The legacy of past droughts induces water-sparingly behaviour in Grüner Veltliner grapevines.

Drought is becoming more frequent and severe in numerous wine-growing regions. Nevertheless, limited research has examined the legacy of recurrent droughts, focusing on leaf physiology and anatomy over consecutive seasons. We investigated drought legacies (after 2 years of drought exposure) in potted grapevines, focusing on stomatal behaviour under well-watered conditions during the third year. Vines were subjected for two consecutive years to short- (SD) or long-term (LD) seasonal droughts, or well-watered conditions (WW). In the third year, all plants were grown without water limitation. Water potential and gas exchange were monitored throughout the three seasons, while leaf morpho-anatomical traits were measured at the end of the third year. During droughts (1st and 2nd year), stem water potential of SD and LD plants fell below -1.1 MPa, with a consequent 75% reduction in stomatal conductance (gs ) compared to WW. In the 3rd year, when all vines were daily irrigated to soil capacity (midday stem water potential ~ -0.3 MPa), 45% lower values of gs were observed in the ex-LD group compared to both ex-SD and ex-WW. Reduced midrib vessel diameter, lower leaf theoretical hydraulic conductivity, and smaller stomata were measured in ex-LD leaves compared to ex-SD and ex-WW, likely contributing to the reduced gas exchange. Our findings suggest that grapevines exposed to drought may adopt a more water-conserving strategy in subsequent seasons, irrespective of current soil water availability, with the degree of change influenced by the intensity and duration of past drought events.

The unstable 'clone': evidence from monitoring AFLP-based mutations for short-term clonal genetic variation in two asexual lineages of the grain aphid, Sitobion avenae (F.).

Clones have been in the forefront of biological interest for many years. Even so, open discussions continue to surround the concept of clonality, which has been recently much debated in the scientific literature, both in terms of philosophical meaning as well as empirical determination. Philosophically, the clone is the horizontally produced lineage from a single fertlized egg (e.g. mammals by division of the fertilized egg and representing a single generation) or vertically produced offspring (e.g. aphids representing different successive generations) from a single asexual stem mother (originally for a particular lineage, following hatching of the overwintering sexual egg in the spring); empirically, the aspect of genetic fidelity is also considered important, so-called clones being assumed to have an identical genome among clone mates. In reality of course, such members of a clonal lineage must differ at various regions of the genome, since mutation is a fundamental property of the DNA itself. Yet few studies have so far set out to show this empirically in eukaryotic organisms, which indulge in periods of asexual reproduction, sometimes, as in aphids, over many generations. In the present study, we have investigated asexual lineages of the grain aphid, Sitobion avenae (F.), a global pest of cereals, over five successive generations employing AFLP-PCR molecular techniques. Our main interest was to see how much variation was present in the early generations and if this variation was transmitted through the asexual lineages. By monitoring AFLP-based polymorphisms, we show that, in this aphid species, of a total of 110 individuals from two lineages tested (termed SA and SB), random mutations (band deletions, more rarely additions) were apparent from the third generation onwards, and although some mutations were found to be transmitted transgenerationally, others were rarely transmitted through the particular lineages they were detected in. Using Arlequin v. 2.0, average gene diversity within the lineages was found to be 0.024 ± 0.013 and 0.031 ± 0.016 for SA and SB, respectively. It was also found from the rearing of the lineages that one lineage, SA, was more fecund than the other lineage, SB, over the five generations (N = 818 vs. N = 358 total stem mothers plus nymphs for the two lineages, respectively).

Expression of putative expansin genes in phylloxera (Daktulosphaira vitifoliae Fitch) induced root galls of Vitis spp.

Grape phylloxera (Daktulosphaira vitifoliae Fitch) is a serious global pest in viticulture. The insects are sedentary feeders and require a gall to feed and reproduce. The insects induce their feeding site within the meristematic zone of the root tip, where they stay attached, feeding both intra- and intercellularly, and causing damage by reducing plant vigour. Several changes in cell structure and composition, including increased cell division and tissue swelling close to the feeding site, cause an organoid gall called a nodosity to develop. Because alpha expansin genes are involved in cell enlargement and cell wall loosening in many plant tissues it may be anticipated that they are also involved in nodosity formation. To identify expansin genes in Vitis vinifera cv. Pinot noir, we mined for orthologues genes in a comparative analysis. Eleven putative expansin genes were identified and shown to be present in the rootstock Teleki 5C (V. berlandieri Planch. x V. riparia Michx.) using specific PCR followed by DNA sequencing. Expression analysis of young and mature nodosities and uninfested root tips were conducted via quantitative real time PCR (qRT-PCR). Up-regulation was measured for three putative expansin genes (VvEXPA15, -A17 and partly -A20) or down-regulation for three other putative genes (VvEXPA7, -A12, -A20) in nodosities. The present study clearly shows the involvement of putative expansin genes in the phylloxera-root interaction.

Evaluation of different derivatisation approaches for gas chromatographic-mass spectrometric analysis of carbohydrates in complex matrices of biological and synthetic origin.

Gas chromatographic analysis of complex carbohydrate mixtures requires highly effective and reliable derivatisation strategies for successful separation, identification, and quantitation of all constituents. Different single-step (per-trimethylsilylation, isopropylidenation) and two-step approaches (ethoximation-trimethylsilylation, ethoximation-trifluoroacetylation, benzoximation-trimethylsilylation, benzoximation-trifluoroacetylation) have been comprehensively studied with regard to chromatographic characteristics, informational value of mass spectra, ease of peak assignment, robustness toward matrix effects, and quantitation using a set of reference compounds that comprise eight monosaccharides (C(5)-C(6)), glycolaldehyde, and dihydroxyacetone. It has been shown that isopropylidenation and the two oximation-trifluoroacetylation approaches are least suitable for complex carbohydrate matrices. Whereas the former is limited to compounds that contain vicinal dihydroxy moieties in cis configuration, the latter two methods are sensitive to traces of trifluoroacetic acid which strongly supports decomposition of ketohexoses. It has been demonstrated for two "real" carbohydrate-rich matrices of biological and synthetic origin, respectively, that two-step ethoximation-trimethylsilylation is superior to other approaches due to the low number of peaks obtained per carbohydrate, good peak separation performance, structural information of mass spectra, low limits of detection and quantitation, minor relative standard deviations, and low sensitivity toward matrix effects.

Analysis of genetic variation within clonal lineages of grape phylloxera (Daktulosphaira vitifoliae Fitch) using AFLP fingerprinting and DNA sequencing.

Two AFLP fingerprinting methods were employed to estimate the potential of AFLP fingerprints for the detection of genetic diversity within single founder lineages of grape phylloxera (Daktulosphaira vitifoliae Fitch). Eight clonal lineages, reared under controlled conditions in a greenhouse and reproducing asexually throughout a minimum of 15 generations, were monitored and mutations were scored as polymorphisms between the founder individual and individuals of succeeding generations. Genetic variation was detected within all lineages, from early generations on. Six to 15 polymorphic loci (from a total of 141 loci) were detected within the lineages, making up 4.3% of the total amount of genetic variation. The presence of contaminating extra-genomic sequences (e.g., viral material, bacteria, or ingested chloroplast DNA) was excluded as a source of intraclonal variation. Sequencing of 37 selected polymorphic bands confirmed their origin in mostly noncoding regions of the grape phylloxera genome. AFLP techniques were revealed to be powerful for the identification of reproducible banding patterns within clonal lineages.

Ecological and genetic aspects of grape phylloxera Daktulosphaira vitifoliae (Hemiptera: Phylloxeridae) performance on rootstock hosts.

Performance and genetic variability of clonal lineages derived from one Californian and one German population of grape phylloxera, Daktulosphaira vitifoliae Fitch were studied on their natal grape rootstock host and on three novel hosts over four generations in an aseptic dual culture system. The ability of D. vitifoliae to adapt to new hosts was measured by changes in fitness (rm) over four generations. The performance of a given clonal lineage changed over successive generations, depending upon the host plant and the phylloxera group. Analysis of amplified fragment length polymorphism-polymerase chain reaction (AFLP-PCR) banding patterns from 40 individual parthenogenetic D. vitifoliae revealed equal levels of genetic variation both among the four clonal lineages analysed and within the different generations of one lineage. Analysis of molecular variance (AMOVA) showed no significant differences between the D. vitifoliae lineages reared on different host plants, nor was a correlation between host performance and genotype found.

Genetic structure of an introduced pest, grape phylloxera (Daktulosphaira vitifoliae Fitch), in Europe.

A model for the genetic structure of grape phylloxera populations in Europe was developed using hierarchical sampling techniques and AFLP-PCR (amplified fragment length polymorphism--polymerase chain reaction) methodology. One-hundred three European and 6 North American phylloxera populations were studied. An additional European sampling set comprising 60 samples was analyzed to study regional subdivision. The populations grouped into two clusters loosely correlated with collection site location. Phylloxera populations collected from northern (above lat 43 degrees) geographic regions were significantly different from southern (below 43 degrees) populations. The northern cluster was more heterogeneous than the southern cluster, possibly reflecting holocyclic versus anholocyclic reproduction. Microgeographic scales of phylloxera genetic structure displayed as much variation within as among host plants. The host plant did not affect the genetic structure of European phylloxera as revealed in two independent experiments.