spectrometer HyPlant. HyPlant has an unprecedented spectral resolution, which allows for the first time quantifying sun‐induced fluorescence fluxes in physical units according to the Fraunhofer Line Depth Principle that exploits solar and atmospheric absorption bands. Maps of sun‐induced fluorescence show a large spatial variability between different vegetation types, which complement classical remote sensing approaches. Different crop types largely differ in emitting fluorescence that additionally changes within the seasonal cycle and thus may be related to the seasonal activation and deactivation of the photosynthetic machinery. We argue that sun‐induced fluorescence emission is related to two processes: (i) the total absorbed radiation by photosynthetically active chlorophyll; and (ii) the functional status of actual photosynthesis and vegetation stress.     

Next‐generation sequencing (NGS)‐based identification of induced mutations in a doubly mutagenized tomato (Solanum lycopersicum) population

The identification of mutations in targeted genes has been significantly simplified by the advent of TILLING (Targeting Induced Local Lesions In Genomes), speeding up the functional genomic analysis of animals and plants. Next‐generation sequencing (NGS) is gradually replacing classical TILLING for mutation detection, as it allows the analysis of a large number of amplicons in short durations. The NGS approach was used to identify mutations in a population of Solanum lycopersicum (tomato) that was doubly mutagenized by ethylmethane sulphonate (EMS). Twenty‐five genes belonging to carotenoids and folate metabolism were PCR‐amplified and screened to identify potentially beneficial alleles. To augment efficiency, the 600‐bp amplicons were directly sequenced in a non‐overlapping manner in Illumina MiSeq, obviating the need for a fragmentation step before library preparation. A comparison of the different pooling depths revealed that heterozygous mutations could be identified up to 128‐fold pooling. An evaluation of six different software programs (camba , crisp , gatk unified genotyper , lofreq , snver and vipr ) revealed that no software program was robust enough to predict mutations with high fidelity. Among these, crisp and camba predicted mutations with lower false discovery rates. The false positives were largely eliminated by considering only mutations commonly predicted by two different software programs. The screening of 23.47 Mb of tomato genome yielded 75 predicted mutations, 64 of which were confirmed by Sanger sequencing with an average mutation density of 1/367 Kb. Our results indicate that NGS combined with multiple variant detection tools can reduce false positives and significantly speed up the mutation discovery rate.     

Photosynthetic activity and proteomic analysis highlights the utilization of atmospheric CO2 by Ulva prolifera (Chlorophyta) for rapid growth

Free‐floating Ulva prolifera is one of the causative species of green tides. When green tides occur, massive mats of floating U. prolifera thalli accumulate rapidly in surface waters with daily growth rates as high as 56%. The upper thalli of the mats experience environmental changes such as the change in carbon source, high salinity, and desiccation. In this study, the photosynthetic performances of PSI and PSII in U. prolifera thalli exposed to different atmospheric carbon dioxide (CO₂) levels were measured. Changes in photosynthesis within salinity treatments and dehydration under different CO₂ concentrations were also analyzed. The results showed that PSII activity was enhanced as CO₂ increased, suggesting that CO₂ assimilation was enhanced and U. prolifera thalli can utilize CO₂ in the atmosphere directly, even when under moderate stress. In addition, changes in the proteome of U. prolifera in response to salt stress were investigated. Stress‐tolerance proteins appeared to have an important role in the response to salinity stress, whereas the abundance of proteins related to metabolism showed no significant change under low salinity treatments. These findings may be one of the main reasons for the extremely high growth rate of free‐floating U. prolifera when green tides occur.     

NPQ(T): a chlorophyll fluorescence parameter for rapid estimation and imaging of non‐photochemical quenching of excitons in photosystem‐II‐associated antenna complexes

In photosynthesis, light energy is absorbed by light‐harvesting complexes and used to drive photochemistry. However, a fraction of absorbed light is lost to non‐photochemical quenching (NPQ) that reflects several important photosynthetic processes to dissipate excess energy. Currently, estimates of NPQ and its individual components (q_E, q_I, q_Z and q_T) are measured from pulse‐amplitude‐modulation (PAM) measurements of chlorophyll fluorescence yield and require measurements of the maximal yield of fluorescence in fully dark‐adapted material (F_m), when NPQ is assumed to be negligible. Unfortunately, this approach requires extensive dark acclimation, often precluding widespread or high‐throughput use, particularly under field conditions or in imaging applications, while introducing artefacts when F_m is measured in the presence of residual photodamaged centres. To address these limitations, we derived and characterized a new set of parameters, NPQ_(T), and its components that can be (1) measured in a few seconds, allowing for high‐throughput and field applications; (2) does not require full relaxation of quenching processes and thus can be applied to photoinhibited materials; (3) can distinguish between NPQ and chloroplast movements; and (4) can be used to image NPQ in plants with large leaf movements. We discuss the applications benefits and caveats of both approaches.     

Plant growth‐promoting effects of native Pseudomonas strains on Mentha piperita (peppermint): an in vitro study

Plant growth‐promoting rhizobacteria (PGPR) affect growth of host plants through various direct and indirect mechanisms. Three native PGPR (Pseudomonas putida) strains isolated from rhizospheric soil of a Mentha piperita (peppermint) crop field near Córdoba, Argentina, were characterised and screened in vitro for plant growth‐promoting characteristics, such as indole‐3‐acetic acid (IAA) production, phosphate solubilisation and siderophore production, effects of direct inoculation on plant growth parameters (shoot fresh weight, root dry weight, leaf number, node number) and accumulation and composition of essential oils. Each of the three native strains was capable of phosphate solubilisation and IAA production. Only strain SJ04 produced siderophores. Plants directly inoculated with the native PGPR strains showed increased shoot fresh weight, glandular trichome number, ramification number and root dry weight in comparison with controls. The inoculated plants had increased essential oil yield (without alteration of essential oil composition) and biosynthesis of major essential oil components. Native strains of P. putida and other PGPR have clear potential as bio‐inoculants for improving productivity of aromatic crop plants. There have been no comparative studies on the role of inoculation with native strains on plant growth and secondary metabolite production (specially monoterpenes). Native bacterial isolates are generally preferable for inoculation of crop plants because they are already adapted to the environment and have a competitive advantage over non‐native strains.     

Quantification of plant surface metabolites by matrix‐assisted laser desorption–ionization mass spectrometry imaging: glucosinolates on Arabidopsis thaliana leaves

The localization of metabolites on plant surfaces has been problematic because of the limitations of current methodologies. Attempts to localize glucosinolates, the sulfur‐rich defense compounds of the order Brassicales, on leaf surfaces have given many contradictory results depending on the method employed. Here we developed a matrix‐assisted laser desorption–ionization (MALDI) mass spectrometry protocol to detect surface glucosinolates on Arabidopsis thaliana leaves by applying the MALDI matrix through sublimation. Quantification was accomplished by spotting glucosinolate standards directly on the leaf surface. The A. thaliana leaf surface was found to contain approximately 15 nmol of total glucosinolate per leaf with about 50 pmol mm^?2 on abaxial (bottom) surfaces and 15–30 times less on adaxial (top) surfaces. Of the major compounds detected, 4‐methylsulfinylbutylglucosinolate, indol‐3‐ylmethylglucosinolate, and 8‐methylsulfinyloctylglucosinolate were also major components of the leaf interior, but the second most abundant glucosinolate on the surface, 4‐methylthiobutylglucosinolate, was only a trace component of the interior. Distribution on the surface was relatively uniform in contrast to the interior, where glucosinolates were distributed more abundantly in the midrib and periphery than the rest of the leaf. These results were confirmed by two other mass spectrometry‐based techniques, laser ablation electrospray ionization and liquid extraction surface analysis. The concentrations of glucosinolates on A. thaliana leaf surfaces were found to be sufficient to attract the specialist feeding lepidopterans Plutella xylostella and Pieris rapae for oviposition. The methods employed here should be easily applied to other plant species and metabolites.     

Direct analysis of pollen fitness by flow cytometry: implications for pollen response to stress

Sexual reproduction in flowering plants depends on the fitness of the male gametophyte during fertilization. Because pollen development is highly sensitive to hot and cold temperature extremes, reliable methods to evaluate pollen viability are important for research into improving reproductive heat stress (HS) tolerance. Here, we describe an approach to rapidly evaluate pollen viability using a reactive oxygen species (ROS) probe dichlorodihydrofluorescein diacetate (i.e. H₂DCFDA‐staining) coupled with flow cytometry. In using flow cytometry to analyze mature pollen harvested from Arabidopsis and tomato flowers, we discovered that pollen distributed bimodally into ‘low‐ROS’ and ‘high‐ROS’ subpopulations. Pollen germination assays following fluorescence‐activated cell sorting revealed that the high‐ROS pollen germinated with a frequency that was 35‐fold higher than the low‐ROS pollen, supporting a model in which a significant fraction of a flower's pollen remains in a low metabolic or dormant state even after hydration. The ability to use flow cytometry to quantify ROS dynamics within a large pollen population was shown by dose‐dependent alterations in DCF‐fluorescence in response to oxidative stress or antioxidant treatments. HS treatments (35°C) increased ROS levels, which correlated with a ~60% reduction in pollen germination. These results demonstrate the potential of using flow cytometry‐based approaches to investigate metabolic changes during stress responses in pollen.     

Reverse genetics in complex multigene operons by co‐transformation of the plastid genome and its application to the open reading frame previously designated psbN

Reverse genetics approaches have contributed enormously to the elucidation of gene functions in plastid genomes and the determination of structure–function relationships in chloroplast multiprotein complexes. Gene knock‐outs are usually performed by disrupting the reading frame of interest with a selectable marker cassette. Site‐directed mutagenesis is done by placing the marker into the adjacent intergenic spacer and relying on co‐integration of the desired mutation by homologous recombination. These strategies are not applicable to genes residing in large multigene operons or other gene‐dense genomic regions, because insertion of the marker cassette into an operon‐internal gene or into the nearest intergenic spacer is likely to interfere with expression of adjacent genes in the operon or disrupt cis‐elements for the expression of neighboring genes and operons. Here we have explored the possibility of using a co‐transformation strategy to mutate a small gene of unknown function (psbN) that is embedded in a complex multigene operon. Although inactivation of psbN resulted in strong impairment of photosynthesis, homoplasmic knock‐out lines were readily recovered by co‐transformation with a selectable marker integrating >38 kb away from the targeted psbN. Our results suggest co‐transformation as a suitable strategy for the functional analysis of plastid genes and operons, which allows the recovery of unselected homoplasmic mutants even if the introduced mutations entail a significant selective disadvantage. Moreover, our data provide evidence for involvement of the psbN gene product in the biogenesis of both photosystem I and photosystem II. We therefore propose to rename the gene product ‘photosystem biogenesis factor 1′ and the gene pbf1.     

Light‐dependent reversible phosphorylation of the minor photosystem II antenna Lhcb6 (CP24) occurs in lycophytes

Evolution of vascular plants required compromise between photosynthesis and photodamage. We analyzed representative species from two divergent lineages of vascular plants, lycophytes and euphyllophytes, with respect to the response of their photosynthesis and light‐harvesting properties to increasing light intensity. In the two analyzed lycophytes, Selaginella martensii and Lycopodium squarrosum, the medium phase of non‐photochemical quenching relaxation increased under high light compared to euphyllophytes. This was thought to be associated with the occurrence of a further thylakoid phosphoprotein in both lycophytes, in addition to D2, CP43 and Lhcb1‐2. This protein, which showed light intensity‐dependent reversible phosphorylation, was identified in S. martensii as Lhcb6, a minor LHCII antenna subunit of PSII. Lhcb6 is known to have evolved in the context of land colonization. In S. martensii, Lhcb6 was detected as a component of the free LHCII assemblies, but also associated with PSI. Most of the light‐induced changes affected the amount and phosphorylation of the LHCII assemblies, which possibly mediate PSI–PSII connectivity. We propose that Lhcb6 is involved in light energy management in lycophytes, participating in energy balance between PSI and PSII through a unique reversible phosphorylation, not yet observed in other land plants.     

Desiccation‐induced changes in photochemical processes of photosynthesis and spectral reflectance in Nostoc commune (Cyanobacteria,Nostocales) colonies from polar regions

Cyanobacterium Nostoc commune is a species highly resistant against desiccation. In this study, we investigated changes in photochemical processes of photosynthesis and spectral reflectance indices during controlled desiccation of the colonies from Antarctica. In a dehydration process, water potential (WP) reached ?3 MPa and values of potential (F _v/F _m) and effective quantum yields (ΦPSII) of photosystem II were kept to high value until 90% of water was lost from the colony, and these values decreased rapidly by further loss of water. This indicates that the colony loses water mostly from the exopolysaccharidic envelope, not from cells during the initial part of dehydration (relative water content, RWC = 100–10%). Other suggestions of inhibition of photosynthetic processes after 90% loss of water were the increase of the chlorophyll fluorescence parameter F _p/F _s. The F _m′ was higher than F _m in hydrated colonies because of state transition which change energy distribution between PS I and PS II, but decreased to same level as F _m in dehydrated colonies. The Normalized Difference Vegetation Index (NDVI) and Photochemical Reflectance Index (PRI) showed concave‐ and convex‐curvilinear relationship with RWC, respectively. The changes of NDVI values were, however, statistically insignificant. PRI values were predominantly below 0 because of phycobiliprotein involvement. These results were compared with the same species in the Arctic region. This is, according to our best knowledge, the first measurement of changes in spectral reflectance indices during desiccation of cyanobacteria.     

Resistance gene enrichment sequencing (RenSeq) enables reannotation of the NB‐LRR gene family from sequenced plant genomes and rapid mapping of resistance loci in segregating populations

RenSeq is a NB‐LRR (nucleotide binding‐site leucine‐rich repeat) gene‐targeted, Resistance gene enrichment and sequencing method that enables discovery and annotation of pathogen resistance gene family members in plant genome sequences. We successfully applied RenSeq to the sequenced potato Solanum tuberosum clone DM, and increased the number of identified NB‐LRRs from 438 to 755. The majority of these identified R gene loci reside in poorly or previously unannotated regions of the genome. Sequence and positional details on the 12 chromosomes have been established for 704 NB‐LRRs and can be accessed through a genome browser that we provide. We compared these NB‐LRR genes and the corresponding oligonucleotide baits with the highest sequence similarity and demonstrated that ~80% sequence identity is sufficient for enrichment. Analysis of the sequenced tomato S. lycopersicum ‘Heinz 1706’ extended the NB‐LRR complement to 394 loci. We further describe a methodology that applies RenSeq to rapidly identify molecular markers that co‐segregate with a pathogen resistance trait of interest. In two independent segregating populations involving the wild Solanum species S. berthaultii (Rpi‐ber2) and S. ruiz‐ceballosii (Rpi‐rzc1), we were able to apply RenSeq successfully to identify markers that co‐segregate with resistance towards the late blight pathogen Phytophthora infestans. These SNP identification workflows were designed as easy‐to‐adapt Galaxy pipelines.     

Natural products in Glycyrrhiza glabra (licorice) rhizome imaged at the cellular level by atmospheric pressure matrix‐assisted laser desorption/ionization tandem mass spectrometry imaging

The rhizome of Glycyrrhiza glabra (licorice) was analyzed by high‐resolution mass spectrometry imaging and tandem mass spectrometry imaging. An atmospheric pressure matrix‐assisted laser desorption/ionization imaging ion source was combined with an orbital trapping mass spectrometer in order to obtain high‐resolution imaging in mass and space. Sections of the rhizome were imaged with a spatial resolution of 10 μm in the positive ion mode, and a large number of secondary metabolites were localized and identified based on their accurate mass and MS/MS fragmentation patterns. Major tissue‐specific metabolites, including free flavonoids, flavonoid glycosides and saponins, were successfully detected and visualized in images, showing their distributions at the cellular level. The analytical power of the technique was tested in the imaging of two isobaric licorice saponins with a mass difference of only 0.02 Da. With a mass resolving power of 140 000 and a bin width of 5 ppm in the image processing, the two compounds were well resolved in full‐scan mode, and appeared with different distributions in the tissue sections. The identities of the compounds and their distributions were validated in a subsequent MS/MS imaging experiment, thereby confirming their identities and excluding possible analyte interference. The use of high spatial resolution, high mass resolution and tandem mass spectrometry in imaging experiments provides significant information about the biosynthetic pathway of flavonoids and saponins in legume species, combing the spatially resolved chemical information with morphological details at the microscopic level. Furthermore, the technique offers a scheme capable of high‐throughput profiling of metabolites in plant tissues.     

Three‐dimensional (3‐D) fluorescence spectroscopy analysis of the fluorescent dissolved organic matter released by the marine toxic dinoflagellate Alexandrium catenella exposed to metal stress by zinc or lead

We investigated the effects of zinc or lead on growth and on exudation of fluorescent dissolved organic matter (FDOM) by the marine toxic dinoflagellate Alexandrium catenella (Whedon & Kofoid) Balech. The species was exposed to increasing free zinc (1.34 × 10^?7 M–3.98 × 10^?6 M) or lead (5.13 × 10^?9 M–1.82 × 10^?7 M) concentra‐tions. Low metal levels ([Zn²⁺] = 1.34 × 10^?7 M; [Pb²⁺] = 5.13 × 10^?9 M) had no effect on cell growth. Toxic effects were observed from higher metal contamination ([Zn²⁺] = 3.98 × 10^?6 M; [Pb²⁺] = 6.54 × 10^?8 M), as a conversion of vegetative cells into cysts. Analysis of the released FDOM by three‐dimensional (3‐D) fluorescence spectroscopy was achieved, using the parallel factor analysis (PARAFAC). The PARAFAC modeling revealed four components associated with two contributions: one related to the biological activity; the other linked to the organic matter decomposition in the culture medium. The C1 component combined a tryptophan peak and characteristics of humic substances, whereas the C2 component was considered as a tryptophan protein fluorophore. The two others C3 and C4 components were associated with marine organic matter production. Relea‐sed fluorescent substances were induced by low ([Zn²⁺]= 1.34 × 10^?7 M; [Pb²⁺] = 5.13 × 10^?9 M) and moderate ([Zn²⁺] = 6.21 × 10^?7 M; [Pb²⁺] = 2.64× 10^?9 M) metal concentrations, suggesting the activation of cellular mechanisms in response to metal stress, to exudate FDOM that could complex metal cations and reduce their toxicity toward A. catenella cells.     

Nitrogen and carbon contents and δ15N and δ13C signatures in six bryophyte species: assessment of long‐term deposition changes (1980–2010) in Spanish beech forests

In this study we used recent (2010) and herbarium material (1980) of six bryophyte species to assess long‐term atmospheric deposition in natural forested areas in northern Spain. For this purpose, tissue nitrogen and carbon content, as well as δ¹³C and δ¹⁵N signatures of samples of Hypnum cupressiforme, Polytrichastrum formosum, Leucobryum juniperoideum, Rhytidiadelphus loreus, Homalothecium lutescens and Diplophyllum albicans were analysed and comparisons made between years and species. In addition, the usefulness of each of the six species was evaluated. The range of values observed was similar to that in other studies carried out in rural areas. Significantly lower values were found in 2010 for N (H. cupressiforme), δ¹⁵N (R. loreus and D. albicans), C (R. loreus) and δ¹³C (all except L. juniperoideum). Our natural areas are thus now less influenced by atmospheric pollutants than they were, most probably due to changes in some traditional local activities. Differences were observed between species for all the four parameters studied, so different species must not be analysed together. Finally, R. loreus and H. lutescens seem to be good bioindicators, sensitive even with a few samples, although further studies are needed to corroborate their usefulness.     

Universal fractionation of lignin–carbohydrate complexes (LCCs) from lignocellulosic biomass: an example using spruce wood

It is of both theoretical and practical importance to develop a universally applicable approach for the fractionation and sensitive lignin characterization of lignin–carbohydrate complexes (LCCs) from all types of lignocellulosic biomass, both natively and after various types of processing. In the present study, a previously reported fractionation approach that is applicable for eucalyptus (hardwood) and flax (non‐wood) was further improved by introducing an additional step of barium hydroxide precipitation to isolate the mannan‐enriched LCC (glucomannan‐lignin, GML), in order to suit softwood species as well. Spruce wood was used as the softwood sample. As indicated by the recovery yield and composition analysis, all of the lignin was recovered in three LCC fractions: a glucan‐enriched fraction (glucan‐lignin, GL), a mannan‐enriched fraction (GML) and a xylan‐enriched fraction (xylan‐lignin, XL). All of the LCCs had high molecular masses and were insoluble or barely soluble in a dioxane/water solution. Carbohydrate and lignin signals were observed in ¹H NMR, ¹³C CP‐MAS NMR and normal‐ or high‐sensitivity 2D HSQC NMR analyses. The carbohydrate and lignin constituents in each LCC fraction are therefore believed to be chemically bonded rather than physically mixed with one another. The three LCC fractions were found to be distinctly different from each other in terms of their lignin structures, as revealed by highly sensitive analyses by thioacidolysis‐GC, thioacidolysis‐SEC and pyrolysis‐GC.     

Dispersal of Monochamus galloprovincialis (Col.: Cerambycidae) as recorded by mark–release–recapture using pheromone traps

The spread of the pine wood nematode (PWN), Bursaphelenchus xylophylus (Nematoda; Aphelenchoididae), the causal agent of the pine wilt disease, is greatly constrained to the dispersal of its vectors, long‐horned beetles of the Monochamus genus. Disease spread at global and regional scales has been mainly caused by human‐mediated transport, yet at a local scale, the short‐ and long‐distance dispersal behaviour of the beetles determine colonization dynamics. Three mark–release–recapture experiments using commercial traps and lures allowed the parameterization of the dispersal kernel under two landscape fragmentation scenarios for the only known European PWN vector, Monochamus galloprovincialis. The respective release of 171 and 353 laboratory‐reared beetles in continuous pine stands in 2009 and 2010 resulted in 36% and 28% recapture rates, yet, at a fragmented landscape in 2011, only 2% of the released 473 individuals could be recaptured. Recaptures occurred as soon as 7–14 days after their release, in agreement with the requirement of sexual maturation to respond to the pheromone–kairomone attractants. Data from the first two experiments were fitted to one mechanical and two empirical dispersal models, from which the distance dispersal kernels could be computed. Derived estimated radii enclosing 50% and 99% of dispersing M. galloprovincialis under continuous pine stands ranged between 250–532 m and 2344–3495 m depending on the replicate and choice of model. Forecasted recaptures in 2011 resulted in a moderate underestimation of long‐distance dispersal, probably influenced by the high degree of habitat fragmentation. In addition, trapping parameters such as the effective sampling area (0.57–0.76 ha) or the seasonal sampling range (426–645 m) could be derived. Observed results, derived dispersal kernels and trapping parameters provide valuable information for the integrated pest management of PWD. Furthermore, estimated dispersal distances indicate that ongoing clear‐cut measures for eradication in the European Union are likely ineffective in stopping the vectors dispersal.