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Field Studies Using a Recombinant Mycoinsecticide (Metarhizium anisopliae) Reveal that It Is Rhizosphere Competent


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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 105 propagules/g at depths of 0 to 2 cm to 103 propagules/g after several months. However, the densities of GMa remained at 105 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.
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APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Dec. 2002, p. 6383–6387 Vol. 68, No. 12
0099-2240/02/$04.000 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
propagules/g at depths of 0 to 2 cm to 10
propagules/g after several months. However,
the densities of GMa remained at 10
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.
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:
chosen for the eld 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 eld 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 ne sandy loam. The rectangular 0.2-ha
eld site was designed to allow for efcient maintenance and the detection of any
dispersal of recombinant fungus outside the connes of the plot. The plot
consisted of two 0.05-ha fungal application areas, each consisting of seven rows
of cabbages separated by a ve-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 eld 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
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 sufcient.
Transfer of the fungus by mechanical means was minimized by using a eld test
design and eld test protocol that included the buffer zone and tool and footwear
Collection of soil samples. Before the start of the experiment and daily (rst
week), weekly (rst 2 months), and at monthly intervals thereafter, soil samples
were taken at dened 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 briey in 0.05% Tween 80, serial dilutions were
made, and 0.1-ml portions were spread on each of two to ve plates of selective
medium per dilution. The detection limit was less than 20 CFU per g of soil.
After range nding 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) (␣⫽
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 lamentous
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 signicantly from
those exhibited by the input transgenic strains. The growth rate, colony mor-
phology, and level of conidial production were tested as described previously
Monitoring nontarget arthropods. During the course of the eld 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
uorescence. 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 briey in acetone followed by 95%
ethanol to remove surface-associated fungal propagules. The individual (identi-
ed 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.
Effects on the indigenous culturable fungal microora. 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
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
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 identied two genotypic classes based on electro-
phoretic phenotypes (10). Based on assigned genotypes (10),
14 of the colonies belonged to class 20 (eld strain 1). The
remaining 6 (eld 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 lamentous 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
signicant 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
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 signicantly 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 microora 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-
Soil persistence monitoring. At 1 h following application,
the differences in the titer of GMa (mean 2.45 10
standard deviation [SD 6.8 10
] CFU/g) and GPMa (mean
2.09 10
[SD 5.2 10
] CFU/g) were not signicant.
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 rst week, these
strain differences were not signicant (F 2.9, P 0.05).
Differences between the strains became signicant 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
, (SD 1.00 10
) CFU/g to
2.00 10
(SD 5.29 10
) CFU/g, respectively. During the
same period, titers of GPMa declined by 70% from 1.53 10
(SD 4.16 10
) to 4.76 10
(SD 1.15 10
) CFU/g.
Reduced tness and survivability could reasonably be derived
from deleterious effects of the additional genetic modica-
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
Stability under eld 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 amplication 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 ufer, 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 eld 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
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 quantied 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.
GPMa, although with a more rapid decline in spore titers,
reecting its reduced persistence.
Samples from the buffer and fallow zones contained no
transgenic M. anisopliae or contained insufcient 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
[SD 2.5 10
] 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 u-
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 difculty 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 eld 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 () rhizosphere of cabbage
roots, 4 months after application. Error bars indicate SD.
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 rst year. These included four
species of carabids (Amara and Stenolophus spp.), other bee-
tles including predatory rove beetles (Staphylinidae), ve 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 unidentied 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
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 signicant, since time increases the possibility of
adaptation for increased tness (7). It cannot be assumed,
therefore, that either strain will die out because of current
reduced tness. 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 tness of a genetically engineered pathogen that
persists in nature is difcult 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.
This work was supported by the USDA risk assessment program
We thank Mark Spiknall and the agricultural technicians at the
Upper Marlboro Research Station for their comprehensive technical
assistance in planting and maintaining the eld 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.
1. Bidochka, M. J. 2001. Monitoring the fate of biocontrol fungi, p. 193218. In
T. M. Butt, C. Jackson, and N. Morgan, (eds.), Fungal biocontrol agents:
progress, problems and potential. CAB International, Wallingford, United
2. Butt, T. M., C. Jackson, and N. Magan. 2001. Introduction-fungal biological
control agents: progress, problems and potential, p. 18. In T. M. Butt, C.
Jackson, and N. Morgan (ed.), Fungal biocontrol agents: progress, problems
and potential. CAB International, Wallingford, United Kingdom.
3. Goettel, M. S., and G. D. Inglis. 1997. Fungi: hyphomycetes, p. 367394. In
L. Lacey, (ed.), Manual of techniques in insect pathology. Academic Press,
Inc., San Diego, Calif.
4. Goettel, M. S., R. J. St. Leger, S. Bhairi, M. K. Jung, B. R. Oakley, D. W.
Roberts, and R. C. Staples. 1990. Pathogenicity and growth of Metarhizium
anisopliae stably transformed to benomyl resistance. Curr. Genet. 17:129
5. Hajek, A. E., I. Delalibera, and M. L. McManus. 2000. Introduction of exotic
pathogens and documentation of their establishment and impact, p. 339370.
In L. A. Lacey and H. K. Kaya (ed.), Field manual of techniques in inver-
tebrate pathology. Kluwer Academic Press, Dordrecht, The Netherlands.
6. Milner, R. J. 1992. Selection and characterization of strains of Metarhizium
anisopliae for control of soil insects in Australia, p. 200207. In C. I. Lomer
and C. Prior (ed.), Biological control of locusts and grasshoppers. CAB
International, Wallingford, United Kingdom.
7. Mundt, C. C. 1995 Models from plant pathology on the movement and fate
of new genotypes of microorganisms in the environment. Annu. Rev. Phy-
topathol. 33:467488.
8. Parke, J. L. 1991. Root colonization by indigenous and introduced micro-
organisms, p. 3342. In D. L. Keister and P. B. Cregan (ed.), The rhizosphere
and plant growth. Kluwer Academic Publishers, Dordrecht, The Nether-
9. Roberts, D. W., and A. E. Hajek. 1992 Entomopathogenic fungi as bioinsec-
ticides, p. 144159. In G. F. Leatham (ed.), Frontiers in industrial mycology.
Chapman & Hall, New York, N.Y.
10. St. Leger, R. J., B. May, L. L. Allee, R. C. Frank, R. C. Staples, and D. W.
Roberts. 1992 Genetic differences in allozymes and in formation of infection
structures among isolates of the entomopathogenic fungus Metarhizium
anisopilae. J. Invertebr. Pathol. 60:89101.
11. St. Leger, R. J., M. J. Bidochka, and D. W. Roberts. 1995. Co-transformation
of Metarhizium anisopliae: by electroporation or using the gene gun to pro-
duce stable GUS transformants. Curr. Genet. 131:289294.
12. St. Leger, R. J., L. Joshi, M. J. Bidochka, and D. W. Roberts. 1996. Con-
struction of an improved mycoinsecticide over-expressing a toxic protease.
Proc. Natl. Acad. Sci. USA 93:63496354.
13. St. Leger, R. J. 2001. Development and testing of genetically improved
mycoinsecticides, p. 229239. In J. Gressel, T. Butts, G. Harman, A.
Pilgeram, R. St. Leger, and D. Nuss (ed.), Enhancing biocontrol agents and
handling risks. Proceedings of a NATO Advanced Research Workshop, 915
June, 2001. IOS Press, Amsterdam, The Netherlands.
14. St. Leger, R. J., and S. Screen. 2001. Prospects for strain improvement of
fungal pathogens of insects and weeds, p. 219238. In T. M. Butt, C. Jackson,
and N. Morgan (ed.), Fungal biocontrol agents: progress, problems and
potential. CAB International, Wallingford, United Kingdom.
15. Suzuki, N., L. M. Geletka, and D. L. Nuss. 2000 Essential and dispensable
virus-encoded replication elements revealed by efforts to develop hypovi-
ruses as gene expression vectors. J. Virol. 74:75687577.
16. Whipps, J. M. 2001. Microbial interactions and biocontrol in the rhizo-
sphere. J. Exp.Bot. 52:487511.
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 uorescent 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).
... Apart from their taxonomy and phylogeny, most of the research studies related to EPF have focused either on their development as biological control agents [45] or their mode of action [46]. Some EPF may colonize plants [46] and the rhizosphere [47,48], extending beyond direct fungi-insect interactions, which requires more investigations to better understand the EPF ecology [48]. ...
... Apart from their taxonomy and phylogeny, most of the research studies related to EPF have focused either on their development as biological control agents [45] or their mode of action [46]. Some EPF may colonize plants [46] and the rhizosphere [47,48], extending beyond direct fungi-insect interactions, which requires more investigations to better understand the EPF ecology [48]. ...
... To improve the virulence of EPF, genetic engineering of these microbial actors will offer an opportunity for a deeper understanding of the molecular machinery and the secondary metabolites regulating host-fungal pathogen interactions, therefore improving their tolerance to environmental stress. For example, the upregulation of genes encoding for the endogenous cuticle-degrading protease Pr1 in Metarhizium and the CHIT1 gene that encodes chitinase in B. bassiana improved the virulence of both fungi [48,103]. Furthermore, transcriptomic studies on insects infected with B. bassiana and Metharizium spp. ...
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Entomopathogenic fungi (EPF) are cosmopolitan species of great interest in pest management due to their ability to cause epizooty in soil-dwelling and aboveground insects. Besides their direct effect against a wide host range of serious agricultural insect pests, such as aphids, a major emphasis has been placed on investigating the impact of EPF with endophytic traits (EIPF) on aboveground tripartite interactions between host plants, herbivores and beneficial insects. However, despite their valuable role in biocontrol processes, there is still more to explore about their diverse potential as ecofriendly biological control agents. Herein, we provide an overview of the meaningful role and faced challenges following the use of EPF and EIPF to control aphids.
... Indeed, EIPF are able to establish inside root tissues without causing significant symptoms of infection in plants [5,6] while simultaneously surrounding the root surface [7]. Metarhizium robertsii (Metchnikoff) Sorokin (1883) is an important species used as a commercial microbial biological control agent because of its direct parasitism toward insect pests [8], its potential to colonize the plant rhizosphere [9,10] as well as its ability to establish as an endophyte [11][12][13]. Its root colonization potential is an important feature in order to optimize its capacity as a biological control agent and as a stimulator of plant health. ...
... Metarhizium spp. strains were previously demonstrated to be rhizosphere and root endosphere colonizers of diverse plant species from annual to wild flowers, grasses, annual to perennial crops, shrubs and trees [6,9,16,24,29,[59][60][61][62] with subsequent stimulation of the root growth in some cases [7,63]. The potential for association of M. robertsii with roots has been supported by studies of Metarhizium spp. ...
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Characterizing the association of endophytic insect pathogenic fungi (EIPF) with plants is an important step in order to understand their ecology before using them in biological control programs. Since several methods are available, it is challenging to identify the most appropriate for such investigations. Here, we used two strains of Metarhizium robertsii: EF3.5(2) native to the French vineyard environment and ARSEF-2575-GFP a laboratory strain expressing a green fluorescent protein, to compare their potential of association with non-grafted grapevine Vitis vinifera. Three methods were used to evaluate the kinetics of rhizosphere and grapevine endospheric colonization: (i) Droplet Digital (ddPCR), a sensitive molecular method of M. robertsii DNA quantification in different plant parts, (ii) culture-based method to detect the live fungal propagules from plant tissues that grew on the medium, (iii) confocal imaging to observe roots segments. Both strains showed evidence of establishment in the rhizosphere of grapevines according to the culture-based and ddPCR methods, with a significantly higher establishment of strain EF3.5(2) (40% positive plants and quantified median of exp(4.61) c/μL) compared to strain ARSEF-2575-GFP (13% positive plants and quantified median of exp(2.25) c/μL) at 96–98 days post-inoculation. A low incidence of association of both strains in the grapevine root endosphere was found with no significant differences between strains and evaluation methods (15% positive plants inoculated with strain EF3.5(2) and 5% with strain ARSEF-2575-GFP according to culture-based method). ddPCR should be used more extensively to investigate the association between plants and EIPF but always accompanied with at least one method such as culture-based method or confocal microscopy.
... The persistence and biological activity of EF are also promoted in the rhizosphere (Hu and St Leger 2002;Pava-Ripoll et al. 2011;Wyrebek et al. 2011;Barelli et al. 2016;McKinnon et al. 2018) (Fig. 1). The rhizosphere is the narrow zone of soil that is influenced by root secretions that can contain an enormous diversity of microbes (Mendes et al. 2011). ...
... Indeed, an adaptation mechanism as a rhizosphere competent organism has been reported for Metarhizium anisopliae (Mets.) Sorokin (Ascomycota: Hypocreales), which expresses a specific subset of genes induced by plant root exudates different from the one expressed during infection of the arthropod hosts (Bruck 2005;Hu and St Leger 2002;Pava-Ripoll et al. 2011). ...
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Biocontrol with hypocrealean entomopathogenic fungi (EF) is a key tool to develop Integrated Pest Management (IPM) programs for the progressive replacement of synthetic chemical insecticides with more environmentally friendly pest control measures. These fungi stand out among entomopathogens not only for their contact mechanism of infection through the arthropod integument, but also for developing close associations with plants including the endophytic lifestyle and rhizosphere competence that can enable them to make broader contributions to IPM and crop production. Anyhow, the interaction of EF with the plants incorporates multitrophic complexity at different levels including insect pests, plants, and their natural enemies. The aim of the present review was to gather and summarize all available data on multitrophic interactions of EF. These fungi can influence both the chemical ecology of host-plant selection by insect pests and the host or prey selection by parasitoid or predators, respectively. Moreover, EF treatments are compatible with natural enemies in terms of safety and effectiveness, which could allow biocontrol strategies for their synergistic application in IPM programs. A comprehensive understanding of the impact of these multitrophic interactions in longer term, farm-level real-life biocontrol implementation studies will provide new opportunities in plant protection and production.
... Nevertheless, Mawarda et al. (2020) reported that 30% of studies using fingerprinting-based methods (e.g., TRFLP, DGGE, and TGGE) did not show any consistent effect of inoculation, pointing to a likely methodological limitation. In the case of EPF, the potential effects on the soil microbiome after the application were found to vary (Hu and St. Leger, 2002;Schwarzenbach et al., 2009;Hirsch et al., 2013;Mayerhofer et al., 2017Mayerhofer et al., , 2019. The results of this study point to similar conclusions, as the modifications observed in the OTU number and intensity were generally of limited effect, not consistent in the two studied fields and across the seasons considered. ...
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Introduction: The multifunctionality of microorganisms, including entomopathogenic fungi, represents a feature that could be exploited to support the development, marketing, and application of microbial-based products for plant protection. However, it is likely that this feature could affect the composition and dynamics of the resident soil microorganisms, possibly over a longer period. Therefore, the methodology utilized to evaluate such impact is critical for a reliable assessment. The present study was performed to evaluate the impact of strains of Beauveria brongniartii and Beauveria bassiana on soil bacterial and fungal communities using an approach based on the terminal restriction fragment polymorphism (T-RFLP) analysis. Materials and methods: Soil samples in the vicinity of the root system were collected during a 3-year period, before and after the bioinocula application, in two organic strawberry plantations. Specific primers were used for the amplification of the bacterial 16S rRNA gene and the fungal ITS region of the ribosome. Results and discussion: Data of the profile analysis from T-RFLP analysis were used to compare the operational taxonomic unit (OTU) occurrence and intensity in the inoculated soil with the uninoculated control. With regard to the impact on the bacterial community, both Beauveria species were not fully consistently affecting their composition across the seasons and fields tested. Nevertheless, some common patterns were pointed out in each field and, sometimes, also among them when considering the time elapsed from the bioinoculum application. The impact was even more inconsistent when analyzing the fungal community. It is thus concluded that the application of the bioinocula induced only a transient and limited effect on the soil microbial community, even though some changes in the structure dynamic and frequency of soil bacterial and fungal OTUs emerged.
... No Metarhizium epizootics have been reported in any scarab populations. Despite this, Metarhizium has been shown to persist in the soil, particularly in the rhizosphere, for years after application [133][134][135][136][137], bolstering its potential as an effective biocontrol agent. ...
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Injury and control costs for the invasive scarab Japanese beetle (Family Scarabeidae, Popillla japonica) alone is estimated at $450 million per year in the U.S. Chemical controls are commonly used to control scarab pests, but concerns about human safety and negative impacts on beneficial and non-target organisms, such as pollinators, are increasingly driving the market towards less toxic and more environmentally friendly management options. Microbial entomopathogens are excellent candidates for biopesticides and biocontrol agents. Although microbial pesticides currently make up only 1–2% of the insecticide market, the discovery and development of new microbes are increasing. Microbial products are non-toxic to humans and most are species-specific, reducing non-target effects. While some are slow-acting, others provide rapid control and some can be as efficacious as chemical insecticides, particularly when used in combination. Another major advantage of microbial controls is that many can persist in the environment, and become biocontrol agents, providing long-term control and reducing costs. This article provides a summary of the microbial entomopathogens that are known to infect scarab beetle species including bacterial, fungal, viral, microsporidian, and protozoan taxa, as well as the existing formulations and their efficacy. Lesser-known microbial species are also discussed as potential future controls. We also discuss the development of new techniques for improving efficacy, such as genetic engineering, synergistic interactions, auto-dissemination strategies, and improved formulations.
... In turn, the host plant provides the fungus with photosynthetic compounds [19]. This association of Metarhizium plus plant can be endophytic [3] and through rhizosphere competence [20]. Endophytic fungi develop within plant tissues without causing any noticeable symptoms of disease in the plant [21,22]. ...
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Metarhizium species can be mutualistic symbionts of plants. They are able to colonize roots, promote plant growth and provide protection against pests. We previously found Metarhizium robertsii and M. brunneum associated with coffee roots in a diversified coffee system. Here, we investigated whether these fungi, when inoculated in coffee seedlings, can associate with roots, improve seedling growth and indirectly protect against the coffee leaf miner (CLM) Leucoptera coffeella (Lepidoptera: Lyonetiidae). We performed a greenhouse experiment with coffee seedlings using suspensions of each Metarhizium species applied as soil drenches to potted seedlings. We also challenged these plants with CLM infestation (two adult couples per plant). We recovered Metarhizium spp. from most of the seedling roots 43 days after fungal inoculation. Plants inoculated with M. robertsii showed a 30% leaf area increase compared to the control. Both isolates promoted protection against CLM in coffee seedlings, reducing the percentual of leaf area mined and prolonging CLM development time by two days versus controls. Besides this protection provided by Metarhizium, M. robertsii also improves seedling growth. Therefore, these Metarhizium species could be considered for the development of inoculants for coffee seedlings.
... For this reason, fungal adaptation, especially in agroecosystems, has been viewed as a crucial attribute that needs to be evaluated for any entomopathogenic fungal (EPF) candidates before their field application. Some Metarhizium species, for example, M. anisopliae, M. brunneum and M. robertsii, demonstrate a strong association in the soil environment in terms of rhizosphere colonization [19,20] and endophytic establishment in plant roots, for example, in cassava [21] and in tomato [22]. Fungal-plant interactions, such as endophytism or rhizosphere competence, can confer crop protection against root herbivores [20] and even result in the suppression of root herbivores by altering the gene expression of the host plant [23]. ...
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Metarhizium anisopliae infects and kills a large range of insects and is a promising biocon-trol agent to manage soil insects, such as wireworm in sweetpotato. The presence of other soil microbes , which exhibit competitive fungistasis, may inhibit the establishment of M. anisopliae in soil. Microbially depleted soil, for example, sterilized soil, has been shown to improve the resporulation of the fungus from nutrient-fortified M. anisopliae. Prior to planting, sweetpotato plant beds can be disinfected with fumigants, such as Metham ® , to control soil-borne pests and weeds. Metham ® is a broad-spectrum soil microbial suppressant; however, its effect on Metarhizium spp. is unclear. In the research presented here, fungal resporulation was examined in Metham ®-fumigated soil and the infectivity of the resulting granule sporulation was evaluated on mealworm, as a proxy for wire-worm. The fungal granules grown on different soil treatments (fumigated, field and pasteurized soil) resporulated profusely (for example, 4.14 × 10 7 (±2.17 × 10 6) conidia per granule on fumigated soil), but the resporulation was not significantly different among the three soil treatments. However, the conidial germination of the resporulated granules on fumigated soil was >80%, which was significantly higher than those on pasteurized soil or field soil. The resporulated fungal granules were highly infective, causing 100% insect mortality 9 days after the inoculation, regardless of soil treatments. The results from this research show that the fungal granules applied to soils could be an infective inoculant in sweetpotato fields in conjunction with soil fumigation. Additional field studies are required to validate these results and to demonstrate integration with current farming practices .
... B. brangroftii showed non-pathogenicity over warmblooded animals and not even a single negative report over decades of use [133,134]. Investigations into the behavior of a transgenic M. anisopliae ARSEF 1080 strain in the soil under field conditions revealed that the transgenic strain did not suppress the culturable native fungal microbiota [135]. M. anisopliae was first experimented with inhalation on mice, guinea pigs, and rats, resulting in no evidence of allergy [136][137][138]. ...
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Chemical pesticides have an adverse impact on non-target organisms, and it leads to biodiversity loss, loss of food safety, development of insect resistance and resurgence in newer areas. All these have led scientists to create more ecofriendly alternatives, such as the use of entomopathogenic fungi against insect pests. Entomopathogenic fungus is a promising alternative to chemical insecticides that provides biological plant protection against insect pests in a sustainable pest control approach. Insect-infecting fungi are now classified into 90 genera and roughly 800 entomopathogenic fungal species have been documented. However, most commercial mycoinsecticides target just three genera: Beauveria bassiana, Metarhizium anisopliae, and Isaria fumosoroseus. They cause about 60 percent of insect diseases. These fungi are key contributors to soil insect population dynamics. Hence, entomopathogenic fungi are important biocontrol agents against insect populations. Insect-infecting fungi are found in several distinct groupings. Insect fungal pathogens include those from the phyla Chytridiomycota, Zygomycota, Oomycota, Ascomycota, and Deuteromycota, which are known to be the best entomopathogens against various insect pests. Entomopathogenic fungi kill or inactivate insects by attacking and infecting their insect hosts. Entomopathogenic fungi are soil-dwelling fungi that infect and kill insects by breaching their cuticle. Most insect-infecting fungi work through penetration. Entomopathogens produce these extracellular enzymes (protease and lipase) and toxins in their adaptive response. Together with a mechanical process via appressoria growth, these enzymes break the insect cuticle and enter the body of the insect to infect and kill it by getting their nourishment from the insect tissues. On the other hand, insects have developed many defense against these fungal pathogens. Insect pests are effectively killed by the soil fungus, Beauveria bassiana, and are easy to use in the field. Now mass manufacturing of new fungal formulations are possible. Further, modern genetic engineering and biotechnology approaches may assist in increasing the bioactivity of entomopathogenic fungi. This chapter discusses entomopathogenic fungi and their detailed usage description in the current scenario. It also explains the mode of infection, approaches, plans, and policies for entomopathogenic fungi.
... Species in the genus Metarhizium (Ascomycota, Clavicipitaceae) and Beauveria (Ascomycota, Cordycipitaceae) are widespread fungal pathogens of insects. These entomopathogens proliferate in the rhizosphere but can also live as endophytes inside plants, where they mainly colonize the root tissue [1][2][3][4]. Due to these characteristics, entomopathogens are potential candidates for the control of soil-borne insect pests. Not only can the fungi infect insects that approach the root system in search for food, but as endophytes they may also induce changes in the plant that negatively affect insect performance [5,6]. ...
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Entomopathogenic fungi infect insects via spores but also live inside plant tissues as endophytes. Frequently, colonization by entomopathogens provides plants with increased resistance against insects, but the mechanisms are little understood. This study investigated direct, local, and systemic root-mediated interactions between isolates of the fungus Metarhizium brunneum and larvae of the cabbage root fly (CRF) Delia radicum attacking Brassica napus plants. All fungal isolates infected CRF when conidia were present in the soil, leading to 43-93% mortality. Locally, root-associated M. brunneum isolates reduced herbivore damage by 10-20% and in three out of five isolates caused significant insect mortality due to plant-mediated and/or direct effects. A split-root experiment with isolate Gd12 also demonstrated systemic plant resistance with significantly reduced root collar damage by CRF. LC-MS analyses showed that fungal root colonization did not induce changes in phytohormones, while herbivory increased jasmonic acid (JA) and glucosinolate concentrations. Proteinase inhibitor gene expression was also increased. Fungal colonization, however, primed herbivore-induced JA and the expression of the JA-responsive plant defensin 1.2 (PDF1.2) gene. We conclude that root-associated M. brunneum benefits plant health through multiple mechanisms, such as the direct infection of insects, as well as the local and systemic priming of the JA pathway.
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Helicoverpa armigera is a key insect pest of tomatoes reducing drastically yields. The effect of the endophytic colonization of tomato plants by Beauveria bassiana using leaf spray as an inoculation method on damage and survival of H. armigera was assessed in a screen house. Two B. bassiana isolates (Bb 115 and Bb 11) and two tomato varieties (a local variety Tounvi and an improved variety Padma) were included in the study. The adaxial and abaxial leaf surfaces were sprayed at a concentration of 107 conidia/ml and 109 conidia/ ml for each isolate and each of the two tomato varieties. Thirty days after inoculation, five discs of tomato leaf and tomato root were cut for each isolate, each concentration per isolate and for each variety. The samples were incubated at room temperature (28˚C ± 2˚C) and periodically checked for fungal growth. Larval survival was checked and a damage assessment was done on tomato flowers and the leaves. The results show that the lowest Mean Survival Times (MSTs) were recorded on larvae feeding on plants inoculated with Bb 11 (4.2 ± 0.8 days against 11.5 ± 0.2 days for control). Compared to the other treatments, low damage rates of the flowers of the improved variety inoculated with Bb 11 at 109 conidia/ml were recorded from the 6th Day After Inoculation (DAI). This rate remains low until the end of treatment. Overall flower damage was lower than leaf damage. The results showed large differences in pathogenicity, with most endophytic isolate belonging to Bb 11 when inoculated at 109 conidia/ml using the leaf spraying technique. Data were discussed with regard to the use of endophytism B. bassiana in an integrated tomato pest control approach.
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This book presents topics on the development, improvement and commercialization of fungi for the biological control of pests, weeds and diseases which are of economic importance. Common themes such as production, formulation and application of technologies, biosafety, risk assessment and registration requirements are all covered. The book attempts to bring together scientists, industry and government agencies involved in all aspects of fungal biological control agents for the first time.
This book presents topics on the development, improvement and commercialization of fungi for the biological control of pests, weeds and diseases which are of economic importance. Common themes such as production, formulation and application of technologies, biosafety, risk assessment and registration requirements are all covered. The book attempts to bring together scientists, industry and government agencies involved in all aspects of fungal biological control agents for the first time.
This book presents topics on the development, improvement and commercialization of fungi for the biological control of pests, weeds and diseases which are of economic importance. Common themes such as production, formulation and application of technologies, biosafety, risk assessment and registration requirements are all covered. The book attempts to bring together scientists, industry and government agencies involved in all aspects of fungal biological control agents for the first time.
Root colonization is defined as the proliferation of microorganisms in, on, or around roots. It includes dispersal of microorganisms from a source of inoculum to the actively growing root, and multiplication or growth in the rhizosphere. Soil physical, chemical, and biological factors have been shown to affect root colonization, but few phenotypic attributes of plants and microorganisms which contribute to successful root colonization have been identified. Quantitative studies on the distributon of root colonists in time and space are needed to develop mathematical models that describe and predict the root colonization process. This would enable more effective management of rhizosphere populations to achieve biological control of soilborne disease or to enhance plant growth.
Publisher Summary This chapter discusses isolation, culture, and production of order Hyphomycetes. Hyphomycetes are filamentous fungi that reproduce by conidia generally formed aerially on conidiophores arising from the substrate. Most entomopathogenic Hyphomycetes are facultative pathogens and are relatively easily grown in pure culture on defined or semidefined media. Entomopathogenic Hyphomycetes may be harvested directly from insect cadavers on which the fungus has already sporulated. Another isolation method requires the homogenization of cadavers followed by dilution plating of the homogenate on an appropriate selective medium. Selective media are frequently used for the isolation of entomopathogenic Hyphomycetes. Inhibition of contaminant fungi is more problematic than bacteria, and fungal contaminants are invariably a problem when attempting to isolate entomopathogenic Hyphomycetes from soil. It is found that although most entomopathogenic Hyphomycetes will produce blastospores in submerged culture, specific parameters need to be evaluated and adjusted for every strain studied for optimum blastospore production. An approach for bioassay of entomopathogenic fungi against fourth instar nymphs of the silverleaf whitefly is also elaborated.
As early as 900 A.D., it was known in the Orient that fungi could grow in insects (Steinhaus, 1975). The pioneering work of Bassi with Beauveria bassiana in silkworms in 1834 proved that fungi could actually cause infectious diseases in insects. From the 1880s through the early 1900s, the spectacular epizootics caused by entomopathogenic fungi—fungi-infecting insects—led to studies of their potential use for pest control. Interest in fungi as pest control agents waned, however, as chemical insecticides were used more frequently. More recently, owing to the myriad difficulties that have been gradually encountered in the development and use of chemical insecticides, the field of biological control has been undergoing a renaissance. In particular, our knowledge of entomopathogenic fungi is at present increasing rapidly.
The potential of β-glucuronidase as a molecular marker for studying the environmental microbiology of entomopathogenic fungi was assessed. Metarhizium anisopliae was stably co-transformed with plasmids (pNOM102 and pBENA3) containing the β-glucuronidase and benomyl resistance ( β-tubulin) genes, using both electroporation and biolistic delivery systems, and it was confirmed that the expressed phenotypes were not exhibited by ten randomly chosen indigenous North-American isolates. In spite of random and multiple integrations, the co-transformants showed normal growth rates and retained their pathogenicity to insects. β-Glucuronidase activity in the co-transformants was used to detect histochemically the presence of fungal hyphae in infected host insects (Bombyx mori) and thus provides a practical means of marking genetically engineered pathogens for field trials.