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.     

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.     

钙肥对葡萄根瘤蚜生长发育和繁殖的影响

【目的】为明确葡萄所需的大量元素钙对葡萄根瘤蚜 Daktulosphaira vitifoliae(Fitch）生长发育和繁殖的影响。【方法】在温室中,设置不同 Ca⁽²⁺⁾浓度（0、2 和 4mmol·L(2+)浓度（0、2 和 4mmol·L^(-1)）的钙全营养液于葡萄全生育期浇灌葡萄,评价离体根段对葡萄根瘤蚜的存活率、若蚜、成蚜寿命和繁殖力的影响。【结果】随着钙离子浓度的增加,葡萄根瘤蚜在葡萄根上的 1 龄若蚜历期缩短 0.80 倍以上,成蚜历期增加超过 1.88倍,寿命显著延长超过 1.18 倍,产卵量提高 3.34 倍以上。与 0 mmol·L(-1)）的钙全营养液于葡萄全生育期浇灌葡萄,评价离体根段对葡萄根瘤蚜的存活率、若蚜、成蚜寿命和繁殖力的影响。【结果】随着钙离子浓度的增加,葡萄根瘤蚜在葡萄根上的 1 龄若蚜历期缩短 0.80 倍以上,成蚜历期增加超过 1.88倍,寿命显著延长超过 1.18 倍,产卵量提高 3.34 倍以上。与 0 mmol·L^(-1) Ca(-1) Ca⁽²⁺⁾和 2 mmol·L(2+)和 2 mmol·L^(-1) Ca(-1) Ca⁽²⁺⁾处理相比,葡萄根瘤蚜在 4 mmol·L(2+)处理相比,葡萄根瘤蚜在 4 mmol·L^(-1) Ca(-1) Ca⁽²⁺⁾处理下的葡萄根上,内禀增长率 r_m、周限增长率 λ 和净增殖率 R_0均最高,分别为（0.191 ± 0.003）、（1.210 ± 0.004）和（169.070 ± 11.897)(P < 0.05）,且世代发育历期显著缩短（P <0.05）。特定年龄龄期存活率 S_(xj)、种群特定年龄繁殖力 m_x和种群特定年龄繁殖值 l_xm_x在 4mmol·L(2+)处理下的葡萄根上,内禀增长率 r_m、周限增长率 λ 和净增殖率 R_0均最高,分别为（0.191 ± 0.003）、（1.210 ± 0.004）和（169.070 ± 11.897)(P < 0.05）,且世代发育历期显著缩短（P <0.05）。特定年龄龄期存活率 S_(xj)、种群特定年龄繁殖力 m_x和种群特定年龄繁殖值 l_xm_x在 4mmol·L^(-1)Ca(-1)Ca⁽²⁺⁾处理种群中保持较高水平,随着钙浓度降低,葡萄根瘤蚜的各生命参数明显降低。【结论】钙元素对葡萄根瘤蚜的生长发育和繁殖能力具有促进作用,葡萄根瘤蚜发生葡萄园更应合理使用钙肥。     

An in vitro assessment of phylloxera (Daktulosphaira vitifoliae Fitch) (Hom., Phylloxeridae) life cycle

Phylloxera single founder lineages were maintained in an aseptic, dual-culture system to investigate the phylloxera life cycle, focusing on holocycle induction. Holocyclic morphs were developed on the grape rootstock 41B and they produced vital sexuales. The morphological development of sexual stages was studied and an assessment of genetic variation within a single founder lineage was performed. Twenty individual phylloxera of differing stages (apterous parthenogenetic, pre-sexual nymphs, sexuparae and sexuals) were genetically fingerprinted using amplified fragment length polymorphism-polymerase chain reaction technology. Four individuals displayed differing banding patterns, exhibiting three different independent genotypes. No influence of the morphal stage on these patterns could be detected. This study did not achieve phylloxera matings. However, the system could serve as a tool for future studies, performing controlled and systematic crosses and back-crosses to examine Mendelian genetics.     

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.     

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.     

Genome-wide identification and characterization of simple sequence repeat loci in grape phylloxera, Daktulosphaira vitifoliae

A genome-wide sequence search was conducted to identify simple sequence repeat (SSR) loci in phylloxera, Daktulosphaira vitifoliae, a major grape pest throughout the world. Collectively, 1524 SSR loci containing mono-, di-, tri-, tetra-, penta-, and hexanucleotide motifs were identified. Among them, trinucleotide repeats were the most abundant in the phylloxera genome (34.4%), followed by hexanucleotide (20.4%) and dinucleotide (19.6%) repeats. Mono-, tetra- and pentanucleotide repeats were found at a frequency of 1.3, 11.2 and 12.9%, respectively. The abundance and inherent variations in SSRs provide valuable information for developing molecular markers. The high levels of allelic variation and codominant features of SSRs make this marker system a useful tool for genotyping, diversity assessment and population genetic studies of reproductive characteristics of phylloxera in agricultural and natural populations.     

我国根瘤蚜mtDNA COⅠ遗传多样性与系统发育

通过线粒体DNA COⅠ多态性研究了我国根瘤蚜Daktulosphaira vitifoliae Fitch的遗传分化与系统发育,并将我国根瘤蚜单倍型与GenBank中已发表的85个单倍型进行了聚类分析。结果表明：序列中A,C,T,G 4种核苷酸的比例分别为34.8%,15.8%,39.2%和10.2%, 29 个变异位点中单一多态位点 12 个,简约信息位点17 个。确定了 13 种单倍型,检测5种单倍型,其中上海群体单倍型相对丰富,且上海葡萄根瘤蚜群体与其他3个群体之间没有共享单倍型。同时上海群体与其他3个群体的遗传距离最大（0.039~0.040）,Nm最小（0.02）,在分子系统发育树和单倍型网络图上为独立分支,说明我国根瘤蚜至少有两个独立的起源。     

Evidence for host-associated clones of grape phylloxera Daktulosphaira vitifoliae (Hemiptera: Phylloxeridae) in Australia

Grape phylloxera, Daktulosphaira vitifoliae Fitch, is an important pest of grapevines (Vitis vinifera L.) (Vitaceae). Using microsatellite DNA markers it was demonstrated strong associations can exist between D. vitifoliae asexual lineages and vine host type within a vineyard. Also, in excised root bioassays, D. vitifoliae collected from three regions where different genotypic classes predominated showed host-specific differences in life table parameters of reproductive rate and intrinsic rate of increase. Lastly, comparisons of mitochondrial DNA (cytochrome oxidase I) sequences revealed that D. vitifoliae in Australia have paraphyletic origins and fall into two clades partially related to vine host usage. These findings indicate introduction of separate lineages of D. vitifoliae which have close host associations and as such, have important implications for management of this pest in Australia.     

根瘤蚜侵染不同抗性葡萄对根结形成及植株营养消耗的影响

为探讨不同抗性水平的葡萄砧木和品种被葡萄根瘤蚜Daktulosphaira vitifoliae Fitch侵染形成根结的状况及其对植株营养消耗的影响,采用盆栽方式接种根瘤蚜,测定了葡萄根结数量、质量及碳水化合物和脯氨酸含量等指标。结果表明：砧木5BB,1103P,SO4,3309C,101-14Mgt被根瘤蚜侵染后均不形成根结;140Ru,Lot和110R仅形成少量根结而不能形成根瘤;砧木贝达、栽培品种赤霞珠、巨峰和达米娜不仅能够形成根结和根瘤,而且根系腐烂程度严重。越敏感的品种形成的根结数量和体积越大,达米娜、巨峰和赤霞珠根结重占根重比例分别高达40.02％,37.08％和35.36％,百个根结质量分别是140Ru的4.4,3.5和433倍。3个形成根结的砧木的枝条生长量平均减少16.5％,而4个高感品种的生长量平均减少43％。根结质量与根结内的营养积聚呈正相关,其中根结质量与淀粉含量增加量的相关系数达0.94,与可溶性糖含量增加量的相关系数达0.81,与游离脯氨酸含量增加量的相关系数达0.93。     

Phylogeography in a galling insect, grape phylloxera, Daktulosphaira vitifoliae (Phylloxeridae) in the fragmented habitat of the Southwest USA

Aim The Southwest USA is characterized by fragmented habitat for a range of species in a manner analogous to island systems. This can restrict gene flow among populations and may promote genetic differentiation and the evolution of endemic populations or species among the forested terrestrial islands. The region has experienced cycles of cooling and warming throughout the Pleistocene and the current isolation among populations may date back only c. 11,000 years. Thus, some studies have found genetic structure that predates the current distribution of organisms. The effect of habitat fragmentation on genetic structure was tested using DNA sequences in a highly specialized herbivorous insect, grape phylloxera, Daktulosphaira vitifoliae, that exhibits poor dispersal capacity and natal‐host associated mating that would seem to predispose it to genetic differentiation. Location Southwestern USA. Methods Phylogenetic trees and statistical parsimony networks were estimated from 458 bp of the mitochondrial cytochrome oxidase I gene sampled from 12 populations in Arizona and New Mexico. Results There was no evidence that populations were differentiated at the level of canyon or mountain range but significant structure was found at the larger level of geographic region. The pattern was confounded by the polyphyly of Southwest (SW) grape phylloxera relative to eastern populations and the coexistence of two highly divergent lineages within populations. Main conclusions An early split of grape phylloxera into the SW has resulted in some differentiation between populations in the Transition and Sonoran + Chihuahuan geographic regions. It is hypothesized that this differentiation has been overlaid by a more recent introduction of haplotypes from midwestern USA populations that occur primarily in the Transition region. The importance of sampling broadly is stressed.     

Evaluating resistance of some rootstocks to grape phylloxera with in vitro and excised root testing systems

Summary The life table of a local strain of grape phylloxera was determined to evaluate the resistance of the most commonly used rootstocks: Rul40, R99, and 3309C, and one local variety ‘Helwani’. The study was carried out by applying both in vitro dual culture and small root pieces testing systems. The results showed that there was a great variation in percentage mortality of immature stages, number of eggs laid, adult fertility, oviposition period, and developmental time between all tested rootstocks and the local variety, regardless of the applied testing systems. Based on the population prediction, Helwani would be a suitable host as it would be susceptible to the destructive insects in the field and the rootstocks would be resistant. However, Rul 40 rootstock was more susceptible than R99 and 3309C rootstocks.     

水稻内生优势成团泛菌GFP标记菌株的性质与标记丢失动力学

为研究内生细菌对宿主植物侵染定殖的机理和其共生生物学作用 ,对水稻内生优势成团泛菌 (Pantoeaagglomerans)YS19与绿色荧光蛋白 (GFP)标记的YS19B ::gfp菌株的生长动力学进行了比较研究 ,探讨了成团泛菌YS19B ::gfp的标记稳定性和荧光性质 .标记菌株与野生型菌株相比 ,最大比生长速率和最大生物量仅减小 12 4 %和 6 % ,代时延长 14 0 % .成团泛菌YS19B ::gfp在指数期连续传代培养 10 0代后 ,GFP标记的保持率为 89 1% ,建立了标记菌株在有标记丢失存在时的生长动力学模型 :dX+ dt =μ+ (1-p)X+ ,解析出细胞分裂时标记丢失的概率p =9 75 6× 10 -7,确定了方程的模型参数 .标记菌株的荧光光谱在激发波长为 4 0 0nm时 ,最大发射波长为 5 0 8nm ,与供体菌株完全相同 .在LB培养基上生长时 ,成团泛菌YS19B ::gfp的GFP产生时间在指数期末期到稳定期较快 ,并于培养至 2 0h时达到最高 ,同时单位菌体生物量的荧光强度也达到最大 .结果说明 ,在GFP标记后成团泛菌YS19B ::gfp的生长仅受到较小影响 ,不致对成团泛菌的生理活动造成大的改变 ,同时由于该菌对宿主的侵染能力比其它内生细菌要强得多 ,因而该菌对植物的侵染活性影响也较小 ,该菌仍然可以保持其内生优势地位 .该标记的稳定性比较高 ,荧光产生正常 ,很适     

(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.     

Isolation of endophytic diazotroph Pantoea agglomerans and nondiazotroph Enterobacter asburiae from sweetpotato stem in Japan

AIMS: To isolate and identify diazotrophic endophytes in the stem of Japanese sweetpotato cv. Koganesengan. METHODS AND RESULTS: Surface-sterilized and thinly sliced (1-2 mm) sweetpotato stem samples were incubated in test tubes with semi-solid modified Rennie (MR) medium. The test tubes were assayed for acetylene reduction activity (ARA) 5 days after incubation at 30 degrees C. Twelve isolates were obtained from MR plates inoculated with a loop of semi-solid MR medium from ARA+ tubes. However, ARA test showed that only nine isolates were diazotrophic and three were nondiazotrophic strains. Using the API 20E diagnostic kit, four diazotrophic isolates were identified as strains of Pantoea spp. and five isolates as Klebsiella spp. The nondiazotrophic bacteria were strains of Enterobacter spp. A diazotrophic isolate Pantoea sp. MY1 and nondiazotrophic isolate Enterobacter sp. MY2 were identified to the species level by full sequence analysis of 16S rRNA gene. The results showed that MY1 had 99.2% similarity to Pantoea agglomerans ATCC 27155 and MY2 had 99.5% similarity to Enterobacter asburiae ATCC 35953. CONCLUSION: The stem of sweetpotato cv. Koganesengan was colonized by diazotrophic endophyte P. agglomerans and nondiazotrophic endophyte E. asburiae. SIGNIFICANCE AND IMPACT OF THE STUDY: This study is an essential step toward understanding the ecology and interaction between endophytic bacteria and sweetpotato.     

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.     

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.     

Reproductive mode of grape phylloxera (Daktulosphaira vitifoliae, Homoptera: Phylloxeridae) in Europe: molecular evidence for predominantly asexual populations and a lack of gene flow between them.

The genetic structure of European grape phylloxera populations, Daktulosphaira vitifoliae (Homoptera: Phylloxeridae), was analyzed using 6 polymorphic microsatellite markers. Genetic diversity data of 6 populations originating from northern and southern European viticultural regions was assessed for geographic differences, and the structure of 2 additional populations was examined in more detail, focusing on specific host plant and habitat characteristics. To test for "signatures" of clonal reproduction, different population genetic measures were applied to the data obtained from these populations. A total of 195 multilocus genotypes were detected in 360 individuals tested. Significant deviations from Hardy-Weinberg equilibrium, negative FIS values (from -0.148 to -0.658 per population), and the presence of multicopy genotypes revealed that the current major reproductive mode at each of the locations tested was asexual. The high genotypic diversity detected within and among populations, however, together with the occurrence of unique D. vitifoliae genotypes, indicates sexual recombination events took place, probably prior to the multiple introductions into Europe. The absence of overlapping genotypes between the sampling sites suggests low migration rates among the populations studied and implies that the main mode of insect dispersal is through infested plant material carried by human agency. The specific features of European D. vitifoliae habitats are illustrated to discuss the role of habitat and life cycle in the genetic structure of this globally important pest aphid species.