A draft genome of field pennycress (Thlaspi arvense) provides tools for the domestication of a new winter biofuel crop

Field pennycress (Thlaspi arvense L.) is being domesticated as a new winter cover crop and biofuel species for the Midwestern United States that can be double-cropped between corn and soybeans. A genome sequence will enable the use of new technologies to make improvements in pennycress. To generate a draft genome, a hybrid sequencing approach was used to generate 47 Gb of DNA sequencing reads from both the Illumina and PacBio platforms. These reads were used to assemble 6,768 genomic scaffolds. The draft genome was annotated using the MAKER pipeline, which identified 27,390 predicted protein-coding genes, with almost all of these predicted peptides having significant sequence similarity to Arabidopsis proteins. A comprehensive analysis of pennycress gene homologues involved in glucosinolate biosynthesis, metabolism, and transport pathways revealed high sequence conservation compared with other Brassicaceae species, and helps validate the assembly of the pennycress gene space in this draft genome. Additional comparative genomic analyses indicate that the knowledge gained from years of basic Brassicaceae research will serve as a powerful tool for identifying gene targets whose manipulation can be predicted to result in improvements for pennycress.     

Sunflower as a trap crop for the European tarnished plant bug (Lygus rugulipennis)

The European tarnished plant bug, Lygus rugulipennis (Heteroptera: Miridae), inflicts serious damage to glasshouse crops, including cucumber, in which it causes distorted foliage, dead growing points and malformed fruits. In a research involving commercial growers, we tested the possibility of using a trap crop to control L. rugulipennis. We screened the attraction of sunflower and lucerne as trap crops using an olfactometer. Adults (females and males) were more attracted to the odour of either flowering sunflower or lucerne than flowering cucumber. In a glasshouse trial, potted flowering sunflowers were evaluated as a trap crop placed at the ends of each cucumber row. Although the trap crop showed much higher attractiveness than the cucumber crop, this effect was not sufficient to provide an acceptable level of control according to the commercial growers. The growers suggested developing artificial sunflower odour as a more efficient lure for removal trapping. As a first step in this development, we tested the attraction of the headspace odour collected from sunflower in olfactometer trials. Sunflower headspace attracted a higher number of adults compared to a blank or a flowering cucumber. The sunflower odour was analysed by gas chromatography coupled to mass‐spectrometry to determine compounds possibly involved in L. rugulipennis attraction. The chemical analysis of the plant odour showed a well‐defined differentiation between sunflower and cucumber, with a number of monoterpenes released exclusively by sunflower. This, plus an emission rate from sunflower being at least four times more abundant, opens the possibility of using synthetic sunflower volatiles to attract L. rugulipennis within a cucumber background.     

Efficient and Stable Transformation of Lactuca sativa L. cv. Cisco (lettuce) Plastids

Transgenic plastids offer unique advantages in plant biotechnology, including high-level foreign protein expression. However, broad application of plastid genome engineering in biotechnology has been largely hampered by the lack of plastid transformation systems for major crops. Here we describe the development of a plastid transformation system for lettuce, Lactuca sativa L. cv. Cisco. The transforming DNA carries a spectinomycin-resistance gene (aadA) under the control of lettuce chloroplast regulatory expression elements, flanked by two adjacent lettuce plastid genome sequences allowing its targeted insertion between the rbcL and accD genes. On average, we obtained 1 transplastomic lettuce plant per bombardment. We show that lettuce leaf chloroplasts can express transgene-encoded GFP to ~36% of the total soluble protein. All transplastomic T0 plants were fertile and the T1 progeny uniformly showed stability of the transgene in the chloroplast genome. This system will open up new possibilities for the efficient production of edible vaccines, pharmaceuticals, and antibodies in plants.     

The potential of lentil (Lens culinaris) as a grain legume crop in the UK: an assessment based on a crop growth model

A crop growth model developed in Canterbury, New Zealand was used to assess the potential of lentil (Lens culinaris) as a grain legume crop in the UK. The model was validated using five sowing dates at Durham (54.77°N, 1.58°W) in 1999. Predicted time to flowering was within 7 days of actual time to flowering and predicted seed yields were within 9% of actual yields. Actual yields ranged from 1.40 to 1.65 t ha‐¹. Seed was of high quality. The model was used to predict rate of development and yields of spring and autumn sown lentils at eight sites along a transect from NW Scotland (Stornoway, 58.22°N, 6.32°W) to SE England (East Mailing, 51.28°N, 0.45°E) chosen to encompass important environmental gradients in the UK. In general, for a 1 May sowing with 150 or 250 mm plant available water (PAW) and a 1 October sowing with 150 mm PAW, predicted mean values over the period 1987–95 for maximum crop growth rate, maximum leaf area index, radiation intercepted, total dry matter produced and seed yield were closely positively related to monthly mean values for mean daily air temperature and increased along the transect from NW to SE UK. Time to flowering generally decreased along the transect from NW to SE UK ranging from 28 June to 9 July and from 20 May to 14 June with the May and October sowings respectively. For the 1 May sowing with 250 mm PAW, predicted mean seed yield ranged from 1.00 to 1.90 t ha‐1. For all sites, yield was very stable over the 9 yr period. For the 1 May sowing with 150 mm PAW, predicted mean seed yield ranged from 0.97–1.23 t ha‐1. Yields for the four more southerly sites were more variable at the lower PAW and, in exceptionally dry years, were substantially lower than average. For these sites, autumn sowing increased seed yields in exceptionally dry years and gave similar or greater mean seed yields to spring sowing with 250 mm PAW. For East Mailing, predicted yields for autumn sowing were on average 2.78 t ha‐1. Also, for Stornoway, because of its relatively high overwinter temperatures, the model predicted substantial increases in yield with autumn sowing. It is concluded that lentil has considerable potential as a grain legume crop in the UK.     

Edge distributions of Ceutorhynchus assimilis and its parasitoid Trichomalus perfectus in a crop of winter oilseed rape (Brassica napus)

The edge distributions of Ceutorhynchusassimilis and its parasitoid Trichomalusperfectus within a crop of winter oilseed rape were investigated between May and July using yellow water-traps arranged on a grid system. A randomisation test, which compared the observed arrangement of trap counts with permuted arrangements, was used to quantify the observed tendency of C. assimilisto congregate at the edges as it migrated into the crop; this tendency declined during the season. Trichomalus perfectus were distributed at the edge of the crop only during the early phase of its immigration in May, which occurred later than immigration of its host. It is considered that this simple means of quantifying insect spatial distribution could prove a useful tool in precision targeting of insecticides. This revised version was published online in July 2006 with corrections to the Cover Date.     

Diploid potato (<Emphasis Type="Italic">Solanum tuberosum</Emphasis> L.) as a model crop to study transgene expression

This paper presents a method of Agrobacterium-mediated transformation for two diploid breeding lines of potato, and gives a detailed analysis of reporter gene expression. In our lab, these lines were also used to obtain tetraploid somatic hybrids. We tested four newly prepared constructs based on the pGreen vector system containing the selection gene nptII or bar under the 35S or nos promoter. All these vectors carried gus under 35S. We also tested the pDM805 vector, with the bar and gus genes respectively under the Ubi1 and Act1 promoters, which are strong for monocots. The selection efficiency (about 17%) was highest in the stem and leaf explants after transformation with pGreen where nptII was under 35S. About half of the selected plants were confirmed via PCR and Southern blot analysis to be transgenic and, depending on the combination, 0 to 100% showed GUS expression. GUS expression was strongest in multi-copy transgenic plants where gus was under Act1. The same potato lines carrying multi-copy bar under Ubi1 were also highly resistant to the herbicide Basta. The suggestion of using Agrobacterium-mediated transformation of diploid lines of potato as a model crop is discussed herein.     

DNA shuttling between plasmid vectors and a genome vector: systematic conversion and preservation of DNA libraries using the Bacillus subtilis genome (BGM) vector

The combined use of the contemporary vector systems, the bacterial artificial chromosome (BAC) vector and the Bacillus subtilis genome (BGM) vector, makes possible the handling of giant-length DNA (above 100 kb). Our newly constructed BGM vector efficiently integrated DNA prepared in the BAC vector. A BAC library comprised of 18 independent clones prepared from mitochondrial DNA (mtDNA) of Arabidopsis thaliana was converted to a parallel BGM library using the new BGM vector. The effectiveness of the combined use of the vector systems was confirmed by the stable recovery of all 18 DNAs as BAC clones from the respective BGM clones. We show that DNA in BGM was stably preserved at room temperature after spore formation of the host B.subtilis. Rapid and stable shuttling between Escherichiacoli and the B. subtilis host, combined with spore-mediated DNA storage, may facilitate the long-term and low-cost preservation and the transportation of DNA resources.     

A Comparison of Auxotrophic and Wild Strains of Sclerotinia sclerotiorum Used as a Mycoherbicide Against Californian Thistle ( Cirsium arvense )

Two auxotrophic mutant strains of Sclerotinia sclerotiorum were tested in the greenhouse for pathogenicity on Cirsium arvense (Californian thistle) with and without amino acid amendments. An arginine auxotrophic mutant, with an amendment of the amino acid, followed an identical disease progress curve to that of the wild strain of the pathogen from which it was derived. However, when deprived of the amino acid amendment it was still highly pathogenic. A leucine auxotrophic mutant demonstrated poor pathogenicity without a leucine amendment, but improved pathogenicity with the addition of the amino acid. However, both of these treatments were inferior to the two wild strains tested and the arginine auxotroph with and without amendments. A field experiment was conducted on C. arvense stems in permanent pasture to compare the pathogenicity of amended auxotrophic strains and wild strains of S. sclerotiorum applied as a granule in a wheat-based carrier. The two wild strains gave significant reductions in thistle cover within 3 months of treatment, and subsequent reductions in thistle stem height and density during the following season. There was no evidence that the auxotrophic strains reduced thistle cover in the season the treatments were applied, but they did reduce subsequent stem density in the following spring. To determine disease carry-over associated with the wild and auxotrophic strains of the pathogen, rape was planted into subplots over the next three consecutive seasons. Despite substantial populations of sclerotia being present in the soil, especially in the first season after treatment of the thistles, no disease of rape caused by S. sclerotiorum was detected over the three seasons in any of the plots. Sclerotium populations of S. sclerotiorum in the soil declined by over 50% between 20 and 32 months after treatment, but there was no decline over the subsequent 12 months. The trial demonstrated that the auxotrophic strains were less field fit compared with the wild strains and that the presence of inoculum and a susceptible host to S. sclerotiorum were not the only prerequisites for disease development. It was concluded that use of a trap crop following treatment is not a suitable method for determining the risk of using this pathogen as a mycoherbicide in pasture.     

Distributions and interactions of the stem miners Psylliodes chrysocephala and Ceutorhynchus pallidactylus and their parasitoids in a crop of winter oilseed rape (Brassica napus)

The within‐field spatio‐temporal distributions and relationships of two pest insects with stem‐mining larvae, Psylliodes chrysocephala (L.) (Coleoptera: Chrysomelidae) and Ceutorhynchus pallidactylus (Marsham) (Coleoptera: Curculionidae), and their larval endoparasitoids, Tersilochus microgaster (Szépligeti) and Tersilochus obscurator Aubert (Hymenoptera: Ichneumonidae), respectively, were studied in a crop of winter oilseed rape [Brassica napus L. (Brassicaceae)]. The insects were sampled, at different life stages, at 40 spatially referenced points within the oilseed rape in 1998–99 and in the following crop of winter wheat. Distributions were analysed and compared using SADIE (Spatial Analysis by Distance IndicEs) and by tests of edge distribution. Tersilochus microgaster emerged from overwintering by the end of March and T. obscurator from mid April to mid May 2000. Tersilochus microgaster parasitized 10.8% of P. chrysocephala larvae, peak parasitism occurring in May. The distribution of each species was patchy and irregular. Psylliodes chrysocephala and its parasitoid were closely associated, but new generation C. pallidactylus and T. obscurator were less so, probably because of host mortality caused by the parasitoid. The two host–parasitoid pairs showed distributions that were polarized with respect to each other. The genesis of these spatial patterns is discussed in relation to the influence of wind direction, plant size, and interspecific interactions. The potential of these parasitoids as biocontrol agents in integrated pest management is discussed. Opportunities for conservation biocontrol in oilseed rape offered by spatio‐temporal targeting of insecticides to avoid parasitoids and by reduced soil tillage are examined.     

Trophic impacts of a rolled-rye cover crop on the lettuce aphid (Nasonovia ribisnigri) and associated insect fauna in cultivated Histosols

Cultivated peatland (Histosol) in Southern Québec (Canada) is a rapidly declining non-renewable resource used to grow most Canadian lettuce (Lactuca sativa L., Asteraceae). Rolled-rye (Secale cereale L., Poaceae) cover crop is one of the conservation practices proposed to reach a more sustainable lettuce production, but the overall impact on the agroecosystem remains poorly studied in Histosols. We assessed multiple effects of rolled-rye cover crop on the trophic chain associated with the lettuce aphid, Nasonovia ribisnigri (Mosley) (Hemiptera: Aphididae), a major pest of lettuce. During one growing season and through two consecutive lettuce crops, we monitored in situ the impacts of rolled-rye cover crop on insect fauna and lettuce quality. We used visual scouting and yellow pan traps to assess plant colonization by N. ribisnigri, its natural enemies, and alternative prey. Exclusion cage experiments were also conducted to measure aphid fitness and population growth. Under greenhouse conditions, following cover crop removal, we examined potential lingering effects of rye within the soil on lettuce plants and N. ribisnigri. In situ experiments showed that rolled-rye cover crop has the potential to inhibit N. ribisnigri field colonization and recruit natural enemies and alternative prey in the first lettuce crop. Rye also reduced aphid fitness as well as lettuce foliar amino acid concentration and weight. For the second lettuce crop, an almost 50% reduction in N. ribisnigri abundance was observed with the use of rolled-rye cover crop. In greenhouse experiments, no persistent effect of rye was observed on the quality of lettuce grown with soil collected under a cover crop, nor on the fitness of aphids inoculated on these lettuces. This study highlights the diversity of trophic effects rolled-rye cover crop may have on lettuce production in Histosols and the potential of rolled-rye cover crop as a cultural practice to reduce aphid populations. Mechanisms at play while underlining agronomic challenges regarding proper rye termination must be further explored to maintain high-performing lettuce yields.     

High-quality genomes reveal significant genetic divergence and cryptic speciation in the model organism Folsomia candida (collembola)

The collembolan Folsomia candida Willem, 1902, is widely distributed throughout the world and has been frequently used as a test organism in soil ecology and ecotoxicology studies. However, it is questioned as an ideal “standard” because of differences in reproductive modes and cryptic genetic diversity between strains from various geographical origins. In this study, we obtained two high-quality chromosome-level genomes of F. candida, for a parthenogenetic strain (named FCDK, 219.08 Mb, 25,139 protein-coding genes) and a sexual strain (named FCSH, 153.09 Mb, 21,609 protein-coding genes), reannotated the genome of the parthenogenetic strain reported by Faddeeva-Vakhrusheva et al. in 2017 (named FCBL, 221.7 Mb, 25,980 protein-coding genes) and conducted comparative genomic analyses of the three strains. High genome similarities between FCDK and FCBL based on synteny, genome architecture, mitochondrial and nuclear gene sequences suggest that they are conspecific. The seven chromosomes of FCDK are each 25%–54% larger than the corresponding chromosomes of FCSH, showing obvious repetitive element expansions and large-scale inversions and translocations but no whole-genome duplication. The strain-specific genes, expanded gene families and genes in nonsyntenic chromosomal regions identified in FCDK are highly related to the broader environmental adaptation of parthenogenetic strains. In addition, FCDK has fewer strain-specific microRNAs than FCSH, and their mitochondrial and nuclear genes have diverged greatly. In conclusion, FCDK/FCBL and FCSH have accumulated independent genetic changes and evolved into distinct species after 10 million years ago. Our work provides important genomic resources for studying the mechanisms of rapidly cryptic speciation and soil arthropod adaptation to soil ecosystems.     

The effects of a winter cover crop on Diabrotica virgifera (Coleoptera: Chrysomelidae) populations and beneficial arthropod communities in no-till maize

The effects of an autumn-planted, spring-killed, grass cover crop (Elymus trachycaulus [Link] Gould ex Shinners) on populations of Diabrotica virgifera virgifera LeConte and its predator community were evaluated in South Dakota maize fields over two seasons. Abundance and size of D. virgifera larvae and adults and sex ratio of adults were measured in maize produced under two treatments (i.e., a winter cover crop or bare soil), as were maize root damage and the abundance and diversity of the predator communities collected on the soil surface and in the soil column. First and second instars and adults of D. virgifera were similarly abundant in the two treatments, but third instars were significantly fewer in maize planted after a winter cover crop. Larvae developed at different rates in the two treatments, and second instars were significantly smaller (head capsule width and body length) in the maize planted after a cover crop. First and third instars and adults were of similar size in the two treatments, and adult sex ratios were also similar. Although initially similar, predator populations increased steadily in the cover-cropped maize, which led to a significantly greater predator population by the time D. virgifera pupated. There was significantly less root damage in the cover-cropped maize. Predator communities were similarly diverse in both treatments. Predator abundance per plot was significantly and negatively correlated with the abundance of third instars per plot. Clearly, winter cover crops reduce D. virgifera performance and their damage to the crop, and we suspect that this reduction is caused by both environmental effects of the treatment on D. virgifera size and development, and of increased predation on the third instars of the pest. Additional data on the impact of cover crops on actual predation levels, grain yield and quality, and farmer profitability, and correlations among pest performance, crop characteristics, and predator populations and behaviors are key components of this system that remain to be addressed.     

Application of a probabilistic model for analysing the abortion of seeds and pods in winter oilseed rape (Brassica napus)

In winter oilseed rape (WOSR), only a subset of ovules can develop into seeds in the majority of pods. Any difficulty during the process of seed production may result in seed or pod abortion. This study aimed to reproduce the process of seed development in WOSR based on a limited number of parameters. As a result of the complexity of the developmental patterns of WOSR, it is challenging to identify the roles of various factors that influence seed production using an experimental approach. Here, we present a stochastic probabilistic model of seed development. The generalised least squares method was implemented to estimate the model parameters using the experimental data. Experiments were done in Grignon (France) in 2008 and 2009. The variations in the parameters were analysed according to the following four factors: year, pod rank, inflorescence position and ramification‐clipping treatment. The year had no effect on the number of ovules per ovary (μ) and the probability of seed viability (p). The proportion of effective pollen grains (k) significantly decreased with pod rank at the end of the main stem. Inflorescence position influenced the number of ovules per ovary (μ: 30.8–33.8 from top to bottom) and the parameter k. The mean number of seeds per pod on the main stem and the bottom ramification along the stem was larger than the other ramifications within one plant. Ramification‐clipping treatment increased the number of ovules per ovary (μ: 31 for control plants and 32 for clipped plants), the parameter k and the number of seeds per pod (p). This effect could be due to the competition for assimilates between the pods and seeds. Furthermore, the distribution parameters of the pollen number per stigma (m) remained stable, and the probability of pod survival (Bo) varied with different factors, including the year, pod rank and inflorescence position. Our results indicate that pollen germination is a factor that determines final seed number. This model can identify the impact of each of the factors that lead to the abortion of seeds and pods in WOSR, which include the position effect, assimilate competition and pollination limitation. However, further studies on the pollination process in WOSR should help to refine this model.     

Transposable elements in Coffea (Gentianales: Rubiacea) transcripts and their role in the origin of protein diversity in flowering plants

Transposable elements are major components of plant genomes and they influence their evolution, acting as recombination hot spots, acquiring specific cell functions or becoming part of protein-coding regions. The latter is the subject of the present analysis. This study is a report on the annotation of transposable elements (TEs) in expressed sequences of Coffea arabica, Coffea canephora and Coffea racemosa, showing the occurrence of 383 ESTs and 142 unigenes with TE fragments in these three Coffea species. Based on selected unigenes, it was possible to suggest 26 putative proteins with TE-cassette insertions, demonstrating a likely contribution to protein variability. The genes for two of those proteins, the fertility restorer (FR) and the pyrophosphate-dependent phosphofructokinase (PPi-PFKs) genes, were selected for evaluating the impact of TE-cassettes on host gene evolution of other plant genomes (Arabidopsis thaliana, Oryza sativa and Populus trichocarpa). This survey allowed identifying a FR gene in O. sativa harboring multiple insertions of LTR retrotransposons that originated new exons, which however does not necessarily mean a case of molecular domestication. A possible transduction event of a fragment of the PPi-PFK beta-subunit gene mediated by Helitron ATREPX1 in Arabidopsis thaliana was also highlighted.     

Denitrification and N mineralization from hairy vetch (Vicia villosa Roth) and rye (Secale cereale L.) cover crop monocultures and bicultures

N mineralization, N immobilization and denitrification were determined for vetch, rye and rye-vetch cover crops using large packed soil cores. Plants were grown to maturity from seed in cores. Cores were periodically leached, allowing for quantification of NO₃ ⁻ and NH₄ ⁺ production, and denitrification incubations were conducted before and after cover crop kill. Gas permeable tubing was buried at two depths in cores allowing for quantification of N₂O in the soil profile. Cover crops assimilated most soil N prior to kill. After kill, relative rates of N mineralization were vetch > rye-vetch mixture > fallow > rye. After correcting for N mineralization from fallow cores, net N mineralization was observed in vetch and rye-vetch cores, while net N immobilization was observed in rye cores. Denitrification incubations were conducted 5, 15 and 55 days after kill, with adjustment of cores to 75% water filled pore space (WFPS). The highest denitrification was observed in vetch cores 5 days after kill, when soil NO₃ ⁻ and respiration rates were high. Substantially lower denitrification was observed on subsequent measurement dates and in other treatments probably due to either limited NO₃ ⁻ or organic carbon in the soil. On day 5, 3%, 23%, 31% and 31% of the N₂O was recovered in the headspace of fallow, vetch, rye and rye-vetch cores, respectively. The rest was stored in the soil profile. In a field study using intact soil cores, denitrification rates also peaked 1 week after cover crop kill and decreased significantly thereafter. Results suggest greater potential N losses from vetch than rye or rye-vetch cover crops due to rapid N-mineralization in conjunction with denitrification and potential leaching, prior to significant crop N-assimilation. This revised version was published online in June 2006 with corrections to the Cover Date.     

The ground beetle fauna (Coleoptera:Carabidae) of abandoned fields, as related to plant cover, previous management and succession stage

This study describes differences in species richness, diversity and composition of Carabidae in gradients from recently abandoned, non-grazed fields over stages of overgrowth into forest on formerly agricultural land in a large, sandy outwash plain, south Sweden. Totally 80 pitfall traps, (4 succession stages, each represented by 4 sites; 5 traps per site) installed on 29 March 2006 were emptied continuously until 1 November. Succession stages were: 7–10 y old fallows after cereals with thin and low vegetation of small perennial and annual herbs (Ia), 7–10 y old fallows abandoned as lay with a rich plant cover of broad-leaved grasses and herbs (Ib), 20–25 y old fallows with a shrub layer of colonising pine and narrow-leaved grasses (II), and ca 80 y old pine stands planted on originally cultivated ground with a rich shrub layer but lacking herbaceous plants (III). A total of 14,068 individuals of 71 carabid species were captured. Species richness was highest in stage Ib, whereas Shannon species diversity was highest in Ia. Both species richness and diversity were lowest in III, sites II being intermediate. Total number of individuals captured site⁻¹ was low in III, being highest in Ib. Mean body weight and total dry mass of species, however, increased with succession stage. Amara and Harpalus species were most common in Ia but important also in Ib, with large differences in species composition between the two stages. These genera were almost lacking in III, where Carabus spp. and Pterostichus niger dominated. The share of Calathus was highest in II, where C. fuscipes played a dominating role. P. versicolor dominated in Ib, whereas P. lepidus was quite common in all non-forest stages. Duration and intensity of capturing activity necessary to find most species of the sites are discussed. Many scarce or rare species in south Scandinavia were captured, mainly in Ib. Abandoned non-grazed fields are important hibernating and breeding refuges for many carabids. Using extensive and non-expensive management this ought to be considered as an additional alternative in environment conservation policy, which now usually recommends economically subsidised grazing on set-aside land.     

Green Fluorescent Protein (GFP) as a Reporter Gene for the Plant Pathogenic Oomycete Phytophthora ramorum

ABSTRACT. Transgenic Phytophthora ramorum strains that produce green fluorescent protein (GFP) constitutively were obtained after stable DNA integration using a polyethylene glycol and CaCl₂-based transformation protocol. Green fluorescent protein production was studied in developing colonies and in different propagules of the pathogen to evaluate its use in molecular and physiological studies. About 12% of the GFP transformants produced GFP to a level detectable by a confocal laser scanning microscope. Green fluorescent protein could be visualized in structures with vital protoplasm, such as hyphal tips and germinating cysts. In infection studies with Rhododendron , one of the GFP expressing strains showed aggressiveness equal to that of the corresponding non-labelled isolate. Thus, GFP could be used as a reporter gene in P. ramorum . Limitations of the technology are discussed.