Plant Research International Research Articles

First report of Ralstonia pseudosolanacearum causing bacterial wilt on Rosa sp. in Greece.

In March 2021, a sample of nine-month-old, non-grafted, diseased rose (Rosa sp.) plants was sent by a grower to the Benaki Phytopathological Institute for examination. The plants exhibited symptoms of dieback with black necrosis of pruned shoots, brown discoloration of shoot and root vascular tissues, and whitish slime exudation on cutting wounds of the shoots. The symptoms resembled those caused by Ralstonia pseudosolanacearum (Tjou-Tam-Sin et al. 2016). According to the sample's information sheet, the sample had been collected in a commercial greenhouse rose crop for cut flowers with a 10% disease incidence in the area of Troizinia-Methana (Regional Unit of Islands, Greece). Microscopic examination of symptomatic shoot and root vascular tissues revealed masses of bacterial cells streaming out of them. Sections of symptomatic tissues were suspended in water and in the resulting suspension, bacteria of the R. solanacearum species complex (RSSC) were detected by an indirect immunofluorescence (IF) assay using polyclonal antibodies (Plant Research International, the Netherlands) and a qPCR assay (RS-I-F/RS-II-R primers, RSP-55T probe) (Vreeburg et al. 2016). Furthermore, colonies with typical characteristics of RSSC were isolated from vascular tissues of shoots and roots on non-selective (NA) and semi-selective (mSMSA) media (EPPO 2022), and their identification as RSSC was confirmed by the above-mentioned IF and qPCR assays. Also, the isolates were assigned to: i) biovar 3, based on their ability to metabolize three disaccharides (maltose, lactose, D(+) cellobiose) and three hexose alcohols (mannitol, sorbitol, dulcitol) producing acid (EU 2006) and ii) phylotype I, by multiplex conventional PCR (Opina et al. 1997; Fegan and Prior 2005). A representative isolate was selected for sequencing part of the genes: 16S rDNA (1464bp), mutS (729bp) and egl (795bp) with GenBank Accession Nos. OR102443, OR683617 and OR702781, respectively. Blast analysis of these sequences showed 100% identity with those of various RSSC strains (e.g. GenBank Ac. Nos. CP025741.1, CP021762.1, MF141029.1, respectively). The obtained egl sequence conforms with the characteristics of phylotype I based on the DNA barcoding tool (EPPO 2021) and is 100% identical to that of the Dutch strain PD7216 (MF141029.1) reported to be sequevar I-33 (Bergsma-Vlami et al. 2018). The pathogenicity of two isolates was tested by inoculating: i) tomato seedlings (cv. 'Belladona') at their stem between the cotyledons and the first true leaf (EU 2006) and b) rose plants (cv. 'Aqua' and 'Papa Meilland') at their shoot base (Tjou-Tam-Sin et al. 2016), with bacterial suspensions in water (108 cfu/ml). The inoculated plants were maintained at a day/night temperature about 28/20°C with tomato plants exhibiting leaf wilting (7-17 dpi) and rose plants exhibiting chlorosis and necrosis of leaves (17 dpi). The pathogen was re-isolated on mSMSA from both artificially infected plant species and identified by the IF assay described above, thus fulfilling Koch's postulates. This is the first diagnosis in Greece of: i) rose plants infected by a Ralstonia species and ii) a crop infected by R. solanacearum phylotype I that corresponds to the R. pseudosolanacearum species (EPPO 2022). Official phytosanitary measures imposed in the affected area include an annual survey of rose crops for the presence of this pathogen, aiming at an early detection and prevention of its spread in such a highly valued ornamental crop.

First Report of the Occurrence of the Bacterial Spot Disease of Stone Fruits Caused by Xanthomonas arboricola pv. pruni on Almond in Greece.

In 2021, two samples of almond (Prunus dulcis (Mill) Webb) shoots with symptoms resembling those caused by Xanthomonas arboricola pv. pruni (Xap), were examined at the Benaki Phytopathological Institute. The first sample was collected in June from a 0.4-ha orchard of fifteen-year-old almond trees (cv. 'Texas') with 40% disease incidence, in the Regional Unit of Serres (Northern Greece). Leaves exhibited, mainly at their tip and margins, small, angular, necrotic spots with chlorotic halo, often coalesced into larger necrotic lesions which fell out leaving leaves with a 'shot-hole' like appearance. Fruits displayed dark brown, sunken, corky, gum oozing lesions and shoots developed dark brown, elongated, slightly sunken lesions. Bacterial streaming from the marginal areas of necrotic lesions was observed microscopically. On the lesions of fruits, leaves and shoots, Xap was detected by immunofluorescence assay (IF) using polyclonal antibodies (Plant Research International, the Netherlands) and two qPCR assays (Garita-Cambronero et al. 2017; Palacio-Bielsa et al. 2011). Eight Xanthomonas-like isolates obtained on the SP agar (Hayward 1960) and Nutrient agar (Schaad et al. 2001) culture media were Gram-negative, oxidase negative, strictly aerobic, sensitive to 0.1% w/v TTC, hydrolysing gelatin and Tween 80 but not starch, and also inducing hypersensitive response in tomato plants, as expected for Xap (Schaad et al. 2001). Isolates' identification was confirmed by the IF and the two qPCR assays cited above, as well as a conventional PCR (Pothier et al., 2011). Infiltration of a suspension (107 cfu/ml) of one isolate into five leaves of a two-year-old almond tree cv. 'Texas', and also into five detached leaves from the same tree (Randhawa and Civerolo 1985), caused necrotic spots on all inoculated leaves (10 inoculation sites/leaf), after a four day incubation period at 25oC under high humidity. The Xap reference strain NCPPB 3877 and sterile water were used as positive and negative controls, respectively. The pathogen was reisolated from necrotic spots of the inoculated leaves and identified by IF and two qPCR assays, as previously. The second sample was collected by a grower in September from a 3.7-ha orchard of five-year-old almond trees (cv. 'Tuono') exhibiting 50% disease incidence, in the Regional Unit of Fthiotida (Central Greece). Leaves and fruits showed symptoms similar to those described for the first sample, except that, lesions on fruits, which were at a stage of advanced mesocarp dehydration, were raised. Five Xap isolates were obtained from symptomatic leaves and fruits, and their pathogenicity on almond was confirmed, as in the first sample. Furthermore, sequences of PCR products using primers targeting the 16S-rDNA (Lane 1991;Lane et al., 1985), gyrB (Parkinson et al. 2007) and ftsX (Pothier et al. 2011) genes of two Xap isolates, one from fruit- and one from leaf-necrotic lesions of the first sample, were searched against the NCBI GenBank database, revealing that the obtained sequences of 16S-rRNA (OP412487; OP412488), gyrB (OP467593; OP467594) and ftsX (OP467595; OP467596) genes were 100% identical to the corresponding genomic regions of the Xap strains IVIA 2626.1 (CP076628.1) and CITA 33 (CP076701.1). This is the first report on the presence of Xap in Greece. As these Xap outbreaks have occurred in regions with extensive almond cultivation, a crop of great economic importance for Greece, measures for its eradication have already been advised.

Genotype by Environment interactions for egg number and egg weight of five dual-purpose chicken breeds in different zones of Oromia region in Ethiopia

The African Chicken Genetic Gain (ACGG) project (https://africacgg.net/) aims at backyard poultry optimization by commercial dual-purpose breeds introduction into Africa. To measure benefits, genotype by environment interaction (GxE) analysis provides guidance while predicting environmental effects on production traits of breeds. A survey among Ethiopian poultry smallholders showed egg sale being the most important purpose of keeping village chickens in Oromia. Data was available about laying of 894 ACGG chickens in Oromia. Hence current research questions were: 1) Does GxE take place? 2) Which breed performs best regarding laying and in which environment within Oromia? Traits investigated were egg number and egg weight of five breeds (S-RIR, Sasso, Horro, Kuroiler and Koekoek) located in three zones (East Hararge, East and West Shoa) and 5 districts (Adami Tulu, Bako Tibe, Dano, Dugda and Haromaya) in Oromia. Observations were taken as group measure performing weighted analyses. GxE was only present for egg number with magnitude strongest for zone. S-RIR performed best for both traits in both environments, except Kuroiler performing better in East Shoa for egg number and Koekoek for egg weight. This indicates success of crossbreed S-RIR. Sasso and Horro performed worst supported by previous research for Horro but not Sasso. Low precipitation in East Shoa caused bigger distance in egg number predictions, being higher for S-RIR and lower for Horro and Sasso compared to West Shoa. Apart from these final conclusions, social context of breeding and data collection difficulties should not be forgotten. Just like relevance of other performance trait analyses.

An Experiment In Breeding Cut Roses For The Indonesian Highlands

BIOBREES is a co-operative scientific programme between Indonesia and The Netherlands, of which the rose part aims at breeding cut roses adapted to the tropical highland. Within that scope four direct plant characters of 62 cut rose genotypes in different physiological stages were studied. The genotypes originated from cross-breeding in 1995 and successive selection for cut rose properties in populations at Plant Research International, Wageningen, The Netherlands, in 1996. Plant stages stuAn died were (i) superior adult seedlings in Wageningen in 1996, (ii) clones grafted onto Natal Briar both in Wageningen and Cipanas in 1997. Clonal Plant Research International, Wageningen, Plant Research International, Wageningen, clonal plants in Cipanas had significantly shorter shoots, more thorns, smaller flowers and fewer petals than the adult seedlings in Wageningen. Clonal plants in Wageningen, however, had significantly longer shoots; more thorns, and larger flowers with more petals than the seedlings in Wageningen. Despite differences in level of expression, for each character the absence of genotype-location interaction was ascertained. The expression of characters as influenced by light (both quantitative and qualitative), temperature and cultivation is discussed in relation to selection of cut roses in the temperate zone, which are adapted to the tropical highland.

REVIEW ON BROWN ROT (RALSTONIA SOLANACEARUM RACE 3, BIOVAR 2, PHYLOTYPE IIB) EPIDEMIOLOGY AND CONTROL IN THE NETHERLANDS SINCE 1995: A SUCCESS STORY OF INTEGRATED PEST MANAGEMENT

SUMMARY The disease brown rot of potato, caused by the bacterium Ralstonia (Pseudomonas) solanacearum (Rsol), Race 3, biovar 2 (R3b2), was found for the first time in 1992 in a potato field in The Netherlands and caused an outbreak in the warm summer of 1995 that appeared to be connected to use of contaminated irrigation water as in other outbreaks in western Europe at that time. The Dutch Plant Protection Service (PPS) immediately took action and started to test all traded seed for (latent) infections, applied strict control measures upon positive detections, and started an intensive survey of surface water contamination. In later years the control measures and testing procedures for Rsol in different substrates, laid down in an EU Directive, were followed. The PPS also conducted intensive applied research (in cooperation with the University of Wageningen, Plant Research International, and foreign research Institutions, also in the framework of an EU-SMT project on the EU brown rot testing method to unravel the complex epidemiology of the pathogen and to improve its detection and identification. These actions have led over a ca. 15-year period to a drastic reduction, actually a functional eradication (only one single finding in 2009/2010 and 2010/2011), of the disease from the production system. A main factor in the combat was a nation-wide irrigation ban for seed potatoes since 2005. The PPS coordinated two EU-funded projects, where the UK, The Netherlands, Belgium and France assisted Egypt in implementing a brown rot safe potato production system. In this project substantial epidemiological research was conducted. This article wants to be a reflection on the work done and the results obtained by the PPS and has a glance into the future, where it will be indicated that the persisting presence of Rsol in surface water necessitates an enduring alert and actively maintained control and survey system. The main lessons learned are: stay away, if

First Report of Xanthomonas arboricola pv. pruni in Ornamental Prunus laurocerasus in the Netherlands.

In 2008, Dutch ornamental plant growers observed a leaf spot of cherry laurel (Prunus laurocerasus) at a greater incidence (5 to 50%) than the usual sporadic level (<1%). For advice on disease control, ~5 to 10% of these growers contacted Dutch regulatory officials. In November and December 2008, six symptomatic samples from northern and southern parts of the Netherlands were submitted for diagnosis. Leaf spots were chlorotic, most had a necrotic brown center with a distinct margin, and the spots readily abscised, resulting in a "shot-hole" appearance. Leaf spots from the samples were surface sterilized (2 s in 70% vol/vol alcohol), blotted dry on tissue paper, chopped into pieces (1 to 2 mm in diameter), and incubated for 30 min in 10 mM phosphate-buffered saline (PBS) (1). A 20-μl aliquot of extract per sample was streaked by dilution plating on four plates of yeast peptone glucose agar medium (1), and the plates were incubated for 2 to 3 days at 28°C. Isolations from all six samples yielded Xanthomonas-like colonies. After purification, characterization of all six isolates revealed oxidative, nonfermentative metabolism of glucose by rod-shaped, gram-negative bacterial cells. All six isolates were identified as Xanthomonas arboricola pv. pruni based on biochemical tests (1), fatty acid analysis (4), and immunofluorescence (IF) using polyclonal antibodies (Plant Research International, the Netherlands). Pathogenicity was tested on potted peach plants (cvs. Peregrine and Vaes Oogst) and on detached leaves of P. laurocerasus (cv. Novita) (1). The six field isolates from 2008 were each inoculated (108 CFU/ml) onto four leaves per plant of each of two peach plants (replicates). As positive control treatments, two reference strains (ATCC 19312 and PD740) were each inoculated onto the same number of leaves and plants, and as a noninoculated negative control treatment, leaves of two peach plants were treated with sterile 10 mM PBS buffer (1). All leaves inoculated with the six field isolates and the two reference strains developed typical bacterial spot symptoms in 3 to 4 weeks. Negative control plants showed no symptoms. The detached leaf assay performed with the same treatments on each of two leaves (replicates) showed identical results. The bacterium was reisolated from leaf spots associated with each of the eight symptomatic treatments and identity of the reisolates was confirmed by IF. Additionally, genotypic variation of 35 Dutch isolates of X. arboricola pv. pruni was assessed by BOX-PCR assay with the BOX A1R primer set (3), and Gyrase B gene sequencing (2). Both methods revealed 100% homology among the 35 isolates, suggesting a single, recent introduction of X. arboricola pv. pruni into the Netherlands. In a 2009 survey to assess distribution of the disease in the Netherlands, X. arboricola pv. pruni was found in 41 fields. Infected hosts included P. laurocerasus cvs. Otto Luyken, Rotundifolia, Novita, Etna, Anbri, Herbergii, Mischeana, and Caucasia. X. arboricola pv. pruni is a quarantine organism in countries affiliated under the EPPO (European and Mediterranean Plant Protection Organization). Phytosanitary measures were taken to prevent movement of infested plants from nurseries where X. arboricola pv. pruni was detected.

PRI-CAT: a web-tool for the analysis, storage and visualization of plant ChIP-seq experiments

Although several tools for the analysis of ChIP-seq data have been published recently, there is a growing demand, in particular in the plant research community, for computational resources with which such data can be processed, analyzed, stored, visualized and integrated within a single, user-friendly environment. To accommodate this demand, we have developed PRI-CAT (Plant Research International ChIP-seq analysis tool), a web-based workflow tool for the management and analysis of ChIP-seq experiments. PRI-CAT is currently focused on Arabidopsis, but will be extended with other plant species in the near future. Users can directly submit their sequencing data to PRI-CAT for automated analysis. A QuickLoad server compatible with genome browsers is implemented for the storage and visualization of DNA-binding maps. Submitted datasets and results can be made publicly available through PRI-CAT, a feature that will enable community-based integrative analysis and visualization of ChIP-seq experiments. Secondary analysis of data can be performed with the aid of GALAXY, an external framework for tool and data integration. PRI-CAT is freely available at http://www.ab.wur.nl/pricat. No login is required.

QBOL: a new EU project focusing on DNA barcoding of Quarantine organisms

In 2009 a new three year EU funded project (QBOL) started on DNA barcoding of important plant pests. An international consortium of 20 partners (universities, research institutes, and phytosanitary organizations) from around the world, coordinated by Plant Research International (Wageningen, the Netherlands), will collect DNA barcodes from many plant pathogenic quarantine organisms, store these sequences in a database accessible over the internet, develop a DNA bank and train end‐users. All these activities should help National Plant Protection Services in the correct identification and detection of plant pathogenic quarantine organisms.

Biotechnologizing Jatropha for local sustainable development

This article explores whether and how the bio- technologization process that the fuel-plant Jatropha curcas is undergoing might strengthen local sustainable develop- ment. It focuses on the ongoing efforts of the multi-stake- holder network Gota Verde to harness Jatropha within local small-scale production systems in Yoro, Honduras. It also looks at the genomics research on Jatropha conducted by the Dutch research institute Plant Research International, specifically addressing the ways in which that research can assists local development in Honduras. A territorial approach is applied for analysis employing a three domain concept (local sustainable biotechnological development) of territory, technology and re-territorialization. The article suggests that, although the biotechnologization process (through genomics) of Jatropha within the socio-technical framework of the institute and multi-stakeholder networks is an ongoing process--and different trajectories are, therefore, still open--the process can, nevertheless, strengthen local sustainable development.

IDENTIFICATION AND MAPPING OF A NEW APPLE SCAB RESISTANCE GENE

Here we report the identification of a new resistance gene (Vd3) against apple scab (Venturia inaequalis) from the apple selection 1980-015-25 of the breeding program at Plant Research International. This accession also contains the Vf gene. We mapped Vd3, using SSR and DArT markers, on linkage group 1, at a distance of 6 cM from Vf gene, but in repulsion phase to Vf. Based on pedigree analysis and resistance tests, it could be deduced that 1980-015-25 had inherited Vd3 from the founder D3. This gene provides resistance to the highly virulent EU-NL-24 strain of the race 7 of V. inaequalis. This strain has overcome the resistance from both Vf and Vg. However, Vd3 has been not effective against the majority of other V. inaequalis strains we used in our disease tests.

Identification and mapping of the novel apple scab resistance gene Vd3

Apple scab, caused by the fungal pathogen Venturia inaequalis, is one of the most devastating diseases for the apple growing in temperate zones with humid springs and summers. Breeding programs around the world have been able to identify several sources of resistance, the Vf from Malus floribunda 821 being the most frequently used. The appearance of two new races of V. inaequalis (races 6 and 7) in several European countries that are able to overcome the resistance of the Vf gene put in evidence the necessity of the combination of different resistance genes in the same genotype (pyramiding). Here, we report the identification and mapping of a new apple scab resistance gene (Vd3) from the resistant selection “1980-015-25” of the apple breeding program at Plant Research International, The Netherlands. This selection contains also the Vf gene and the novel V25 gene for apple scab resistance. We mapped Vd3 on linkage group 1, 1 cM to the south of Vf in repulsion phase to it. Based on pedigree analysis and resistance tests, it could be deduced that 1980-015-25 had inherited Vd3 from the founder “D3.” This gene provides resistance to the highly virulent EU-NL-24 strain of race 7 of V. inaequalis capable of overcoming the resistance from Vf and Vg.

First Report of Wisteria vein mosaic virus on Wisteria sinensis in New Zealand.

Wisteria vein mosaic virus (WVMV) is a member of the Potyvirus genus. The virus has been reported in Wisteria spp. in Australia, China, the United States, and a number of European countries (2). In 2006, several W. sinensis plants with mottling and mosaic symptoms were observed in a commercial plant nursery in Whenuapai, north of Auckland, New Zealand. These plants had been propagated from a nursery in the New Plymouth area of New Zealand. Sap from the symptomatic Wisteria plants was examined with an electron microscope and elongated and flexuous potyvirus-like particles approximately 750 nm long were observed. RNA was extracted from the symptomatic plants with a Qiagen RNeasy Plant Mini Kit (Doncaster, Australia). The RNA was initially tested using general potyvirus primers, PV1/SP6 (4) and U335 (3), with the cycling conditions of 94°C for 5 min followed by 40 cycles of 94°C for 45 s, 50°C for 45 s, 72°C for 90 s, and a final extension of 72°C for 7 min. The polymerase chain reaction (PCR) product (695 bp) was directly sequenced (GenBank Accession No. EU580146) and a BLAST search in GenBank showed 98% nucleotide identity with WVMV (GenBank Accession no. AF484549). The RNA was then tested using WVMV-specific primers, WVMVF1 and WVMVR1, and the published cycling conditions (2). PCR amplicons of 701 bp were obtained. PCR products were directly sequenced (GenBank Accession No. EU308592), and a BLAST search in GenBank showed 98% nucleotide identity with published sequences of WVMV (GenBank Accession Nos. AF484549 and AY656816). In addition, RNA was extracted from the original isolate of WVMV that was reported in the Netherlands (1; supplied by R. van der Vlugt, Plant Research International) and the RNA was amplified using the WVMV-specific primer pair. The sequence obtained from PCR amplicons of the type isolate (GenBank Accession No. EU308593) showed a 98% nucleotide identity with the New Zealand WVMV isolate and with published sequences of WVMV (as shown above). From the symptomatology, particle morphology, and nucleotide sequences, it is concluded that WVMV is present in New Zealand. The distribution of the virus in New Zealand is not known, but the affected plants at the New Plymouth nursery may have been imported into New Zealand as many as 30 years ago. Although WVMV infection can reduce the quality of commercial plants, the disease is not economically significant in New Zealand.

Saroja Raman, Agricultural Sustainability: Principles, Processes and Prospects

Dr. Raman has worked for over thirty-Wve years at agricultural universities in India where she compiled the information published in the book. A foreword written by Dr. M.S. Swaminathan describes the “green revolution” as the product of an interaction between crop genotypes and agronomic management with particular reference to soil fertility enhancement and irrigation water availability. He expresses the hope that the book will be read and used for launching an Evergreen Revolution in agriculture. The author deWnes sustainable agriculture as the maintenance of the long-term productivity of a system in an environmentally conserving and safe manner coupled with economic viability, social justice and equity for the grower. Here we recognize the three P’s usually associated with sustainability: the P for Planet (environment), People (well-being) and proWt (for all involved in agricultural supply chains). The book Agricultural Sustainability however associates sustainability with three other less descriptive but more activating P’s of Principles, Processes and Prospects. This is also how the book is organized in three parts. The Wrst part deals with the historic evolution of the concept of sustainability that really became an issue because of uncontrolled population growth required increasing demand for food. DiVerent agricultural systems in the developed, fairly developed (e.g., India) and least developed countries are compared to illustrate that sustainability is a context-based issue and that not a generic globally applicable sustainable production system can be generated. The second part explains that the availability of resources—soil, water, minerals, energy, biodiversity, chemicals— and their proper management are key to promote agricultural sustainability. The role of science and technology as a facilitator for sustainable use of resources is treated. Here attention is given to the need to measure agricultural sustainability in a quantitative way although the author Wnds it “a complex and nebulous task”. The third P of Prospects is dealt with in the last section where vision documents of international agencies (FAO, IFPRI, World Bank) are portrayed with emphasis on poverty reduction and the importance of preventing soil degradation and enhancing income generation to assure livelihood for the farming community to ensure sustainable food security and minimum human development standards. This is illustrated with case studies from India and China. The book is very readable and treats the subject with great accessibility. The subjects is A. J. Haverkort (&) Plant Research International, Wageningen University and Research Centre, P. O. Box 16, Wageningen, 6700 AA, The Netherlands e-mail: anton.haverkort@wur.nl

Plant–fungal symbiosis en gros and en détail

Plant–fungal symbiosis <i>en gros</i> and <i>en détail</i>

DEVELOPMENT OF A GREENHOUSE SYSTEM FOR TROPICAL LOWLAND IN INDONESIA

Wageningen UR - A&F (former IMAG) developed together with Plant Research International (PRI) and the Dutch industries Rovero Systems B.V. and Plasthill B.V. and the Indonesian company PT East West Seed Indonesia a new greenhouse system for the production of high quality horticultural products in the tropics. In this study plastic-covered greenhouses are designed which are particularly adapted to the regional circumstances of tropical lowland in Indonesia. The greenhouse design has enough resistance against local wind loads, has high natural ventilation, but is at the same time mainly closed for insects by using insect nets. The airflow and the temperatures in the greenhouse are simulated by CFD software during design stage to optimize greenhouse size, shape and the ventilation openings. A new plastic film covering is developed. The new covering consists of a 200 µm polyethylene film with a lifetime of about 3 years in Indonesia. The film contains a UV-block pigment and has highly light diffusing properties. Additionally film prototypes are developed, which selectively reflect near infrared radiation (NIR) with the objective to reach a cooling effect inside the greenhouse. Six identical greenhouse prototypes are produced in The Netherlands and built in the isle of Java in Indonesia in spring 2003. In the greenhouses tomatoes are cultivated and observed. Additionally climate measurements are carried out to evaluate the new designed greenhouses.

Assessing morphological degradation in running waters using Blackfly communities (Diptera, Simuliidae): Can habitat quality be predicted from land use?

Based on benthic macroinvertebrate samples from 57 sites of streams in the European Central Highlands (Ecoregion 9; Illies (1978). Limnofauna Europaea, Stuttgart), the composition and the abundance of Simuliid species were analysed in relation to hydromorphological and land use parameters. Sampling sites were located at two stream types differing in catchment geology, stream morphology and channel width. Land use data were taken from the official German information system for cartography and topography (ATKIS) and the German River Habitat Survey. Land use categories suitable to describe the sampling sites were ‘agricultural land’, ‘urban’ and near-natural areas. Hydromorphological parameters of the sites were recorded using the AQEM site protocol (AQEM Consortium (2002). Manual for the application of the AQEM system. A comprehensive method to assess European streams using benthic macroinvertebrates, developed for the purpose of the Water Framework Directive. EVK1-CT1999-00027, Version 1.0. Available via the Internet from www.aqem.de). Relevant parameters controlling Simuliid distribution in streams were the mineral substrates and the biotic microhabitats. Blackfly larvae and pupae were sampled at each site for 15 min. Statistical analysis was performed by CANOCO ® (Ter Braak & Smilauer (1997). CANOCO Version 4.5. Biometrics Plant Research International, Wageningen, The Netherlands) using redundancy analysis (RDA). Our results show a stream type-specific composition of the Blackfly fauna. The analyses reveal sensitivity of the Simuliid species to morphological degradation, which is indicated by the shift in the longitudinal zonation of the Simuliid communities. Especially, Prosimulium hirtipes (Fries, 1824) and Simulium argyreatum Meigen, 1838 are typical representatives of headwater streams. While they seem to indicate undisturbed conditions of this stream type and react sensitively to the degradation of stream habitats, Simulium ornatum Meigen, 1818 and Simulium equinum (Linnaeus, 1758) are more tolerant to stream degradation. On catchment scale, ‘% natural forest’ and ‘% agricultural land use’ illustrate the degree of degradation of the two selected stream types. ‘Average stream depth’ and ‘relation riffles/pools’ account for hydromorphological degradation reflected by Simuliid species on the smaller reach scale. The analysis of habitat quality revealed that ‘% woody debris’ represents an important parameter of morphological degradation reflected by the Blackfly community.

Clavibacter michiganensis subsp. michiganensis

<i>Clavibacter michiganensis</i> subsp. <i>michiganensis</i>

First Report of Narcissus mosaic virus Infecting Crocus spp. Cultivars in the Netherlands.

A survey to identify virus diseases affecting Crocus spp. in the Netherlands was conducted during April 2004. Crocus spp. (cvs. Flavus, Pick-wick, Remembrance, and Grand Maitre) with symptoms suggestive of virus infection (stunting, yellowing, necrosis, and flower color breaking) were collected from several fields in the Breezand and Lisse districts in northern and southern Netherlands, respectively. All samples were tested for the presence of six known crocus-infecting viruses (1,2) using enzyme-linked immunosorbent assay (ELISA) and reverse transcription-polymerase chain reaction (RT-PCR) assays. The ELISA assay was performed with the following polyclonal and monoclonal antibodies: Iris severe mosaic virus (ISMV); Tobacco rattle virus (TRV) isolates F, Y, and J obtained from the Applied Plant Research Institute, Lisse, Netherlands; Arabis mosaic virus; Cucumber mosaic virus from the Plant Research International Institute, Wageningen, Netherlands; Iris yellow spot virus (IYSV) from the Virology Department at Wageningen University, Netherlands; and the potyvirus group-specific monoclonal antiserum from the DSMZ, Braunschweig, Germany. All samples that tested positive with a potyvirus antiserum were further tested for the presence of Bean yellow mosaic virus (BYMV) using a BYMV-specific antiserum. Serological results obtained indicated that BYMV, detected with the potyvirus antiserum and BYMV-specific antiserum, and ISMV were the most commonly encountered viruses. Tobacco necrosis virus (TNV) and TRV were only found occasionally, whereas IYSV, was not detected in any of the samples tested. To study the presence of viruses not yet reported, total RNA was extracted and tested with a RT-PCR assay with carlavirus, potexvirus, necrovirus (R. Miglino, unpublished), and potyvirus (3) genus-specific oligonucleotides. In accordance with the ELISA results, PCR amplicons were obtained with the potyvirus, TNV, and TRV primer sets. Furthermore, a 280-bp amplicon corresponding to the expected size was amplified in a RT-PCR assay performed on total RNA with a potexvirus genus-specific primer set. The reverse primer (5'-AGC ATG GCG CCA TCT TGT GAC TG-3') was located upstream in the conserved viral replicaseencoding region at position 4254-4231 of Narcissus mosaic virus (NMV) RNA genome (Genbank Accession No. D13747) and the forward primer (5'-CTG AAG TCA CAA TGG GTG AAG AA-3') was located downstream at position 3969-3992. Sequence homology using BLAST analysis of the cloned and sequenced PCR product showed 98% identity with NMV. Although the virus has a very narrow host range, the results of this study may have a significant impact on the crocus industry in the Netherlands. To our knowledge, this is the first report of NMV infecting crocus.

CLEAN VECTOR TECHNOLOGY FOR MARKER-FREE TRANSGENIC FRUIT CROPS

Marker-free transgenic crops confer several advantages over transgenic crops equipped with selection genes coding e.g. for antibiotic resistance. Firstly, the European Union has prepared a guidance document for risk assessment of GM-crops to be introduced in the environment (E.U. Joint Working Group on Novel Foods and GMO’s, 2003). In this document based on compliance to consumer demands the EU encourages to “avoid or minimise the inclusion of superfluous transgenes or sequences”. EU thus promotes the use of clean vector systems. Secondly, the number of selection genes allowing the preferential growth of transformed cells and tissues is limited. Often a gene transfer protocol for a specific crop or even a cultivar depends on the use of one specific selectable marker gene. Hence, stacking of genes within the same transgenic line is difficult once a selectable marker gene has been introduced. If these marker genes can be removed, the subsequent introduction of the next gene-of-interest is greatly facilitated. At Plant Research International a system has been developed for specific elimination of any introduced DNA/gene sequences using site-specific recombination combined with selection for successful removal using a negative selection system. Completely marker-free transgenic plants have been obtained using a model vector, both in an efficient transformation system (strawberry) as well as in a non-efficient transformation system (apple). Frequencies were more than adequate. Presently a versatile vector set providing a choice of several selectable markers and carrying a multiple cloning site for receiving cassettes of the gene-of-interest is available for application in, amongst others, fruit crops.

Synchytrium endobioticum

<i>Synchytrium endobioticum</i>