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. schachtiipopulation 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. schachtiifemale 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 wereFusarium oxysporum,Fusariumsp. nov., andDactylella oviparasitica.Paecilomyces lilacinusand some non-identi®ed fungi occurred less frequently. Suppressiveness was transferred at a rate of one cyst from suppressive soil amended to 110 g ofH. schachtii-infested conducive soil. Heat treatment of suppressive soil for 30 min at 558C eliminatedH. schachtiisuppressiveness and reduced F. oxysporumpopulations to the detection level.q2001 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 suita-ble 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; RodrõÂ-guez-KaÂbana and Morgan-Jones, 1988). A particularly well-documented example of soil suppressiveness against a plant-parasitic nematode was the study of a population density
decline ofHeterodera avenaeWoll. in England (Kerry et al.,
1980). In suppressive soil,Nematophthora gynophilaKerry
and Crump destroyed young females ofH. avenaebefore they
could mature to cysts (Crump and Kerry, 1977). In addition,
Verticillium chlamydosporiumGoddard parasitized the eggs of this nematode. The concerted parasitic activity of both fungi was considered the main factor in the natural population
suppression ofH. avenae(Kerry et al., 1980).
Recently, we have shown that soil suppressiveness
against Heterodera schachtii Schm. at a ®eld site at the
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-detect-able levels by soil fumigation and it was transfernon-detect-able. 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 suppres-sive 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 ofH. schachtiiwas 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 matterq2001 Elsevier Science Ltd. All rights reserved. PII: S 0 0 3 8 - 0 7 1 7 ( 0 0 ) 0 0 1 0 8 - 5
www.elsevier.com/locate/soilbio
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 napusL., the soil in the trial area had an initialH. schachtii population density of 18 eggs g21soil. The area was chiseled to a depth of 45 cm, disked, and NPK fertilizer
(336 kg ha21, 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 ha21) (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 30-cm 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) ofH. 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
at220 to230 kPa. After one month, one 5-week-old
seed-ling of Swiss chard (Beta vulgarisL. subsp. cicla(L.) W.
Koch cv. Large White Ribbed, Lockart Seeds Inc., Stock-ton, CA) was planted into the center of each planting site. Additional fertilizer solution was applied via overhead
sprinkling as needed (total: 234 kg ha21Miracle 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. ha21) 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 Fish-er'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 w21) with silica sand.
Portions of this soil±silica mix were fumigated with methyl
iodide (500 kg ha21) (Becker et al., 1998).
2.2.1. H. schachtii female population development in root observation chambers
Suppressive 9E soil was mixed with methyl
iodide-fumi-gated ®eld soil (1:9, dry w w21) and placed into
custom-made 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 observa-tion 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 ofH. schachtii.
Each root observation chamber was fertilized with 100 ml
nutrient solution (Miracle Gro 10 g 3.79 l21of water, 15%
N, 30% P, 15% K) twice per week. Starting three weeks
after infestation, the females ofH. schachtiivisible 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 hand-picked from each chamber and examined for fungal infesta-tion as described below. In addiinfesta-tion, 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
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. schachtiieggs 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 kg21). Cysts containing fungi were enumerated.
The frequencies of infested cysts were expressed as percen-tage of the total number of cysts examined per root
observa-tion 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 w21) of methyl iodide-fumigated 9E ®eld soil
(500 kg ha21) and sandy soil. The sandy soil was infested
with cysts containing ca. 50,000 eggs ofH. schachtiiin the
®rst experiment and ca. 70,000 eggs ofH. schachtiiin 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% suppres-sive 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
emer-gence 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 oven-dry weights were determined.
2.2.4. Heat fractionation of soil micro¯ora
Nematode-suppressive 9E soil (405 g, moisture content:
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).
Fusar-ium oxysporum Schlecht. was enumerated on Komada's medium (1975) that was prepared without the micro-nutri-ents.
Heat-treated soils were mixed 1:9 (dry w w21) with
methyl iodide-fumigated soil from theH. schachtii
-suppres-sive 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
repli-cations. 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 l21 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. schachtiigenerations, 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. schachtiicysts 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 P0:05 if the treatment Fhad a P#
0:05:The test was performed atP0:10 if the probability
for the treatmentFwas between 0.10 and 0.05.
3. Results
3.1. Field trial
At 600 DD the number ofH. schachtiicysts g21soil in the
A. Westphal, J.O. Becker / Soil Biology & Biochemistry 33 (2001) 9±16
Fig. 1.H. schachtiipopulation 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 atP0:05:
1Tops of Swiss chard were cut off at 5 cm stubble. Bars represent standard error
n6:
3rd 4th 5th 6th 7th 8th 9th 10th
0 300 600 900 1200 1500 1800
Observation week post inoculation
suppressive
conducive
F
emales
of
H.
schachtii
per
580
cm
2
* *
* *
*
Fig. 2. Female populations ofH. schachtiion mustard-greens roots in suppressive and conducive soil in root observation chambers. *Signi®cant differences were determined atP0:05 with Fisher's protected LSD.
suppressive plots was signi®cantly higher than in the condu-cive plots (Fig. 1A). At DD 750, 900 and 1050 the popula-tion densities were not signi®cantly different in the suppressive and conducive plots. Beginning at 1200 DD,
the numbers of cysts g21soil 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 g21
soil remained low in the suppressive plots. The overall
increase of nematode cysts g21 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; P0:1826: 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
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 condu-cive 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-iden-ti®ed fungal species in low frequencies. D. oviparasitica
was most frequently isolated in the ®rst experiment from
infectedH. schachtiieggs, whileF. oxysporumand
Fusar-iumsp. 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.
A. Westphal, J.O. Becker / Soil Biology & Biochemistry 33 (2001) 9±16
Table 1
Percentage of females and cysts of H. schachtiiwith fungal infestation in root observation chambers means^SE;n3 in both experiments)
Soil status First experimenta Second experimentb
Females Cysts Cysts
Suppressive 2:0^2:0 67.0^11:0 34:6^0:8
Conducive 0:7^0:7 4.0^3:1 7:2^7:2
Pfor treatmentF 0.4950 0.0062 0.0200
a H. schachtiispecimens 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. schachtiicysts 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.
Table 2
Amendment of conduciveH. schachtii-infested soil with cysts from different sources in comparison with non-amended infested soil means^SE;n4 in both experiments). Cysts were collected from suppressive 9E ®eld soil or from the corresponding root observation chambers
Transfer source Experiment 1 Experiment 2
Root dry weight Cysts g21soil Eggs per cyst Root dry weight Cysts g21soil Eggs per cyst
Non-amended (conducive) 3:5^0:3 1.8^0.1 51.2^9.8 1.5^0.2 4.2^0.3 51.7^12.9 Cysts, conducive soil (rootbox) 3:1^0:4 1.7^0.1 52.7^10.3 1.9^0.2 3.9^0.2 63.5^11.9 Cysts, suppressive soil (rootbox) 3:0^0:1 1.2^0.1 52.6^7.1 2.2^0.3 2.8^0.1 34.5^4.7 Cysts, 1% suppressive 9E ®eld
soil
3:3^0:3 1.4^0.1 76.6^11.0 2.2^0.5 2.4^0.2 43.3^4.7
1% Suppressive ®eld 9E soil 2:9^0:3 1.4^0.0 66.9^7.5 1.9^0.3 2.7^0.2 44.9^8.1
LSD P0:05 0.3 0.50
3.1.3. Transfer of suppressiveness with cysts from suppressive and conducive root observation chambers
In both transfer experiments the numbers of cysts g21soil
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 g21after 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 g21soil 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. oxysporumcfu
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
experi-ment, 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 ofH. schachtii were a major
target of nematode-suppressiveness in the presented experi-ments. In the ®eld trial, the number of eggs per cyst was lower in the suppressive than in the conducive plots begin-ning 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 g21 soil in the
suppressive plots might have been the consequence of the suppressiveness on the nematode eggs, since the time inter-val between the difference in the number of eggs per cyst
(DD 750) and the difference in the number of cysts g21soil
(DD 1200) was suf®cient for one nematode generation to
occur. The number of cysts g21soil 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. schachtiicysts 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-suppressive-ness.
The transfer attempt with cysts from the suppressive soil
was based on the transferability ofH. schachtii
suppressive-ness with portions of suppressive 9E soil (Westphal and Becker, 2000). As little as 1% of suppressive soil transferred
and establishedH. schachtii suppressiveness in fumigated
®eld plots. Cysts, which had developed in suppressive soil in observation chambers, transferred nematode-suppressive-ness 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 theH. schachtiicyst as the primary
target of the nematode suppressiveness. It has been
suggested that certain stages of the life cycle ofH. 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 experi-ments, 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 suppressH. schachtiipopulations.
The ability to spread throughout the soil from the source of inoculum or the current food base is an important char-acteristic of successful antagonists. It was shown earlier that
certain fungal egg parasites can infectG. 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
estab-lishment ofH. 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 nega-tive effects of fungal culture ®ltrates on nematode hatch and/ or mobility have been shown (Mani and Sethi, 1984; Sikora
et al., 1990), in situ production and ef®cacy of such meta-bolites 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.
oxysporumpopulations in a fusarium wilt-suppressive soil (Rouxel et al., 1977) and that the reintroduction of resident
F. oxysporum strains can reestablish the
wilt-suppressive-ness (Rouxel et al., 1979). In our trials, F. oxysporumand
Fusarium sp. nov. were frequently isolated from cysts developed in suppressive 9E soil and suppressiveness was
eliminated at the same thermal fractionation step at whichF.
oxysporumwas 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
pathogeni-city 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
popu-lations likeGloboderaspp. andH. 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
Meloido-gyne 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). WhileD.
oviparasi-tica parasitized H. schachtii eggs in vitro (Stirling and
Mankau, 1979) its potential to suppress sugar beet cyst
nematode populationsin vivohas 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
popula-tion development in the second generapopula-tion ofH. schachtii
between conducive and suppressive soil. However, the high degree of egg parasitism almost exclusively in the suppres-sive test variant suggested that the main interference with the population development preceded these observations. Earlier tests had already indicated that the number of infec-tive 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 parasitizeH. schachtii eggs. But
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®ll-ment of the requireful®ll-ments 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 assis-tance.
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