Genetic diversity in the blackberry rust pathogen, Phragmidium violaceum, in Europe and Australasia as revealed by analysis of SAMPL

Indigenous to Europe, the blackberry rust fungus Phragmidium violaceum was introduced to Australia and subsequently appeared in New Zealand, with the most recent authorised introductions to Australia specifically for the biological control of European blackberry. Markers for ‘selective amplification of microsatellite polymorphic loci’ (SAMPL) were developed for studying the population genetics of P. violaceum. Modification of one of the two SAMPL primers with a HaeIII adapter (H) revealed significantly greater levels of genetic variation than primers used to generate AFLPs, the latter revealing little or no variation among 25 Australasian and 19 European isolates of P. violaceum. SAMPL was used to describe genetic variation among these 44 isolates of P. violaceum from 51 loci generated using primer pairs (GACA)₄ + H–G and R1 + H–G. The European isolates were more diverse than Australasian isolates, with 37 and 22 % polymorphic loci, respectively. Cluster analysis revealed geographic clades, with Australasian isolates forming one cluster separated from two clusters comprising the European isolates. However, low bootstrap support at these clades suggested that Australian isolates had not differentiated significantly from European isolates since the first record of P. violaceum in Australia in 1984. In general, the results support two hypotheses. First, that the population of P. violaceum in Australia was founded from a subset of individuals originating from Europe. Second, that P. violaceum in New Zealand originated from the Australian population of P. violaceum, probably by wind dispersal of urediniospores across the Tasman Sea. The application of SAMPL markers to the current biological control programme for European blackberry is discussed.     

The collection and selection in Europe of isolates of Phragmidium violaceum (Uredinales) pathogenic to species of European blackberry naturalised in Australia

Phragmidium violaceum is a rust fungus with potential for the biological control of European blackberry (Rubus fruticosus) in Australia. The collection, selection, purification and multiplication in Europe of isolates of the rust is described. Species of European blackberry naturalised in Australia showed different levels of susceptibility when inoculated with a pool of 15 isolates highly pathogenic to Rubus procerus. Selection of individual isolates on the four most widespread blackberry species showed that only two of the isolates would be required to obtain the best attack on these four species.     

Dynamics of Introduced Populations of Phragmidium violaceum and Implications for Biological Control of European Blackberry in Australia

Phragmidium violaceum causes leaf rust on the European blackberry (Rubus fruticosus L. aggregate). Multiple strains of this pathogen have been introduced into southern Australia for the biological control of at least 15 taxa of European blackberry, a nonindigenous, invasive plant. In climates conducive to leaf rust, the intensity of disease varies within and among infestations of the genetically variable host. Genetic markers developed from the selective amplification of microsatellite polymorphic loci were used to assess the population genetic structure and reproductive biology of P. violaceum within and among four geographically isolated and diseased infestations of the European blackberry in Victoria, Australia. Despite the potential for long-distance aerial dispersal of urediniospores, there was significant genetic differentiation among all populations, which was not associated with geographic separation. An assessment of multilocus linkage disequilibrium revealed temporal and geographic variation in the occurrence of random mating among the four populations. The presence of sexual spore states and the results of genetic analyses indicated that recombination, and potentially random migration and genetic drift, played an important role in maintaining genotypic variation within populations. Recombination and genetic differentiation in P. violaceum, as well as the potential for metapopulation structure, suggest the need to release additional, genetically diverse strains of the biocontrol agent at numerous sites across the distribution of the Australian blackberry infestation for maximum establishment and persistence.     

Host-specificity of Uromyces heliotropii, a fungal agent for the biological control of common heliotrope (Heliotropium europaeum) in Australia

Common heliotrope, Heliotropium europaeum (Boraginaceae), a summer annual, is a serious weed of pastures in Australia causing poisoning in sheep and other grazing animals. The weed is native to Mediterranean and Middle Eastern regions of Eurasia where it is attacked by several arthropods and pathogens including the rust fungus Uromyces heliotropii. The host-specificity of a virulent and effective strain of the rust from Turkey has been tested by inoculating 96 plants of importance to the Australasian region using both microscopic and macroscopic observations of the reaction of host and non-host plants. The test plants included several members of the Boraginaceae and related families as well as species of Heliotropium native to Australia. The infection was mostly limited to European species of Heliotropium. U. heliotropii was thus found to represent a safe introduction and has recently been introduced to Australia for the biological control of common heliotrope.     

Unusual filamentous virus-like particles in symptomless blackberry associated with severe leaf curling in the virus indicator Rubus macraei, and anomalous data using a degenerate badnavirus primer in PCR of Rubus species

Electron microscopy of ultrathin sections of leaves of symptomless Himalaya Giant blackberry and of the virus indicator species, Rubus macraei, showing severe leaf curl symptoms following graft inoculation with scions from this blackberry, detected highly flexuous virus‐like particles with an unusual ‘beaded’ structure. Such particles were restricted to a few vascular cells and were distinct from P‐protein common in some such cells. This virus, provisionally named Hawaiian rubus leaf curl virus (HRLCV), symptomlessly infected a wide range of Rubus species and cultivars. Badnavirus‐like bacilliform particles were observed in some cells of a single R. macraei plant showing leaf curl symptoms following graft inoculation with the causal agent of this disease symptom from Himalaya Giant blackberry after passage through red raspberry, but not in any other material. PCR with primer sets for the badnaviruses Rubus yellow net virus and Gooseberry veinbanding associated virus, showed that no Rubus sources studied contained these viruses. However, using a sequence‐specific primer set designed from the sequence of the product generated with a badnavirus degenerate primer set, a specific product was amplified from healthy plants of all of 16 raspberry cultivars and two Rubus species, but not from 16 blackberry cultivars (including cv. Himalaya Giant). All of these sources were free from viruses known to occur in Rubus. Sequence analysis of this product showed no homology with any known badnavirus, or with any other published sequences. It seems most likely therefore that a region of the raspberry genome has been amplified using the degenerate badnavirus primer set and that it is absent from the blackberry genome.     

An Evaluation of the Rust FungusGymnoconia nitensas a Potential Biological Control Agent for AlienRubusSpecies in Hawaii

The rust fungusGymnoconia nitensinfects blackberry (Rubus argutus) systemically in regions of the continental United States, producing bright yellow–orange masses of spores on newly developing floricanes during springtime. In tests to determine the suitability of this rust as a biological control agent forR. penetransin Hawaii, a species now thought to be conspecific withR. argutus,rooted cuttings of the Hawaiian plants were grown at North Carolina State University, inoculated, and observed. Other introduced weedyRubusspp. in Hawaii, includingR. ellipticus, R. rosifolius,andR. glaucus,as well as the two endemic speciesR. hawaiensisandR. macraei,also were inoculated. No species ofRubusare of commercial importance in Hawaii, but the protection of the native species, of whichR. macraeiis rare, was of utmost concern. The native Hawaiian species did not survive well in North Carolina in this study, however. Later availability of a plant pathogen containment laboratory in Hawaii enabled similar tests to be conducted at that facility. In addition to the above species,R. spectabilis(salmonberry), a species native to the Pacific Northwest with which the HawaiianRubusspp. are thought to share a common ancestor, was inoculated in Hawaii. Infection withG. nitensunder natural field conditions becomes apparent only when sporulation occurs on floricanes the second year following infection. However, experimental inoculation led to early responses of chlorotic leaf flecking and puckering, leaf and stem contortion, and stem gall formation, indicating the sensitivity ofR. penetrans(=R. argutus),R. hawaiensis,andR. macraeito this rust. Apparent systemic infection also resulted in sporulation on one plant ofR. macraei.Ability to attack the endemic species suggests thatG. nitenswould not be suitable for release in Hawaii as a biological control agent, at least on the islands with populations of the native species.     

Purification and properties of blackberry chlorotic ringspot, a new virus species in subgroup 1 of the genus Ilarvirus found naturally infecting blackberry in the UK

A mechanically transmissible virus was isolated from Bedford Giant blackberry plants showing chlorotic mottling and ringspot symptoms growing in Scotland. It infected several herbaceous test plants, many of them symptomlessly. This virus was also transmitted to several Rubus species and cultivars by graft inoculation with scions from the field‐infected Bedford Giant plant. Most grafted plants were infected symptomlessly, but Himalaya Giant blackberry and the hybrid berry Tayberry developed symptoms similar to those in the infected Bedford Giant plant. In the sap of infected Chenopodium quinoa, the virus lost infectivity when diluted 10^?4 but not 10^?3, after 6 h and 48 h when kept at 20°C and 4°C, respectively, but was infective for more than 8 days when kept at ?15°C. Preparations of purified virus from infected C. quinoa or spinach sedimented as three major nucleoprotein components and consisted of quasi‐isometric particles that varied in size from 24 to 32 nm in diameter and that were not penetrated by negative stain. Such virus particle preparations contained a major polypeptide of ca 28 kDa and three single‐stranded RNA species of estimated size 3.2, 2.8 and 2.1 kb. The complete sequence of the largest RNA (RNA 1, 3478 nt) and the partial sequence of the other RNAs (1863 and 2102 nt long, respectively) were determined and compared with sequences in databases. These findings, together with the biological and biochemical properties of this virus, indicate that it should be regarded as a distinct species in subgroup 1 of the genus Ilarvirus even though it was serologically unrelated to existing members of this subgroup. The virus showed a very distant serological relationship with prune dwarf virus (PDV) but differed significantly from it in the amino acid sequence of its coat protein, experimental host range and symptomatology and was unrelated to PDV at the molecular level. The virus, tentatively named blackberry chlorotic ringspot virus, is therefore a newly described virus and the first ilarvirus found naturally infecting Rubus in the UK.     

Genomic characterisation and the detection of raspberry chromatin in polyploid Rubus

 This paper reports genomic in situ hybridization (GISH) and fluorescent in situ hybridization (FISH) data for chromosomes of raspberry (Rubus idaeus 2n=2x=14), blackberry (Rubus aggregate, subgenus Eubatus. 2n=2–12x=14–84) and their allopolyploid derivatives used in fruit breeding programmes. GISH was used to discriminate labelled chromosomes of raspberry origin from those of blackberry origin in allopolyploid hybrid plants. The raspberry chromosomes were labelled by GISH at their centromeres, and 1 chromosome was also labelled over the short arm. In one allopentaploid plant a chromosome carried a terminal signal. Karyotype analysis indicated that this is a blackberry chromosome carrying a raspberry translocation. GISH analysis of an aneuoctaploid blackberry cv ‘Aurora’ (2n=8x=58) showed that both whole and translocated raspberry chromosomes were present. The basic Rubus genome has one ribosomal DNA (rDNA) locus, and in all but one case all levels of ploidy had the expected multiples of rDNA loci. Interestingly, in the blackberry cv ‘Aurora’, there were only six sites, two less than might be predicted from its aneuoctaploid chromosome number. Our results highlight the potential of GISH and FISH for genomic designation, physical mapping and introgression studies in Rosaceous fruit crops. Received: 20 February 1998 / Accepted: 12 May 1998     

Mycoplasma-like bodies associated with Rubus stunt disease

Mycoplasma-like bodies were found by electron microscopy in the sieve tubes of blackberry, loganberry and raspberry plants showing symptoms of Rubus stunt disease, but not in those of normal plants. Watering diseased blackberry plants with aureomycin caused remission of the symptoms and greatly decreased the number of mycoplasma-like bodies.     

The host specificity ofHartigia albomaculatus (Hym.: Cephidae] and its potential effectiveness in the biological control of European blackberry

The host specificity of the cephid stem-borerHartigia albomaculatus (Stein), was studied to determine its suitability for the biological control of European blackberry (Rubus fruticosus L. agg.) in Australia. Field observations indicated that the insect was specific toR. fruticosus, however laboratory tests showed that it can attack some cultivatedRubus and garden rose (Rosa spp.) varieties. Raspberry (R. idaeus L.) the most importantRubus crop in Australia, was not attacked, nor were 4Rubus spp. endemic to Australia. Observations in the Montpellier area, France, indicate thatH. albomaculatus has no significant detrimental effect on the vegetative reproduction of its host.     

Foliar indumentum in central‐European Rubus species (Rosaceae) and its contribution to the systematics of the group

The foliar indumentum of 94 taxa of Rubus has been surveyed by scanning electron microscopy. The analysed species belonged to five subgenera. Rubus subgen. Rubus with its three sections and 21 series was the most numerously represented. In all studied species, indumentum was present, but the species differed more or less from each other with respect to the density of the indumentum on the abaxial leaf surface. Three main hair types were present: simple eglandular (unbranched) trichomes, branched eglandular trichomes, and very short secretory uni‐seriate trichomes; subtypes were distinguished according to trichome size. Apart from investigating separate traits, we described character patterns for the best represented and homogeneous series: Discolores, Subthyrsoidei, Rhamnifolii and Rubus. Other large groups (Sylvatici, Glandulosi and Micantes) were more diverse.     

Genetic relationships among wild and cultivated blackberries (Rubus caucasicus L.) based on amplified fragment length polymorphism markers

Abstract

Rubus is a large genus of flowering plants in the rose family, Rosaceae, subfamily Rosoideae. The blackberries, as well as various other Rubus species with mounding or rambling growth habits, are often called brambles. Little information is available on the genetic diversity of wild-grown blackberries. The objective of this study was to determine the genetic relationships among nine promising (high-yield capacity, free of pest and diseases, better fruit traits) wild blackberry (Rubus caucasicus L.) selections and the well-known cultivar, “Chester” by using amplified fragment length polymorphism (AFLP) markers. Genotypes were evaluated with three selective primer-enzyme combinations, producing a total of 223 AFLP fragments with 53% polymorphism ratio. Clustering of genotypes using unweighted pair-group method of arithmetic average cluster analysis clearly separated groups of wild blackberry genotypes while the variety “Chester” was clustered independently. Wild selections represented a distinct germplasm source on the basis of the estimated genetic distance among them. Genetic diversity data from this study will be helpful in using and exploiting the wild genetic material for breeding purposes as well as for further research.     

Behavioral analysis of learning and memory in Anastrepha fraterculus

We evaluate the influence of prior exposure to artificial substrate for oviposition on learning and memory in the fruit fly Anastrepha fraterculus (Wiedemann) (Diptera: Tephritidae). Some females were previously exposed to artificial fruits made of water, agar, and blackberry [Rubus spec. (Rosaceae)] or guava [Psidium guajava L. (Myrtaceae)] pulp for 48 and 72 h. We also studied adult flies exposed for 72 h to essential oil of lemongrass [Cymbopogon citratus (DC) Stapf, Poaceae] and adult flies from larvae exposed to the oil. Control females were naive with respect to these experimental substrates. Prior experience with blackberry‐based artificial fruits resulted in an increase in the number of punctures and deposited eggs by A. fraterculus, and memory lasted for up to 72 h. On the other hand, fly behavior was independent of exposure to guava‐based substrate. Prior exposure of 1‐ or 15‐day‐old females to artificial substrate with lemongrass oil modified innate substrate selection behavior. The scent of lemongrass oil during the larval stage modified innate oviposition responses of adult A. fraterculus. The study shows that A. fraterculus females are able to learn and retain information through chemical stimuli released by both host (blackberry and guava) and non‐host (lemongrass) species, and they can use olfactory memory obtained during the larval stage to select oviposition sites.     

Taxonomy and phylogeny of Puccinia lagenophorae: a study using rDNA sequence data, morphological and host range features

Puccinia lagenophorae is a rust fungus originating from Australasia which has spread throughout the world. A phylogenetic analysis of taxa related to this species was performed using rDNA (LSU, ITS) sequence data. The analyses revealed a well-supported cluster including all specimens of P. lagenophorae. By evaluating morphological and sequence data, the species is taxonomically re-defined and a list of synonyms is provided. Puccinia distincta on Bellis perennis, a species recently separated from P. lagenophorae, P. saccardoi, a species on the Goodeniaceae, and P. byliana, a species so far only known from South Africa, are reduced to synonymy in P. lagenophorae, as are several other species. Our analysis indicates that P. lagenophorae is likely not derived from the northern hemisphere species P. obscura, but from a species from Australia host-alternating between Asteraceae (aecial host) and Cyperaceae/Juncaceae (telial host). Another related species, P. stylidii (on Stylidium sp., Stylidiaceae) may have been derived from the same parental species as P. lagenophorae. From ontogenetical and morphological studies, the presence of pycnia could not be confirmed in the life cycle of this species, and the width of the pedicel of teliospores at the point of attachment was found to be highly variable and not a taxonomic character. The number of known host species is approximately 150, including 41 new host plants recorded herein.     

Incidence and genetic diversity of raspberry bushy dwarf virus (RBDV) in Rubus spp. in Turkey

Raspberry bushy dwarf virus (RBDV), recently renamed to Idaeovirus rubi, is one of the most common viruses infecting Rubus species worldwide but there is still a limited number of genome sequences available in the GenBank database and the majority of the sequences include partial sequences of RNA-1 and RNA-2. The distribution and incidence of RBDV in main raspberry and blackberry growing provinces in Turkey were monitored during 2015–2019 and 537 Rubus spp. samples were tested by both DAS-ELISA and RT-PCR. Among the tested samples, 36 samples tested positive for RBDV by DAS-ELISA and 67 samples by RT-PCR. There was relatively low nucleotide diversity among the Turkish isolates. Turkish isolates shared 93%–97.7%, 84.3%–98.9%, and 85%–99.2% nucleotide sequence identities with available sequences in the GenBank, in partial RNA-1, movement protein (MP) and coat protein (CP) genes, respectively. In the phylogenetic tree constructed for RNA-1, MP, and CP sequences, all Turkish raspberry isolates were clustered in a distinct clade. However, the blackberry isolates showed considerable variation in nucleotide sequences and were placed in three distinct groups. The divergent blackberry isolates showed high variability in MP (84.5%–89.3%) and CP (85.5%–89.7%) regions and were placed in a distinct group. The rest of blackberry isolates clustered together with sweet cherry RBDV isolates adjacent to the grapevine clade or together with raspberry isolates. The comparative analysis conducted on three RNA segments of RBDV highlighted the high sequence diversity of Turkish RBDV isolates. This study also emphasizes the importance of regular monitoring of RBDV infections in Turkey, with special regard to those Rubus spp. and grapevine accessions employed in conservation and selection programmes. In particular, the presence of new RBDV genetic variants and infection of Rubus species must be taken into account to choose a correct detection protocol and management strategy.     

Influences of blackberry margins on population dynamics of Drosophila suzukii and grape infestation in adjacent vineyards

Understanding the dynamics of pest insect populations in relation to the presence of non‐crop habitats and infestation levels of adjacent crops is essential to develop sustainable pest management strategies. The invasive pest species Drosophila suzukii (Diptera: Drosophilidae) is able to utilize a broad range of host plants. In viticulture, scientific risk assessment for D. suzukii has only recently started and studies assessing the effects of field margins containing wild host plants on D. suzukii population dynamics and on infestation risks in adjacent vineyards are lacking. Thus, in a one‐year field study, the role of different field margins on fly abundance and crop infestation in adjacent vineyards of Vitis vinifera, variety “Pinot Noir,” were investigated. Different monitoring methods were conducted to assess fly distribution, sex ratio and grape infestation in 14 vineyards adjacent to field margins containing either blackberry (BB) Rubus spp. or non‐host (NH) plants. Our results show that blackberries strongly enhanced D. suzukii abundance within field margin vegetation all year long, whereas fly abundance in vineyards adjacent to BB margins was just enhanced in some seasonal periods. Moreover, the influence of BB margins was limited by distance. However, high fly numbers in BB field margins did result in zero egg infestation of “Pinot Noir” berries. These results may have important implications for winegrowers to make efficient management decisions: regardless of high abundance of adult D. suzukii, only grape berry monitoring can assess the actual infestation risk and the potential need to take management action.     

Chromosome numbers of European blackberries (Rubus subg.Rubus,Rosaceae)

Chromosome numbers are presented for 32 collections of 29 European blackberry species (Rubus subg.Rubus) from Germany. One species is triploid (2n = 21), 27 species are tetraploid, (2n = 28), and one species is pentaploid (2n = 35). Chromosome numbers are reported for the first time ofR. adspersus, R. amisiensis, R. calvus, R. conothyrsoides, R. contractipes, R. demissus, R. elegantispinosus, R. ferocior, R. foliosus, R. hypomalacus, R. leucandrus, R. nemorosus, R. platyacanthus, R. praecox, R. rhombifolius, andR. rhytidophyllus. Chromosome numbers forR. dasyphyllus, R. gelertii, R. glandithyrsos, R. lamprocaulos, R. lindebergii, R. macrophyllus, R. montanus, R. muenteri, R. pedemontanus, R. polyanthemus, R. senticosus, R. silvaticus, andR. vigorosus are confirmed.     

Isolation of 44 polymorphic microsatellite loci in three host races of the phytopathogenic fungus Microbotryum violaceum

We report the development of 44 microsatellite markers in three host races of the fungus Microbotryum violaceum, a sexually transmitted disease of the Caryophyllaceae. An enrichment protocol was used to isolate microsatellite loci from three strains, collected, respectively, from the plant species Gypsophila repens, Dianthus sylvestris, and Silene vulgaris. Polymorphism and cross‐amplification were explored with 32 isolates of M. violaceum, collected on 12 different plant species in natural populations.     

Melting pot of biodiversity: first insights into the evolutionary patterns of the Colchic bramble flora (Rubus subgenus Rubus,Rosaceae)

The Caucasus is a biodiversity hotspot of global significance, containing a number of highly diverse and species‐rich plant taxa. The region is also thought to be an important evolutionary hotspot for Rubus subgenus Rubus (brambles). However, Caucasian brambles have only been poorly studied to date and our knowledge of their evolutionary mechanisms, systematics and taxonomic variability remains rudimentary. Therefore, the objectives of this study were to shed light on the evolution, diversity and reproduction modes of Rubus in one of the two Caucasian glacial refugia, Colchis. Flow cytometry measurements were used to estimate DNA ploidy, a flow cytometric seed screen was conducted to determine reproduction mode and Sanger sequencing of two non‐coding plastid regions was used to reveal phylogenetic patterns. The most common ploidy level was tetraploid, followed by diploid and (rarely) triploid. Intra‐individual variation in reproduction mode was low, as the morphoseries Glandulosi and Radula exhibited strict sexuality and other taxa were mostly apomictic. A few exceptions were observed that deserve further attention, e.g. sexuality induced hypothetically by haploid pollen or by environmental conditions, a high proportion of triploid embryos or polyspermy. Plastid haplotype variability revealed specific, ancient evolutionary patterns with limited involvement of extant diploid taxa and recent isolation from European brambles. We provide the first insight into the variability and evolution of Colchic brambles, which serves as a starting point for further systematic and evolutionary studies.