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To understand how plants defend themselves against aphids, the transcriptome of several natural Arabidopsis accessions and a T-DNA knockout mutant of the WRKY22 transcription factor were studied.

The new phytologist 213(3), 1346-1362 (2017). doi:10.1111/nph.14220, Published by Wiley-Blackwell, Oxford [u.a.]

Plantago asiatica mosaic virus (PlAMV) is a member of the genus Potexvirus and has an exceptionally wide host range. It causes severe damage to lilies. Here we report on the complete nucleotide sequences of two new Japanese PlAMV isolates, one from the eudicot weed Viola grypoceras (PlAMV-Vi), and the other from the eudicot shrub Nandina domestica Thunb. (PlAMV-NJ). Their genomes contain five open reading frames (ORFs), which is characteristic of potexviruses. Surprisingly, the isolates showed only 76.0-78.0 % sequence identity with each other and with other PlAMV isolates, including isolates from Japanese lily and American nandina. Amino acid alignments of the replicase coding region encoded by ORF1 showed that the regions between the methyltransferase and helicase domains were less conserved than other regions, with several insertions and/or deletions. Phylogenetic analyses of the full-length nucleotide sequences revealed a moderate correlation between phylogenetic clustering and the original host plants of the PlAMV isolates. This study revealed the presence of two highly divergent PlAMV isolates in Japan.

Two laboratory performance studies with 21 and 11 participants were carried out for passive sampling of nonpolar chemicals in water, using silicone samplers that were deployed for 7 wk and 13 wk at 2 river sites in the Netherlands. Target analytes were polychlorinated biphenyls, polycyclic aromatic hydrocarbons (PAHs), polybrominated diphenyl ethers (PBDEs), hexachlorobutadiene, hexachlorobenzene, and a number of performance reference compounds (PRCs). Calculation of aqueous concentrations based on prescribed input values and a prescribed uptake model was also included. Between-laboratory coefficients of variation (CVs) in the analysis of target compounds were approximately 20% at concentrations of 100ngg-1 and approximately 100% at concentrations of 0.01ngg-1, which was similar to previous results for the analysis of biota samples. The analysis of PRCs yielded water sampling rates with a between-laboratory CV of 18% to 30%. The sampling rate model showed a nearly perfect match with the consensus values of retained PRCs. The implications of the present study for future interlaboratory exercises are discussed.

The 2013 EU ban of three neonicotinoids used in seed coating of pollinator attractive crops was put in place because of concern about declining wild pollinator populations and numbers of honeybee colonies. It was also concluded that there is an urgent need for good field data to fill knowledge gaps. In the meantime such data have been generated. Based on recent literature we question the existence of recent pollinator declines and their possible link with the use of neonicotinoids. Because of temporal non-coincidence we conclude that declines of wild pollinators and of honeybees are not likely caused by neonicotinoids. Even if bee decline does occur and if there is a causal relationship with the use of neonicotinoids, we argue that it is not possible on such short term to evaluate the effects of the 2013 ban. In order to supply future debate with realistic (field) data and to discourage extrapolating the effects of studies using overdoses that are not of environmental relevance, we propose - in addition to field studies performed by the chemical industry - to use the 'semi-field worst case' treated artificial diet studies approach to free flying colonies in the field. This kind of study may provide realistic estimates for risk and be useful to study realistic interactions with non-pesticide stressors.

EU agriculture is currently in transition from conventional crop protection to integrated pest management (IPM). Because biocontrol is a key component of IPM, many European countries recently have intensified their national efforts on biocontrol research and innovation (R&I), although such initiatives are often fragmented. The operational outputs of national efforts would benefit from closer collaboration among stakeholders via transnationally coordinated approaches, as most economically important pests are similar across Europe. This paper proposes a common European framework on biocontrol R&I. It identifies generic R&I bottlenecks and needs as well as priorities for three crop types (arable, vegetable and perennial crops). The existing gap between the market offers of biocontrol solutions and the demand of growers, the lengthy and expensive registration process for biocontrol solutions and their varying effectiveness due to variable climatic conditions and site-specific factors across Europe are key obstacles hindering the development and adoption of biocontrol solutions in Europe. Considering arable, vegetable and perennial crops, a dozen common target pests are identified for each type of crop and ranked by order of importance at European level. Such a ranked list indicates numerous topics on which future joint transnational efforts would be justified.

In western Europe, Pectobacterium carotovorum subsp. brasiliense is emerging as a causal agent of blackleg disease. In field experiments in the Netherlands, the virulence of this pathogen was compared with strains of other Dickeya and Pectobacterium species. In 2013 and 2014, seed potato tubers were vacuum infiltrated with high densities of bacteria (106 CFU mL−1) and planted in clay soil. Inoculation with P. carotovorum subsp. brasiliense and P. atrosepticum resulted in high disease incidences (75–95%), inoculation with D. solani and P. wasabiae led to incidences between 5% and 25%, but no significant disease development was observed in treatments with P. carotovorum subsp. carotovorum, D. dianthicola or the water control. Co-inoculations of seed potatoes with P. carotovorum subsp. brasiliense and D. solani gave a similar disease incidence to inoculation with only P. carotovorum subsp. brasiliense. However, co-inoculation of P. carotovorum subsp. brasiliense with P. wasabiae resulted in a decrease in disease incidence compared to inoculation with only P. carotovorum subsp. brasiliense. In 2015, seed potatoes were inoculated with increasing densities of P. carotovorum subsp. brasiliense, D. solani or P. atrosepticum (103–106 CFU mL−1). After vacuum infiltration, even a low inoculum density resulted in high disease incidence. However, immersion without vacuum caused disease only at high bacterial densities. Specific TaqMan assays were evaluated and developed for detection of P. carotovorum subsp. brasiliense, P. wasabiae and P. atrosepticum and confirmed the presence of these pathogens in progeny tubers of plants derived from vacuum-infiltrated seed tubers.

Highlight The transcription factor WRKY22 increases susceptibility to aphids in Arabidopsis via the suppression of salicylic acid signalling., Aphids induce many transcriptional perturbations in their host plants, but the signalling cascades responsible and the effects on plant resistance are largely unknown. Through a genome-wide association (GWA) mapping study in Arabidopsis thaliana, we identified WRKY22 as a candidate gene associated with feeding behaviour of the green peach aphid, Myzus persicae. The transcription factor WRKY22 is known to be involved in pathogen-triggered immunity, and WRKY22 gene expression has been shown to be induced by aphids. Assessment of aphid population development and feeding behaviour on knockout mutants and overexpression lines showed that WRKY22 increases susceptibility to M. persicae via a mesophyll-located mechanism. mRNA sequencing analysis of aphid-infested wrky22 knockout plants revealed the up-regulation of genes involved in salicylic acid (SA) signalling and down-regulation of genes involved in plant growth and cell-wall loosening. In addition, mechanostimulation of knockout plants by clip cages up-regulated jasmonic acid (JA)-responsive genes, resulting in substantial negative JA–SA crosstalk. Based on this and previous studies, WRKY22 is considered to modulate the interplay between the SA and JA pathways in response to a wide range of biotic and abiotic stimuli. Its induction by aphids and its role in suppressing SA and JA signalling make WRKY22 a potential target for aphids to manipulate host plant defences.

Fusarium head blight (FHB) mainly caused by Fusarium graminearum is a devastating disease that causes extensive yield and quality losses to wheat in humid and semi-humid regions of the world. The biocontrol effect of two bacterial strains on FHB incidence, severity and deoxynivalenol (DON) accumulation in wheat was evaluated in field trials during 2010 and 2011 at Marcos Juarez, Córdoba province, Argentina. Bacillus subtilis RC 218 and Brevibacillus sp. RC 263 applied at anthesis period were evaluated through several combinations of cell type, strains, inoculum density (104 and 106cfu/ml) and physiological modification. A significant and consistent biocontrol effect on FHB severity and DON contamination was observed in all the evaluated treatments during both 2010 and 2011 field trials. Reduction in FHB severity ranged 62-76% and 42-58% for 2010 and 2011 field trials, respectively. When evaluating the effect of the combined strains (104+104 and 106+106cfu/ml), a better biocontrol effect was observed in 2010 field trial. After biocontrol treatments, no DON accumulation was observed in wheat heads; meanwhile in control plots an average of 1372μg/kg DON was detected during the two trials. FHB incidence was significantly reduced by biocontrol treatments during the 2010 field trial but not during the 2011 field trial. The results showed the effectiveness of the two formulated biological control agents in reducing both FHB severity and DON accumulation by F. graminearum under semi controlled field conditions. Fil: Palazzini, Juan Manuel. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas, Fisicoquímicas y Naturales. Departamento de Microbiología e Inmunología. Cátedra de Micología; Argentina Fil: Alberione, Enrique. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Córdoba. Estación Experimental Agropecuaria Marcos Juárez; Argentina Fil: Torres, Adriana Mabel. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas, Fisicoquímicas y Naturales. Departamento de Microbiología e Inmunología. Cátedra de Micología; Argentina Fil: Donat, Christina. Bioferm-gmbh; Austria Fil: Köhl, Jürgen. Wageningen University And Research Centre; Países Bajos Fil: Chulze, Sofia Noemi. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas, Fisicoquímicas y Naturales. Departamento de Microbiología e Inmunología. Cátedra de Micología; Argentina

Since 1992 elm trees have been treated with a biological control product Dutch Trig® to protect them from infection by Ophiostoma novo-ulmi causing Dutch elm disease (DED). The active ingredient of the biocontrol product consists of the fungus Verticillium albo-atrum strain WCS850. A conidiospore suspension of this fungus is injected into the vascular system of elm trees at a height of 1.3 m. This biocontrol product prevents healthy elm trees from fungal infection transmitted by elm bark beetles. Dutch Trig®, however, does not protect already infected trees or trees connected with diseased trees via root grafts. Since 2010, only 0.1 % of the injected elms became infected with DED through beetle transmission and an additional 0.4 % of the treated elms were infected through root contact in the Netherlands. Regression analysis considering all injected elm trees in the Netherlands since 1992 indicated that DED infection through beetle transmission had significantly decreased during the 24 years application of Dutch Trig®. In 2015, approximately 28,300 trees in five countries (Netherlands, USA, Germany, Canada and Sweden) were treated with Dutch Trig® to protect valuable susceptible elm trees, mainly in urban environments.

In augmentative biological control (ABC), invertebrate and microbial organisms are seasonally released in large numbers to reduce pests. Today it is applied on more than 30 million ha worldwide. Europe is the largest commercial market for invertebrate biological control agents, while North America has the largest sales of microbials. A strong growth in use of ABC, particularly of microbial agents, is taking place in Latin America, followed by Asia. The current popularity of ABC is due to (1) its inherent positive characteristics (healthier for farm workers and persons living in farming communities, no harvesting interval or waiting period after release of agents, sustainable as there is no development of resistance against arthropod natural enemies, no phytotoxic damage to plants, better yields and a healthier product, reduced pesticide residues [well below the legal Maximum Residue Levels (MRLs)], (2) professionalism of the biological control industry (inexpensive large scale mass production, proper quality control, efficient packaging, distribution and release methods, and availability of many (>440 species) control agents for numerous pests), (3) a number of recent successes showing how biological control can save agricultural production when pesticides fail or are not available, (4) several non-governmental organizations (NGOs), consumers, and retailers demanding pesticide residues far below the legal MRLs, and (5) policy developments in several regions of the world aimed at reduction and replacement of synthetic pesticides by more sustainable methods of pest management. We are convinced, however, that ABC can be applied on a much larger area than it is today. We plead in the short term for a pragmatic form of agriculture that is adaptable, non-dogmatic and combines the sustainability gain from all types of agriculture and pest management methods. We then propose to move to “conscious agriculture”, which involves participation of all stakeholders in the production and consumer chain, and respects the environment and resource availability for future generations. Were “conscious agriculture” to be considered a serious alternative to conventional farming, ABC would face an even brighter future.

Male sexual aggregations are a common territorial, mating-related or resource-based, behaviour observed in diverse organisms, including insects such as thrips. The influence of factors such as plant substrate, time of day, and geographic location on aggregation of thrips is uncertain, therefore we monitored the dispersion of male and female bean flower thrips (BFT), Megalurothrips sjostedti (Trybom) (Thysanoptera: Thripidae), on cowpea, Vigna unguiculata (L.) Walp. (Fabaceae), over three cowpea growth stages and across three cowpea-growing areas of Kenya. Our results indicated that for all the crop growth stages, the density of BFTs varied over the time of day, with higher densities at 10:00, 13:00, and 16:00 hours than at 07:00 hours. Thrips densities did not differ among blocks at the budding stage, but they did at peak flowering and podding stages. Dispersion indices suggested that both male and female BFTs were aggregated. Active male aggregation occurred only on green plant parts and it varied across blocks, crop stages, and locations. Similarly, active female aggregation was observed in peak flowering and podding stages. Such active aggregation indicates a semiochemical or behaviour-mediated aggregation. Identification of such a semiochemical may offer new opportunities for refining monitoring and management strategies for BFT on cowpea, the most important grain legume in sub-Saharan Africa.

Pink ear rot of maize caused by Fusarium verticillioides, Fusariumproliferatum and Fusariumgraminearum can lead to severe yield losses and contamination of grain with a range of mycotoxins. Maize stalks colonized by Fusarium spp. are the main primary inoculum source for Fusarium incited epidemics in maize or other susceptible crops grown in rotation. The colonization of individual maize stalks originating from fields in The Netherlands, Italy and Nigeria by Fusarium spp. was quantified using specific TaqMan-PCR assays. Highest values were found for F. graminearum and Fusariumavenaceum in Dutch samples, for F. graminearum and FUM producing species (F. verticillioides and F. proliferatum) in Italian samples and FUM producing Fusarium spp. in Nigerian samples. The increase in Fusarium spp. colonization under field conditions during a period of 3-6 months after harvest of the maize crops varied considerably between individual stalks. The fungal and bacterial diversity was analyzed for sub-sets of stalks with high versus low increase of Fusarium colonization. Bacterial taxonomic groups such as Bacillus, Curtobacterium, Erwinia, Flavobacterium, Novosphingobium, Pantoea, Sphingomonas, Rahnella and Staphylococcus and fungal taxonomic groups such as Acremomium sp., Cryptococcus flavescens, Cryptococcus zeae, Leptosphaeria sp. and Microdochium bolleyi were more abundant in the stalks with lower increase in pathogen colonization. Such fungal and bacterial groups associated with 'suppressive stalks' may be antagonistic to Fusarium spp. and a source of candidate strains for the selection of new biological control agents.

International audience; Agriculture in the twenty-first century faces the challenge of meeting food demands while satisfying sustainability goals. The challenge is further complicated by climate change which affects the distribution of crop pests (intended as insects, plants, and pathogenic agents injurious to crops) and the severity of their outbreaks. Increasing concerns over health and the environment as well as new legislation on pesticide use, particularly in the European Union, urge us to find sustainable alternatives to pesticide-based pest management. Here, we review the effect of climate change on crop protection and propose strategies to reduce the impact of future invasive as well as rapidly evolving resident populations. The major points are the following: (1) the main consequence of climate change and globalization is a heightened level of unpredictability of spatial and temporal interactions between weather, cropping systems, and pests; (2) the unpredictable adaptation of pests to a changing environment primarily creates uncertainty and projected changes do not automatically translate into doom and gloom scenarios; (3) faced with uncertainty, policy, research, and extension should prepare for worst-case scenarios following a “no regrets” approach that promotes resilience vis-à-vis pests which, at the biophysical level, entails uncovering what currently makes cropping systems resilient, while at the organizational level, the capacity to adapt needs to be recognized and strengthened; (4) more collective approaches involving extension and other stakeholders will help meet the challenge of developing more robust cropping systems; (5) farmers can take advantage of Web 2.0 and other new technologies to make the exchange of updated information quicker and easier; (6) cooperation between historically compartmentalized experts in plant health and crop protection could help develop anticipation strategies; and (7) the current decline in skilled crop protection specialists in Europe should be reversed, and shortcomings in local human and financial resources can be overcome by pooling resources across borders.

Western flower thrips (WFT), Frankliniella occidentalis (Pergande) (Thysanoptera: Thripidae), is a major pest of horticultural crops worldwide. The search for alternative pest management techniques has resulted in increasing interest in the use of kairomones and other behaviour-modifying chemicals to mitigate the impact of this pest. In this study, we determined whether the origin of populations, feeding history, and/or genotype influence the response of WFT to the thrips kairomone lure methyl isonicotinate (MI) in a Y-tube olfactometer study. Four New Zealand thrips populations were tested: (1) from a commercial glasshouse capsicum crop, (2) from a long-established laboratory colony (>222 generations) kept on chrysanthemums, (3) from a laboratory colony (6-9 generations) kept on French dwarf beans, and (4) thought to be a separate cryptic non-pest species from outdoor yellow tree lupins, Lupinus arboreus Sims (Fabaceae). In the laboratory tests, significantly more WFT from all four populations chose the MI-laden arm of a Y-tube olfactometer when it contained 1 μl methyl isonicotinate (61.3-73.2%) compared with the blank no-odour arm. No differences in response to MI were found between the two laboratory and the one glasshouse WFT populations. Both laboratory populations and the greenhouse population belonged to the 'glasshouse pest' genotype of WFT. However, the cryptic non-pest WFT genotype responded more strongly to MI than any of the other populations, although the response was only significantly stronger than that of the long-established laboratory population. Significant differences were also found among populations in the average time taken for thrips to make a choice to enter either arm of the Y-tube olfactometer, with the cryptic non-pest lupin genotype taking the shortest time, followed by thrips from the capsicum glasshouse. The results are discussed with respect to the variability in olfactory perception and olfactory behaviour within a species and the relevance to the use of such a kairomone lure in pest management programmes.

The number of bees, amount of brood and haemolymph vitellogenin titre are parameters to establish the vitality of a honey bee colony. Increasing numbers of bees during summer until autumn; increasing amounts of brood in spring towards summer followed by a decrease; and low haemolymph vitellogenin titres in foraging bees and high vitellogenin titres in nurse bees and winter bees are typical courses over the bee season. In contrast to the number of bees and amount of brood, determined at the colony level, vitellogenin is determined in individual bees. To turn the factor vitellogenin from the mean of individual data into a colony parameter, a protocol was developed to determine the colony fraction vitellogenin in a pooled haemolymph sample of 30 bees, sampled from a defined location in the colony. This colony fraction vitellogenin is a parameter for honey bee colony vitality.

Zymoseptoria tritici is an economically important pathogen of wheat. However, the molecular basis of pathogenicity on wheat is still poorly understood. Here, we present a global survey of the proteins secreted by this fungus in the apoplast of resistant (cv. Shafir) and susceptible (cv. Obelisk) wheat cultivars after inoculation with reference Z. tritici strain IPO323. The fungal proteins present in apoplastic fluids were analyzed by gel electrophoresis and by data-independent acquisition liquid chromatography/mass spectrometry (LC/MSE) combined with data-dependent acquisition LC–MS/MS. Subsequent mapping mass spectrometry-derived peptide sequence data against the genome sequence of strain IPO323 identified 665 peptides in the MSE and 93 in the LC–MS/MS mode that matched to 85 proteins. The identified fungal proteins, including cell-wall degrading enzymes and proteases, might function in pathogenicity, but the functions of many remain unknown. Most fungal proteins accumulated in cv. Obelisk at the onset of necrotrophy. This inventory provides an excellent basis for future detailed studies on the role of these genes and their encoded proteins during pathogenesis in wheat.

Thrips are small insects that cause direct damage and some transmit viruses to agricultural and horticultural crops and forest trees. Current thrips management requires a significant input of agrichemicals, which creates risks for workers, consumers and the environment. As well, a number of thrips species are of significant quarantine concern. New thrips management strategies are urgently needed to supplement existing tools such as chemical and biological pesticides. For example, semiochemical-based technologies could serve as additional management tools, since they have proved their efficiency at controlling many large insect pests. However, these technologies have been under-exploited for small insects such as thrips. To address this issue, an international network was initiated in 2012, the focus of this Europe Australasian Thrips Semiochemical Network (EATS) is to develop semiochemical-based tools for management of thrips. The progress made in this field is reported in the following paper.

As an alternative to MeBr fumigation a 48h Controlled Atmosphere Temperature Treatment (CATT) was developed and scaled up by Wageningen UR in cooperation with the Dutch plant propagating association Plantum. This results in an excellent de-infestation and 99.8 % mortality of the strawberry tarsonemid mite (Phytonemus pallidus). This non-chemical and sustainable method provides a healthy production of highly qualified strawberry runners in the field. From 2009 CATT is up scaled to a commercial level and widely applied by Dutch nurseries. In 2011 this CATT method was successfully modified to eradicate also the root knot nematode Meloidogyne hapla (>99.7% mortality), which was not effectively controlled by MeBr fumigation. For an effective killing of the root knot nematodes, temperature must be raised to 40 ºC. In several experiments the optimum conditions for a high mortality of both tarsonemids and nematodes was studied. This leads into an adapted CATT of 20 hours at a temperature of 35 ºC and 50 % CO2 followed by 20 hours at a temperature of 40 ºC. In 2012 this adapted CATT was successfully upgraded and tested under field conditions. Additional research in 2013 leads to the conclusion that cross infection of plants by the bacterial Q-disease Xanthomonas fragariae during CATT treatment is unlikely

Apple scab, caused by Venturia inaequalis, is the most important disease in apple production, reducing yield and quality of fruit. Control of apple scab in commercial orchards currently depends on multiple applications of fungicides. The potential of the antagonistic isolate Cladosporium cladosporioides H39, originating from a sporulating colony of V. inaequalis, to control apple scab development was tested in eight trials during 2 years in orchards in Eperjeske (Hungary), Dabrowice (Poland), and Bavendorf (Germany) planted with different cultivars. Treatments were conducted as calendar sprays or after infection periods. Additional trials in an orchard in Randwijk (The Netherlands) focused on the effect of timing of antagonist application before or after infection periods. The overall results of the field trials consistently showed—for the first time—that stand-alone applications of the antagonist C. cladosporioides H39 can reduce apple scab in leaves and fruit. This was demonstrated in an organic growing system as well as in conventional orchards by spray schedules applied during the primary or the summer season. In both systems, the same control levels could be reached as with common fungicide schedules. Efficacies reached 42 to 98% on leaf scab incidence and 41 to 94% on fruit scab. The antagonist was also effective if applied one or even several days (equivalent to approximately 300 to 2,000 degree h) after infection events in several field trials and a trial conducted in Randwijk with single-spray applications at different intervals before or after infection events. Better understanding of the biology of the antagonist will help to further exploit its use in apple scab control.

The aim of our study was to explore whether honeybees (Apis mellifera L.) could be used as a reliable alternative to the standard mechanical devices for monitoring of air quality, in particular with respect to the concentration of the heavy metals cadmium (Cd), lead (Pb) and vanadium (V). We therefore tested whether the concentrations of these metals in adult honeybees and in ambient air were positively correlated, and whether differences in concentration between locations were similar for bees and air. On the basis of our measurements, conducted over a two-month period at three distinct locations in the Netherlands with each three replicate honeybee colonies placed next to mechanical monitoring devices, we concluded that a significant positive relationship between the concentrations in bees and in air could only be established for V. Also, only in the case of V, the differences between the three locations in mean concentration were similar for bees and air. Both outcomes were probably due to the relatively large range over which the concentrations of V varied, both in bees and in air, as compared to Cd and Pb. However, for V, as well as for Cd and Pb, the concentrations in ambient air were about two orders of magnitude below the established air quality standards. We therefore conclude that in the Netherlands, both variation and levels of the atmospheric concentrations of these metals are too low to establish a relationship between the concentration in bees and in air that is useful to present honeybees as an alternative to mechanical devices in monitoring of air pollution. However, in countries with larger variation and higher levels of the atmospheric concentrations of these metals, further exploration of the potential of honeybees in biomonitoring of air pollution may be worthwhile

Fungal plant pathogens, such as Zymoseptoria tritici (formerly known as Mycosphaerella graminicola), secrete repertoires of effectors to facilitate infection or trigger host defence mechanisms. The discovery and functional characterization of effectors provides valuable knowledge that can contribute to the design of new and effective disease management strategies. Here, we combined bioinformatics approaches with expression profiling during pathogenesis to identify candidate effectors of Z.¿tritici. In addition, a genetic approach was conducted to map quantitative trait loci (QTLs) carrying putative effectors, enabling the validation of both complementary strategies for effector discovery. In¿planta expression profiling revealed that candidate effectors were up-regulated in successive waves corresponding to consecutive stages of pathogenesis, contrary to candidates identified by QTL mapping that were, overall, expressed at low levels. Functional analyses of two top candidate effectors (SSP15 and SSP18) showed their dispensability for Z.¿tritici pathogenesis. These analyses reveal that generally adopted criteria, such as protein size, cysteine residues and expression during pathogenesis, may preclude an unbiased effector discovery. Indeed, genetic mapping of genomic regions involved in specificity render alternative effector candidates that do not match the aforementioned criteria, but should nevertheless be considered as promising new leads for effectors that are crucial for the Z.¿tritici–wheat pathosystem

[EN] The Ophioviridae is a family of filamentous plant viruses, with single-stranded negative, and possibly ambisense, RNA genomes of 11.3-12.5 kb divided into 3-4 segments, each encapsidated separately. Virions are naked filamentous nucleocapsids, forming kinked circles of at least two different contour lengths. The sole genus, Ophiovirus, includes seven species. Four ophioviruses are soil-transmitted and their natural hosts include trees, shrubs, vegetables and bulbous or corm-forming ornamentals, both monocots and dicots. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the taxonomy of the Ophioviridae, which is available at http://www.ictv.global/report/ophioviridae., Production of this summary, the online chapter and associated resources was funded by a grant from the Wellcome Trust (WT108418AIA).

Recent major losses of managed honeybee, Apis mellifera, colonies at a global scale have resulted in a multitude of research efforts to identify the underlying mechanisms. Numerous factors acting singly and/or in combination have been identified, ranging from pathogens, over nutrition to pesticides. However, the role of apiculture in limiting natural selection has largely been ignored. This is unfortunate, because honeybees are more exposed to environmental stressors compared to other livestock and management can severely compromise bee health. Here, we briefly review apicultural factors that influence bee health and focus on those most likely interfering with natural selection, which offers a broad range of evolutionary applications for field practice. Despite intense breeding over centuries, natural selection appears to be much more relevant for the health of managed A. mellifera colonies than previously thought. We conclude that sustainable solutions for the apicultural sector can only be achieved by taking advantage of natural selection and not by attempting to limit it. Recent major losses of managed honeybee, Apis mellifera, colonies at a global scale have resulted in a multitude of research efforts to identify the underlying mechanisms. Numerous factors acting singly and/or in combination have been identified, ranging from pathogens, over nutrition to pesticides. However, the role of apiculture in limiting natural selection has largely been ignored. This is unfortunate, because honeybees are more exposed to environmental stressors compared to other livestock and management can severely compromise bee health. Here, we briefly review apicultural factors that influence bee health and focus on those most likely interfering with natural selection, which offers a broad range of evolutionary applications for field practice. Despite intense breeding over centuries, natural selection appears to be much more relevant for the health of managed A. mellifera colonies than previously thought. We conclude that sustainable solutions for the apicultural sector can only be achieved by taking advantage of natural selection and not by attempting to limit it

Background The Fusarium oxysporum species complex (FOSC) contains several phylogenetic lineages. Phylogenetic studies identified two to three major clades within the FOSC. The mitochondrial sequences are highly informative phylogenetic markers, but have been mostly neglected due to technical difficulties. Results A total of 61 complete mitogenomes of FOSC strains were de novo assembled and annotated. Length variations and intron patterns support the separation of three phylogenetic species. The variable region of the mitogenome that is typical for the genus Fusarium shows two new variants in the FOSC. The variant typical for Fusarium is found in members of all three clades, while variant 2 is found in clades 2 and 3 and variant 3 only in clade 2. The extended set of loci analyzed using a new implementation of the genealogical concordance species recognition method support the identification of three phylogenetic species within the FOSC. Comparative analysis of the mitogenomes in the FOSC revealed ongoing mitochondrial recombination within, but not between phylogenetic species. Conclusions The recombination indicates the presence of a parasexual cycle in F. oxysporum. The obstacles hindering the usage of the mitogenomes are resolved by using next generation sequencing and selective genome assemblers, such as GRAbB. Complete mitogenome sequences offer a stable basis and reference point for phylogenetic and population genetic studies.

Two laboratory performance studies with 21 and 11 participants were carried out for passive sampling of nonpolar chemicals in water, using silicone samplers that were deployed for 7 wk and 13 wk at 2 river sites in the Netherlands. Target analytes were polychlorinated biphenyls, polycyclic aromatic hydrocarbons (PAHs), polybrominated diphenyl ethers (PBDEs), hexachlorobutadiene, hexachlorobenzene, and a number of performance reference compounds (PRCs). Calculation of aqueous concentrations based on prescribed input values and a prescribed uptake model was also included. Between-laboratory coefficients of variation (CVs) in the analysis of target compounds were approximately 20% at concentrations of 100 ng g−1 and approximately 100% at concentrations of 0.01 ng g−1, which was similar to previous results for the analysis of biota samples. The analysis of PRCs yielded water sampling rates with a between-laboratory CV of 18% to 30%. The sampling rate model showed a nearly perfect match with the consensus values of retained PRCs. The implications of the present study for future interlaboratory exercises are discussed. Environ Toxicol Chem 2016;9999:1–6

The 2013 EU ban of three neonicotinoids used in seed coating of pollinator attractive crops was put in place because of concern about declining wild pollinator populations and numbers of honeybee colonies. It was also concluded that there is an urgent need for good field data to fill knowledge gaps. In the meantime such data have been generated. Based on recent literature we question the existence of recent pollinator declines and their possible link with the use of neonicotinoids. Because of temporal non-coincidence we conclude that declines of wild pollinators and of honeybees are not likely caused by neonicotinoids. Even if bee decline does occur and if there is a causal relationship with the use of neonicotinoids, we argue that it is not possible on such short term to evaluate the effects of the 2013 ban. In order to supply future debate with realistic (field) data and to discourage extrapolating the effects of studies using overdoses that are not of environmental relevance, we propose - in addition to field studies performed by the chemical industry - to use the 'semi-field worst case' treated artificial diet studies approach to free flying colonies in the field. This kind of study may provide realistic estimates for risk and be useful to study realistic interactions with non-pesticide stressors.

International audience; EU agriculture is currently in transition from conventional crop protection to integrated pest management (IPM). Because biocontrol is a key component of IPM, many European countries recently have intensified their national efforts on biocontrol research and innovation (R&I), although such initiatives are often fragmented. The operational outputs of national efforts would benefit from closer collaboration among stakeholders via transnationally coordinated approaches, as most economically important pests are similar across Europe. This paper proposes a common European framework on biocontrol R&I. It identifies generic R&I bottlenecks and needs as well as priorities for three crop types (arable, vegetable and perennial crops). The existing gap between the market offers of biocontrol solutions and the demand of growers, the lengthy and expensive registration process for biocontrol solutions and their varying effectiveness due to variable climatic conditions and site-specific factors across Europe are key obstacles hindering the development and adoption of biocontrol solutions in Europe. Considering arable, vegetable and perennial crops, a dozen common target pests are identified for each type of crop and ranked by order of importance at European level. Such a ranked list indicates numerous topics on which future joint transnational efforts would be justified.

Mycotoxins including aflatoxins, deoxynivalenol, fumonisins and ochratoxin A are among the main fungal secondary metabolites detected as natural contaminants in South America in different commodities such as peanuts (aflatoxins), cereals (deoxynivalenol and fumonisins) or grapes (ochratoxin A). Different strategies including crop rotation, tillage practices, fungicide application and planting less susceptible cultivars are used in order to reduce the impact of these mycotoxins in both food and feed chains. The development of fungicide resistance in many fungal pathogens as well as rising of public concern on the risks associated with pesticide use led to the search for alternative environmentally friendly methods. Biological control of plant pathogens and toxigenic fungi offers an alternative that can complement chemical control in the frame of an integrated pest management to reduce the impact of mycotoxins in the food and feed chains. The advances made in Argentina on reducing the impact of toxigenic fungi and mycotoxins in peanut, grapes and cereals using the biocontrol strategy are summarised. Native bacteria, yeasts and filamentous fungi have been selected to evaluate them as potential biocontrol agents. Field trials showed that Bacillus subtilis RC 218 and Brevibacillus sp. RC 263 were effective at reducing deoxynivalenol accumulation in wheat. The application of Clonostachys rosea isolates on wheat stubble reduced Fusarium colonisation on the stubble. Bacillus amyloliquefaciens and Microbacterium oleovorans showed good activity to control both Fusarium verticillioides growth and the accumulation of fumonisins at pre-harvest stage in maize. Control of toxigenic Aspergillus flavus and aflatoxin accumulation in peanuts was achieved using a native atoxigenic Aspergillus flavus strain based on competitive exclusion of the toxigenic strains. Kluyveromyces thermotolerans strains were used as biocontrol agents to reduce the impact of Aspergillus section Nigri and ochratoxin A accumulation in grapes. Fil: Chulze, Sofia Noemi. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas, Fisicoquímicas y Naturales. Departamento de Microbiología e Inmunología. Cátedra de Micología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza; Argentina Fil: Palazzini, Juan Manuel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza; Argentina. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas, Fisicoquímicas y Naturales. Departamento de Microbiología e Inmunología. Cátedra de Micología; Argentina Fil: Torres, Adriana Mabel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza; Argentina. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas, Fisicoquímicas y Naturales. Departamento de Microbiología e Inmunología. Cátedra de Micología; Argentina Fil: Barros, Germán Gustavo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza; Argentina. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas, Fisicoquímicas y Naturales. Departamento de Microbiología e Inmunología. Cátedra de Micología; Argentina Fil: Ponsone, Maria Lorena. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza; Argentina. Instituto Nacional de Tecnología Agropecuaria; Argentina Fil: Geisen, R.. Max Rubner-Institut; Alemania Fil: Schmidt Heydt, M.. Max Rubner-Institut; Alemania Fil: Köhl, J.. Wageningen UR. Plant Research International, Wageningen; Países Bajos

Late blight is an important disease in potato that is caused by the oomycete Phytophthora infestans. In the past, Solanum demissum late blight resistance (R) genes were introgressed into cultivated potato (Solanum tuberosum). Eleven of these resistant plants were selected to characterize the virulence spectrum of individual P. infestans isolates and to monitor the dynamics of virulence in P. infestans populations. These plants are referred to as the Mastenbroek and Black differential sets. It has long been assumed that each differential plant contained one single R gene. In the current study and previous studies, however, most Mastenbroek differential plants were shown to harbor multiple R gene(s), which blurs virulence typing of late blight isolates. In order to acquire more accurate virulence profiles, we extended the Mastenbroek differential set with Solanum spp. plants harboring reduced R gene complexity and with plants containing recently identified R genes from related but different Solanum species. In addition, a differential set of ten Genetically Modified (GM) plants harboring single late blight R genes in the same genetic background (Desiree). By analyzing the virulence spectra of recently collected isolates using both newly described differential sets, we found that the GM Desiree differential set was more accurate for isolate virulence typing than the conventional (extended) differential set. Besides, the GM Desiree differential set was shown to be useful as trap plants to isolate novel P. infestans strains and to monitor virulence towards particular R genes in P. infestans populations `on site´. Legislative restrictions are, however, limiting the use of the GM Desiree differential set.

n 2011, two polytunnel greenhouse experiments were conducted on seed production farms, one under conventional conditions in the South-West (Rilland) and the other under organic conditions in the East (Voorst) of the Netherlands, to study transmission routes of Xanthomonas campestris pv. campestris (Xcc), that can result in seed infections of cauliflower plants (Brassica oleracea var. botrytis). Each experiment consisted of three plots. Before head formation, plants in the two outside plots were spray-inoculated with either Xcc strain IPO 3076 or IPO 3078, and plants in the central plot were sprayed with water. A colony of honeybees (Apis mellifera) was placed in each tunnel to provide pollination. At Rilland, incidences of black rot were low, and seed infections were not detected in a plating assay, although peduncles were infected in a high percentage of the plants. At Voorst, black rot symptoms developed in almost all Xcc-inoculated plants, resulting in plant losses. The incidence of plants with infected seeds in the plot inoculated with Xcc IPO 3076 was higher than in the two other plots. Identification of Xcc isolates by BOX-PCR revealed that Xcc IPO 3076 was transmitted to the other plots of the experiment. Xcc IPO 3078, however, was only recovered from plants inoculated with this strain. The routes of seed infection for Xcc and then transmission of the pathogen are discussed

Dit artikel omvat de jaarverslagen van de volgende KNPV-werkgroepen over 2013: Bodempathogenen en bodemmicrobiologie; Fusarium; Oömyceten; Onkruidkunde; Botrytis; Nematoden; Fytobacteriologie; Gewasbescherming en Maatschappelijk Debat; Jongeren.

Dit artikel is een compilatie van het Wageningen-UR PRI rapport 606 ’Factoren die het foerageergedrag van honingbijen bepalen (deel I)’. In dit rapport wordt het haalgedrag van de honingbij beschreven: hoe wordt het bepaald en wat wordt verzameld en hoe. Daarnaast is in het rapport een drachtplantenlijst opgenomen (deel II). Hoe bijen drachten bezoeken is interessant voor de bijenhouder en van wezenlijk belang voor het inzetten van honingbijen voor bestuiving en voor het interpreteren van uitkomsten in studies waarin bijenvolken gebruikt worden voor het aantonen van plantenziekten en milieuverontreinigingen.

Inwinteren is het slotstuk van het bijenjaar. Het is een soort “Grande Finale” na een seizoen hard werken met de bijen. We geven onze bijen voldoende voedsel om de winter goed door te komen en in het voorjaar weer fris en vrolijk aan de slag te kunnen gaan. Gelukkig is het eenvoudig mits er aan enkele voorwaarden voldaan wordt. Waarom winteren we onze bijen in? Simpelweg omdat we de dure wintervoorraad (honing) die de bijen in de zomer opgebouwd hebben geheel of gedeeltelijk afnemen en daar een goedkoop alternatief voor in de plaats geven (suiker)

International audience; Modern agriculture often involves the use of pesticides to protect crops. These substances are harmful to target organisms (pests and pathogens). Nevertheless, they can also damage non-target animals, such as pollinators and entomophagous arthropods. It is obvious that the undesirable side effects of pesticides on the environment should be reduced to a minimum. Western honey bees (Apis mellifera) are very important organisms from an agricultural perspective and are vulnerable to pesticide-induced impacts. They contribute actively to the pollination of cultivated crops and wild vegetation, making food production possible. Of course, since Apis mellifera occupies the same ecological niche as many other species of pollinators, the loss of honey bees caused by environmental pollutants suggests that other insects may experience a similar outcome. Because pesticides can harm honey bees and other pollinators, it is important to register pesticides that are as selective as possible. In this manuscript, we describe a selection of methods used for studying pesticide toxicity/selectiveness towards Apis mellifera. These methods may be used in risk assessment schemes and in scientific research aimed to explain acute and chronic effects of any target compound on Apis mellifera.

International audience; In 2016, the order Mononegavirales was emended through the addition of two new families (Mymonaviridae and Sunviridae), the elevation of the paramyxoviral subfamily Pneumovirinae to family status (Pneumoviridae), the addition of five free-floating genera (Anphevirus, Arlivirus, Chengtivirus, Crustavirus, and Wastrivirus), and several other changes at the genus and species levels. This article presents the updated taxonomy of the order Mononegavirales as now accepted by the International Committee on Taxonomy of Viruses (ICTV).

Black Sigatoka or black leaf streak disease, caused by the Dothideomycete fungus Pseudocercospora fijiensis (previously: Mycosphaerella fijiensis), is the most significant foliar disease of banana worldwide. Due to the lack of effective host resistance, management of this disease requires frequent fungicide applications, which greatly increase the economic and environmental costs to produce banana. Weekly applications in most banana plantations lead to rapid evolution of fungicide-resistant strains within populations causing disease-control failures throughout the world. Given its extremely high economic importance, two strains of P. fijiensis were sequenced and assembled with the aid of a new genetic linkage map. The 74-Mb genome of P. fijiensis is massively expanded by LTR retrotransposons, making it the largest genome within the Dothideomycetes. Melting-curve assays suggest that the genomes of two closely related members of the Sigatoka disease complex, P. eumusae and P. musae, also are expanded. Electrophoretic karyotyping and analyses of molecular markers in P. fijiensis field populations showed chromosome-length polymorphisms and high genetic diversity. Genetic differentiation was also detected using neutral markers, suggesting strong selection with limited gene flow at the studied geographic scale. Frequencies of fungicide resistance in fungicide-treated plantations were much higher than those in untreated wild-type P. fijiensis populations. A homologue of the Cladosporium fulvum Avr4 effector, PfAvr4, was identified in the P. fijiensis genome. Infiltration of the purified PfAVR4 protein into leaves of the resistant banana variety Calcutta 4 resulted in a hypersensitive-like response. This result suggests that Calcutta 4 could carry an unknown resistance gene recognizing PfAVR4. Besides adding to our understanding of the overall Dothideomycete genome structures, the P. fijiensis genome will aid in developing fungicide treatment schedules to combat this pathogen and in improving the efficiency of banana breeding programs., Author Summary Black Sigatoka or black leaf streak disease, caused by the ascomycete fungus Pseudocercospora fijiensis, inflicts huge costs on banana producers, due to crop losses and expenses for disease control. The global banana export trade relies on Cavendish clones that are highly susceptible to P. fijiensis. Sustainable production of the world’s number one fruit requires a better understanding of host resistance and sophisticated management of fungicide resistance in the pathogen. In the P. fijiensis genome sequence we identified an effector that induced an HR-like necrosis on a resistant banana accession but not on a susceptible cultivar. If validated, this assay may be useful for identifying resistance in banana breeding programs. We also used the genomic sequence to develop highly polymorphic molecular markers for analyzing P. fijiensis field populations and identified a strong enrichment (nearly 100%) for fungicide resistance markers in fungicide-treated banana plantations compared to untreated wild-type populations. This rapid evolution of fungicide resistance poses an immediate threat to sustainable banana production.

Background Piercing-sucking insects cause severe damage in crops. Breeding for host-plant resistance can significantly reduce the yield losses caused by these insects, but host-plant resistance is a complex trait that is difficult to phenotype quickly and reliably. Current phenotyping methods mainly focus on labor-intensive and time-consuming end-point measurements of plant fitness. Characterizing insect behavior as a proxy for host-plant resistance could be a promising time-saving alternative to end-point measurements. Results We present a phenotyping platform that allows screening for host-plant resistance against Western flower thrips (WFT, Frankliniella occidentalis (Pergande)) in a parallel two-choice setup using automated video tracking of thrips behavior. The platform was used to establish host-plant preference of WFT with a large plant population of 345 wild Arabidopsis accessions and the method was optimized with two extreme accessions from this population that differed in resistance towards WFT. To this end, the behavior of 88 WFT individuals was simultaneously tracked in 88 parallel two-choice arenas during 8 h. Host-plant preference of WFT was established both by the time thrips spent on either accession and various behavioral parameters related to movement (searching) and non-movement (feeding) events. Conclusion In comparison to 6-day end-point choice assays with whole plants or detached leaves, the automated video-tracking choice assay developed here delivered similar results, but with higher time- and resource efficiency. This method can therefore be a reliable and effective high throughput phenotyping tool to assess host-plant resistance to thrips in large plant populations. Electronic supplementary material The online version of this article (doi:10.1186/s13007-016-0102-1) contains supplementary material, which is available to authorized users.

The plant virus genus Torradovirus was created to contain two new species of whitefly-transmitted, tomato-infecting plant picorna-like viruses: Tomato torrado virus and Tomato marchitez virus. So far, research on torradoviruses has focused mainly on the description of the viruses and the diseases they cause, the determination of viral properties and nucleic acid sequences, and the development of detection methods. Evidence has been found for the transmission of torradoviruses by three whitefly species in a semipersistent, stylet-borne manner.Key concepts included in this chapter:Discovery of TorradovirusesProperties of TorradovirusesTransmissionFuture Research

In June 2008, a surveillance study for metals in honeybees was performed in the Netherlands. Randomly, 150 apiaries were selected. In each apiary, five colonies were sampled. Per apiary, the hive samples were pooled. The apiary sample was analysed for Al, As, Ba, Cd, Co, Cr, Cu, Li, Mn, Mo, Ni, Sb, Se, Sn, Sr, Ti, V and Zn. All metals could be detected in all apiaries. As, Li, Sb, Sn and V were detected in part of the apiaries. The overall picture showed a regional pattern. In apiaries in the east of the Netherlands, Al, Ba, Cr, Mn, Mo, Ni, Se and Ti are found in higher concentrations compared to the west. In-region variation was demonstrated, indicating local effects. The vicinity of the apiaries was mapped afterwards and characterised as land uses of >50 % agricultural area, >50 % wooded area, >50 % urban area and mixed land use within a circle of 28 km2 around the apiary. The results indicated that in apiaries located in >50 % wooded areas, significantly higher concentrations of Al, Ba, Cd, Cr, Cu, Li, Mn, Mo, Ni, Sb, Sr, Ti and Zn were found compared to agricultural, urban and mixed land use areas. We conclude that (1) the ratio between metal concentrations varies per region, demonstrating spatial differences, and (2) there is in-region local variation per metal. The results indicate the impact of land use on metal concentrations in honeybees. For qualitative bioindication studies, regional, local and land use effects should be taken into account.

This chapter provides an overview of the possibilities of genetic modification (GM) potato breeding in general and specifically to combat the most important disease, late blight. Potato plants are vulnerable to a number of pests and diseases. An enigmatic question concerns whether individual Rpi genes can confer sufficient broad-spectrum resistance to impart durability. The presence of Rpi genes or transcripts as determined using molecular markers does not warrant their functional expression. Late blight resistance resources from crossable species can be deployed for intragenic or cisgenic breeding strategies. The chapter presents and discusses a pipeline for cisgenic late blight resistance breeding that was developed at Wageningen UR Plant Breeding. If only two Rpi genes are present, often isolates can be identified that overcome recognition mediated by one Rpi gene but not by the other. The late blight-resistant GM potato which was probably closest to commercialization was “Fortuna”.

Bio-sampling is a function of bio-indication. Bio-indication with honeybee colonies (Apis mellifera L) is where the research fields of environmental technology and apiculture overlap. The honeybees are samplers of the environment by collecting unintentionally and simultaneously, along with nectar, pollen, water and honeydew from the flowers or on the leaves, other matter (in bio-indication terms: target matter) and accumulating this in the colony. Collected target matter, in this thesis heavy metals, the plant pathogens Erwinia pyrifoliae and Erwinia amylovora and the soil pollutant γ-HCH, is collected from the colony by subsampling. Subsampling the honeybee colony is done by taking and killing bees from the hive (sacrificial) or by collecting target matter from the bee’s exterior without killing the bee (non-sacrificial). In environmental technology terms the application of the honeybee colony is a Passive Sampling Method (PSM). In this thesis the possibilities and restrictions of the PSM honeybee colony are explored. Bio-indication is a broad research field with one common factor: a living organism (bio) is applied to record an alteration of the environment (indication). The environment may be small such as a laboratory or big such as an ecosystem. Alterations in the organism may vary from detecting substances foreign to the body to mortality of the organism. In environmental technology the concept Source-Path-Receptor (SPR) is applied to map the route of a pollutant. It describes where in the environment the pollution is, how it moves through the environment and where it ends. This environment is the same environment of all living organisms, ergo also honeybees. Honeybees depend on flowers for their food. In the SPR concept, a flower can be a source, path or receptor. Along with collecting pollen, nectar, water and honeydew, target matter is collected by honeybees. Each honeybee functions as a micro-sampler of target matter in the environment, in this case the flower. Each honeybee is part of a honeybee colony and in fact the honeybee colony is the bio-sampler. The honeybee colony is a superorganism. The well-being of the colony prevails over the individual honeybee. Food collection is directed by the colony’s need. Foragers are directed to the most profitable food sources by the bee dance and food exchange (trophallaxis). The result of this feature is that mainly profitable sources are exploited and poor food sources less or not at all. During the active foraging period hundreds to thousands of flowers are visited daily. The nectar, pollen, water and honeydew plus the unintentionally collected target matter is accumulated in the honeybee colony. In order to obtain target matter the colony must be subsampled. This is done by picking bees from the hive-entrance (hive-entering bees) or inside the hive (in-hive bees) and processing them for analysis (sacrificial). This is the most commonly applied method. However, it is possible to subsample the colony without picking and processing the bees by collecting target matter from the hive-entering bee’s exterior (non-sacrificial). For non-sacrificial subsampling of the honeybee colony the Beehold device with the sampling part Beehold tube has been developed. The results of bio-indication with honeybee colonies are qualitative and indicative for follow up study (Chapter 1). Six bio-indication studies with honeybee colonies for bio-indication of heavy metals, the plant pathogens Erwinia pyrifoliae and Erwinia amylovora and the soil pollutant γ-HCH are presented. Chapter 2 describes how the concentration of eighteen heavy metals in honeybees fluctuate throughout the period of July, August and September (temporal) at the study sites: the city of Maastricht, the urban location with an electricity power plant in Buggenum and along the Nieuwe Waterweg at Hoek van Holland (spatial). A number of the metals have not been previously analysed in honeybees. To study whether honeybees can be used for bio-indication of air pollution, the concentrations of cadmium, vanadium and lead were compared to concentrations found in honeybees. The honeybee colonies were placed next to the air samplers. Only significant differences of metal concentrations in the ambient air also show in honeybees. This was the case with vanadium in ambient air and honeybees. The spatial and temporal differences of cadmium and lead were too futile to demonstrate a correspondence (Chapter 3). In a national surveillance study in 2008 the concentration of eighteen metals in honeybees has been analysed. The results showed a distinct regional pattern. Honeybees in the East of the Netherlands have higher concentrations of heavy metals compared to the bees in the West. Besides regional differences local differences were also recorded. An approximate description of the land use around 148 apiaries (> 50% agriculture, > 50% wooded area, > 50% urban area and mixed use) indicated the impact of land use on metal concentrations in honeybees. In areas with > 50% wood significantly higher concentrations of heavy metals were detected (Chapter 4). Subsampling of the honeybee colonies in Chapter 2, 3 and 4 was done sacrificially. In the studies presented in Chapter 5, 6, and 7 the honeybee colonies were subsampled non-sacrificially or simultaneously non-sacrificially and sacrificially. The plant pathogen E. pyrifoliae causes a flower infection in the strawberry cultivation in greenhouses. In greenhouse strawberry cultivation honeybees are applied for pollination. In Chapter 5 the combination pollination / bio-indication by honeybee colonies is studied. This proved to be a match. E. pyrifoliae could be detected on in-hive bees prior to any symptom of the infection in the flowers. In the Beehold tube, the bacterium was detected at the same time as the first tiny symptoms of the infection. In Chapter 5 the principles on which the Beehold tube is based are presented and discussed. The plant pathogen E. amylovora causes fireblight in orchards. The combination pollination / bio-indication has also been applied in this study performed in Austria in 2013. It is known that E. amylovora can be detected on honeybees prior to any symptom in the flower or on the fruit tree. A fireblight outbreak depends on flowering period, humidity and temperature. In 2013 no fireblight infection emerged in the orchards where the study was performed. Therefore, the bacterium could not be detected on the honeybees. γ-HCH (Lindane) is one of the soil pollutants in the Bitterfeld region in Saxony-Anhalt in Germany. It is the result of dumping industrial waste around the production locations. Although γ-HCH is bound to soil particles there is a flux to groundwater and surface water. Consequently, the pollution may end up in the sediments of the streambed and flood plains. The study objective was to investigate the hypothetic route of γ-HCH from polluted soil (source), via soil erosion and atmospheric deposition (route) to the receptor (flowering flowers) by detecting γ-HCH in the Beehold tube. Although on average over 17000 honeybees passed through the Beehold tube daily for a maximal period of 28 days, no γ-HCH has been detected. The pollen pattern in the Beehold tube revealed where the bees collected the food (Chapter 7). The application of the honeybee colony has pros and cons. Distinctive pros are many micro samplers, the extensive collection of matter (both food and target matter) and the accumulation in the colony. For successful bio-indication with honeybee colonies, determining factors are: the target matter, location of the target matter, distance between target matter and the honeybee colony, individual or pooled subsampling, the minimal sampling frequency and sample size, and sacrificial or non-sacrificial subsampling applied solely or in combination. Taking bees from a colony impacts upon the colony’s performance and consequently the passive sampling method. Based on a long-years’ experience and inter-collegial discussion it is stated that 3% of the forager bees (hive-entering) and 1.5% of the in-hive bees can be sampled safely without impacting upon the colony. This restriction does not apply when carrying out non-sacrificial subsampling of the honeybee colony (Chapter 8). Performing bio-indication with honeybee colonies has more applications than have been exploited so far. Further research can make a change. In particular I mention here the combination of pollination and bio-indication and the application of non-sacrificial subsampling solely or in combination with sacrificial subsampling. Everywhere Apiculture is practiced (all over the world except the polar areas) bio-indication with honeybee colonies can be applied in a simple, practical and low cost way.

De rol van de bodem als schatkamer voor nieuwe, nuttigeen natuurlijke producten wordt opnieuw bevestigddoor de ontdekking van nog onbekende genen inLysobacter-bacteriën. Onderzoek van Wageningen URen NIOO toont aan dat deze genen of genclusters waarschijnlijkcoderen voor nog onbekende anti-microbiëlestoffen.