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.     

An assessment method for the quantification of Daktulosphaira vitifoliae (Fitch) (Hem., Phylloxeridae) populations in the field

Abstract:  Grape phylloxera is a plant parasite that has started to become a pest again in European viticulture in spite of the use of grafted rootstocks. Since the middle of the 1980s, there have been various reports of vine damage caused by phylloxera. It was shown that in some cases there is no direct correlation between the occurrence of phylloxera and vine damage. In this context, phytopathogenic microorganisms, especially fungi, are mentioned in literature. To investigate the possible interactions it is necessary to be able to assess phylloxera populations in the field, e.g. to correlate phylloxera abundance with the vigour of the vines. Because of the non-homogenous distribution of the grape roots in the soil and of phylloxera on the roots, a method that meets these conditions was established. For the root sampling, a method that ensures reproducible numbers and a high probability of root detection was developed. The second component of the method is a ranking system for the visual assessment of the populations. For the validation of the assessment system, the absolute number of phylloxera feeding sites, all phylloxera instars and phylloxera eggs were related to the weight of fresh and dry roots. The statistical analysis has shown a significant differentiation between the assessment classes (P ≤ 0.01). Furthermore, a correlation ( R ² = 0.732) between the phylloxera populations, assessed with this method, and soil temperatures could be shown. Such a correlation was shown in various studies and, therefore, supports the validity of the assessment system presented in this paper.     

烟草水浸提液对葡萄根瘤蚜侵染危害的控制效应

【目的】检疫害虫葡萄根瘤蚜Daktulosphaira vitifoliae已经在我国多个地区葡萄园发现为害, 直接影响葡萄生长; 前期实验发现烟草水浸提液可有效控制离体根上根瘤蚜的存活率, 本研究进一步开展盆栽验证, 探讨烟草水浸提液对葡萄根瘤蚜侵染的控制效应及葡萄植株生长抑制的缓解作用。【方法】以两年生温室盆栽‘巨峰’自根苗为实验材料, 接种根瘤蚜虫卵35 d后进行烟草水浸提液处理, 处理后测定供试植株的根结数、生长量、根系活力、叶片光合速率及叶绿素荧光等指标。【结果】20 mg/mL和50 mg/mL的烟草水浸提液对根瘤蚜均有一定程度的抑制作用, 施药21 d后, 葡萄根部的新增根结数分别比侵染对照下降了42.9%和52.8%。烟草水浸提液处理还在一定程度上缓解了根瘤蚜侵染对植株的光合能力和荧光的抑制作用。20 mg/mL和50 mg/mL烟草水浸提液处理的株高分别比侵染对照提高了57.1%和7.7%; 根瘤蚜侵染降低了土壤中真菌、细菌和放线菌的数量, 烟草水浸提液处理进一步降低了真菌和放线菌的数量, 降低了由微生物带来的次生侵染几率。【结论】烟草水浸提液对葡萄根瘤蚜的侵染具有控制效果, 并可对葡萄根瘤蚜侵染已造成的寄主危害有一定的缓解作用。     

Grape phylloxera (Daktulosphaira vitifoliae) – a review of potential detection and alternative management options

The management options for grape phylloxera, Daktulosphaira vitifoliae, a monophagous insect pest of Vitis species are reviewed. Although in a worldwide context, grape phylloxera is managed predominantly by the use of resistant rootstocks developed through conventional breeding of hybrid crosses of American Vitis species, this management aspect is largely excluded from the review so that emerging technologies in the field of detection, quarantine and alternative management are discussed. In some viticulture regions of the world, where grape phylloxera's geographic distribution is limited (e.g. Australia), the pest is managed through a combination of surveillance, detection and quarantine. Although some alternative management options for grape phylloxera exist they have received relatively limited research attention because of the relative success of resistant rootstocks. The resilience of resistant rootstocks as the primary management option could also be challenged in the future by host‐plant interactions with diverse grape phylloxera clonal lineages and by potential impacts of climate change on both grapevine and grape phylloxera distribution. A range of control options exist which could be integrated into an improved management system for grape phylloxera. Priority areas for future evaluation and further development include early detection techniques, investigation into the use of biological control agents and development of an integrated approach to grapevine phylloxera management.     

Clonal reproduction and population genetic structure of grape phylloxera, Daktulosphaira vitifoliae, in Australia

The grape phylloxera, Daktulosphaira vitifoliae, is a viticultural pest that in the past has devastated vineyards worldwide, yet little is known about this insect's biology. The genetic structure of Australian populations of grape phylloxera and its mode of reproduction were studied following the development of four polymorphic microsatellite loci. Insects were collected from 28 vineyards, with a total of 361 insects included in the study. The majority of vineyards were infested by functionally parthenogenetic lineages of grape phylloxera that inhabit the root system and there was little support for the traditionally described holocyclic life cycle for this species. Clonal diversity was limited in all of the vineyard regions, with the exception of the Rutherglen region. A multiple founder scenario or occasional sex may contribute to diversity within the Rutherglen region. Leaf galling populations comprised classes distinct from the common genotypic classes identified on the roots, suggesting limited exchange between these groups. Implications for the management of D. vitifoliae are discussed.     

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.     

Pantoea agglomerans‐associated bacteria in grape phylloxera (Daktulosphaira vitifoliae,Fitch)

1 Grape phylloxera lack intracellular symbionts, but the leaf‐galling form appears to be associated with a single microbial species. 2 16S and 18SrDNA sequences were used for identification of symbiotic material. 3 A single bacterial species, closely related to Pantoea agglomerans, was identified in adult parthenogenetic individuals, their eggs and leaf gall tissue of several populations. 4 A 16S rDNA primer pair was designed to test grape phylloxera populations more specifically for the presence of P. agglomerans. 5 16S rDNA sequences of the identified bacteria were very similar to already‐known secondary symbionts occurring in aphids, thrips and other insects. 6 The identified bacteria were culturable on simple media, which demonstrates that the relationship between grape phylloxera and P. agglomerans is not as firm as that of the obligately endosymbiotic Buchnera aphidicola and other aphids.     

(A)sexual reproduction – a review of life cycles of grape phylloxera, Daktulosphaira vitifoliae

Since its emergence as a pest of grapevine about 150 years ago, studies on the life cycle and mode of reproduction of grape phylloxera ( Daktulosphaira vitifoliae Fitch) (Homoptera: Phylloxeridae) have been of considerable scientific interest and importance for applied viticulture. The life cycle of grape phylloxera, as described in the mid to late nineteenth century, is still subject to discussion and confusion. In this article, we aim to provide for the first time an overview of the reported life cycle strategies of grape phylloxera by summarizing documented observations, the results of controlled field and laboratory assays, and the results of molecular genetic analyses. Thus, we present a detailed morphological comparison of grape phylloxera morphs and provide clarification of these by suggesting a simplified nomenclature as well as revising errors found in the published literature. Moreover, recent molecular markers studies, which further enhance our understanding of the impact of sexual recombination on grape phylloxera, are described and discussed.     

Variability in grape phylloxera preference and performance on canyon grape (Vitis arizonica)

We tested the deme-formation hypothesis experimentally with four populations of leaf-galling grape phylloxera, Daktulosphaira vitifoliae, and its host, canyon grape (Vitis arizonica). An experiment designed to examine preference and performance showed that phylloxera populations did not significantly prefer their original host relative to other hosts in the percent available leaves galled. There were significant herbivore population effects (P<0.01), host effects (P<0.001), and population x host interaction effects (P<0.001). Herbivore populations had different colonizing abilities (performance, as measured in the mean number of galls per leaf) on an individual host (P<0.001), but there was no host effect. Host genotype significantly affected phylloxera performance, measured as survivorship (P<0.01), but a phylloxera population did not necessarily have higher survivorship on its original host. Differences in fecundity, an-other measurement of performance, were due to intrinsic differences among herbivore populations (P<0.05), and not related to host genotype. There was no correlation between distance from a phylloxera population in the field and a host's susceptibility to attack. There was a significant positive relationship between levels of infestation on a clone in the field and its susceptibility to colonization experimentally (P<0.05), suggesting inherent differences in host resistance and susceptibility. These results did not support the deme-formation hypothesis. In a second experiment, host clone x water treatment interactions affected phylloxera survivorship (P<0.05) and fecundity (P<0.05). We conclude that host genotype x environment interactions may prevent sessile, parthenogenetic herbivores from locally adapting to individual host genotypes.     

Changes in grape phylloxera abundance in ungrafted vineyards

To examine seasonal changes in the abundance of grape phylloxera Daktulosphaira vitifoliae (Fitch), several sampling methods were tested at vineyards in Victoria, Australia. At a recently infested site, changes detected by root assessment, trunk trapping, and emergence trapping were closely correlated, although the largest numbers of grape phylloxera were obtained using traps that collected phylloxera emerging from soil. This trapping technique was further used to investigate changes in grape phylloxera numbers across three different sites from southeastern Australia as well as in three consecutive seasons at the same vineyard. Grape phylloxera numbers decreased as vines deteriorated; a single peak of emergence occurred in every summer. Size and timing of emergence peaks varied between sites and also between vine blocks within a site. The number of grape phylloxera trapped was correlated with degree-days. Monitoring soil temperature may provide a way of timing control options against grape phylloxera and a way of identifying peak periods when phylloxera detection surveys should be completed or when grape phylloxera are at the highest risk of spreading among vineyards.     

根瘤蚜侵染对不同抗性葡萄品种根系超微结构的影响

【目的】探讨不同抗性葡萄品种对葡萄根瘤蚜Daktulosphaira vitifolia Fitch侵染后的组织结构响应。【方法】以砧木140Ru (Vitis rupestris×V. berlandieri)和栽培品种赤霞珠Vitis vinifera cv. Cabernet Sauvignon离体三级根及140Ru盆栽苗为试材接种根瘤蚜卵, 接种两周后取刺吸位点组织作超薄切片并观察超微结构变化, 取盆栽140Ru不同时期根结测定多酚含量。【结果】超微结构显示, 砧木140Ru根系周皮层最外层细胞壁的厚度（1 031.25 nm）及周皮层细胞层数（6～7层）显著高于赤霞珠品种的周皮层细胞厚度（543.75 nm）及层数（3～4层）, 140Ru根系韧皮部酚类物质含量比赤霞珠高出35％; 侵染后, 砧木140Ru周皮层细胞第3层以内的细胞壁加厚并积聚大量的多酚类物质, 140Ru葡萄新根被根瘤蚜侵染后多酚含量呈升高趋势, 在接种20 d时是对照的2.4倍; 赤霞珠粗根被根瘤蚜侵染后薄壁细胞中细胞质变浓, 出现大量淀粉粒, 线粒体及内质网数量增多。【结论】砧木140Ru的周皮层组织结构较赤霞珠不利于根瘤蚜口针穿刺, 被根瘤蚜侵染后发生了不利于根瘤蚜侵染取食的变化。     

Strong gene flow and lack of stable population structure in the face of rapid adaptation to local temperature in a spring-spawning salmonid, the European grayling (Thymallus thymallus)

Gene flow has the potential to both constrain and facilitate adaptation to local environmental conditions. The early stages of population divergence can be unstable because of fluctuating levels of gene flow. Investigating temporal variation in gene flow during the initial stages of population divergence can therefore provide insights to the role of gene flow in adaptive evolution. Since the recent colonization of Lake Lesjaskogsvatnet in Norway by European grayling (Thymallus thymallus), local populations have been established in over 20 tributaries. Multiple founder events appear to have resulted in reduced neutral variation. Nevertheless, there is evidence for local adaptation in early life-history traits to different temperature regimes. In this study, microsatellite data from almost a decade of sampling were assessed to infer population structuring and its temporal stability. Several alternative analyses indicated that spatial variation explained 2-3 times more of the divergence in the system than temporal variation. Over all samples and years, there was a significant correlation between genetic and geographic distance. However, decomposed pairwise regression analysis revealed differing patterns of genetic structure among local populations and indicated that migration outweighs genetic drift in the majority of populations. In addition, isolation by distance was observable in only three of the six years, and signals of population bottlenecks were observed in the majority of samples. Combined, the results suggest that habitat-specific adaptation in this system has preceded the development of consistent population substructuring in the face of high levels of gene flow from divergent environments.     

An experimental assessment of genetic tagging and founder representation in hatchery-reared red drum (Sciaenops ocellatus) used in stock enhancement

Multiplexed microsatellite markers were evaluated as genetic tags for red drum ( Sciaenops ocellatus ) juveniles used in stock enhancement. Offspring were generated from spawns of nine sets of five broodfish (three dams and two sires) in individual brood tanks. Intensive sampling, by trawling, at 2, 7, 8, 10 and 11 days after release of ∼192 500 hatchery-raised fingerlings resulted in recovery of a total of 310 fingerlings. All parents and recovered offspring were genotyped for variation at 30 microsatellites combined for simultaneous assay in six multiplex panels. An optimal combination employing three of the six multiplex panels allowed unambiguous parentage assignment of all recovered offspring. Only 21 of 52 possible dam x sire combinations were represented among recovered offspring. The founder equivalent ( f _e) of the recovered offspring was 8.7 vs the expected f _e of 36.0 (95% CI = 33.3–38.4) if reproductive success was randomly distributed among breeders. The significantly lower founder equivalent translated into reduced genetic diversity among the recovered offspring and may reflect differing contributions of individual broodfish to spawning events, differing productivity among brood tanks, and/or variable survival of families during early larval and/or juvenile stages.     

Metarhizium anisopliae, a potential agent for the control of grape phylloxera

This investigation deals with the control effects of the insect pathogenic fungus Metarhizium anisopliae var. anisopliae on Daktulosphaira vitifoliae. In pot experiments, the soil surrounding phylloxera-infected grapes was inoculated with barley colonised with M. anisopliae. After thirty-two days, ineight of ten Metarhizium-applied pots nofresh phylloxera infections could be observed.In two of ten plants, a few fresh nodositiessingly occupied with phylloxera or phylloxeraeggs could be found. In all untreated plants,fresh nodosities with either single (two of sixplants) or multiple (four of six plants)occupation with phylloxera could be observed.M. anisopliae could be re-isolated in aconcentration of <1 × 10³ CFU g^$minus;1 soil dry weight from those pots with phylloxera-infected plants that had been treated with the fungus. The potential role of M. anisopliae in grape phylloxera management is discussed.     

Seasonal capacity of attached and detached vineyard roots to support grape phylloxera (Homoptera: Phylloxeridae)

Field experiments were conducted to evaluate population densities and survival, developmental rate, and fecundity of grape phylloxera, Daktulosphaira vitifoliae (Fitch), as influenced by root attachment or detachment from mature, field-grown, Vitis vinifera L. grapevines through the growing season. Experiments were performed using artificial infestations of California biotype A grape phylloxera. Thirty-day bioassays on attached- and detached-roots were repeated monthly from May to September in 1997 (cultivar 'Carignane') and April to September in 1998 (cultivar 'Thompson Seedless'). The bioassays showed that attached roots had lower population densities than detached roots in all months of both years. Densities varied by month, tending to be higher in spring than in summer. Of the population parameters studied, survival was most influenced by attachment condition, being higher on detached than on attached roots by up to 25-fold. These results imply the importance of vine-related mortality factors to grape phylloxera population density. Influence of root attachment condition on developmental rate and fecundity was not uniform across bioassay months for either year; however, in the four out of 21 assays where there was a significant difference it favored detached roots by twofold. Fruit harvest resulted in higher survival in the July assay but not for assays in August and September; however, neither developmental rate nor fecundity was affected by harvest in any ofthe assays. We conclude that mortality rather than nutritional factors are most limiting for field populations on susceptible vines. This work suggests that detachment of roots as occurs with root girdling by root pathogens may increase grape phylloxera populations on infested, susceptible vines. These results imply that excised root bioassays over-estimate grape phylloxera virulence and underestimate rootstock resistance.     

根瘤蚜对不同抗性葡萄的选择性及葡萄根系挥发性物质的鉴定

为了探讨不同抗性水平砧木及品种对根瘤蚜吸引及驱避效应以及鉴定挥发性物质组分,采用离体根段培养法测定葡萄根瘤蚜Phylloxera viticola Fitch对砧木5BB,140Ru及栽培品种巨峰的选择性,发现根瘤蚜对栽培品种巨峰的根段具有很强的选择性,至接种后3 d,67.13％的根瘤蚜优先选择巨峰;而对5BB和140Ru的选择性却很差,分别有15.29％和17.58％的根瘤蚜选择5BB及140Ru。根瘤蚜在不同品种根段上的生长发育和繁殖也存在明显差异。对砧木5BB和巨峰根系的挥发性组分进行测定,发现2个品种挥发物独有组分数量均较高,巨峰独有组分为23个,占总成分数的48.94％,5BB的独有组分为32个,占总成分数的57.14％。5BB和巨峰的共有组分为24个,主要成分均为亚油酸甲酯和反油酸甲酯,但含量有所不同,其中,5BB以倍半萜类的丁香醇及脂肪酸甲酯类的亚油酸甲酯,反油酸甲酯,棕榈油酸甲酯含量较高,高于巨峰中相应物质含量的4.23%~6.46％,而巨峰以邻苯二甲酸二丁酯及胆甾烷含量较高,分别高于5BB相应组分含量的6.81％和1.07％。结果说明葡萄根瘤蚜对不同抗性水平砧木及品种确实存在不同的选择性,并且不同抗性水平的砧木及品种的挥发性物质存在显著差异。     

Inferring recruitment history from spatial genetic structure within populations of the colonizing tree Albizia julibrissin (Fabaceae)

Comparative analyses of spatial genetic structure (SGS) among species, populations, or cohorts give insight into the genetic consequences of seed dispersal in plants. We analysed SGS of a weedy tree in populations with known and unknown recruitment histories to first establish patterns in populations with single vs. multiple founders, and then to infer possible recruitment scenarios in populations with unknown histories. We analysed SGS in six populations of the colonizing tree Albizia julibrissin Durazz. (Fabaceae) in Athens, Georgia. Study sites included two large populations with multiple, known founders, two small populations with a single, known founder, and two large populations with unknown recruitment histories. Eleven allozyme loci were used to genotype 1385 individuals. Insights about the effects of colonization history from the SGS analyses were obtained from correlograms and Sp statistics. Distinct differences in patterns of SGS were identified between populations with multiple founders vs. a single founder. We observed significant, positive SGS, which decayed with increasing distance in the populations with multiple colonists, but little to no SGS in populations founded by one colonist. Because relatedness among individuals is estimated relative to a local reference population, which usually consists of those individuals sampled in the study population, SGS in populations with high background relatedness, such as those with a single founder, may be obscured. We performed additional analyses using a regional reference population and, in populations with a single founder, detected significant, positive SGS at all distances, indicating that these populations consist of highly related descendants and receive little seed immigration. Subsequent analyses of SGS in size cohorts in the four large study populations showed significant SGS in both juveniles and adults, probably because of a relative lack of intraspecific demographic thinning. SGS in populations of this colonizing tree is pronounced and persistent and is determined by the number and relatedness of founding individuals and adjacent seed sources. Patterns of SGS in populations with known histories may be used to indirectly infer possible colonization scenarios for populations where it is unknown.     

Fine-scale genetic structure of grape phylloxera from the roots and leaves of Vitis

Patterns of variation at microsatellite loci suggest that root populations of the pest grape phylloxera (Daktulosphaira vitifoliae) are largely parthenogenetic in Australian vineyards. To investigate reproduction in leaf galling phylloxera and the association between these individuals and phylloxera on roots, we examined in detail genetic variation in phylloxera from a vineyard block. Some genotypes found on leaf galls within this block were not present on roots, whereas others spanned both zones. There was no evidence that genotypes on roots were the product of sexual reproduction in leaf galls. mtDNA variation was not associated with the location of the phylloxera clones. The spatial distribution of genotypes within a root population was further investigated by intensively sampling phylloxera from another vineyard block. Join-count spatial autocorrelation statistics were used to explore fine-scale spatial structure. Clones were nonrandomly distributed within the block and there was evidence that the distribution of clones followed rows. These findings suggest firstly that there is limited dispersal of root and leaf feeding phylloxera, and secondly that factors, other than vine host, are likely to be important and contribute to clonal structure within populations.     

Electrophoretic 'keys' for the identification of parasitoids (Hymenoptera: Braconidae: Aphelinidae) attacking Sitobion avenae (F.) (Hemiptera: Aphididae)

Polyacrylamide gel electrophoresis was used to construct electrophoretic 'keys' for identifying larval, pupal and adult hymenopterous parasitoids of Sitobion avenae (F.). In general, for each parasitoid and enzyme system tested, all life stages gave similar banding patterns. Although five enzyme systems were tested, esterase was found to be the most useful single system. Using laboratory-reared parasitoids and staining for esterase activity, all species examined, except Aphidius rhopalosiphi and A. ervi could be readily distinguished with the aid of the appropriate key.     

部分葡萄品种和砧木抗葡萄根瘤蚜性能鉴定

为了检测我国葡萄品种和砧木对中国生态型葡萄根瘤蚜Daktulosphaira viticola (Fitch) 的抗性,在实验室条件下采用离体根接种法鉴定了10个葡萄砧木、18个葡萄栽培品种,测定了根瘤蚜存活率、发育历期、产卵量、种群龄期结构指数和成虫大小等指标。结果表明： 砧木品种SO4,5BB,1103P,420A,Lot,101-14MG,3309C,140Ru和110R抗根瘤蚜,接种后蚜虫陆续死亡,尤其是SO4,5BB,1103P和420A对根瘤蚜高抗,使根瘤蚜滞育仅发育至1龄。我国当作砧木使用的贝达不抗根瘤蚜,接种后蚜虫存活率始终保持在38.82％～47.5%,日产卵量达9.01;欧美杂交种中的康克、康拜尔和卡它巴对根瘤蚜稍有抗性,根瘤蚜日产卵量在6.00～6.85粒之间,其中康克、康拜尔葡萄上蚜虫的存活率随时间的延长而明显降低,由最初的60％下降至25％～30％; 欧美杂交种中的黑虎香和白香蕉虽然在一定程度上能够限制根瘤蚜的存活和发育进程,但对根瘤蚜的繁殖量没有影响。所有供试欧亚种和欧美杂交种的栽培品种都对根瘤蚜高度敏感,接种后第29 d 蚜虫存活率仍保持在40％～60％,且前后变化较小,日产卵量变化于7～12.35粒之间,总产卵量均在190粒以上。由此可见,我国供试栽培品种均不抗根瘤蚜,在根瘤蚜疫区需要用抗性砧木嫁接苗建园。