Vector-Mediated
Transmission of Plant Pathogens
Edited by Judith K. Brown
CHAPTER 6
Dutch Elm Disease and Elm Bark Beetles: Pathogen–Insect Interaction Massimo Faccoli University of Padua Legnaro, Italy
third species, O. himal-ulmi Brasier & M. D. Mehrotra, which also causes DED, was identified in the Himalayas (Brasier and Mehrotra, 1995), but its presence has not yet been reported in Europe or North America.
Alberto Santini
Infection cycle and disease symptoms
Institute for Sustainable Plant Protection National Research Council of Italy Sesto Fiorentino, Florence, Italy
DED has a complex infection cycle involving three organisms: the pathogenic fungus, the host tree, and vector insects (Fig. 6.2). The disease is spread by various species of elm bark beetles (Coleoptera: Curculionidae, Scolytinae). Callow, i.e., sexually immature, beetles emerge in spring from the bark of dying infected elms and fly to the crowns of healthy elms to feed at the crotches of young twigs. Infected beetles contaminate healthy elms by depositing the pathogen spores into feeding wounds, which are in direct contact with the host’s vascular tissues. Spores germinate into a growing mycelium and reach the xylem, where the pathogen moves into the vessels through a yeast multiplication phase (Webber and Brasier, 1984). Later, the beetles move to dying elms to lay eggs in the inner bark of trunks or branches, which provides an ideal environment for both larval development (Rudinsky, 1962) and pathogen fructification (Webber and Brasier, 1984). The new, contaminated
Ophiostoma ulmi (sensu lato) and Dutch Elm Disease: An Overview Taxonomy of Ophiostoma ulmi s.l. During the last century, elm (Ulmus spp.) populations suffered major losses worldwide, with the near-total disappearance of adult trees in some areas as a result of Dutch elm disease (DED), caused by the ascomycete Ophiostoma ulmi (sensu lato), one of the most aggressive pathogens known in plant pathology. Two pandemics occurred. The first, caused by O. ulmi (Buisman) Nannf., began in Europe in the 1910s (Schwarz, 1922) and rapidly devastated elm populations in Europe and, 20 years later, in North America (Brasier, 2000; Guries, 2001). Around 1940, the disease declined in Europe (Brasier, 1990; Mittempergher, 1989). A few years later, in the mid 1900s, a second and more destructive pandemic caused the widespread destruction of mature elms in Europe, Asia, and North America (Brasier and Kirk, 2001; Gibbs and Brasier, 1973). This second, still-active pandemic is caused by a different species, the highly virulent O. novo-ulmi Brasier (Brasier, 1991), which has almost totally replaced O. ulmi (Fig. 6.1). Two subspecies of O. novo-ulmi are known: O. novo-ulmi subsp. novo-ulmi, previously known as the Euro-Asiatic race (EAN), and O. novo-ulmi subsp. americana, previously known as the North American race (NAN) (Brasier, 1979; Brasier and Kirk, 2001). Since the 1980s, hybrids of these two subspecies have been detected. These hybrids, whose pathogenicity does not differ from that of their parent subspecies (Santini et al., 2005b), are now spreading across the continents (Brasier and Kirk, 2010). A
Fig. 6.1. Taxonomy of Ophiostoma ulmi (sensu lato) with different species and subspecies. EAN = Euro-Asiatic race, and NAN = North American race. (© APS)
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beetles emerging from the bark complete the cycle. Infection may also occur through root anastomoses between infected and healthy trees (Webber and Brasier, 1984). Initial external symptoms of DED include crown discoloration and leaf wilting. Symptoms then spread along branches centrally, affecting the entire crown, and the tree dies rapidly (Fig. 6.3). A typical internal symptom of the disease is the formation of a brown ring in the infected sapwood (Fig. 6.4) caused by the formation of tyloses and gels in the xylem vessels (Et-Touil et al., 2005; Ouellette et al., 2004a,b; Rioux et al., 1998; Stipes and Campana, 1981), resulting in their obstruction (Newbanks et al., 1983; Zimmermann and McDonough, 1978). A systemic infection drastically reduces the hydraulic conductivity in the functional xylem, resulting in a severe wilt syndrome that may kill the tree rapidly (MacHardy and Beckman, 1973).
Host Resistance to DED There is considerable variation among elm species in resistance to DED. Asian elms are usually the most resistant (Smalley and Guries, 2000; Smalley and Kais, 1966; Ware, 1995), while most North American elms are highly susceptible and European species are moderately to very susceptible (Dunn, 2000; Gibbs, 1978). Resistance to DED is associated strictly with the host’s capacity to quickly localize the infection, preventing the pathogen from spreading in the vascular system (Sinclair et al.,
1975) and reaching the cambium (Bonsen et al., 1985; Shigo and Tippet, 1981). Reactions to infection may include vessel closing by tyloses (Fig. 6.4), embolisms, accumulation of pectin and hemicelluloses (Elgersma, 1982; Ouellette and Rioux, 1992; Rioux et al., 1998; Shigo, 1982), synthesis of chemicals, such as phytoalexin-like sesquiterpenes (Duchesne et al., 1985; Jeng et al., 1983; Sticklen et al., 1991), and formation of histological barriers typically containing phenols and suberin (Et-Touil et al., 2005; Ouellette et al., 2004a,b; Rioux and Ouellette, 1991a,b).
Elm Bark Beetles as Vectors of Ophiostoma ulmi s.l. Bark beetles belonging to the genus Scolytus Geoffroy are the main vectors of O. ulmi s.l. (Webber and Brasier, 1984). Although about 10 species of Scolytus live on elms, the large and small elm bark beetles, S. scolytus (F.) and S. multistriatus (Marsham), respectively, are the most common and important species spreading the pathogen worldwide (Faccoli, 2001, 2004; Webber, 1990, 2000; Webber and Brasier, 1984; Webber and Gibbs, 1989; Webber and Kirby, 1983).
Host trees and host selection Many species of elms have been recorded as potential hosts of elm bark beetles, both in the insects’ native distribution
Fig. 6.2. Life cycle of the elm bark beetle and spread of Dutch elm disease. (© APS)
Dutch Elm Disease and Elm Bark Beetles: Pathogen–Insect Interaction • 75 ranges and in countries into which they have been introduced (Balachowsky, 1949; Michalski, 1973; Pfeffer, 1995; Stark, 1952; Wood and Bright, 1992). Elm bark beetles attack trees that are dying, stressed, or weakened, e.g., by drought, disease, pruning, or defoliation. Mature beetles identify potentially suitable hosts by detecting a blend of volatiles released by damaged or diseased elms (Meyer and Norris, 1967; Pearce et al., 1975). Following initial attacks by the pioneer beetles, most of the insect populations find the suitable hosts in response to a blend of aggregation pheromones released by conspecific insects (Faccoli, 2004; Pearce et al., 1975; Wood, 1982), assuring whole-bark colonization (Peacock et al., 1971).
Bark colonization and insect development Elm bark beetles usually lay eggs in the phloem of weakened trees. After finding a suitable host, the elm bark beetle female bores an entrance hole through the tree bark and creates a small nuptial chamber in the phloem, where mating occurs (Fransen, 1939a; Svihra and Clark, 1980). Each mated female excavates a maternal tunnel (or egg gallery) in the phloem in which eggs are laid along both sides. The maternal galleries run parallel to the wood fibers without ramifications (Agrios, 1988; Burdekin, 1979; Sinclair and Campana, 1978). Construction of the maternal tunnels and egg laying take about 3 weeks (Betrem, 1929). Larvae hatch about 1 week after oviposition and immediately begin to bore characteristic larval galleries in an orthogonal direction from the maternal ones (Agrios, 1988;
Fig. 6.3. Symptoms of Dutch elm disease. (Courtesy A. Santini–© APS)
Balachowsky, 1949; Burdekin, 1979) (Fig. 6.5C). Larval galleries of Scolytus spp. are 60–150 mm long on average and only rarely cross each other (Betrem, 1929; Buisman, 1932; Kletecka, 1996; Manojlovic and Sivcev, 1995). Larval tunnels become wider as the larvae develop and move away from the maternal gallery (Betrem, 1929). Larvae feed in the phloem for about 30 days, passing through five developing instars before becoming fully grown (Fransen, 1939a). Shortly before pupation, mature larvae bore a pupal chamber in the external part of the sapwood, where they metamorphose first into pupae (Webber, 1990; Webber and Brasier, 1984) and after about 2 weeks into adults (Zanta and Battisti, 1990). The new, callow adults then emerge from the bark of the host tree through a hole excavated directly from the pupal chamber (Kletecka, 1996). After emergence, adults fly and disperse, looking for healthy trees on which to carry out sexual maturation feeding. Elm bark beetles may be monovoltine or bivoltine. Under favorable climatic conditions, there are usually two generations per year (Della Beffa, 1949), the first starting in late spring (May–June) and emerging in late summer (August–September), the second beginning in autumn (September), overwintering as larvae, and emerging in the following spring (Betrem, 1929; Buisman, 1932; Fransen, 1939a; Lanier and Peacock, 1981; Zanta and Battisti, 1990).
Maturation feeding As in all monogamous bark beetle species, before reproducing in the bark of dying trees, the newly emerged callow adults need a period of sexual maturation feeding in crotches of 2- to 3-year-old twigs of healthy and vigorous elms (Fig. 6.5A and B). The maturation feeding lasts a few days, during which the insects excavate short tunnels (2–4 cm long) in the twig phloem and sapwood (Fig. 6.5A and B) (Fransen, 1939a; Webber and Brasier, 1984). Twig feeding, which is a prerequisite for sexual maturation of callow adults, is associated with restoration of the beetle reserves (food and water) (Fransen, 1939a; Heybroek et al., 1982; Lanier and Peacock, 1981; Lunderstadt and Rohde, 1993; Svihra and Clark, 1980). Although Scolytus spp. prefer twigs on the upper part of the crown (Svihra and Clark, 1980; Webber and Kirby, 1983), feeding tunnels can be found in almost any young, sappy bark (Fransen, 1939a; Lanier and Peacock, 1981; Webber and Kirby, 1983). Scolytus spp. can stay in the feeding tunnels for up to 13 days (Fransen, 1939a). When sexual maturity is reached, adults fly away, looking for weakened trees on which to lay eggs and start a new generation.
Pathogen–Insect–Host Interactions and Transmission Mechanisms History of studies on O. ulmi s.l. and elm bark beetle interactions
Fig. 6.4. Elm shoot infected by Ophiostoma ulmi s.l. Note the brown ring in the infected sapwood caused by the formation of tyloses in the xylem vessels. (Courtesy A. Santini–© APS)
Pathogen transmission received a lot of attention in the past (Webber and Brasier, 1984). Some authors considered the wind to be important in spreading fungal spores from infected plants to tissues of healthy trees exposed by wounds or pruning (Smucker, 1935; Westerdijk and Buisman, 1929). This hypothesis, however, was soon disproved because anemochoral dispersion would be too casual and generic and could not ensure the ideal growing conditions needed by the pathogen for rapid development (Goidanich and Goidanich, 1937).
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Fig. 6.5. Symptoms on elm trees at different stages of the elm bark beetle life cycle. A and B, Maturation feeding by callow adults on 2- to 3-year-old elm twig crotches; C, maternal and larval galleries of Scolytus multistriatus on Ulmus procera (note the central and vertical maternal gallery and lateral larval galleries); and D, dried twigs killed by Ophiostoma ulmi s.l. infection resulting from maturation feeding of bark beetles on healthy elms. (Courtesy M. Faccoli–© APS)
Dutch Elm Disease and Elm Bark Beetles: Pathogen–Insect Interaction • 77 Others have suggested that rain is mainly responsible for the spread of conidia, allowing them to reach the leaf stomata (Schwarz, 1922). However, Marchal (1927) proposed that bark beetles could be vectors of O. ulmi s.l. as they migrated between infected and healthy elms. Although the pathogen may be transmitted effectively in several ways (Schwarz, 1922; Smucker, 1935; Westerdijk and Buisman, 1929), elm bark beetles belonging to the genus Scolytus were found to be the most efficient vectors (Basset et al., 1992; Battisti et al., 1994; Collins et al., 1936; Faccoli, 2004; Faccoli and Battisti, 1997; Faccoli et al., 1998; Favaro and Battisti, 1993; Fransen, 1931; Jacot, 1934, 1936; Marchal, 1927; Webber, 1990; Webber and Brasier, 1984). The definitive demonstration of Marchal’s theory was provided by Fransen (1931) and Fransen and Buisman (1935), who showed pathogen presence and proliferation in the short galleries bored by elm bark beetles in healthy plants during maturation feeding. Many other vectors were later identified, mainly mites and insects living on elms, but none as effective as bark beetles (Collins et al., 1936; Jacot, 1934, 1936).
Behavioral interactions: Role of insect behavior in the transmission pathways The success of pathogen–insect interactions results mainly from the characteristic reproductive behavior of the elm bark beetle species, which, however, depends largely on the occurrence of O. ulmi-infected trees. Callow adults that emerge from infected trees carrying pathogen conidia on their bodies contaminate healthy trees through their feeding activity, facilitating consequent development and movement of the pathogen within the wood vessels (Fig. 6.5D) (Burdekin, 1979; Fransen and Buisman, 1935; Gibbs, 1974; Goidanich, 1936; Goidanich and Goidanich, 1937; Webber and Brasier, 1984; Webber and Kirby, 1983). Basset et al. (1992) showed that contact for at least 72 h between infected beetles and xylem can be sufficient for pathogen transmission. This is known as the “pathogenic phase” of the disease (Gibbs and Smith, 1978; Lea, 1977). The canopy of every tree may host several dozen callow adults, and the process can be repeated for several years; thus, healthy trees growing close to elms infected by O. ulmi s.l. and infested by elm bark beetles have a very high risk of infection. This transient phase is very important for pathogen spread because it is the only way for the fungus to reach and infect isolated trees. Maturation feeding and pathogen infection weaken trees, making them attractive for mature reproductive adults of the following insect generations, which will be looking for dying, i.e., diseased, elms in which to lay eggs beneath the bark of the trunk and main branches. During bark infestation and excavation during the mating period, the mature adults again infect the hosts with the conidia carried on their tegument, fulfilling their role as vectors for a second time. Bark colonization by insects and pathogen on already infected elms (from the twigs) is known as the “saprophytic phase” of the disease (Gibbs and Smith, 1978; Lea, 1977). In this respect, maternal galleries and the pupal chambers are an ideal microenvironment for both fungal growth and sporulation (Webber and Brasier, 1984) (Fig. 6.6). Interestingly, in this phase, two fungal clones having different origins, one from previous years during maturation feeding and the other from the more recent bark colonization, may meet in the same tree. The emerging offspring developed in the phloem of infected trees become new vectors of fungal conidia, and the dispersal cycle starts again (Webber and Brasier, 1984). Not every wound
caused by maturation feeding results in pathogen transmission (Fransen, 1939a). Parker et al. (1941) reported that 13% of all cases resulted in tree infection, whereas Webber and Brasier (1984) found that about 30% of feeding wounds were infected by O. ulmi s.l. Xylem infection may be a result either of a primary and direct spore transfer from the beetle into the xylem vessels or, more likely, of a secondary infection resulting from earlier pathogen colonization of the feeding wound followed by subsequent growth into the xylem tissues (Buisman, 1932).
Biological interactions: Symbiosis between insect and pathogen The insect–pathogen relationship can work only if the insects newly emerged from infected trees are efficient pathogen vectors, and this depends on biological interactions between the insect and the pathogen that allow the fungus to contaminate the newly emerging beetles. Mature adults looking for suitable hosts on which to breed are still vectors of fungal conidia, which are inoculated under the host bark. Here, a phloem lesion produced by pathogen growth progressively expands from the insect penetration holes. However, since pathogen development is slower than larval feeding, at the initial stage of infection there is no physical contact between fungus and larvae. Nevertheless, O. ulmi s.l. may grow through the whole gallery system, and the reunion of pathogen and vector occurs in the pupal chambers (Webber and Kirby, 1983) when larvae stop feeding and await metamorphosis. Since the outer, or thin, bark dries out quickly, sporulation of O. ulmi s.l. occurs less frequently in pupal chambers located in these regions compared with those in the moist inner bark (Buchel and Cornelissen, 2000). The location of the pupal chambers differs among species and thus strongly influences the spore load of the emerging beetles (Webber and Kirby, 1983). Contamination of the new adult beetles is complete by the time of beetle emergence (Buchel and Cornelissen, 2000). Pathogen transmission can also result from fungal spores ingested by newly formed adults just before their emergence from the bark of infested trees (Pfeffer, 1979), although the percentage of spores occurring in the insect gut is much lower than that of spores found on the tegument (Parker et al., 1941). Similarly, the number of conidia potentially occurring in the adult gut as a result of larval feeding on infected hosts is irrelevant. Fransen
Fig. 6.6. Black perithecia of Ophiostoma ulmi s.l. producing the conidia vectored by elm bark beetles. (Courtesy A. Santini–© APS)
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(1931) observed that the gut of larvae of S. scolytus and S. multistriatus contains fecal pellets that can be contaminated with fungal spores. During insect metamorphosis, the midgut and posterior gut of the mature larva are expelled twice from the pupa; thus all the ingested conidia are lost. Transmission of O. ulmi s.l. from larvae via pupae to the new adult beetles does not therefore occur directly, although the fecal pellets can contaminate the pupal chamber (Fransen, 1931).
Chemical interactions: Attraction of infected trees to insects Because only a small percentage of elms in a forest are stressed or dying, i.e., susceptible to attack by Scolytus spp., the insects must be able to identify the few potentially suitable hosts in a vast natural landscape (Byers, 1996). Elms affected by DED—and therefore suitable for insect colonization—release a blend of volatile terpene-derived chemicals composed mainly of α-cubebene, which makes the diseased trees recognizable to mature beetles (Meyer and Norris, 1967; Pearce et al., 1975). This “primary attraction” was also demonstrated by Gore et al. (1977), who reported that the quantity of sesquiterpene and α-cubebene released by elms increased as the tissues were degraded. Besides terpenes, several lignin intermediates and phenols isolated from infected trees have been recognized as feeding stimulants and short-range attractants for elm bark beetles of the genus Scolytus (Baker et al., 1968; Meyer and Norris, 1967, 1974). Water content of the host tissues also appears to be an important stimulus for beetles to start boring (Von Keyserlingk, 1980). After the initial attack, most beetles find the host suitable in response to a mixture of both volatiles released by the tree and pheromones released by conspecifics (Wood, 1982). S. scolytus and S. multistriatus produce an aggregation pheromone composed of 4-methyl-3-heptanol (threo and erythro isomers) and 2,4-dimethyl-5-ethyl-6,8-dioxabicyclo-octane (commonly known as α-multistriatin) (Faccoli, 2004; Pearce et al., 1975). This pheromone, released by both males and females (Blight et al., 1978a), concentrates the insect population on a recently infested tree. Aggressive Scolytus spp., such as S. scolytus and S. multistriatus, generally tolerate defensive plant chemicals better than do nonaggressive species (Krokene, 1994). However, the aggregation pheromone assures whole-bark infestation, helping the insects to overcome tree defenses by mass attack (Peacock et al., 1971). Beetles respond differently to a pheromone depending on its ratio to host volatiles (Blight et al., 1978b), reflecting the current colonization state of the host tree.
the spores of the associated fungi (Batra, 1963; Beaver, 1989; Francke-Grosmann, 1956a,b, 1963a,b, 1967; Lévieux et al., 1991; Six, 2003). Used more generally, the term mycangia may refer to any structure involved in transport and protection of fungi, regardless of whether glandular cells are present (Kirisits, 2004; Six, 2003; Whitney, 1982). As previously mentioned, bark beetle species living on elms are intimately associated with ophiostomatoid fungi of the genus Ophiostoma, and the insect–pathogen relationship seems not to be casual (Kirisits, 2004). Although the conidia are not located in specific mycangia (Webber and Kirby, 1983), they are lodged in small pits excavated at the bases of the hairs on the insect elytra (Fig. 6.7) (Barbosa and Wagner, 1989; Faccoli, 1995; Francke-Grosmann, 1963a). Glandular cell ducts occur at the bases of these cavities (Fig. 6.7), which produce viscous substances involved in adherence of the fungal conidia on the hairs and bristles of beetle tegument and prevent spore dehydration (Francke-Grosmann, 1963a; Manion, 1981). Sex-related morphological features, such as setae on the beetle frons (Faccoli et al., 1998), and specific breeding preferences also influence the number of spores carried by the beetles (Basset et al., 1992; Favaro and Battisti, 1993; Webber, 1990). Nevertheless, the association of O. ulmi and O. novo-ulmi with Scolytus spp. is relatively recent (beginning of the last century), and this short period of coevolution has probably not been long enough to allow the insects to differentiate specific morphological struc-
Morphological interactions and coevolutionary considerations Both bark beetles and their associated fungi have evolved morphological adaptations to ensure maintenance of symbiosis from generation to generation. The most common strategy of the insects to assure effective dispersal of their associated fungi is a specialized structure in the tegument associated with gland or secretory cells and used for the storage, transport, and transmission of fungi (Kirisits, 2004). These structures, called “mycangia” or “mycetangia” (Batra, 1963; Beaver, 1989; Berryman, 1989; Francke-Grosmann, 1967), consist of large tubes, pouches, pits, crevices, or cavities in the tegument lined with glandular cells producing secretions that protect and preserve
Fig. 6.7. Mycangia associated with elytral bristles (short) and hairs (long) of Scolytus pygameus. Note the small holes in the mycangia associated with gland and secretory cells used for conidial storage and maintenance. (Courtesy M. Faccoli–© APS)