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APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Dec. 2002, p. 6383–6387 Vol. 68, No. 12
0099-2240/02/$04.00⫹0 DOI: 10.1128/AEM.68.12.6383–6387.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.
Field Studies Using a Recombinant Mycoinsecticide (Metarhizium
anisopliae) Reveal that It Is Rhizosphere Competent
Gang Hu and Raymond J. St. Leger*
Department of Entomology, University of Maryland, College Park, Maryland 20742
Received 1 April 2002/Accepted 29 August 2002
In the summer of 2000, we released genetically altered insect-pathogenic fungi onto a plot of cabbages at a
field site on the Upper Marlboro Research Station, Md. The transformed derivatives of Metarhizium anisopliae
ARSEF 1080, designated GPMa and GMa, carried the Aequorea victoria green fluorescent protein (gfp) gene
alone (GMa) or with additional protease genes (Pr1) (GPMa). The study (i) confirmed the utility of gfp for
monitoring pathogen strains in field populations over time, (ii) demonstrated little dissemination of transgenic
strains and produced no evidence of transmission by nontarget insects, (iii) found that recombinant fungi were
genetically stable over 1 year under field conditions, and (iv) determined that deployment of the transgenic
strains did not depress the culturable indigenous fungal microflora. The major point of the study was to
monitor the fate (survivorship) of transformants under field conditions. In nonrhizosphere soil, the amount of
GMa decreased from 10
5
propagules/g at depths of 0 to 2 cm to 10
3
propagules/g after several months. However,
the densities of GMa remained at 10
5
propagules/g in the inner rhizosphere, demonstrating that rhizospheric
soils are a potential reservoir for M. anisopliae. These results place a sharp focus on the biology of the soil/root
interphase as a site where plants, insects, and pathogens interact to determine fungal biocontrol efficacy,
cycling, and survival. However, the rhizospheric effect was less marked for GPMa, and overall it showed
reduced persistence in soils than did GMa.
Biocontrol experiments with fungi have often produced in-
consistent results, and this has deterred commercial develop-
ment (14). However, many fungi are amenable to genetic mod-
ification for purposes of enhancing utility for disease control,
insect and plant pest management, or bioremediation. In such
cases, genetically engineered fungi may provide environmen-
tally preferred alternatives to current chemical-based control
strategies. Much attention has focused on the ascomycete en-
tomopathogen Metarhizium anisopliae. It is widely applied
abroad, was recently registered for use in the United States
and Europe (2), and offers particular promise as a suppressive
agent for many soil insect pests that would otherwise provide a
particular challenge to pest control specialists (6, 9). The ad-
dition and expression of pesticidal genes in M. anisopliae is
quite straightforward and was used to genetically engineer a
strain that overexpresses toxic proteases and kills insects faster
than the wild type dose in laboratory tests (12).
This technology has potential for pest control (13), but there
is an inherent uncertainty about the efficacy, survivability, and
environmental risk posed by any introduced or engineered
fungus because of our lack of knowledge about the fate of
fungal genotypes at the population and ecosystem levels (1, 5).
To achieve successful, reproducible, and safe (from the risk
management point of view) biological control, we need to be
able to study the ecology of the transformed genotype. After
extensive laboratory analysis to test potential risks, including
acquisition and evaluation of host range information, we were
granted approval (38567-NMP-R) from the Biopesticides and
Pollution Prevention Division of the EPA Office of Pesticide
Program to conduct a planned release in a field of cabbage
plants. The approval constrained us to establishing the tech-
nology required to monitor the fate of genetically enhanced M.
anisopliae and to using this technology to determine the po-
tential of engineered strains to establish and disperse over
1-year test period. This was achieved by combining conven-
tional techniques used by soil microbiologists and ecologists
with gfp (encoding green fluorescent protein) as a molecular
marker. Since root exudates stimulate the growth of bacterial
and fungal populations and the rhizosphere is of great impor-
tance to plant health and fertility (16), we focused on it as a
potential refuge for transgenic fungi that could increase their
persistence in the environment.
MATERIALS AND METHODS
Fungi and host. The wild-type M. anisopliae strain ARSEF1080 was originally
isolated from larvae of the cabbage looper (Trichoplusia ni: Noctuidae, Lepidop-
tera) in Florida. Allozyme analysis identified M. anisopliae strain 1080 as belong-
ing to genotypic class 14, which is rare in North America (10). The recombinant
strain gpd-Pr1-4 contains four copies of the Pr1a subtilisin gene under control of
the constitutive gpd promoter from Aspergillus nidulans (12).
Transformation. The wild-type strain and gpd-Pr1-4 were transformed with
pEGFP-CP (obtained from Don Nuss, Center of Agricultural Biotechnology,
University of Maryland, College Park, Md. Plasmid EGFP-CP carries the gene
for EGFP1 (a variant of the green fluorescent protein) under control of the
glyceraldehyde 3-phosphate dehydrogenase (gpd) promoter from Cryphonectria
parasitica (15).
Transformation was performed using a previously established protocol (12)
with the modification that inoculated plates were incubated at 28°C for 30 h and
transformants visible under a fluorescence microscope were rescued using a glass
pipette. Transformants were purified by generating single-spore colonies, and
these were subcultured on potato dextrose agar five times to confirm stability.
CHEF (clamped homogeneous electric field) gel analysis employing a CHEF-
DR-III apparatus (Bio-Rad) was used as described previously (11) to identify
transformants carrying the pr1 and egfp1 genes at unlinked locations, i.e., on
different chromosomes, so as to allow effective recovery of recombination events.
Fluorescent transformant progeny of wild-type (GMa) and gpd-Pr1-4 (GPMa)
* Corresponding author. Mailing address: 4112 Plant Science Build-
ing, University of Maryland, College Park, MD 20742-4454. Phone:
(301) 405-5402. Fax: (301) 314-9290. E-mail: rl106@umail.umd.edu.
6383
chosen for the field trial had parent-type growth rate, colony morphology, level
of conidial production, and relative virulence as determined by standard labo-
ratory protocols (12).
Fungal release. The field site was located in the University of Maryland Upper
Marlboro research farm, Upper Marlboro, Md. It is a frequently cultivated
(tilled) site, and the soil is a Monmouth fine sandy loam. The rectangular 0.2-ha
field site was designed to allow for efficient maintenance and the detection of any
dispersal of recombinant fungus outside the confines of the plot. The plot
consisted of two 0.05-ha fungal application areas, each consisting of seven rows
of cabbages separated by a five-row buffer. A barren, plant-free zone surrounded
the subplots, and a low-maintenance fallow zone outside the plot was also
monitored for marked (recombinant) fungus through the field tests. The cabbage
plants (var. Early Flat Dutch) were sprayed on a low-wind day (14 June 2000)
with a water-based application containing 0.01% Silwet L77 (Loveland Indus-
tries, Greeley, Colo.) at a rate of 10
13
spores per ha. The ground and the plants
in each row were sprayed with a backpack-mounted hydraulic sprayer (18-in.
spray band). Application area 1 received GPMa; application area 2 received
GMa. Because the purpose of this study was not quantitative, i.e., we were not
attempting to compare virulence between GMa and GPMa, a single experimen-
tal plot for each was deemed sufficient.
Transfer of the fungus by mechanical means was minimized by using a field test
design and field test protocol that included the buffer zone and tool and footwear
disinfestation.
Collection of soil samples. Before the start of the experiment and daily (first
week), weekly (first 2 months), and at monthly intervals thereafter, soil samples
were taken at defined depths using a 1-cm soil core sampler from 50, 20, and 15
evenly spaced locations within the application zones, buffer zone, and fallow
zone, respectively. Soil samples from the innermost rows of the application areas
were taken at 4 to 5 cm from the cabbage tap root as well as alongside the tap
root (0 to 1 cm) to check for uneven distribution and persistence of spores close
to the rhizosphere (vicinity of the root). Subsamples of soil were used for
dry-weight determination.
Soil samples were stored for up to 3 days at 4°C before the propagules of M.
anisopliae were quantitated by using Veens semiselective agar medium (3). Soil
samples (1 g) were sonicated briefly in 0.05% Tween 80, serial dilutions were
made, and 0.1-ml portions were spread on each of two to five plates of selective
medium per dilution. The detection limit was less than 20 CFU per g of soil.
After range finding experiments, only dilutions near those likely to produce
countable numbers of CFU (up to 300 per plate) were plated. The medium was
supplemented with hide protein azure to detect constitutive protease production
by GPMa (12) and scanned with UV light to distinguish GFP-expressing recom-
binants from indigenous strains of Metarhizium spp. Proc MIXED was used to
test for differences in rates of decline of spore titers between GMa and GPMa.
Spore count data were transformed to the log scale before analyses. Means were
compared using the Student-Newman-Keuls (SNK) method. All analyses were
carried out using the SAS software package V8.2. (SAS Institute, Cary NC) (␣⫽
0.05).
Indigenous strains of M. anisopliae were characterized by allozyme analysis,
which allows a large number of strains to be analyzed for recombination events,
which we do by assigning a genetic basis to electrophoretic banding patterns (10).
Cycloheximide was omitted from the Veens medium to study the abundance and
composition of fungal populations other than M. anisopliae (total filamentous
fungi).
Rhizosphere competence. Samples taken alongside the root by using a 1-cm
core borer may contain nonrhizospheric bulk soil that will cause the rhizospheric
titer to be underestimated. Therefore, 4 months after planting, eight randomly
selected cabbage plants from each application site were cut off above the soil and
root samples with adhering soil (rhizosphere samples) were taken. Roots were
sectioned into 2-cm segments, and the segments were shaken to collect soil
adhering loosely to the roots (outer rhizosphere). To collect soil adhering after
shaking but subject to removal by washing (inner rhizosphere), roots were
weighed and ultrasonicated (15 s) in sterile water. Outer and inner rhizospheric
suspensions were plated onto Veens medium for plate assays. Subsamples of the
rhizospheric suspensions were used for dry-weight determinations. To sample
rhizoplane M. anisopliae, root segments were further washed (10 times), air
dried, weighed, and placed on Veens medium.
Monitoring strain stability. Integrative transformants are very stable when
grown for long periods in the absence of selection in pure culture under labo-
ratory conditions (4, 11). However, stability may be different in a complex
environment, in which case we reasoned that it would be unlikely for two
unlinked markers (Pr1 and GFP) to be lost at once. There should usually be at
least one marker remaining to positively distinguish a transformant from a native
organism and detect recombination. To determine whether fungi retain the
marker elements in their original form, we screened M. anisopliae isolates re-
covered from the application sites for any examples that have lost GFP but
retained constitutive expression of Pr1 or for GFP-expressing strains demon-
strating one or more phenotypic characteristics that differ significantly from
those exhibited by the input transgenic strains. The growth rate, colony mor-
phology, and level of conidial production were tested as described previously
(12).
Monitoring nontarget arthropods. During the course of the field tests, 50 pit
fall traps embedded in the soil in and around the application sites were used to
collect nontarget arthropods, particularly carabid beetles (important predators).
These were maintained in the laboratory to determine if disease developed, and
healthy as well as infected insects were analyzed for the presence of the marked
fungus. Insects were placed in petri dishes containing Veens agar medium.
Fungal growth over the medium was examined under UV illumination for GFP
fluorescence. We anticipated that background levels would be high within the
sprayed areas. Consequently, a representative portion (10%) of nontarget insects
recovered from these areas were washed briefly in acetone followed by 95%
ethanol to remove surface-associated fungal propagules. The individual (identi-
fied to species) insects were squashed and placed on Veens agar medium to
detect internalized transgenic M. anisopliae spores and mycelia. These experi-
ments were designed to determine the extent to which transgenic M. anisopliae
strains can be recovered from insects (including nonhosts) within an intense
deployment area in comparison to the extent found in the surrounding and
remote sampling areas, i.e., to determine the potential of insect-mediated dis-
persal to nontargeted deployment areas.
RESULTS AND DISCUSSION
Effects on the indigenous culturable fungal microflora. Be-
fore the application, soil samples from the top 3 cm were
collected from locations within the application areas, buffer
zone, and surrounding fallow zone. Veens medium minus cy-
cloheximide was used to sample total fungal populations. Each
fallow location contained at least 10
6
propagules/g comprising
30 or more fungal species. By contrast, the cultivated buffer
and application areas were comparatively impoverished and
contained only seven species with a total CFU of less than 10
4
propagules/g. Veens medium containing cycloheximide grew
M. anisopliae from only three sites with a mean at these sites of
212 ⫾ 24 propagules/g. Allozyme analysis of 20 colonies picked
at random identified two genotypic classes based on electro-
phoretic phenotypes (10). Based on assigned genotypes (10),
14 of the colonies belonged to class 20 (field strain 1). The
remaining 6 (field strain 2) differed only at the glutathione
reductase locus, which demonstrated a mobility of 121 com-
pared to the 100 shown by class 20. For several months after
spraying with transformants, the indigenous strains of M.
anisopliae were infrequently detected on plates. However, this
would be accounted for by the large initial dilution (dilution
factor 100) required to obtain countable numbers of GMa and
GPMa. Sampling in the spring of the second year of bulk
(nonrhizosphere) soil revealed that the original three locations
still contained mixed populations of the two indigenous strains.
Thus, there is no evidence of a detrimental effect on indige-
nous populations from introducing GMa and GPMa.
Total filamentous fungal populations in soil 0 to 1 cm from
the roots was analyzed using repeated measurements analysis
of variance (ANOVA) to determine whether the development
of fungal populations is affected by the application of trans-
genic M. anisopliae (interaction between time and treatment).
On the Veens medium minus cycloheximide, there was no
significant difference in the total rhizospheric fungal popula-
tions or its composition in either GPMa- or GMa-treated
plants compared to the untreated plants in the buffer zone. In
6384 HU AND ST. LEGER APPL.ENVIRON.MICROBIOL.
fact, with (Fig. 1) or without (data not shown) application of
GMA, the population levels of the three most frequently iso-
lated genera, Paecilomyces, Penicillium, and Aureobasidium,
did not change significantly over time.
With the caveat that not all fungi are culturable, these re-
sults indicate that there is minimal risk of the engineered
fungus displacing naturally occurring fungi. Given the impov-
erished fungal microflora observed in the cultivated compared
to the noncultivated land the impact of introduced microor-
ganisms in general is likely to be minor compared to that of
common agricultural practices such as plowing or crop rota-
tion.
Soil persistence monitoring. At 1 h following application,
the differences in the titer of GMa (mean ⫽ 2.45 ⫻ 10
5
,
standard deviation [SD ⫽ 6.8 ⫻ 10
4
] CFU/g) and GPMa (mean
⫽ 2.09 ⫻ 10
5
[SD ⫽ 5.2 ⫻ 10
4
] CFU/g) were not significant.
The data set for subsequent decline in spore numbers in the
vicinity of cabbage roots (0 to 1 cm) behaved as piecewise
regressions (Fig. 2). Thus, while soil titers of GMa and GPMa
declined by 56 and 73%, respectively, in the first week, these
strain differences were not significant (F ⫽ 2.9, P ⬎ 0.05).
Differences between the strains became significant from day 10
(F ⫽ 7.8, P ⬍ 0.05). Thus, titers of GMa between 4 months
(October) and 10 months (April of the second year) were
reduced by 30% from 2.96 ⫻ 10
4
, (SD ⫽ 1.00 ⫻ 10
3
) CFU/g to
2.00 ⫻ 10
4
(SD ⫽ 5.29 ⫻ 10
3
) CFU/g, respectively. During the
same period, titers of GPMa declined by 70% from 1.53 ⫻ 10
4
(SD ⫽ 4.16 ⫻ 10
3
) to 4.76 ⫻ 10
3
(SD ⫽ 1.15 ⫻ 10
3
) CFU/g.
Reduced fitness and survivability could reasonably be derived
from deleterious effects of the additional genetic modifica-
tions, compared to the situation for transgenic fungi expressing
GFP only. If so, then M. anisopliae may not just persist in the
soil in a dormant state but characteristics for soil survival may
include gene expression that can be interfered with by plasmid
integration.
Stability under field conditions. Closely related, therefore,
to the issue of population dynamics is the question whether an
intensive deployment protocol into complex microbial commu-
nities will promote the generation and amplification of altered
transgenic M. anisopliae strains. Growth rates of colonies iso-
lated from application areas were similar to those of the input
transgenic strains. In some cases the isolated colonies differed
from their progenitors in producing fluffier, raised colonies and
fewer conidia. In each case, the original phenotype returned
after three serial propagations on Sabouraud dextrose agar,
consistent with experimental and physiological variations
rather than genetic differences. During the course of this study,
more than 50,000 colonies were examined, and all fungi ex-
pressing GFP in application area 1 were also constitutive pro-
ducers of Pr1 and vice versa. This indicates stability of plasmid
DNA in the chromosomes and provides no evidence for re-
combination. At 10 months after spraying, allozyme analysis
was performed on 100 colonies chosen at random from loca-
tions also containing field strains 1 and 2. No interisolate vari-
ability was detected with each of the eight enzymes possessing
electromorphic forms characteristic of genotypic class 14, the
class to which strain 1080 belongs (10). In particular, none of
the eight loci displayed symmetrical three-banded phenotypes
characteristic of heterozygotes or two banded electromorphs,
which would be evidence of mixed alternatively homozygous
cultures.
Spatial distribution of transgenic M. anisopliae. Soil samples
were taken at 4 to 5 cm as well as alongside cabbage tap roots
(0 to 1 cm) to check for uneven distribution of spores. For
GMa, the ratio of fungi alongside the root to 4 to 5 cm from
the root increased from 1.4:1 (1 month postinoculation) to
about 3:1 (4 months postinoculation) (Fig. 3). The value re-
mained high after the cabbage plants were killed by frost (8
months), indicating that GMa was persisting on the decaying
organic matter. The rhizosphere effect is also apparent in
FIG. 1. Mean soil titers of propagules of Paecilomyces farinosus,
(Œ) Penicillium spp. (●), and Aureobasidium pullulans (■) in the ap-
plication area treated with M. anisopliae GMa. Soil samples were taken
within 1 cm from cabbage plant roots at depths of 0 to 2 cm, and fungal
propagules were quantified on Veens medium minus cycloheximide.
Error bars indicate SD.
FIG. 2. Changes in the soil titer of GMa () and GPMa (●), over
300 days, within 1 cm of cabbage plant roots at depths of 0 to 2 cm.
Spore count data were transformed to the log scale. The lines repre-
sent the model outcome of the population decline of GMa (dashed
line) and GPMA (solid line). The analysis was conducted using Proc
MIXED, SAS. Error bars indicate SD.
V
OL. 68, 2002 RECOMBINANT ENTOMOPATHOGENS 6385
GPMa, although with a more rapid decline in spore titers,
reflecting its reduced persistence.
Samples from the buffer and fallow zones contained no
transgenic M. anisopliae or contained insufficient numbers to
be detected using dilution plate counts.
Rhizosphere competence. To further analyze rhizosphere
competence, samples of soil were taken directly from roots 4
months after application of GMa. A four-fold-larger popula-
tion of fungal propagules was observed in the inner rhizo-
sphere soil than in the outer rhizosphere soil at the top 2 cm of
the root base. This suggests that close proximity to the root and
its exudates is involved in the rhizospheric effect. The titer of
GMa at the root base (3.1 ⫻ 10
5
[SD ⫽ 2.5 ⫻ 10
4
] CFU) was
close to the original inoculum load. Most other studies using
fungi known to be good root colonizers show a decline, per-
haps because the initial population added is to large for the
carrying capacity of the root (8). Evidently, soil in the vicinity
of plant roots provides a refuge for M. anisopliae from factors
in the environment that reduce fungal titer.
The colonization of roots by GMa in the outer and inner
rhizosphere of roots formed a gradient, with the rhizosphere
effect decreasing with increasing depth (Fig. 4). The presence
of fungal propagules more than 10 cm from the stem in the
inner but not the outer rhizosphere implies some degree of
vertical movement along the roots through fungal growth or
cracks in the soil or via percolating water. When unwashed
root segments were placed on Veens medium, growth of flu-
orescent fungus was observed from roots up to 10 cm from the
stem. In spite of this proximity, a lot of the fungus could be
removed from the roots by serial washings (Fig 5). The patchy
distribution of fungal colonies on washed roots compared with
the total coverage of unwashed roots implies weak adhesion by
most propagules.
If rhizosphere competence is a general phenomenon among
insect pathogens, its impact on plant ecology could be consid-
erable and it possesses implicit coevolutionary implications. In
addition, most efforts employing M. anisopliae for biocontrol
have ignored habitat preferences and survival outside the host.
Evidently, factors associated with soil dwelling may be even
more critical in the selection of an isolate than virulence per se
(1). However, rhizosphere competence also imposes potential
risks since it might increase the difficulty of eliminating a
pathogen following unanticipated and deleterious environ-
mental effects.
Monitoring of naturally occurring insects. A natural infes-
tation of Pieris rapae occurred on the cabbages. Eight of 20 and
6 of 20 third-instar or older P. rapae larvae collected from
application areas 1 and 2, respectively, within 10 days of the
application died from infections with GPMa and GMa, respec-
tively. A majority of 43 P. rapae larvae collected from the test
sites 1 month following application died of bacterial infections
and parasitoids and further analysis of these was not possible.
However, three larvae died from fungal infections and pro-
duced spores of GMa. Flea beetles (Alticinae) collected from
cabbages up to 1 week after spraying also had spores of GMa
and GPMa on their surfaces, but none of 30 beetles main-
tained for 10 days in the laboratory on cabbage seedlings suc-
FIG. 3. Effect of proximity to cabbage roots on the persistence of M. anisopliae under field conditions. Mean soil titers of propagules of M.
anisopliae GMa and GPMa at depths of 0 to 2 cm are shown. Error bars indicate SD.
FIG. 4. Mean number of propagules of M. anisopliae GMa at dif-
ferent depths in the outer (■) and inner (䊐) rhizosphere of cabbage
roots, 4 months after application. Error bars indicate SD.
6386 HU AND ST. LEGER APPL.ENVIRON.MICROBIOL.
cumbed to overt fungal infection. More than 3,000 arthropods
were collected from pit fall traps in and around the application
sites in the summer of the first year. These included four
species of carabids (Amara and Stenolophus spp.), other bee-
tles including predatory rove beetles (Staphylinidae), five spe-
cies of ants, and many types of aphids, springtails, spiders, and
mites. About 5% of the arthropods monitored under labora-
tory conditions died of a variety of overt fungal infections and
27% died of septicemia or other unidentified causes, but we
did not detect external or internalized M. anisopliae in healthy,
sick, or dead insects. These results suggest the potential of
insect-mediated dispersal to nontargeted deployment areas is
low.
Conclusion. The results of this trial do not suggest any safety
concerns to using GMa or GPMa that would detract from their
being environmentally preferred alternatives to current chem-
ical-based control strategies. However, their survival into the
second year is significant, since time increases the possibility of
adaptation for increased fitness (7). It cannot be assumed,
therefore, that either strain will die out because of current
reduced fitness. There was no evidence for phenotypic insta-
bility of the introduced fungi, but this might not be expected if
genetic changes with clear phenotypes follow the punctuated-
equilibrium model of evolution, with long periods of apparent
stability punctuated by large infrequent changes. Given that
the long-term fitness of a genetically engineered pathogen that
persists in nature is difficult to predict, it is all the more essen-
tial to establish technologies such as the use of gfp that will
permit informed risk assessment through monitoring the fate
of marked strains.
ACKNOWLEDGMENTS
This work was supported by the USDA risk assessment program
(CSREES-99331208284).
We thank Mark Spiknall and the agricultural technicians at the
Upper Marlboro Research Station for their comprehensive technical
assistance in planting and maintaining the field site. Several under-
graduate students from the 2000 and 2001 BSCI105 course at the
University of Maryland, particularly Franklin Johnson, Jason Lloyd,
and Jordan Newmark, participated in collecting and analyzing samples.
We thank Mary Christman, Department of Agriculture, University of
Maryland, for help with statistical analyses.
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fungal pathogens of insects and weeds, p. 219–238. In T. M. Butt, C. Jackson,
and N. Morgan (ed.), Fungal biocontrol agents: progress, problems and
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FIG. 5. UV micrographs of 2- to 5-cm-deep cabbage roots from
application area 2 (4 months after spraying with GMa) placed on
Veens medium for 48 h and showing growth by fluorescent M. aniso-
pliae. The roots were shaken free of rhizosphere soil (A) or, in addi-
tion, ultrasonicated and subjected to a series of 10 water washes (B),
which removed the inner rhizosphere and most but not all fungal
propagules (indicated by arrows).
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