Vector Transmission of Xylella Fastidiosa: Applying Fundamental Knowledge to Generate Disease Management Strategies

Home , Acrogonia, Machaerotidae, Xylella fastidiosa, Xyphon

SPECIAL FEATURE ON INSECT TRANSMISSION OF PLANT PATHOGENS Vector Transmission of Xylella fastidiosa: Applying Fundamental Knowledge to Generate Disease Management Strategies

RODRIGO P. P. ALMEIDA,1 MATTHEW J. BLUA,2 JOA˜ O R. S. LOPES,3 4 AND ALEXANDER H. PURCELL

Ann. Entomol. Soc. Am. 98(6): 775Ð786 (2005) ABSTRACT Xylella fastidiosa is a xylem-limited bacterium transmitted to plants by xylem sap- feeding insects. This pathogen has a wide host range, causing disease in crops such as grape, citrus, almond, and coffee; ornamental plants; and trees. Sharpshooter leafhoppers are the major vectors of X. fastidiosa to crops of economic importance. Transmission characteristics include the lack of a latent period, no transstadial or transovarial transmission, persistence in adults, and multiplication in the foregut of vectors. Various factors inßuence vector transmission of X. fastidiosa, including the distribution and density of bacterial populations in host plants, insect host range and plant preference, season of inoculation, and climatic conditions. The ecology of vectors can affect epidemics, as demonstrated by the large increase in PierceÕs disease of grapevine incidence in California after the introduction of Homalodisca coagulata (Say). Disease control strategies should incorporate basic knowledge of transmission biology, vector ecology, and other interactions involved in X. fastidiosa diseases. We discuss basic aspects of X. fastidiosa transmission by vectors, the ecology of insects in relation to transmission and disease spread, and how basic research can be applied to the development of management strategies for a X. fastidiosa disease.

KEY WORDS Hemiptera, Cicadellidae, bacteria, plant disease, bioÞlm

INSECTS THAT INGEST PRIMARILY xylem sap belong to four (Fig. 1). Epidemics of citrus variegated chlorosis families within the Hemiptera: Cicadellidae (subfam- (CVC) in Brazil and new outbreaks of PierceÕs disease ily Cicadellinae, sharpshooter leafhoppers), Cercopi- (PD) of grapevines in California driven by Homalo- dae (spittlebugs), Machaerotidae (tube-building disca coagulata (Say) (Blua et al. 1999, Hopkins and spittlebugs), and Cicadidae (cicadas). In general, Purcell 2002), and the availability of genome se- these insects cause little damage to plants. Some ci- quences of four strains of X. fastidiosa (Simpson et al. cadas can damage foliage or debilitate plants by suck- 2000; Bhattacharyya et al. 2002a,b; Van Sluys et al. ing signiÞcant amounts of xylem sap when large 2003), are among the major reasons for such emerging broods emerge, whereas spittlebugs can affect pasture interest. This recent research has rapidly advanced grasses or sugarcane. Sharpshooters rarely damage knowledge of X. fastidiosa biology and epidemiology. plants directly (Andersen et al. 2003) but are eco- Recent reviews evaluate various aspects of X. fastid- nomically important as vectors of the plant pathogenic iosa diseases, including genomics (Van Sluys et al. bacterium Xylella fastidiosa. Grape, almond, citrus, 2002), vector ecology and disease epidemiology coffee, peach, and many forest tree species are among (Redak et al. 2004), and case studies (Purcell and Feil a large list of economically important plants in which 2001, Hopkins and Purcell 2002). We focus this review X. fastidiosa induces disease (Purcell 1997). Many on X. fastidiosa transmission by vectors, the ecology of other agronomic, ornamental, and wild plants are also the interactions, and how this knowledge can be in- hosts of the pathogen, most without expressing symp- corporated into disease management strategies. toms (Hopkins and Purcell 2002). X. fastidiosa Transmission Characteristics. Initial Worldwide, research involving X. fastidiosa and its studies on PD in vineyards suggested that the diseaseÕs vectors has increased dramatically in recent years etiological agent was an insect-borne virus (Hewitt et al. 1946). After Newton PierceÕs observations in the late 1800s and the devastation caused by the disease to 1 Department of Plant and Environmental Protection Sciences, southern California vineyards (Pierce 1892), interest University of Hawaii at Manoa, Honolulu, HI 96822. 2 Department of Entomology, University of California, Riverside, in PD diminished until an outbreak of the disease in CA 92521. the Central Valley of California during the 1930s and 3 Departamento de Entomologia, Fitopatologia and Zoologia Agrõ´- early 1940s (Hewitt et al. 1949). Researchers during cola, ESALQ/Universidade de Sa˜o Paulo, SP, Piracicaba, SP 13418- the 1940s identiÞed sharpshooter leafhoppers and 900, Brazil. 4 Department of Environmental Science, Policy and Management, spittlebugs as vectors (Houston et al. 1947; Severin University of California, Berkeley, CA 94720. 1949, 1950).

0013-8746/05/0775Ð0786$04.00/0 ᭧ 2005 Entomological Society of America 776 ANNALS OF THE ENTOMOLOGICAL SOCIETY OF AMERICA Vol. 98, no. 6

vector competency (Purcell and Finlay 1979). Longer access periods result in higher transmission efÞciency. X. fastidiosa is irregularly distributed in infected tissues (Hopkins 1989), thus longer plant access time may increase chances of the vector probing infected vessels. Because X. fastidiosa must attach to the foregut of vectors in a fast moving environment with ßuid speed of 5Ð10 cm/s (Purcell et al. 1979), longer access periods also can increase the number of cells ingested by the insect, and, proportionally, the opportunities for attachment to occur. By contrast, two other factors may be associated with the increase in inoculation efÞciencies over time. First, attached cells in the foregut would have more opportunities to detach from the cuticle and be inoculated into a susceptible vessel with Fig. 1. Number of publications retrieved after a ÔXylellaÕ term was used as a keyword to search the ISI Web of Knowl- longer access times relative to shorter ones. Second, it edge (Web of Science) database in December 2004. Solid may be that cells are frequently inoculated into plants, line (empty circles) represents number of hits with ÔTitleÕ but most die before proliferating and generating dis- only option selected; dashed line (solid squares) are results ease. The number of cells inoculated and the proba- for general ÔTopicÕ search. bility of establishment would increase with access time. Both factors could operate independently. The basis of the transmission mechanism may have some The identiÞcation of a group of vectors considered similarities with that of semipersistently transmitted xylem feeders suggested that the etiological agent was (foregut-borne) viruses (Nault 1997). limited to xylem, as was later demonstrated by exper- Vector Foregut and Its Relationship to Trans- imentally blocking vector transmission to xylem tis- mission. Sharpshooters have typical hemipteran sues with a metal foil barrier (Houston et al. 1947). mouthparts adapted for feeding on liquids. The max- Davis et al. (1978) identiÞed X. fastidiosa as the eti- illary stylets interlock to form parallel food and sali- ological agent of PD. Vector ability seems to require vary canals. The mandibular stylets surround and sup- only that the insect is a xylem sap-feeding specialist. port the maxillary stylets and cut through plant Although phloem and mesophyll feeders have been tissues during probing. The mouthpart morphology of tested for X. fastidiosa transmission to plants (Purcell H. coagulata has been described in detail previously 1980), including leafhopper species known to pene- (Leopold et al. 2003). Sap enters the stylets and is trate and ingest from xylem, e.g., Euscelidius variegatus transferred to the precibarium, a narrow tube con- (Kirschbaum) (Naito 1977), none of these insect necting the food canal to the cibarium (pumping groups transmitted the pathogen. In contrast, most chamber). The precibarial valve, located about mid- Cicadellinae species tested in sufÞcient numbers were way in the precibarium, opens to admit ingested sap proven to be vectors. Observations that X. fastidiosa and closes to prevent backßow while food is pumped has little, if any, species speciÞcity within xylem-feed- to the gut. Backus and McLean (1982, 1983) described ing specialists (Purcell 1989) suggest that distinctive the morphology of the precibarial valve and a likely probing behaviors or foregut morphological charac- ingestion mechanism. The precibarium canal contin- teristics shared by xylem feeders enable bacterial ues after the valve and ends in the cibarium. The transmission. cibarium connects the precibarium to the esophagus X. fastidiosa is unique in its transmission character- or true mouth. From the esophagus, food is pushed istics among vector-borne plant pathogens in that it through the cardiac/esophageal valve into the midgut. does not require a latent period yet is persistently Sap ingestion is regulated by the precibarial and transmitted. Severin (1949) showed that adult sharp- cardiac (esophageal) valves and the cibarium dilator shooters remained infective for long periods. How- musculature, which originates at the insect clypeus ever, the lack of transstadial transmission or a latent and inserts along a medial line distal to the elastic period (Purcell and Finlay 1979) strongly suggested cibarial diaphragm (Purcell et al. 1979). Contraction that the pathogen is inoculated into plants from the of the dilator muscle raises the diaphragm to create foregut of vectors, the cuticle of which is shed at each negative pressure in the cibarium chamber, sucking in molt. Although the bacterium multiplies in the foregut ßuid from the food source (e.g., xylem vessel) via the (Purcell et al. 1979, Hill and Purcell 1995), which stylets to the cibarium. Negative pressure collapses explains the persistence of transmission in adults, very shut the cardiac valve, serving as a one-way check few live bacterial cells in the vectorÕs foregut are valve that opens with positive pressure and closes with required for transmission (Hill and Purcell 1995), an negative pressure from the cibarium. Relaxation of observation that agrees with the lack of a latent period. the dilator muscle collapses the top of the cibarium Freitag (1951) demonstrated the lack of X. fastidiosa chamber, pushing ßuid into two directions, along a transovarial transmission. groove on the ßoor of the cibarium, toward the esoph- The length of both acquisition and inoculation agus and cardiac valve and the precibarial valve. Pos- access periods affects transmission efÞciency, or itive pressure opens the cardiac valve to admit ßuid November 2005 ALMEIDA ET AL.: VECTOR TRANSMISSION OF X. fastidiosa 777 into the midgut. Small amounts of ßuid present distally attached to the precibarium and inoculate them into to the precibarial valve and in the food canal could be a host plant. The use of electronic means to elucidate pushed back into plants during the closure of the sharpshooter probing behaviors will provide useful precibarial valve. information on the mechanisms of pathogen acquisi- Studies documenting the relationship between tion and inoculation (Almeida and Backus 2004, X. fastidiosa distribution in the foregut of vectors and Backus et al. 2004). its transmission to plants have only been conducted a Bacterial Colonization of Host Plant and Its Rela- few times (e.g., Purcell et al. 1979). Newman et al. tionship to Transmission. X. fastidiosa transmission is (2004) showed a correlation between bacterial pres- the result of three events: acquisition from a source ence in the precibarium of vectors and its transmission plant, attachment and retention to vectorÕs foregut, to plants. Likewise, Almeida and Purcell (unpublished and detachment and inoculation into a new host. A data) found a strong correlation between transmission successful transmission event also includes develop- to plants and bacterial presence in the precibarium. ment of an infection (i.e., multiple cycles of bacterial Furthermore, X. fastidiosa also was frequently ob- multiplication) after inoculation. Although factors af- served in the precibarium by using scanning (Brlansky fecting acquisition are relatively well understood, et al. 1983) and confocal microscopy with a strain primarily the effect of different source plants and expressing a green-ßuorescent protein (Newman et al. bacterial populations within them on acquisition ef- 2003). Þciency, little is known about inoculation and persis- Based on microscopy studies, X. fastidiosa forms a tence in plants. bioÞlm on the foregut of vectors, where cells are Bacterial populations within host plants affect ac- polarly attached to the cuticle in a monolayer (Purcell quisition efÞciency from grape (Hill and Purcell et al. 1979, Brlansky et al. 1983, Newman et al. 2004). 1997). In general, higher numbers of live bacteria Polar attachment may increase the amount of nutri- found in plants (using culturing methods for quanti- ents cycling through the bioÞlm. Interestingly, the Þcation) have been associated with higher acquisition bacterial growth seems to be most dense where ßuid efÞciency. The minimum acquisition threshold in grape velocities along the cuticle are likely most rapid, i.e., was 104 colony forming units (CFU)/g of plant tissue, in the narrowest portions of the foregut, such as the and acquisition efÞciency increased with increasing bac- precibarium, or a groove in the ßoor of the cibarium terial numbers (Hill and Purcell 1997). Transmission chamber leading from the precibarium to the esoph- efÞciency for CVC vectors is low, usually Ͻ10% trans- agus, or the irregular grooves in the cibarial chamber mission efÞciency even with long acquisition and inoc- internal to the insertions of the cibarial dilator muscle. ulation periods (reviewed in Redak et al. 2004). Esti- Although bioÞlm formation may be a characteristic of mated X. fastidiosa live populations in symptomatic X. fastidiosa colonization of vectors, it is not required citrus leaves were Ϸ106 CFU/g, 100- to 1000-fold lower for successful transmission because inoculation can than in grapes (Almeida et al. 2001, Oliveira et al. 2002). occur before there is sufÞcient time for a bioÞlm to Bacterial populations within almonds are also lower form, as evidenced by the lack of latent period. Details (Ϸ107 CFU/g) (Almeida and Purcell 2003a). Corre- on detachment of cells from the foregut for inocula- spondingly, transmission of X. fastidiosa from almond tion of healthy plants have not been addressed; al- to almond by H. coagulata was less efÞcient (Ϸ5% per though it can be hypothesized that leafhopper behav- insect per day) than from grape to grape (Ϸ20% per ior may induce or facilitate such detachment. insect per day) (Almeida and Purcell 2003b, c). Non- Requirements for a X. fastidiosa Transmission Hy- persistent X. fastidiosa hosts, in which X. fastidiosa even- pothesis. Although vector transmission of X. fastidiosa tually die out (Purcell and Saunders 1999), could func- would seem to be a relatively simple matter of bac- tion as signiÞcant reservoirs of bacterial inoculum only terial attachment and detachment from the foregut, if they harbor large numbers of vectors. However, numerous observations and experimental Þndings they are usually not considered of major epidemiological show that transmission is a complex process with many importance. requirements. Transmission also can be analyzed X. fastidiosa–Vector Molecular Interactions. The based on results of biological experiments conducted recent surge in molecular research on X. fastidiosa has mostly under laboratory conditions, for which a mech- provided new tools and approaches to study bacterial anistic hypothesis must accommodate existing data. transmission. The development of techniques to ge- Thus, any hypothesis must consider: 1) lack of latent netically transform X. fastidiosa is arguably one of the period, 2) inoculum persistence over time, 3) trans- most important achievements in recent years (Mon- mission to plants with xylem sap under negative ten- teiro et al. 2001, Neto et al. 2002, Guilhabert and sion or under positive root pressure (dormant grape- Kirkpatrick 2003). Site-speciÞc mutants have enabled vines), 4) lack of transmission after molting, 5) spatial studies on bacterial attachment to various surfaces, distribution of X. fastidiosa in the foregut of vectors, bioÞlm formation, and regulation of several pathways and 6) the failure of sap-sucking insects that are not associated with pathogenesis in plants (Souza et al. xylem specialists (but none the less probe xylem) to 2003). Use of reporter genes, such as a strain of transmit. Successful transmission to plants with posi- X. fastidiosa expressing green ßuorescent protein, has tive root pressure suggests that vector probing behav- facilitated microscope observations and real-time ior is related to inoculation (Almeida et al. 2005). analysis of bacterial biology (Newman et al. 2003). Thus, a speciÞc probing behavior may dislodge cells Linking these studies to transmission experiments 778 ANNALS OF THE ENTOMOLOGICAL SOCIETY OF AMERICA Vol. 98, no. 6 undoubtedly will provide important insight into by feeding and physiological processes that allow ac- X. fastidiosaÕs mechanisms of attachment, persistence, quisition, retention, and inoculation of X. fastidiosa, as and detachment on the vectorÕs cuticle. X. fastidiosa discussed in previous sections, the importance of vec- mutants have been created to disable targeted genes tors in natural spread of the diseases is inßuenced eliminated by different transformation methods; how- mainly by ecological attributes such as habitat and ever, most mutants have not been tested for insect host selection, vector density and mobility, and spatial transmissibility. Although it is possible to mechani- and temporal distribution (Purcell 1985). cally inoculate mutants into plants to test for patho- Habitat and Host Selection. Habitat and host pref- genicity, adding a vector component to such studies is erences limit the economic importance of many vec- of interest. After disabling the rpfF gene of X. fastid- tors of bacterial pathogens (Rosenberger 1982). Less iosa, which is necessary for the bacterium to produce than one-third of the 43 documented vector species of a cell-to-cell signal, Newman et al. (2004) found that X. fastidiosa are associated with crop disease epidem- the rpfF mutant caused more severe disease when ics (Redak et al. 2004). The most important vector mechanically inoculated into grapevines but was species for spreading X. fastidiosa diseases are gener- greatly impaired for vector transmission, probably be- ally those most often found on or near the affected cause of its inability to attach and colonize the vectorÕs crops. Among the vectors associated with PD in the foregut. The availability of microarrays for gene ex- United States, H. coagulata is now considered the most pression studies (Souza et al. 2003) will be useful to important because of its high mobility, extreme identify genes associated with X. fastidiosaÕs coloni- polyphagy, and wide distribution on various crops that zation of insect vectors. Because of limitations in the are susceptible to X. fastidiosa (Redak et al. 2004), number of cells that are present in an insect foregut, despite its relatively inefÞcient transmission of X. fas- obtaining enough bacterial nucleic acids directly from tidiosa (Almeida and Purcell 2003c). Graphocephala insect heads for such tests may be challenging. The use atropunctata (Signoret) is also an important PD vec- of ßow cell and other artiÞcial systems with surfaces tor, but it is somewhat restricted to coastal areas of that mimic sharpshooterÕs cuticle may be a good al- California, where vineyards are bordered by riparian ternative for these studies. The comparison of various woods, which serve as overwintering habitats for this X. fastidiosa genomes and the use of nontransmissible sharpshooter (Purcell 1975). Likewise, Draecula- isolates also should permit researchers to identify cephala minerva Ball and Xyphon fulgida Nottingham genes required for successful vector transmission, as are more closely associated with epidemics in vine- has already been done for plant pathogenicity (Koide yards neighboring irrigated pastures and alfalfa Þelds et al. 2004). in the Central Valley of California (Hewitt et al. 1942). Vector Ecology as a Basis for Spread of X. fastidiosa Of 11 sharpshooters known to transmit X. fastidiosa in Diseases. The development of X. fastidiosa diseases is Brazilian citrus, Þve [Acrogonia citrina Marucci & determined by multiple interactions among several Cavichioli, Bucephalogonia xanthophis (Berg), Di- elements that constitute the pathosystems, including lobopterus costalimai Young, Macugonalia leucomelas host plants, vectors, environment, and the pathogen. (Walker), and Oncometopia fascialis (Signoret)] are The interactions of vectors with the other elements considered to be key vectors for CVC spread. These are essential for survival and spread of the bacterium Þve species are commonly found on citrus (Paiva et al. among host plants. In both natural and agricultural 1996), which seems to be the major source of inocu- systems, X. fastidiosa spread depends on suitable xy- lum for CVC spread (Laranjeira et al. 1998). In con- lem-feeding vectors. Because of the low vector spec- trast, Ferrariana trivittata (Signoret) and Plesiommata iÞcity and high diversity of Cicadellinae species in corniculata Young are two vector species that are very tropical and temperate regions of the Americas, some abundant on weedy grasses in citrus orchards but are crops affected by X. fastidiosa (e.g., grapevine, al- not associated with CVC epidemics and are rarely mond, and citrus) are visited by numerous hopper found on the citrus trees (Paiva et al. 1996, Lopes species that can act as vectors. Up to 27, 22, and 11 1999). species of sharpshooters and spittlebugs have been Vector preference inßuences the density of insects reported as vectors associated with PD, alfalfa dwarf and their residence time on plants; thus, the prefer- disease, and CVC, respectively (Redak et al. 2004). ence of particular vector species for feeding on hosts Given the broad group speciÞcity for vector transmis- of X. fastidiosa should increase the probability of in- sion (Purcell 1989), these numbers probably depend fection. Vector preference expressed in Þeld and lab- more on how many species have been tested than on oratory studies, however, failed to explain differences how many species are capable of transmitting X. fas- in PD incidence among grape cultivars of differing tidiosa. susceptibility. Instead, the plantÕs rate of overwinter Despite the large number of potential vectors, how- recovery and the speed of bacterial colonization after ever, only a few species are likely to play a signiÞcant initial infection seemed to be more important (Purcell role in any particular crop disease in a region. The 1985). Vector probing behavior on plants also may be relevance of speciÞc sharpshooter vectors for spread- critical for acquisition and inoculation success. Several ing X. fastidiosa diseases depends not only on their factors can inßuence vector preference, including competence to transmit the pathogen but also on plant age, nutritional status, and pathogen infection. ecological interactions with host plants and the envi- B. xanthophis is the prevalent sharpshooter species in ronment. Although vector competence is determined citrus nurseries and groves up to 2 yr old (Roberto et November 2005 ALMEIDA ET AL.: VECTOR TRANSMISSION OF X. fastidiosa 779 al. 2000, Yamamoto et al. 2001), whereas other vectors levels of disease were obtained when date of inocu- associated with CVC prefer older trees (Gravena et al. lation was considered in this model. Because the 1998). The preference for young citrus plants makes sharpshooter G. atropunctata usually inoculates X. fas- this sharpshooter an important vector, because bac- tidiosa in the tips of grapevine shoots (Purcell 1975), terial inoculation in small trees enhances the proba- infections that take place later than May are much less bility and speed of systemic infection in the trunk, likely to move down to woody tissues and to persist which precludes plant recovery by pruning. In addi- after winter pruning (Feil et al. 2003). This shows that tion, its propensity to inoculate nursery trees allows vectors must occur in sufÞcient numbers when con- long-distance spread of the pathogen through conditions are favorable for establishment of chronic in- taminated nursery stocks. Nutritional quality of hosts fections, which is during the spring. In central Florida, also impacts sharpshooter preference. Because the Oncometopia nigricans (Walker) is thought to be a concentration of nutrients in plant xylem sap is very more important vector than H. coagulata because it low and can vary signiÞcantly depending on many develops larger populations on grapevines in early factors such as season, time of the day, fertilization, spring (Adlerz and Hopkins 1979). Likewise, the abil- and irrigation (Andersen and Brodbeck 1991, ity to overwinter as adults in riparian woods adjacent Andersen et al. 1992, Brodbeck et al. 1993), sharp- to vineyards makes G. atropunctata a suitable vector to shooters are likely to select plant species that offer a establish infections of X. fastidiosa in grapevines early nutritionally balanced or rich diet at a given time. in the spring in California (Purcell 1975). The appar- Attractiveness and acceptance of infected plants by ent absence of diseases caused by X. fastidiosa in sharpshooter vectors are relevant for bacterial acqui- Europe may be because of the lack of vectors that sition and disease spread. The yellowish coloration overwinter as adults that could establish early season caused by infection of certain viruses and mollicutes infections capable of surviving the following winter. can make host plants more attractive to vectors Vector species that overwinter as eggs or nymphs (Peterson 1973, Ajayi and Dewar 1983). A choice would not be capable of spreading the bacterium study revealed that the level of infection by X. fastid- among plants until after this critical spring period iosa inßuenced the selection of diseased citrus plants (Purcell 1997). by sharpshooters. The vectors tend to reject severely The effect of vector density on probability of in- infected plants with typical CVC symptoms, whereas fection is also dependent on characteristics of the crop symptomless infected plants are equally accepted in system (Purcell 1985). For long-lived perennial crops, relation to healthy citrus (Marucci et al. 2005). This such as grapes and citrus that are planted in low den- information supports the hypothesis that pathogenic- sities, even low vector numbers should result in high ity resulted from extensive vessel plugging is not a levels of disease incidence after a few years of expo- favorable outcome for vector acquisition and mainte- sure. Disease levels are more likely to be related to nance of X. fastidiosa in the environment (Newman et vector densities for short-lived annual crops with high al. 2004). plant densities (Purcell 1985). For CVC, in which Preference for young growth of shoots is a common inefÞcient vectors are present at relatively low pop- behavior among various sharpshooters (Purcell 1975, ulations in citrus groves, it is likely that changes in Marucci et al. 2004) and may be important for inoc- vector density have a greater impact on probability of ulation success. In several grapevine cultivars, older infection (Lopes 1999). foliage was more resistant to infection by X. fastidiosa Vector dispersal capacity is an important factor in after inoculation by sharpshooters than young leaves disease spread. It is a determinant of vector population (Purcell 1981). However, the feeding preferences of density (Taylor and Taylor 1977) and pathogen dis- H. coagulata for woody stems may allow the establish- tribution among host plants and vectors (Purcell ment of late season infections that are more likely to 1985). There is little or no information available on persist through the winter (Purcell and Feil 2001). It dispersal abilities for many sharpshooter vectors. also was shown that H. coagulata can transmit X. fas- H. coagulataÕs active movements among several hab- tidiosa to dormant vines during the winter (Almeida itats and host plants allow rapid spread of X. fastidiosa et al. 2005). This particular behavior of H. coagulata over a wide area, whereas movements of G. atropunc- extends signiÞcantly the time frame during which tata are more limited to riparian vegetation and neigh- grapevines are susceptible to chronic infections boring vineyards (Blua et al. 2001, Redak et al. 2004). (Redak et al. 2004). Some spittlebugs are able to transmit the PD pathogen Vector Density and Mobility. It is reasonable to with relatively high efÞciencies (12Ð65%) (Severin assume that probability of infection relates to vector 1950). However, this group of vectors is not consid- abundance. However, it was noted that variables such ered relevant for PD epidemics in California because as vector density and propensity to transmit X. fastid- it is not observed on grapevines under Þeld conditions; iosa are not adequate to explain the levels of PD usually does not disperse widely or rapidly (DeLong incidence in California (Purcell 1985). A theoretical and Severin 1950; Redak et al. 2004), and overwinters model based on estimates of sharpshooter density per in the egg stage, producing adults only after the time plant, percentage of infective individuals, and trans- of year when chronic infection can be established. mission efÞciency predicted much higher levels of Vector species not considered important in crops infection than the disease incidence observed under may still play a role in maintaining the infection cycle the assumed conditions (Purcell 1981). More realistic outside the affected crops (Purcell 1982). Outside 780 ANNALS OF THE ENTOMOLOGICAL SOCIETY OF AMERICA Vol. 98, no. 6 sources of inoculum seem to be particularly important 100Ð200 m from the vineyard edge (Purcell 1974). when epidemics are generated mainly by primary Third, researchers noticed that the exponential in- spread, as it is observed in northern coastal areas and crease of PD from year to year, which should occur in the Central Valley of California, where adjacent where transmission from vine to vine, i.e., secondary riparian woods, alfalfa Þelds, and pastures serve as spread, was important. In contrast, PD spread by na- major reservoirs of X. fastidiosa for PD spread into tive vectors was mainly from outside of vineyards into vineyards (Hewitt et al. 1942, Purcell 1975). For ex- the vineyard (primary spread) but not among grape- ample, some spittlebugs vectors not found on grapevines (Feil and Purcell 2001). Although the degree to vines are fairly common on several herbs and shrubs which grapevines are infected as a result of primary that grow nearby vineyards and alfalfa Þelds in Cali- versus secondary spread is not clear, secondary spread fornia (DeLong and Severin 1950); thus, they may has the potential to occur much more rapidly than contribute to maintain bacterium inoculum in weedy primary spread. Secondary spread generates a log- hosts. linear relationship between disease spread and time, Association of vectors with diverse habitats border- whereas primary spread is relatively constant (linear) ing affected crops allows colonization of a wider range over time (Van der Plank 1963). of host plants that contribute to keep sufÞcient inoc- Importantly, recent studies showed that H. coagu- ulum reservoirs for primary spread of the bacterium lata can inoculate dormant grapevines in the winter (Purcell 1985) and might allow survival of both vector (Almeida et al. 2005), when these insects are com- and bacterium during unfavorable periods in the crop. monly observed feeding on grape (Purcell and Feil Because X. fastidiosa interacts with diverse vector 2001). H. coagulataÕs ability to feed on and inoculate groups and colonizes a broad array of plant taxa (Hop- X. fastidiosa into woody grapevine tissue, on which kins 1989, Redak et al. 2004), there is probably selec- native vectors do not feed, may be responsible for tive pressure for the bacterium to maintain genes for secondary spread if such infections during summer attachment and colonization in the xylem of variable through fall establish chronic PD (Almeida and Pur- hosts as well as in the foregut of distinct vectors. The cell 2003c). Summer infections of grape by native high diversity of species and polyphagy in Cicadelli- vectors in California seldom cause infections that sur- nae may increase the opportunities for the bacterium vive the following winter (Feil et al. 2003). The over- to adapt to new host plants and habitats and promote winter recovery of infections of summer-inoculated the evolution of new bacterial strains. vines largely explains the lack of secondary spread in Development of X. fastidiosa Spread Control Strat- California vineyards (Hopkins and Purcell 2002). egies, a Case Study. Since Pierce (1892) Þrst investi- Given the evidence that secondary spread seems to be gated what eventually came to be called PD, the main important where H. coagulata is present, a hypothesis characteristics of PD epidemics in California have worth testing is whether summer infections estab- remained relatively unchanged. Exceptions to this in- lished by H. coagulata overwinter successfully. clude new outbreaks associated with plantings in new A necessary extension of PD research is to use new areas and the typical cyclical nature of PD (Hewitt et information in conjunction with knowledge of crop- al. 1949). Yet, the major biological components in the ping systems to generate economically sound disease system have remained static for Ͼ100 yr in California. management strategies. Under development are novel PD epidemiology in California changed drastically in disease management tactics for deployment at land- the mid-1990s with the introduction of a new vector, scape, vineyard, and grapevine scales. These tactics H. coagulata (Blua et al. 1999). H. coagulata is a well focus on reducing the numbers of H. coagulata over a known and important vector of several X. fastidiosa- large area, to keep them out of vineyards, manipulat- caused diseases in the southeastern United States, in- ing their interactions with grapevines, and manipulat- cluding PD, and precludes the successful culture of ing the interactions between grapevines and X. fas- Vitis vinifera L. and Vitis labrusca L. in that region tidiosa. (Hopkins 1989). This leafhopper was Þrst detected in Ecology of X. fastidiosa Spread to Grapevines by southern California in 1989 (Sorensen and Gill 1996) H. coagulata in California. Understanding the details and was implicated in the spread of a new X. fastidiosa of the spread of X. fastidiosa by H. coagulata to disease of oleander in southern California, oleander grapevines is useful to identify research needs, math- leaf scorch, in the mid-1990s (Purcell et al. 1999). ematically model epidemiology, and develop control In 1997, H. coagulata was Þrst implicated in the methods and an overall PD management strategy. It is spread of PD in the wine grape-growing area of Te- important to consider not only the interacting organ- mecula in Riverside County of southern California isms in the crop (i.e., grapevines, X. fastidiosa, and (Blua et al. 1999). Initial observations of the epidemic H. coagulata) but also the effects of the environment, indicated large differences from epidemics of PD including the larger agricultural community. This is present before in California. First, vector sources particularly important because of the large reproduc- mainly consisted of citrus, not previously known to be tive host range (Mizell and French 1987, Turner and important to PD vectors. Second, the distance with Pollard 1959) and wide dispersion potential of which disease was detected from vector sources was H. coagulata (Blua and Morgan 2003). hundreds of meters from the edge of the vineyard The larger agricultural community provides plant (Perring et al. 2001) relative to native California vec- hosts of X. fastidiosa and H. coagulata. Plant hosts of tors that spread disease mainly to grapevines within PD strains of X. fastidiosa are important to the epi- November 2005 ALMEIDA ET AL.: VECTOR TRANSMISSION OF X. fastidiosa 781 demic in proportion to their use as feeding hosts for the larger agricultural community and biocontrol and vectors. Pathogen host plants include annuals, peren- insecticides to reduce H. coagulata in citrus and vine- nials, herbaceous, and woody plants as well as native, yards (Wendel et al. 2002, Hix et al. 2003). Systemic feral, agricultural, and ornamental plants (Freitag insecticides, especially neonicotinoids, and repellents, 1951, Purcell and Saunders 1999). Likewise, this agri- such as kaolin, a formulation of aluminum silicate, are cultural community also provides hosts for H. coagu- used in vineyards. Besides impinging on interactions lata. As with pathogen hosts, hosts for the vector are between H. coagulata and grapevines, they disrupt the best considered a continuum from plants that are not H. coagulataÐX. fastidiosa interface by impacting vec- fed on, such as Eriogonum fasciculatum (Benth.) tor orientation, host determination, and feeding be- (buckwheat), a common native plant, to citrus, an havior (Puterka 2002, Tubajika et al. 2003). Another excellent feeding and overwintering host on which tactic gaining interest to impact the H. coagulata– H. coagulata reproduces (Adlerz 1980, Mizell and X. fastidiosa interface is the removal of diseased French 1987). Citrus seems to be the single most grapevines (Hashim and Hill 2003). Currently, there important host that contributes to high population are no tactics available to interrupt the grapevineÐ densities of H. coagulata in southern California. X. fastidiosa interface. Plants that have access to water during the summer, In the H. coagulata–X. fastidiosa system, tactics used most typically via irrigation in California, seem to be to manage PD spread by H. coagulata are costly, not the most important for H. coagulata. Natural plant completely effective, and not all are sustainable. Be- communities in southern California provide hosts dur- cause grapevines are perennial, the use of these tactics ing the winter rainy season and in riparian areas. Some to “hold back the tide” may eventually prove to be nonriparian native plants, such as Toyon, Heteromeles ineffective. With current tactics, an important chal- arbutifolia (Lindl.) M. Roemer, allow feeding and lenge looms in sustaining the management of H. co- reproduction (M.J.B., unpublished data). Riparian na- agulata in citrus, where its economic threshold far tive plants as a group, however, are generally in a more exceeds that of grapevines. Inoculative releases of hydrated state than nonriparian natives and are likely parasitoids (e.g., Gonatocerus sp.) of H. coagulata eggs more acceptable as hosts for H. coagulata. likely can maintain sharpshooter populations below Residential landscaping provides a plethora of irri- the economic threshold for citrus yet allow citrus to gated feeding and reproductive hosts that can serve as produce population densities that are dangerous to reservoirs of H. coagulata and X. fastidiosa. The grape nearby vineyards. A major problem for effective bio- growing-area of Ontario, CA, that has recently suf- logical control with these egg parasitoids is that their fered signiÞcant outbreaks of PD spread by H. coagu- populations decline precipitously from September lata in the Þrst time in their Ͼ80-yr-old history of wine through the winter, when egg production by H. co- grape-growing (M.J.B., unpublished data), has no agulata is low. Thus, the parasites have a difÞcult time large blocks of citrus like the other areas. Rather, it has surviving winters, and only a small proportion of Þrst numerous residences with various reproductive and generation (spring) sharpshooter eggs are parasitized. feeding hosts of H. coagulata surrounding the vine- In the second generation, rates of parasitization fre- yards. The larger agricultural community provides an quently were Ͼ90% in some areas (Morgan et al. opportunity for interactions with other vectors that 2001). DifÞculties in rearing H. coagulata, and conse- may be important to the spread of X. fastidiosa in quently rearing egg parasites, make inundative re- grapevines. An otherwise small proportion of infected leases implausible. grapevines in a vineyard generated by native vectors An Overview of PD Management Tactics under would be a key source from which the pathogen is Development. One likely consequence of the current spread by H. coagulata among grapevines in the vine- H. coagulataÐPD management system is that it will yard. The main interacting characters involved in the allow growers to maintain vineyards until more effec- new PD epidemic in southern California are grape- tive or sustainable tactics can be developed. The dis- vine, X. fastidiosa, and H. coagulata. V. vinifera is an cussion below clariÞes management tactics for PD excellent host of X. fastidiosa that supports the growth spread by H. coagulata on the basis of interrupting of a high density of bacterial cells. For 2Ð4 yr, or until two-way interactions related to pathogen transmission the vine declines severely, such plants can be an ex- and disease spread: vectorÐpathogen and vectorÐ cellent acquisition host for vector transmission. plant. Some of the tactics under development may Current Management of PD Spread by H. coagu- impinge on plant-pathogen interactions as well. lata. PD epidemics can be managed based on disrup- Interrupting the H. coagulata–Grapevine Interaction. tion of the interactions among the main characters Improvements in traditional tactics and novel ap- involved, including the agricultural community. Ab- proaches are under development. They include the solute disruption of any single interaction would com- use of improved insecticides, especially soil-applied pletely diminish disease spread. Yet, absolute disrup- systemics and insect growth regulators, that are com- tion of any interaction is unlikely, so combining patible with biocontrol and do not affect most non- multiple tactics that partially interrupt more than one target species because of their method of application interaction is the current management strategy. or activity (Toscano and Castle 2002, Akey et al. 2002, Tactics used to reduce the spread of PD by Redak and Bethke 2003). Further studies are optimiz- H. coagulata on a local level target vectors primarily. ing the deployment of insecticides in vineyards and These tactics include the use of biocontrol agents in key crops that are H. coagulata reproductive hosts 782 ANNALS OF THE ENTOMOLOGICAL SOCIETY OF AMERICA Vol. 98, no. 6

(e.g., citrus) to reduce its population densities in the sharpshooter feeding does not necessarily translate agricultural community that can impact vineyards into a similar reduction in disease spread. Field studies (Akey et al. 2002, Toscano and Castle 2002). Research examining the impact of neonicotinoids on the spread continues to improve biocontrol by searching for and of PD in a 1000-grapevine experimental vineyard collecting additional H. coagulata egg parasitoids showed a reduction in disease because of neonicoti- (Hoddle and Triapitsyn 2003) and examining the im- noid treatment by Ϸ36% relative to untreated controls pact of entomopathogenic fungi, particularly to target (Redak and Blua 2002). Vector pressure in this ex- overwintering H. coagulata adults in citrus (Kaya perimental vineyard was large relative to outbreaks in 2003). commercial vineyards. After one season of exposure, A screen barrier is being developed as a tactic to 69% of the untreated control grapevines expressed impede the movement of H. coagulata into vineyards symptoms of PD, indicating a large primary spread or plant nurseries (Blua and Redak 2003). Investiga- event. After the next season, all treatments had high tions of H. coagulata ßight height revealed a potential levels of PD, and there were no statistical differences. of a 5-m-high screen barrier to reduce their movement It is likely that neonicotinoid treatment would have a from citrus by 97Ð99% (Blua and Morgan 2003, Blua larger impact on secondary spread than primary and Redak 2003). Further studies indicate that 70% of spread, because vectors would be exposed to treated H. coagulata placed on a platform near the barrier ßew vines twice; once during feeding for acquisition and away from it. Only 6% of individuals placed on the once during feeding for inoculation. For primary barrier ßew over it, and none walked over it or more spread, exposure to treated grapevines occurs only than a few centimeters (Blua and Redak 2003). The during inoculation. disadvantages to a barrier tactic include the high cost Another treatment examined in the experiment of materials and installation, but the barriers should described above is applications of kaolin to grape- last at least 10 yr. The greatest advantage to a barrier vines, creating a “particle Þlm” that disrupts insect is its compatibility with other tactics such as insecti- behavior (Puterka 2002, Tubajika et al. 2003). The cides and biocontrol. overall effect of kaolin is to reduce acquisition of Interrupting the H. coagulata–X. fastidiosa Interac- X. fastidiosa from infected grapevines and the ability tion. The advent of new systemic insecticides, espe- of H. coagulata to inoculate noninfected vines. Treat- cially neonicotinoids, has spurred research focused on ments with kaolin, either alone or in combination with developing tactics directed against the H. coagulataÐ neonicotinoids, had 50Ð57% less PD than untreated X. fastidiosa interface. Breaking this interaction in- controls. Civerolo (2001) reported similar results. Part volves altering the behavior of the vector related to X. of the impact of kaolin likely involves masking visual fastidiosa transmission or altering surface sites at the cues that H. coagulata relies on in host Þnding, be- cellular level that allow bacterial adhesion to the vec- cause treatments make grapevines look white. Studies torÕs foregut. show that through a season Ϸ50% fewer H. coagulata Field studies showed that application of a neonic- were caught in kaolin-treated grapevines compared otinoid insecticide slowed the rate of PD spread even with control plots (Redak and Blua 2002). This does in the presence of a high H. coagulata infestation not likely account for the reduction in PD observed, (Krewer et al. 2002), suggesting that the insecticide so other factors are likely signiÞcant, including plant was operating on the transmission process and not surface interactions that mask host tactile cues and just decreasing sharpshooter population density. direct effects of kaolin that irritate sharpshooters. Currently in California, restrictions on the amount of Logically, removing infected grapevines should re- neonicotinoids used per unit area do not allow high duce secondary (vine-to-vine) spread of X. fastidiosa. enough levels to ensure high H. coagulata mortality However, this tactic is only worthwhile if secondary throughout the year. But sublethal effects still may spread of the pathogen is important. Removing in- be important to disease transmission if they impact fected grapevines when PD epidemics involved native H. coagulata feeding behavior. Field studies by M.J.B. California vectors did not signiÞcantly reduce disease (unpublished data) reveal that three different neo- (Hewitt et al. 1949). The explanation for the ineffec- nicotinoids in grapevines blocked feeding by tiveness of rouging diseased vines is that in traditional H. coagulata, as measured by the production of ex- PD, only primary spread by vectors entering the vine- creta, for 6 wk after application. For one neonicotinoid yard from outside is important in establishing chronic examined further, a substantial decrease in feeding disease. Disease removal is currently being reinvesti- was observed 11 mo after treatment on Chardonnay gated as a PD management tactic where H. coagulata grapevines, whereas feeding was blocked on Cabernet is present, however, because of the possible impor- Sauvignon grapevines. Interestingly, grapevine vari- tance of secondary spread of chronic PD by this vector ety also affected feeding, with individuals on Char- (Hashim and Hill 2003). Unfortunately, by the time donnay producing Ϸ4 times the excreta as individuals grapevines show disease symptoms, they may have on Cabernet Sauvignon in the same period. It is not been point sources for the spread of X. fastidiosa for known whether decreasing H. coagulata feeding may months. Research involving a strain of X. fastidiosa reduce their ability to acquire X. fastidiosa from in- that causes CVC in Brazil revealed that vectors prefer fected grapevines that are treated or inoculate healthy plants that do not show disease symptoms (Marucci et treated grapevines. al. 2005). Also, H. coagulata abundance was lower on However, this neonicotinoid-induced reduction in peach trees showing symptoms of phony peach dis- November 2005 ALMEIDA ET AL.: VECTOR TRANSMISSION OF X. fastidiosa 783 ease compared with control trees (Mizell and French complex issue dependent on, among other factors, 1987). Thus, the greatest beneÞt of the removal of grape variety, yield, region, year, relative contribution diseased plants would be achieved if infection could of primary and secondary spread for the vineyard in be determined soon after inoculation or at least before question, and for wine grapes, whether a vineyard is symptom production. owned and operated by a winery. Second, X. fastidiosa Genetically induced interruption of the H. coagu- is a pathogen of a few plants but a commensal of many. lataÐX. fastidiosa interface is being investigated by Thus, many plants, including wild plants, ornamentals, manipulating endophytic bacteria to block transmis- and crops may be long-term point sources from which sion. One line of investigation is focusing on geneti- the pathogen is acquired by vectors. Third, H. coagu- cally altering Acaligenes xylosoxidans denitriﬁcans, an lata is relatively long-lived, and once it acquires X. H. coagulata gut bacterium that also is found in the fastidiosa as an adult, it maintains the capacity to xylem of grapevines and other plants (Lauzon and inoculate plants through the remainder of its life. This Miller 2002). The objective is to insert genes coding vector has a large host range (Turner and Pollard 1959, for antibody fragment that interacts with the surface Mizell and French 1987) and disperses widely in short of X. fastidiosa cells, rendering them incapable of hopping ßights that tend to enhance the spread of X. adhering to the H. coagulata mouthparts/foregut fastidiosa (Blua and Morgan 2003). H. coagulata also (Lampe and Miller 2003). This tactic would tend to overwinters as adults and is frequently observed feed- block secondary transmission, but it might be ineffec- ing on dormant grapevines that Almeida et al. (2005) tive for primary spread because primary spread in- showed may be at risk for inoculation. Finally, H. volves acquisition from nongrapevine sources that coagulata seems to spread X. fastidiosa from vine to would not be treated with the transgenic bacterium. vine, thus causing a very rapid epidemic once patho- Integrating Management Tactics. The most robust gen sources are in the vineyard, but this is a hypothesis and sustainable management strategy to combat PD that needs to be tested rigorously because of its disease associated with H. coagulata should use tactics that control implications. Transgenic techniques hold disrupt multiple interactions, without reducing the great promise to produce future PD control tactics. maximum efÞcacy of each. Reliance on a single tactic But gene-conferred protection should not have spec- that is adequate to reduce disease spread below the iÞcity that would allow protection to break down if a economic threshold may be worthwhile and cost-ef- closely related strain of X. fastidiosa challenged the fective, but at a greater risk. At the foundation of system. The issue of regulatory and public acceptabil- current management for PD spread by H. coagulata is ity of transgenic tactics, however, will be a key as to control of the spread of this new vector to areas of whether successful methods could ever be applied. California that are not infested. The California De- partment of Food and Agriculture (CDFA 2004) im- posed strict regulations on the nursery industry in References Cited California to curtail movement of H. coagulata via nursery stock transported to noninfested counties. Adlerz, W. C. 1980. Ecological observations of two leafhop- CDFA also traps to monitor H. coagulata movement pers that transmit the PierceÕs disease bacterium. Proc. throughout the state and attempts to eradicate new, Fla. State Hortic. Soc. 93: 115Ð120. localized infestations by repeated insecticide treat- Adlerz, W. C., and D. L. Hopkins. 1979. Natural infectivity of two sharpshooter vectors of PierceÕs disease of grape ments. in Florida USA. J. Econ. Entomol. 72: 916Ð919. Even if a solution to PD is developed, diseases in- Ajayi, O., and A. M. Dewar. 1983. The effect of barley yellow duced in other plants by different strains of the patho- dwarf virus on Þeld populations of the cereal aphids, gen can create new problems. Of particular concern is Sitobion avenae and Metopolophium dirhodum. Ann. Appl. the spread of X. fastidiosa-induced diseases other than Biol. 103: 1Ð11. PD, such as almond leaf scorch, alfalfa dwarf, and Akey, D. H., M. Blua, and T. J. Henneberry. 2002. Control oleander leaf scorch. Recent studies show that various of immature and adult glassy-winged sharpshooters: ornamentals are currently supporting different strains evaluation of biorational and conventional insecticides. of X. fastidiosa in California, some of which are asso- pp. 133Ð135. In M. A. Tariq, S. Oswalt, P. Blincoe, and T. Esser [eds.], Proceedings of CDFA PierceÕs disease ciated with disease symptoms (Wong et al. 2004). research symposium, 15Ð18 December 2002, Coronado, These studies also have conÞrmed the presence of CA. Copeland Printing, Sacramento, CA. mulberry leaf scorch (Kostka et al. 1986), a disease not Almeida, R.P.P., and E. A. Backus. 2004. Stylet penetration previously identiÞed in the state (Wong et al. 2004). behaviors of Graphocephala atropunctata (Signoret) Concerns are high that other diseases caused by dif- (Hemiptera, Cicadellidae): EPG waveform characteriza- ferent strains of X. fastidiosa, such as those causing tion and quantiÞcation. Ann. Entomol. Soc. Am. 97: 838Ð CVC (Lee et al. 1991) and phony peach disease 851. (Turner and Pollard 1959), may spread to new regions. Almeida, R.P.P., and A. H. Purcell. 2003a. Biological traits of The California epidemic of PD in grapevines spread Xylella fastidiosa strains from grapes and almonds. Appl. Environ. Microbiol. 69: 7447Ð7452. by H. coagulata has several aspects that make it par- Almeida, R.P.P., and A. H. Purcell. 2003b. Homalodisca co- ticularly difÞcult to manage. From an economic per- agulata (Hemiptera, Cicadellidae) transmission of Xylella spective, at some proportion of infected vines, main- fastidiosa to almond. Plant Dis. 87: 1255Ð1259. taining the vineyard or orchard is not proÞtable. An Almeida, R.P.P., and A. H. Purcell. 2003c. Transmission of economic threshold for H. coagulata-spread PD is a Xylella fastidiosa to grapevines by Homalodisca coagulata 784 ANNALS OF THE ENTOMOLOGICAL SOCIETY OF AMERICA Vol. 98, no. 6

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