5
Vector–Pathogen Interaction

The relationship between pathogens and their vectors can range from highly specific associations, in which the pathogen depends on a single species of vector for transmission, to more general associations, in which the pathogen can be transmitted by a wide range of vector taxa. But transmission often requires highly specific pathogen transport mechanisms in the vector (Gray and Banerjee, 1999; van den Heuvel et al., 1999). In the case of the interaction between Xylella fastidiosa (Xf) and its vectors, relatively little is known about the mechanism of transmission. The pathogen can be transmitted by most sharpshooter leafhoppers and spittlebugs, and probably by most xylem-feeding homopterans, but the efficiency of transmission varies among vectors and plant hosts. Thirty-nine species of sharpshooter in the subfamily Cicadellinae have been demonstrated to transmit Xf, as have 4 species of spittlebug and 1 cicada (Redak et al., 2004). However, many species, particularly those found only in the tropics, have not been tested for the ability to transmit, and it is highly likely that more vectors could be identified (Redak et al., 2003). Data suggest that members of the sharpshooter tribe Cicadellini transmit more efficiently than do members of the Proconiini tribe (Almeida and Purcell, 2003; Krugner et al., 1998; Redak et al., 2003). In California, the blue-green sharpshooter (BGSS), Graphocephala atropunctata (Signovet), is in the Cicadellini; the glassy-winged sharpshooter (GWSS) Homalodisca coagulata (Say) is a Proconiine sharpshooter.



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California Agricultural Research Priorities Pierce’s Disease 5 Vector–Pathogen Interaction The relationship between pathogens and their vectors can range from highly specific associations, in which the pathogen depends on a single species of vector for transmission, to more general associations, in which the pathogen can be transmitted by a wide range of vector taxa. But transmission often requires highly specific pathogen transport mechanisms in the vector (Gray and Banerjee, 1999; van den Heuvel et al., 1999). In the case of the interaction between Xylella fastidiosa (Xf) and its vectors, relatively little is known about the mechanism of transmission. The pathogen can be transmitted by most sharpshooter leafhoppers and spittlebugs, and probably by most xylem-feeding homopterans, but the efficiency of transmission varies among vectors and plant hosts. Thirty-nine species of sharpshooter in the subfamily Cicadellinae have been demonstrated to transmit Xf, as have 4 species of spittlebug and 1 cicada (Redak et al., 2004). However, many species, particularly those found only in the tropics, have not been tested for the ability to transmit, and it is highly likely that more vectors could be identified (Redak et al., 2003). Data suggest that members of the sharpshooter tribe Cicadellini transmit more efficiently than do members of the Proconiini tribe (Almeida and Purcell, 2003; Krugner et al., 1998; Redak et al., 2003). In California, the blue-green sharpshooter (BGSS), Graphocephala atropunctata (Signovet), is in the Cicadellini; the glassy-winged sharpshooter (GWSS) Homalodisca coagulata (Say) is a Proconiine sharpshooter.

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California Agricultural Research Priorities Pierce’s Disease STRATEGIES OF INTERFERENCE Several approaches to interfering with the interaction between the vector and the pathogen have been proposed for controlling the spread of Pierce’s disease (PD). In theory, interference could be targeted at one or more of three stages: at the acquisition of the bacteria by the vector, during attachment and replication of the bacteria in the vector, or during the inoculation of the bacteria to a healthy host. Most of those approaches are experimental, and their effectiveness has not been demonstrated for other insect-vectored disease systems. However, several strategies are worth considering in the context of long-term management of PD: feeding disruption, inhibition by other bacteria of Xf attachment in the vector, and inhibition of transmission of PD strains of Xf by other strains by paratransgenesis. PATHOGEN TRANSMISSION The characteristics of Xf transmission by GWSS are similar to those of other known leafhopper vectors of Xf, although GWSS is a less efficient vector than is BGSS, a primary vector in Northern California. Leafhopper vectors acquire Xf through feeding on the xylem of host plants, and the bacteria replicate in the mouthparts (Purcell et al., 1979). Although Xf can be acquired by adults and nymphs, it is lost during the molting process—a fact that suggests that the bacteria attach to the foregut, because the foregut lining is shed in molting. Scanning-electron microscopy of leafhopper vectors shows bacteria attached to the cibarial pump and the lining of the esophagus in the foregut (Purcell et al., 1979). Adults that acquire the bacteria can continue to transmit throughout their lifetime (Severin, 1979). Although there is currently no strong evidence of gender differences in transmission (Redak et al., 2003), differences have been shown for other leafhopper-transmitted pathogens and could have significant effects on epidemiology (Beanland et al., 1999). Adult GWSS can acquire Xf from infected plants and inoculate healthy plants in less than an hour of access time on a plant. There is no evidence for latent period between acquisition of the bacteria and the ability to transmit it (Almeida and Purcell, 2003). The rate of successful inoculation increases with increased time on the plant, but acquisition efficiency does not increase after 6 hours. Nymphs and newly molted adults transmit more efficiently than older adults do. Almeida and Purcell (2003) reported, in their experiments, that a maximum of 20% of individual leafhoppers acquired and transmitted Xf. In contrast, experiments with BGSS indicated an average transmission efficiency of 90% (Purcell and Finlay, 1979). More work demonstrated 68% efficiency of Xf acquisition from infected grape and 56–99% inoculation efficiency to grape, depending on the number of days post acquisition (Hill and Purcell, 1995). One striking difference between the two major vectors is that GWSS feeds on woody tissues throughout the year and can transmit Xf to stems that are more than 2 years old, but BGSS transmits only to green shoots (Almeida and Purcell, 2003).

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California Agricultural Research Priorities Pierce’s Disease For GWSS there is no significant difference in transmission to woody stems or to green shoots, and transmission to dormant vines also is apparent. That pattern has important implications for virus epidemiology: it shows a polycyclic pattern of disease spread that contrasts with the general pattern of monocyclic spread of PD in California (see Chapter 1). Therefore, to develop ecologically based management strategies that rely on economic thresholds and to arrive at an understanding of pathogen epidemiology, the committee recommends the following: Recommendation 5.1. Research should be done on the transmission biology of the disease system, including acquisition from and inoculation to alternative hosts and acquisition from and inoculation to dormant grapevines (Category 2). Transmission efficiency is influenced also by the population of Xf in host plants. In experiments by Hill and Purcell (1997), higher bacterial densities in source plants led to greater rates of transmission. Despite the evidence that the number of bacteria acquired by the vector can dictate subsequent transmission, the relationship between number of bacteria in vector heads and transmission efficiency is not well understood. Efficient transmission of Xf requires less than 100 cultivable bacteria for the BGSS (Hill and Purcell, 1995), and no clear relationship between cultivable bacteria in the head and transmission has been identified for GWSS at a detection threshold of about 265 colony-forming units per head (Almeida and Purcell, 2003). Those findings, and the rapidity with which vectors can transmit the bacteria, suggest that the threshold density of bacteria necessary for transmission is very low. Unfortunately, this implies that not strategy that involves feeding disruption is likely to be successful in preventing transmission. Similarly, rapid transmission makes it difficult to use insecticides effectively to control disease spread. Attachment and Replication in Vectors The precise mechanism by which Xf attaches to the sharpshooter mouthparts is not well understood. Processes that have been identified as important in attachment and replication in plants and in media include attachment to cell walls by ionic bonds (Leite et al., 2002), production of a polysaccharide gum (Silva et al., 2001), and formation of biofilm (Marques et al., 2002). The pH and nutrient content of the substrate could also influence bacteria replication (S.M. Fry et al., 1994), so xylem chemistry could be important for replication in the vector. Attachment in the vector can pose challenges for the bacteria; the rate of sap uptake by sharpshooters GWSS is extremely high. Average midstream velocities within the food canal of the vectors’ mouthparts have been reported to be nearly 10 cm/s for BGSS (Purcell et al., 1979) and at least 50 cm/s for GWSS (Andersen et al., 1992). Polysaccharide fibers produced

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California Agricultural Research Priorities Pierce’s Disease by bacteria are thought to function in attachment in other turbulent habitats, such as streams with rapid flow and animal guts (Costerston and Irwin, 1981). Identification of the mechanisms of attachment and replication in the vector could lead to strategies that decrease the probability of attachment or that reduce the replication rate. A reduction in bacterial population in the vector’s foregut should lead to a lower efficiency of transmission. Although the number of bacteria needed for successful transmission is quite low, presumably there is a threshold below which no transmission takes place. Research to determine that threshold-using methods more sensitive than culturing, such as polymerase chain reaction–based methods, would be useful (Almeida and Purcell, 2003). Therefore, the committee makes the following recommendation: Recommendation 5.2. Research should be done on the determinants of transmission efficiency, including attachment and reproduction of Xf in GWSS (Category 2). Inhibition of Xylella Transmission by Other Bacteria There has been speculation that the transmission of Xf could be reduced by the presence of other microbes within the vector. Almeida and Purcell (2003) cultured several microbes, mostly bacteria, from the head capsules of GWSS and suggested that those microbes could compete with Xf for attachment sites in vectors’ mouthparts. Preliminary identification of common bacteria in the foregut and midgut of GWSS also has been done by Peloquin et al., (2002), but no one has addressed the competitive relationships between those bacteria and Xf. That will be a necessary step toward evaluating the potential for biological control of PD through interfering with the attachment and replication of Xf in the vector. Obviously, because attachment occurs in the foregut, it would be most useful to target bacteria there, rather in than the midgut. Bextine and colleagues (2004) have begun developing a plant-based delivery system to introduce endosymbiotic bacteria into GWSS. Such a system would be useful if an appropriate biological-control agent were available. It is useful to consider the possibility of interference between two strains of Xf within the vector. That strategy is similar to the cross-protection that has been used successfully for some viral diseases, but it focuses on interference within the vector rather than within the host plant. Work with virus transmission by insect vectors has shown that the acquisition of one strain of a virus can reduce acquisition or the transmission of a second strain. For instance, acquisition of the MAV strain of the barley yellow dwarf virus by aphid vectors markedly reduces the transmission of the PAV strain by those insects (Gildow and Rochow, 1980). Similarly, there is some evidence that the acquisition of a spiroplasma (a plant-pathogenic mollicute) by a leafhopper vector interferes with the replication and transmission of another mollicute (Maramorosch, 1958). The mechanism interference is not well understood, but the process can be reliable.

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California Agricultural Research Priorities Pierce’s Disease Preliminary results by Costa and Cooksey (Proposal to CDFA, March 2003) suggest that there may be some degree of inhibition between the PD and oleander leaf scorch strains of Xf within GWSS, but the effect appears to be small. It would be interesting to pursue that idea using other strains of Xf, including nonpathogenic ones, as well as other species of bacteria. Although the research is interesting from a biological perspective, the committee concluded that biological control of bacterial vascular pathogens, particularly of perennial crops, has generally shown little success in the field. Thus the committee views this as Category 4 research. Transgenic Interference of Transmission There are two potential transgenic approaches to interfering with interactions between vector and pathogen to reduce successful transmission. The first is genetic manipulation of vectors to reduce transmission competence. That approach requires the development of efficient transformation of the targeted vector species, the identification of pathogen-specific molecules that impair competence, and the development of a mechanism to drive the competence-reducing molecules through the vector population (Beatty, 2000). Proposed mechanisms include transposable elements, densoviruses, and bacterial symbionts of the vectors such as Wolbachia. Research on genetic modification of vectors has focused on vectors of human pathogens, particularly mosquito vectors of malaria, and mosquitoes have been transformed to express antiparasitic genes that make them inefficient vectors (Ito et al., 2002), although genetic modification often reduces fitness (Catteruccia et al., 2003). Driving the competence-reducing molecules through the vector population is likely to take decades, during which time some of the vectors remain competent. Consequently, in the case of malaria control, the ecological hurdles could well exceed the technical hurdles to success (Spielman et al., 2001). It is likely that the barriers to genetic transformation of PD vectors as a means of disease control would be equally daunting. The second transgenic approach focuses on genetic engineering of bacterial symbionts of vectors to express and release transgene products that damage the pathogen. That process has been called “paratransgenesis.” In the Chagas disease system, researchers have engineered the bacterial symbiont Rhodococcus rhodnii to express and release transgene products into insect tissues that are damaging to the disease agent (Durvasula et al., 1997). The most promising drive mechanism in this system is the coprophagic behavior of the insect vector which should promote the dispersal of the recombinant symbiont (Durvasula et al., 1999). However, as in the case of malaria, effective disease control is mainly in the future. Using paratransgenesis to manage PD clearly would be a long-term strategy, and one in which the likelihood of success is limited. Although some progress toward transformation of GWSS endosymbionts has been made (Lampe and Miller, 2002), the committee views this as Category 4 research. In

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California Agricultural Research Priorities Pierce’s Disease addition to its scientific uncertainty, there are ecological and regulatory barriers to success that are at least as significant as any technical barriers. PATHOGENS AND VECTOR FITNESS Many studies have shown that plant pathogens transmitted by insects can significantly affect vector fitness (for review, see A. G. Power, 1992). Those effects can be direct or indirect, mediated by changes in host plant nutrition induced by virus infection of the host (Box 5-1). Indirect effects can result because virus infection often increases the amino acid content of the phloem (Matthews, 1981), thereby improving the nutritional value of plans for phloem-feeding insects. However, differences in nitrogen mobilization in different host species in response to virus infection can lead to enhanced or diminished vector fitness (Blua et al., 1994; A. G. Power, 1992). Aphids have received the most attention; fewer studies have examined the responses of leafhoppers or whiteflies to plant infection status. However, no studies have isolated unambiguously the direct effects of a pathogen on vector fitness from the indirect effects mediated through the host. BOX 5-1 Pathogen Effects on Vector Survivorship and Reproduction There have been no studies of the effects of Xf on GWSS survivorship, but some work has demonstrated the effects of viruses on leafhopper survivorship on infected plants. For example, R. E. Hunt and Nault (1990) reported increased survivorship of the leafhopper Graminella nigrifrons (Forbes) on plants infected with maize chlorotic dwarf virus. However, the longevity of the green rice leafhopper Nephotettix virescens (Distant) was reduced on rice plants infected with tungro viruses (Khan and Saxena, 1985). Tungro-infected plants had higher concentrations of free sugars, but lower concentrations of soluble amino acids, but the effects on vectors, whether positive or negative, could be limited to those that feed on phloem. It is not clear whether virus-induced changes in xylem would similarly influence vector survivorship. Host plant infection with plant pathogenic mollicutes (phytoplasmas and spiroplasmas) also can lead to greater survival of leafhoppers (Beanland et al., 2000; Madden and Nault, 1983; Madden et al., 1984; Maramorosch, 1960; Purcell, 1988), despite the fact that the pathogens typically propagate in vector as well and host plant alike. In the case of the corn stunt spiroplasma and the maize bushy stunt phytoplasma, fitness of the primary, most efficient vector is improved by acquisition of the pathogen, whereas the fitness of occasional vectors or those with low efficiency is impeded (Madden and Nault, 1983; Madden et al., 1984). Corn leafhoppers (Dalbulus maidis; DeLong and Walcott) survive better at cool temperatures when they are infected either with corn stunt

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California Agricultural Research Priorities Pierce’s Disease spiroplasma (Ebbert and Nault, 1994) or with maize bushy stunt phytoplasma (Moya-Raygoza and Nault, 1998). Thus the effects of plant pathogens on leafhopper survival can be complex and environment dependent. Most research on aphid vectors shows higher reproductive rates on plants infected with viruses than healthy plants, although some studies have shown negative effects of plant infection on fecundity (reviewed in A. G. Power, 1992). These conflicting results are probably attributable to differences among host plants in nitrogen mobilization in response to plant infection with a diversity of viruses (A. G. Power, 1992; Blua et al., 1994). Studies of whiteflies that are vectors also have shown higher fecundity on virus-infected hosts (Mayer et al., 2002; McKenzie, 2002), although Costa and colleagues (1991) reported differing effects of virus infection on whiteflies in different hosts and detected no correlation for amino acid concentration and whitefly reproductive rate. In one study of leafhoppers, the fecundity and population growth of the green rice leafhopper were lowered when the leafhoppers fed on rice plants infected with tungro viruses than they were when the insects ate healthy rice plants (Khan and Saxena, 1985). As noted above, the work so far has involved phloem-feeding vectors, and the effect of virus infection on xylem-feeders is not documented. Recommendation 5.3. A subset of studies of the vector should explore the effects of Xf on vector survivorship, fecundity, and population growth rates (Category 2). INFECTION AND VECTOR BEHAVIOR Like the effects of pathogens on vector fitness, effects on vector behavior can be either direct or indirect, the latter mediated by changes in host plant nutrition or morphology induced by virus infection of the host. The evidence for direct effects of insect-transmitted pathogens on vectors is limited, but several studies have documented indirect effects in which vector behavior was influenced by the infection status of the host plant. Most of those studies have been done with aphids (Box 5-2). The preference of sharpshooters for plants infected with Xf is not known, but a few studies of leafhoppers have shown vector preference for plants infected with viruses or phytoplasmas (Box 5-3). Bennett (1967) reported that beet leafhoppers preferentially colonized, and performed better on, beet plants infected with beet curly top virus than they did on healthy beets. Similarly, aster leafhoppers prefer carrot plants infected with the aster yellows phytoplasma over

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California Agricultural Research Priorities Pierce’s Disease BOX 5-2 Vector Attraction to Infected Plants Aphids are likely to show greater preference for infected plants than for healthy plants (e.g., P. B. Baker, 1960; Castle et al., 1998; Eckel and Lampert, 1996; Eigenbrode et al., 2002; Fereres et al., 1999; Macias and Mink, 1969), although that pattern is not completely consistent (A. G. Power, 1992). Beetle vectors also have been reported to prefer virus-infected host plants (Musser et al., 2003). In many cases, the preference appears to be driven by a visual attraction to yellowing, senescent plants, and many viruses cause symptoms similar to those of senescence. The “leaf scorch” symptoms of PD can also appear similar to senescence in some grape varieties, suggesting that vectors could be attracted to infected vines. In one interesting study, Castle and colleagues (1998) compared the preference of aphids for virus-free potatoes and those infected with potato leafroll virus (PLRV), potato virus X, and potato virus Y. Earlier studies had shown that aphid population growth rates were higher on potatoes infected with PLRV, which depends on aphids for transmission, than they were on virus-free plants or on plants infected with the other viruses, that are not obligately transmitted by aphids (Castle and Berger, 1993). Augmenting the earlier study, Castle and colleagues (1998) demonstrated that significantly more aphids settled on hosts infected with the obligately aphid-transmitted PLRV than on either virus-free plants or on plants infected with the other viruses. Recent studies by Eigenbrode and colleagues (2002) indicate that volatile compounds from PLRV-infected potatoes are involved in the attraction and arrestment of aphid vectors of the virus, and that those compounds could similarly influence the behavioral responses of other vectors to infected hosts. healthy plants (Peterson, 1973) and have better fitness on infected carrot and aster plants than they exhibit on healthy plants (Beanland et al., 2000; Peterson, 1973). Recommendation 5.4. A subset of studies of the vector should explore the effects of Xf on vector behavior, including movement and attraction to infected hosts (Category 2).

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California Agricultural Research Priorities Pierce’s Disease BOX 5-3 Implications for Epidemiology Epidemiologic models can be useful for evaluating the potential of various management strategies to control the spread of plant pathogens (Jeger and Chan, 1995). In the case of insect-transmitted pathogens, such models suggest that effects on vector fitness that lead to higher rates of population growth on infected plants could significantly affect rates of pathogen spread (Holt et al., 1997; Zhange et al., 2000). One model of the whitefly-transmitted African cassava mosaic virus is based on substantial field data (Holt et al., 1997) which showed that the rate of spread was sensitive both to vector population dynamics (abundance, birth rate, mortality) and to virus transmission rates (inoculation and acquisition). When virus infection of hosts leads to increased vector fecundity, spatial aggregation of vectors is promoted (Zhang et al., 2000). Models predict that vector aggregation should have a dual effect. Within the infected crop, it should reduce the effective contact rate between vector and host and thus lead to lower disease incidence than would be predicted without aggregation. On the other hand, it could cause increased emigration rates of infective vectors to other hosts in the area (Zhang et al., 2000). Most of the models described here are based on systems in which the vector insect reproduces within the crop. However, when the incidence of disease depends primarily on the immigration of vectors from alternative hosts that act as reservoirs of pathogens and of vectors, other models show that disease incidence is largely insensitive to vector mortality unless the vector population is extremely small, and therefore insecticide treatment is ineffective (Holt et al., 1999). To evaluate the potential of targeting various aspects of the interaction between Xf and its vectors for control, it is essential to understand the population dynamics of vectors inside and outside of the vineyard. Epidemiologic models have predicted that vector preference behavior will have a significant influence on the rate of pathogen spread, and conventional wisdom postulated that vector preference for infected hosts over healthy ones would promote the spread of disease (e.g., Irwin and Thresh, 1990; Matthews, 1991). However, the results of several recent epidemiologic models suggest that often the reverse is true (McElhany et al., 1995; Real et al., 1992). That is, a preference for healthy plants often leads to greater rates of pathogen spread, because an infective vector is less likely to “waste” a visit to a plant that is already infected. However, the effect of vector preference depends on the frequency of infected plants in the population and on whether the transmission system is persistent, nonpersistent, or semipersistent. Models predict that pathogens with even moderate persistence are likely to have higher rates of spread by vectors that prefer healthy plants at most disease frequencies (McElhany et al., 1995). Given the persistence of Xf in adult sharpshooters, vector preference for healthy grape plants could lead to faster spread of PD.