Annu. Rev. Phytopathol. 1996. 34:13151 Copyright c 1996 by Annual Reviews Inc.

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FASTIDIOUS XYLEM-LIMITED BACTERIAL PLANT PATHOGENS

Annu. Rev. Phytopathol. 1996.34:131-151. Downloaded from www.annualreviews.org by Universidad de Costa Rica on 11/02/10. For personal use only.

Alexander H. Purcell
Department of Environmental Science, Policy and Management, University of California, Berkeley, California 94720-3112

Donald L. Hopkins
University of Florida, Central Florida Research and Education Center, Leesburg, Florida 34748
KEY WORDS: Xylella, Clavibacter xyli, Pseudomonas syzygii, leaf scorch, endophyte

ABSTRACT
Numerous bacteria have been isolated from within plants, and many reported from xylem, but only three species of xylem-limited bacteria (XLB) that are fastidious in cultural requirements, are plant pathogens, and exclusively occupy xylem, have been well characterized. Two XLB, Xylella fastidiosa and Pseudomonas syzygii, are transmitted by sucking insects that feed on xylem sap but are not transmitted mechanically from plant to plant. In contrast, Clavibacter xyli is mechanically transmitted to plants by cutting tools. All of these XLB occupy a highly specialized yet diverse ecological niche: the water-conducting systems of an extremely wide range of plant hosts. A variety of detection methods are available as diagnostic aids; each method has advantages and disadvantages; no single method is best for all uses. Molecular and genetic comparisons of strains of XLB lag behind progress being made for many other plant-pathogenic bacteria, but such studies are needed to answer important questions: (a) How do XLB move from cell to cell within plants? (b) What are the physiological and genetic bases of plant host specicity for XLB? (c) Why are only xylem-feeding specialists vectors of X. fastidiosa (and probably P. syzygii), when many leafhoppers feed regularly (but not continuously) on xylem?

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INTRODUCTION
Xylem-limited bacteria (XLB), as we dene them for this review, are endophytic bacterial parasites that live in plants exclusively in xylem cells or tracheary elements. There are numerous reports of bacteria classied as endophytic because they are isolated from surface-sterilized plant parts (60, 67), but such bacteria usually have not been characterized as to which internal tissues are colonized. This denition of XLB excludes bacteria such as Erwinia stewartii and many other species that have a systemic phase in xylem tissues but also inhabit other tissues (111). This denition of XLB currently describes the following fastidious bacterial pathogens: Xylella fastidiosa (144), Pseudomonas syzygii (125), and Clavibacter xyli (33). Insect vectors transmit the rst two species (119); no insect vectors are known for C. xyli (33, 56). Diseases caused by X. fastidiosa and C. xyli t early concepts of plant viruses because they were undetected causal agents that colonized the plant vascular system systemically and were transmissible by grafting and by insect vectors (56, 69). Early researchers concluded that the causal agents were viruses (55, 70), an example of how the strong inuences of prevailing paradigms can limit or cloud scientic investigation.

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XYLEM-INHABITING BACTERIA AND THE DISEASES THEY CAUSE

Various strains of X. fastidiosa cause most known XLB plant diseases. Since the last major review of X. fastidiosa, diseases caused by this bacterium have been newly described for citrus variegated chlorosis (12, 19, 66, 98, 126), a leaf scorch of live oak (105), and pear leaf scorch (100). Besides causing disease, X. fastidiosa colonizes numerous plant species without provoking symptoms or noticeable distress. Studies during the 1950s (45) demonstrated that over 100 plant species harbored the Pierces disease virus. Vector transmission of the pathogen from symptomless plants to grape or alfalfa indicator plants indicated the presence of the causal agent in the source plant. The use of in vitro culture (80, 81, 123) or serological assays (81, 123, 134, 146) has expanded this list of symptomless hosts. Similarly, the causal agent of ratoon stunting disease, C. xyli subsp. xyli has other host plants (92, 131). C. xyli subsp. cynodontis, the Bermuda grass stunting disease pathogen (28), colonizes maize (7, 115, 125) and develops large populations with no visual symptoms, and numerous other plants if mechanically inoculated (94, 131). Epidemiological clues suggest that P. syzygii, which causes Sumatra disease of cloves, may have originated from forest plants (101). If true, P. syzygii could also have a wide host range.

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The causal role of X. fastidiosa in citrus blight is controversial. The bacterium has been isolated from citrus trees with blight symptoms and caused blight symptoms when inoculated into greenhouse plants (79, 86), However, the bacterium could not be consistently isolated from trees with blight (82), and eld-grown plants inoculated with X. fastidiosa did not develop blight. Recently, inoculations of mature citrus trees in a grove with virulent strains of the Pierces disease bacterium increased the incidence of citrus blight, supporting the hypothesis that X. fastidiosa has a causal role (87). Since the review of diseases caused by X. fastidiosa in 1989 (80), citrus variegated chlorosis disease (98) has been proven to be caused by a strain of X. fastidiosa that has rapidly spread to a high percentage of citrus trees in Brazil (20, 66). Recently, X. fastidiosa detected by culture and polymerase chain reaction has been associated with a rapidly spreading disease of oleander that appeared in southern California (AH Purcell, unpublished data).

OCCURRENCE AND MOVEMENT IN PLANTS

The conclusion that XLB are restricted to xylem is based mainly on microscopic examinations of diseased plants. Examples are X. fastidiosa in grape (108, 110), peach (48), citrus (17, 127), and other trees (68); C. xyli in cane (141, 148), Bermuda grass (34), and maize (42); and P. syzygii in clove (8). X. fastidiosa occasionally may be found in the intercellular spaces of the xylem (57). For X. fastidiosa, graft transmission of the pathogen requires inclusion of xylem tissues (70, 90, 142, 147), blocking access to grapevine xylem with metal foil beneath the bark prevents vector transmission (88). The XLB are cell or tissue specic; moreover, they also tend to accumulate in specic plant parts. This often varies with the host and is related to the type of symptoms expressed. In the leaf scorch diseases, bacteria accumulate fastest and reach the highest concentrations in symptomatic leaf veins and petioles (68, 76). In diseases with die-back and general decline, such as caused by infection of live oak by X. fastidiosa, bacteria tend to accumulate in the branches or trunk of the tree and often may be detected only in the sector of the tree with symptoms (105). In diseases with stunting as the primary symptom, such as phony peach (1, 47, 146) and ratoon stunting disease (29, 133), bacteria accumulate primarily in either the roots or basal part of the main stem. The XLB grow in xylem uid, which contains the lowest concentration of organic compounds (sources of available energy) of any plant tissue; however, it contains a diversity of amino acids, organic acids, and inorganic ions, predominantly in monomeric form that are essential nutrients for the bacteria (reviewed in 114, 124). Glutamine and asparagine occur in disproportionately high concentrations, consistent with the apparent growth media requirement of

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X. fastidiosa for high concentrations of glutamine and organic acids (18, 35, 38). The quantity and composition of xylem sap vary with plant age, season of the year, time of day, location in the plant, and general plant health (2, 5, 114, 124). Many similar stress factors (plant senescence, overproduction of fruit, other diseases, drought, and root pruning) favor disease production by X. fastidiosa (77, 85). Manipulation of xylem chemistry could present new approaches to control of diseases caused by XLB. XLB must have special mechanisms to concentrate and absorb nutrients from the environment. Aggregates of X. fastidiosa appeared to be attached to vessel walls by extracellular strands produced by the bacteria (89, 107, 110) that are usually most abundant at the ends of the bacterial rods. The strands resemble the polysaccharide bers that constitute the glycocalyx (25), which is thought to attach bacteria in a variety of turbulent habitats such as rapidly owing streams or animal guts. The negatively charged polysaccharide bers of the glycocalyx may function as an ion-exchange substrate, binding nutrient ions to the bacterial aggregate or conserving and concentrating digestive enzymes released by the bacteria for action against the host tissue (27, 38, 80). The basic and important question of how XLB spread within the xylem system is unanswered. The use of reporter genes to aid in detecting the location of XLB within living plants would facilitate in vivo studies of their movements within plants. Micrographs of vascular tissues from various plant organs colonized by X. fastidiosa show that movement of the bacterium from cell to cell is restrained by xylem pit membranes (for example, 27, 68, 104, 107, 110). The movement of bacteria through pit membranes is presumed but unproved. The movement of XLB from vessel to vessel suggests that these bacteria produce enzymes that degrade the pit membrane. In one study (78), Pierces disease strains of X. fastidiosa did not produce any of the degradative enzymes when grown in liquid media amended with substrates that might induce the production of pectolytic, proteolytic, or cellulolytic enzymes. Pectins seem to make up a signicant fraction of occlusions in grapevines (51), but pectinases are not present in cultures of X. fastidiosa (78). However, a highly virulent strain of X. fastidiosa produced twice as much protease that degraded azocasein and gelatin as did a weakly virulent strain (49). This suggests that X. fastidiosa can produce enzymes that could degrade and breach the pit membrane. Perhaps X. fastidiosa produces degradative enzymes in plants but not in culture, or the glycocalyx and bacterial aggregates may function in conserving and concentrating the catabolic enzymes at the site of action. This idea is supported by the frequent concurrent loss of virulence and aggregation characteristics of X. fastidiosa strains (80). Symptoms in plants appear to depend on the rate and extent of colonization by XLB. Leaf scorch symptom in grapevines with Pierces disease required a

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threshold bacterial population of approximately log 6 bacteria per cm of leaf vein for symptom development (78). Xylella fastidiosa attains higher populations in susceptible cultivars than in more resistant species and cultivars of grapevines (50), although numbers of cultivable bacteria were similar during early infection (< 8 weeks) among all cultivars examined. Average populations of X. fastidiosa were approximately 10 times greater in petioles and 100 to 1000 times greater in stems of the susceptible cultivar French Colombard than of the more resistant cultivars Carlos and Noble. The relative population density of C. xyli subsp. xyli in extracts from sugarcane was positively correlated with degree of cultivar susceptibility to ratoon stunting disease (29, 63). Pathogen density in basal stalk internodes was directly correlated with differences in cultivar yield due to ratoon stunting disease (29). Sugarcane cultivars resistant to ratoon stunting disease may restrict the intraxylar spread of C. xyli subsp. xyli in resistant plants (29, 133). Resistant plants have more profuse branching of xylem elements and fewer elements that pass through the node without terminating. Colonization of vascular tissues by the bacterium was most extensive in lower, more mature internodes, and the number of infected vascular bundles and pathogen density estimates within successive internodes both declined distally from the bases of stalks (29, 30). Infected vascular tissue also was more extensive in the inner, more mature stalk tissues and less extensive in the outer, less mature tissues next to the epidermis. Different plant species probably vary enormously in their importance as source plants for vector spread of X. fastidiosa, depending upon whether the bacteria spread systemically. Plants that support systemic movement can preserve and expand inoculum during periods of low vector abundance. X. fastidiosa multiplied in California mugwort (Artemisia douglasiana) and water grass (Echinochloa cruz-galli) at or near (< 1 cm) the point of infection but did not move systemically to other parts of the plant (73). Localized concentrations of viable bacterial cells were adequate for vectors to acquire X. fastidiosa from the inoculated plants (71). The relationship of bacterial populations to disease symptoms suggests that bacterial toxins are probably not involved in disease symptoms caused by X. fastidiosa (reviewed in 58, 80). Concentrations of plant hormones in grapevines were inuenced by infection with X. fastidiosa (58), and exogenous applications of plant growth hormones suppressed disease symptoms as well as bacterial populations in peach (46) and grapevine (77). Bacterial populations can be very high long in advance of symptoms in symptomless tissues such as for young stems of clove in Sumatra disease (8) or in grapevine with Pierces disease (73, 77, 85). Symptoms of water stress in grapevines with Pierces disease, such as marginal leaf scorch, appear to be closely related to cumulative chronic blockage of the xylem transport system by the occlusion of tracheary

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elements by tyloses produced by plants (41), gums, or bacterial masses (51, 84, 108). The cohesion model of xylem transport suggests that bacteria within a xylem stream under tension should precipitate the cavitation of colonized vessels (27, 152), but there is no direct evidence that this occurs with XLB. In fact, the multiplication and movement of XLB within xylem vessels suggest the opposite. However, the eventual formation of embolisms within colonized vessels rather than direct blockage of water ow by occlusions may cause water stress symptoms of leaf scorch diseases (103).
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DETECTION METHODS
The sensitivity and reliability of methods for detection of XLB in plants and insects, and of differentiating subspecies or strains have important implications for research and management of these pathogens, especially because of the many reported and undoubtedly even more numerous unreported plant species that are symptomless hosts of X. fastidiosa and C. xyli. Detection is a fundamental component of programs to establish seed stock free of C. xyli subsp. xyli for control of ratoon stunting disease in sugarcane (56). Effective and economically feasible detection methods, especially for plants with low bacterial populations, are essential for quarantines. Microscopy has been an essential but insensitive and not always reliable method of detection. Over a period of 90 years, numerous investigators of Pierces disease failed to detect a candidate pathogen using microscopy (41, 70). Microscopy of alfalfa with alfalfa dwarf disease revealed an association with bacterialike bodies, in most but not all diseased plants examined (143), but these observations were overlooked or disregarded for the following 35 years. Many common botanical stains used on grapes with symptoms of Pierces disease or alfalfa with symptoms of alfalfa dwarf (41) do not differentiate X. fastidiosa from matrix material in which they are often embedded in plants. Hematoxylin (50), carbolfuschin (143), or Giemsa stains (121) are satisfactory for differentially staining bacteria embedded in intraxylar matrixes in plants. Transmission electron microscopy and scanning electron microscopy (reviewed in 80) have been used to locate X. fastidiosa in plant hosts and insect vectors (reviewed in 119). Fluorescent antibody (13, 15, 48) or immunogold (11) labeling has been useful in detecting X. fastidiosa in vectors and plants. Immunogold labeling also has been applied to C. xyli with scanning electron microscopy (26). Enzymelinked immunosorbent assays (ELISA) have been used to identify or conrm new plant and insect hosts of X. fastidiosa (20, 54, 81, 122, 123, 130, 150, 151). Important limitations of ELISA using polyclonal antibodies for X. fastidiosa are low sensitivities (71, 73, 109, 122) and false positive readings (71, 73). Cross-

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absorption of antisera with extracts from plants free of X. fastidiosa reduced false positive readings (71, 73). Tissue blotting by immunostaining of C. xyli cells on lters to assess sugarcane for colonization by this XLB was faster and more sensitive than ELISA (31, 64). Molecular techniques for detection include DNA-DNA hybridization and immunoblotting for X. fastidiosa in citrus (9, 97) and polymerase chain reaction (PCR) for grapevine (43, 106). Most studies of PCR employed cultured cells, and inhibitors of PCR occur in grape (106) and probably other plants. PCR detected less than 100 X. fastidiosa cells per sample, which was similar to threshold values obtained by in vitro culture (73). Using cloned and sequencespecic RAPPD-PCR products, a set of PCR primers was developed that are specic for strains of X. fastidiosa that cause citrus variegated chlorosis (113). The chief disadvantages of PCR are the inability to estimate the viability or population density of XLB. The chief disadvantages of assays using in vitro culture are the time needed for colonies of slow-growing XLB to appear and the constant threat of contamination.

MOLECULAR BIOLOGY Molecular Phylogeny of XLB

The original description of X. fastidiosa (144) grouped 25 XLB strains isolated from 10 different plant species within a single bacterial species. All strains were very similar phenotypically and genotypically, with 51 to 53 mol% guanine plus cytosine and DNA relatedness of at least 85%. The more difcult question was their phylogenetic relationship to other bacteria. Early speculations on an association with the order Rickettsiales were based on morphology and nutritional fastidiousness (57, 75, 83). Phony peach strains (80) were rst isolated with modications of media developed for Rochalimaea and Legionella spp. (32, 145). However, DNA from a Pierces disease strain was > 85% related to two other strains of XLB, but essentially unrelated to DNA of other bacteria including 18 strains of Legionella (145). Reactions of gene probes prepared from L. pneumophila and Escherichia coli with DNA from these three XLB strains were ve to ten times lower than with DNA from the Enterobacteriaceae and Legionellaceae, indicating a lack of relationship to either of these families. Comparison of 16S rRNA sequences by similarity and signature analysis related these XLB to the xanthomonads but warranted status in a new genus, Xylella (144).

Molecular Genetics and Strain Relationships

Although all strains of X. fastidiosa are grouped as a single species (144), they differ in host range (80, 81, 129), pathogenicity (49, 76, 89), nutritional

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fastidiousness (52, 81, 86), and DNA homology (2123, 36, 93, 145). Obviously, X. fastidiosa consists of more than one pathovar or subspecies. Adding to the confusion is the probability that genetically different strains of the bacterium produce the same symptoms in a common host. Resolution of these problems is essential for progress in research on the ecology and epidemiology of these bacteria. Host range studies are too costly in time and labor to be useful in determining whether a bacterial strain from a newly discovered host is identical to reference strains or is a new pathovar. Molecular techniques, such as restriction endonuclease ngerprinting, restriction fragment-length polymorphisms (RFLPs), and the randomly amplied polymorphic DNA (RAPD) technique, have begun the process of differentiating strains of X. fastidiosa. In DNA homology studies, Pierces disease strains were distinct from strains causing phony disease of peach, plum leaf scald, and periwinkle wilt (93). Pulsed eld electrophoresis was used for restriction endonuclease ngerprinting of strains of X. fastidiosa from grape, plum, oak, sycamore, and goldenrod (99). After restriction with NotI and SI, similarity coefcients of fragments of strains associated with Pierces disease had a high genetic homogeneity. The DNA of the strains from other hosts (only one or two strains per host) had diverse restriction patterns, indicating genetic heterogeneity. RFLP analysis with two restriction enzymes and 12 probes from a grape strain of X. fastidiosa and 12 from a plum strain conrmed that strains associated with Pierces disease, alfalfa dwarf, and almond leaf scorch comprise a closely related Pierces disease taxonomic group (22). Of the six other strains in the study, the ragweed strain was in the Pierces disease group, and the two mulberry strains were close to this group. The plum and elm strains were very similar and distinct from the Pierces disease group and the periwinkle strain. Two separate studies utilizing RAPDs suggested several similarity groups among the strains evaluated (23, 112). The closer relationship of an oak leaf scorch cluster (seven strains) to the Pierces disease cluster than to plum leaf scorch and periwinkle strains (23) was somewhat surprising, considering growth rates and nutritional requirements of the strains. Also of interest in one RAPD study, Pierces disease strains were more similar to a ragweed strain than to an almond strain (112). Two citrus variegated chlorosis strains were only distantly related to the other X. fastidiosa strains. These two studies suggested several distinct strain groupings by the plant hosts in which they were virulent: the oak group, the grape-alfalfa-almond-ragweed group, the mulberry group, the plumelm group, and the citrus group. These preliminary results must be followed by comparisons with many more strains from each group and from more hosts. Cross-pathogenicity data need to be correlated with the molecular data.

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The lack of symptoms in plants systemically colonized by high populations of C. xyli subsp. cynodontis prompted commercial development attempts to use genetically transformed strains of C. xyli to control pest insects that feed on xylem tissues (39, 42). The CryIA(c) gene from Bacillus thuringiensis has been integrated into C. xyli for production of insect-toxic proteins in maize colonized by the bacteria (96, 136). Colonization of maize seed or seedlings by recombinant strains of C. xyli subsp. cynodontis reduced tunneling by the European corn borer by 85% but did not signicantly increase net yields (135).
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GENETIC ENGINEERING FOR CONTROL

Most of the economically important hosts of XLB are long-lived perennials requiring long periods of time for fruit production and conventional breeding for resistance. Traditional cultivars of wine grapes are crucial to marketing the crop. This makes genetic engineering for improving resistance of popular cultivars a speculative but promising alternative to conventional breeding. The genes for several antibacterial peptides from arthropods and mammals have been identied and cloned (95). In transgenic tobacco, the modied cecropin Shiva-1 increased resistance to Pseudomonas solanacearum by delaying symptom production and decreasing plant mortality (91). Somatic embryos of V. vinifera Thompson Seedless have been transformed with the lytic peptide Shiva-1 gene (R Scorza, JM Cordts, DJ Gray, D Ramming, R Emershad, & D Gonsalves, personal communications). Transformed grape plants were produced from these embryos, and PCR amplication of DNA from leaves of these plants demonstrated the presence of the Shiva-1 gene. The effectiveness and safety of this gene for resistance to Pierces disease has not yet been determined but demonstrates that the technology for gene transfer can be applied to grapes.

VECTOR TRANSMISSION
It should not be surprising to nd that sucking insects that feed on xylem sap are vectors of X. fastidiosa and P. syzygii. It is somewhat surprising, however, that not all XLB are so transmitted. For example, C. xyli does not appear to be transmitted by insect vectors (7). It is also unexpected and as yet unexplained why sucking insects that feed occasionally on xylem sap are not vectors (119). Understanding the mechanism of vector transmission is valuable not only to identify vectors: It also is important in evaluating cultivar resistance and eld or epidemiological data. The documented characteristics of insect vector transmission of X. fastidiosa are that (a) virtually all sucking insects that feed predominantly on xylem sap are potential vectors (44); but (b) vector species may vary greatly in their

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competence or transmission efciency (119, 128, 137). (c) There is a very short, if any, latent period (120); (d) vectors retain the potential to transmit for indenitely long periods following acquisition by feeding (120, 128), but (e) infectivity is lost by molting (120). Finally, (f) vectors acquire and inoculate X. fastidiosa with similar efciencies for the same acquisition or inoculation access period, respectively (120). The transmission characteristics of X. fastidiosa, plus observations of carpetlike mats of bacterial cells adhering to various portions of vectors foreguts (14, 15, 121) led to the hypothesis that X. fastidiosa is transmitted from the foregut by infective insects. If X. fastidiosa multiplies in the foregut, transmission could persist until the bacteria are lost during molting by the shedding of the cuticular lining of the foregut. Because adults do not molt, they remain infective indenitely. Recent experiments to estimate the relationship between the numbers of viable cultivable X. fastidiosa cells in vector heads to the transmission efciency of an efcient vector-plant combination (Graphocephala atropunctata and V. vinifera) found that above a low (< 100 cells) initial threshold of bacterial cell density, there was no obvious correlation between inoculation efciency and how many viable cells could be recovered from homogenates of vectors heads, although transmission efciency increased within the rst week following acquisition (72). Efcient transmission of X. fastidiosa required less than 100 cultivable cells per insect head, and populations of bacteria in the head greater than this low threshold number did not increase transmission efciency. The sucking pump in a sharpshooters foregut (cibarial pump) can contain many thousands of bacterial cells (15, 72, 150), but some transmitting insects had few or no visible (121) or cultivable bacteria. A plausible explanation is that the bacteria are transmitted from a much smaller area, the precibarium, that is just anterior to the much larger cibarial pump chamber (72). This proposed transmission mechanism would explain how xylem sap-feeders acquire X. fastidiosa. Such a seemingly simple mechanism would also explain why virtually all xylem sap-feeding insect species that have been tested as vectors of X. fastidiosa have been proven to be vectors, even though they belong to different families of hemipterans that have independently evolved xylem sapfeeding behavior. For example, the prediction that cicadas could be vectors because they are xylem feeders was conrmed experimentally (AH Purcell, unpublished data). One shortcoming of the proposed transmission mechanism is that it does not explain why other sap-feeding insects that regularly contact the xylem are not vectors (119). Other species of leafhoppers (Hemiptera: Cicadellidae) fall into the category of occasional xylem feeders (reviewed in 114, 119). Species such as pear psylla also ingest from xylem elements (138). After feeding on maize plants colonized by Clavibacter xyli, the leafhopper

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Euscelidius variegatus acquired over 105 cells per insect (AH Purcell, unpublished data). This indicates that E. variegatus ingests considerable amounts of xylem sap. Yet hundreds of E. variegatus failed to transmit X. fastidiosa in tests on almond (116). Insects that feed on xylem sap survive and reproduce on what is probably the most nutritionally and energetically dilute food source for any group of terrestrial animals (4, 16, 124). Consequently, xylem feeders typically ingest quantities of xylem sap that are many times their body volume. Sharpshooter leafhoppers have amazingly high digestive efciencies, over 99% for most amino acids (3). Another obstacle for xylem sap-feeders is that the xylem sap is often under negative tension relative to the atmosphere (152). This substantially increases the energy required to extract food from plants (124). Xylem tensions that are too high simply cause xylem feeders to stop feeding. Sharpshooters stopped feeding during high temperatures, when xylem water potentials caused by the high rates of transpiration are more than 2.4 MPa (16). Negative pressure in the xylem stream might aid momentary reversals of ow within the food canal of xylem sap-feeders, leading to the introduction of bacteria into the xylem stream, but there is no experimental evidence for this role. The high rates of sap uptake by sharpshooter leafhoppers imply that average midstream velocities within the food canal of vectors stylets must be extremely high, at least 50 cm/s for the leafhopper Homaladisca coagulata (4) or nearly 10 cm per second for Graphocephala atropunctata (121). This is the equivalent of about 50,000 bacterial cell lengths per second. It is thus not surprising to rarely see microorganisms other than X. fastidiosa in the food canals, precibaria, or sucking pumps of sharpshooter leafhoppers. The previously discussed requirements for bacterial attachment and nutrition in plants may apply equally to bacterial colonization of the hydrodynamically turbulent foreguts in vector insects. An unexplained mystery is how xylem-sucking insects penetrate xylem elements and suck in large quantities of xylem sap without disrupting the active functioning of the penetrated elements. A widely accepted view is that such disruptions should cause xylem cells to cavitate (ll with gas) when the xylem stream is under negative pressures (152), as discussed previously for bacterial invasion of xylem. The continued ow of sap through xylem elements may be crucial to effective vector transmission of XLB (119). On the other hand, simple needle puncture efciently transmits X. fastidiosa to grape (50, 76), alfalfa, and other plants (37, 81). Vector transmission of P. syzygii appears to be similar to that of X. fastidiosa, but with some evidence of a short but required latent period (40), unlike vector transmission of X. fastidiosa (120). The tube-building spittlebug

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Hindola striata (Hemiptera: Machaerotidae) readily acquired P. syzygii by feeding through articial membranes on suspensions of cultured bacterial cells and later transmitted the bacterium to clove test plants (6). In contrast to X. fastidiosa and P. syzygii, there is no evidence of vector transmission of C. xyli, despite its efcient transmission from plant to plant in sugarcane (132) and Bermuda grass (7) by cutting implements. Xylem-sap feeders acquired over log 5 cultivable cells per leafhopper when fed on maize or Bermuda grass colonized by C. xyli subsp. cynodontis, yet did not transmit the bacterium to susceptible plants. This is a spectacular example of why an insect that carries a large amount of a plant pathogen cannot be considered a vector until it can be shown to transmit the pathogen. Chewing insects such as grasshoppers, caterpillars, and leaf beetles also did not transmit C. xyli subsp. xyli from infected plants to healthy plants. Grasshoppers transmitted C. xyli subsp. xyli only by minimizing the time between feeding acquisition and inoculation access and by articially coating insect mandibles with concentrated bacteria (7). Grasshoppers quickly lost viable C. xyli subsp. xyli from their mouthparts while feeding on uninfected plants, so that only those plants that were fed on briey by grasshoppers became infected.

EPIDEMIOLOGY
Because the bacterial nature of these pathogens and their strain differences were overlooked for so long, and because relatively few researchers have studied them, many basic features of their epidemiology remain to be investigated. On a global scale, it is curious that Pierces disease apparently has not established in grape-growing regions of Europe and Asia. All the other major viticultural scourges originating from the Americaspowdery mildew, downy mildew, and grape phylloxerabecame established in Europe, probably as a result of importing wild grapes as botanical curiosities (53). Many thousands of wild grape cuttings were exported from the southeastern United States to Europe during the last century for use as resistant rootstocks against phylloxera (53). This should have provided ample opportunities for X. fastidiosa to be introduced. Once in place, these rootstocks would have provided inoculum for transmission by xylem sap-feeders, which are not rare in European vineyards (119). Reports of an episodic occurrence of Pierces disease in France (10) were based on ELISA assays, but not denitive culture of X. fastidiosa. Within North America, both Pierces disease and phony disease of peach only occur in the warmer winter climate areas of the eastern and western United States (74, 75, 117). The diseases extend further north, for example, in the milder climates at lower altitudes near oceans. The resistance to Pierces disease of grape species endemic to the southern states and the Pierces disease-susceptibility

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of species such as V. labrusca and V. riparia, which are endemic to northern latitudes (102), is indirect evidence that this type of distribution has persisted on an evolutionary time scale. Pierces disease may limit the southern extension of these Vitis species. Bacterial leaf scald diseases of forest trees such as oaks appear to extend into cooler winter climates than Pierces disease or phony disease and have been veried as far north as Kentucky (65). The reasons hypothesized for winter climate limitation have been discussed previously (75, 119). The spread of Pierces disease in California vineyards has been proposed as a classic example of a simple interest disease (140) that spreads to a crop from outside sources. For this to be true, vine-to-vine spread of the disease must be negligible, yet experimental evidence is abundant that vector transmission to grape can be extremely efcient (118, 128). A proposed explanation is that vectors cannot acquire X. fastidiosa from grapevines until it is too late in the growing season to establish infections that will survive the subsequent winter (118). In contrast, another disease caused by X. fastidiosa, citrus variegated chlorosis, appears to be spreading as a compound interest disease in Brazilian citrus (59). The initial data do not indicate whether this exponential spread occurs rst within another plant population and then to citrus or mostly from tree to tree within citrus groves. Phony peach disease was described as an epidemic wave that began in southern Georgia. Within less than 50 years, the disease spread for hundreds of kilometers to its current boundaries (137). The ease of mechanical transmission of C. xyli subsp. xyli in sugarcane creates rapid secondary (within crop) spread of ratoon stunting disease in susceptible cultivars of sugarcane. Sugarcane cultivars resistant to ratoon stunting disease have lower concentrations of C. xyli subsp. xyli cells in expressed sap than do susceptible cultivars (29, 30, 61, 62), and the effective dosage required for infection is lowest in susceptible cultivars and highest in resistant cultivars (61, 62). As a consequence, resistant cane cultivars produce less inoculum and have a higher threshold of infection than susceptible cultivars. At a certain level of plant resistance, C. xyli populations could be too low in resistant plants to infect other resistant plants (24, 31), a condition dened as population immunity (139). Clavibacter xyli subsp. xyli infects Bermuda grass, but does not occur (or is too rare to detect) in this common weed in cane elds (34). The opposite is true for C. xyli subsp. cynodontis. The explanation may be that each plant species has population immunity to the least adapted subsp. of C. xyli. By analogy, for X. fastidiosa to persist within plant communities, either some plants must support multiplication and systemic movement over extended time periods or rates of vector transmission establishing nonsystemic infections must keep pace with rates of loss of plant tissues colonized by bacteria.

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NEW PLANT DISEASES CAUSED BY XLB

We can condently predict that new diseases caused by XLB will continue to appear. Some of these will be diseases that have long been present but overlooked as being caused by pathogens, as were bacterial leaf scorches of forest trees caused by X. fastidiosa. In other cases, the movement of new pathogen species or strains or the establishment of crops in new regions will allow for new encounter diseases (149). Finally, we think that it is likely that strains may evolve to colonize new hosts. The conditions that would select for this in X. fastidiosa are monocultures of genetically uniform plants that can be nonsystemically infected by X. fastidiosa, and high populations of efcient vectors. This describes the current situation of many crops in the tropical and subtropical Americas. Localized, nonsystemic infections by XLB (73) and other bacteria (60, 67) may be very common in many plant species. The genesis, by a mutation or recombination, of a strain of X. fastidiosa capable of systemic movement in the crop could create sufcient conditions for epidemic spread, depending on vector abundance. If virulence is related to bacterial population levels in plants, disease epidemics would result where XLB can multiply to populations high enough to cause disease.

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CONCLUDING REMARKS
The three species of XLB considered in this review are taxonomically diverse. They share a distinctive ability to multiply within xylem and move systemically within certain plant species, often without causing easily noticed symptoms. The most extensively studied XLB, X. fastidiosa, occurs as a complex mixture of pathovars that will be challenging to characterize. Do P. syzygii or other yet unrecognized XLBs in this genus have a similar potential for host diversity? Clavibacter xyli strains characterized so far are less variable, suggesting more recent associations with their hosts. Clavibacter xyli subsp. xyli spreads to sugarcane or C. xyli subsp. cynodontis to Bermuda grass mainly, if not solely, by mechanical wounding, whether by machete or lawnmower. Human cultivation of sugarcane or Bermuda grass is the sustaining force in the synanthropic persistence of C. xyli in nature. In contrast to C. xyli, both X. fastidiosa and P. syzygii depend upon insect transmission to move from plant to plant and perhaps depend upon a variety of alternative hosts to persist in natural ecosystems. Disease epidemics for these pathogens may arise chiey in the encounters of new crops or plantings with endemic pathogens. But the increased long-distance commerce in plants over the past two centuries in the Americas may have added introduced strains of X. fastidiosa as an important feature of current and future epidemics. Why

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such movements have not yet introduced virulent X. fastidiosa strains to other continents is unknown, so no predictions can be made that introductions will not become established in new regions. Quarantines for systemic pathogens such as X. fastidiosa tend to focus on single crops. For example, quarantines directed against strains of X. fastidiosa virulent to grape focus on indexing or assaying grapevines to prevent introductions of this bacterium. It would seem far more likely that introductions of X. fastidiosa would arrive in symptomless but systemic hosts. For this purpose, sensitive detection methods able to discriminate pathovars or strain differences would be necessary. ACKNOWLEDGMENTS We thank MJ Davis for helpful comments and suggestions to an earlier draft and colleagues who provided reprintsand up-to-date information on their research.
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