Sharpshooter Leafhoppers Research Articles

Dynamics of Xylella fastidiosa and plum leaf scald: Insights from a long‐term study in Brazilian orchards

AbstractXylella fastidiosa is one of the most dangerous plant‐pathogenic bacteria globally, affecting various host species. Plum leaf scald (PLS) is the primary disease affecting plum trees in Brazil, with plant eradication efforts significantly increasing in recent years. This study aims to elucidate the disease dynamics and its vector in three monitored orchards from 2010 to 2015 and associate the pattern of pathogen dispersal with mechanical pruning. The incidence was lower in Porto Amazonas, starting at 0% and reaching 14% after 5 years. In contrast, the orchards in Araucaria and Palmeira showed an initial incidence around 5% in the first year, exceeding 80% by the end of the study. Sharpshooter leafhoppers, the vectors, were collected at ground and plant canopy level during the last 3 years. In 2013, more leafhoppers were found near the ground, but their numbers equalized across heights in the following years. Kernel smoothing revealed spatial dependence, and an autologistic model indicated an aggregated infection pattern with increased PLS risk for neighbouring plants. The border effect at the epidemic's onset was linked to pathogen entry via external vectors. The disease's progression along rows was attributed to effective mechanical pathogen transmission through pruning. Therefore, in regions with several summer prunings, it is recommended to sanitize pruning shears to prevent pathogen transmission. Constant monitoring and control of the vector are essential for effective disease management.

The vector regulation hypothesis: dynamic competition between pathogen and vector behaviors constrains Xylella fastidiosa biofilm development in sharpshooter foreguts.

Xylella fastidiosa (Xf) bacteria form biofilm on the cuticular surfaces of the functional foregut (precibarium and cibarium) of its vectors, xylem fluid-ingesting sharpshooter leafhoppers and spittlebugs. While much is known about Xf biofilm development and maturation in vitro, little is known about these processes in vectors. Real-time (RT)-PCR was used to quantify Xf genomes daily in the functional foreguts of blue-green sharpshooters, Graphocephala atropunctata, over 7 days of exposure to infected grapevines. Scanning electron microscopy (SEM) was used to examine Xf biofilm formation at 4 and 7 days of that time course. PCR showed populations building and reducing over a 4-day cycle. SEM revealed that foreguts at 4 days showed variability in quantity and location of bacterial attachment. Only early-stage biofilm formation occurred in low-turbulence areas of the cibarium, while high-turbulence areas of the cibarium and precibarium had rare but older, more developed macro-colonies. Biofilm was almost absent at 7 days but left behind adhesive material and remnants of prior colonization. Evidence supports the hypothesis that bacterial colonization was repeatedly interrupted and constrained by the vector. Behaviors such as egestion and enzymatic salivation likely can loosen and eject Xf biofilm, perhaps when profuse biofilm interferes with ingestion. Thus, vector acquisition of Xf is a dynamic and stochastic process of interactions between bacteria and insects. We further hypothesize for future testing that the insect can regulate this interaction. A deep understanding of Xf acquisition will aid the ongoing development of grapevine resistance to vector transmission of xylellae diseases.IMPORTANCEXylella fastidiosa (Xf) is one of the most destructive invasive plant pathogens in the world, able to hijack new vectors when it invades a region; yet the temporal interplay of bacterial colonization and insect behavior is unknown. This paper describes important findings about the process of Xf biofilm formation and maturation in a vector, contrasting similarities and differences with such formation in vitro. Results support the hypothesis that the behavior of the vector constrains and may regulate Xf biofilm formation, in dynamic competition with the bacterium. The data from this paper partly explain why Xf is so successful at invasion. Because the bacterium can be acquired and inoculated very quickly, it can move readily from old to new vectors and host plants in all-new environments. Our findings are relevant to biosecurity decisions because they demonstrate the importance of identifying potential vector species in the Xylella invasion front.

Definitive elucidation of the inoculation mechanism of Xylella fastidiosa by sharpshooter leafhoppers

Definitive elucidation of the inoculation mechanism of Xylella fastidiosa by sharpshooter leafhoppers

DC‐EPG assisted comparison of European spittlebugs and sharpshooters feeding behaviour on grapevine

AbstractXylem‐feeding is apparently the only requirement making an insect a competent vector of the bacterium Xylella fastidiosa, an organism responsible for the devastation of the Southern Italian olive forest and nowadays considered one of the most feared threats to agriculture and landscape in Europe, including vineyards. Here, we used the direct current‐electrical penetration graph (DC‐EPG) technique to compare and describe the feeding behaviour on grapevine of four xylem‐feeding species considered candidate vectors of X. fastidiosa widespread in Europe, namely two spittlebugs (the meadow spittlebug Philaenus spumarius and the spittlebug Neophilaenus campestris) and two sharpshooter leafhoppers (the rhododendron leafhopper Graphocephala fennahi and the green leafhopper Cicadella viridis). We created a standard for the analysis of EPG waveforms recorded with a DC‐EPG device, describing feeding activities performed by these insects from stylet insertion into the plant to withdrawal. This standard, along with freely available software, has been developed to harmonize the calculation of feeding behavioural parameters in xylem‐feeders. The most relevant differences between the two vector taxa were the probing frequency and the dynamics of xylem ingestion. Sharpshooters tended to perform significantly more probes than spittlebugs. In contrast, the latter spent longer times in low‐frequency xylem ingestion, characterized by scattered contractions of the cibarial dilator muscle interspersed with periods of pump inactivity. Cicadella viridis was the species displaying the highest frequency of the electrical pattern found to be associated with X. fastidiosa inoculation in spittlebugs (Xe). Feeding behavioural data presented here represent an important step forward for deepening our knowledge of xylem‐sap feeding insects' interaction with both the host plants and the bacterium they transmit.

Fluid dynamic simulations at the interface of the blue-green sharpshooter functional foregut and grapevine xylem sap with implications for transmission of Xylella fastidiosa.

Xylella fastidiosa is a multi-continental, lethal, plant pathogenic bacterium that is transmitted by sharpshooter leafhoppers (Insecta: Hemiptera: Cicadellidae: Cicadellinae) and adult spittlebugs (Hemiptera: Aphrophoridae). The bacterium forms biofilms in plant xylem and the functional foregut of the insect. These biofilms serve as sources of inoculum for insect acquisition and subsequent inoculation to a healthy plant. In this study, 3D fluid dynamic simulations were performed for bidirectional cibarial propulsion of xylem sap through tube-like grapevine xylem and an anatomically accurate model of the functional foregut of the blue-green sharpshooter, Graphocephala atropunctata. The analysis supports a model of how fluid dynamics influence X. fastidiosa transmission. The model supports the hypothesis that X. fastidiosa inoculation is mostly driven by detachment of bacteria from the foregut due to high-velocity flow during egestion (outward fluid flow from the stylets). Acquisition occurs by fluid dynamics during both egestion and ingestion (fluid uptake through the stylets and swallowing). These simulation results are supported by previously reported X. fastidiosa colonization patterns in the functional foregut and sharpshooter stylet probing behaviors. The model indicates that xylem vessel diameter influences drag forces imposed on xylem wall-adherent bacteria; thus, vessel diameter may be an important component of the complex transmission process. Results from this study are directly applicable to development of novel grapevine resistance traits via electropenetrographic monitoring of vector acquisition and inoculation behaviors.

First sharpshooter species proven as vectors of Xylella fastidiosa subsp. multiplex in Prunus salicina trees in Brazil

Plum leaf scald (PLS), caused by the vector-borne bacterium Xylella fastidiosa, is a major obstacle to the expansion of plum crops in Brazil. In other affected crops, this pathogen is naturally transmitted mainly by sharpshooter leafhoppers (Hemiptera: Cicadellidae: Cicadellinae). Despite the importance of PLS, the vector species responsible for bacterial spread in plums remain unknown. This study, therefore, aimed to determine the X. fastidiosa transmission ability of three sharpshooter species commonly found in plum orchards in southern and southeastern Brazil: Macugonalia cavifrons (Stål), Macugonalia leucomelas (Walker), and Sibovia sagata (Signoret). After a 72-h acquisition access period to plum trees infected with X. fastidiosa subsp. multiplex (sequence type 67), the insects were transferred for an inoculation access period of 96 h to 10–20 healthy Prunus salicina plants for each sharpshooter species, using four insects per plant. Bacterial infection in the plants was verified 7 months after inoculation by polymerase chain reaction. Results showed that the three sharpshooter species were able to transmit X. fastidiosa to plums, with mean transmission efficiencies by single insects of 16%, 14%, and 18% for M. cavifrons, M. leucomelas, and S. sagata, respectively. To our knowledge, this study is the first to identify vector species and prove the transmission of a PLS strain of X. fastidiosa subsp. multiplex by sharpshooters in Brazil.

Functional foregut anatomy of the blue\u2013green sharpshooter illustrated using a 3D model

Sharpshooter leafhoppers (Hemiptera: Cicadellidae: Cicadellinae) are important vectors of the plant pathogenic bacterium Xylella fastidiosa Wells et al. (Xanthomonadales: Xanthomonadaceae). This pathogen causes economically significant diseases in olive, citrus, and grapes on multiple continents. Bacterial acquisition and inoculation mechanisms are linked to X. fastidiosa biofilm formation and fluid dynamics in the functional foregut of sharpshooters, which together result in egestion (expulsion) of fluids likely carrying bacteria. One key X. fastidiosa vector is the blue–green sharpshooter, Graphocephala atropunctata (Signoret, 1854). Herein, a 3D model of the blue–green sharpshooter functional foregut is derived from a meta-analysis of published microscopy images. The model is used to illustrate preexisting and newly defined anatomical terminology that is relevant for investigating fluid dynamics in the functional foregut of sharpshooters. The vivid 3D illustrations herein and supplementary interactive 3D figures are suitable resources for multidisciplinary researchers who may be unfamiliar with insect anatomy. The 3D model can also be used in future fluid dynamic simulations to better understand acquisition, retention, and inoculation of X. fastidiosa. Improved understanding of these processes could lead to new targets for preventing diseases caused by X. fastidiosa.

Review of the EPG Waveforms of Sharpshooters and Spittlebugs Including Their Biological Meanings in Relation to Transmission of Xylella fastidiosa (Xanthomonadales: Xanthomonadaceae).

Electropenetrography (EPG) is one of the most rigorous methods to study stylet probing behaviors of piercing-sucking insects whose mouthparts move invisibly inside hosts. EPG is particularly useful for identifying vector behaviors that control transmission (acquisition, retention, and inoculation) of plant pathogens, comparing those behaviors among vector species, and aiding in development of novel vector and disease management tactics. Xylella fastidiosa (Wells et al.) is a gram-negative, invasive bacterium native to the Americas, where it is the causal agent of lethal scorch-type diseases such as Pierce's disease of grapevines. Xylella fastidiosa is transmitted by sharpshooter leafhoppers (Hemiptera: Cicadellidae: Cicadellinae) and spittlebugs (Hemiptera: Aphrophoridae). Despite over 75 yr of study, details of the inoculation mechanism of X. fastidiosa were unknown until the advent of EPG research with sharpshooters. Herein, the following topics are presented: 1) review of key EPG principles and waveforms published to date, emphasizing sharpshooters and spittlebugs; 2) summary of present understanding of biological meanings of sharpshooter waveforms; 3) review of mechanisms of transmission for X. fastidiosa illuminated by EPG; and 4) recommendations of the most useful waveform categories for EPG use in future, quantitative comparisons of sharpshooter stylet probing on various treatments such as infected versus uninfected plants, resistant varieties, or insecticide treatments. In addition, new work on the functional anatomy of the precibarial valve is discussed in the context of X. fastidiosa transmission and EPG waveforms. Also, the first block diagram of secondary, signal-processing circuits for the AC-DC EPG is published, and is discussed in relation to EPG signals appearances and meanings.

Do Sharpshooters From Around the World Produce the Same EPG Waveforms? Comparison of Waveform Libraries From Xylella fastidiosa (Xanthomonadales: Xanthomonadaceae) Vectors Kolla paulula (Hemiptera: Cicadellidae) From Taiwan and Graphocephala atropunctata From California.

When an exotic invasive species is a vector-borne plant pathogen, vector feeding behavior must be studied to identify potential host plant range and performance of specialized pathogen transmission behaviors. The most rigorous tool for assessing vector feeding behavior is electropenetrography (EPG). Xylella fastidiosa Wells et al. is a gram-negative bacterium native to the Americas, where it is the causal agent of lethal scorch-type diseases such as Pierce's disease (PD) of grapevines. In 2002, a PD strain of X. fastidiosa invaded Asia for the first time, as confirmed from grape vineyards in Taiwan. Kolla paulula (Wallker), a native Asian species of sharpshooter leafhopper, was found to be the primary vector in Taiwanese vineyards. This study used an AC-DC electropenetrograph to record stylet probing behaviors of K. paulula on healthy grapevines. The main objective was to create an EPG waveform library for K. paulula. Waveform description, characterization of R versus emf components (electrical origins), and proposed biological meanings of K. paulula waveforms are reported. In addition, comparison of K. paulula waveforms with those from the most efficient, native vector of X. fastidiosa in California vineyards, Graphocephala atropunctata, is also reported. Overall, both species of sharpshooters had similar-appearing waveforms. Five new findings were identified, especially that the previously described but rare waveform subtype, B1p, was extensively produced in K. paulula recordings. Sharpshooter waveforms from species worldwide share a high degree of similarity. Thus, EPG methods can be rapidly applied to potential vectors where X. fastidiosa is newly introduced.

An overview on the worldwide vectors of Xylella fastidiosa

The Gram-negative bacterium Xylella fastidiosa causes disease in a number of important crops and plants within natural ecosystems. Most notable among these diseases are Pierce’s disease of grapevine, Citrus Variegated Chlorosis, Almond Leaf Scorch, Oleander Leaf Scorch and Olive Quick Decline Syndrome. In response to substantial economic losses, extensive research efforts have been underway over the past several decades in North and South America. Recently, X. fastidiosa has been detected in several countries of Europe and Asia, likely appearing as a result of global trading of plant material. Once established in a new region, X. fastidiosa spread is dependent on the obligate transmission by xylem-sap feeding insects. Sharpshooter leafhoppers (Hemiptera: Cicadellidae: Cicadellinae) are the best-studied group of vectors of this bacterium; however the list of known vector species is expanding. As X. fastidiosa moves into new regions, the combination of an introduced pathogen with existing or introduced vectors carries the potential of increasing economically important disease problems in the local context. By reviewing ecological and biological information of known X. fastidiosa vectors and the epidemiology of local X. fastidiosa outbreaks, this paper aims at increasing our understanding of vector– X. fastidiosa interactions.

Certain applied electrical signals during EPG cause negative effects on stylet probing behaviors by adult Lygus lineolaris (Hemiptera: Miridae)

This study is the first to fully evaluate whether electrical signals applied to large insects during electropenetrography (EPG; also called electrical penetration graph) negatively affect insect behavior. During EPG, electrical signals are applied to plants, and thus to the gold-wire-tethered insects feeding on them. The insect completes an electrical circuit whose changes in voltage reflect the insect’s stylet probing/penetration behaviors, recorded as waveform output. For nearly 50 years of EPG science, evidence has supported that there are no or negligible effects on tiny insects from applied electricity during EPG. Recently however, EPG studies of large-bodied hemipterans such as heteropterans and sharpshooter leafhoppers have been published. The wider stylet diameters of such large insects cause them to have lower inherent resistances to applied signals compared with smaller insects, conveying more electrical current. The present study asked whether such increased currents would affect insect stylet probing, by comparing Lygus lineolaris behaviors on pin-head cotton squares using an AC-DC electropenetrograph. Effects of AC or DC applied signals were separately examined in two factorial studies, each comparing four input resistor (Ri) levels (106, 107, 108 and 109 Ω) and four applied voltage levels (2, 60, 150 and 250 mV). Results showed that changes in both probing and non-probing behaviors were indeed caused by changing signal type, Ri level, or applied voltage. Negative effects on feeding were numerically greater overall for DC than AC applied signals, perhaps due to muscular tetany from DC; however, AC versus DC could not be statistically tested. Results strongly support the need for flexible Ri and applied voltage levels and types, to tailor instrument settings to the size and special needs of each insect subject. Our findings will facilitate further EPG studies of Lygus spp., such as host plant resistance or insecticidal assays/bioassays to assess mode of action and appropriate dosage. It is hoped that this study will also inform EPG studies of similar, large heteropterans in the future.

Anatomy and Ultrastructure of Male Reproductive System in Two LeafhoppersCicadella viridisandKolla paulula(Hemiptera: Auchenorrhyncha: Cicadellidae)

The morphology and ultrastructure of the male reproductive system were investigated in two species of sharpshooter leafhoppers, Cicadella viridis (L.) and Kolla paulula (Walker), using light microscopy and transmission electron microscopy. The gross morphology and ultrastructure of the male reproductive system in the two species is similar to those of other Cicadellidae and related Auchenorrhyncha. The two species differ in the number and shape of testicular follicles, the shape of the seminal vesicle, and the size and color of the accessory gland. The testicular follicle consists of the epithelium lying on a thick basal lamina surrounded by a connective sheath, and a wide lumen filled with germ cells. Large numbers of vesicles and granular materials are observed in the epithelial cell. The two seminal vesicles close-set under a common tunica are composed of the basal lamina, the thick muscular-connective sheath, a mono-layered epithelium, and the vesicular lumen. The vesicles and granules are extensive in the cytoplasm of the flattened epithelial cell, whose apical surface usually has numerous microvilli. The accessory gland is made up of thin muscular-connective sheath, the basal lamina, the epithelium, and the tubular lumen. The epithelial cell with secretory granules has abundant rough endoplasmic reticulum and small vesicles, and its cytoplasm abounds with arrays of three types of granules. Spermiogenesis and sperm ultrastructure of K. paulula was also observed, whose sperm can be distinguished from those of other Cicadellidae by the accessory bodies and mitochondrial derivatives with gap region.

The current status of the quick decline syndrome of olive in southern Italy

The quick decline syndrome of olive (OQDS) is a disease that appeared all of a sudden some years ago in a restricted area near the city of Gallipoli (Ionian coast of the Salento peninsula, southern-east Italy) and began spreading through the heavily olive-grown countryside of lower Salento. Xylella fastidiosa, a quarantine pathogen of American origin previously undetected in the European Union territory, except for two unconfirmed records from Kosovo and Turkey, proved to be consistently associated with symptomatic trees. X. fastidiosa is a Gram-negative bacterium that invades and multiplies in the xylem vessels of infected hosts, from which it is acquired by xylem-feeding insect vectors (belonging to Auchenorrhyncha, including cicadellids sharpshooter leafhoppers group, Cicadellidae, Cicadellinae), and aphrophorids (cercopids and spittlebugs, Cercopidae) and transferred to other plants. The Salentian strain of X. fastidiosa, denoted CoDiRO, was obtained in axenic culture. Its genome, a DNA molecule ca. 2.5 million base-pairs in size, was sequenced and identified as a genotype of X. fastidiosa subsp. pauca molecularly identical to an isolate of the same subspecies from Costa Rica. In nature, strain CoDiRO infects a number of woody and shrubby hosts but not grapevines and citrus and is mainly transmitted by Philaenus spumarius (meadow spittlebug), a froghopper quite common in the Salento area where it thrives primarily on olive. Since OQDS eradication and sanitation of infected olives are unfeasible, strategies have been envisaged for restraining the spread of the pathogen and its vector within the boundaries of the currently infected zone.

Lineage-Specific Patterns of Genome Deterioration in Obligate Symbionts of Sharpshooter Leafhoppers.

Plant sap-feeding insects (Hemiptera) rely on obligate bacterial symbionts that provision nutrients. Some of these symbionts are ancient and have evolved tiny genomes, whereas others are younger and retain larger, dynamic genomes. Baumannia cicadellinicola, an obligate symbiont of sharpshooter leafhoppers, is derived from a relatively recent symbiont replacement. To better understand evolutionary decay of genomes, we compared Baumannia from three host species. A newly sequenced genome for Baumannia from the green sharpshooter (B-GSS) was compared with genomes of Baumannia from the blue-green sharpshooter (B-BGSS, 759 kilobases [kb]) and from the glassy-winged sharpshooter (B-GWSS, 680 kb). B-GSS has the smallest Baumannia genome sequenced to date (633 kb), with only three unique genes, all involved in membrane function. It has lost nearly all pathways involved in vitamin and cofactor synthesis, as well as amino acid biosynthetic pathways that are redundant with pathways of the host or the symbiotic partner, Sulcia muelleri. The entire biosynthetic pathway for methionine is eliminated, suggesting that methionine has become a dietary requirement for hosts. B-GSS and B-BGSS share 33 genes involved in bacterial functions (e.g., cell division, membrane synthesis, metabolite transport, etc.) that are lost from the more distantly related B-GWSS and most other tiny genome symbionts. Finally, pairwise divergence estimates indicate that B-GSS has experienced a lineage-specific increase in substitution rates. This increase correlates with accelerated protein-level changes and widespread gene loss. Thus, the mode and tempo of genome reduction vary widely among symbiont lineages and result in wide variation in metabolic capabilities across hosts.

Identification of three potential insect vectors of Xylella fastidiosa in southern Italy.

In order to identify potential vectors of Xylella fastidiosa in olive orchards in Puglia (southern Italy), Hemiptera insects were collected from October to December, 2013, in olive orchards with high incidences of X. fastidiosa associated with “rapid decline” symptoms. The study focused on species in the Auchenorrhyncha (sharpshooter leafhoppers and froghoppers or spittlebugs), a group that includes known vectors of X. fastidiosa . Adults of three species, i.e. Philaenus spumarius L. (Aphrophoridae), Neophilaenus campestris Fallen (Aphrophoridae) and Euscelis lineolatus Brulle (Cicadellidae) were captured, from which total DNA was extracted and assayed by PCR using three sets of specific primers designed for X. fastidiosa detection. Results of PCR showed that 38 out of a total of 84 tested insects were positive for X. fastidiosa , i.e. eight (of 20) P. spumarius , 14 (of 18) N. campestris and 16 (of 46) E. lineolatus . PCR amplicons of the RNA polymerase sigma-70 factor gene from six specimens (two of each insect species) were sequenced. The sequences obtained were 99.3‒99.4% identical. BlastN analyses demonstrated these sequences to be similar to those of X. fastidiosa isolates from olive OL-X and OL-G reported from Puglia, whereas they displayed distant molecular identity (always less than 98%) with X. fastidiosa subspecies from other countries. The detection of X. fastidiosa in P. spumarius and, for the first time, in N. campestris and E. lineolatus (which, unlike the others, is a phloem feeder), indicates potential vectoring roles of these insects for the spread of the bacterium in Puglia. Further investigations and specific infectivity trials are required to definitively determine the roles of these insects as effective vectors of this pathogen.

Egg parasitoids (Hymenoptera: Mymaridae and Trichogrammatidae) of Kolla paulula (Walker) (Hemiptera: Cicadellidae) in Taiwan

Pseudoligosita nephotetticum (Mani) (Hymenoptera: Trichogrammatidae) and Gonatocerus (Cosmocomoidea) sp. (Hymenoptera: Mymaridae) have been identified for the first time as egg parasitoids of the sharpshooter leafhopper Kolla paulula (Walker) (Hemiptera: Cicadellidae: Cicadellinae: Cicadellini) in Taiwan. K. paulula has been recently identified as a candidate vector of the phytopathogenic bacterium Xylella fastidiosa, the causative agent of Pierce's disease of grapes and similar diseases of other affected plants in Taiwan, and thus has become of particular economic importance. Also provided is a summary of the known records of egg parasitoids (Mymaridae and Trichogrammatidae) of other leafhoppers from the tribe Cicadellini in the world, as well as taxonomic notes on P. nephotetticum.

New and Simple Methods for Studying Hemipteran Stylets, Bacteriomes, and Salivary Sheaths in Host Plants

Abstract Many hemipteran insects are important agricultural pests because they cause direct feeding damage to their host plants and / or transmit plant disease agents including viruses and bacteria. Microscopic and behavioral studies on five hemipteran species from four families (Psyllidae, Aphididae, Cicadellidae, and Aleyrodidae) showed that their exuviae (molted skins) normally had either fully or partially extended stylets in a feeding-like position. In most cases these stylets were still partially embedded in their host plants after ecdysis, which indicated that plant-feeding hemipteran nymphs use their stylets to anchor themselves to host plants during molting. This phenomenon was used here to study the stylet length and ultrastructure in exuviae of various instars, which is normally more difficult in nymphs than in adults because of the fragility and smaller size of nymphs. Additionally, autofluorescence was used for studying the hemipteran salivary sheaths of nymphs and adults in their host plants. This method is based on fixation of free hand sections of plant parts on which hemipteran insects have been feeding, then mounting and examination of these sections with epifluorescence or confocal microscopy. No embedding, microtomy, or staining is necessary for this method that makes it much faster and simpler than other methods. Autofluorescence was also used to study the location and size of bacteriomes/mycetomes (organs containing symbionts) in hemipteran eggs and nymphs. The above methods were applied successfully with the Asian citrus psyllid (Diaphorina citri Kuwayama), melaleuca psyllid (Boreioglycaspis melaleucae Moore), oleander aphid (Aphis nerii Boyer de Fonscolomb), the whitefly Bemisia tabaci (Gennadius), and/or the glassy-winged sharpshooter leafhopper (Homalodisca vitripennis Germar).

DETECTION AND IDENTIFICATION METHODS AND NEW TESTS AS USED AND DEVELOPED IN THE FRAMEWORK OF COST 873 FOR BACTERIA PATHOGENIC TO STONE FRUITS AND NUTS

The quarantine pathogen Xylella fastidiosa (EPPO A1 list), a xylem-inhabiting, vector-transmitted, Gramnegative, very slow growing bacterium, was cultured and properly described in 1987 in the USA as the cause of Pierce’s disease (PD) of grapevine (Vitis vinifera) and of phony peach disease (PPD) in peach (Prunus persica) and in 1993 in Brazil as the cause of citrus variegated chlorosis (CVC) in sweet orange (Citrus sinensis). Moreover, it was found that the bacterium also causes a number of so-called leaf scorch diseases in Prunus spp. [including almond leaf scorch (ALS) in P. armeniaca and plum leaf scald (PLS) in P. domestica], ornamental trees, Coffea arabica (CLC, in Brazil, isolated in 1995), and many other plant species (see under ‘Host range’). Many wild plants, including grasses, sedges and trees may carry the pathogen without showing symptoms. All these diseases are quite destructive, giving substantial yield losses. They occur mainly in tropical/subtropical areas, although leaf scorch diseases also occur in much colder climates, e.g. oak leaf scorch in eastern North America, as far north as Canada. Several pathogenic genotypes have been described, which are often host-specific (e.g., the strain will not cause disease if introduced to peach or plum). The following subspecies are currently known: (i) Xylella fastidiosa subsp. fastidiosa (erroneously named X. f. subsp. piercei) strains from cultivated grape, alfalfa, almond (two), and maple; (ii) X. f. subsp. multiplex, strains from peach, elm, plum, pigeon grape, sycamore, and almond; (iii) X. f. subsp. pauca, strains from citrus and probably those of coffee; (iv) X. f. subsp. sandyi, strains from Nerium oleander. X. f. subsp. taske was described from the ornamental tree Chitalpa tashkentensis on the basis of one gene sequence only and its status as a separate subspecies is therefore not ultimately established. Vectors are sharpshooter leafhoppers (Hemiptera:Cicadellidae) and froghoppers or spittlebugs (Hemiptera: Cercopidae), the most important of which in the USA include Cuerna costalis and Graphocephala versuta (PPD), Draculacephala minerva (green sharpshooter); G. atropunctata (blue-green sharpshooter, formerly Hordnia circellata) and Homalodisca vitripennis (formerly H. coagulata, glassy-winged sharpshooter, GWSS) (PD). CVC vectors in Brazil include Acrogonia terminalis, Dilobopterus costalimai and Oncometopia fascialis. Local possible vectors for Europe are Cicadella viridis and Philaenus spumarius (meadow spittle bug), as well as all other members of the family Cercopidae and the leafhopper subfamily Cicadellinae. The bacterium is, as far it is known, still absent from Europe. For more general information on the bacterium, epidemiology, control, etc. see Purcell (1997), Hopkins and Purcell (2002), EPPO/OEPP (1992), Chatterjee et al. (2008), Janse and Obradovic (2010 ), and http://www.cnr. berkeley.edu/xylella/ In order to study the disease and train on diagnosis in a pro-active way, the EU COST873 project organised in October 2010 a workshop specifically on X. fastidiosa detection and identification with excellent support and participation of two outstanding experts in the field, Prof. A.H. Purcell and Prof. R.P.P. Almeida from the University of California at Berkeley (USA). The workshop was hosted by IAM, Bari, Italy (http://cost873.ch/5_activites/ meeting_detail.php?ID=28). Most of the methods presented here have been found useful by the US experts over the years and were practised during the workshop.

Understanding the effects of multiple sources of seasonality on the risk of pathogen spread to vineyards: vector pressure, natural infectivity, and host recovery

Seasonality plays an important role in the dynamics of infectious disease. For vector‐borne pathogens, the effects of seasonality may be manifested in the variability in vector abundance, vector infectiousness, and host‐infection dynamics over the year. The relative importance of multiple sources of seasonality on the spread of a plant pathogen, Xylella fastidiosa, into vineyards was explored. Observed seasonal population densities of the primary leafhopper vector, Graphocephala atropunctata, from 8 years of surveys in northern California were incorporated into a model of primary spread to estimate the risk of pathogen infection under different scenarios regarding seasonality in vector natural infectivity (i.e. constant or increasing over the season) and grapevine recovery from infection (i.e. none or seasonal recovery). The extent to which local climatic conditions affect risk estimates via differences in vector abundance was investigated. Seasonal natural infectivity, seasonal recovery, and especially the combination, reduced (up to 8‐fold on average) within‐season and cumulative yearly estimates of pathogen spread. Estimated risk of infection also differed greatly among years due to large differences in vector abundance, with wet and moderate winter and spring conditions favouring higher G. atropunctata abundance. Seasonal variation of the pathogen–vector interaction may play an important role in the dynamics of disease in vineyards, reducing the potential prevalence from what it could be in their absence. Moreover, climate, by affecting sharpshooter leafhopper abundance or activity, may influence Pierce’s disease dynamics.

Diseases of Important Crops, a Review of the Causal Fastidious Prokaryotes and Their Insect Vectors

Phytopathogenic fastidious prokaryotes are plant pathogens that either resist to grow in any available bacterial culture media or require specific or enriched media to grow. They include Xylella fastidiosa, Leifsonia xyli subsp. xyli, L. xyli subsp. cynodontis and Clavibacter michiganensis subsp. sepedonicus and C. michiganensis subsp. michiganensis that reside in xylem and spiroplasmas, phytoplasmas and Candidatus Liberibacter spp. that reside in phloem. X fastidiosa is the causal agent of more than 19 diseases; among them Pierce's disease of grape and citrus variegated chlorosis are two major maladies that cause serious economic loss on wine and citrus juice industry. L. xyli subsp. xyli, and L. xyli subsp. cynodontis are associated with ratoon stunting disease of sugarcane and Bermuda grass stunting respectively and C. michiganensis subsp. sepedonicus with bacterial ring rot in potato and C. michiganensis subsp. michiganensis with bacterial tomato canker. Spiroplasmas are the causal agents of citrus stubborn, corn stunt and periwinkle diseases. Phytoplasmas are associated with more than 500 diseases worldwide. Ca. Liberibacter spp., are the causal agents of citrus Huanglongbing or citrus greening, zebra chip disease of potato and others. General characteristics of X fastidiosa including (i) its scientific classification, host ranges and diseases incited, and cell shape and size, (ii) specific and enriched media for X fastidiosa, (iii) symptoms induced by X fastidiosa, (iv) geographic distribution of X. fastidiosa, and (v) the insect vectors that transmit the diseases will be discussed. Pierce's disease is the limiting factor for the establishment of wine industry for the entire southeastern United States from Texas to the Carolinas along the gulf coast of Mexico. Recent introduction of the glassy-winged sharpshooter leafhoppers in California has threatened the winery industry of California. The successful isolation of X. fastidiosa from the tissues with citrus variegated chlorosis (CVC) symptoms followed by the identification of the major insect vectors provided crucial information for citrus growers and citrus juice industry to deal with the CVC crisis in Brazil. The biological characteristics of the three phloem-limited prokaryotes, namely spiroplasmas, phytoplasmas and Ca. Liberibacter spp., and the diseases they induce and their vectors will be discussed. Most plant pathogenic prokaryotes do not require an active insect vector to spread them from plants to plants, while X. fastidiosa, Ralstonia syzygii, Ca. Liberibacter spp., phytoplasmas, and spiroplasmas do. To date among all known vectors, the single most successful insects vectoring the diseases belong to the Order of Hemiptera. In the past three decades, researches have emphatically addressed the biology, ecology, vector relationships and epidemiology of crop diseases caused by plant pathogenic prokaryotes which were well documented in numerous review articles. Herein a review of the significance of the insect vectors of fastidious prokaryotes that cause diseases of important crops is to be addressed.