SOME PROPERTIES OF THE BACTERIAL WETWOOD (WATERMARK) IN SALIX SACHALINENSIS CAUSED BY ERWINIA SALICIS
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IAWA Journal, Vol. 23 (2), 2002: 179–190
SOME PROPERTIES OF THE BACTERIAL WETWOOD (WATERMARK)
IN SALIX SACHALINENSIS CAUSED BY ERWINIA SALICIS
by
Yasuaki Sakamoto1 & Atsushi Kato 2
SUMMARY
Some properties of bacterial wetwood (watermark) in Salix sachalinensis
caused by Erwinia salicis were investigated. Vessel-ray parenchyma and
interfibre pit membranes were often damaged or absent. Hence, they
could serve as effective pathways for water transport resulting in water
accumulation. Osmotic potentials (φ π) were substantially lower than
those in healthy sapwood. 13 C-NMR spectroscopy revealed that E.
salicis produced levan in the watermark, which is suspected to be a
causal agent of low φ π. These data support the concept that levan pro-
duction decreases φ π, leading to water accumulation.
Key words: Bacterial wetwood, watermark, Erwinia salicis, Salix sach-
alinensis, pit membranes, osmotic potential, 13 C-NMR spectroscopy,
levan.
INTRODUCTION
Watermark disease is a serious wilt disease in willows (Salix spp.) caused by the
bacterium Erwinia salicis Chester 1939 (Young et al. 1996). When affected branches
or trunks were cut, a distinct, watery reddish brown or brownish black stained zone
(watermark) was seen. This formed a circumferential stain in sapwood (A-3 in Fig. 1)
and almost covered the whole transverse section in seriously affected trees (A-1, 2,
and 4 in Fig. 1). The relationship between inhibition of water conduction and water-
mark in affected trees has been discussed in an earlier paper (Sakamoto & Sano 2000).
The watermark had an abnormally high moisture content compared to surrounding
regions (Sakamoto & Sano 2000). This kind of wood tissue is called wetwood (Panshin
& De Zeeuw 1980). Wetwood is responsible for substantial losses of wood and en-
ergy and considerable production expenditures in the forest product industry (e.g.,
Ward & Pong 1980). A mechanism for water accumulation in wetwood has yet to be
elucidated. However, several kinds of bacteria in the xylem tissues are frequently
associated with wetwood (e.g., Hartley et al. 1961; Bauch et al. 1979; Ward & Pong
1980; Ward & Zeikus 1980; Schink et al. 1981a, b; Rishbeth 1982; Murdoch & Cam-
1) Hokkaido Research Center, Forestry and Forest Products Research Institute (FFPRI),
Hitsujigaoka 7, Toyohira-Ku, Sapporo 062-8516, Japan.
2) Forestry and Forest Products Research Institute (FFPRI), Tsukuba Norin P.O. Box 16,
Tsukuba, Ibaraki 305-8687, Japan.
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Fig. 1. Transverse views of the sample woods. – H-1 & 2 = healthy woods. – A-1– 4 = affected
woods. H indicates heartwood; the other stained zone in A-1– 4 is the watermark.
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pana 1983; Schink & Ward 1984; Scott 1984; Warshaw et al. 1985; Lee 1988), and
the term “bacterial wetwood” has been applied in such cases (Murdoch & Campana
1983). Only a few studies regarding pathological interaction between bacteria and
bacterial wetwoods have been performed. Carter (1945) and Seliskar (1950) consid-
ered wetwoods to result from bacterial infection, although their attempts to produce
wetwood by bacterial inoculation was not conclusive (quoted in Knutson 1973).
In the case of watermark disease, successful inoculation tests of E. salicis to pro-
duce watermark in willows (Sakamoto et al. 1999) have satisfied Koch’s postulates.
Hence, water accumulation in watermarked wood is obviously due to pathological
interaction between E. salicis and affected tissues.
Most genera of phytopathogenic bac-
teria, including Erwinia, have been
known to produce mucoid growths when
cultured on high-sucrose media (Krieg
& Holt 1984; Maloy & Murray 2001).
Extracellular polysaccharide (EPS) is the
main component of these growths.
Bacterial EPS’s have been reported to
play important roles in producing dis-
ease symptoms, including water-soak-
ing symptoms (e.g., Leigh 1992; Denny
1995). Erwinia salicis produces copious
amounts of EPS slime (mucoid growth)
when cultured on high-sucrose media
(Fig. 2) (Sakamoto et al. 1999). Chemi-
cal and functional analyses of this EPS Fig. 2. Slime of the EPS (arrows) on a high-
have not been carried out. However, if sucrose medium produced by Erwinia salicis
this mucoid growth is also produced in (after two days of incubation at room tempera-
the watermark, it should play an impor- ture).
tant role in making tissues water-soaked.
In this paper we describe some anatomical, physiological and chemical properties
of watermark in Salix sachalinensis Fr. Schm. as a kind of bacterial wetwood.
MATERIALS AND METHODS
Sample collections
Sampling was performed on Salix sachalinensis trees from the natural forests of
Kamikawa, in the mountainous area of central Hokkaido (the northern island in Ja-
pan) on 27 June 2000. Cylindrical specimens (approximately one metre in length)
were excised from the trunks of four affected trees (Tree no.: A-1– 4) and two healthy
trees (Tree no.: H-1 & 2) (Fig. 1). The average DBH of the trees was 8.1 cm. After
excision, both cut surfaces of the specimens were wrapped with polyvinyl sheets to
prevent dehydration. They were transported to the laboratory and then divided into
small specimens (approximately 30 cm in length).
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Fig. 3. Examples of preparations of samples for moisture content measurement: above = H-1;
below = A-1.
Moisture content
The moisture content (MC) of healthy and affected disks was measured. Sample
preparation and calculation of MC were conducted according to procedures described
by Sakamoto and Sano (2000). In brief, wood strips including pith were removed
from cylindrical specimens and then serially divided into 10 pieces (Fig. 3). MC was
calculated based on the weight of the oven-dried wood.
Scanning electron microscopy
For scanning electron microscopy, five wood blocks (approximately 5 × 5 × 5 mm)
were taken from separate regions of each specimen. They were freeze-dried accord-
ing to the methods described by Sano and Fukazawa (1994). After drying, blocks
were split radially or tangentially with razor blades, and two halves of each specimen
were affixed to stubs with carbon adhesive tape (DTM 9101; JEOL DATUM Ltd.) and
coated with gold using a ion sputter coater (FC-1100; JEOL). Coated samples were
observed with a scanning electron microscope (SEM; JSM-5600LV; JEOL) at 10 or
15 kV.
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Osmotic potential
Osmotic potentials (φπ) of the liquid exuded from the watermarks, from small disks
(approximately 5 mm in diameter, 2 mm in thickness) of the sapwood surrounding
the watermarks and the sapwood of the healthy trees, were measured by a dew-point
microvoltmeter (HR-33T; WESCOR Inc., USA) equipped with a sample chamber
(C-52-SF; WESCOR Inc., USA). The liquid from twenty separate regions in the water-
marks, from eight separate regions in sapwood surrounding the watermarks, and eight
separate regions in sapwood of the healthy trees were measured.
Polysaccharide in watermark
1) Isolation of EPS from bacterial culture
The isolate of E. salicis (No.: ys-43 in Sakamoto et al. 1999), which had been
stored in 10% skimmed milk at -80 °C, was streaked on the high-sucrose medium
developed by Dye (1968) and incubated at 25 °C for 2 days. Crude EPS slime on the
plate medium was collected and dissolved in 50 ml of distilled water. The liquid was
passed through filter paper (ADVANTEC No. 4A; Toyo Roshi Kaisha, Ltd.) and ex-
tracted with ethyl acetate. The water layer was concentrated to 10 ml, and then ap-
proximately 100 ml of ethanol was added. The collected precipitate was washed by
acetone, dissolved in distilled water, freeze-dried, and then collected. Dried weight of
the EPS was measured.
2) Isolation of polysaccharide from watermark
Small specimens (approximately 30 cm in length) of affected and healthy wood
were stored in a refrigerator at -30 °C until used. The samples (10 g fresh weight)
were excised from the watermark (Tree no.: A-1– 4) and healthy sapwood (Tree no.:
H-1 & 2). Water extracts of milled samples were purified by the same procedure as in
the case of the crude EPS described above. Dried weights of the polysaccharides were
measured.
3) 13C-NMR spectroscopy
Samples of the EPS fraction from bacterial culture, the polysaccharide fraction
from watermark (Tree no.: A-2), and healthy sapwood (Tree no.: H-1) were dissolved
in deuterium oxide and subjected to 13 C-NMR spectroscopy using ALPHA-500 (JEOL)
operated at 125 MHz. Commercial levan (β-2,6-D-fructofuranan) (WAKO Pure Chemi-
cal Industries, Ltd.) served as the reference standard.
RESULTS
Moisture content
Moisture contents of healthy versus affected woods are shown in Table 1 & 2.
Within the watermark, the values of MC in each piece differed to some extent. How-
ever, they were generally higher than the values in the surrounding sapwood and the
sapwood in healthy trees, as previously reported (Sakamoto & Sano 2000).
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Table 1. Comparison of moisture contents (%) among samples of the healthy wood and
affected wood.
Piece no.:
Tree no. 1 2 3 4 5 6 7 8 9 10
H–1 81 51 51 126a 124a 132 113a 55 57 86
H–2 101 62 50 101a 124a 127a 82a 59 73 103
A–1 115b 108b 105b 55b 138a,b 150a,b 145b 104b 124b 124b
A–2 134b 82b 78 131a,b 164a,b 168a,b 161b 172b 178b 167b
A–3 121b 108b 73 86a 139a 145a 122a 68 72b 91
A–4 127 158b 181b 183b 193a,b 191a,b 171b 171b 153b 91
a) Pieces containing heartwood — b) Pieces containing watermark.
Table 2. Comparison of average moisture contents (%) among each category.
Average of the pieces in:
H–1—2 Unaffected sapwood 69.1
H–1—2 Unaffected heartwood 116.1
A–1—4 Watermarked sapwood 134.4
A–2—4 Non-watermarked sapwood 88
A–1—2 & A–4 Watermarked heartwood 162.1
Fig. 4a & b. SEM photographs of the vessel-wall surfaces between vessels and ray paren-
chyma cells. Pit membranes are often heavily incrusted or absent. Scale bar for a = 5 µm, for
b = 10 µm.
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Fig. 5a & b. SEM photographs of a complementary pair of surfaces between wood fibres. Pit
membranes are often absent on both faces of pit pairs (arrows). Scale bars = 10 µm.
Scanning electron microscopy
In tissues of the watermark, some of the vessels were plugged with masses of
bacteria. However, plugged vessels were not evenly distributed within the watermark;
some regions had many plugged vessels, while some regions did not. Vessel-ray pa-
renchyma pit membranes were heavily incrusted, damaged, or often absent (Fig. 4).
Figure 5 shows a complementary pair of interfibre surfaces in which the pit mem-
branes are absent.
In contrast to watermarked tissues, appreciable incrustations were rarely observed
in tissues of healthy sapwood. Also, vessel-ray parenchyma and interfibre pit mem-
branes typically remained intact.
Osmotic potential
The results of φπ measurements are shown in Table 3. Substantial decrease in the
mean values of φπ was observed in the watermark (-0.34 MPa in average) compar-
ed to values of surrounding sapwood (-0.06 MPa in average) and healthy sapwood
(-0.04 MPa in average).
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Table 3. Measured osmotic potential (φ π) of the watermark and the surrounding sap-
wood of the affected trees and the sapwood of the healthy trees.
Tree no. Type of tissue φ π (MPa)
H–1 sapwood -0.04a -0.1 – 0
H–2 sapwood -0.04a -0.1 – 0
A–1 watermark -0.37b -1.16 – -0.05
sapwood -0.04a -0.1 – 0
A–2 watermark -0.4b -1.45 – -0.05
sapwood -0.05a -0.05 – 0
A–3 watermark -0.07b -0.1 – -0.05
sapwood -0.04a -0.1 – 0
A–4 watermark -0.53b -1.39 – -0.05
sapwood -0.1a -0.21 – 0
a) Means for 8 replications — b) Means for 20 replications.
Polysaccharide in watermark
The dried weight of the EPS fraction from the bacterial culture was 72.0 mg. The
dried weights of the polysaccharide fraction from watermark and healthy sapwood
are listed in Table 4.
The signals in the 13 C-NMR spectrum of the commercial levan appeared in the
range of 60–110 ppm (arrowheads in Fig. 6a). These signals completely agreed with
signals in the spectrum of the EPS fraction (arrowheads in Fig. 6b). Thus, the EPS
fraction was identified as levan. The spectrum of the polysaccharide fraction of the
watermark (Fig. 6d) showed the same signals as the EPS fraction and commercial
levan, while the spectrum of healthy sapwood (Fig. 6c) did not. This fact obviously
indicated that the watermark contained levan; on the contrary, healthy sapwood did
not.
Table 4. Dried weight (mg) of the polysaccharide fraction from watermarked sapwood
and healthy sapwood.
Watermarked sapwood Healthy sapwood
A–1 40.3 a H–1 31.0 b
A–2 39.1 a H–2 31.3 b
A–3 32.3 a
A–4 36.3 a
a) Means for 3 replications — b) Means for 2 replications.
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a
ppm
110 100 90 80 70
b
ppm
110 100 90 80 70
c
ppm
110 100 90 80 70
d
ppm
110 100 90 80 70
Fig. 6. 13 C-NMR spectroscopy. – a = commercial levan. – b = EPS from the bacterial culture.
– c = polysaccharide from healthy sapwood (H-1). – d = polysaccharide from the watermark
(A-2). – Arrowheads indicate the signals of levan.
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DISCUSSION
This study is the first report dealing with the direct detection of levan production by
phytopathogenic bacteria in affected wood tissues.
A previous report (Sakamoto & Sano 2000) revealed that masses of Erwinia salicis
were only found in watermark and not in surrounding or healthy sapwood of Salix
sachalinensis. Except for levan, 13 C-NMR spectroscopy revealed that both water-
mark and healthy sapwood contained the same kinds of polysaccharides (not identified
in this study). This indicated that the increment of dried weight of the polysaccharide
fraction of the watermark was due to levan, which was produced by E. salicis. The
low φπ in the watermark can be attributed to a large extant to levan, which acts as the
osmotically active material. Furthermore, as was shown in the previous report
(Sakamoto & Sano 2000), masses of E. salicis were often found in the lumina of
tyloses. This indicated that the tyloses were often collapsed and did not block the
vessel lumina from water invasion. The present study revealed that the vessel-ray
parenchyma and interfibre pit membranes were often damaged, and could therefore
serve as effective pathways for the accumulation of water in the watermark. These
data support the concept that levan production decreases φ π, leading to water accu-
mulation through those pathways.
Relatively low φ π has also been found in bacterial wetwood of Ulmus americana
L., and several kinds of dissolved cations and organic acids (such as K, Ca, and acetic
acids) have been suspected to be the causal factors (Murdoch et al. 1987). These sub-
stances in the watermark were not chemically analyzed in this study; however, we
suspect that they are also involved in the low φ π in the watermark.
The “osmotic potential concept” is based on the premise of the existence of a semi-
permeable “membrane” between the watermark and sapwood area (Zimmermann
1983). In many cases it has been reported that wetwoods are surrounded by narrow
dry zones that separate them from sapwood (e.g., Bauch et al. 1975; Coutts & Rish-
beth 1977; Worrall & Parmeter 1982). In the case of some conifer xylems, Coutts
(1977) hypothesized that the dry zones were caused by gas emboli formed in tracheids
as a result of metabolic events in gradually dying ray parenchyma. In addition,
Sakamoto and Sano (2000) showed that some parts of outer layers of the watermark
were also regarded as dry zones. Zimmermann (1983) hypothesized that microorgan-
isms of the wetwood cause deposition of semipermeable materials within the wall
and intercellular spaces, surrounding the wetwood area with a semipermeable mem-
brane. The nature of the dry zone of the watermark has not been studied yet, although
there is a possibility that this zone acts as a semipermeable membrane.
Tables 1, 2 and 3 show significant differences of MC and φ π between the water-
mark and healthy sapwood, although variation of the data within the watermark was
rather large. This fact may reflect the distribution patterns of the masses of E. salicis
in the watermark, which may alter the physiological and chemical properties of each
measured region.
The hemicellulolytic and pectolytic activities of the bacteria might account for the
degradation of pit membranes characteristic of wetwood (Ward & Zeikus 1980). Hemi-
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cellulolytic activity in the initially aerobic atmosphere of inner sapwood might weaken
pit membranes and, perhaps, alter the cell wall matrix, resulting in the absorption and
retention of water (Scott 1984). Schink et al. (1981a, b) demonstrated the destruction
of vessel-ray parenchyma pit membranes in wetwoods by pectin-degrading bacteria.
In the case of watermark, Wong and Preece (1978) reported a marked increase of
cellulase in the affected wood tissues by E. salicis. They also reported the in vitro
production of pectinolytic enzymes by the bacterium. These facts suggested that ana-
tomical changes in vessel-ray parenchyma and interfibre pit membranes were due to
enzymatic activities produced by the bacterium.
This study clarified some properties of the watermark as a kind of bacterial wet-
wood. There is a need to study the processes and mechanisms of water accumulation
in more detail. It also presents relevant information to elucidate properties of bacte-
rial wetwood in other tree species.
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