Leptographium Species a Component of Loblolly Pine Decline
L. Eckhardt1, J. Jones1, N. Hess2 and E. Carter3
of Plant Pathology and Crop Physiology, Louisiana Agricultural Experiment Station, Louisiana State University Agricultural Center, Baton Rouge, LA; 2US Forest Service, Forest
Health Protection, Pineville, LA; 3US Forest Service, Southern Research Station, Auburn University, AL.
ABSTRACT
Fungi Recovered from Roots, Soil, and Insects
Fungus
Graphium
Hylastes salebrosus (2004)
94
57
Hylastes tenuis (796)
83
48
Pachylobius picivorus (325)
98
49
Hylobius pales (125)
70
26
Xyleborinus saxeseni (28)
56
13
48
+a
Dendroctonus frontalis (29)
+a
+a
Xylosandrus crassiusculus* (9)
67
0
+a
+a
Corthylus punctatissimus* (7)
29
+a
Monarthrum mali* (7)
42
+a
Ips spp (24)
Rhizophagus spp* (8)
Graphium
Ophiostoma
Leptographium
Leptographium terebrantis, L. procerum, Graphium
spp. and Ophiostoma spp. have been recovered from
sample plots where the disease is present.
400
350
300
2
250
200
150
1
100
50
0
1
2
3
Leptographium Incidence Rating
INTRODUCTION
As the incidence of Leptographium spp. increases,
vegetation density decreases and the mean number of
insects increases (P<0.05).
6
35
Resin (gm)
4
Plot Selection. During 1999, thirty-nine plots were identified on ten
different sites located in Choccolocca State Park, the Talladaga National
Forest Shoal Creek Ranger District, the Oakmulgee Ranger D istrict and
on Gulf State Paper Company property to represent a range of decline
symptoms, age classes and management objectives. The loblolly pines
on 32 of the plots showed obvious symptoms of decline; 7 plots had
only mild or no symptoms of decline.
Root Collection. Roots were collected using the modified two-root
excavation method of Otrosina et al. (4) in which 3 dominant/codominant symptomatic trees nearest to plot center were selected. Roots
were stored at 4C for a period of < 5 days before processing. Roots
were cut into pieces, the pieces rinsed in tap water, surface sterilized in
a 10% commercial bleach 10% ethanol for 1 min, rins ed in tap water for
3 min and blotted dry. Samples were plated (4 pieces/plate, 20
plates/sample) on a selective medium (CSM A) [2% malt extract agar
(M EA) containing 800 µg/ml of cycloheximide and 200 µg /ml of
streptomycin sulfate] (5) to isolate Leptographium species. Plates were
incubated at 25C for 7-14 d, and then Leptographium-like isolates were
subcultured by transferring hyphal tips and spore heads of each isolate
to sterile plates of M EA (w/o antibiotics). Subcultured isolates were
placed on cleared V-8 agar slants and stored at 8C.
300
250
2
200
150
1
100
50
0
0
Symptomatic
Asy mptomatic
Loblolly Decline Foliar Symptoms
Loblolly decline foliar symptoms were correlated
with increased number of insects trapped, increased
Leptographium incidence and vegetation density
ratings (P<0.05).
Of the eleven species of insects trapped (Table 1),
seven have been previously associated with
Leptographium, but the other four (Xylosandrus
crassiusculus, an unidentified Rhizophagus spp.,
Corthylus punctatissimus (Zimmermann) and
Monarthum mali (Fitch)) have not. Both C.
punctatissimus and M. mali are considered
hardwood pests which breed in maple and
dogwood, both of which were common in our
plots, but M. mali has been observed on pines. M.
mali is commonly considered as a gum pest in the
gulf states in green lumber and fresh logs.
Presently, Hylastes spp. are being reared in bolts
to confirm their ability to vector Leptographium
and sustain larval growth within galleries.
REFERENCES
1.
30
25
20
2
15
10
5
0
Livingston, W.H., Mangini, A.C., Kinzer, H.G., and Mielke, M.E.
1983. Association of root disease with bark beetles (Coleoptera:
Scolytidae) in Pinus ponderosa in New Mexico. Plant Dis. 67:
674-676.
2.
Healthy Root
Initial
0
0
1
2
Ferrel, G.T ., and Parmeter, J.R., Jr. 1989. Interactions of root
disease and bark beetles. In Proceedings of the Symposium on
Research and Management of Annosus Root Disease
(Heterobasidion annosum) in Western North America. USDA
Forest Service Gen. Tech. Rep., PSW-116, Berkley, CA.
Herbert, H.D., Miller, D.L., and Partridge, A.D. 1975. Interaction
of bark beetles (Coleoptera: Scolytidae) and root-rot pathogens in
grand fir in northern Idaho. Can. Ent. 107: 899-904.
Foliage Transparency Rating
& Crown Density Rating
40
MATERIALS AND METHODS
3
Plots with lower vegetation density ratings also
had higher insect populations and a higher
incidence of Leptographium. While the control
plots had a lowe r incidence of Leptographium,
they also had a lower insect population and a
higher vegetation density rating. Loblolly decline
foliar symptoms were correlated with increased
number
of
insects
trapped,
increased
Leptographium incidence and vegetation density
ratings (P<0.05). There was also a direct
correlation between resin flow (tree vigor), foliage
transparency and crown density to the incidence
of Leptographium. Although cause and effect
cannot be determined, the higher the
Leptographium incidence and insect populations,
along with decreased vegetation on a plot, the less
healthy the trees.
More data, particularly
experimental data, are needed to determine the
etiology of the decline syndrome.
50
45
This study is a work in progress and is aimed
at evaluating whether or not the incidence of
Leptographium and its insect vectors (along
with other physical and chemical factors) are
correlated with loblolly pine decline.
Insect species trapped and the percent of
Leptographium and Graphium spp. recovered.
*Not previously associated with a Leptographium
spp. +a Isolations and counts still being conducted.
Mean Number of Insects
Vegetation Density Rating
3
0
Leptographium species can function as
saprobes, i.e., to cause disease in stressed
trees (3). These fungi may also predispose
otherwise healthy trees to lethal attacks by
bark beetles. However, this theory is still
highly controversial, due to the extremely
complex relationships between the insects
and the development of disease.
DISCUSSION
-% insects with fungi-
0
Several fungi in the genus Leptographium are
important nationally and internationally as
pathogens of conifers. Considerable evidence
links the occurrence of root diseases and
bark beetle infestations in conifer mortality
in the United States (1), and the combined
impact of root disease and bark beetle attack
results in severe losses of pine resources (2).
Most Leptographium species are known for
their association with blue-stain of sapwood
in conifers.
Insects (#)
Le ptographium
Mean Number of Insects
Leptographium species are associated with
root-feeding bark beetles and weevils that
attack living trees and are commonly
present during pine decline and mortality
throughout the United States. The biological
basis for these relationships is variable, and
the relative importance of the beetles and
fungi in the death of trees is still unclear. In
the southeastern United States, loblolly pine
stands affected by root disease are more
likely to contain Leptographium species
within their root systems and appear to be
more vulnerable to attack by southern pine
beetle
(Dendroctonus
frontalis)
than
apparently healthy stands.
A study to
determine the role of Leptographium species
and their vectors in the etiology of loblolly
decline has been initiated. A series of plots
in central Alabama have been selected which
exhibit
a range of symptoms.
Leptographium species have been isolated
from the roots and soil of the loblolly trees,
as well as trapped insects. Soil samples have
been taken from each plot for analysis of
physical and chemical characteristics.
Above-ground symptoms have been assessed
in accordance
with Forest Health
Monitoring standards. Resin samples were
taken to evaluate tree vigor. Stand
vegetation density was also determined for
all plots. We are evaluating whether or not
these factors are correlated with loblolly
pine decline.
Leptographium and Graphium spp. associated
with insects that infest Pinus spp.
RESULTS
Vegetation Density Rating &
Leptographium Incidence Rating
1Department
Staining of
Infected Root
3
Leptographium Incidence Rating
As the incidence of Leptographium spp. increases,
resin flow decreases, foliage transparency rating
increases and crown density rating decreases (P<0.05).
Soil Collection. Soil collections were made using a soil auger from
around selected lateral roots of the 3 dominant/co-dominant symptomatic
trees nearest to center of the central plot in a specific collection pattern.
Samples from each root were placed in separate plastic bags and stored
at 4C until processed. Each soil sample was thoroughly mixed, and a
representative 10g subsample w as taken. All visible root fragments were
removed from the subsample using a sieve, and the subsample was then
suspended in 40 ml of sterile 0.5% water agar. One ml of this
suspension was spotted onto 5 plates and five more were swirled to
distribute the sample evenly across plates containing 10 ml of CSM A.
Plates were incubated at 25C for 7-14 d, and Leptographium isolates
were subcultured by transferring hyphal tips and spore heads of each
isolate to sterile plates of M EA (w/o antibiotics). Subcultured isolates
were placed on cleared V-8 agar s lants at 8C.
Insect Collection. Pitfall traps were capture crawling insects for 8 wks
to determine potential vectors for Leptographium organisms. Traps were
placed so that entrance holes were slightly above ground. Each trap was
baited with two 8 ml glass vials, one containing 95% alcohol and one
containing turpentine, and two cut pine stems (adapted from (6)). Traps
were checked and insects collected weekly. The insects were placed in
sterile polyethylene specimen cups at 4C until isolations could be made
(<4 d). Ins ects were counted, speciated and rolled non-destructively
across CSM A and M EA agar plates. Plates were incubated at 25C for 714 d, and then Leptographium colonies were subcultured by transferring
hyphal tips and spore heads of each colony to sterile M EA plates.
Subcultured species were placed on agar slants at 8C.
Infected Root
Initial
Leptographium procerum inoculated seedling roots
compared to uninoculated roots. Inoculated seedling
roots have no root hairs and root initials have stopped
growing. Staining is evident in all roots of inoculated
seedlings and all had infection sites at all root initials.
Control seedlings had two times as many root initials as
did inoculated seedlings.
Leptographium Species Characterization. Cultures are being singlespored and grown on M EA in the dark for comparison to species
described in the literature. Isolates were also grown out on different
media (PDA, CM A, M EA + 40% sucrose, V-8, Water agar + P ine chips)
in the light and dark to accentuate morphological differences. The
isolates were grown on M EA and punches were taken from the outer
edge of the colony and placed in the center of a new plate with tweezers
(2 plates/isolate); they were then incubated at room temperature under a
12-12 photo-period and in the dark. Isolates were classified into
morphological groups and placed on silica gel slants for long term
storage.
Vegetation Density Ratings. Vegetation was rated by the number of
stems (stems >2.54 cm and <10.16 cm) in the plot. Vegetation was
categorized as follows: Light=0 to 20 stems; M oderate=21-40 stems; and
Heavy=41+ stems. Plots with vines and thicket-type vegetation were
rated visually on a comparable scale.
Crown Ratings. Forest Health M onitoring (FHM ) uses crown/damage
indicators to describe
relative tree health. Tree/Stand measurements
Crown
taken included: species,
dbh, age, growth increment, and basal area. The
Outline
crown damage indicators included: live crown ratio, crown light
exposure, crown position, crown density, crown dieback, and foliage
transparency.
FHM Posters home page
| FHM 2002 Posters
Christiansen, E., Waring, R.H., and Berryman, A.A. 1987.
Resistance of conifers to bark beetle attack: searching for general
relationships. For. Ecol. Manage. 22: 89-106.
Hess, N.J., Otrosina, W.J., Jones, J.P., Goddard, A.J., and
Walkinshaw, C.H. 1999. Reassessment of loblolly pine decline on
the Oakmulgee District, Talladega National Forest, Alabama.
Report No. 99-2-03. Pineville, LA: U.S. Dept. of Ag., Forest
Service Forest Health Protection. p.12
3.
Wingfield, M.J., Capretti, P., and MacKenzie, M.
1988.
Leptographium spp. as pathogens of conifers, an international
perspective. In Leptographium root diseases on conifers. Edited
by T .C. Harrington and F.W. Cobb, Jr. APS Press, St. Paul, MN.
pp. 113-128.
4.
Otrosina, W.J., Hess, N.J., Zarnoch, S.J., Perry, T .J., and Jones, J.P.
1997.Blue-stain fungi associated with roots of southern pine trees
attacked by the southern pine beetle, Dendroctonus frontalis. Plant
Dis. 81: 942-945.
5.
Hicks, B.R., Cobb, F.W., Jr., and Gersper, P.L. 1980. Isolation of
Ceratocystis wagneri from forest soil with a selective medium.
Phytopathology, 70: 880-883.
6.
Klepzig, K.D., Raffa, K.F., and Smalley, E.B. 1991. Association
of an insect-fungal complex with red pine decline in Wisconsin.
For. Sci. 37: 1119-1139.
7.
Hessbur g, P.F., and Hansen, E.M. 2000. Infection of Douglas-fir
by Leptographium wageneri. Can. J. Bot. 78:1254-1261.
Statistical Analysis. Plots were analyzed by categorizing incidence of
Leptographium-like fungi on plots as follows: 0=target fungi were
recovered from none of the three trees sampled on the plot; 1=target fungi
were recovered from one of the three sampled trees on the plot; 2=target
fungi were recovered from two of the three trees sampled on the plot; and
3=target fungi was recovered from all the trees sampled on the plot. The
incidence of Leptographium-like fungi w as compared to the live crown
ratio data using ANOVA. Correlation analysis was used to compare
insects, incidence of L eptographium, and vegetation density.
Pathogenicity Experiments. Ninety bare-root seedlings were inoculated
immediately after lifting (dormant), and 180 were potted in the
greenhous e and inoculated after four and eight w eeks. Inoculations were
accomplished by dipping the seedling roots in a Leptographium slurry
(adapted from (7)). Inoculated trees were replanted in 2.0L containers
containing a Jiffymix planting medium. Pots were randomized in the
greenhous e and watered from above three times each week for 9 weeks.
Seedlings were destructively sampled 34, 44, 54, 64, and 74 days after
inoculation, with six pots sampled each time, including pots with
seedlings showing even the slightest indication of wilting. At the time of
destructive sampling, seedlings were removed from the pots, gently
washed, wrapped in a w et paper towel, and cold stored in a plastic bag at
4C for no longer than 24 hr until dissection. Dissections were made
under a binocular dissecting microscope. Tissue was repeatedly misted
with a spray bottle containing a sterile dilute solution of distilled water
and lemon juice to keep the wood moist and discourage phenolic
discoloration. Ninety seedlings transplanted to pots for 4 and 8 weeks
each, w ill be treated as described above.