Fire Blight: History, Biology, and Management

CHAPTER 6

Growth and Physiology of Erwinia amylovora Morphology and Cultural Characteristics

reported as gram-negative, short rods with rounded ends, which are motile by means of many peritrichous flagella.

In 1927, Bryan published the earliest good photographs, clearly showing the peritrichous flagella of the bacterium (Figure 12A). In Arkansas, Rosen (1926), however, disagreed, after mistakenly finding only a single polar flagellum, and believed that the organism should have been placed in the genus Phytomonas. Several years later, Rosen (1938) showed that bacteria derived from exudate were enveloped in slimy capsules of a nonproteinaceous, stainless material, which was surrounded by an extreme outer layer.

Since the first brief descriptions of the morphological characters of Micrococcus amylovorus by Burrill in 1882 and 1883 (Chapter 1), several investigators have studied the fire blight bacterium in more detail. The morphological characters of Erwinia amylovora are summarized in Table 4. The wild- or normal-type cell ranges from 0.6 to 2.5 µm in length and from 0.5 to 1.2 µm in width (average 1.1–1.6 × 0.6–0.9 µm). These cells have usually been

Figure 12. Bacterial cells of Erwinia amylovora. A, Cells isolated from pear, apple, and hawthorn and stained with Casares-Gil’s flagella stain. B, Single virulent cell with abundant peritrichous flagella (18,000×). C, Scanning electron micrograph of wild-type virulent cells (19,500×). (A reprinted from Bryan, 1927; B and C reprinted, by permission, from P. Y. Huang, 1969)

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84 • Chapter 6 In Great Britain, Billing (1960) examined 150 isolates of E. amylovora and distinguished between the “typical” dominant type of colony, displaying characteristic markings on Yeastrel peptone agar plates incubated at 30ºC (86ºF), and “atypical” colonies, which were relatively featureless and less opaque. Phase-contrast microscopy with the use of India ink demonstrated that the cells of typical strains had small capsules, some atypical strains had none, and the remainder had capsules of varying proportions.

At the University of Missouri, Huang and Goodman studied the morphology of E. amylovora in great detail (P. Y. Huang, 1969, 1974; P. Y. Huang and Goodman 1970, 1971).

They used electron microscopy to show the characteristic rounded ends and numerous peritrichous flagella of the cells (Figure 12B and C). Ultrathin sections of wild-type, virulent E. amylovora cells, prepared at room temperature, revealed two separate electron-dense layers in their walls (Figure 13A–C). When cells were fixed at 4ºC (39ºF), three layers were visible. Silva and Sousa (1973) found that these cell walls were almost indistinguishable when uranyl acetate and calcium were omitted in their procedure.

In addition to the small, wild-type cells, E. amylovora also produces long, filamentous cells. In Missouri, Voros and Goodman (1965) reported that the filamentous cells were 7.0–

Growth and Physiology of Erwinia amylovora • 85 35.0 µm long, with a width similar to that of the wild-type cell.

Intermediate cells were 2.0–7.0 µm long and were reported to be nonmotile. Also, the filamentous cells were as virulent and phage-sensitive as the wild-type cells, whereas many produced minicells (Figure 13E) (P. Y. Huang and Goodman,

1970). Huang (1969) reported the size of the minicells as 0.3– 0.8 µm in diameter, but serial sections showed no evidence of any nuclear material. Both types of cells divided similarly, with cell division preceded by the invagination of cell membranes and then cell wall constriction (Figure 13B and D).

Figure 13. Ultrastructure of Erwinia amylovora. A, Single virulent cell (54,000×). B, Early stage of cell division (55,900×). C, Enlarged part of cell wall in B, showing double-track structure (118,250×). D, Advanced stage of cell division, with cell membranes of daughter cells entirely formed before the completion of cell wall constriction (57,000×). E, Filamentous cell with minicell attached (36,000×). (Reprinted, by permission, from P. Y. Huang and Goodman, 1970, 1971)

86 • Chapter 6 In Canada, Gibbins et al. (1976) made a study of the ultrastructure of the cell envelope of E. amylovora strain NCPPB595, using the freeze-fracture technique. This technique exposed the outer regions of the cell envelope at the level of the plasma membrane (Figure 14A). Thus, they were able to expose four planes in this region, and concluded that the plasma membrane at the site of the fracture must be devoid of included particles. In France, Laurent et al. (1987) observed modifications of the cell ultrastructure, following preparation of E. amylovora strains on two different culture media.

Besides the wild- and filamentous-type cells, some cells of E. amylovora are avirulent and thus can form avirulent isolates. They usually appear rather rough in culture and are often referred to as “rough” isolates (Ark, 1937; Goodman et al., 1962). In California, Ark (1934a, 1934b, 1937) observed the dissociation of E. amylovora in culture from the normal, smooth (S) colony to the rough (R) forms upon aging of the cultures. He found the R type avirulent on some susceptible shrubs and only slightly virulent on green pear fruit and succulent tips of pear seedlings. P. Y. Huang (1974) reported only a few well-separated craters on the surface of

Figure 14. A, Freeze-fractured exponential phase cell of Erwinia amylovora NCPPB595: a, convex surface exposed by probable cleavage of outer membrane; b, outer surface of cytoplasmic membrane (particulate); c, outer surface of cytoplasmic membrane (nonparticulate); d, surface revealed by cleavage of cytoplasmic membrane; arrow indicates direction of metal deposition; scale bar = 0.1 nm. B, Colony of avirulent E. amylovora on crystal violet medium, with only a few craters. C, Single avirulent cell of E. amylovora with few peritrichous flagella (18,000×). D, Plaque morphology of phage PEal halo and nonhalo (arrow) after 48 h of incubation at 27ºC (81ºF) with E. amylovora 110; scale bar = 1.0 cm. E, Cell of E. amylovora parasitized by Bdellovibrio bacteriovorus (26,200×). (A courtesy L. N. Gibbins; B and C reprinted, by permission, from P. Y. Huang, 1974; D reprinted, by permission, from Ritchie and Klos, 1977; E reprinted, by permission, from Stolp and Starr, 1963)

Growth and Physiology of Erwinia amylovora • 87 colonies of an avirulent strain of E. amylovora (Figure 14B), in contrast to the colonies produced by virulent strains. Single cells of an avirulent strain had only a few peritrichous flagella (Figure 14C), compared to cells of a virulent strain.

In their external morphology and ultrastructure, Huang observed no remarkable differences between the two types except for the precocious flagellar development of the virulent strain. After 26 to 48 h of incubation, the avirulent strain formed small, butyraceous colonies with dark red centers, whereas virulent strains formed fluidal white colonies with small, bright pink centers.

When E. amylovora is isolated from blighted host tissue, nearly pure cultures may be obtained when the surface has been properly sterilized and laboratory conditions are aseptic (Keil and van der Zwet, 1972a). Bacterial growth from small sections of host tissue is usually a characteristic smooth, creamy white (Figure 15A). When this growth is streaked on standard culture media, single colonies should be small, round, and white, with a typical glistening shine (Figure 15B).

In California, Miller and Schroth (1970, 1972) developed a selective medium for isolating E. amylovora and described the colony morphology as having characteristic dark orange centers, smooth peripheries, and translucent margins (Figure 15D and Plate 111).

Goldberg and Morgan (1954) observed lumps in the cytoplasm of E. amylovora cells treated with noninhibitory concentrations of streptomycin. These lumps were thought to be any solid constituent of the cytoplasm, including nuclei, lipids, or chromatin material.

In Missouri, Crosse and Goodman (1973) observed characteristic craters when colonies grown on a high-sucrose medium were examined under oblique light at maximum 30× magnification. Later, P. Y. Huang (1974) reported more numerous craters, especially in colonies of virulent isolates (Figure 15C), and observed that several of these craters fused and formed irregular bowl-shaped depressions.

In Canada, Dueck and Quamme (1973) reported that E. amylovora colonies on a high-sucrose medium appeared convex and “uniquely striated” when viewed with transmitted incandescent light after 48–72 h of incubation at 28ºC (82.5ºF). Some of the colonies from pear were described as being more cone-shaped than convex, having darker striations in the center, and having no striations at the margins (Figure 12E). They observed the cratered appearance only in very young bacterial colonies.

In California, Moore and Hildebrand (1966) subjected bacterial cells from ooze to electron microscopy and found that they were essentially nonvacuolated, whereas vacuoles were common in cultured cells. They suggested that dif-

Figure 15. Colony characteristics of virulent isolates of Erwinia amylovora. A, Growth surrounding sections of infected pear shoots plated on nutrient yeast dextrose agar (NYDA). B, Small, round, white, glistening colonies of E. amylovora on NYDA, isolated from an oozing pear canker. C, Single colony on crystal violet medium, with numerous characteristic craters on the surface. D, Smooth colonies with dark orange centers and translucent margins on selective medium. E, Colony isolated from pear, with dark striations in the center and a smooth margin. (C reprinted, by permission, from P. Y. Huang, 1974; D reprinted, by permission, from Miller and Schroth, 1972; E reprinted, by permission, from Dueck and Quamme, 1973)

Fruit Blight (apple)

26. Advanced blight of a Jonathan apple, with a reddish margin along the necrotic area.

28. Infected Red Rome apples with advanced coloration, following delayed blossom infection in Maryland.

30. Profuse ooze production on a young Jonathan apple in Michigan. (Courtesy of A. L. Jones, Michigan State University, East Lansing)

27. Advanced blight of a Granny Smith apple, with a green margin along the necrotic area.

29. Bacterial tendrils forming from lenticels of a Rome Beauty apple in Utah.

31. Natural infection of immature Idared apples in Yugoslavia. (Courtesy of V. Gavrilovic, Institute for Plant Protection, Belgrade, Yugoslavia)

Fruit Blight (pear)

32. Early fruit infection, resulting in mum足 mified fruit.

33. Severe oozing on a Bartlett pear, following hail damage in Maryland.

34. Advanced blight of Moonglow pears, with green rings around the necrotic areas.

35. Profuse ooze production on a Stark足 rimson pear, following hail in Michigan.

(Courtesy of M. Danilovich, Michigan State University Extension Service, Hart)

37. Branch of Starkrimson pear with blighted fruit (left) and apparently healthy fruit (right).

36. Advanced blight of DeVoe pear in Maryland, with purple discoloration.

Leaf Blight

38. Spread of blight infection from the petiole into the leaf midrib and shoot, following injury caused by simulated hail.

39. Spread of blight infection through the leaf midrib and into side veins.

40. Advanced leaf blight of Rome apple in Pennsylvania.

41. Pyracantha leaves with angular lesions following invasion from the stem. (Courtesy of E. Billing, East Malling Research Station, East Malling, Kent, U.K.)

42. Necrosis of the midrib of an apple leaf. (Courtesy of Institut National de la Recherche Agronomique, Angers, France)

Limb and Trunk Blight

44. Flies visiting the sticky, moist surface of a canker. (Courtesy of S. V. Beer, Cornell University, Ithaca, N.Y.)

43. Streaking of brown ooze on the central leader of a pear tree.

45. Early sign of bacterial ooze on the trunk of an apple tree.

46. Bacterial ooze on the bark of a pear tree.

47. Trunk blight at the base of a Paulared apple tree, spreading into attached root suckers.