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Soil Biology & Biochemistry 33 (2001) 9±16
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Components of soil suppressiveness against Heterodera schachtii
A. Westphal, J.O. Becker*
Department of Nematology, University of California, Riverside, CA 92521, USA
Received 18 November 1999; received in revised form 1 May 2000; accepted 16 May 2000

Abstract
Heterodera schachtii populations were introduced into nematode-suppressive and conducive ®eld plots and were monitored for
2550 degree-days (DD). At 1200 DD, H. schachtii population densities signi®cantly increased in conducive versus suppressive plots, up
to 14-fold at termination of the trial. In greenhouse experiments with the same soil, H. schachtii female population densities were similar in
suppressive and conducive soil in the ®rst nematode generation, but remained low in the suppressive soil compared to signi®cant increase in
conducive soil in the second generation. At termination of the experiment, ca. one third of the cysts, but no females from suppressive soil
were infested with fungi, whereas fungal-infested females and cysts were rarely found in conducive soil. The most common fungi isolated
from infested cysts were Fusarium oxysporum, Fusarium sp. nov., and Dactylella oviparasitica. Paecilomyces lilacinus and some nonidenti®ed fungi occurred less frequently. Suppressiveness was transferred at a rate of one cyst from suppressive soil amended to 110 g of H.
schachtii-infested conducive soil. Heat treatment of suppressive soil for 30 min at 558C eliminated H. schachtii suppressiveness and reduced
F. oxysporum populations to the detection level. q 2001 Elsevier Science Ltd. All rights reserved.
Keywords: Biological control; Dactylella oviparasitica; Fusarium oxysporum; Nematode parasitic fungi; Sugar beet cyst nematode

1. Introduction

Several cyst nematode-suppressive soils have been identi®ed (Kerry, 1988). Such soils are typically characterized by a
relatively low population of the cyst nematode and its inability
to increase despite the presence of a susceptible host and suitable environmental conditions. Fungal egg, female and/or cyst
parasites have repeatedly been associated with cyst nematode
population decline (Kerry et al., 1980; Heijbroek, 1983;
Crump and Kerry, 1987; Chen et al., 1996), and many fungi
have been isolated from nematode cysts (Tribe, 1977; RodrõÂguez-KaÂbana and Morgan-Jones, 1988). A particularly welldocumented example of soil suppressiveness against a plantparasitic nematode was the study of a population density
decline of Heterodera avenae Woll. in England (Kerry et al.,
1980). In suppressive soil, Nematophthora gynophila Kerry
and Crump destroyed young females of H. avenae before they
could mature to cysts (Crump and Kerry, 1977). In addition,
Verticillium chlamydosporium Goddard parasitized the eggs
of this nematode. The concerted parasitic activity of both fungi
was considered the main factor in the natural population
suppression of H. avenae (Kerry et al., 1980).
Recently, we have shown that soil suppressiveness
against Heterodera schachtii Schm. at a ®eld site at the
* Corresponding author. Tel.: 11-909-787-2185; fax: 11-909-787-3719.
E-mail address: ole.becker@ucr.edu (J.O. Becker).

agricultural research station of the University of California,
Riverside was of a biological nature (Westphal and Becker,
1999). The soil suppressiveness was reduced to non-detectable levels by soil fumigation and it was transferable. It
established in fumigated ®eld plots within the ®rst cropping
season when 1% of suppressive soil was incorporated into
the top 10-cm soil layer. The transfer of 10% suppressive
soil resulted in an even faster establishment of nematode
suppressiveness. In effect, the amended soil was as suppressive as the original source of the transfer soil (Westphal and
Becker, 2000).
The objective of this research was to determine which stage
in the life cycle of H. schachtii was the primary target of the
suppressiveness. Furthermore, we characterized the thermal
sensitivity of the soil suppressiveness in order to focus our
search for cyst nematode antagonists responsible for the
phenomenon. Potential antagonists were isolated from
infested cysts and identi®ed. Preliminary results of this study
have been published (Westphal and Becker, 1998).

2. Materials and methods
2.1. Field trial

The sugar beet cyst nematode suppressive site 9E was
located at University of California Ð Riverside,

0038-0717/01/$ - see front matter q 2001 Elsevier Science Ltd. All rights reserved.
PII: S 0038-071 7(00)00108-5

10

A. Westphal, J.O. Becker / Soil Biology & Biochemistry 33 (2001) 9±16

Agricultural Operations, Riverside, CA. The soil type was a
Hanford ®ne sandy loam soil (60.9% sand, 29.6% silt and
9.5% clay, pH 8). The objective of the trial was to compare
the population dynamics of introduced H. schachtii in
untreated, suppressive versus methyl bromide-fumigated,
conducive plots. In the end of March 1996, the trial was
designed as a randomized split-block with two treatments
and six replications. After a green-manure crop of canola,
Brassica napus L., the soil in the trial area had an initial H.
schachtii population density of 18 eggs g 21 soil. The area

was chiseled to a depth of 45 cm, disked, and NPK fertilizer
(336 kg ha 21, 15% N, 15% P, 15% K) was incorporated
with a cultimulcher. For each replication, there were two
seedbeds (0.75 m seedbed center-to-center spacing, 5.1 m
long), which were separated from the next replication by
one border seedbed. Designated conducive plots were
covered with 0.03-mm plastic tarp and hot-gas fumigated
with methyl bromide (336 kg ha 21) (Davidson, 1957). The
tarps were removed after 5 days, all plots were rototilled and
were kept moist with a low-volume drip irrigation system.
Six weeks later, greenhouse-reared H. schachtii were
introduced into planting sites of both non-fumigated and
fumigated plots. Ten planting sites were established at 30cm intervals along the center line of each seedbed. From
each planting site a 10-cm-deep soil core was taken with a
bucket auger (diameter: 7.6 cm). The soil was placed into a
plastic bag which contained 250 g of a sandy potting soil
infested with ca. 446 cysts (ca. 50,000 eggs) of H. schachtii.
The nematodes had been raised in this potting soil on sugar
beets in the greenhouse. After thoroughly mixing, the
infested soil was replaced into the original hole. The plots

were irrigated with a low-volume drip irrigation system and
monitored with tensiometers to keep the soil water potential
at 220 to 230 kPa. After one month, one 5-week-old seedling of Swiss chard (Beta vulgaris L. subsp. cicla (L.) W.
Koch cv. Large White Ribbed, Lockart Seeds Inc., Stockton, CA) was planted into the center of each planting site.
Additional fertilizer solution was applied via overhead
sprinkling as needed (total: 234 kg ha 21 Miracle Gro, 15%
N, 30% P, 15% K, Scotts Miracle Products Inc., Port
Washington, NY). Insect control was conducted with
spray applications of imidachloprid (52.5 g a.i. ha 21) as
needed.
Starting at degree-day 600 (DD, 88C basal temperature;
Curi and Zmoray, 1966) two randomly selected plants per
plot were harvested every 150 DD and later in the season
every 300 DD. Foliar growth was determined by taking dry
weights. Roots and soil from the respective planting sites
were recovered with a bucket auger (diameter: 7.6 cm).
Adhering soil and cysts were shaken and washed from the
root systems and mixed into the corresponding soil sample.
Subsamples of 350 g soil were used for cyst extraction with
a modi®ed Fenwick ¯otation can (Caswell et al., 1985). The

extraction ef®ciency from moist soil was determined as ca.
80%. H. schachtii cysts and eggs were counted and the
arithmetic means of the two subsamples per plot were

used for ANOVA followed by mean separation with Fisher's Protected LSD. At DD 1350, the Swiss chard foliage
was cut to ca. 5-cm stubble to allow new growth and to
prolong the cropping period. The trial was terminated in
February 1997 (DD 2550).
2.2. Greenhouse experiments
All soil used were sampled from the upper 10-cm of the
suppressive ®eld 9E and passed through a screen with 6-mm
openings and mixed 10:1 (dry w w 21) with silica sand.
Portions of this soil±silica mix were fumigated with methyl
iodide (500 kg ha 21) (Becker et al., 1998).
2.2.1. H. schachtii female population development in root
observation chambers
Suppressive 9E soil was mixed with methyl iodide-fumigated ®eld soil (1:9, dry w w 21) and placed into custommade rectangular polyacrylic root observation chambers
…27 cm £ 23:5 cm £ 2:5 cm†: Previously it was shown that
amendment of 10% suppressive 9E soil to fumigated 9E soil
established soil suppressiveness (Westphal and Becker,

2000). Methyl iodide-fumigated ®eld soil was placed in
the remaining root observation chambers as the conducive
treatment. The two treatments with three replications were
arranged in a completely randomized design. One side of the
chamber was translucent to allow non-destructive observation of the root systems. Fifteen seeds of mustard-greens
(Brassica juncea (L.) Czern cv. Florida Broadleaf, Lockhart
Seeds Inc., Stockton, CA) were planted per chamber and
thinned after emergence to ®ve seedlings per chamber.
The chambers were watered twice a day with tap water
and the soil moisture content was adjusted weekly to 11%.
The translucent side of the root observation chambers was
covered with aluminum foil and faced downward at a 458
angle. The experiment was incubated in a greenhouse at
24 ^ 38C and ambient light. After three weeks, each of
the chambers was infested with 15,000 J2 of H. schachtii.
Each root observation chamber was fertilized with 100 ml
nutrient solution (Miracle Gro 10 g 3.79 l 21 of water, 15%
N, 30% P, 15% K) twice per week. Starting three weeks
after infestation, the females of H. schachtii visible on the
root surface were counted in weekly intervals. Ten weeks

after infestation, the visible root length per root chamber
was determined using a line intersection method (Tennant,
1975). Fifty individual cysts and females each were handpicked from each chamber and examined for fungal infestation as described below. In addition, cysts from the
chambers were used for transfer studies as described
below. The root observation chamber experiment was
conducted twice.
2.2.2. Fungal infestation of females and cysts from
suppressive and conducive soils
The picked females and cysts were examined in two
ways. In the ®rst experiment, single nematode specimens

A. Westphal, J.O. Becker / Soil Biology & Biochemistry 33 (2001) 9±16

were put in a drop of water on a microscope slide and
smashed with a cover slip. Individual female and cyst
contents were rated for visible fungal infestation. The
results were expressed as percentage of the H. schachtii
female or cyst population infested. H. schachtii eggs from
the suppressive chambers, which were ®lled with fungal
hyphae, were placed on water agar (2%) to isolate fungi.

Cysts from the second observation chamber experiment
were surface sterilized (3 min in 25% commercial bleach
solution, 5.25% sodium hypochlorite), plated onto water
agar (2%) and examined for fungal growth. After about
20 days the cysts were recovered, broken and the content
spread onto water agar (2%) amended with rifampicin
(100 mg kg 21). Cysts containing fungi were enumerated.
The frequencies of infested cysts were expressed as percentage of the total number of cysts examined per root observation chamber. Fungal isolates were hyphal-tipped,
subcultured and identi®ed.
2.2.3. Transfer of suppressiveness with cysts
At the end of each root observation chamber experiment,
cysts from the suppressive and conducive soil were picked
from the three replicates, pooled and used to amend H.
schachtii-infested conducive soil at approximately one
cyst per 110 g soil. This soil was a 2:1 soil mix (dry
w w 21) of methyl iodide-fumigated 9E ®eld soil
(500 kg ha 21) and sandy soil. The sandy soil was infested
with cysts containing ca. 50,000 eggs of H. schachtii in the
®rst experiment and ca. 70,000 eggs of H. schachtii in the
second experiment. The amendment ratio was equivalent to

the number of cysts present in 1% suppressive 9E soil. A
non-amended treatment, the amendment with 1% suppressive 9E soil and the amendment with the cysts from 1% of
suppressive 9E soil were included as comparisons.
After application of the different amendments, the soil
was placed into 350-ml styrofoam cups and adjusted to a
moisture content of 11%. The experimental design was a
randomized complete block with ®ve replicates. The pots
were incubated in the greenhouse at 24 ^ 38C; ambient light
and watered with a low-volume irrigation system with tap
water (Neta®m Irrigation Inc., Fresno, CA). After one
month, the soils were permitted to dry to a soil moisture
content of ca. 7% to make them mixable. Each soil sample
was placed into a plastic bag, mixed thoroughly, and a 345-g
subsample was ®lled back into the styrofoam cup and was
adjusted to 15% moisture. Mustard-greens (cv. Florida
Broadleaf) was seeded into each pot and thinned after emergence to one plant per pot. After 12 weeks at 24 ^ 38C at
ambient light, the plant tops were cut off. The soil with the
contained cysts was washed into the modi®ed Fenwick
¯otation can for cyst extraction. The nematode cysts and
the contained eggs were counted. Plant top and root ovendry weights were determined.

11

7.5%) was placed in plastic bags and heat-treated in a water
bath for effective 30 min in a series of 45, 50, 55, 60 or 658C
and a second series of 50, 60, 70, 80 or 908C. Subsamples of
the heat-treated soils were either directly dilution plated
onto modi®ed Pseudomonas media (Sands and Rovira,
1970) (10 ml glycerol, 1.5 g K2HPO4 anhydrous, 1.5 g
MgSO4´7H2O, 10 g proteose peptone No. 3, 20 g agar in
1 l water, and 45 mg novobiocin and 45 mg penicillin G
diluted in methanol, added after autoclaving) to determine
the number of colony forming units (cfu) of ¯uorescent
pseudomonads, or air-dried, screened through a USS
number 40 screen and used for dilution plating on modi®ed
selective fungal media. Pythium spp. Pringsheim were
enumerated on a selective medium described by Mircetich
(1971) (10 mg CaCl2 17 g cornmeal agar, 10 mg
MgSO4´7H2O, 20 g sucrose, 1 mg ZnCl2, 23 g agar, 1 l
water, and 100 mg PCNB, 50 mg rifampicin, 10 mg rose
bengal diluted in methanol, added after autoclaving). Fusarium oxysporum Schlecht. was enumerated on Komada's
medium (1975) that was prepared without the micro-nutrients.
Heat-treated soils were mixed 1:9 (dry w w 21) with
methyl iodide-fumigated soil from the H. schachtii-suppressive 9E ®eld. The soil-mixes were placed in 15-cm pots and
adjusted to a soil moisture content of 8%. The pots were
incubated in the greenhouse at ambient light and at 24 ^
38C in a randomized complete block design with ®ve replications. One 5-week-old seedling of Swiss chard (cv. Large
White Ribbed) was planted into each pot. The experiments
were watered with a low-volume drip irrigation system with
tap water. Each plant was fertilized with 50 ml nutrient
solution (Miracle Gro, 10 g 3.79 l 21 of water) weekly.
Two weeks after planting, the pots were infested with
7500 J2 of H. schachtii. Eleven weeks after infestation,
after ca. two H. schachtii generations, the experiment was
terminated. Nematode cysts and soil were shaken and
washed from the roots and mixed with the remaining pot
content. Subsamples of 350 g of soil from each pot were
used for cyst extraction. H. schachtii cysts and eggs were
counted. Oven dry weights of the plant tops and the roots
were determined.
2.3. Data analysis
All data from individual experiments were subjected to
analysis of variance with SuperANOVA (Abacus Concepts,
1989, Berkeley, CA). Fisher's Protected LSD was used to
separate means at P ˆ 0:05 if the treatment F had a P #
0:05: The test was performed at P ˆ 0:10 if the probability
for the treatment F was between 0.10 and 0.05.
3. Results
3.1. Field trial

2.2.4. Heat fractionation of soil micro¯ora
Nematode-suppressive 9E soil (405 g, moisture content:

At 600 DD the number of H. schachtii cysts g 21 soil in the

12

A. Westphal, J.O. Becker / Soil Biology & Biochemistry 33 (2001) 9±16

Females of H. schachtii per 580 cm2

Fig. 1. H. schachtii population densities during 230 days (2550 DD, basal temperature: 88C) under Swiss chard in suppressive and conducive ®eld plots.
*Signi®cant difference according to Fisher's protected LSD at P ˆ 0:05: 1Tops of Swiss chard were cut off at 5 cm stubble. Bars represent standard error
…n ˆ 6†:

*

1800

*

suppressive
1500

conducive

1200

*

900
600
300

*
*

0
3rd

4th

5th

6th

7th

8th

9th

10th

Observation week post inoculation
Fig. 2. Female populations of H. schachtii on mustard-greens roots in suppressive and conducive soil in root observation chambers. *Signi®cant differences
were determined at P ˆ 0:05 with Fisher's protected LSD. 1A total area of 580 cm 2 was monitored. The results of one representative experiment are presented.
Bars represent standard error …n ˆ 3†:

13

A. Westphal, J.O. Becker / Soil Biology & Biochemistry 33 (2001) 9±16
Table 1
Percentage of females and cysts of H. schachtii with fungal infestation in root observation chambers …means ^ SE; n ˆ 3 in both experiments)
Soil status

Suppressive
Conducive
P for treatment F

First experiment a

Second experiment b

Females

Cysts

Cysts

2:0 ^ 2:0
0:7 ^ 0:7
0.4950

67.0 ^ 11:0
4.0 ^ 3:1
0.0062

34:6 ^ 0:8
7:2 ^ 7:2
0.0200

a

H. schachtii specimens from the root surface of mustard greens, smashed on a microscope slide and rated for the presence of fungal hyphae. Percentage
infested specimens of total number examined.
b
H. schachtii cysts from the root surface of mustard-greens were broken open after surface sterilization and plated on water agar. Cyst content with fungal
growth was rated as positive count. Percentage infested specimens of total number examined.

suppressive plots was signi®cantly higher than in the conducive plots (Fig. 1A). At DD 750, 900 and 1050 the population densities were not signi®cantly different in the
suppressive and conducive plots. Beginning at 1200 DD,
the numbers of cysts g 21 soil in the conducive plots were
signi®cantly higher than those in the suppressive plots (Fig.
1A). During the length of the trial, the numbers of cysts g 21
soil remained low in the suppressive plots. The overall
increase of nematode cysts g 21 soil from the ®rst to the
last sampling was 1.6-fold in the suppressive plots versus
21-fold in the conducive plots (Fig. 1A). The number of
eggs per cyst was higher in the conducive soil throughout
the monitoring time, although non-signi®cant at DD 600
and DD 2550 (Fig. 1B). The top dry weights of Swiss
chard were signi®cantly higher in the conducive plots at
the ®rst, second and seventh sampling, but not signi®cantly
different from the suppressive plots at other sampling times
(Fig. 1C).
3.1.1. H. schachtii female population development in root
observation chambers
At the three ®rst monitoring times, the numbers of
females were not signi®cantly different in the suppressive
and the conducive soils, but starting at the fourth sampling
date, the number of females in the conducive soil was up to
7-fold higher than in the suppressive soil (Fig. 2). There
were no signi®cant differences in the root length (cm) per
observation area in the suppressive versus the conducive

soil in the ®rst experiment (suppressive: 3438 ^ 180;
conducive: 3066 ^ 145; P ˆ 0:1826†: The root length
(cm) was signi®cantly shorter in the suppressive soil than
in the conducive soil in the second experiment (suppressive:
1431 ^ 84; conducive: 1905 ^ 117; mean ^ SE; P ˆ
0:0303†: The top and root dry weights of the mustard-greens
in both experiments were not signi®cantly different (data not
shown).
3.1.2. Fungal infestation of females and cysts from
suppressive and conducive soil
Many cysts from the suppressive soil were infested in
both experiments, either determined by the presence of
fungal hyphae (experiment 1) or by fungal growth on agar
medium (experiment 2). In contrast, cysts from the conducive soil were almost free of fungal infestation (Table 1).
Fungal species isolated from within cysts included, in the
order of decreasing frequencies, F. oxysporum, Fusarium
sp. nov., Dactylella oviparasitica Stirling and Mankau,
Paecilomyces lilacinus(Thom) Samson, and other non-identi®ed fungal species in low frequencies. D. oviparasitica
was most frequently isolated in the ®rst experiment from
infected H. schachtii eggs, while F. oxysporum and Fusarium sp. nov. were the most frequently detected fungi in the
second root observation chamber experiment. P. lilacinus
and other fungal species were isolated at low frequencies.
Cyst nematode females were rarely found infested in either
suppressive or conducive soil.

Table 2
Amendment of conducive H. schachtii-infested soil with cysts from different sources in comparison with non-amended infested soil …means ^ SE; n ˆ 4 in
both experiments). Cysts were collected from suppressive 9E ®eld soil or from the corresponding root observation chambers
Transfer source

Non-amended (conducive)
Cysts, conducive soil (rootbox)
Cysts, suppressive soil (rootbox)
Cysts, 1% suppressive 9E ®eld
soil
1% Suppressive ®eld 9E soil
LSD …P ˆ 0:05†
P for treatment F

Experiment 1

Experiment 2

Root dry weight

Cysts g 21 soil

Eggs per cyst

Root dry weight

Cysts g 21 soil

Eggs per cyst

3:5 ^ 0:3
3:1 ^ 0:4
3:0 ^ 0:1
3:3 ^ 0:3

1.8 ^ 0.1
1.7 ^ 0.1
1.2 ^ 0.1
1.4 ^ 0.1

51.2 ^ 9.8
52.7 ^ 10.3
52.6 ^ 7.1
76.6 ^ 11.0

1.5 ^ 0.2
1.9 ^ 0.2
2.2 ^ 0.3
2.2 ^ 0.5

4.2 ^ 0.3
3.9 ^ 0.2
2.8 ^ 0.1
2.4 ^ 0.2

51.7 ^ 12.9
63.5 ^ 11.9
34.5 ^ 4.7
43.3 ^ 4.7

2:9 ^ 0:3
0.3
0.489

1.4 ^ 0.0

66.9 ^ 7.5

1.9 ^ 0.3
0.50
0.183

2.7 ^ 0.2

44.9 ^ 8.1

0.002

0.274

, 0.001

0.161

14

86:9 ^ 9:1
76:3 ^ 15:4
95:7 ^ 12:1
157:0 ^ 14:5
166:9 ^ 20:1
184:4 ^ 17:9
40.5
,0.001
1:0 ^ 0:1
0:8 ^ 0:1
2:1 ^ 0:6
5:3 ^ 0:4
5:6 ^ 0:5
4:6 ^ 0:6
1.4
,0.001
3:7 ^ 0:4
3:3 ^ 1:0
5:0 ^ 1:0
5:9 ^ 1:1
6:3 ^ 0:8
7:5 ^ 0:5
2.2
0.015

0:3 ^ 0:1
0:5 ^ 0:1
0:8 ^ 0:2
1:7 ^ 0:4
1:8 ^ 0:2
2:2 ^ 0:0
0.6
,0.001

Eggs per cyst
Cysts g 21 soil
Root dry (g)
g

Top dry (g)

H. schachtii populations
Harvest weights

3:8 ^ 0:1
2:9 ^ 0:3
1:4 ^ 0:4
0:0 ^ 0:0
0:0 ^ 0:0
0:0 ^ 0:0
0.6
,0.001

soil)

F. oxysporum (cfu £ 10
soil)

23

226:7 ^ 17:7
222:2 ^ 26:7
6:7 ^ 3:2
0:0 ^ 0:0
0:0 ^ 0:0
1:1 ^ 1:1
39.9
,0.001
9:5 ^ ^4:0
5:1 ^ 1:1
0:0 ^ 0:0
0:0 ^ 0:0
0:0 ^ 0:0
0:0 ^ 0:0
4.9
0.002
Control
458C
508C
558C
608C
658C
LSD …P ˆ 0:05†
P for treatment F

Pseudomonas
spp.
(cfu £ 10 24 g
soil)

Pythium spp. (cfu g

21

Precropping±posttreating monitoring
Heat treatment

Table 3
Effect of heat treatment of H. schachtii-suppressive soil on microbial groups and consecutive H. schachtii population development under Swiss chard in a greenhouse trial …means ^ SE; n ˆ 4†

A. Westphal, J.O. Becker / Soil Biology & Biochemistry 33 (2001) 9±16

3.1.3. Transfer of suppressiveness with cysts from
suppressive and conducive root observation chambers
In both transfer experiments the numbers of cysts g 21 soil
were signi®cantly higher in the non-amended control than in
soil amended with cysts from the suppressive soil from root
observation chambers, cysts from the suppressive 9E ®eld
soil or 1% suppressive 9E soil (Table 2). Meanwhile, the
®nal number of cysts g 21 after amendment with cysts from
conducive root observation chambers was not signi®cantly
different from that in the non-amended treatment. There
were no signi®cant differences in the numbers of eggs per
cyst or in the root dry weights in either experiment (Table
2).
3.1.4. Heat fractionation of soil micro¯ora
In the ®rst experiment, the number of cysts g 21 soil and
the number of eggs per cyst were signi®cantly lower in the
untreated control and in the 45 and 508C treatments than in
the 55, 60 and 658C treatments (Table 3). F. oxysporum cfu
were signi®cantly reduced after the 458C treatment in
comparison to the non-heat-treated control and reduced to
near the detection level at 558C (Table 3). In the ®rst experiment, the plant top dry weights increased with increasing
preseason heat treatment (Table 3) but not in the second
experiment (data not shown). The root dry weights were
signi®cantly higher in the 55, 60 and 658C treatments than
in the 45, 508C treatments and the non-heated control (Table
3). Heat treatments of the soil in the second experiment
resulted in very similar results concerning nematode and
microbial population levels (data not shown).
4. Discussion
The eggs within the cysts of H. schachtii were a major
target of nematode-suppressiveness in the presented experiments. In the ®eld trial, the number of eggs per cyst was
lower in the suppressive than in the conducive plots beginning at DD 750. The number of cysts was signi®cantly
higher in the conducive than in the suppressive plots starting
at DD 1200. The low number of cysts g 21 soil in the
suppressive plots might have been the consequence of the
suppressiveness on the nematode eggs, since the time interval between the difference in the number of eggs per cyst
(DD 750) and the difference in the number of cysts g 21 soil
(DD 1200) was suf®cient for one nematode generation to
occur. The number of cysts g 21 soil remained at a low level
in the suppressive plots, which is another con®rmation for
the nematode-suppressiveness of this soil.
The results of the root observation chambers con®rmed
the results of the ®eld trial. The number of females in the
conducive chambers was signi®cantly higher than in the
suppressive chambers in the second generation of H.
schachtii. In the ®rst half of the experiment, the number
of females did not statistically differ in suppressive and
conducive soils. Apparently, the inoculated J2 were not

A. Westphal, J.O. Becker / Soil Biology & Biochemistry 33 (2001) 9±16

measurably hindered by the suppressiveness to develop into
®rst-generation females. Frequent fungal infestations of the
H. schachtii cysts were observed in the suppressive soil. In
contrast, females from the suppressive soil were almost free
of fungal infestation. Fungal parasitism often increases
when cyst nematodes mature (Gintis et al., 1983). The
presence of fungi in the cysts from the suppressive soil in
comparison to almost no infestation in females and cysts
from the conducive soil suggests that fungal cyst parasites
may be major components in this nematode-suppressiveness.
The transfer attempt with cysts from the suppressive soil
was based on the transferability of H. schachtii suppressiveness with portions of suppressive 9E soil (Westphal and
Becker, 2000). As little as 1% of suppressive soil transferred
and established H. schachtii suppressiveness in fumigated
®eld plots. Cysts, which had developed in suppressive soil in
observation chambers, transferred nematode-suppressiveness to the same extent as 1% of suppressive 9E soil or
the extracted cysts from 1% of suppressive 9E soil. This
emphasized the role of the H. schachtii cyst as the primary
target of the nematode suppressiveness. It has been
suggested that certain stages of the life cycle of H. schachtii
could be suitable as transmitters for nematode antagonists.
Nicolay and Sikora (1989) concluded that the cysts in their
experiments were not the main site of fungal multiplication,
but they assumed that cysts can serve as survival agents as
well as propagation units for egg parasites. In our experiments, the cysts did act as a survival base and inoculum
source for suppressiveness. As few as one cyst from a
suppressive soil per 110 g of infested conducive soil was
suf®cient to suppress H. schachtii populations.
The ability to spread throughout the soil from the source
of inoculum or the current food base is an important characteristic of successful antagonists. It was shown earlier that
certain fungal egg parasites can infect G. pallida eggs in
0.5±1.0 cm distances during a two-week incubation in a
non-sterile soil mix (Sikora et al., 1990). In our transfer
experiments, the suppressiveness was effective throughout
a much larger soil volume. Spread of the suppressiveness in
a 1-cm range in each dimension from each transferred cyst
would cover only ca. 6.7 % of the pot volume. Potentially
not only fungal egg parasites were transferred with the cysts,
but any organisms associated with the cysts, some of which
may have not been detected by isolation procedures. The
much longer incubation time and the fact that fumigated soil
was used as potting medium probably supported the establishment of H. schachtii suppressiveness. At this time, we
have not determined if the suppressiveness was based on
organisms parasitizing the nematodes or if toxic metabolites
produced by suppressive organisms played a role in H.
schachtii suppression. Production of nematotoxic
compounds by fungal nematode antagonists has been
suggested to occur (Meyer et al., 1990). But although negative effects of fungal culture ®ltrates on nematode hatch and/
or mobility have been shown (Mani and Sethi, 1984; Sikora

15

et al., 1990), in situ production and ef®cacy of such metabolites have not been demonstrated.
The results from the heat treatment experiments further
supported the possible role of fungi in the suppressiveness.
The thermal fractionation technique we used can help to
reduce the surviving microbiota to the principal antagonists
(Baker and Roistacher, 1957) or at least to narrow the ®eld
of potential candidates. It has been shown that temperature
treatment at 558C for 30 min can reduce resident F.
oxysporum populations in a fusarium wilt-suppressive soil
(Rouxel et al., 1977) and that the reintroduction of resident
F. oxysporum strains can reestablish the wilt-suppressiveness (Rouxel et al., 1979). In our trials, F. oxysporum and
Fusarium sp. nov. were frequently isolated from cysts
developed in suppressive 9E soil and suppressiveness was
eliminated at the same thermal fractionation step at which F.
oxysporum was reduced to near detection level. While this is
by far not an unambiguous proof for the involvement of
Fusarium spp. in the soil suppression, F. oxysporum is a
known fungal parasite of cyst nematodes. F. oxysporum
was isolated from H. schachtii populations in California
(Nigh et al., 1980) and in other parts of the world (Crump,
1987; Qadri and Saleh, 1990). Furthermore, the pathogenicity of certain strains of F. oxysporum to nematode eggs
were demonstrated (Nigh et al., 1980). Strains of that fungus
were also found associated with other cyst nematode populations like Globodera spp. and H. glycines (Goswami and
Rumpenhorst, 1978; Carris et al., 1989; Meyer et al., 1990;
Crump and Flynn, 1995; Chen et al., 1996). More recently,
strains of endophytic, non-pathogenic F. oxysporum had
been suggested as biocontrol organisms against Meloidogyne incognita (Kofoid and White) Chitwood (Hallmann
and Sikora, 1994). The other often isolated fungus from
9E soil D. oviparasitica was ®rst described as a parasite
of M. incognita in root-knot nematode-suppressive peach
orchards (Stirling and Mankau, 1979). While D. oviparasitica parasitized H. schachtii eggs in vitro (Stirling and
Mankau, 1979) its potential to suppress sugar beet cyst
nematode populations in vivo has not yet been shown.
Although these studies did not conclusively identify the
cause of the soil suppressiveness, they have advanced our
search for the responsible organism/s considerably. The
suppressive effect became evident by differences in population development in the second generation of H. schachtii
between conducive and suppressive soil. However, the high
degree of egg parasitism almost exclusively in the suppressive test variant suggested that the main interference with
the population development preceded these observations.
Earlier tests had already indicated that the number of infective juveniles at the beginning of the second generation was
much lower in suppressive than in conducive soil (Westphal
and Becker, 2000). The isolated fungi will therefore be
tested for their ability of parasitize H. schachtii eggs. But
perhaps even more exciting was the ®nding that soil
suppressiveness can entirely be transferred with cysts that
had developed in suppressive soil. This will allow us in

16

A. Westphal, J.O. Becker / Soil Biology & Biochemistry 33 (2001) 9±16

continuation of this project to focus exclusively on the
micro¯ora located in those cysts.
Acknowledgements
The article is a portion of a dissertation by the ®rst author
submitted to the University of California in partial ful®llment of the requirements for the PhD degree. We thank the
Departments of Nematology and Plant Pathology as well as
the agricultural research station, University of California,
Riverside for their support. We thank the Department of
Soil and Environmental Sciences, UC Riverside, the TriCal
Inc., and the Drip-In Irrigation Co. for technical support.
The ®rst author was in part supported by a German
Academic Exchange Service-Graduate Scholarship
(DAAD, HSP II/AUFE) and the Storkan-Hanes Foundation.
We thank J. Darsow and A. de Bever for technical assistance.
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