René Sforza

New mealybug species vectoring Grapevine leafroll-associated viruses-1 and -3 (GLRaV-1 and -3)

Many grape viruses, such as filamentous Grapevine leafroll-associated viruses in the Closteroviridae family, are spread primarily through infected propagating material. However, there is increasing evidence that leafroll disease are spread in the field by insect vectors, namely mealybugs and other scale insects. This study was carried out in the northern wine-growing regions of France where Grapevine leafroll-associated virus-1 and -3 (GLRaV-1 and -3) are the most widespread grape Ampelovirus species. The vineyards were inspected for presence of mealybug and scale insects and grapes infected by GLRaV-1 and -3. Mealybugs, Heliococcus bohemicus, Phenacoccus aceris (Pseudococcidae) and the soft scale insect Parthenolecanium corni (Coccidae), were capable of a transmission efficiency of 14%, 23% and 29% respectively. GLRaV-1 and -3 infections that resulted from virus transmission were confirmed with DAS-ELISA using polyclonal antibodies. This is the first report of GLRaV-1 and -3 transmission by mealybug and coccid species in France, and the first report of the ability of H. bohemicus and Phenacoccus aceris to transmit these viruses to grapevines. The relevance of these findings with regards to maintenance of virus-free grapevine stocks and to control leafroll spread in commercial vineyards is discussed.

Biology and Management of Mealybugs in Vineyards

Economic losses resulting from vineyard mealybug infestations have increased dramatically during the past decade. In response, there has been a cosmopolitan effort to improve control strategies and better understand mealybug biology and ecology, as well as their role as vectors of plant pathogens. Mealybugs are named for the powdery secretions covering their bodies. The most important vineyard mealybugs belong to the subfamily Pseudococcinae (Hardy et al. 2008). Although numerous mealybug species are found in vineyards, this chapter will cover only those that have risen to the level of primary pest. These are the grape mealybug, Pseudococcus maritimus (Ehrhorn), obscure mealybug, Pseudococcus viburni (Signoret), long-tailed mealybug, Pseudococcus longispinus (Targioni Tozzetti), citrophilus mealybug, Pseudococcus calceolariae (Maskell), vine mealybug, Planococcus ficus (Signoret), citrus mealybug, Planococcus citri (Risso), pink hibiscus mealybug, Maconellicoccus hirsutus (Green), and the newly identified Gills mealybug, Ferrisia gilli Gullan. Meanwhile in Brazil and India, Dysmicoccus brevipes (Cockerell) and Xenococcus annandalei Silvestri respectively, feed on vine roots. Collectively, these species will be referred to as the vineyard mealybugs, although their host range is diverse and many are pests of other agricultural crops and ornamental plants (McKenzie 1967; Ben-Dov 1995).

Evidence for multiple introductions of Centaurea stoebe micranthos (spotted knapweed, Asteraceae) to North America

Invasive species’ success may depend strongly on the genetic resources they maintain through the invasion process. We ask how many introductions have occurred in the North American weed Centaurea stoebe micranthos (Asteraceae), and explore whether genetic diversity and population structure have changed as a result of introduction. We surveyed individuals from 15 European native range sites and 11 North American introduced range sites at six polymorphic microsatellite loci. No significant difference existed in the total number of alleles or in the number of private alleles found in each range. Shannon–Weaver diversity of phenotype frequencies was also not significantly different between the ranges, while expected heterozygosity was significantly higher in the invasive range. Population structure was similar between the native range and the invasive range, and isolation by distance was not significant in either range. Traditional assignment methods did not allocate any North American individuals to the sampled European populations, while Bayesian assignment methods grouped individuals into nine genetic clusters, with three of them shared between North America and Europe. Invasive individuals tended to have genetically admixed profiles, while natives tended to assign more strongly to a single cluster. Many North American individuals share assignment with Romania and Bulgaria, suggesting two separate invasions that have undergone gene flow in North America. Samples from three other invasive range sites were genetically distinct, possibly representing three other unique introductions. Multiple introductions and the maintenance of high genetic diversity through the introduction process may be partially responsible for the invasive success of C. stoebe micranthos.

When invasion increases population genetic structure: a study with Centaurea diffusa

Biological invasions offer excellent systems to study the evolutionary processes involved in introductions of species to new ranges. Molecular markers can reveal invasion histories and the effects of introductions on amounts and structuring of genetic variation. We used five polymorphic microsatellite loci to elucidate genetic diversity and population structure between native range and introduced range populations of a prominent North American rangeland weed, Centaurea diffusa (Asteraceae). We found that the total number of alleles and the number of private alleles was slightly higher in the native Eurasian range, and that allelic richness did not differ between the ranges, indicating overall levels of diversity were similar in Eurasia and North America. It therefore seems unlikely that this invasion has been affected by genetic bottlenecks or founder effects. Indeed, results of assignment tests suggest that multiple introductions have contributed to North America’s C. diffusa invasion. Additionally, assignment tests show that both Eurasian and North American sites had a strong pattern of mixed genetic ancestry. This mixed assignment corresponded to a lack of geographic population structure among Eurasian samples. The lack of population structure in the native range conflicts with general expectations and findings to date for invasion genetics, and cautions that even species’ native ranges may show signs of recent ecological upheaval. Despite the mixed assignments, North American samples showed strong population structure, suggesting that the invasion has been characterized by long-range dispersal of genetically distinct propagules across the introduced range.

What Magnitude Are Observed Non-Target Impacts from Weed Biocontrol?

A systematic review focused by plant on non-target impacts from agents deliberately introduced for the biological control of weeds found significant non-target impacts to be rare. The magnitude of direct impact of 43 biocontrol agents on 140 non-target plants was retrospectively categorized using a risk management framework for ecological impacts of invasive species (minimal, minor, moderate, major, massive). The vast majority of agents introduced for classical biological control of weeds (>99% of 512 agents released) have had no known significant adverse effects on non-target plants thus far; major effects suppressing non-target plant populations could be expected to be detectable. Most direct non-target impacts on plants (91.6%) were categorized as minimal or minor in magnitude with no known adverse long-term impact on non-target plant populations, but a few cacti and thistles are affected at moderate (n = 3), major (n = 7) to massive (n = 1) scale. The largest direct impacts are from two agents (Cactoblastis cactorum on native cacti and Rhinocyllus conicus on native thistles), but these introductions would not be permitted today as more balanced attitudes exist to plant biodiversity, driven by both society and the scientific community. Our analysis shows (as far as is known), weed biological control agents have a biosafety track record of >99% of cases avoiding significant non-target impacts on plant populations. Some impacts could have been overlooked, but this seems unlikely to change the basic distribution of very limited adverse effects. Fewer non-target impacts can be expected in future because of improved science and incorporation of wider values. Failure to use biological control represents a significant opportunity cost from the certainty of ongoing adverse impacts from invasive weeds. It is recommended that a simple five-step scale be used to better communicate the risk of consequences from both action (classical biological control) and no action (ongoing impacts from invasive weeds).

Development of a Multiplex PCR for Identification of Vineyard Mealybugs

ABSTRACT A simple molecular tool was developed and tested to identify seven mealybug species found in North American vineyards: Pseudococcus maritimus Ehrhorn, Pseudococcus viburni (Signoret), Pseudococcus longispinus (Targioni-Tozzeti), Pseudococcus calceolariae (Maskell), Planococcus ficus (Signoret), Planococcus citri (Risso), and Ferrisia gilli Gullan. The developed multiplex PCR is based on the mitochondrial cytochrome c oxidase subunit one gene. In tests, this single-step multiplex PCR correctly identified 95 of 95 mealybug samples, representing all seven species and collected from diverse geographic regions. To test the sensitivity, single specimen samples with different Pl. ficus developmental stages (egg to adult female and adult male) were processed PCR and the resulting output provided consistent positive identification. To test the utility of this protocol for adult males caught in sex baited pheromone traps, Pl. ficus adult males were placed in pheromone traps, aged at a constant temperature of 26 ± 2°C, and processed with the multiplex each day thereafter for 8 d. Results showed consistent positive identification for up to 6 d (range, 6–8 d). Results are discussed with respect to the usefulness of this molecular tool for the identification of mealybugs in pest management programs and biosecurity of invasive mealybugs.

Weed biological control in the European Union: from serendipity to strategy

Biological control of weeds is a globally recognised approach to the management of some of the most troublesome invasive plants in the world. Accidental introductions of agents accounted for all weed biological control agent establishments in the European Union until 2010, but these examples include some current or emerging control successes both large and small, from the redistribution of the weevil Stenopelmus rufinasus Gyllenhal (Coleoptera: Curculionidae) for the control of small outbreaks of Azolla filiculoides Lam. (Azollaceae), to the large scale control provided by the cochineal insect Dactylopius opuntiae (Cockerell) (Hemiptera: Dactylopiidae), used against some problematic prickly pears (Opuntia spp. (Cactaceae)), and the ragweed beetle Ophraella communa LeSage (Coleoptera: Chrysomelidae), against common ragweed, Ambrosia artemisiifolia L. (Asteraceae), which are providing benefits to an increasing number of Member States of the European Union. Recent programmes involving the intentional introduction of biological control agents against target weeds including Fallopia japonica (Hout.) Ronse Decr. (Polygonaceae), Impatiens glandulifera Royle (Balsaminaceae) and Acacia longifolia (Andrews) Willd (Fabaceae) show a shift from luck to judgement in the European Union. The inclusion of new weed targets on the European Invasive Species Regulation should lead to a growth in the profile and use of biological control which would be assisted by the publication of any successes from the few intentional introductions covered in this paper.

Inferring the complex origins of horticultural invasives: French broom in California

Investigating the origins of invasive populations provides insight into the evolutionary and anthropogenic factors underlying invasions, and can inform management decisions. Invasive species introduced for horticultural purposes often have complex origins typified by multiple introductions of species, cultivars, and genotypes, and interspecific and intraspecific hybridizations in introduced ranges. Such complex introduction histories may result in complex genetic signatures in the invaded range, making inferences about origins difficult, particularly when all putative sources cannot be sampled. In this study, we inferred the origins of the invasive French broom complex in California using 12 nuclear microsatellite markers. We characterized the genetic diversity and population structure of invasive and horticultural brooms in their invaded range in California and of Genista monspessulana in its native Mediterranean range. Overall, no significant differences in allelic richness, observed heterozygosity, inbreeding, or genetic structure were observed between the invaded and native ranges, but differences existed among populations within ranges. Bayesian STRUCTURE analysis revealed three genetic clusters in the French broom complex. Nearly all native G. monspessulana assigned highly to a single cluster. Many invasives assigned to a second cluster that contained Genista canariensis, Genista stenopetala, and ornamental sweet broom, and the remaining invasives assigned to a third cluster that also contained some G. monspessulana individuals from Sardinia and Corsica. Admixture between the second and third clusters was detected. Approximate Bayesian Computation analysis of six alternative scenarios supported the hypothesis that some invasive French broom is derived from an unsampled population branching from ornamental sweet broom. A combination of factors, including multiple introductions, escapes from cultivation, and inter-taxon hybridization, likely contribute to the invasive success of French broom in California and may have important implications for management, in particular biological control.

Determining the geographic origin of invasive populations of the mealybug Planococcus ficus based on molecular genetic analysis

Determining the most likely source of an invasive pest species might help to improve their management by establishing efficient quarantine measures and heading the search of efficient biological control agents. Planococcus ficus is an invasive mealybug pest of vineyards in Argentina, California, Mexico, Peru and South Africa. This mealybug pest had a previously known geographic distribution spanning southern Europe, the Middle East, and parts of northern Africa. In North America, Pl. ficus was first discovered in the early 1990s and soon thereafter in Mexico. To determine the origin of invasive populations in North America, Pl. ficus from California and Mexico were compared with material throughout its presumptive native range in the Mediterranean region, as well as material collected from an older invasion in South Africa and recently invaded Argentina. From each sample location, genomic DNA was sequenced for the nuclear internal transcribed spacer one (ITS1) and the mitochondrial cytochrome c. oxidase one (CO1). Phylogenetic analyses of CO1, ITS1 and concatenated CO1 and ITS1 data-sets using Bayesian and neighbor-joining analysis support two major divisions: a European grouping (Europe, Tunisia, Turkey) and a Middle Eastern grouping (Israel and Egypt). The invasive populations in Argentina and South Africa align with the European group and the invasive populations in North America align with the Middle Eastern group, with one Israel sample aligning closely with the North American clade, suggesting that Israel was the origin of those populations.

Preliminary results of a survey on the role of arthropod rearing in classical weed biological control.

The rearing of arthropods is an essential but sometimes neglected and underestimated part of a classical weed biological control programme. Success in rearing is usually a pre-requisite to conducting host-specificity tests, obtaining enough individuals for initial field release or, later, for large-scale implementation. Although most biological control researchers can list situations where agent development has been stopped or slowed due to rearing difficulties, failures seldom get reported in the literature, thus preventing us from gauging the extent and relevance of rearing issues. To rectify this, a questionnaire was developed to investigate the prevalence of rearing problems in weed biological control programmes and to classify their occurrence according to a list of variables (e.g. taxonomy, biological features, genetic issues and researcher/programme attributes). The questionnaire was sent to 80 researchers from eight countries; 65% responded, generating 79 useful responses. Results confirm that, of the challenges faced in programmes, rearing is the most prevalent (56% out of ten possible general problem categories). The most common rearing problems encountered were conditions that were not conducive to mating and/or oviposition (30% of reported arthropod cases) or development (22% of reported arthropod cases). Our results identify key areas for rearing improvement, thus contributing to increased weed biological control project successes.