Ten Challenges to Understanding and Managing the Insect-Transmitted, Xylem-Limited Bacterial Pathogen Xylella fastidiosa.

An unprecedented plant health emergency in olives has been registered over the last decade in Italy, arguably more severe than what occurred repeatedly in grapes in the United States in the last 140 years. These emergencies are epidemics caused by a stealthy pathogen, the xylem-limited, insect-transmitted bacterium Xylella fastidiosa. Although these epidemics spurred research that answered many questions about the biology and management of this pathogen, many gaps in knowledge remain. For this review, we set out to represent both the U.S. and European perspectives on the most pressing challenges that need to be addressed. These are presented in 10 sections that we hope will stimulate discussion and interdisciplinary research. We reviewed intrinsic problems that arise from the fastidious growth of X. fastidiosa, the lack of specificity for insect transmission, and the economic and social importance of perennial mature woody plant hosts. Epidemiological models and predictions of pathogen establishment and disease expansion, vital for preparedness, are based on very limited data. Most of the current knowledge has been gathered from a few pathosystems, whereas several hundred remain to be studied, probably including those that will become the center of the next epidemic. Unfortunately, aspects of a particular pathosystem are not always transferable to others. We recommend diversification of research topics of both fundamental and applied nature addressing multiple pathosystems. Increasing preparedness through knowledge acquisition is the best strategy to anticipate and manage diseases caused by this pathogen, described as "the most dangerous plant bacterium known worldwide."

'Sharpshooter' in Botanic Garden: the tale of a rare plant-insect interaction.

Here we report a unique plant-insect interaction between the leafhopper Aloka depressa (tribe Phlogisini) and the host liana, Diploclisia glaucescens, from a Botanic Garden located at the southern edge of Western Ghats in India. Field observations and SEM micrographs were employed to derive evidences on this rare plant-insect interaction. 20-Hydroxyecdysone (20E), insect moulting hormone, was detected and quantified in the host plant D. glaucescens using HPTLC-densitometry. 20E was isolated and characterized from D. glaucescens using column chromatography, 1H-, 13C-NMR and HR-MS. 20E was also detected in A. depressa excrement using HPTLC-densitometry. The leafhopper A. depressa is functioning as a 'sharpshooter' drawing nutrients from the host liana, D. glaucescens, and flinging the waste fluid as droplets through their tail ends. SEM micrographs of A. depressa revealed its external morphological features, characteristic of a sharpshooter. We quantified 20E (0.44-1.44%, dry wt.) in various parts of D. glaucescens. 20E (1.47%, dry wt.) was also detected in the excrement of A. depressa. This plant (D. glaucescens)-insect (A. depressa) association crucially is not damaging the host liana. Considering the diseases caused by sharpshooting leafhoppers in the Americas, this association and the survival of the host plant (D. glaucescens) is illustrating a unique plant-insect interaction.

Transcriptome and population structure of glassy-winged sharpshooters (Homalodisca vitripennis) with varying insecticide resistance in southern California.

BACKGROUND: Homalodisca vitripennis Germar, the glassy-winged sharpshooter, is an invasive insect in California and a critical threat to agriculture through its transmission of the plant pathogen, Xylella fastidiosa. Quarantine, broad-spectrum insecticides, and biological control have been used for population management of H. vitripennis since its invasion and subsequent proliferation throughout California. Recently wide-spread neonicotinoid resistance has been detected in populations of H. vitripennis in the southern portions of California's Central Valley. In order to better understand potential mechanisms of H. vitripennis neonicotinoid resistance, we performed RNA sequencing on wild-caught insecticide-resistant and relatively susceptible sharpshooters to profile their transcriptome and population structure. RESULTS: We identified 81 differentially expressed genes with higher expression in resistant individuals. The significant largest differentially expressed candidate gene linked to resistance status was a cytochrome P450 gene with similarity to CYP6A9. Furthermore, we observed an over-enrichment of GO terms representing functions supportive of roles in resistance mechanisms (cytochrome P450s, M13 peptidases, and cuticle structural proteins). Finally, we saw no evidence of broad-scale population structure, perhaps due to H. vitripennis' relatively recent introduction to California or due to the relatively small geographic scale investigated here. CONCLUSIONS: In this work, we characterized the transcriptome of insecticide-resistant and susceptible H. vitripennis and identified candidate genes that may be involved in resistance mechanisms for this species. Future work should seek to build on the transcriptome profiling performed here to confirm the role of the identified genes, particularly the cytochrome P450, in resistance in H. vitripennis. We hope this work helps aid future population management strategies for this and other species with growing insecticide resistance.

Using X-ray Micro-Computed Tomography to Three-Dimensionally Visualize the Foregut of the Glassy-Winged Sharpshooter (Homalodisca vitripennis).

Dissecting the heads of small insects belonging to the order Hemiptera for detailed anatomical investigation with light or scanning electron microscopy is difficult, time-consuming, and destructive, often resulting in sample preparation artifacts. Nevertheless, the structural details of these insects often hold critical information regarding their interactions with bacterial pathogens. For example, the glassy-winged sharpshooter (GWSS) is an efficient vector of the bacterium Xylella fastidiosa, the causal agent of Pierce's disease in grape vines, but the foregut of this species is exceptionally difficult to dissect. Here, we describe a simple, non-destructive method to investigate the structure of the anterior gut of GWSS using high-resolution micro-computed X-ray tomography (microCT). The use of microCT eliminates the need for destructive dissection and reveals the morphology of small insects in three dimensions, allowing the user to virtually dissect the sample. The use of microCT imaging is a promising and powerful tool in the entomological sciences for studying the structures of vector insects, especially for difficult-to-dissect regions such as the foregut.

Improved draft reference genome for the Glassy-winged Sharpshooter (Homalodisca vitripennis), a vector for Pierce's disease.

Homalodisca vitripennis (Hemiptera: Cicadellidae), known as the glassy-winged sharpshooter, is a xylem feeding leafhopper and an important agricultural pest as a vector of Xylella fastidiosa, which causes Pierce's disease in grapes and a variety of other scorch diseases. The current H. vitripennis reference genome from the Baylor College of Medicine's i5k pilot project is a 1.4-Gb assembly with 110,000 scaffolds, which still has significant gaps making identification of genes difficult. To improve on this effort, we used a combination of Oxford Nanopore long-read sequencing technology combined with Illumina sequencing reads to generate a better assembly and first-pass annotation of the whole genome sequence of a wild-caught Californian (Tulare County) individual of H. vitripennis. The improved reference genome assembly for H. vitripennis is 1.93-Gb in length (21,254 scaffolds, N50 = 650 Mb, BUSCO completeness = 94.3%), with 33.06% of the genome masked as repetitive. In total, 108,762 gene models were predicted including 98,296 protein-coding genes and 10,466 tRNA genes. As an additional community resource, we identified 27 orthologous candidate genes of interest for future experimental work including phenotypic marker genes like white. Furthermore, as part of the assembly process, we generated four endosymbiont metagenome-assembled genomes, including a high-quality near complete 1.7-Mb Wolbachia sp. genome (1 scaffold, CheckM completeness = 99.4%). The improved genome assembly and annotation for H. vitripennis, curated set of candidate genes, and endosymbiont MAGs will be invaluable resources for future research of H. vitripennis.

Insect-to-insect horizontal transmission of a phytoreovirus in the absence of an infected plant host.

Vertical transmission of Homalodisca vitripennis reovirus (HoVRV) from glassy-winged sharpshooter (GWSS, Homalodisca vitripennis (Germar)) females to progeny occurred in laboratory assays at frequencies too low (2.6%-15.4%) to account for HoVRV incidence (90-100%) in field populations resident in citrus. Because citrus is immune to HoVRV and no plant host is known, horizontal transmission of HoVRV from insect-to-insect was evaluated. Exposure of colony-reared, virus-free test nymphs to HoVRV-infected source adults held in the same cage for 10 days on virus-immune cowpea resulted in HoVRV transmission (13.3%-30.7%) to test nymphs. HoVRV was not transmitted when exposure was indirect and required passive movement of virions through the xylem of immune citrus seedlings. Collectively, these results demonstrate direct insect-to-insect horizontal transmission of HoVRV, providing a plausible explanation for high incidence of HoVRV in GWSS field populations in the absence of efficient vertical transmission or a plant host.

Inoculum reduction and vector control on the temporal progress of citrus variegated chlorosis incidence.

BACKGROUND: Citrus variegated chlorosis (CVC) is an important citrus disease caused by the sharpshooter-transmitted bacterium Xylella fastidiosa subsp. pauca. Information about the efficacy of its disease management is bounded to either inoculum reduction or vector control. This study aimed to assess the role of different frequencies of roguing of symptomatic trees combined with different chemical control programs for the sharpshooters on CVC temporal progress. The experimental area was set up in October 2005 and had a 3 × 3 factorial design. Symptomatic tree removal intervals were every 28, 56, and 112 days to May 2012. Vector control treatments were no control, program A (PA), and program B (PB). Contact insecticides were sprayed every 28 (PA) or 14 (PB) days. Trees within both programs were treated with systemic and foliar insecticides to October 2008. After this, systemic insecticides were discontinued and only foliar treatments were applied. The number of diseased trees and the sharpshooter population were assessed to April 2015. RESULTS: Detection of the first symptomatic trees was similar for all treatments and occurred, on average, in month 50 after planting. Sharpshooter population and cumulative disease incidence were similar for all roguing intervals and between vector control programs, differing in plots with no control. We observed a reduction over 85% in disease incidence for both PA and PB compared with no control. CONCLUSION: These results support systematic control of sharpshooters as an efficient strategy for reducing CVC progress. © 2021 Society of Chemical Industry.

Insecticide resistance in California populations of the glassy-winged sharpshooter Homalodisca vitripennis.

BACKGROUND: The glassy-winged sharpshooter, Homalodisca vitripennis (Germar), is a primary vector of Pierce's disease of grapes in California. Systemic imidacloprid treatments have been the mainstay of area-wide treatment programs that were established in the Central Valley (Kern and Tulare Counties) and Southern California (Riverside County) during the 1990s to combat the pest. The programs helped to suppress populations on citrus, a major sharpshooter host, to levels that significantly reduced migration into adjacent vineyards. However, beginning in 2012, there has been a resurgence of glassy-winged sharpshooter populations in Kern and Tulare counties, and hitherto successful treatment strategies have not been as effective. This study investigated the possibility that insecticide resistance was a contributing factor to the population resurgence. RESULTS: Topical application bioassays detected high levels of resistance to imidacloprid in Kern and Tulare populations, and lower levels of resistance (perhaps due to cross-resistance) to the foliar neonicotinoid acetamiprid (20-fold), the pyrethroid fenpropathrin (7.4-fold), and the butenolide flupyradifurone (4-fold). Samples of glassy-winged sharpshooters from citrus groves under organic management also exhibited high levels of imidacloprid resistance. CONCLUSION: The long-term use of imidacloprid has selected for resistance in glassy-winged sharpshooters. The most resistant populations also exhibited resistance to the foliar neonicotinoid acetamiprid, the pyrethroid fenpropathrin, and the butenolide flupyradifurone. High levels of imidacloprid resistance in insects sampled from organic groves indicate that resistant insects are migrating from nearby conventional groves, which could compromise the control of sharpshooters in organic systems with insecticides affected by cross-resistance. © 2021 Society of Chemical Industry.

Two new species of Balacha (Insecta: Hemiptera: Cicadellidae: Cicadellinae) from Brazil with comments on their phylogenetic position and biogeography.

The sharpshooter genus Balacha Melichar, 1926 has seven recognized species and is distributed in southern South America (Argentina, Brazil, Paraguay, and Uruguay). Here, two new Brazilian species of this genus are described and illustrated, one (B. ancora sp. nov.) from Serra do Caraça and Serra do Cipó (Minas Gerais State) and another (B. nigroflava sp. nov.) from Ponta Grossa (Paraná State) and Passo Fundo (Rio Grande do Sul State). Based on 67 morphological characters, we carried out a parsimony analysis to estimate the phylogenetic position of the two new species. Using implied weighting in TNT, a single topology within Balacha was obtained with k values that varied from 1.599 to 7.412. In this topology, the two new taxa appeared within the clade of black Balacha species. A biogeographical analysis (S-DIVA) suggested that the ancestral area of distribution of Balacha was the Atlantic Forest domain. Balacha species were so far known to have only Eryngium (Apiaceae) species as host plants. Here we report a new host plant for the genus: B. ancora sp. nov. specimens were collected on Actinocephalus polyanthus (Eriocaulaceae). Balacha similis and B. rubripennis are newly recorded from Argentina and specimens of the latter have a very distinct color pattern from the typical Brazilian specimens. Finally, a key to species and maps showing the known distribution of the genus are given (several state records of species are also provided).

Redescription of Pseudophera heveli Kramer (Hemiptera, Cicadellidae) with the first description of the female.

Pseudophera heveli Kramer is redescribed from Monteverde, Costa Rica. The female is described for the first time. Fifteen images of the species are provided, including genitalia.

XadA2 Adhesin Decreases Biofilm Formation and Transmission of Xylella fastidiosa subsp. pauca.

Xylella fastidiosa is a vector-borne bacterium that causes diseases in many plants of economic interest. The bacterium-vector initial interactions involve bacterial membrane-bound adhesins that mediate cell attachment to the foregut of insect vectors. We investigated the role of the afimbrial adhesin XadA2 in the binding and biofilm formation of X. fastidiosa subsp. pauca to vector surfaces in vitro, as well as its potential to disrupt pathogen transmission. We showed that XadA2 has binding affinity for polysaccharides on sharpshooter hindwings, used as a proxy for the interactions between X. fastidiosa and vectors. When in a medium without carbon sources, the bacterium used wing components, likely chitin, as a source of nutrients and formed a biofilm on the wing surface. There was a significant reduction in X. fastidiosa biofilm formation and cell aggregation on vector wings in competition assays with XadA2 or its specific antibody (anti-XadA2). Finally, pathogen acquisition and transmission to plant were significantly reduced when the vectors acquired X. fastidiosa from an artificial diet supplemented with anti-XadA2. These results show that XadA2 is important in mediating bacterial colonization in the insect and that it could be used as a target for blocking X. fastidiosa transmission.

Description of a new, unusual species of Diestostemma Amyot & Serville (Hemiptera, Cicadellidae) from Ecuador.

Diestostemma bicristata sp. nov., is described from Napo Province, Ecuador. It is unusual for the genus in lacking a visible white powdery covering, having raised, weakly reticulate veins on the forewing, a short metathoracic femur (half length of the metathoracic tibia), and is the only species with a double-crested pronotum.

Fluid dynamics in the functional foregut of xylem-sap feeding insects: A comparative study of two Xylella fastidiosa vectors.

Xylem sap sucking insects are adapted to ingest fluids under tension. Although much has been learned about such feeding strategy, this adaptation still poses several unresolved questions, including how these insects ingest against strong xylem sap tension. Xylem sap-feeding insects are vectors of the plant pathogenic xylem-limited bacterium Xylella fastidiosa. This bacterium colonizes the cuticular lining of the foregut of vectors in a persistent manner. We used micro-computed tomography and scanning electron microscopy to investigate the foregut morphometry of two X. fastidiosa vector species: Philaenus spumarius and Graphocephala atropunctata (Hemiptera: Aphrophoridae and Cicadellidae, respectively). On the basis of morphometric data, we built a hydrodynamic model of the foregut of these two insect species, focusing on the precibarium, a region previously shown to be colonized by X. fastidiosa and correlated with pathogen acquisition from and inoculation to plants. Our data show that space in the P. spumarius functional foregut could potentially harbor twice as many cells as similar space in G. atropunctata, although the opposite trend has been observed with biological samples. Average flow velocity of ingested fluid depended on the percentage of the cibarium volume exploited for suction: if the entire volume were used, velocities were in the range of meters per second. In contrast, velocities on the order of those found in the literature (about 10 cm/s) were attained if only 5% of the cibarium volume were exploited. Simulated bacterial colonization of the foregut was analyzed in relation to hydrodynamics and pressure needed for insects to ingest. Our model is designed to represent the diameter reduction of the food canal in both insect species when infected with X. fastidiosa. Results indicated that full bacterial colonization significantly increased the mean sap-sucking flow velocity. In particular, the colonization increased the maximum section-averaged velocity in the G. atropunctata more than two times and the net pressure needed to mantain the flow in the precibarium when colonized is relevant (about 0.151 MPa) if compared to a standard xylem sap tension (1 MPa). Bacterial colonization also influenced the sucking process of the G. atropunctata, by hindering the formation of a recirculation zone (or eddy), that characterizd the flow in the distal part of the precibarium when bacteria were absent. On the other hand, considering the pressure the insect must generate to feed, X. fastidiosa colonization probably influences fitness of the G. atropunctata more than that of P. spumarius.

Two new species of Hanshumba from Southeastern Brazil and a key to males of the genus (Insecta: Hemiptera: Cicadellidae: Cicadellini)

ABSTRACT The genus Hanshumba Young, 1977 is recorded from Southeastern and Southern Brazil (Atlantic Forest) and currently includes only three species. Here we describe and illustrate, based mainly on features of the male terminalia, two new species from State of Espírito Santo, Municipality of Santa Teresa: H. setifera sp. nov. and H. teresa sp. nov. The former can be distinguished by the male pygofer and anal tube with large processes bearing numerous setae and the aedeagus with pair of dentiform processes on median portion, whereas the latter has three pairs of longitudinal flanges on the aedeagal shaft. A key to males of the genus is added and its taxonomic status is briefly discussed.

Juliaca Melichar, 1926 (Hemiptera: Cicadellidae: Cicadellini): description of a new species from Southeastern Brazil and redescriptions of J. sertigerula (Jacobi, 1905) and J. xanthogramma (Signoret, 1854) comb. nov.

A new species of the sharpshooter genus Juliaca Melichar, 1926, J. nigra sp. nov., from Southeastern Brazil (State of Espírito Santo) is described based on specimens collected on a coffee plantation. In addition, J. sertigerula (Jacobi, 1905), an Andean species, and Tettigonia xanthogramma Signoret, 1854, from Southeastern Brazil (states of Minas Gerais and Rio de Janeiro), are redescribed. The former species was not adequately illustrated and the latter was not treated in the most recent monograph on the Cicadellini. The identity of T. xanthogramma is elucidated and it is transferred to Juliaca (J. xanthogramma comb. nov.).

Mating disruption of Homalodisca vitripennis (Germar) (Hemiptera: Cicadellidae) by playback of vibrational signals in vineyard trellis.

BACKGROUND: Glassy-winged sharpshooter (GWSS), Homalodisca vitripennis (Germar) (Hemiptera: Cicadellidae) is an important vector of the bacterium Xylella fastidiosa, the causal agent of Pierce's disease of grapevine. Area-wide insecticide applications have suppressed GWSS populations for ∼ 25 years, but reduced levels of insecticide susceptibility have been reported. Therefore, alternative methods of control are needed. Objectives of this study were to evaluate the efficacy of playback of vibrational mating communication signals for disrupting mating of GWSS in a natural vineyard setting and to evaluate spectral properties of signal transmission through vineyard trellis. RESULTS: Playback reduced mating of GWSS on grapevines. A total of 28 (of 134) male-female pairs mated in the control treatment (silence) and only one (of 134) pair mated when treated with the vibrational signal playback. Playback of vibrational signals through vineyard trellis was affected by distance from the signal source, with frequency composition being the highest at the source and lowest on vines positioned away from the source. Frequency composition in canes housing test insects decreased exponentially as distance from the source increased, whereas the relative amplitude of analyzed frequencies decreased linearly. CONCLUSION: Although further studies are needed prior to method implementation, data from this study continue to support integration of vibrational mating disruption with current methods to suppress GWSS populations. Published 2018. This article is a U.S. Government work and is in the public domain in the USA.

Descriptions of seven new Acrogonia species from South America (Hemiptera: Cicadellidae: Cicadellinae: Proconiini).

Seven new species of the economically important sharpshooter genus Acrogonia Stål are described and illustrated: A. falcata sp. nov. (French Guiana and state of Amazonas, Brazil), A. felixi sp. nov. (department of Loreto, Peru), A. quintasi sp. nov. (state of Pará, Brazil), A. distincta sp. nov. (state of Amazonas, Brazil), A. dentata sp. nov. (department of San Martin in Peru, French Guiana, and states of Amazonas, Mato Grosso, Pará, and Rondônia in Brazil), A. luizi sp. nov. (province of Pastaza in Ecuador and state of Amazonas in Brazil), and A. lobulata sp. nov. (province of Orellana, Ecuador). In addition to the external morphology, color pattern, and male genitalia, detailed descriptions and illustrations of the female genitalia are provided for three of the new species (A. dentata, A. luizi, and A. lobulata). Females of the other four new species are unknown. Acrogonia includes now 39 species, being among the most species-rich genera of the Proconiini.

Descriptions of eight new Acrogonia species from the Amazon region, including peculiar features of the female genitalia (Hemiptera: Cicadellidae: Cicadellinae: Proconiini).

Eight new species of the agriculturally important sharpshooter genus Acrogonia Stål, 1869 are described and illustrated from Brazil (one of them also recorded from Peru): A. amazonensis sp. nov. (state of Amazonas), A. clarae sp. nov. (states of Amazonas and Amapá), A. barbara sp. nov. (state of Mato Grosso), A. youngi sp. nov. (department of Madre de Dios in Peru and state of Acre), A. albertoi sp. nov. (state of Pará), A. tenuis sp. nov. (state of Mato Grosso), A. filiformis sp. nov. (states of Pará and Mato Grosso), and A. interrupta sp. nov. (state of Mato Grosso). In addition to the external morphology, color pattern and male genitalia, this paper includes detailed descriptions and illustrations of the female genitalia of three new species (A. barbara, A. filiformis and A. tenuis). The first ovipositor valvulae of A. tenuis are quite peculiar because they bear a large basal lobe and a projection on the median portion of the ventral margin, both displaying setae. Females of the other five new species are not known.

Transmission of phage by glassy-winged sharpshooters, a vector of Xylella fastidiosa.

Xylella fastidiosa subsp. fastidiosa (Xff) is the causal agent of Pierce's Disease (PD) of grapevines and is vectored by the glassy-winged sharpshooter (GWSS, Homalodisca vitripennis). Previously we have reported the development of a bacteriophage (phage) based biocontrol system for PD, but no information on insect transmission of phages has been reported. Here we communicate that laboratory reared GWSSs fed on cowpea plants (Vigna unguiculata subsp. unguiculata) harboring the virulent phage Paz were able to uptake of phage efficiently when the phage was present in high concentration, but were inefficient in transfer to plants.

Three new species of Fonsecaiulus (Hemiptera, Cicadellidae, Cicadellini) from Brazil and key to species of the genus.

Three new sharpshooter species of the genus Fonsecaiulus Young, 1977 are described and illustrated from specimens collected in the Brazilian Atlantic Forest, Fonsecaiulus rectangularis and Fonsecaiulus guttiformis, and in the Brazilian Cerrado, Fonsecaiulus filiformis. The descriptions are based on features from the external morphology, color pattern, and male and female genital structures. Comparisons of the three new taxa with the remaining six Fonsecaiulus species are provided. An identification key to males of all known species of the genus is given.