Phylloxera Populations Research Articles

Assaying the potential benefits of thiamethoxam and imidacloprid for phylloxera suppression and improvements to grapevine vigour

Grape phylloxera ( Daktulosphaira vitifoliae, Fitch) is an encroaching problem for the Australian wine industry with four new outbreaks recorded in the past 5 years. Whilst quarantine- and phylloxera-resistant rootstocks are the recommended long-term management strategies, there are at present no short-term options for recently infested vineyards. In this study, an assay for assessing the impact of the systemic insecticides thiamethoxam (Actara ®; two applications at a concentration of 2000 ppm active ingredient) and imidacloprid (Confidor ®; one and two applications at a concentration of 2000 ppm active ingredient) on suppression of phylloxera populations on grapevine roots was developed. Results from in vitro and in planta trials showed that both insecticides resulted in reduced phylloxera numbers and improved vine vigour. Imidacloprid was found to have a greater effect on phylloxera suppression and vine vigour improvement. Therefore imidacloprid has the potential to be successfully used for phylloxera suppression in the field, but will require further validation under field conditions before recommendations can be made. The assay developed provides a screening procedure for future chemical testing against phylloxera.

Consequences of insect herbivory on grape fine root systems with different growth rates

Herbivory tolerance has been linked to plant growth rate where plants with fast growth rates are hypothesized to be more tolerant of herbivory than slower-growing plants. Evidence supporting this theory has been taken primarily from observations of aboveground organs but rarely from roots. Grapevines differing in overall rates of new root production, were studied in Napa Valley, California over two growing seasons in an established vineyard infested with the sucking insect, grape phylloxera (Daktulosphaira vitifoliae Fitch). The experimental vineyard allowed for the comparison of two root systems that differed in rates of new root tip production (a 'fast grower', Vitis berlandieri x Vitis rupestris cv. 1103P, and a slower-growing stock, Vitis riparia x Vitis rupestris cv. 101-14 Mgt). Each root system was grafted with a genetically identical shoot system (Vitis vinifera cv. Merlot). Using minirhizotrons, we did not observe any evidence of spatial or temporal avoidance of insect populations by root growth. Insect infestations were abundant throughout the soil profile, and seasonal peaks in phylloxera populations generally closely followed peaks in new root production. Our data supported the hypothesis that insect infestation was proportional to the number of growing tips, as indicated by similar per cent infestation in spite of a threefold difference in root tip production. In addition, infested roots of the fast-growing rootstock exhibited somewhat shorter median lifespans (60 d) than the slower-growing rootstock (85 d). Lifespans of uninfested roots were similar for the two rootstocks (200 d). As a consequence of greater root mortality of younger roots, infested root populations in the fast-growing rootstock had an older age structure. While there does not seem to be a trade-off between potential growth rate and relative rate of root infestation in these cultivars, our study indicates that a fast-growing root system may more readily shed infested roots that are presumably less effective in water and nutrient uptake. Thus, differences in root tip production may be linked to differences in the way plants cope with roots that are infested by sucking insects.

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

Abstract1 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.

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.

New Simple Sequence Repeat Loci for the Study of Grape Phylloxera (Daktulosphaira vitifoliae) Genetics and Host Adaptation

Vineyard damage due to grape phylloxera, <i>Daktulosphaira vitifoliae</i> Fitch, has been controlled by resistant rootstocks for over 100 years. There are now a wide range of rootstocks used in California vineyards since the collapse of AXR#1. To study the effect of this rootstock diversity on phylloxera genetics and possible host adaptation, a set of microsatellite primers were developed to augment the four produced by Corrie and colleagues (2002). In order to develop more microsatellite loci to improve the sensitivity and effectiveness of these markers for use in genetic diversity and rootstock adaptation studies, a subtractive-based hybridization strategy was used to construct microsatellite enriched genomic libraries from grape phylloxera DNA. Fifty loci were identified for primer design. Nineteen produced good PCR products, seven of which reliably detected polymorphisms across the 32 grape phylloxera populations tested. These seven SSR loci were used to distinguish genetic diversity in California and European grape phylloxera populations. Results confirm the utility of these loci for analyzing genetic diversity, “finger-printing” strains, and studying host associations. A significant deviation from the Hardy-Weinberg equilibrium for the tested California populations suggests that parthenogenesis is perhaps the primary, if not only, reproductive system in California.

Effect of Soil Type on the Establishment of Grape Phylloxera Colonies in the Pacific Northwest

Soils from the Pacific Northwest were investigated to determine if soil type affects the establishment of new phylloxera populations. Soils included those of the Abegg, Helvetia, and Jory series in Oregon, the Burke series in Washington, and two artificial soils, a soilless mix and a Jory/sand mix. <i>Vitis vinifera</i> cv. Pinot noir vines in each soil were infested with phylloxera eggs originating from the Willamette Valley in western Oregon and the Yakima Valley in eastern Washington. After three months, phylloxera numbers were collected through investigation of soil core samples. Soils from all six soil types supported phylloxera establishment, regardless of population origin. Overall, soil from the Burke series had the lowest percentage of establishment and significantly lower population means. All Oregon soil types and the artificial mixes consistently supported higher population numbers of phylloxera. Based on establishment and proliferation of population numbers in the Burke series soil, we conclude that some soils may provide an environment that suppresses the growth of phylloxera populations. However, none of the soils tested in this study prevented grape phylloxera establishment.

Variation in aggressiveness and genetic diversity of grape phylloxera in Southern France

&lt;p style="text-align: justify;"&gt;The aphid relative &lt;em&gt;Daktulosphaira vitifoliae&lt;/em&gt; (Fitch) (&lt;em&gt;Homoptera : Phylloxeridae&lt;/em&gt;) feeds on the leaves and roots of grapevine (&lt;em&gt;Vitis&lt;/em&gt; spp.). The insect was introduced into France from North America around 140 years ago and caused severe damage until control of the root-infesting form was initiated by grafting European cultivars onto resistant rootstocks. The objective of our research was to analyze both the variation in pathogenicity and the genetic diversity of the insect in southern France. Twenty samples of phylloxera were collected from leaf galls throughout the region. Their aggressiveness was monitored by measuring the reduction in root dry mass of artificially inoculated potted plants. One trial was performed with three rootstocks (41B, 3309C and 140R) and another with the highly susceptible &lt;em&gt;Vitis vinifera&lt;/em&gt; cv. Carignan. No evidence for preference was shown, but aggressiveness varied widely among phylloxera samples, especially in the Carignan trial. Genetic diversity was assessed using 30 Random Amplified Polymorphic DNA (RAPD) markers obtained with 15 primers. Wide diversity was found and all samples corresponded to distinct RAPD phenotypes. The genetic structure of phylloxera population in Southern France was not showed to be influenced either by the location or by the host.&lt;/p&gt;

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 (R2 = 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.

In vitro assessment of grapevine resistance to two populations of phylloxera from Australian vineyards

A perlite-based medium is described for in vitro cocultivation of phylloxera with micropropagated vines. Using this system, as well as cocultivations on excised primary roots, six vine types were screened for resistance to one or two populations of phylloxera (SRU-1 or VWL-1) sourced from different Australian vineyards. When inoculated with VWL-1 phylloxera, V. vinifera cv. Shiraz was rated as susceptible, Ramsey as resistant, Schwarzmann, V. riparia and Börner as highly resistant and V. rotundifolia as immune. When inoculated with SRU-1 phylloxera, both V. vinifera and Schwarzmann were rated as susceptible. The potential use of these in vitro methods for rootstock resistance screening and determination of phylloxera biotypes is discussed. Phylloxera behaviour and root responses observed in vitro are also described.

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.

Influence of Plant Growth Stage on Grape Phylloxera (Homoptera: Phylloxeridae) Populations

To understand the effects of plant growth stage on the performance of grape phylloxera, Daktulosphaira vitifoliae (Fitch), demographic analyses were done using attached roots in a Vitis vinifera L. ‘Chardonnay’ vineyard during the vegetative, mid-ripening, and postharvest periods of grapevines. Survival of first instars was <30%. First instars coincide with the time of gall formation, and their survival was higher during postharvest than during the vegetative and mid-ripening periods. Survival after the second instar did not decrease with development but was stable, generally above 90% for the postharvest period and below 75% for the other experimental periods. Sucrose levels in root phloem parenchyma tissues were highest during postharvest compared with vegetative and mid-ripening periods and reflect a more rapid development during this period but only by a few days. The limiting step of grape phylloxera population growth appears to occur simultaneously with gall formation and is associated with survival. Survival and developmental rate of a virulent grape phylloxera strain were limiting with greenhouse vines of the resistant rootstocks 5C and SO4. Survival and developmental rates were more limiting for attached than detached roots.

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.

Interaction of Phylloxera (Daktulosphaira vitifoliaeFitch) with Grape (Vitisspp.) in Simple Isolation Chambers

A greenhouse-based system to evaluate Daktulosphaira vitifoliae (Fitch) performance on various host plants was modified. Two parthenogenetic generations could be continuously observed and the numbers of reproductive adults and feeding sites (nodosities) determined. Phylloxera performance could be separated into three types. The standard type produced increased numbers of reproducing adults within the first two generations. The second type performed significantly better in the colonizing generation (G1) than the standard type, but had declining reproduction in the second generation (G2). The third type exhibited standard reproduction in G1, but significantly faster and higher reproduction in G2. The isolation chambers were built from 2 L clear plastic water bottles with fine silk-screen tops. These chambers produce quarantine conditions for phylloxera populations, allow continuous monitoring, produce rapid results, and allow inexpensive study of phylloxera performance on selected host plants. Furthermore, the chambers provide the isolation necessary to maintain and rear single-founder phylloxera lines under laboratory conditions. Acknowledgments: We thank Ms. E. J. Frantz for her valuable assistance and Prof. J. Granett (University of California, Davis) for supplying biotype A phylloxera samples. We are grateful for financial assistance provided by the German Research Society (DFG), the California Grape Rootstock Improvement Commission, the California Table Grape Commission, the American Vineyard Foundation, and scholarships provided by the Bundesland Baden-Würtemberg and the German Academic Exchange Service (DAAD).

Genetic structure of an introduced pest, grape phylloxera (Daktulosphaira vitifoliaeFitch), 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°) geographic regions were significantly different from southern (below 43°) 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.Key words: Daktulosphaira vitifoliae, aphid, genetic structure, AFLP-PCR.

Patterns of genetic variation in native grape phylloxera on two sympatric host species.

Random amplified polymorphic DNA (RAPD) markers were used to examine population genetic structure in populations of native grape phylloxera. This research asked: (i) do RAPD markers distinguish two groups corresponding to the two host plant species; and (ii) do RAPD markers distinguish groups according to spatial location, independent of host plant association? Forty-nine phylloxera clones were collected from five pairs of adjacent individuals of two sympatric grape species in five sites along a 145 km transect in Missouri, USA. A high level of polymorphism was observed, with some evidence for structuring between host plant species and no evidence for spatial structuring. An analysis of molecular variance (AMOVA) found that 6.52% of the variance in RAPD banding patterns was attributable to host species and 7.96% of the variance was attributable to spatial location. A cluster analysis did not result in two groups corresponding to the two hosts, or to five groups corresponding to the geographical sites sampled. A Mantel test showed a low correlation between genetic similarity and spatial location. Two of the 93 RAPD markers were nonrandomly associated between the hosts. It is suggested that there may be a small host-mediated effect on genetic variation but stochastic dispersal and a highly heterogeneous environment may be the primary influences on the observed polymorphism.

Genetic structure of an introduced pest, grape phylloxera (&lt;i&gt;Daktulosphaira vitifoliae&lt;/i&gt; 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.

Distribution, Abundance, and Short-Term Persistence of Grape Phylloxera (Homoptera: Phylloxeridae) Populations in Two Regions of the Native Range

Studies of genetic structure in natural populations of herbivores are strengthened when detailed data are available on distribution and relative population size across space and host plants. No studies have documented in depth how grape phylloxera, Daktulosphaira vitifoliae Fitch, a major pest of cultivated grapes, is distributed among its host plants or among sites within regions of its native range. We surveyed the frequency of foliar grape phylloxera, and collected data that described the relative population sizes of grape phylloxera among host plant species, sites, and dates within 2 regions of the native range (Missouri/Arkansas and the southwest United States). Three of 5 grape species surveyed in Missouri/Arkansas had populations of phylloxera. We found that host species in this region were ranked in one year, but not another. We found weak evidence that populations on different host species are regulated independently. Most, but not all, sites with grape habitat in the fragmented topography of the southwest had populations of phylloxera. Their sizes varied widely among sites, but not across dates. We found an extinction rate of 0.23/yr of colonies on vines in the southwest United States The implications of the data for the genetic structure of grape phylloxera populations are discussed.

Differences in Grape Phylloxera-related Grapevine Root Damage in Organically and Conventionally Managed Vineyards in California

Secondary infection of roots by fungal pathogens is a primary cause of vine damage in phylloxera-infested grapevines (Vitis vinifera L.). In summer and fall surveys in 1997 and 1998, grapevine root samples were taken from organically (OMVs) and conventionally managed vineyards (CMVs), all of which were phylloxera-infested. In both years, root samples from OMVs showed significantly less root necrosis caused by fungal pathogens than did samples from CMVs, averaging 9% in OMVs vs. 31% in CMVs. Phylloxera populations per 100 g of root did not differ significantly between OMVs and CMVs, although there was a trend toward higher populations in OMVs. Soil parameters, percent organic matter, total nitrogen, nitrate, and percent sand/silt/clay also did not differ significantly between the two regimes. Cultures of necrotic root tissue showed significantly higher levels of the beneficial fungus Trichoderma in OMVs in 1997 but not in 1998, and there were significantly higher levels of the pathogens Fusarium oxysporum and Cylindrocarpon sp. in CMVs in 1998 but not in 1997. Implications for further research and for viticulture are discussed.

178 Differences in Grape Phylloxera-related Grapevine Root Damage in Organically and Conventionally Managed Vineyards in California

Secondary infection of roots by fungal pathogens is a primary cause of vine damage in phylloxera-infested grapevines. In summer and fall surveys in 1997 and 1998, grapevine root samples were taken from organically managed vineyards (OMVs) and from conventionally managed vineyards (CMVs), all of which were phylloxera-infested. In both years, root samples from OMVs showed significantly less fungal pathogen-caused root necrosis than samples from CMVs, averaging 9% in OMVs and 31% in CMVs. There was no significant difference in phylloxera populations per 100 g of root between OMVs and CMVs, although there was a trend toward higher populations in CMVs. Soil characteristics, percent organic matter, total nitrogen, nitrate, and percent sand/silt/clay were not significantly different between the two regimes. Cultures of necrotic root tissue showed significantly higher levels of the benefical fungus Trichoderma in OMVs in 1997 but not in 1998, and there were significantly higher levels of the pathogens Fusarium oxysporum and Cylindrocarpon spp. in CMVs in 1998 but not in 1997. Implications for further research and viticulture are discussed.

Genetic Structure in Native Populations of Grape Phylloxera (Homoptera: Phylloxeridae)

Random amplified polymorphic DNA (RAPD) markers were used to study genetic structure and diversity in native grape phylloxera populations growing on Vitis arizonica Englemann in central Arizona and on V. riparia Michaux in New York. RAPD data from the Arizona collections were clustered into 3 subpopulations, whereas data from the New York collections were not clustered, which reflected topographic features and the distribution of the sampled vines. Similarity coefficients of the 2 collection areas had similar ranges (0.89–1.0). The similarity coefficient between the Arizona and New York collections was 0.62. Analyses of molecular variance were used to partition the variance in genetic distances, and confirmed the results of the dendrogram clustering. The clustering of the Arizona populations is likely the result of gene flow restriction caused by geographic isolation. Greater diversity was expected among the Arizona populations. That diversity levels were similar suggests that other factors, such as inbreeding or past population history, must play a role in the relatively low level of diversity found in Arizona.