Publications
Halik, S. and D.R. Bergdahl. 1994. Long-term survival of
Bursaphelenchus xylophilus in living Pinus sylvestris
in an
established plantation. European Journal of Forest Pathology
24:357-363.
Scots pines (Pinus sylvestris) in a 20-year-old plantation
in
northern Vermont, USA, were inoculated with the pinewood nematode
(Bursaphelenchus xylophilus) in 1987. To determine how long
B.
xylophilus would survive after inoculation, the trees were
periodically observed and sampled for the nematode up to the end of
1993. The nematode was still found in living, healthy-appearing
pines 6 years after inoculation.
Bergdahl, D.R. and S. Halik. 1993. Persistence of
Bursaphelenchus
xylophilus in living Pinus sylvestris. Phytopathology
83:242
(Abstr.).
A total of 100, 20-year-old Scots pines (Pinus sylvestris)
were
inoculated with a Scots pine isolate of Bursaphelenchus
xylophilus
(pinewood nematode=PWN) to evaluate persistence of the nematode in
the host tree. Ten trees were inoculated on each of 10 dates
between 6/1 and 9/14, 1987. Two inoculation wounds were made with
a drill bit in the main stem of each tree and approximately 30,000
nematodes were inoculated per wound. In addition, 10 trees were
inoculated on each of 3 dates with a nematode-free solution. All
trees were visually evaluated annually and sampled periodically
between 1987 and 1992. B. xylophilus was extracted from
asymptomatic living trees for up to 5 years after inoculation as
well as from dead trees but not from controls. The PWN was most
frequently extracted from trees inoculated on 7/7 and 9/14,
1987.
Halik, S. and D.R. Bergdahl. 1992. Survival and infectivity of
Bursaphelenchus xylophilus in wood chip-soil mixtures.
Journal of
Nematology 24:495-503.
To determine the effect of soil environment on the life stages and
total numbers of Bursaphelenchus xylophilus,
nematode-infested wood
chips alone and mixed with soil were incubated at 12 and 20 C.
Nematodes were extracted at 2-week intervals for 12 weeks. Numbers
of nematodes and percentage of third-stage dispersal larvae were
greater at 12 C and in chips without soil. Percentage of juveniles
of the propagative cycle was greater at 20 C and in chips with
soil. Although B. xylophilus survived in chips with soil
for 12
weeks, nematode numbers and life stage percentages changed little
over time. To determine if B. xylophilus was capable of
infecting
wounded roots, infested and uninfested chips were mixed with soil
in pots with white and Scots pine seedlings. Trees were maintained
at 20 and 30 C and harvested at mortality or after 12 weeks. Only
seedlings treated with infested chips contained nematodes. In
field experiments, planted seedlings were mulched with infested
chips to determine if nematodes would invade basal stem wounds.
Among these trees, Scots pine was more susceptible than white or
red pines to infection and mortality.
Bergdahl, D.R., S. Halik, J. Tomminen, and H. Akar. 1991.
Frequency of infestation of Monochamus notatus and M.
scutellatus
by Bursaphelenchus xylophilus in Vermont. Phytopathology
81:120
(Abstr.).
Adult Monochamus notatus (Mn) and M. scutellatus (Ms)
were
collected from two locations in central and northern Vermont from
6/4-9/1, 1988. On the day of collection, beetles were identified
to sex, sectioned and placed in distilled water for 24 hours to
extract dauerlarvae of Bursaphelenchus xylophilus (Bx).
There was
no difference in frequency of infestation of the two beetle species
by Bx (Mn=51% and Ms=56%) but infested Ms carried a greater mean
number of dauerlarvae (5450 vs 595). Frequency of infestation was
independent of sex for each beetle species and there was no
difference in mean number of dauerlarvae carried between sexes
within each species of Monochamus. Frequency of infestation
of
male Ms was dependent on time and the mean number of dauerlarvae
carried by male Ms appeared to decrease after the first month.
There was no difference in frequency of infestation or mean number
of dauerlarvae carried for Ms females.
Tomminen, J., S. Halik and D.R. Bergdahl. 1991. Incubation
temperature and time effects on life stages of Bursaphelenchus
xylophilus in wood chips. Journal of Nematology
23:477-484.
Wood chips of Pinus strobus inoculated with Bursaphelenchus
xylophilus were incubated at 3, 12, 30, or 40 C during
intervals of
47, 82, and 130 days to determine the effects of incubation
temperature and time on total number of nematodes and occurrence of
each life stage. Nematodes did not survive at 40 C; the greatest
number of nematodes was maintained at 3 C. The number and
percentage of juveniles in the propagative cycle were greatest at
3 C after 47 days, but the percentage was greatest at 30 C after
130 days. More third-stage dispersal larvae, with percentages as
high as 85%, were extracted at 3 and 12 C than at 30 C by the end
of the study. Dauer larvae were extracted from the chips but
percentages never exceeded 5%. The percentage of adults was
greater at 30 C than at 3 and 12 C after 82 and 130 days. When a
1-week heat treatment of 30 C was applied to samples at 3 and 12 C,
numbers and percentages of adults increased. Percentages of dauer
larvae increased very slightly when the heat treatment was applied
after 47 days.
Halik, S. and D.R. Bergdahl. 1990. Development of
Bursaphelenchus
xylophilus populations in wood chips with different moisture
contents. Journal of Nematology 22:113-118.
Bags of Pinus strobus wood chips with moisture contents of
38, 92,
164, and 217% (oven dry weight) were inoculated with
Bursaphelenchus xylophilus and incubated at 30 C in order to
determine the effect of wood moisture on nematode population
development. Nematodes were extracted after 2, 4, 8, and 12 weeks.
Population levels were greatest in wood chips with a moisture
content of 38% and decreased successively with each higher moisture
content. In chips with the three lower moisture contents,
populations peaked at 2 weeks, but at 217% moisture, they peaked at
8 weeks. By 12 weeks, nematode populations had declined in wood
chips with 92 and 164% moisture contents. The fungi most
frequently isolated from the wood chips were Alternaria,
Fusarium,
Gliocladium, Graphium, Penicillium,
Trichoderma, and Mucorales.
Halik, S. 1990. Survival of Bursaphelenchus xylophilus
in wood
chips in soil and potential for infesting roots of pine seedlings.
M.S. thesis, University of Vermont, Burlington, VT, 64pp.
To determine the effect of soil environment on pinewood nematode
life stages and population level, nematode-infested wood chips
alone and mixed with soil were incubated at 12 and 20 C. Nematodes
were extracted at 2-week intervals for 12 weeks. Nematode
population level was greater in chips without soil, and both
population level and percentage of dispersal larvae were greater at
12 C. Percentage of propagative stage juveniles was greater in
chips with soil. Although nematodes survived for 12 weeks in chips
with soil, neither population level nor proportions of life stages
changed over time.
To determine if the nematode was capable of infesting wounded
roots, infested and uninfested chips were mixed with soil in pots
with white and Scots pine seedlings. Trees were maintained at 20
and 30 C and harvested at mortality or after 12 weeks. Planted
seedlings were mulched with infested chips to determine if
nematodes would invade basal stem wounds. More seedlings treated
with infested chips died and contained nematodes, but there was no
difference between temperature effects. Of planted seedlings,
Scots pine was more susceptible to infestation and mortality than
were white or red pines.
To observe the infection process of the pinewood nematode in root
tissue, sections of roots of white pine seedlings were wounded and
inoculated with nematodes. Inoculated roots were prepared for
scanning electron microscopy and the wound surfaces scanned for
evidence of nematode penetration. To investigate the histological
associations of the pinewood nematode in root tissue, infested
roots of white pine seedlings were sectioned and stained for light
microscopy. Nematodes appeared to enter roots primarily through
cortex and phloem, either intercellularly or via resin canals and
were observed infesting all woody root tissues. Parenchyma cell
contents were granular and stained brown or were completely
destroyed.
Bergdahl, D.R. 1988. Impact of pinewood nematode on North
America: Present and future. Journal of Nematology
20:260-265.
Bursaphelenchus xylophilus, pinewood nematode (PWN), is the
most
serious pest of pine forests in Japan, but in North America its
role in pine wilt disease is still being studied. The PWN is known
to infest many species of Pinus, with P. nigra, P.
sylvestris, and
P. thunbergii the most susceptible in the eastern United
States.
Because of its potential, several European countries (Finland,
Norway, and Sweden) and Korea have established embargoes against
the importation of coniferous wood from regions of the world known
to be infested with the PWN. Although the PWN is not considered an
economic pest in North American forests, the recent embargoes have
established an impact on current forest management practices and an
economic impact on North American export trade.
Tomminen, J., S. Halik, and D.R. Bergdahl. 1988. Dauerlarvae
of
Bursaphelenchus xylophilus formed in wood chips of Pinus
strobus.
Nematologica 34:298 (Abstr.).
Twenty plastic bags each containing 200 g of wood chips of Pinus
strobus L. (eastern white pine) were inoculated with an isolate
of
Bursaphelenchus xylophilus (Steiner & Buhrer 1934) Nickle
1970
(pine wood nematode) from that host. All bags were incubated at 30
C for four weeks after which the bags were divided into four
treatments and incubated as follows: Treatment 1 was incubated an
additional 10 weeks at 30 C. Treatment 2 was incubated an
additional 10 wk at 30 C and another 8 wk at 12 C after which 3
bags were extracted. The remaining two bags were extracted after
one additional week at 30 C. Temperatures for treatment 3 were
gradually reduced during a 3 week period from 30 C to 12 C and then
incubated 7 more weeks. Three bags were then sampled and the
remaining 2 were extracted after an additional 2 days at 30 C.
Incubation temperature for treatment 4 was decreased to 12 C as in
treatment 3 but then incubated 8 wk at that temperature.
Temperature was further reduced to 3 C and after 8 wk three bags
were extracted. The remaining 2 bags were sampled after an
additional week at 30 C. To recover the nematodes the wood chips
were extracted using a modified version of the Baermann funnel
technique. The nematodes were then evaluated to determine the
presence of dauerlarvae. These were recovered only from wood chips
which had been incubated at either 3 or 12 C followed by 1 week of
incubation at 30 C. The two highest percentages of dauerlarvae
were 30 and 21 percent from treatment temperatures of 3 and 12 C,
respectively.
Halik, S. and D.R. Bergdahl. 1987. Infestation of wounded
roots
of Pinus strobus by Bursaphelenchus xylophilus from
contaminated
wood chips in soil. Phytopathology 77:1615 (Abstr.).
White pine (Pinus strobus) wood chips were inoculated with
an
isolate of Bursaphelenchus xylophilus from that host and
incubated
at 30 C for 8 wks. After incubation, the average wood moisture
content (mc) was about 130% based on oven dry weight (ODW) and the
nematode population was about 50/g ODW. Uninfested wood chips
(130% mc) were used as a control. For each treatment,
approximately 150 g of wood chips were mixed with soil in each of
12 one liter pots. Wounds were made at 3 locations on the roots of
24 (12 per treatment) five-year-old white pines by scraping the
bark to expose xylem tissue. Seedlings were potted in these
chip-soil mixtures and maintained in the greenhouse up to 12 wks at
18-29 C. Seven of 12 seedlings treated with nematode-infested
chips
wilted and B. xylophilus was extracted from roots and stems.
Histological studies showed B. xylophilus only in tissues of
inoculated seedlings.
Bergdahl, D.R. and S. Halik. 1987. The pine wood nematode
associated with conifer mortality in the northeastern United
States. Pp. 46-49 in M.J. Wingfield, ed. Pathogenicity of
the pine
wood nematode. St. Paul, MN: American Phytopathological Society
Press.
Bursaphelenchus xylophilus was first found in the
northeastern
United States (Vermont) in dead and dying eastern larch, red pine,
and Scots pine in October 1979 and has since been found
in eastern white pine and in the exotic species
Japanese larch and ponderosa pine. The nematode is usually
recovered in relatively low numbers from dead or dying trees, thus
the primary cause of conifer
mortality in Vermont does not appear to be solely the result of
pine wood nematode infestation. The nematode is believed to be one
important component of a highly integrated biological disease
complex which includes: the nematode, insect vectors
(Monochamus
spp.), insect associates (bark beetles), wood staining fungi
(Ceratocystis spp.), other pathogenic fungi, and
environmental
stress factors such as low soil moisture and high temperature.
Results of seedling inoculations in a growth chamber have shown
that isolates of B. xylophilus from either eastern larch or
red
pine are pathogenic to both hosts. Inoculations of potted and
planted seedlings in the field have caused limited mortality.
Inoculations of larger trees have not resulted in mortality,
however inoculated branches of red and Scots pines have displayed
some mortality. Histological observations of B. xylophilus
in red
pine have shown nematodes abundant in the longitudinal and radial
resin canals of the xylem and only occasionally present in the
bark. In eastern larch, the nematode has not been observed in the
xylem but has been found near the cambium and in the phloem, cortex
and resin canals of the outer bark.
Halik, S. and D.R. Bergdahl. 1986. Population dynamics of
Bursaphelenchus xylophilus in wood chips of Pinus
strobus.
Phytopathology 76:653 (Abstr.).
Freshly cut eastern white pine (Pinus strobus L.) wood was
chipped,
placed in plastic bags (325 g ea.) and inoculated with an isolate
of Bursaphelenchus xylophilus (Steiner and Buhrer) Nickle
from P.
strobus. All inoculated and uninoculated (control) chips were
placed in a growth chamber at 30 C for up to 8 wk. Nematodes were
extracted at 2 wk intervals using a modification of the Baermann
funnel technique. Populations of B. xylophilus increased
about 30X
after 8 wk. Histological studies showed juvenile nematodes present
in resin canals and tracheids of wood chips. Wood staining fungi
were more abundant on uninoculated chips than on nematode-infested
chips.
Bergdahl, D.R., D.L.K. Smeltzer, and S.S. Halik. 1985.
Components
of a conifer wilt disease complex in the northeastern United
States. Pp. 152-155 in V.H. Dropkin, ed. Proceedings of the
United
States-Japan Seminar: The resistance mechanisms of pines against
pine wilt disease. University of Missouri, Columbia, MO.
Mortality of eastern larch has been observed in many areas of the
northeastern United States including Maine, New Hampshire, New York
and Vermont. Symptoms include a rapid discoloration and wilting
of the foliage, followed by death of the tree. This mortality has
also been observed to occur sporadically in other conifers
including: European larch, red spruce, ponderosa pine, eastern
white pine and Scotch pine. Trees naturally infested with B.
xylophilus are also commonly infested with bark beetles, blue
staining fungi, and the root rotting fungus Inonotus
tomentosus. The bark beetle Dendroctonus simplex is
commonly
associated with dead and dying larch and the bark beetle Ips
pini,
with pines. Monochamus scutellatus is the only pine sawyer
beetle
that has been trapped from diseased Scotch pine and M.
carolinensis
and M. notatus have been trapped from dead white pine.
Species of
Verticicladiella, Leptographium, Pesotum and
Ceratocystis have been
isolated from blue-stained wood of eastern larch infested with
B.
xylophilus. Another nematode (Aphelenchoides sp.) is
commonly
extracted from conifers showing symptoms of wilt. Seedlings of
eastern larch inoculated in a growth chamber with
Aphelenchoides
sp. have shown some wilt and mortality but not as consistently or
as rapidly as in B. xylophilus inoculations.
Aphelenchoides sp.
are extracted in low numbers near the point of inoculation and
histological studies show the nematode in the cambium and bark
tissues.
Bergdahl, D.R. and D.L.K. Smeltzer. 1981. Histological
observations of Bursaphelenchus xylophilus in symptomatic
tissues
of Larix laricina and Pinus resinosa. Phytopathology
72:257
(Abstr.).
Eastern larch (Larix laricina) and red pine (Pinus
resinosa) were
inoculated in the greenhouse with the pine wood nematode
(Bursaphelenchus xylophilus). Symptomatic tissues were
excised,
fixed and stored in FAA before sectioning on a freezing microtome.
Sections of wood were stained with safranin/cotton blue in
glycerine/alcohol before examination for nematodes. Nematode
populations were higher in tissues of red pine than in eastern
larch. In red pine, nemas were abundant in longitudinal and radial
resin canals of the xylem, but only occasionally observed in
tissues of the bark. Nemas in eastern larch were not observed in
the xylem tissues, but were found in the cambial region and in the
phloem, cortex and resin canals of the bark.
