Hotson (1916) noticed that, in fire blight infections in pear trees, some shoots and branches showed a reddish colour in the sapwood and sometimes in the pith. Such branches were not more seriously affected than hundreds of others that did not show this peculiarity. The red colour was evident 12–18 inches beyond where the bark showed discoloration. For disease control, the grower needed to cut beyond the red streak. The streaks were never found on normal branches or shoots, only where there was evidence of blight. The pathogen was isolated from red streaks, but never in the abundance found in diseased bark. No bacteria were found towards the lower limits of the red colour. This was in contrast to affected bark tissue, where the pathogen was found regularly beyond the discoloured tissue. The red streak was found 6 or 8 ft from the tip of a branch, but no bacteria were found there. Nearer the tip of the branch, however, often 2 ft from it, the bacteria were plentiful. Sometimes, in branches of the preceding year’s growth that were over an inch in diameter, the streak could be detected readily. In such cases, the infection could be traced to a small lateral branch, bacteria gaining access to the xylem tubes near the tip and then working their way into the larger branch. The colouration extended beyond the region of the organisms. When tips of healthy branches were cut under water and immediately placed in a bottle of eosin, a 4-oz bottle was emptied within a few hours. To a certain distance, the colouring matter was evenly distributed through the sapwood, wood and pith, but it had travelled further in the sapwood than in the pith. There was practically no trace of it in the phloem tissues of the bark. Thus, liquids might pass readily in the sapwood from the tip of a branch backward, at least when weather was extremely warm and dry. Rosen (1929) cited several early observers who occasionally found the pathogen in the symptomless sapwood. He believed that vascular invasion was far more common than was generally believed, but he did not investigate the problem in detail.
Shaw (1934) inoculated apple shoots 1·0 cm from the tip by pricking growth from a slope culture with a needle. Shoots were examined visually for external signs of disease and, in some cases, microscopically and by culturing the pathogen. Shaw found that the pathogen was commonly present in the xylem and pith at considerable distances towards the basal ends of shoots beyond cork layers. It was not found in the cortex, phloem or cambium at the basal ends. Movement out of the xylem there appeared to occur in only seven out of about 800 shoots on potted plants in the greenhouse. Cortical blight occurred at the base of a leaf petiole towards the basal end of the shoots beyond the cork layer. R. A. Lelliott (personal communication) studied the persistence of E. amylovora in young pear trees growing in soil in a polythene tunnel. Blight that developed, after spraying trees with inoculum, was cut out and the trees then remained symptomless. Three years after inoculation, microscopic examination showed that many bacteria were present deep in the wood. When cultured, they proved to be typical, pathogenic E. amylovora.
Crosse et al. (1972) examined various aspects of the entry of E. amylovora into xylem vessels when leaves on growing apple shoots on young trees in greenhouse experiments were cut to expose veins. During daylight hours, shoots will be transpiring and there will be negative pressure in the xylem vessels, so drops of inoculum applied to the cut ends are likely to be sucked into the vessels. The tension will vary according to leaf age, shoot maturity and the conditions of cultivation of the plant. Tension will tend to be low when soil moisture levels are high and when transpiration rates are low, for example, at night. There was no direct evidence of the depth of penetration of the bacteria but, using stained bacterial suspensions, bacteria were observed in stained xylem vessels up to 3·0 mm below the cut edge of the leaf. It was certain that some penetrated much deeper, possibly as much as 30 mm. Following inoculation, typical blight symptoms often appeared at the shoot tip within 6–12 days. Whether or not the bacteria responsible for these symptoms migrated to the shoot tip in the xylem vessels was not determined. When drops of inoculum were applied to the cut ends of leaves, the drops disappeared within 5 min, except in the case of the youngest unfolded leaves where they persisted longer and some of the inoculum could be lost through spillage. However, in later experiments using cut petioles of L0 leaves and inoculation at 09·00–10·00 h on a sunlit day, droplets were absorbed within 2–4 min (Goodman & White, 1981). Inoculation of the youngest leaves was most likely to give rise to shoot-tip infection. This occurred with 61% of shoots inoculated at L0, 47% at L1 and 11% at L2. Inoculation at L3–L5 did not lead to blight at the shoot tips. To give rise to typical symptoms there, the pathogen must have invaded cortical parenchyma at some stage. The authors claimed in the abstract of their paper that there was evidence that the bacteria migrated in the xylem vessels to the shoot axis (the stem) but their microscopic evidence did not go beyond a petiole whose leaf age was not stated.
In field and greenhouse tests on the fire blight susceptibility of 92 apple cultivars, Aldwinckle & Preczewski (1976) often observed discoloured streaks in the xylem. Grafted trees were trained to a single shoot to ensure uniformity. The shoot tips were inoculated using a needle just above the youngest unfolded leaf, thus favouring cortical parenchyma invasion. Fire blight lesions were measured 6 weeks after inoculation when visible development of blight had ceased. Many individual shoots had longitudinal dark brown streaks in the stele extending further than the visible cortical lesions to a varying extent. Histological studies indicated that the streaks were confined to the primary xylem. Bacteria were seen there and pathogenic E. amylovora were isolated from such tissues. Discoloured streaks were more common in resistant than in susceptible cultivars. The bacteria were presumed to have gained access to the primary xylem vessels of the stem at the time of needle inoculation. This type of damage would not be normal under field conditions, but it was suggested that entry to xylem vessels near the shoot tip might sometimes occur as a result of other types of damage, e.g. during storms, or, in nurseries during budding, grafting or pruning.
Momol et al. (1998), using the same inoculation method as Aldwinckle & Preczewski (1976), investigated the possibility that the pathogen might migrate directly from an infected scion into the rootstock. In one series of experiments where scion shoots were inoculated in July, the pathogen was later detected in the rootstocks of five out of six plants, but no symptoms developed. The migration route was not investigated, but the pathogen had moved through stems up to 3 years old in either the bark or in the sapwood. Their statement that their results ‘supported previous findings of rapid internal movement in woody tissue’ (no reference given) does not seem justified by the evidence presented.
Suhayda & Goodman (1981a) considered fire blight to be a vascular wilt disease and that bacterial multiplication in the xylem vessels was the primary event required for the development of wilting symptoms and subsequent spread of the pathogen in host tissue. One objective of their work was to determine the distribution and rate of movement of virulent and avirulent strains of E. amylovora in apple shoot tissue using 32P-labelled bacteria. Only the results obtained with the virulent strain are considered here. Migration in the petioles was studied by placing a bacterial suspension on the cut petiole surface immediately after excision. Movement of the pathogen in 5-cm-long apple shoots was also examined by immersing the cut stem bases in labelled inoculum. In petioles (as might be expected) some bacteria were rapidly drawn into xylem vessels and moved 5 mm in 20 min. Most of the inoculum remained at the site of inoculation. In the cut shoots, the pathogen was judged to have moved up the stem at a rate of 34 mm h−1. It was concluded that leaf damage that exposes xylem vessels could be an important factor in the infection process under natural conditions. These findings are in line with those of Crosse et al. (1972).
The objectives of another study by Suhayda & Goodman (1981b) were: to determine the tissue in which bacterial proliferation occurred initially, to relate the proliferation to extracellular polysaccharide (EPS) production by virulent strains and to determine whether or not there was a link between EPS production and vascular occlusion. Apple shoot stems were inoculated 5 cm below the shoot apex on the assumption that this would give an equal opportunity for infection to occur in the intercellular spaces of the cortical parenchyma as well as in xylem vessels. That distance suggests a region well below L0 where the cortical parenchyma cells might no longer be expanding rapidly and not be highly susceptible to infection. It is not clear how many shoots were examined nor their degree of maturity; they were described only as young shoots on trees. Using transmission electron microscopy, the pathogen was seen to colonize xylem vessels and, 48 h after inoculation, the vessels were blocked. This coincided with signs of wilt in the inoculated shoots. The authors concluded that the blocking material was largely EPS, that this probably restricted water flow in the vessels and that this was responsible for the wilting. There was no evidence of multiplication of the pathogen in the cortical parenchyma beyond the wound site. The authors concluded that: ‘The site of early proliferation and systemic migration is in mature xylem vessels rather than in the cortex and pith as reported in the earlier literature.’ The weakness of this argument is that entry to xylem vessels was likely at the site of inoculation and the cortical tissue may not have been highly susceptible there. To test their hypothesis, they did not make parallel observations at less mature inoculation sites. Several questions arise. Were their microscopic studies sensitive enough to detect migration in the cortical parenchyma to the shoot tip by very small numbers of bacteria? Does this study justify the dismissal of earlier studies where younger tissue would have been inoculated? Wilting is not the only symptom associated with shoot blight; early signs include ooze production and necrosis as well as wilt, which are associated with colonization of cortical parenchyma, where invasion of xylem vessels may not have occurred.
Goodman & White (1981) wanted to find an explanation for early observations by Huang & Goodman (1976) and others concerning the parenchyma cells surrounding the xylem vessels. Inoculum drops were placed on cut leaf petioles (L0) at 09·00–10·00 h on a sunlit day so that drops were absorbed in 2–4 min. After 24 h, plasmolysis of xylem parenchyma cells was seen using electron microscopy. This was most common in the cells that bordered the xylem vessels containing bacteria. At this time there were no external signs of disease on the petioles. At 48 h, protoplasts of the xylem parenchyma cells were further aggregated and distorted. Diminished turgor in several files of parenchyma cells allowed adjacent xylem vessels to twist and rupture. This released bacteria into what had been intercellular space that had developed from the collapse of the xylem parenchyma and lysigenous cavities developed. There was then rapid growth and movement of the bacteria. The authors suggested that the loss of turgor in the parenchyma cells was the result of plasmolysis induced by EPS production by the pathogen. Visual signs of petiole reddening and necrosis usually appeared 72 h after inoculation. Plasmolysis of xylem parenchyma was also reported by Bachmann (1913). This report showed how the pathogen may escape from xylem vessels in young petiole tissue, but not from vessels near the base of a shoot or in older invaded stems.
In Germany, Bogs et al. (1998) used small apple seedlings in a growth chamber. Such delicate tissue enabled them to detect the pathogen marked with genes for bioluminescence and fluorescence. Leaves were inoculated by cutting off the tips with contaminated scissors, puncturing the petiole with a needle or by applying filter paper discs soaked in inoculum to avoid damage. Bacteria were observed in xylem vessels; they later broke out into intercellular spaces. The pathogen moved from inoculation areas as far as the roots. Entry to leaf tissue occurred at the base of leaf hairs when inoculum was applied without damage. How the results relate to orchard tree infections is uncertain. Unfortunately, this approach can only be used with very young tissue.