Ultrastructural changes associated with June Yellows in strawberry and with leaf yellowing symptoms of viral and genetic origin in Fragaria, Rubus and Ribes

Light and electron microscopy was used to study the ultrastructural effects of June Yellows (JY) in leaves of strawberry. Four cultivars of strawberry, affected to different extents by JY, were compared with healthy (JY-free) cv. Cambridge Favourite and with strawberry infected with strawberry crinkle rhabdovirus, Fragaria vesca infected with strawberry mottle virus (SMotV), raspberry and black currant showing virus-induced yellowing and with strawberry and raspberry showing chaemeric yellow sectors in the leaves. Except for isometric virus-like particles detected in SMotV-infected F. vesca, no virus-like particles or structures of other pathogenic agents were found in any of the tissues examined. Leaf cells of JY-affected strawberry showed severe disruption of chloroplasts and plasmalemma, whorls of membranous vesicles and decreased vacuole size. The extent and severity of these abnormalities increased with increased severity of JY symptoms but, even in leaves with mild JY symptoms, chloroplast abnormalities were obvious. In the most severely affected leaves, the cells lacked discrete vacuoles and extensive hypertrophy was seen in other organelles such as nuclei and mitochondria. Few, if any, ultrastructural abnormalities were observed in virus-infected strawberry or F. vesca, or in chaemeric leaves of strawberry and raspberry. By contrast, in raspberry and black currant with yellowed leaves caused by virus infection, the cells showed enlarged chloroplasts, decreased vacuole size and vesicle formation. However, chloroplast enlargement and disruption in this material seemed due to increased size of starch grains which were largely absent from swollen chloroplasts of JY-affected strawberry. The ultrastructural abnormalities observed in JY-affected strawberry are, therefore, not inconsistent with the possibility that a pathogenic agent may be involved in the condition.     

The effect of heat treatment and meristem-tip culture on June Yellows in strawberry

Because there is some evidence that June Yellows (JY) of strawberry may be caused by a pathogenic agent, combinations of heat treatment and meristem-tip culture that are known to eliminate some viruses from tissues were used in attempts to cure affected Cambridge Favourite strawberry plants from JY. None of 397 propagants derived from JY-affected plants subjected to various combinations of these treatments were freed from JY. Indeed, all propagants showed more obvious JY symptoms than the parent plants from which they were derived, suggesting that such treatments may be useful for detecting incipient JY in symptomless strawberry stocks.     

Microsatellite markers for Fragaria from ‘Strawberry Festival’ expressed sequence tags

We present 37 microsatellite primer pairs developed from a cDNA library of Fragaria ^xananassa Duch. cv. Strawberry Festival. Polymorphism was high and the number of presumptive alleles of 13 expressed sequence tag–simple sequence repeats (EST–SSRs) in 70 strawberry cultivars ranged from five to 32 per primer pairs, averaging 16.1. Cross‐species amplification was also high and ranged from 89% in Fragaria vesca L. to 100% in the progenitor species of octoploid strawberry, Fragaria chiloensis (L.) Duch. and Fragaria virginiana Duch.     

Isolation and characterization of polymorphic microsatellites in diploid strawberry (Fragaria vesca L.) for mapping,diversity studies and clone identification

This study reports the development and characterization of 10 polymorphic microsatellite primer pairs in wild strawberry (Fragaria vesca). The primers were designed from a genomic library enriched for di‐, tri‐ and tetranucleotide repeats from F. vesca‘Reugen’. They showed single locus polymorphism in a set of nine F. vesca accessions; two to six alleles were detected per locus. The level of polymorphism in F. vesca was surprisingly low, although three pairs of primers were sufficient to distinguish between most accessions.     

Identification of flowering genes in strawberry, a perennial SD plant

Background  

We are studying the regulation of flowering in perennial plants by using diploid wild strawberry (Fragaria vesca L.) as a model. Wild strawberry is a facultative short-day plant with an obligatory short-day requirement at temperatures above 15°C. At lower temperatures, however, flowering induction occurs irrespective of photoperiod. In addition to short-day genotypes, everbearing forms of wild strawberry are known. In 'Baron Solemacher' recessive alleles of an unknown repressor, SEASONAL FLOWERING LOCUS (SFL), are responsible for continuous flowering habit. Although flower induction has a central effect on the cropping potential, the molecular control of flowering in strawberries has not been studied and the genetic flowering pathways are still poorly understood. The comparison of everbearing and short-day genotypes of wild strawberry could facilitate our understanding of fundamental molecular mechanisms regulating perennial growth cycle in plants.     

Three wild Lithuanian strawberry species and their pollinators

The European wild strawberry species Fragaria vesca L., Fragaria viridis Weston, and Fragaria moschata Weston are sympatric and bloom concurrently, introducing the possibilities of cooperation and competition for pollinators. We studied the pollination biology of strawberries that were grown in rectangular plots at a field site near Vilnius city in Lithuania. The number of open flowers, the number of insects visiting flowers, and the plot area covered by strawberry plants were recorded. Wild strawberries were visited by solitary bees (38.5%), flies (31.4%), and other hymenopterans (21%; ants accounted for approximately 98% of this group). Coleopterans, lepidopterans and honey bees together represented less than 10% of the insects recorded on strawberries. Fragaria viridis and F. moschata flowers were more attractive to pollinators than those of F. vesca. The main visitors of F. viridis were small solitary bees; those of F. moschata were ants, while F. vesca flowers were visited by solitary bees, ants and flies in comparable numbers. We discuss competition for pollinators and the possible impact of climate fluctuation on strawberry-pollinator interactions.     

Reduced clonal reproduction indicates low potential for establishment of hybrids between wild and cultivated strawberries (Fragaria vesca × F. × ananassa)

The genus Fragaria (Rosaceae) contains 24 species, including hybrid species such as the garden strawberry (Fragaria × ananassa Duch.). Natural hybridization between Fragaria species has repeatedly been reported, and studies on the hybridization potential between F. × ananassa and its wild relatives have become increasingly important with the outlook for genetically modified garden strawberries. In Europe, a candidate species for hybridization with garden strawberries is the common woodland strawberry (Fragaria vesca L.). Although a previous field survey indicated that the potential for hybridization between F. vesca and F. × ananassa is low, it is not clear whether the lack of natural hybrids is caused by known pre- and postzygotic barriers, or whether hybrid plants lack the fitness to establish in natural F. vesca populations. We grew different F. vesca and F. vesca × F. × ananassa hybrid clones with and without competition in a greenhouse and assessed biomass production, clonal reproduction, and sexual reproduction of plants. While some hybrid clones exceeded F. vesca in biomass production, general clonal reproduction was much lower and delayed in hybrids. Furthermore, hybrids were sterile. These results demonstrate a mechanism by which the general lack of F. vesca × F. × ananassa hybrids in natural habitats can be explained, in addition to the known low hybridization potential between garden and woodland strawberries. We conclude that hybrids have a competitive disadvantage against co-occurring F. vesca plants due to inferior and delayed clonal reproduction, and that the potential for hybrid establishment under natural conditions is low.     

Seed germination of Fragaria vesca L. From atypical ecotopes of West Siberia

Seed formation and seed germination rate were established to be essentially higher in Fragaria vesca L. populations growing in the ecotopes which are atypical for wood strawberry than in the standard ecotopes for the species. Having equal capabilities for cross-pollination and self-pollination, the plants exhibit higher level of xenogamy than autogamy when growing in atypical ecotopes. Xenogamy predomination promotes the gene diversity of the population, thus ultimately providing realization of the ecological plasticity of the species.     

Growth rates of rhizosphere microorganisms depend on competitive abilities of plants and N supply

Abstract

Plant‐microbial interactions under N‐limiting conditions are governed by competitive abilities of plants for N. Our study aimed to examine how two plant species of strawberry, Fragaria vesca L. (native species) and Duchesnea indica (Andrews) Focke (an invasive plant in central Europe), growing in intra‐specific and inter‐specific competition alter the functions of rhizosphere microorganisms in dependence on N availability. By intra‐specific competition at low N level, a 2.4‐fold slower microbial‐specific growth rate was observed under D. indica characterized by smaller root biomass and lower N content in roots compared with F. vesca. By inter‐specific competition of both plants at low N level, microbial growth rates were similar to those for D. indica indicating that plants with stronger competitive abilities for N controls microbial community in the rhizosphere. Since a high N level smoothed the differences between plant species in root and microbial biomass as well as in microbial growth rates under both intra‐specific and inter‐specific competition, we conclude that competitive abilities of plant species were crucial for microbial growth in the rhizosphere only under N imitation.     

EST‐SSR markers from Fragaria vesca L. cv. Yellow Wonder

Fourteen microsatellite primer pairs were developed from a cDNA library of heat‐treated seedlings of Fragaria vesca cv. yellow wonder. Transferability to 13 species of Fragaria ranged from 71% in diploid species F. gracilis Losinsk., F. iinumae Makino, F. nilgerrensis Schltdl. ex J. Gay and F. nipponica Makino, to 100% in octoploid domestic strawberry and its progenitors. Polymorphism was high in polyploid Fragaria species. However, polymorphism and heterozygosity of eight EST‐SSRs (expressed sequence tag–simple sequence repeats) was low in 14 F. vesca genotypes.     

Development of an efficient transformation method by Agrobacterium tumefaciens and high throughput spray assay to identify transgenic plants for woodland strawberry (Fragaria vesca) using NPTII selection

Key message

We developed an efficient Agrobacterium -mediated transformation method using an Ac/Ds transposon tagging construct for F. vesca and high throughput paromomycin spray assay to identify its transformants for strawberry functional genomics.

Abstract

Genomic resources for Rosaceae species are now readily available, including the Fragaria vesca genome, EST sequences, markers, linkage maps, and physical maps. The Rosaceae Genomic Executive Committee has promoted strawberry as a translational genomics model due to its unique biological features and transformability for fruit trait improvement. Our overall research goal is to use functional genomic and metabolic approaches to pursue high throughput gene discovery in the diploid woodland strawberry. F. vesca offers several advantages of a fleshy fruit typical of most fruit crops, short life cycle (seed to seed in 12–16 weeks), small genome size (206 Mbb/C), small plant size, self-compatibility, and many seeds per plant. We have developed an efficient Agrobacterium tumefaciens-mediated strawberry transformation method using kanamycin selection, and high throughput paromomycin spray assay to efficiently identify transgenic strawberry plants. Using our kanamycin transformation method, we were able to produce up to 98 independent kanamycin resistant insertional mutant lines using a T-DNA construct carrying an Ac/Ds transposon Launchpad system from a single transformation experiment involving inoculation of 22 leaf explants of F. vesca accession 551572 within approx. 11 weeks (from inoculation to soil). Transgenic plants with 1–2 copies of a transgene were confirmed by Southern blot analysis. Using our paromomycin spray assay, transgenic F. vesca plants were rapidly identified within 10 days after spraying.     

Agrobacterium-mediated transformation of `Alpine' Fragaria vesca, and transmission of transgenes to R1 progeny

Agrobacterium-mediated transformation was used to stably introduce β-glucuronidase (gus) and neomycin phosphotransferase (nptII) marker genes into `Alpine' Fragaria vesca FRA 197, a diploid (2n = 2x = 14) strawberry. R0 generation transformants derived from a single clump of kanamycin-resistant callus were vegetatively propagated. The presence of the gus and nptII genes in five clonal R0 runner plants was confirmed by PCR. Southern analysis suggested two sites of nptII insertion. When R1 generation seedlings obtained via self-fertilization of R0 plants were tested by histochemical assay, 591 were GUS positive and 39 were GUS negative. The R1 segregation data fit a 15 : 1 ratio (0.5 > P > 0.25), consistent with the independent segregation of two transgene insertion loci. These results demonstrate the suitability of `Alpine' F. vesca for transgene research in strawberry. Received: 20 March 1997 / Revision received: 29 July 1997 / Accepted: 3 September 1997     

High-efficiency transformation of the diploid strawberry (Fragaria vesca) for functional genomics

Fragaria vesca L., a diploid (2n=2x=14) relative of the commercial octoploid strawberry, is an attractive model for functional genomics research in Rosaceae. Its small genome size, short reproductive cycle, and facile vegetative and seed propagation make F. vesca a promising candidate for forward and reverse genetics experiments. However, the lack of a high-efficiency transformation protocol required for systematic production of thousands of T-DNA insertional mutant lines and high-throughput gene validation is a major bottleneck. We describe a new transformation procedure that uses leaf explants from newly unfolded trifoliate leaves obtained from stock plants 6–7 weeks after seed germination, co-cultivation with Agrobacterium strain GV3101, and stringent selection on MS medium containing 4 mg l⁻¹ hygromycin. Using this protocol we achieved 100% transformation efficiency for 6 of 14 F. vesca accessions tested. Accession PI 551572 was determined to be the best candidate for a model in F. vesca functional genomics research, as it showed the greatest propensity for callus formation, transformation, shoot regeneration, ex vitro establishment, and plant growth, requiring only 14–15 weeks to complete its life cycle in different seasons in the greenhouse.     

Isolation and characterization of microsatellite loci in Fragaria

This study reports the development and characterization of 20 microsatellite primer pairs in wild strawberry Fragaria vesca. One hundred primers were obtained from an AC‐enriched library developed in the cultivar ‘Ilaria’. A set of eight F. vesca genotypes was used to detect the polymorphism resulting in an average of 7.0 alleles, an average observed heterozygosity of 0.32 and an average expected heterozygosity of 0.73. Nineteen (95%) of the primers also amplified the cultivated octoploid strawberry Fragaria×ananassa.     

EST‐derived polymorphic microsatellites from cultivated strawberry (Fragaria×ananassa) are useful for diversity studies and varietal identification among Fragaria species

Microsatellite or simple sequence repeat markers derived from expressed sequence tags (ESTs) provide genetic markers within potentially functional genes, which could be very useful for breeding programs. To date, the development of microsatellite markers in the genus Fragaria has focused mainly on Fragaria vesca. However, most of the interests of breeding programs relate to specific characteristics of cultivated strawberry. Here, we describe a set of 10 EST‐derived microsatellites from Fragaria × ananassa. These markers showed high levels of polymorphism within strawberry cultivars and among different Fragaria species, indicating their potential for genetic studies not only on strawberry but also in other species within the genus.     

Further studies on double-stranded RNA species from raspberry plants infected with aphid-borne viruses

Analysis by electrophoresis in polyacrylamide gels, followed by silver staining, of dsRNA extracted from many samples of raspberry leaves infected with raspberry leaf mottle virus (RLMV) and/or raspberry leaf spot virus (RLSV) failed to detect reliably any significant quantities of dsRNA species in excess of 1·0 × 10⁶mol. wt. This contrasts with results reported from Canada where three dsRNA species of estimated mol. wt 2·6 × 10⁶1·6 × 10⁶and 1·1 × 10⁶were consistently associated with infection with RLSV but none were associated with RLMV. However, in Scotland, four dsRNA species of estimated mol. wt 2·4 × 10⁶1·6 × 10⁶0·7 × 10⁶and 0·3 × 10⁶were detected in raspberry infected with apple mosaic ilarvirus. These results suggest that the dsRNA species reported from Canada are not those of RLSV but are probably those of a second virus, possibly an ilarvirus, which occurs together with RLSV and/or induces similar symptoms. A few samples from plants infected with RLMV and RLSV contained very small amounts of two dsRNA species of estimated mol. wt 4·7 × 10⁶and 4·5 × 10⁶. It is not known whether these species are those of RLMV and RLSV.     

Agamospermy in outbred crosses of <Emphasis Type="Italic">Fragaria vesca</Emphasis> L. (Rosaceae)

The seed progeny obtained in European wood strawberry Fragaria vesca L. (2n = 2x = 14) via outbred crosses proved to include matromorphic diploid plants, whose proportion varied from 10.0 to 21.0%. To allow their identification, the crosses were performed using recessive dihomozygotes (variety Alexandria) as a maternal form and dominant dihomozygotes as a paternal form. Generation of parthenogenetic plants in ourbred crosses with known genetic markers suggested pseudogamous agamospermy. The question is discussed as to whether seed development via agamospermy is possible in wild-growing F. vesca populations of Siberia. The capability of forming some seeds via agamospermy is considered to result from successful adaptation of the F. vesca reproductive system to stressful growth conditions.     

Development and characterization of polymorphic microsatellite markers from Fragaria viridis,a wild diploid strawberry

To date, the development of microsatellite (SSR) markers in the genus Fragaria has focused on F. vesca. However, further species are thought to have contributed to the complex allo‐octoploid genome of the cultivated strawberry, F.×ananassa. Here, we present 22 new SSR markers developed from the diploid species F. viridis. Twenty‐one of the primer pairs amplified polymorphisms in six F. viridis accessions, with an average of 4.95 alleles per primer pair and an average expected heterozygosity of 0.68. Fourteen of these primer pairs, and a locus monomorphic in F. viridis, amplified polymorphic alleles in the parents of a F. vesca mapping population.     

Natural occurrence of Azospirillum brasilense in strawberry plants

Azospirillum species are free-living nitrogen-fixing bacteria commonly found in soil and in association with roots of different plant species. For their capacity to stimulate growth they are known as plant growth-promoting bacteria (PGPB). In this work, we demonstrate the natural occurrence and colonization of different parts of strawberry plants by Azospirillum brasilense in the cropping area of Tucumán, Argentina. Although bacteria isolations were carried out from two strawberry cultivars, e.g., Camarosa and Pájaro, attempts were successful only with the cultivar Camarosa. Whereas different strains of Azospirillum were isolated from the root surface and inner tissues of roots and stolons of the cultivar Camarosa, we have not obtained Azospirillum isolates from the cultivar Pájaro. After microbiological and molecular characterization (ARDRA) we determined that the isolates belonged to the species A. brasilense. All isolates showed to have the capacity to fix nitrogen, to produce siderophores and indoles. Local isolates exhibited different yields of indoles production when growing in N-free NFb semisolid media supplemented or not with tryptophan (0.1 mg ml⁻¹). This is the first report on the natural occurrence of A. brasilense in strawberry plants, especially colonizing inner tissues of stolons, as well as roots. The local isolates showed three important characteristics within the PGPB group: N₂-fixation, siderophores, and indoles production.     

Metabolite profiling reveals novel multi-level cold responses in the diploid model Fragaria vesca (woodland strawberry)

Winter freezing damage is a crucial factor in overwintering crops such as the octoploid strawberry (Fragaria × ananassa Duch.) when grown in a perennial cultivation system. Our study aimed at assessing metabolic processes and regulatory mechanisms in the close-related diploid model woodland strawberry (Fragaria vesca L.) during a 10-days cold acclimation experiment. Based on gas chromatography/time-of-flight-mass spectrometry (GC/TOF-MS) metabolite profiling of three F. vesca genotypes, clear distinctions could be made between leaves and non-photosynthesizing roots, underscoring the evolvement of organ-dependent cold acclimation strategies. Carbohydrate and amino acid metabolism, photosynthetic acclimation, and antioxidant and detoxification systems (ascorbate pathway) were strongly affected. Metabolic changes in F. vesca included the strong modulation of central metabolism, and induction of osmotically-active sugars (fructose, glucose), amino acids (aspartic acid), and amines (putrescine). In contrast, a distinct impact on the amino acid proline, known to be cold-induced in other plant systems, was conspicuously absent. Levels of galactinol and raffinose, key metabolites of the cold-inducible raffinose pathway, were drastically enhanced in both leaves and roots throughout the cold acclimation period of 10 days. Furthermore, initial freezing tests and multifaceted GC/TOF-MS data processing (Venn diagrams, independent component analysis, hierarchical clustering) showed that changes in metabolite pools of cold-acclimated F. vesca were clearly influenced by genotype.