Phylogeography and conservation perspectives of an endangered macaronesian endemic: Picconia azorica (Tutin) Knobl. (Oleaceae)

The Azores provide an excellent field test for research activities aimed at developing conservation strategies for endangered tree species. In this work, the urgency to promote Picconia azorica conservation programs addressed (a) insights into the biotaxonomy of the species (including an evaluation of its origin and relationships with the only congeneric species of P. azorica: P. excelsa from the Canary and Madeira islands) and (b) the evaluation of P. azorica genetic diversity. Plastid DNA sequence analysis and molecular markers (RFLP and SSR) were used for this purpose. Phylogenetic data suggest the monophyly of Picconia and support a late Miocene divergence of the two species. Three polymorphic cpSSR loci allowed the identification of five different haplotypes in P. azorica. Uniqueness and relictuality of lineages are presented and discussed. Picconia azorica intra-specific diversity patterns revealed low genetic diversity and a weak genetic structure, which could result from long-lasting ecological stability and efficient inter-island seed movement that have been severely affected in recent times. The species survival is at risk, and we suggest management practices focusing on ex situ and in situ conservation units based on eco-genetic data. Additional measures contributing to mild erosion of the genepool and to remove barriers to seed dispersal are indicated.     

First record of Ektaphelenchoides pini associated with Ips sexdentatus on Pinus nigra laricio in Italy

In February 2015, an unexpected windstorm downed five hectares of a European black pine Pinus nigra subsp. laricio forest formation located close to Vallombrosa, Florence (Central Italy). In the following spring, an extensive survey was conducted in the area. Felled trees, stumps and all the suitable plant material were screened for the presence of the pinewood nematode (PWN), Bursaphelenchus xylophilus, by sampling wood and bark. Bark beetles were then collected from the gallery systems on the inner side of bark samples and observed in the laboratory. The following bark beetles were morphologically identified: Ips sexdentatus, Orthotomicus erosus, O. laricis and Pityogenes bidentatusa. The dissection of Ips sexdentatus allowed the extraction of numerous nematodes that were morphologically and molecularly identified as Ektaphelenchoides pini. Conversely, only few nematode specimens were isolated from either pine bark or wood. These individuals could be only molecularly identified and belonged to an undescribed nematode taxon. Even though no PWN was recorded in the investigated sites, our survey allowed the detection of a new association between E. pini and I. sexdentatus on P. nigra.     

Activity changes of glyoxalase system enzymes and glutathione-S-transferase in the bivalve mollusc Scapharca inaequivalvis exposed to the organophosphate chlorpyrifos

The present report was aimed to study possible changes in the activity of glutathione-dependent enzymes glyoxalase I (G I) and glyoxalase II (G II), forming the glyoxalase system, and glutathione-S-transferase in tissues (gills, foot, and digestive gland) of the mediterranean bivalve mollusc Scapharca inaequivalvis, after a 4- or 15-day exposure to a sublethal concentration (0.1 ppm) of the organophosphate pesticide chlorpyrifos (CPF). Control experiments were also performed on untreated animals. G I activity decreased in gills and foot, and increased in the digestive gland after 4 days of CPF exposure. Glyoxalase II activity in the gills decreased after a 15 days exposure to CPF, was unchanged in the foot and increased after 15 days in the digestive gland. Glutathione-S-transferase activity, which is involved in the detoxification of xenobiotics, increased significantly in all tissues after CPF exposure. The results suggest that sublethal CPF exposure of S. inaequivalvis does not compromise the cellular redox balance in these tissues.     

Elimination of in vitro bacterial contaminants in shoot cultures of ‘MRS 2/5’ plum hybrid by the use of <Emphasis Type="Italic">Melia azedarach</Emphasis> extracts

The antimicrobial activity of leaf and callus extracts of Melia azedarach was tested on in vitro shoot cultures of the peach rootstoch ‘MRS 2/5’ (Prunus cerasifera × Prunus spinosa) that were heavily contaminated with Sphingomonas paucimobilis (Sp) and Bacillus circulans (Bc). The extracts were filter-sterilised and added at 0%, 1%, 5%, 10% and 20% to a modified Murashige and Skoog proliferation medium previously autoclave-sterilised. Up to about 17% shoots died with 10–20% extract, except for Sp-contaminated shoots, whose survival was reduced to 50% after treatment with 20% extract. No shoots died with 1% to 5% supplement. The undiluted leaf extract showed bactericidal activity on plated Sp and Bc isolates. The homogenates of shoots randomly collected from treated cultures were processed for bacterial colony counting. Thus the 10% supplement was the best treatment for ridding Bc-contaminated cultures of bacteria (although 5% had a similar bactericidal effect), and allowing shoot growth and proliferation comparable to controls at the fifth subculture on a standard medium, while 20% extract was needed to eliminate Sp, and could induce higher growth and proliferation rates in surviving shoots than in untreated cultures. Callus extract was ineffective. The bactericidal activity of the leaf extract seemed attributable to a synergistic effect of azadirachtin with other unidentified compounds present in the extract.     

Reference Intervals of Serum Protein Concentrations from Clinically Healthy Female Ragusana Donkeys (Equus asinus) Determined by Cellulose Acetate Electrophoresis

The aim of this study was to evaluate total serum protein concentration measured using biuret reaction and the protein fractions determined using acetate cellulose electrophoresis in Ragusana donkeys (Equus asinus). Blood samples were collected from 68 clinically healthy female donkeys by jugular venipuncture. The serum levels of total proteins were determined using biuret method, and the separation of proteins was performed using acetate cellulose electrophoresis. Coefficients of variation were also calculated for within-assay precision, and were found to be less than 5% for α- and β₁-globulins and 8% or less for albumin, β₂-, and γ-globulins. A total of five protein fractions were separated and quantified: albumin, α-, β₁-, β₂-, and γ-globulins. Data obtained from young and adult subjects were compared using the Mann–Whitney U test. Reference intervals (2.5%-97.5% quantiles) were determined for total proteins (50.0-84.0 g/L), albumin (16.2-36.6 g/L), α-globulins (4.85-19.5 g/L), β₁-globulins (2.25-10.35 g/L), β₂-globulins (3.30-14.85 g/L), γ-globulins (10.0-30.5 g/L), and albumin/globulin ratio (0.41-1.13). In relation to age, statistically significant differences were found in total protein concentration and γ-globulins. The results obtained in the present study contributed to establish reference intervals of serum protein fractions obtained using acetate cellulose electrophoresis in female Ragusana donkeys to be used by practitioners for health control.     

Performance of diagnostic tests for the detection and identification of <Emphasis Type="Italic">Pseudomonas syringae</Emphasis> pv. <Emphasis Type="Italic">actinidiae</Emphasis> (Psa) from woody samples

The aim of this study was to characterise the performance of new molecular methods for the detection and identification of Pseudomonas syringae pv. actinidiae (Psa) and to provide validation data in comparison to the assays mentioned in official diagnostic protocols and being currently used. Eleven molecular tests for the Psa detection were compared in an inter-laboratory comparison where each laboratory had to analyse the same panel of samples consisting of thirteen Psa-spiked kiwifruit wood extracts. Laboratories had to perform also isolation from the wood extracts. Data from this interlaboratory test performance study (TPS) was statistically analysed to assess the performance of each method. In order to provide complete validation data, both for detection and identification, this TPS was supplemented by a further study of identification from pure culture of phylogenetically closely related Pseudomonas spp., Psa, and bacterial strains associated with kiwifruit. The results of both these studies showed that simplex-PCRs gave good results, whereas duplex-PCR and real-time PCR were the most reliable tools for detection and identification of Psa. Nested and multiplex-PCR gave false-positive results. The use of the most reliable detection test is suggested for routine analyses, but when Psa-free status needs to be accurately assessed, it is recommended that at least two detection tests are used. This work provides a wide comparison of the available diagnostic methods, giving new information for a possible revision of the official diagnostic protocols (e.g. European and Mediterranean Plant Protection Organization (EPPO) protocol PM7/120 for the detection of Psa).     

Morphological and molecular identification of potato and cereal cyst nematode isolates from Algeria and their phylogenetic relationships with other populations from distant theirgeographical areas

A nematode survey conducted in 2013 in Algeria, revealed that potato cyst nematodes (PCN) and cereal cyst nematodes (CCN) are widely distributed in several potato and cereal growing regions of the country. Sixteen PCN populations from five localities and five CCN populations from four of these localities were collected and characterized at the morphological and molecular levels. The PCN populations were identified as Globodera rostochiensis and G. pallida occurring separately or in mixed populations. Two species of CCN were detected. Heterodera avenae was found in four localities, whereas H. hordecalis only in one locality in association with H. avenae. The morphological and morphometric identification of PCN and CCN was confirmed by diagnostic ITS-RFLP profiles and sequencing. Phylogenetic analysis of the ITS, D2-D3 expansion domains of the 28S rRNA gene and 18S rRNA gene was made for PCN and CCN populations. Globodera pallida and G. rostochiensis from Algeria show great similarity with European and South American populations. Because of the high divergence among Algerian populations of G. pallida and G. rostochiensis it can be assumed that they were multi-introduced in Algeria. The most divergent population of G. pallida, that formed a well-separated group with some populations from Chile and Peru, suggests a later or independent introduction of this population into Algeria. Heterodera avenae and H. hordecalis formed a well-supported cluster with the corresponding populations.     

Cytochrome c oxidase subunit 1 analysis of <Emphasis Type="Italic">Xiphinema diversicaudatum,X. pachtaicum,X. simile</Emphasis> and <Emphasis Type="Italic">X. vuittenezi</Emphasis> (Nematoda,Dorylaimida)

Genetic analyses using sequences of partial cytochrome c oxidase subunit 1 (cox1) gene of mitochondrial DNA were conducted to determine the extent of genetic variation within and among Xiphinema diversicaudatum, X. pachtaicum, X. simile and X. vuittenezi populations. Pairwise distance among the four species was 22.5 to 31.2%. Four different sequence variants of cox1 were determined among six populations of X. diversicaudatum and three variants among three populations of X. simile. Nucleotide variation was detected at 18 of 414 bp (1.9 to 2.7%) in X. diversicaudatum and 4 of 435 bp (0.2 to 0.9%) in X. simile. All changes were at silent sites. No nucleotide variation was detected within three populations of X. pachtaicum and within three populations of X. vuittenezi.     

Detection of Botryosphaeriaceae species within grapevine woody tissues by nested PCR, with particular emphasis on the Neofusicoccum parvum/N. ribis complex

Several species of Botryosphaeriaceae and Phaeomoniella chlamydospora are common agents of grapevine decline worldwide. Currently, the use of culture independent PCR based techniques for detection of Botryosphaeriaceae within grapevine tissues has been limited to Botryosphaeria dothidea. In the present study, two Botryosphaeriaceae specific nested PCR assays were developed. One with a narrow target range, to detect Neofusicoccum parvum and the closely related species complex (Neofusicoccum parvum/N. ribis sensu Pavlic et al. Molecular Phylogenetics and Evolution 51:259–268, 2009) and another, with a wider range, to detect all 17 species of Botryosphaeriaceae which have been reported as potential wood pathogens of grapevine. The effectiveness of these assays was validated in vivo on naturally infected wood samples collected from standing vines and dormant grafted rooted cuttings commercialized in Italy by different nurseries in different years. All samples were also screened by means of a previously published nested PCR assay specific for Phaeomoniella chlamydospora. It was found that: 1) propagation material may play an important role as source of primary inoculum, not only of Phaeomoniella chlamydospora, as previously reported, but also for members of the Botryosphaeriaceae, among which Neofusicoccum parvum, Botryosphaeria dothidea and Diplodia seriata are the most common, and 2) multiple infections by different species belonging to Botryosphaeriaceae and/or Phaeomoniella chlamydospora occur frequently both in standing vines and propagation material. This last finding supports the hypothesis that at least some of the non-specific symptoms of grapevine decline may be due to the presence of different pathogens within host tissues.     

Agroforestry as a sustainable land use option to reduce wildfires risk in European Mediterranean areas

Agroforestry Systems - Wildfires have always been an integral part of the ecology of many terrestrial ecosystems, but their frequency is increasing in many parts of the world. Wildfires were once a...