EFFECTS OF AMBROSIA BEETLE ATTACK ON CERCIS CANADENSIS1, 2 - Brill
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IAWA Journal, Vol. 23 (2), 2002: 143–160
EFFECTS OF AMBROSIA BEETLE ATTACK ON CERCIS CANADENSIS1, 2
by
Roland R. Dute 3, 8, Michael E. Miller 3, 4, Micheal A. Davis 5 ,
Floyd M. Woods 6 & Kathy S. McLean 7
SUMMARY
Damage caused to Cercis canadensis by the Asian ambrosia beetle and
its associated micro-organisms was investigated as was host response
to infestation. Various micro-organisms were connected with beetle in-
festation but only filamentous fungi exhibited extensive growth. In par-
ticular, species of Ambrosiella were associated with beetle tunnels. Fun-
gal hyphae infected all cell types by growing directly through cell walls,
by penetrating pit membranes, and by traversing perforations. Coloni-
zation of parenchyma was intensive and these cells probably provided
the nutrients for continued hyphal growth. Host cell response to dam-
age included breakdown of the protective layer of some parenchyma
cells and accumulation of polysaccharide gels within vessel members.
Measurements showed no significant difference in ethylene production
by wood samples from infected versus uninfected trees. However, es-
tablished literature indicates that damage-induced ethylene production
was responsible for initiating events that caused vascular blockage by
carbohydrate gel.
Key words: Ambrosia beetle, Ambrosiella, Cercis, wood ultrastructure,
Xylosandrus.
INTRODUCTION
Xylosandrus crassiusculus (Motschulsky), or the Asian ambrosia beetle, is native to
the Old World Tropics (Atkinson et al. 1988). It was first discovered in the continental
United States in 1974 infesting peach trees in South Carolina (Anderson 1974; Bambara
et al. 1998; Ree & Hunter 1995) and has since spread throughout the Southeast from
1) Dedicated to Ray F. Evert upon his retirement.
2) Supported by the Alabama Agricultural Experiment Station, Auburn University, Auburn, AL
36849, U.S.A.
3) Department of Biological Sciences and Agricultural Experiment Station, Auburn University.
4) AU Research Instrumentation Facility, Advanced Microscopy and Imaging Laboratory, Auburn
University.
5) Department of Biological Sciences, University of Southern Mississippi, Hattiesburg, MS, U.S.A.
6) Horticulture Department and Agricultural Experiment Station, Auburn University.
7) Department of Entomology and Plant Pathology and Agricultural Experiment Station, Auburn
University.
8) To whom correspondence should be addressed.
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Maryland to Eastern Texas (Ree & Hunter 1995). In Alabama, the beetle is especially
prevalent in eastern and central counties (Ree & Hunter 1995). Although the number
of tree species attacked is very large, this organism can pose a special problem for
growers of ornamentals and fruit trees throughout the Southeast (Ree & Hunter 1995;
Ree, pers. comm.).
The life cycle of these beetles is similar to that of other ambrosia beetles (Faulds
1977; Roeper & French 1981; Kovach & Gorsuch 1988; Sreedharan et al. 1991; Mesh-
ram et al. 1993). In spring, females tunnel through the bark and into the sapwood of
either healthy or stressed trees (Bambara et al. 1998). As the insects tunnel, they leave
behind toothpick-like cylinders of sawdust which protrude from the trunk of the in-
fested tree (Davis & Dute 1995). Eggs, larvae and pupae are found together in the
same tunnels (Davis & Dute 1995; Bambara et al. 1998). Newly matured females
mate with their male broodmates (who are smaller and do not fly) and then emerge
from the stem to attack another host. Some investigators believe that the same fe-
males can infect more than one tree throughout the summer, whereas other inves-
tigators indicate that two generations may be involved (Bambara et al. 1998). In
Alabama, infestations are reported to occur during September and October as well as
during the spring (Ree & Hunter 1995).
Different species of ambrosia beetles (including X. crassiusculus) maintain an
ectosymbiotic relationship with ambrosia fungi (often of the genus Ambrosiella)
(Roeper & French 1981; Atkinson et al. 1988; Kinuura 1995). The insects possess a
structure known as a mycetangium (Kovach & Gorsuch 1988) or mycangium (Roeper
& French 1981) within which they carry the fungal inoculum. As beetles travel through
galleries, tunnel walls are smeared with this inoculum which proliferates and in turn
provides a food source for beetles and their larvae (Atkinson et al. 1988; Anonymous
1994). Most authors believe that the insects do not feed on the wood of the host, but
Kessler (1974) considers that dead and dying wood cells, caused by fungal growth,
are necessary for larval development. Kovach and Gorsuch (1988) believe that auxil-
iary fungi other than Ambrosiella might serve as a secondary food source.
The typical symptom of beetle infestation of a tree is wilted foliage (Atkinson
et al. 1988; Bambara et al. 1998). Wilting is followed by death of affected stems;
heavy infestation can lead to death of the tree. The mechanism of this wilt is unclear
due to lack of detailed anatomical studies of fungal growth within the host and of host
response to invasion. Faulds (1977) showed that beetle tunnels themselves would not
induce wilting, and Kovach and Gorsuch (1988) have made a good case for the non-
involvement of resident ambrosia fungi. Rather, some authors implicate auxiliary fungi,
such as Fusarium, which are introduced by the beetle along with the ambrosia fungi
(e.g. Kessler 1974).
Faulds (1977) provides the following reasoned discussion of how auxiliary fungi
might induce wilting. Wilting involves blockage of the transpiration stream to the
leaves. What mechanisms could produce interruption of flow within xylem vessels?
Faulds lists the possibilities as gums, tyloses, mycelial strands, or host cell debris.
Jutte (1977), for example, observed tyloses, fungal hyphae and spores as well as gums
to be associated with non-conducting vessels from oak wood infected with oak wilt
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fungus. In addition to these possibilities, wilt fungi in tomato are known to produce
mucilaginous polysaccharides which could occlude vessels (Moore-Landecker 1996).
The objectives of this study were to identify potential fungal pathogens and to
provide an account of fungal growth within the host and the host’s response to infec-
tion, both anatomically and physiologically. Analysis of infection at the anatomical
level was undertaken to clarify the following: a) pathway of infection within the host
tissue, b) interaction of hyphae and wood parenchyma cells (the probable food source
for the pathogen), and c) host response to fungi in the form of gums and/or tyloses.
Analysis of physiology involved the correlation of infection with ethylene produc-
tion.
MATERIAL AND METHODS
Specimens for anatomical studies were collected from Lone Oak Nursery in Hogans-
ville, Georgia, U.S.A., in the spring of 1995, 1999, and 2000. A total of four infested,
young trees and three controls were cut for this project. Specimens for ethylene meas-
urements were collected in the spring of 2000 and for fungal identification in the
spring of 2001.
For transmission electron microscopy (TEM), portions of branches or small trunks
(35– 40 mm diameter) of redbud, Cercis canadensis L., both infected and uninfected
(control), were collected, kept moist, and returned to the laboratory. Slivers of wood
were taken from sapwood of both control and infected specimens in the laboratory.
Some slivers were collected directly from the sites of beetle tunnels, whereas others
were collected from sites 1 to 2 cm vertically removed from a tunnel.
Slivers were briefly floated in 0.1 M sodium cacodylate buffer (pH 7.2) and diced
into small segments which were then placed into vials of cold, one-half strength
Karnovsky’s fixative (Karnovsky 1965) in 0.1 M cacodylate buffer and evacuated for
approximately 15 minutes. Afterward, the fixative was replaced with fresh fluid and
the vials kept at 4 °C overnight. Following this, segments were thoroughly washed in
buffer and postfixed in 1% buffered OsO4 at room temperature for four hours. Dehy-
dration of segments in an ethanol /propylene oxide series was followed by embed-
ment in Spurr’s resin (Spurr 1969). Silver sections were cut with a Leica Ultracut T
and stained with uranyl acetate and lead citrate. Sections were observed with a Zeiss
EM 10 transmission electron microscope operated at 60 kV.
Material for light microscopy (LM) was fixed according to methods used by Dute
et al. (1999). Briefly, segments were fixed in cold 3% glutaraldehyde in 0.05 M potas-
sium phosphate buffer (pH 6.8) overnight. After buffer washes and dehydration to
95% ethanol, material was infiltrated and embedded in JB-4 plastic (Polysciences
Inc., Warrington, PA). Sections of 6 µm were cut with a JB-4 microtome and heat-
fixed to glass slides. Some sections were stained using the periodic acid /Schiff’s
Reaction (PAS) followed by aniline blue-black (ABB). The former stain combination
stains many carbohydrates, including starch, glycogen, and some wall constituents;
the latter substance stains proteins. Other sections were treated with 1% toluidine
blue O (TBO), a metachromatic stain.
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via free accessTable 1. Percent frequency of mycofloral recovery from healthy and ambrosia beetle infested Cercis canadensis trees.
146
Tree* Seg- Ambrosi- A. sp. 2 Fusarium F. oxy- F. laterium Pestalotia Phoma Macropho- Phomop- Penicillium Nigospora Aspergil- Tricoderma
ment ella sp. 1 solani sporum sp. cercidicola ma cercis sis sp. spp.** sp. lus niger harzianum
CK 1 0.00 0.00 0.00 0.00 0.00 0.00 20.00 0.00 0.00 0.00 0.00 0.00 0.00
CK 2 0.00 0.00 0.00 0.00 0.00 6.66 13.33 0.00 6.66 0.00 8.66 0.00 0.00
CK 3 0.00 0.00 0.00 0.00 0.00 0.00 5.83 0.00 5.83 0.00 0.00 0.00 0.00
CK 4 0.00 0.00 0.00 0.00 0.00 0.00 6.25 0.00 6.25 18.75 0.00 0.00 0.00
CK 5 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
I1 1 62.50 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
I1 2 10.50 10.50 0.00 0.00 0.00 0.00 5.26 0.00 0.00 5.26 6.25 0.00 0.00
I1 3 43.75 6.25 0.00 0.00 0.00 18.75 0.00 0.00 0.00 29.42 0.00 0.00 0.00
I1 4 35.29 5.88 0.00 0.00 0.00 0.00 0.00 0.00 14.28 7.14 0.00 0.00 0.00
I1 5 0.00 7.14 0.00 0.00 0.00 14.28 0.00 0.00 0.00 0.00 0.00 14.26 0.00
I2 1 0.00 6.25 0.00 0.00 0.00 16.66 0.00 0.00 0.00 0.00 8.33 8.33 0.00
I2 2 0.00 0.00 0.00 0.00 0.00 12.50 0.00 0.00 6.25 12.5 0.00 6.25 0.00
I2 3 0.00 0.00 0.00 0.00 41.17 5.88 0.00 0.00 17.64 0.00 0.00 0.00 0.00
I2 4 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 8.33 0.00
I2 5 20.00 0.00 0.00 0.00 13.33 0.00 0.00 0.00 33.33 0.00 0.00 0.00 0.00
I3 1 33.33 0.00 0.00 0.00 0.00 0.00 8.33 0.00 0.00 0.00 0.00 8.33 0.00
I3 2 0.00 18.75 0.00 0.00 0.00 56.26 0.00 0.00 0.00 0.00 0.00 0.00 6.25
I3 3 41.66 0.00 0.00 0.00 0.00 8.33 8.33 0.00 0.00 16.66 0.00 0.00 0.00
I3 4 15.78 5.26 0.00 0.00 0.00 43.38 0.00 0.00 0.00 5.26 0.00 15.78 0.00
I3 5 33.33 0.00 0.00 0.00 0.00 0.00 8.33 0.00 8.33 0.00 0.00 8.33 0.00
I4 1 22.22 0.00 0.00 0.00 0.00 11.11 0.00 5.55 0.00 16.66 0.00 0.00 0.00
I4 2 14.28 0.00 0.00 0.00 0.00 28.57 0.00 19.04 0.00 0.00 0.00 0.00 0.00
I4 3 15.78 0.00 0.00 0.00 0.00 21.05 5.26 0.00 0.00 0.00 0.00 0.00 0.00
I4 4 0.00 0.00 4.34 0.00 0.00 12.50 0.00 0.00 0.00 4.16 0.00 0.00 0.00
I4 5 0.00 0.00 17.65 23.52 0.00 0.00 0.00 0.00 0.00 11.76 0.00 11.76 0.00
* CK = healthy control tree; I = trees infested with the ambrosia beetles. — ** Penicillium spp. included P. lilacinum series, P. janthinellum series, and P. oxalicum series.
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Blocks were collected for ethylene determination from three sites per tree from
three uninfected and three heavily wilted individuals. The sites were 1) 0.5 m above
the base of the trunk, 2) 1.0 m above the trunk’s base, and 3) from a randomly se-
lected branch with basal diameter of 1 cm at a point 0.5 m below the branch tip. The
wood from each site was divided into three blocks (total = 27 blocks /treatment) each
of which was put into a separate glass vial with a tightly fitting cap for transport to
Auburn.
Ethylene production was measured by enclosing approximately 200 mg fresh weight
of redbud material in a 22 ml screw-capped vial fitted with a rubber serum stopper
for 24 hours. Internal ethylene produced was measured by gas chromatography (GC).
A 1 ml gas sample was withdrawn from the headspace of the vial with a syringe and
injected into a Varian Model 3400 Gas Chromatograph GC. Ethylene was separated
on a Porepak-Q column (76 cm × 1.6) at 70 °C with helium as the carrier gas and in-
jector and detector temperature set a 150 °C and 250 °C respectively. The concentra-
tion was measured with a flame ionization detector (FID) and rates of ethylene pro-
duced expressed as µl C2 H4 Kg tissue fresh weight -1 h -1.
Fungal identification was made using tree trunks approximately 2.5 cm wide from
trees infested with ambrosia beetles and showing severe wilt. Starting about 1 m high
and moving downward, the trunks were cut into five, 15 cm segments with a hand
saw. The saw was sterilized with 95% ethanol between cuts. The trunk segments were
sealed in plastic bags, kept on ice, and transported to Auburn. Segments were taken
from four infested trees of Cercis canadensis. Segments from an uninfested tree were
provided as controls.
In the laboratory, a thin cross-sectional wafer of wood (1–2 mm thick) was re-
moved from each infested C. canadensis trunk segment at a distance 1 cm above or
below an ambrosia beetle tunnel. Wafers were also removed from each trunk segment
of the control. Each wafer was subdivided into 10 to 25 one-half cm square tissue
sections which were surface sterilized in 95% ethanol for 10 seconds followed by 1%
sodium hypochlorite (NaOCl) for 1 minute. Tissue sections were then aseptically
placed on potato dextrose agar (PDA). Cultures were incubated at 24 °C for 5 to 7
days, during which time fungi growing from the tissues were identified or subcultured
for later identification. Data are presented as percent frequency of recovery of myco-
flora from the tissue sections of a wafer from a given trunk segment (Table 1).
RESULTS
Micro-organisms associated with beetle infection
Figure 1 is an example of a female Xylosandrus crassiusculus. According to inves-
tigators (Atkinson et al. 1988; Bambara et al. 1998) the adult females of this species
are responsible for excavating tunnels into a woody host and introducing pathogenic
micro-organisms.
A diverse set of micro-organisms was associated with beetle tunnels including
bacteria, yeasts, and filamentous fungi (molds). Bacteria were located only in those
cells directly exposed by tunneling activity (Fig. 2) and routinely occurred in clusters,
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Fig. 1. Adult female Xylosandrus cras-
siusculus. — Scale bar = 0.5 mm.
Key to labeling of all figures: A = axial parenchyma cells; B = pit border; D = dictyosome; ER
= endoplasmic reticulum; G = glycogen; H = hypha; L = lipid body; LP = leucoplast; M = pit
membrane; MB = microbody; MT = mitochondrion; N = nucleus; R = ray (cells); S = septal
pore; SH = fungal sheath; SW = secondary wall; V = vacuole; VE = vessel; W = Woronin
body. TEM figures are noted; other figures are LM.
Fig. 2–5 (3 & 4 TEM). Micro-organisms in infected
wood. – 2: Vessel member lumen plugged with micro-
organisms. The vessel member is exposed to the tun-
nel in the direction of the arrow. TLS. Scale bar = 10
µm. – 3: Bacteria with capsular material (arrows). Note
attachments to host cell wall, fungi, and other bacteria.
Scale bar = 1 µm. – 4: Bacteria undergoing fission.
Arrow indicates a developing septum. Scale bar = 0.5
µm. – 5: Yeast cells in vessel lumen exposed during
tunneling. Arrow denotes bud. TLS. Scale bar = 10 µm.
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probably as a result of capsule material surrounding the cells (Fig. 3). This extracel-
lular substance allowed bacteria to adhere not only to one another, but also to the
walls of fungal hyphae and to the walls of host cells. Metabolic activity of the bacte-
ria was indicated by the presence of septa indicating fission (Fig. 4, arrow).
Yeasts, like bacteria, were located only in those cells whose interiors had been
exposed by tunneling activity. Yeasts were easily identified by the light microscope as
they underwent budding (Fig. 5, arrow).
Filamentous fungi represented the most conspicuous form of infection. Branched
hyphae, represented by filaments of different diameter ramified throughout the wood.
Although most common on the inner surface of the beetle tunnel (where they often
occluded vessel lumens – Fig. 6), they were still observed 2 cm vertically from the
passage (greater distances were not investigated). The hyphal mass was represented
by both living and dead filaments. Walls of the former stained pink with PAS, whereas
the latter were brown. Fungal reproductive structures (conidiophores) could be ob-
served within the tunnel (Fig. 7). In some instances, the conidia formed were so nu-
merous as to plug vessels.
Hyphal ultrastructure was consistent among the filaments observed (Fig. 8). Septa
with simple pores were associated with Woronin bodies. PAS-stained granules in the
cytoplasm (seen with light microscopy) corresponded to clusters of glycogen gran-
ules. Fungal cells contained variable numbers of lipid bodies. Vacuolation was also
variable with some of these organelles possibly acting as lysosomes (Fig. 8). Endoplas-
mic reticulum (ER), nuclei, ribosomes, vesicles, and mitochondria were also present.
A sheath was noticeable external to the cell wall of some hyphae (Fig. 8, 9). When
present, it was often especially well developed where a hypha adjoined the lumen
side of a vessel wall but was also found within other host cell types.
Hyphae entered parenchyma cells, vessel members, and fibers. They did so by three
different pathways. Some hyphae manufactured bore holes directly through the com-
pound middle lamella and secondary walls of adjoining cells (Fig. 10). These “infec-
tion threads” were observed with TEM as well as with light microscopy and were as
narrow as 0.39 µm in diameter. A second method of cell entry was through pit mem-
branes and apertures of associated pit pairs (Fig. 11, 12). As in the formation of infec-
tion threads, a hypha passing through a pit pair exhibited the ability to decrease its
diameter as it grew through a pit aperture (Fig. 11). In the case of a bordered pit, the
hypha sometimes expanded in the pit cavity before narrowing again to penetrate the
pit membrane or pit aperture (Fig. 12). The hypha fit tightly in the space it made
through the pit membrane. Finally, hyphae passed from one vessel member to another
through perforation plates. The different pathways were not exclusive to different
hyphae as branches from the same hypha were found to use different pathways to
enter neighboring cells.
Identification of fungi associated with ambrosia beetle infestations is provided in
Table 1. Relative to the controls, infested wood of Cercis canadensis showed the
presence of one species of Ambrosiella van Arx & Hennebert emend. Batra in some
samples from all four trees and a second species of this same genus in certain samples
of three of the four trees. Only two trees contained any recoverable Fusarium species
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Fig. 6–9 (8 & 9 TEM). Fungal hyphae in infected wood. – 6: Hyphal plug in vessel. To the
immediate right is the beetle’s tunnel. TLS. Scale bar = 10 µm. – 7: Fungal reproductive
structures in tunnel cavity. Scale bar = 10 µm. – 8: Sectional view of hyphae showing cyto-
plasmic contents. The vacuoles contain electron-opaque inclusions and might act as lysosomes.
Scale bar = 1 µm. – 9: Hypha attached to inner surface of vessel lumen via sheath. Scale bar =
1 µm.
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Fig. 10–12 (11 & 12 TEM). Pathway of
hyphal growth. – 10: Pathway of hyphae
from cell to cell via fungal bore holes (ar-
rows). TLS. Scale bar = 10 µm. – 11: Hy-
pha penetrating a half-bordered pit pair.
Scale bar = 1 µm. – 12: Fungus traversing
a half-bordered pit pair. Note how the
hypha expands within the pit cavity. Scale
bar = 1 µm.
which included F. solani Mart. Appel & We. emend. Snyd & Hans., F. oxysporum
Schlecht. and F. lateritum Nees. These species are known to cause wilts in various
plants. Other species of fungi common in the infested woods relative to the controls
were a Pestalotia sp., which causes a leaf spot on Cercis canadensis, and the common
secondary invaders Penicillium spp. and Aspergillus niger Tiegh.
Response of wood to infection – Anatomical
All host cell types were penetrated by filamentous fungi, but only parenchyma
cells were living at maturity and therefore killed by infection and /or the act of wound-
ing. Ray cells can serve as an example of the changes that occurred. Rays were both
uni- and multiseriate, and at the level of the light microscope their constituent cells
were highly vacuolate with homogeneous cytoplasm (Fig. 13). Elongate nuclei were
oriented parallel to the cells’ length and contained distinct masses of heterochroma-
tin. The most noticeable feature of these cells was their distinctly pitted secondary
walls (simple pits). At the TEM level the cytoplasmic components were those typical
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Fig. 13–15 (14 & 15 TEM). Components of uninfected ray parenchyma cells. – 13: Uninfected
ray parenchyma cells with distinct simple pits. RLS. Scale bar = 10 µm. – 14: Portion of
uninfected parenchyma cell cytoplasm showing subcellular composition. Scale bar = 1 µm. –
15: Pit region. A protective layer (asterisk) is located within the secondary wall of a paren-
chyma cell adjacent to the lumen of a vessel member. Scale bar = 0.5 µm.
for a parenchyma cell (Fig. 14). Leucoplasts of varied shape were present, some pos-
sessing starch and some containing prolamellar bodies. Cytoplasmic lipid bodies were
common.
Both axial and ray parenchyma cells had a protective layer lining the cell lumen
(Fig. 15). This layer was best developed in those parenchyma cells adjacent to vessel
members, and within those parenchyma cells was thickest on the wall adjacent to the
water-conducting channel.
In infected wood (particularly at the site of the tunnels) it was not uncommon for
all cells of a ray to contain one or more hyphae (Fig. 16). Multiple hyphae within ray
cells were common and sometimes filled the lumen (Fig. 17). The host’s cytoplasm
was very electron opaque, and the various cellular components lost their distinctive
ultrastructure while often becoming vesicular in appearance (Fig. 18). Host cytoplasm
frequently withdrew from the cell wall. Whether these symptoms were the result of
tunnel formation, fungal activities, or a combination of the two was not investigated.
Hyphae were found both within collapsed cytoplasm and between it and the host cell
wall (Fig. 18).
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Fig. 16–18 (17 & 18 TEM). Infected ray cells. – 16: TLS of an infected ray. Hyphae are found
within each ray cell. Scale bar = 10 µm. – 17: TLS of infected ray cell. The cell lumen is
largely occluded with hyphae. Scale bar = 2 µm. – 18: An infected ray cell (TLS) with hyphae
located both without and within the collapsed cytoplasm. Scale bar = 1 µm.
Breakdown of the protective layer of some parenchyma cells in the host was asso-
ciated with the disease complex. The wall layer decomposed into granules which
dispersed throughout the lumen of the parenchyma cell (Fig. 19). These granules also
apparently crossed the pit membranes (Fig. 20) into vessel members where this mate-
rial accumulated along with other debris possibly representing breakdown products
of the fungal hyphae (Fig. 21). This material (as seen with TEM) corresponded in
location to large masses of gums appearing in the vessels during infection (Fig. 22).
As seen with the light microscope these masses impregnated pit membranes and lined
the lumens of associated parenchyma cells (Fig. 23). The mucilaginous material was
at least partly polysaccharide as it gave a slight though distinct response to PAS stain-
ing. Also, location of this substance corresponded to the deep brown staining of rays,
axial parenchyma, and vessel lumens as seen in fresh, infected wood. TEM observa-
tions specifically of these large gum deposits frequently showed a granular substruc-
ture, although fibrils were also noted. Interestingly, at the sites of massive gum accu-
mulation, the protective layer, although of much less electron density than the controls,
remained intact. The granular material either contacted the surface of fungal hyphae
or was kept from doing so by the presence of the fungal sheath (Fig. 24).
Response of wood to infection – Physiological
Ethylene measurements made from infected vs. control stem segments indicated
that ethylene concentration was higher but not significantly so in the latter (p = 0.18).
Twenty-seven infected samples gave a mean of 4.72 µl / kg / h (range = 0.00–23.08),
whereas a similar number of control (uninfected samples) provided a mean of 9.73
µl / kg / h (range = 0.00–38.46).
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DISCUSSION
In this study, fungal identification was made from wood of trees showing severe wilt.
Beetle galleries in the wood were well developed, and it was felt that wafers of wood
taken one cm vertically from the tunnel should contain filamentous fungi associated
with and expanding from the site of original inoculation.
Both infested and control wood segments contained a number of different fungal
species, but Ambrosiella spp., when present, were always associated specifically with
infected tissue. However, a number of infested trunk segments produced no Ambrosiella
upon culturing. These inconsistent results do not necessarily mean that Ambrosiella
was not always present lining the beetle tunnels, rather that it did not always systemi-
cally colonize the host to a distance sufficient to infect the tissue squares of a given
wafer of wood. Fusarium spp., although present, were uncommon, being found only
in some segments of two infested trees. In contrast, similar studies with infested
Lagerstroemia indica (crepe myrtle) wood show a predominance of Fusarium oxy-
sporum over Ambrosiella spp. (unpublished data).
In addition to Ambrosiella and Fusarium, other fungi such as Pestalotia, Penicil-
lium, and Aspergillus were common in some infested host specimens of Cercis. Prob-
ably the species of fungi associated with the tunnels are determined by the propagules
introduced by the beetle, and this might vary from one insect to another. A mixture of
yeasts along with ambrosia fungus cells and other fungi such as Fusarium and Cerato-
cystis has been isolated from tunnels of other species of ambrosia beetles (Batra 1963);
therefore the presence of multiple fungal species in the present study was not sur-
prising, nor was the presence of bacteria. It must be noted, however, that the yeasts
observed by us might represent the yeast-like state of Ambrosiella or of a similar
fungus.
There is considerable controversy regarding the identification of the major infec-
tive agent(s) in ambrosia beetle infestations. Kinuura (1995), in Japan, isolated fungi
from both the mycangia of Xylosandrus crassiusculus and from beetle galleries, and
showed that beetles carried an Ambrosiella sp. almost exclusively during their disper-
sal. Ambrosiella was also localized from infected redbuds in Texas (Ree et al. 1998).
These observations would seem to indicate that this fungus is the pathogen, but this
notion is at odds with other research. Most studies show that infestation of trees by
Fig. 19–24 (19–21, 24 TEM). Degradation of protective layer of parenchyma cells. – 19: An
axial parenchyma cell adjacent to a vessel member in infected wood. Note the breakdown of
the protective layer (asterisk). Scale bar = 1 µm. – 20: A detailed view of a pit pair from the
previous figure. The granular material from the protective layer is present within the pit mem-
brane (arrow). Scale bar = 0.5 µm. – 21: A vessel member filled with granular material from
adjacent parenchyma cells. Scale bar = 2 µm. – 22: XS of infected wood showing a vessel
partially occluded with gum (asterisk) (PAS/ABB staining). Scale bar = 25 µm. – 23: Detailed
view of gums 1) in vessels (asterisks), 2) in pit pairs between vessels (unlabeled arrow), and 3)
between vessels and parenchyma cells (arrowhead). Stained material also lines the lumens of
parenchyma cells. TBO staining. Scale bar = 20 µm. – 24: Granular material largely excluded
from surface of hyphal wall by the sheath. Scale bar = 0.5 µm.
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via free access156 IAWA Journal, Vol. 23 (2), 2002
ambrosia beetles (including X. crassiusculus) is associated with auxiliary fungi, and
it is these fungi which are thought to be responsible for wilting. In particular, Fusarium
spp. are often isolated from diseased wood in trees attacked by X. germanus (Kessler
1974; Anderson & Hoffard 1978; Weber 1982). When introduced into healthy stems,
the Fusarium taken from the vicinity of beetle tunnels proved to be pathogenic (Kessler
1974). Ceratocystis is another fungus associated with ambrosia beetles (Davidson
1979). Sporothrix, a pathogenic fungus associated with the ambrosia beetle Platypus,
was observed to cause wilting and death in a Nothofagus sp. in New Zealand when
inoculated into drill holes (Faulds 1977). In India, X. crassiusculus attacks silver oak
trees on coffee plantations. The fungus, Botryodiplodia theobromae, isolated from
these beetles is thought to cause death of the woody host (Sreedharan et al. 1991). In
1997, Davis and Dute reported preliminary results of culturing fungi found in X. cras-
siusculus-infested wood of Japanese pagoda trees (Sophora japonica), redbud (Cercis
canadensis), and Zelkova serrata. Colonies of Nectria cinnabarina, Phomopsis sp.,
and Fusarium sp. were obtained. Reinfecting host trees with inoculum from Nectria
was unsuccessful. Reinfection with the other two genera was not attempted. Our present
study indicated that, although Fusarium can be an infectious component in some
specimens, it is absent from others. Perhaps wilt is induced by the activities of Am-
brosiella spp. or by the combined activities of the various fungal species introduced
into the tunnel. Of course, we have yet to complete Koch’s postulates by attempting
reinfection with our colonies. Until that is attempted we cannot ascribe the wilting
phenomenon to any species with certainty.
All hyphae observed possessed an ultrastructure common to Ascomycetes and many
Deuteromycetes. This information corresponds with the results of the culture studies
in which all species identified were members of the Deuteromycetes.
Our observation of a sheath surrounding many of the hyphae is of some interest.
Evidence from other studies suggests that the sheath is an integral part of pathogen
function. Highley et al. (1983) hypothesized that decomposition of cellulose by Poria
placenta was aided by the sheath’s ability to transmit and confine the depolymerizing
agent to the surface of the fiber. Ruel & Joseleau (1991) provided experimental evi-
dence showing that the glucan sheath of Phanerochaete chrysosporium not only pro-
vided attachment to the host (Populus wood), but also provided binding sites for the
lignin peroxidases secreted by the hyphae. Breakdown of the sheath would release
these enzymes at the site of attack and provide hydrogen peroxide necessary for en-
zyme action. Such a process might enhance cell wall penetration by the fungi in our
study. Furthermore, once hyphae have entered parenchyma cells, the sheath could be
involved in binding and transport of enzymes involved in utilization of host cell con-
tents. In contrast, the sheath would serve primarily as a means of support /attachment
to the host in empty cells (such as vessel members or fibers). Also, images such as
Figure 24 suggest a protective function, in which the sheath would isolate the hyphal
cytoplasm from possible chemical effects due to host response. The reason for some
hyphae lacking a sheath is unknown, but no special procedures were followed to
preserve and enhance this structure during tissue processing.
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The ability of filamentous fungi in this study to pass from one host cell to another
gave the hyphae access to all types of wood cells. Undoubtedly, parenchyma cells
provided nourishment, whereas vessels, in theory, provided long, vertical unobstructed
pathways for hyphal extension as well as access to numerous paratracheal paren-
chyma cells.
This study indicates that some of the occluding material observed within vessels of
infected wood originated as a breakdown product of the protective layer (PL) of the
surrounding parenchyma cells. This process is not unique to ambrosia beetle infec-
tion, but is common to other trees and results from wounding rather than from fungal
infection per se. Schmitt and Liese (1990) observed reactions to wounding in wood
of Betula pendula and noted that fibrils produced both by parenchyma cell cytoplasm
and the degradation of the PL passed through pit membranes into vessels. An identi-
cal process has been observed in Acacia mangium (Schmitt et al. 1995). A similar
process was found for wounded wood of Fraxinus excelsior (Schmitt et al. 1997)
although no mention was made of PL breakdown. In these instances pit membranes
were modified, allowing extrusion of mucilage material from parenchyma cells into
vessels.
Ultrastructure of secreted material varies from species to species. In B. pendula it
is finely fibrillar (Schmitt & Liese 1990), whereas in A. mangium it is “sometimes
fibrillar” (Schmitt et al. 1995) and in F. excelsior it can be fibrillar or coarsely granu-
lar (Schmitt et al. 1997). In the present study with Cercis, the mucilage is often ob-
served to be finely granular, but vessels of Prunus serrata specimens infested with
ambrosia beetles show finely fibrillar mucilage similar to that of B. pendula (unpub-
lished results). This structural difference might in part reflect a chemical difference,
but it is well known that all such wound-induced mucilages are pectinaceous
(VanderMolen et al. 1977; Weiner & Liese 1995). Pectin composition would explain
the positive response to PAS in the present study. The formation of occluding mate-
rial by the host cells does not preclude a contribution by fungal debris or secretions to
these masses.
Wound responses causing vascular blockages are known to be mediated by ethyl-
ene (Fujino et al. 1983; Morrow & Dute 1999, and literature cited therein). For exam-
ple, Morrow & Dute (1999) noted that wounded rhizomes of Botrychium dissectum,
when incubated in silver thiosulfate (an ethylene inhibitor) did not develop wound
material. The ethylene results from the present investigation are surprising in that we
would have expected ethylene production in infected trees from not only host paren-
chyma cells but also from the fungi (Moore-Landecker 1996). Instead, the amount of
ethylene present in wood blocks from infected trees varied greatly and was somewhat
less, though not significantly so, than the uninfected controls. Since the trees showed
severe wilting at the time of sample collection, much of the ethylene might already
have evolved and ultimately the experimental results may reflect later stages of ethyl-
ene biosynthesis from the branches. Furthermore, the amount of ethylene manufac-
tured would be determined in part by the number of parenchyma cells still alive to
synthesize it.
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via free access158 IAWA Journal, Vol. 23 (2), 2002
As regards the sequence of events, we hypothesize the following scenario. With
beetle infestation and subsequent hyphal growth, ethylene is manufactured by host
parenchyma cells and hyphae. Ethylene stimulates cellulase production by paren-
chyma tissue (Kawase 1979) and leads to breakdown of the PL of parenchyma cells
adjacent to vessels. Wall breakdown could also be mediated by enzymes manufac-
tured and secreted by the hyphae (Moore-Landecker 1996). Particles of the degraded
wall (containing pectins) become hydrated (gel-like) and are extruded through pit
membranes into the lumens of vessel members. The cytoplasmic manufacture of oc-
cluding material must also be stimulated since not all protective layers undergo com-
plete breakdown.
The question remains as to what agent or substance is responsible for wilting of the
host in ambrosia beetle infestations. Faulds (1977) drilled holes into trunks of
Nothofagus fusca to simulate tunnels of the ambrosia beetle Platypus apicalis. Some
trees had their tunnels inoculated with the pathogenic fungus Sporothrix, others with
sterile distilled water. All fungus-inoculated trees wilted and died; controls did not.
Davis and Dute (1997) observed that holes drilled into trunks of Sophora japonica in
order to mimic beetle infestation had no noticeable negative effect on the health of
the trees. It is clear from these studies that symptoms of wilt involved activities of the
fungus. What activities might be involved? A number of processes suggest them-
selves, and they need not be mutually exclusive. One possibility is secretion of wilt-
inducing substances by the hyphae (Moore-Landecker 1996). A second possibility is
blockage of the transpiration stream by large numbers of hyphae. Instances of such
hyphal blockage were observed in Cercis and Prunus in a preliminary study (Davis &
Dute 1995), but complete blockage does not appear in large numbers of vessels. A
third possibility is that mucilaginous occlusions within vessels limit transpiration to
such an extent that wilting occurs. As fungi ramify throughout infected tissue more
mucilaginous material is manufactured and secreted into the vessels than would be
associated with the beetle’s tunnel alone. In other words, hyphal growth extends the
wound response beyond the immediate site of beetle entry. We are presently investi-
gating not only the fungal species associated with hyphal infection, but also how far
the hyphae extend into the wood from individual beetle tunnels. However, Schmitt
and Liese (1990) make an interesting point, stating, “vessel plugging … can … be
induced by an abiotic factor, e.g. the influx of air.” It is known that tyloses do not
form until after air has entered vessel members (Zimmermann 1983). If this fact also
holds true for mucilage (gum) secretion, then water flow in the vessels is already
disrupted prior to polysaccharide deposition. Perhaps air embolisms are spread from
vessel to vessel as hyphae grow.
Rather than disrupt water flow, mucilage is thought by some investigators to oc-
clude vessel members and prevent spread of pathogens (Bonsen & Kucera 1990).
Schmitt et al. (1995) found that wound-induced secretions in vessels of Acacia did
not inhibit subsequent fungal growth; in fact the mucilage was degraded by the hy-
phae. However, we have observed desiccated hyphae within wound-induced material
of Prunus vessels (unpublished observations). Clearly, effectiveness of mucilages as
antimicrobial agents needs investigation.
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