Development of taxon‐specific sequence characterized amplified region (SCAR) markers based on actin sequences and DNA amplification fingerprinting (DAF): a case study in the Phoma exigua species complex

Phoma exigua is considered to be an assemblage of at least nine varieties that are mainly distinguished on the basis of host specificity and pathogenicity. However, these varieties are also reported to be weak pathogens and secondary invaders on non‐host tissue. In practice, it is difficult to distinguish P. exigua from its close relatives and to correctly identify isolates up to the variety level, because of their low genetic variation and high morphological similarity. Because of quarantine issues and phytosanitary measures, a robust DNA‐based tool is required for accurate and rapid identification of the separate taxa in this species complex. The present study therefore aims to develop such a tool based on unique nucleotide sequence identifiers. More than 60 strains of P. exigua and related species were compared in terms of partial actin gene sequences, or analysed using DNA amplification fingerprinting (DAF) with short, arbitrary, mini‐hairpin primers. Fragments in the fingerprint unique to a single taxon were identified, purified and sequenced. Alignment of the sequence data and subsequent primer trials led to the identification of taxon‐specific sequence characterized amplified regions (SCARs), and to a set of specific oligonucleotide combinations that can be used to identify these organisms in plant quarantine inspections.     

A retrospective,dual‐isotope approach reveals individual predispositions to winter‐drought induced tree dieback in the southernmost distribution limit of Scots pine

Winter‐drought induced forest diebacks in the low‐latitude margins of species' distribution ranges can provide new insights into the mechanisms (carbon starvation, hydraulic failure) underlying contrasting tree reactions. We analysed a winter‐drought induced dieback at the Scots pine's southern edge through a dual‐isotope approach (Δ¹³C and δ¹⁸O in tree‐ring cellulose). We hypothesized that a differential long‐term performance, mediated by the interaction between CO₂ and climate, determined the fates of individuals during dieback. Declining trees showed a stronger coupling between climate, growth and intrinsic water‐use efficiency (WUEi) than non‐declining individuals that was noticeable for 25 years prior to dieback. The rising stomatal control of water losses with time in declining trees, indicated by negative Δ¹³C‐δ¹⁸O relationships, was likely associated with their native aptitude to grow more and take up more water (suggested by larger tracheid lumen widths) than non‐declining trees and, therefore, to exhibit a greater cavitation risk. Freeze‐thaw episodes occurring in winter 2001 unveiled such physiological differences by triggering dieback in those trees more vulnerable to hydraulic failure. Thus, WUEi tightly modulated growth responses to long‐term warming in declining trees, indicating that co‐occurring individuals were differentially predisposed to winter‐drought mortality. These different performances were unconnected to the depletion of stored carbohydrates.     

The importance of social information in breeding site selection increases with population size in the Eurasian Griffon Vulture Gyps fulvus

Animals can select breeding sites using non‐social information (habitat characteristics) and social information (conspecific presence or abundance). The availability of both types of information is expected to vary over time during the colonization of a new area, conditioning their use by colonizers. However, if and how both types of information are exploited during the colonization process remains unclear. We hypothesized that non‐social information should be predominant at the beginning of a colonization episode (when conspecific presence is low) and that social information should gain in importance as the colonization progresses. We tested this hypothesis by studying habitat selection by the Griffon Vulture Gyps fulvus, a long‐lived colonial raptor, during a natural colonization process spanning 40 years. In NW Spain, the population showed a sharp increase from 15 breeding pairs in three colonies in the 1970s to 586 breeding pairs in 120 colonies in 2008, expanding its range from 90 km² in the 1970s to 6403 km² in 2008, with directions of expansion following areas rich in nesting cliffs. The main determinants of habitat selection varied over time. Livestock density and the characteristics of nesting cliffs were the main predictors of settlement at the onset of colonization. Breeding density of conspecifics increased its importance over time, having the greatest relative weight in habitat selection later in the colonization process. Our results indicated a prevalent use of non‐social information during the early stages of the colonization and an increasing role of social information as the expansion progressed.     

Metabolite specific effects of solar UV-A and UV-B on alder and birch leaf phenolics

We measured the concentrations of ultraviolet (UV)‐absorbing phenolics varying in response to exclusion of either solar UV‐B or both solar UV‐A and UV‐B radiations in leaves of grey alder (Alnus incana) and white birch (Betula pubescens) trees under field conditions. In alder leaves 20 and in birch leaves 13 different phenolic metabolites were identified. The response to UV exclusion varied between and within groups of phenolics in both tree species. The changes in concentration for some metabolites suggest effects of only UV‐A or UV‐B, which band being effective depending on the metabolite. For some other metabolites, the results indicate that UV‐A and UV‐B affect concentrations in the same direction, while for a few compounds there was evidence suggesting opposite effects of UV‐A and UV‐B radiation. Finally, the concentration of some phenolics did not significantly respond to solar UV. We observed only minor effects on the summed concentration of all determined phenolic metabolites in alder and birch leaves, thus indicating that measuring only total phenolics concentration may not reveal the effects of radiation. Here, we show that the appropriate biological spectral weighting functions for plant‐protective responses against solar UV radiation extend in most cases – but not always – into the UV‐A region and more importantly that accumulation of different phenolic metabolites follows different action spectra. This demonstrates under field conditions that some of the implicit assumptions of earlier research simulating ozone depletion and studying the effects of UV radiation on plant secondary metabolites need to be reassessed.     

Development of simple sequence repeat markers for Botryosphaeria spp. with Fusicoccum anamorphs

We report the development of eight sets of microsatellite markers for the ascomycete fungus and tree pathogen, Botryosphaeria parva. The primers were identified after cloning and sequencing of fragments amplified using simple sequence repeat (SSR) primers. Genome walking was used to determine unknown sequences on either side of new SSRs. The primers were tested and proved useful in nine other Botryosphaeria species that all have Fusicoccum anamorphs, similar to B. parva.     

A rapid PCR method to discriminate between Tomato yellow leaf curl virus isolates

Tomato yellow leaf curl virus (TYLCV) was recently divided into two different species: Tomato yellow leaf curl virus‐Israel (TYLCV‐Is) and Tomato yellow leaf curl virus‐Sardinia (TYLCV‐Sar). There are no rapid methods by which TYLCV viruses may be assigned to either TYLCV‐Is or TYLCV‐Sar species. In the present work, using an extensive alignment of begomovirus sequences, TYLCV‐specific primers were designed and tested which allow the specific amplification of DNA fragments from any isolate of TYLCV. Also, a primer was designed and tested which allows the specific amplification of TYLCV‐Sar. Furthermore, a combination of these primers was selected to develop a duplex PCR method, which has the potential to detect either TYLCV‐Is or TYLCV‐Sar. The PCR methods were also highly effective with minimal sample preparation and allowed direct amplification of TYLCV from infected leaf extracts. This approach may be used in the laboratory as a tool for rapid, large‐scale diagnostics of TYLCV‐infected samples.     

Population fluctuations of mosquitoes in the non-seasonal tropics

Abstract.

• 1 Seasonal and annual fluctuations in abundance of a number of species of mosquitoes were studied in a relatively non-seasonal climate near Almirante, Panama.
• 2 The fluctuations observed were large, but did not have a period of 1 year and should be classified as non-seasonal.
• 3 At each site the various species were not synchronous in their fluctuation pattern, but the fluctuations of each species were synchronous over an area of at least 4 km long, in spite of large differences in habitat.
• 4 Changes in abundance from year to year observed in these mosquitoes are large compared with those of insects, including mosquitoes, in other areas of Panama.
• 5 The importance of such large and unpredictable fluctuations in abundance for planning control measures is discussed.

     

Effects of three modern insecticides, pyriproxyfen, spinosad and tebufenozide, on survival and reproduction of Chrysoperla carnea adults

Three novel insecticides, pyriproxyfen, spinosad and tebufenozide, were evaluated for their effect on survival and reproduction of Chrysoperla carnea adults using two methods of exposure: direct contact and ingestion. Pyriproxyfen and tebufenozide proved to be harmless to adult survival, whereas spinosad 72 h after treatment reduced the number of adults by 39.8% and 87.2% in topical and ingestion treatment at the maximum concentration recommended (800 mg a.i. litre^?1). Fecundity was not affected irrespective of the insecticide or time of application (before or after the onset of oviposition). Concerning fertility, only pyriproxyfen exerted a negative effect on hatching when the eggs were deposited by females treated by ingestion in the post‐oviposition period at the highest concentration tested (150 mg a.i. litre^?1).