New Host Plant Species of Grapevine Virus A Identified with Vector-Mediated Infections.

Grapevine virus A (GVA) is an economically important virus and a member of the genus Vitivirus (family Betaflexiviridae) that causes a range of symptoms with qualitative and quantitative effects on grape production. Wild and domesticated species of Vitis, including hybrids used as rootstocks, are considered important natural hosts of GVA. Mechanical transmission to some herbaceous plant species, graft transmission, and vector transmission from grape to grape by various mealybugs and soft scale insects have been reported. Under laboratory and greenhouse conditions, this study demonstrates the transmission of GVA from grapes to alternative hosts by the vine mealybug (Planococcus ficus). Results of ELISA, end-point one-step RT-PCR, and real-time RT-PCR, and in some cases electron microscopy and genome sequencing, confirmed successful transmission to three new plant species commonly found in Croatian vineyards: velvetleaf (Abutilon theophrasti), redroot pigweed (Amaranthus retroflexus), and field poppy (Papaver rhoeas), along with Chenopodium murale and the previously known host Nicotiana benthamiana, with variable infection rates. Depending on the host species, symptoms in the form of leaf reddening, yellow spots, reduced growth of lateral shoots, systemic vein clearing, foliar deformation and rugosity, and dwarfism were observed in GVA-infected plants, whereas no symptoms were observed in infected plants of A. theophrasti. Reverse transmission from these new hosts to grapevines by Pl. ficus was not successful. These results confirm four new GVA host species and open new research venues.

Comparison of two different host plant genera responding to grapevine leafroll-associated virus 3 infection.

Grapevine leafroll-associated virus 3 (GLRaV-3) is one of the most important viruses of grapevine but, despite this, there remain several gaps in our understanding of its biology. Because of its narrow host range - limited to Vitis species - and because the virus is restricted to the phloem, most GLRaV-3 research has concentrated on epidemiology and the development of detection assays. The recent discovery that GLRaV-3 can infect Nicotiana benthamiana, a plant model organism, makes new opportunities available for research in this field. We used RNA-seq to compare both V. vinifera and P1/HC-Pro N. benthamiana host responses to GLRaV-3 infection. Our analysis revealed that the majority of DEGs observed between the two hosts were unique although responses between the two hosts also showed several shared gene expression results. When comparing gene expression patterns that were shared between the two hosts, we observed the downregulation of genes associated with stress chaperones, and the induction of gene families involved in primary plant physiological processes. This is the first analysis of gene expression profiles beyond Vitis to mealybug-transmitted GLRaV-3 and demonstrates that N. benthamiana could serve as a useful tool for future studies of GLRaV-3-host interactions.

Discriminating between viable and membrane-damaged cells of the plant pathogen Xylella fastidiosa.

Xylella fastidiosa is a plant pathogenic bacterium with devastating consequences to several crops of economic importance across the world. While this pathogen has been studied for over a century in the United States, several aspects of its biology remain to be investigated. Determining the physiological state of bacteria is essential to understand the effects of its interactions with different biotic and abiotic factors on cell viability. Although X. fastidiosa is culturable, its slow growing nature makes this technique cumbersome to assess the physiological state of cells present in a given environment. PMA-qPCR, i.e. the use of quantitative PCR combined with the pre-treatment of cells with the dye propidium monoazide, has been successfully used in a number of studies on human pathogens to calculate the proportion of viable cells, but has less frequently been tested on plant pathogens. We found that the use of a version of PMA, PMAxx, facilitated distinguishing between viable and non-viable cells based on cell membrane integrity in vitro and in planta. Additional experiments comparing the number of culturable, viable, and total cells in planta would help further confirm our initial results. Enhancers, intended to improve the efficacy of PMAxx, were not effective and appeared to be slightly toxic to X. fastidiosa.

Infection and Colonization of Nicotiana benthamiana by Grapevine leafroll-associated virus 3.

Grapevine leafroll disease is an increasing problem in all grape-growing regions of the world. The most widespread agent of the disease, Grapevine leafroll-associated virus 3 (GLRaV-3), has never been shown to infect species outside of the genus Vitis. Virus transmission to several plant species used as model systems was tested using the vine mealybug, Planococcus ficus. We show that GLRaV-3 is able to infect Nicotiana benthamiana. Working with GLRaV-3 infected N. benthamiana revealed distinct advantages in comparison with its natural host Vitis vinifera, yielding both higher viral protein and virion concentrations in western blot and transmission electron microscopy observations, respectively. Immunogold labelling of thin sections through N. benthamiana petioles revealed filamentous particles in the phloem cells of GLRaV-3 positive plants. Comparison of assembled whole genomes from GLRaV-3 infected V. vinifera vs. N. benthamiana revealed substitutions in the 5' UTR. These results open new avenues and opportunities for GLRaV-3 research.

Spatial genetic structure of a vector-borne generalist pathogen.

Vector-borne generalist pathogens colonize several reservoir species and are usually dependent on polyphagous arthropods for dispersal; however, their spatial genetic structure is generally poorly understood. Using fast-evolving genetic markers (20 simple sequence repeat loci, resulting in a total of 119 alleles), we studied the genetic structure of the vector-borne plant-pathogenic bacterium Xylella fastidiosa in Napa Valley, CA, where it causes Pierce's disease when it is transmitted to grapevines from reservoir plants in adjacent riparian vegetation. Eighty-three different X. fastidiosa multilocus microsatellite genotypes were found in 93 isolates obtained from five vineyards, resulting in an index of clonal fraction closer to 0 and a Simpson's genotypic diversity index (D) closer to a maximum value of 1. Moderate values of Nei's gene diversity (H(Nei); average H(Nei) = 0.41) were observed for most of the X. fastidiosa populations. The low Wright's index of genetic diversity among populations calculated by the FSTAT software (Wright's F(ST) index) among population pairs (0.0096 to 0.1080) indicated a weak or absent genetic structure among the five populations; a panmictic population was inferred by Bayesian analyses (with the STRUCTURE and BAPS programs). Furthermore, a Mantel test showed no significant genetic isolation by distance when both Nei (r = -0.3459, P = 0.268) and linearized (r = -0.3106, P = 0.269) indices were used. These results suggest that the riparian vegetation from which vectors acquire the pathogen prior to inoculation of grapevines supports a diverse population of X. fastidiosa.

Geographic distribution and relative abundance of the invasive glassy-winged sharpshooter: effects of temperature and egg parasitoids.

The capacity to predict the geographic distribution and relative abundance of invasive species is pivotal to developing policy for eradication or control and management. Commonly used methods fall under the ambit of ecological niche models (ENMs). These methods were reviewed and shortcomings identified. Weather-driven physiologically based demographic models (PBDMs) are proposed that resolve many of the deficiencies of ENMs. The PBDM approach is used to analyze the invasiveness of the polyphagous glassy-winged sharpshooter (Homalodisca vitripennis [Germar]), a pest native to the southeastern United States and northeastern Mexico that extended its range into California in 1989. Glassy-winged sharpshooter vectors the pathogenic bacterium, Xylella fastidiosa (Wells) that causes Pierce's disease in grape and scorch-like diseases in other plants. PBDMs for glassy-winged sharpshooter and its egg parasitoids (Gonatocerus ashmeadi Girault and G. triguttatus Girault) were developed and linked to a PBDM for grape published by Wermelinger et al. (1991). Daily weather data from 108 locations across California for the period 1995-2006 were used to drive the PBDM system, and GRASS GIS was used to map the simulation results. The geographic distribution of glassy-winged sharpshooter, as observed, is predicted to be largely restricted to the warm areas of southern California, with the action of the two egg parasitoids reducing its abundance >90%. The average indispensable mortality contributed by G. triguttatus is <1%. A temperature-dependent developmental rate model for X. fastidiosa was developed that suggests its geographic range is also limited to the warm inland areas of southern California. Biological control of glassy-winged sharpshooter further decreases the pathogen's relative range. Climate warming scenarios of +2°C and +3°C suggest that the distribution and severity of glassy-winged sharpshooter and X. fastidiosa will increase in the agriculturally rich central valley of California. The utility of holistic analyses for formulating control policy and tactics for invasive species is discussed.

Demography of gut symbiotic and aposymbiotic Nezara viridula L. (Hemiptera: Pentatomidae).

Nezara viridula L. is a highly polyphagous and cosmopolitan pentatomid stink bug. Despite its economic importance, aspects of its biology are poorly understood. N. viridula has one primary bacterium associated with its gastric caeca, which females provide to offspring by smearing it on the surface of eggs during oviposition. We studied the impact of three temperatures and egg mass surface sterilization on N. viridula's nymphal development rate and reproductive performance. Our results show that maintenance of the symbiont is affected both by temperature and egg mass surface sterilization. We detected the symbiont in 100, 84, and 8.3% of the untreated control insects at 20, 25, and 30 degrees C, respectively, by using polymerase chain reaction. In insects originated from surface sterilized egg masses, the symbiont was never detected at 20 or 30 degrees C and was detected in only 1 of 21 insects at 25 degrees C. Nymphal mean development time decreased with increasing temperature, but there were no differences between the sterilized and control treatments. Sterilized insects at 20 degrees C lived longer than insects in any other treatment but never laid eggs. Life table analysis of N. viridula adults showed that net reproductive rate, intrinsic rate of increase, finite rate of increase, and gross reproductive rate were not significantly different among treatments except at 20 degrees C for the surface sterilized treatment. Mean generation time, however, was significantly longer at 20 degrees C (70.96 +/- 4.43 d), regardless of the surface sterilization treatment. Our results highlight the effect that temperature has on the maintenance of this symbiosis and its relationship with N. viridula host's development and reproduction.

Xylella fastidiosa cultivation on a minimal solid defined medium.

A simple defined solid medium containing citrate and succinate, three amino acids (L-glutamine, L-asparagine, and L-cysteine), hemin chloride, potato starch, gellan gum (GelRite), and mineral salts supported the growth of grape strains of Xylella fastidiosa, the bacterial pathogen that causes Pierce's disease of grape. Isolation efficiency from infected grape plant samples, determined by the number of colony forming units recovered, on the defined medium was slightly less ( approximately 10-fold) or indistinguishable from two standard rich media used for culturing X. fastidiosa, PWG and PD3, respectively. The bacterium also grew on media with citrate and L-glutamine as the only carbon and nitrogen sources. Potato starch was not essential for bacterial growth, but no growth was observed on media without hemin chloride. Agar inhibited bacterial growth when used as the gelling agent.