Table 1. Average pre-treatment soil populations of Fusarium, Trichoderma and Pythium spp.
at the Lone Peak Nursery, Draper, Utah’.

Fungus

Treatment Fusarium Trichoderma TIF Ratio” Pythium
MBC® 2180 324 0.149 21
Chloropicrin* 2651 776 0.079 35
Fallow - 1 2585 254 0.098 30
Fallow - 2 2901 481 0.166 28
All Treatments 2579 460 0.178 28

"Values in table are colony-forming units per gram of oven-dry soil.

?Ratio of Trichoderma to Fusarium populations; higher numbers denote more potential for disease
suppressiveness.

*Methyl bromide-chloropicrin applied at 350 Ibs./acre.

“Applied at 300 Ibs./acre.

Table 2. Average pre-sowing soil populations of Fusarium, Trichoderma and Pythium spp.
at the Lone Peak Nursery, Draper, Utah’.

Fungus
Treatment Fusarium Trichoderma TIF Ratio” Pythium
MBC® 67 126 0.125 10)
Chloropicrin* 0 8 0 0
Fallow - 1 1486 667 0.593 3
Fallow - 2 1612 465 0.287 7
All Treatments 791 317 0.399 2

"Values in table are colony-forming units per gram of oven-dry soil.

Ratio of Trichoderma to Fusarium populations; higher numbers denote more potential for disease
suppressiveness.

*Methyl bromide-chloropicrin applied at 350 Ibs./acre.

“Applied at 300 Ibs./acre.

Table 3. Average end-of-first-growing-season populations of Fusarium, Trichoderma and
Pythium spp. at the Lone Peak Nursery, Draper, Utah’.

Fungus
Treatment Fusarium Trichoderma TIF Ratio” Pythium
MBC? 605 485 0.802 19
Chloropicrin* 108 149 1.378 9
Fallow 2163 498 0.2302 149
All Treatments 1159 397 0.343 74

‘Values in table are colony-forming units per gram of oven-dry soil.

*Ratio of Trichoderma to Fusarium populations; higher numbers denote more potential for disease
suppressiveness.

*Methyl bromide-chloropicrin applied at 350 Ibs./acre.

“Applied at 300 Ibs./acre.


rinsed in sterile, distilled water, and placed on
the Fusarium selective agar medium. Plates
were incubated and emerging Fusarium spp.
identified as described previously. Percent of
sampled seedlings and extent of seedling root
colonization by particular Fusarium spp. was
calculated.

At the end of the growing season (November),
seedling density within each replicated block for
each treatment was measured and expressed
as number of seedlings per square meter.
Means for each treatment were compared with a
one-way analysis of variance. Significant
differences among the means were located with
the LSD statistical test. After density
measurements, fifty “average” seedlings from
each of the three treatment areas were collected
for analysis of morphological characters.
Seedling heights (from the groundline to the tip
the terminal bud on the longest stem), diameter
(just above the groundline) and root biomass
(oven-dry weight of all roots below the
groundline) were measured. Means for each
treatment were statistically compared with a
one-way analysis of variance and LSD multiple
comparison test. Significant differences were
expressed at P=0.05.

A final sampling of diseased seedlings was
conducted at the end of the growing season. Six
seedlings displaying root disease symptoms
(leaf necrosis and branch dieback) were
collected from within the test area. Seedling
roots were washed thoroughly and dissected
into pieces about 5 mm in length. Fifteen root
pieces per seedling were selected, surface
sterilized, rinsed and incubated on the selective
Fusarium agar medium. Plates were incubated
and emerging Fusarium spp. identified as
described previously. Percent of sampled
seedlings and extent of seedling root
colonization by particular Fusarium spp. was
calculated.

RESULTS

Soil in the field selected for this evaluation was
populated with relatively high levels of Fusarium

and corresponding low levels of Trichoderma
(table 1). The low T/F ratios probably indicated
low disease suppressiveness. Populations of
Pythium were low throughout the field.

At the time of sowing, Fusarium populations had
been greatly reduced in plots treated with MBC
and completely eliminated in chloropicrin-treated
plots (table 2). Bare fallowing also reduced
Fusarium populations, but not nearly as much as
chemical fumigation and surviving populations
still exceeded expected disease thresholds
(Hildebrand and Dinkel 1988; James et al. 1990,
1996). Trichoderma levels were likewise greatly
reduced by chemical fumigation and either
increased or did not change in fallowed plots.
Pythium populations were eliminated by
chemical fumigation and greatly reduced by
fallowing.

By the end of the growing season, Fusarium
levels had increased to much higher levels in
MBC-treated plots than in those treated only
with chloropicrin (table 3). Fallowed plots also
had higher Fusarium populations by the end of
the growing season. Trichoderma levels also
increased more in MBC-treated plots compared
to chloropicin-treated plots but did not change
much in fallowed plots. Pythium levels increased
much more in fallowed plots but were still at very
low levels in fumigated plots.

Six different Fusarium species were identified
from soil isolates within the test area (table 4).
Initially, F. solani (Mart.) Appel & Wollenw. was
by far the most prominent species; following
treatment, F. solani and F. oxysporum Schlecht.
were detected at similar levels. By the end of the
growing season, F. solani, F. oxysporum and
F. equiseti (Corda) Sacc. were all isolated at
relatively similar levels. Fusarium chlamydo-
sporum Wollenw. & Reinking was_ initially
detected at higher levels than was found
following soil treatments or at the end of the
growing season. The other two species, F.
acuminatum Ell. & Ev. and F. scirpi var.
compactum (Lambotte & Fautr.) Wollenw. were
detected at very low levels during each assay.


Forest Health

Protection

Report 04-14 October 2004
PREPLANT SOIL TREATMENT EFFECTS ON
PRODUCTION OF BARE ROOT BITTERBRUSH SEEDLINGS
LONE PEAK CONSERVATION NURSERY
DRAPER, UTAH

R. L. James and S. Zeidler

ABSTRACT

Three preplant soil treatments were evaluated for their effects on the production of bare root bitterbrush
seedlings at the Lone Peak Conservation Nursery in Draper, Utah. The two fumigation treatments
(standard methyl bromide/chloropicrin [MBC] and chloropicrin alone) reduced levels of soilborne
Fusarium spp. more than bare fallowing with periodic cultivation. At the end of the growing season,
Fusarium spp. had reinvaded beds treated with MBC at higher levels than beds treated with chloropicrin
alone. Soil fumigation treatments resulted in greater seedling density than bare fallowing. Seedlings from
beds treated with chloropicrin had significantly greater heights and diameters at the end of the growing
season than seedlings from beds fumigated with MBC. Results of this evaluation indicate that
chloropicrin by itself is as effective as MBC in controlling soilborne pathogens, resulting in production of
high-quality bitterbrush seedlings. Bare fallowing was not as effective as chemical soil fumigation in
reducing pathogen inoculum in soil or controlling seedling disease.

INTRODUCTION plant soil fumigation with methyl bromide/

chloropicrin (MBC)(66% and 33%, respectively)

Production of high-quality seedlings for orna-
mental and reforestation plantings is an
important priority of the Lone Peak Nursery in
Draper, Utah. Many different hardwood and
conifer species are grown annually at the
nursery; most of these are produced as bare
root seedlings. In order to ensure high seedling
quality by reducing damage from soilborne
pathogens and competition from weeds, growers
at the nursery have routinely implemented pre-

for many years. Although this practice is
expensive, it has generally assured predictable
production of high-quality seedlings in sufficient
numbers to meet demands. However, methyl
bromide has been identified as an important
chemical destroyer of atmospheric ozone (World
Meteorological Association 1995), which limits
ultraviolet light from reaching the earth's surface.
As a result, methyl bromide is currently being
phased out and, for the most part will no longer

United States Forest
Department of Service
Agriculture

Northern

200 East Broadway 2
P.O. Box 7669
Missoula, MT 59807 Nn 4



be produced or used in the United States after
January 2005 (Shaheen 1996; Stone et al.
1995). Therefore, nursery growers need to
develop alternatives to methyl bromide for
preplant soil fumigation. Efforts have been
widespread throughout the U.S. to develop
effective alternatives to methyl bromide in forest
nurseries (Chapman 1992; Hildebrand et al.
2004; James et al. 2004a, 2004b, 2004c; Stone
et al. 1995). It has generally been found that
each nursery is unique in its potential disease
problems and suitable alternatives must be
designed specifically for that nursery. For
example, dazomet, a granular soil fumigant, has
been widely used at several nurseries (Boone
1988; Chapman 1992; James et al. 2004a,
2004b). It is usually effective at some nurseries
(James et al. 1996; Miller and Norris 1970) but
not efficacious at others (James and Beall
1999). Bare fallowing for at least 1 year prior to
sowing, accompanied by periodic tilling to keep
weed populations low and mix soil is often
effective because it leads to reduced organic
matter in soil which tends to limit microbial
survival (Hildebrand et al. 2004; James and
Beall 2000; Stone et al. 1995). Often, potential
plant pathogens are reduced in fallowed fields
because of the lack of food sources (Hildebrand
et al. 2004). When susceptible seedling crops
are introduced into fallowed fields, pathogen
levels may be too low to initiate substantial
disease. The key to effective use of fallowing to
control soilborne pathogens is to ensure that
supplemental organic matter is not added to soil
(Hamm and Hansen 1990; Hansen et al. 1990;
Ramirez-Villapudua and Munnecke 1988). Since
many potential pathogens are very good
saprophytes, they are able to effectively colonize
most sources of organic matter, resulting in
buildup of populations to the point that they can
be damaging to seedling crops (Hamm and
Hansen 1990; James et al. 1996).

Bitterbrush (Purshia tridentata [Pursh] DC) is
one of the most important seedling crops
produced at the Lone Peak Nursery. This plant
is in high demand for wildlife habitat restoration
(Austin and Urness 1983). Seedlings are grown
for 1 year in bare root beds; they are then lifted
in the fall and kept in cold storage until being
shipped to the field, usually the following year.
Root diseases, primarily caused by Fusarium
spp., are major limiting factors in the production
of bare root seedlings, including bitterbrush, at

the nursery (James 2002a, 2002b). These
pathogens initiate a series of diseases including
pre- and postemergence damping-off of young
germinants and root decay of older seedlings
(James et al. 1991, 2004a, 2004b, 2004c). As a
result, stands of seedlings may sometimes be
greatly reduced and quality of surviving
seedlings may be adversely affected (Hilde-
brand et al. 2004; James et al. 1990, 2004a).
Fusarium spp. are well-adapted  soilborne
pathogens that produce resting spores that
remain viable for long time periods in the
absence of suitable host plants (James et al.
1991; Nelson et al. 1983; Smolinska et al. 2003).
Their populations can quickly expand in the
presence of organic matter (Hamm and Hansen
1990; Hansen et al. 1990; Ramirez-Villapudua
and Munnecke 1988) to levels that can cause
diseases on susceptible nursery crops.

Because of the importance of bitterbrush and
the prevalence of Fusarium-associated diseases
at the Lone Peak Nursery, an evaluation was
conducted to determine efficacy of different pre-
plant soil treatments on production of bitterbrush
seedlings.

MATERIALS AND METHODS

This test was conducted in the production field
at the farthest southern end of the nursery.
Three treatments were compared: standard
operational soil fumigation with MBC at 350 Ibs.
/acre; chloropicin alone at 300 Ibs./acre; and
bare fallowing with periodic cultivation for one
growing season prior to sowing. Preplant
treatments were conducted within plots that
were 50 feet in length and the equivalent of one
seedbed in width (approximately 4-5 feet).
Treatments were implemented in a complete
randomized block design and replicated four
times.

Three sets of soil samples were taken to
evaluate effects of soil treatments on
populations of Fusarium, Trichoderma, a group
of common saprophytes and potential anta-
gonists of Fusarium (Knudsen and Bin 1990;
Knudsen et al. 1991; Papavizas 1985), and
Pythium, another important group of soilborne
pathogens that are known to cause disease
problems at some nurseries (James 1982,
1989). The first set was taken prior to treatment
to give an overall assessment of resident


populations of these groups of fungi. The
second set was taken just prior to sowing in
approximately the same locations as the first set
to evaluate preplant treatment effects on fungal
populations. A final set of soil samples was
collected and processed at the end of the
growing season to determine fungal population
response to presence of the bitterbrush crop.

Samples were collected from within each
replicate plot; two samples from near the center
of each plot were collected. Each sample
consisted of three cores of soil which were
mixed together. Each collection consisted of soil
cores taken to a depth of about 8 inches (20
cm). Soil was placed in plastic bags, kept
refrigerated, and transported to the laboratory
for analysis.

Standard soil dilutions (Hildebrand and Dinkel
1988; James et al. 1990, 1996; Stone et al.
1995) were conducted to estimate populations of
Fusarium, Trichoderma, and Pythium spp. Soil
from each sample was initially sieved (2-mm
sieve) to remove rocks, pieces of organic matter,
and soil aggregates. From each sample, an
approximate 5 g subsample was oven dried at
about 100°C for at least 24 h until sample weight
stabilized. Oven-dry weight was then calculated
to provide a standard for sample comparison.
For assays of Fusarium and Trichoderma
populations, 0.05 g of field-moist soil was
combined with 10 ml of 0.3% water agar (WA)
and thoroughly mixed. One milliliter of solution
was placed on each of three plates of selective
agar medium for Fusarium and closely related
fungi (Komada 1975) and spread uniformly.
Trichoderma propagules were also enumerated
on Komada’s medium which readily supports
growth of this fungus unless the medium is
amended with benomyl or lithium chloride
(James et al. 1990, 1996). Plates were
incubated at least 7 days at about 24°C under
diurnal cycles of cool, fluorescent light. Fusarium
and Trichoderma colonies were identified by
their morphology on the selective medium;
populations were expressed as number of
colony-forming units (cfu) per gram of oven-
dried soil (it was assumed that each fungal
colony originated from one propagule). Selected
Fusarium isolates were transferred to carnation
leaf agar (Fisher et al. 1982) and potato
dextrose agar (PDA) for species identification
using the taxonomy of Nelson et al. (1983).

Ratios of Trichoderma to Fusarium populations
were calculated for each treatment; these ratios
may indicate very rough estimates of potential
disease suppressiveness of the soil since
Trichoderma spp. are known antagonists of a
wide range of soilborne plant pathogens,
including Fusarium spp. (Knudsen and Bin 1990;
Knudsen et al. 1991; Papavizas 1985).

For assays of Pythium populations, 0.5 g of soil
was combined with 10 ml of 0.3% WA. One
milliliter of solution was placed on each of three
plates containing another selective medium
consisting of V-8 juice agar amended with the
antibiotics pimaricin, rifamycin, and ampicillin
and the fungicide pentachloronitrobenzene
(James et al. 1990, 1996; Stone et al. 1995).
Plates were incubated in the dark at about 24°C
for 3 days. Pythium colonies were identified on
the basis of their diameter after 3 days (15-20
mm), feathery margin, and growth within rather
than superficially on the agar surface.
Populations were expressed as cfu/g of oven-
dried soil.

To determine background levels of potentially
pathogenic fungi on bitterbrush seed, two lots
[98-310-01 and 00-310-01; 98 and 00 refer to
years of collection], which were used to sow the
test area, were analyzed. Sampled seed was
collected from bulk storage and transferred to
the laboratory for analysis. Three hundred seeds
from each seedlot were incubated on Komada’s
medium and emerging fungi identified to genus
or species using the taxonomy of Barnett and
Hunter (1998) and Booth (1966).

Postemergence disease was evaluated by
collecting samples of recently emerged
seedlings displaying disease symptoms, such as
leaf necrosis. A total of 106 seedlings were
sampled from within the three treatment areas.
Seedlings were transported to the laboratory
and rated for disease severity based on level of
above-ground leaf necrosis. Five numerical
ratings were used: 1 = no leaf necrosis; 2 =
1%-25% leaf area necrosis; 3 = 26%-50% leaf
area necrosis; 4 = 51-75% leaf area necrosis;
5 = greater than 75% leaf area necrosis.
Seedling roots were washed thoroughly and
dissected into pieces about 5 mm in length. Four
root pieces per seedling were randomly
selected, surface sterilized in a 10% bleach
solution (0.525% aqueous sodium hypochlorite),