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Mycol. Res. 109 (11): 1302–1312 (November 2005). f The British Mycological Society
1302
doi:10.1017/S0953756205003746 Printed in the United Kingdom.
Development of a PCR-based diagnostic assay for the
specific detection of the entomopathogenic fungus
Metarhizium anisopliae var. acridum
Susan C. ENTZ1, Dan L. JOHNSON1 and Lawrence M. KAWCHUK2
1
University of Lethbridge, Lethbridge, Alberta T1K 3M4, Canada.
Agriculture and Agri-Food Canada Research Centre, Lethbridge, Alberta T1J 4B1, Canada.
E-mail : [email protected]
2
Received 2 December 2004; accepted 1 July 2005.
The entomopathogenic fungus Metarhizium anisopliae var. acridum is registered as a mycopesticide for acridid control
in Africa and Australia. Traditionally, identification of M. anisopliae var. acridum infection in grasshoppers and locusts
has relied upon development of fungal growth in infected cadavers. Conventional methods of detection of this
entomopathogen in the environment and non-target organisms have been based on culture and bioassay. A PCR-based
method for the detection of M. anisopliae var. acridum was developed. Sequence data from the distinct ITS rDNA
regions facilitated the design of PCR primers that were used in PCR-based diagnostic assays for the detection of fungal
DNA. The amplified sequence was 420 bp in length and specific to M. anisopliae var. acridum. Isolates of M. anisopliae
var. anisopliae and M. flavoviride produced no PCR product with these primers. Other fungal entomopathogens, plant
pathogens, mycopathogens, and soil saprophytes were also not detected by the pathogen-specific primers. The assay was
also effective for the detection of M. anisopliae var. acridum DNA in the presence of soil DNA extracts and in infected
grasshoppers.
INTRODUCTION
Prior to the release of a biocontrol agent, the ability to
identify and to monitor its impact on the target pest,
persistence, and fate in the environment should be
demonstrated (Bidochka 2001). A further issue requiring consideration is differentiation of the introduced
organism from native populations.
Metarhizium anisopliae var. acridum is a hyphomycetous fungus that is pathogenic to grasshoppers
and locusts. It has been commercialized as Green
Muscle1 in Africa (Douthwaite, Langewald & Harris
2000) and as Green Guard1 in Australia (Milner &
Hunter 2001) for the control of acridids. Registration
has also been procured in Madagascar (Lomer et al.
2001), and field tests have been conducted in Brazil
(Magalhães et al. 2001).
Traditionally, identification in the genus Metarhizium
has been through the observation of morphological
features on culture media, microscopic examination of
spores and associated structures, and bioassay of target
hosts, resulting in initial recognition of two species
(M. anisopliae and M. flavoviride) with M. album later
restored as a third. Concern, however, over the demonstration of considerable overlap in ranges of spore
sizes and other features raised uncertainty over the
taxonomic relationships among Metarhizium species
(Bridge et al. 1997). Difficulties in identification are
frequently encountered as different morphologies can
be exhibited under varying environmental and physiological conditions. Spore morphology was previously
observed to vary within the same culture and between
isolates of the genus Metarhizium. Conidia and blastospores can be of variable size and shape (Glare, Milner
& Beaton 1996). There may also be differences in colony morphology between isolates of the same variety
(Milner et al. 2003). Lomer et al. (2001) noted that
M. anisopliae var. acridum cannot be distinguished
from other M. anisopliae varieties on the basis of spore
size and shape.
Earlier, Bridge et al. (1997) had used molecular
characterization to propose recognition of those
isolates associated with acridoid hosts as a single distinctive genotype denoted as M. flavoviride Group 3.
However, a high level of genetic diversity demonstrated by sequence data at the ITS and 28S rDNA
D3 regions in this genus indicated a more complex
resolution at the specific and varietal levels (Driver,
Milner & Trueman 2000). Recognition of M. album as
a separate species was supported by morphologically
based taxonomy and molecular data ; however, polymorphisms in the ITS region for M. anisopliae and
S. C. Entz, D. L. Johnson and L. M. Kawchuk
M. flavoviride suggested further refinement at the
infraspecific level for these two species. Based on ITS
sequence data, representative isolates were assigned to
ten separate clades, four of these varieties of M. anisopliae and five varieties of M. flavoviride, while
M. album was retained in a distinct separate clade.
The acridoid isolates, most previously identified as
M. flavoviride on the basis of conidial and phialide
morphology, clustered as their own distinct clade and
were described as Metarhizium anisopliae var. acridum.
The work by Driver et al. (2000) was one of the first
major indications within Metarhizium that the ovoid
(rather than cylindrical) conidia, more clavate (rather
than cylindrical) conidiogenous cell were not altogether
dependable characters for species-level taxonomy in
this genus (Richard A. Humber, pers. comm.).
Bioassay of target hosts has served as a sensitive
method for the detection of M. anisopliae var. acridum,
although caution must be exercised in the identification
of isolates derived by this technique since acridids can
serve as hosts to M. anisopliae other than M. anisopliae
var. acridum. Moreover, in general, bioassays may
fail in the detection of an isolate due to unfavourable
temperature or target host, and thus may suggest artificially low levels of the pathogen. Notwithstanding,
bioassay has been the method of choice for detection of
M. anisopliae var. acridum in field trials (Lomer et al.
1993, Caudwell & Gatehouse 1996, Delgado et al.
1997a, Langewald et al. 1997, Lomer 1997, Milner
et al. 1997, Magalhães et al. 2000) and for surveys
and screening of virulent isolates (Zimmerman et al.
1994, Bateman et al. 1996, Thomas, Gbongboui &
Lomer 1996, Shah et al. 1998).
Comparative studies of nucleotide sequences of
rRNA genes have provided significant data for analysis
of phylogenetics and taxonomy. White et al. (1990)
introduced the use of PCR methods for the amplification of the ITS regions in nuclear rDNA of the
fungal genome. The ITS sequences are an ideal target
for the development of species-specific primers because
they evolve relatively rapidly and are highly variable in
length and nucleotide content between closely related
species, and sometimes within a species as has been
demonstrated for the genus Metarhizium. The objective
of this study was to use sequence data from the ITS
rDNA regions to develop a PCR-based assay for the
highly specific detection of M. anisopliae var. acridum.
An objective diagnostic assay was required that would
facilitate the differentiation of an introduced strain of
M. anisopliae var. acridum from native populations
of M. anisopliae and M. flavoviride in environmental
samples.
MATERIALS AND METHODS
Fungal isolates and cultivation
The fungal isolates studied are listed in Table 1. All
were propagated and maintained on potato dextrose
1303
agar (PDA). M. anisopliae var. acridum (IMI 330189 ;
commercialized as Green Muscle1 by the Lutte Biologique Contre les Locustes et Sauteriaux ‘LUBILOSA ’
programme) was obtained from the International
Institute of Tropical Agriculture (IITA, Benin).
M. anisopliae var. anisopliae isolates 421 and 4450
and other fungi coded as UAMH were obtained from
the University of Alberta Microfungus Collection and
Herbarium, Edmonton. Metarhizium isolates coded
as ARSEF were obtained from the USDA-ARS Collection of Entomopathogenic Fungal Cultures, Ithaca,
NY, USA. Those coded as LRC and Isaria fumosorosea (PFR-97) were obtained from the Lethbridge
Research Centre (LRC), Lethbridge. Metarhizium
anisopliae var. acridum SP9 and Beauveria bassiana
(GHA 726) were previously obtained from Mycotech
Corporation, Butte, MT. M. anisopliae var. acridum
FI-985 (commercialized as Green Guard1 ) was
procured from Bio-Care Technology, Somersby,
Australia.
Fungal DNA isolation
The procedure of Cenis (1992) was used for fungal
DNA extraction. Briefly, hyphae were used to inoculate
500 ml of potato dextrose broth in a 1.5 ml Eppendorf
tube. Following 3–5 d incubation at 25 x, the mycelial
mat was pelleted by centrifugation for 5 min at 16 000
g, washed with 500 ml 10 mM Tris-HCl, 1 mM EDTA,
pH 8 (TE), and pelleted again. The TE was decanted
and 300 ml of 200 mM Tris-HCl pH 8.5, 250 mM NaCl,
25 mM EDTA, 0.5 % SDS extraction buffer added.
The mycelial mat was hand-ground for 1–2 min with a
conical microtube pestle. Following homogenization,
150 ml of 3 M sodium acetate, pH 5.2, was added. The
suspension was briefly vortexed and placed at x20 x
for 10 min. The microtube was then centrifuged as
previously described, and the supernatant transferred
to a new tube. An equal volume of isopropanol was
added and after incubation at ca 20 x for approximately
10 min, the precipitated DNA was pelleted by centrifugation. The supernatant was removed, and the pellet
washed with 70% ethanol. After another centrifugation and removal of the supernatant, the pellet was
dried before being resuspended in 50 ml of TE and
stored at x20 x. Estimates of DNA quantities were
obtained by electrophoresis in 0.9 % TAE (40 mM Tris
acetate, pH approx. 8.3, containing 1 mM EDTA)
agarose gels containing 10 mg mlx1 ethidium bromide
(Sambrook, Fritsch & Maniatis 1989). PCR amplification with general fungal primers TW81 and AB28
and M. anisopliae var. acridum-specific Mac-ITS-spF
and Mac-ITS-spR primers was performed on 50 ng
DNA.
A positive control was generated by cloning the
PCR product resulting from amplification of M. anisopliae var. acridum IMI 330189 DNA with the TW81
and AB28 primers in vector pGEM1 -T Easy using
the pGEM1 and pGEM1 -T Easy Vector Systems
PCR detection of Metarhizium anisopliae var. acridum
1304
Table 1. List of isolates studied.
Isolate codea
Nameb
Host
Metarhizium spp.
IMI 330189
M. anisopliae var. acridum
Ornithacris cavroisi
(Orthoptera : Acrididae)
Locusta migratoria capito
(Orthoptera : Acrididae)
Austracris guttulosa
(Orthoptera : Acrididae)
Zoonocerus elegans
(Orthoptera : Pyrgomorphidae)
Kraussaria angulifera
(Orthoptera : Acrididae)
Teleogryllus commodus
(Orthoptera : Gryllidae)
Unidentified tettigonid
(Orthoptera : Tettigoniidae)
Unidentified insect larvae
Soil
Soil
Soil
Galleria mellonella
(Lepidoptera : Pyralidae)
Otiorhynchus sulcatus
(Coleoptera: Curculionidae)
Unidentified acridid
(Orthoptera : Acrididae)
SP9
M. anisopliae var. acridum
FI 985
M. anisopliae var. acridum
ARSEF 3391
M. anisopliae var. acridum
ARSEF 6421
M. anisopliae var. acridum
ARSEF 437
M. anisopliae var. anisopliae
ARSEF 727
M. anisopliae var. anisopliae
UAMH 421
UAMH 4450
S54
6W-2
11S-1
M. anisopliae var. anisopliae
M. anisopliae var. anisopliae
M. anisopliae var. anisopliae
M. anisopliae var. anisopliae
M. anisopliae var. anisopliae
ARSEF 1184
M. flavoviride Gams & Rozsypal
ARSEF 2023
M. flavoviride var. minus
Other isolates
GHA 726
UAMH 4756
Beauveria bassiana
UAMH 1656
LRC 2111
LRC 2087
UAMH 772
UAMH 2876
PFR 97
Colletotrichum gloeosporioides
(telomorph Glomerella cingulata)
Emericella nidulans
Fusarium oxysporum
Clonostachys rosea f. catenulata
Hydropisphaera peziza
Isaria farinosa
Isaria fumosorosea
LRC 2176
LRC 2391
LRC 2524
LRC race 1
Penicillium bilaii
Rhizopus sp.
Trichoderma reesei
Verticillium albo-atrum
Melanoplus sanguinipes
(Orthoptera : Acrididae)
Laeliocattleya sp.
Feed
Soil
Soil
Soil
Soil
Phenacoccus solani
(Homoptera : Pseudococcidae)
Soil
Soil
Soil
Solanum tuberosum
Country
of origin
Niger
Madagascar
Australia
Tanzania
Senegal
Australia
Brazil
USA
Canada
Canada
Canada
Canada
France
Galapagos Islands
USA
Canada
Canada
Canada
Canada
Canada
Canada
USA
Canada
Canada
Canada
Canada
a
IMI, International Mycological Institute, Egham; ARSEF, Agriculture Research Service Entomopathogenic Fungus Collection, Ithaca,
NY; UAMH, University of Alberta Microfungus Collection and Herbarium, Edmonton; and LRC, Lethbridge Research Centre, Lethbridge,
Alberta.
b
Name as received.
cloning kit (Promega, Madison, WI). Standard protocols were used for plasmid DNA isolation, buffers, and
electrophoresis techniques (Sambrook et al. 1989).
Correct nucleotide sequence of the cloned product was
confirmed by sequencing (University Core DNA and
Protein Services, University of Calgary) and comparison to the published sequence for M. anisopliae var.
acridum (AF137062; Driver et al. 2000).
Spiking of a simulated soil DNA pool
with Metarhizium anisopliae var. acridum DNA
A simulated soil DNA pool was constructed using
fungal DNA from above, exempting that from Metarhizium anisopliae var. acridum and M. flavoviride
var. minus, at a final concentration of 100 ng mlx1.
The pool consisted of equal proportions of Metarhizium spp. DNA versus non-Metarhizium spp. DNA.
The pool was spiked by addition of 100 ng M. anisopliae var. acridum DNA (concentration 100 ng mlx1) to
900 ng soil DNA pool. Four ten-fold dilutions were
made of the spiked DNA pool using the simulated soil
DNA pool as diluent, representing final concentrations
of 1 ng, 100 pg, 10 pg, and 1 pg mlx1 M. anisopliae var.
acridum DNA. PCR amplifications using the Mac-ITSspF and Mac-ITS-spR primers were performed with
1 ml of each spiked sample.
Inoculation of soil
Spores of Metarhizium anisopliae var. acridum were
applied at various concentrations to a local southern
S. C. Entz, D. L. Johnson and L. M. Kawchuk
1305
(a)
400 bp
(b)
400 bp
Fig. 1. Specificity determination of the PCR assay using the Mac-ITS-spF and Mac-ITS-spR primers and genomic DNA
from various fungal isolates. (a) Lane 1, 100 bp ladder ; Lane 2, Positive control (cloned M. anisopliae var. acridum);
Lane 3, M. anisopliae var. anisopliae UAMH 421; Lane 4, M. anisopliae var. anisopliae UAMH 4450; Lane 5, M. anisopliae
var. anisopliae S54 ; Lane 6, M. anisopliae var. anisopliae 6W-2; Lane 7, M. anisopliae var. anisopliae 11S-1; Lane 8,
M. anisopliae var. anisopliae ARSEF 437; Lane 9, M. anisopliae var. anisopliae ARSEF 727; Lane 10, M. flavoviride Gams
& Rozsypal ARSEF 1184 ; Lane 11, M. flavoviride var. minus ARSEF 2023; Lane 12, M. anisopliae var. acridum IMI 330189;
Lane 13, M. anisopliae var. acridum SP9; Lane 14, M. anisopliae var. acridum FI 985; Lane 15, M. anisopliae var. acridum
ARSEF 3391; Lane 16, M. anisopliae var. acridum ARSEF 6421; and Lane 17, Water. (b) Lane 1, 100 bp ladder ; Lane 2,
Positive control (cloned M. anisopliae var. acridum); Lane 3, Beauveria bassiana ; Lane 4, Isaria farinosa ; Lane 5,
I. fumosoroseus ; Lane 6, Verticillium albo-atrum ; Lane 7, Colletotrichum gloeosporioides ; Lane 8, Clonostachys rosea
f. catenulata ; Lane 9, Trichoderma reesei ; Lane 10, Fusarium oxysporum; Lane 11, Penicillium bilaii ; Lane 12, Emericella
nidulans ; Lane 13, Hydropisphaera peziza ; Lane 14, Rhizopus sp. ; and Lane 15, Water.
Alberta soil (clay-loam). Prior to inoculation, the
soil was examined for Metarhizium spp. according
to Rath, Koen & Yip (1992). Moist soil equivalent
to 20 g oven-dried weight of the soil sample was
added to 200 ml of sterile Ringer’s solution (Oxoid,
Ogdensburg, NY), the suspension shaken on an orbital
shaker at 150 rpm for 30 min at ca 20 x, and then
spread-plated as 0.1 ml of neat or 10x1 dilutions in
Ringer’s solution onto a 100r15 mm Petri dish containing selective media consisting of 3.5 % mycological
agar (Difco, Franklin Lakes, NJ) with 10 mg/ml dodine
(Cyprex 65-W, American Cyanamid, Wayne, NJ),
50 mg/ml chloramphenicol (Sigma-Aldrich, St Louis,
MO), and 200 mg mlx1 cycloheximide (Sigma-Aldrich)
(Liu et al. 1993). Each dilution was plated as five
replicates. Plates were incubated at 25 x for 15 d before
examination for colonies of Metarhizium spp. Also
prior to inoculation, DNA was extracted from 0.25 g
of the soil using the Ultra Clean Soil DNA kit
(MoBio, Carlsbad, CA). Following extraction, the
PCR detection of Metarhizium anisopliae var. acridum
1306
(a)
600 bp
(b)
600 bp
Fig. 2. Amplification of genomic DNA from various fungal isolates with general fungal primers TW81 and AB28.
(a) Lane 1, 100 bp ladder ; Lane 2, Positive control (cloned M. anisopliae var. acridum) ; Lane 3, M. anisopliae var. anisopliae
UAMH 421; Lane 4, M. anisopliae var. anisopliae UAMH 4450 ; Lane 5, M. anisopliae var. anisopliae S54; Lane 6,
M. anisopliae var. anisopliae 6W-2; Lane 7, M. anisopliae var. anisopliae 11S-1; Lane 8, M. anisopliae var. anisopliae
ARSEF 437; Lane 9, M. anisopliae var. anisopliae ARSEF 727; Lane 10, M. flavoviride Gams & Rozsypal ARSEF 1184;
Lane 11, M. flavoviride var. minus ARSEF 2023; Lane 12, M. anisopliae var. acridum IMI 330189; Lane 13, M. anisopliae
var. acridum SP9 ; Lane 14, M. anisopliae var. acridum FI 985; Lane 15, M. anisopliae var. acridum ARSEF 3391; Lane 16,
M. anisopliae var. acridum ARSEF 6421 ; and Lane 17, Water. (b) Lane 1, 100 bp ladder; Lane 2, Positive control (cloned
M. anisopliae var. acridum); Lane 3, Beauveria bassiana ; Lane 4, Isasia farinosa; Lane 5, I. fumosorosea; Lane 6,
Verticillium albo-atrum ; Lane 7, Colletotrichum gloeosporioides ; Lane 8, Clonostachys catenulata; Lane 9, Trichoderma reesei ;
Lane 10, Fusarium oxysporum; Lane 11, Penicillium bilaii ; Lane 12, Emericella nidulans ; Lane 13, Hydropisphaera peziza ;
Lane 14, Rhizopus sp.; and Lane 15, Water.
DNA was then subjected to PCR amplification with
the general fungal TW81 and AB28 primers of Curran
et al. (1994) to confirm successful DNA extraction,
and amplification with a set of primers (Mac-ITS-spF
and Mac-ITS-spR) designed for the specific detection
of M. anisopliae var. acridum DNA.
Spores of M. anisopliae var. acridum (IMI 330189)
were scraped from a PDA plate and resuspended in
0.05 % Tween 20. Spore concentration was estimated
with a hemocytometer and concentrations adjusted to
102, 103, 104, and 105 spores, each in 200 ml of 0.05 %
Tween 20. The spore suspensions were each added
to 0.25 g of soil, followed immediately by soil DNA
extraction using the MoBio Ultra Clean Soil DNA
kit. Extracted DNA (1 ml) was subsequently subjected
to PCR amplification with the Mac-ITS-spF and
Mac-ITS-spR primers.
Inoculation of grasshoppers
Nymphs (second and third instar) of a non-diapausing
strain of Melanoplus sanguinipes (Pickford & Randell
S. C. Entz, D. L. Johnson and L. M. Kawchuk
400 bp
1307
400 bp
Fig. 3. Sensitivity determination of the PCR assay using the
Mac-ITS-spF and Mac-ITS-spR primers and genomic DNA
from Metarhizium anisopliae var. acridum. Lane 1, 100 bp
ladder; Lane 2, 1 ng; Lane 3, 100 pg; Lane 4, 10 pg; Lane 5,
1 pg; Lane 6 ; 100 fg; and Lane 7, Water.
Fig. 4. Detection of Metarhizium anisopliae var. acridum
DNA in a simulated soil DNA pool using PCR primers
Mac-ITS-spF and Mac-ITS-spR. Lane 1, 100 bp ladder;
Lane 2, 10 ng; Lane 3, 1 ng; Lane 4, 100 pg; Lane 5, 10 pg;
Lane 6, 1 pg; Lane 7, Positive control (cloned M. anisopliae
var. acridum) ; and Lane 8, Water.
1969) were collected at random from a laboratory
colony at the Lethbridge Research Centre and placed
individually in sterile 20 ml glass vials stoppered with
a sterile polyurethane foam plug. The experiment
involved a total of 174 insects (30 in the control group,
144 in the treated group) with approximately equal
proportions of males and females in each group. On
the day of inoculation, conidia of M. aniospliae var.
acridum were harvested from a PDA culture (15–20 d
of growth) and resuspended in sunflower oil (Safflo).
Formulation of the inoculum has been previously
described by Johnson et al. (2002). Briefly, the concentration of conidia was estimated with a hemocytometer and adjusted to 5r107 conidia mlx1.
Subsequently, 2 ml aliquots were pipetted onto lettuceleaf wafers (0.7 cm diam), resulting in a dose of
approximately 105 spores per insect (via handling
and feeding). Each grasshopper was confined with one
wafer for 24 h. Control grasshoppers were confined
with wafers containing only sunflower oil. After 24 h
confinement, all grasshoppers were removed and individually housed in 240-ml transparent plastic containers. Throughout the experiment, insects were
exposed to a temperature regime of 24 x/16 x day/night
with a corresponding 16/8 h light/dark photoperiod
under ambient relative humidity (40–55 %). Nymphs
were observed and fed daily with fresh wheat leaves.
Cadavers were removed daily with sterile forceps and
stored in sterile 1.5 ml Eppendorf vials at x20 x prior
to DNA extraction. All treated grasshoppers were
dead by day 8 ; all remaining control grasshoppers
were then killed at x20 x. Viability of conidia was
determined by microscopic examination of germination
following 48 h incubation at 25 x of 2r10-ml replicate
aliquots of the inoculum onto PDA blocks on a
microscope slide.
Grasshopper DNA extraction
The method of Hegedus & Khachatourians (1993)
was modified for the extraction of DNA from infected
and noninfected grasshoppers. Individual nymphs were
macerated in 500 ml of TE with a sterile microtube
pestle for 2–3 min accompanied by vigorous vortexing.
A 25 ml aliquot of the homogenate was removed and
spread on a 60r15 mm Petri dish containing selective media for Metarhizium spp. as described above.
Inoculated agar plates were incubated at 25 x for
confirmation of presence/absence of M. anisopliae
var. acridum colonies (maximum 20 d). The remaining
solution was extracted with an equal volume of
phenol:chloroform (1 :1, v/v) followed by a 10 min
centrifugation at 16 000 g. The upper aqueous phase
was removed and extracted once more with chloroform :isoamyl alcohol (24 : 1, v/v), followed by addition
of 0.1 volume of 3 M sodium acetate, pH 5.2, and one
volume of isopropanol to the aqueous phase. Following incubation at ca 20 x for 10 min, the mixture was
centrifuged, and the supernatant removed. The pellet
was washed with 1 ml of ice-cold 70% ethanol, centrifuged, and the pellet dried briefly. The DNA was
then resuspended in 500 ml of TE containing 2 ml
RNase A (Sigma-Aldrich) and stored at x20 x.
Quantitation of DNA was determined with use of
PCR detection of Metarhizium anisopliae var. acridum
a spectrophotometer (Pharmacia Biotech, Piscataway,
NJ) and 100 ng later subjected to PCR amplification.
PCR amplification
General fungal primers TW81 (5k-GTTTCCGTAGGTGAACCTGC-3k) and AB28 (5k-ATATGCTTAAGTTCAGCGGGT-3k) (Curran et al. 1994) were used
to amplify the region of the ribosomal repeat from
the 3k end of the 16S rDNA to the 5k end of the 28S
rDNA flanking the ITS1, the 5.8S rDNA, and ITS2
sequences, from total fungal DNA. PCR amplifications
were performed in a total volume of 50 ml containing
10 mM Tris, pH 8.4, 50 mM KCl, 1.5 mM MgCl2, 0.05 %
Tween 20, 0.05 % NP40, 0.4 mM of each primer, 25 mM
of each dNTP (Invitrogen, Carlsbad, CA), 2.5 units
Taq DNA polymerase (MBI Fermentas, Hanover,
MD) and template DNA. Negative controls contained
sterile water in place of DNA. DNA amplification was
performed in a GeneAmp1 PCR System 9700 (Applied
Biosystems, Foster City, CA) programmed as follows :
initial denaturation 5 min at 94 x; 30 cycles of: denaturation 1 min at 94 x, annealing 1 min 30 at 55 x, extension 2 min at 72 x; with a final extension 5 min at
72 x. PCR products were analyzed on a 1.5 % TAE
agarose gel with a 100 bp DNA ladder (MBI
Fermentas) included as a size marker.
Primers Mac-ITS-spF (5k-CTGTCACTGTTGCTTCGGCGGTAC-3k) and Mac-ITS-spR (5k-CCCGTTGCGAGTGAGTTACTACTGC-3k) were designed
based on the ITS1 and ITS2 regions of the rDNA
sequence data for M. anisopliae var. acridum (clade
7 ; Driver et al. 2000). Total fungal and soil DNA
and grasshopper DNA from infected and noninfected
insects were used in PCR assays with this primer
combination. Amplifications were performed in a
total volume of 50 ml containing 20 mM Tris, pH 8.3,
50 mM KCl, 1.5 mM MgCl2, 0.1 % Triton X-100, 0.4 mM
of each primer, 25 mM of each dNTP (Invitrogen), 2.5
units Taq DNA polymerase (MBI Fermentas) and
template DNA. As previously noted, negative controls contained sterile water in place of DNA. DNA
amplification was also performed in a GeneAmp1
PCR System 9700 (Applied Biosystems) programmed
as follows : initial denaturation 5 min at 94 x; 30 cycles
of: denaturation 1 min at 94 x, combined annealing
and extension 3 min at 72 x; with a final extension
5 min at 72 x. PCR products were analyzed as previously mentioned.
Nested PCR amplifications were carried out on
grasshopper DNA from infected insects that initially
produced weak products in a single amplification with
the Mac-ITS-spF and Mac-ITS-spR primers. DNA
from infected grasshoppers was amplified in an initial
reaction with the TW81 and AB28 primers using conditions previously described. A second amplification
was then performed with a 1-ml aliquot from the initial
reaction and the Mac-ITS-spF/Mac-ITS-spR primer
combination using conditions described above.
1308
RESULTS
As expected, use of the Mac-ITS-spF and Mac-ITSspR primers in a PCR assay successfully amplified a
420 bp DNA sequence from the total genomic DNA
extracted from Metarhizium anisopliae var. acridum
(Fig. 1a). M. anisopliae var. minus also produced a
420 bp amplification product. Isolates of Metarhizium
anisopliae var. anisopliae and M. flavoviride produced
no amplified product, nor did isolates of Beauveria
bassiana, Isaria fumosorosea, I. farinosa, Verticillium
albo-atrum, Colletotrichum gloeosporioides, Emericella
nidulans, Trichoderma reesei, Fusarium oxysporum,
Clonostachys rosea f. catenulata, Penicillium bilaii,
Hydropisphaera peziza, or an isolate of Rhizopus sp.
(Fig. 1b). In contrast, the TW81 and AB28 primers
produced a varying range (most around 500–600 bp) of
amplified products (Fig. 2a–b) in all isolates tested,
thus confirming successful extraction of PCR-quality
DNA from all fungal species.
The sensitivity of the M. anisopliae var. acridumspecific PCR assay was determined for genomic fungal
DNA extracted from an axenic culture of M. anisopliae
var. acridum. The assay was sensitive enough to detect
approximately 1 pg of genomic DNA (Fig. 3).
The M. anisopliae var. acridium-specific PCR assay
successfully detected M. anisopliae var. acridum DNA
in the presence of a simulated soil DNA pool. A
detection limit of 10 pg was observed, representing
0.001 % of total DNA in the sample (Fig. 4).
M. anisopliae var. acridum spores were detected at
a concentration of 104 spores per 0.25 g of soil (Fig. 5).
Use of general fungal TW81 and AB28 primers in
conjunction with specific Mac-ITS-spF and Mac-ITSspR primers in a nested PCR assay increased the
detection limits to 102 spores per 0.25 g of soil (data
not shown).
The specific assay also successfully detected M. anisopliae var. acridum DNA in each of the 144 infected
grasshoppers. Counts of viable conidia in the inoculum
revealed a germination rate of >90 % at 48 h after
incubation at 25 x. Ecdysis was either completed or
initiated by 65 of the treated nymphs prior to death ;
however, this did not inhibit detection of fungal
DNA. Only 28 of the treated cadavers displayed the
reddish discolouration of the cuticle associated with
infection by M. anisopliae var. acridum. Fungal colonies with M. anisopliae var. acridum morphological
features, namely dark green conidia, were observed on
134 agar plates for the treated group. No growth was
observed on nine plates, and for another plate, overgrowth by Rhizopus sp. interfered with examination
for colonies of M. anisopliae var. acridum.
No amplified products were observed with PCR
assay of the control group, and no colonies of M. anisopliae var. acridum were isolated from any of the
agar plates for the control nymphs. Fig. 6 shows the var.
acridum-specific PCR amplification results for a representative group of infected and noninfected nymphs.
S. C. Entz, D. L. Johnson and L. M. Kawchuk
400 bp
Fig. 5. Detection of Metarhizium anisopliae var. acridum
spores (spore counts per 0.25 g soil) in soil using PCR primers
Mac-ITS-spF and Mac-ITS-spR. Lane 1, 100 bp ladder;
Lane 2, Positive control (cloned M. anisopliae var. acridum);
Lane 3, 102 ; Lane 4, 103 ; Lane 5, 104 ; Lane 6, 105 ; and Lane 7,
Water.
DISCUSSION
The ITS sequences of the rDNA region of the fungal
genome are an ideal target for molecular characterization due to their high copy number and divergence
of sequences between taxa (Pipe et al. 1995). We
exploited these features to design Metarhizium
anisopliae var. acridum-specific DNA primers. The
primers were used in a PCR-based assay to amplify
a 420 bp sequence with genomic DNA extracted
from M. anisopliae var. acridum. A 420 bp product
observed after amplification of M. flavoviride var.
minus DNA was also expected as this species has been
recognized as M. anisopliae var. acridum by Driver
et al. (2000). The ability to produce an amplified
product specific to M. anisopliae var. acridum supports the concept of divergence between taxa and
also corroborates the theme of divergent evolutionary
lines within the genus Metarhizium (Driver et al.
2000). Although representatives from only two other
clades of Metarhizium were evaluated, the high sequence variability of M. anisopliae var. acridum in
comparison with other clades combined with the
highly stringent composition of the synthesized sequences support the specificity of the Mac-ITS-spF
and Mac-ITS-spR primers.
The fungal genera other than Metarhizium analyzed
in this study encompassed a range of entomopathogenic, phytopathogenic, mycopathogenic, and soil
saprophytic organisms. Several of the genera have
previously been isolated from southern Alberta soils
(Inglis et al. 1998). Verticillium albo-atrum and
1309
400 bp
Fig. 6. Detection of Metarhizium anisopliae var. acridum
DNA in infected grasshoppers using PCR primers
Mac-ITS-spF and Mac-ITS-spR. Lane 1, 100 bp ladder;
Lane 2, M. anisopliae var. acridum (positive control) ; Lane 3,
DNA from uninfected grasshopper ; Lane 4, DNA from
uninfected grasshopper ; Lane 5, DNA from grasshopper
infected with M. anisopliae var. acridum (4 days postinfection) ; Lane 6, DNA from grasshopper infected with
M. anisopliae var. acridum (5 days post-infection) ; Lane 7,
DNA from grasshopper infected with M. anisopliae var.
acridum (6 days post-infection) ; and Lane 8, Water.
Colletotrichum gloeosporioides are phytopathogens
(Domsch, Gams & Anderson 1980, Evans, Greaves &
Watson 2001). Others, such as Isaria (syn. Paecilomyces p.p.), are entomopathogenic (Inglis et al. 2001).
One of these other entomopathogens, Beauveria
bassiana, was selected due to its nature as an acridid
pathogen (Johnson & Goettel 1993). Clonostachys
spp., Trichoderma spp., and Fusarium spp. have been
identified as pathogens of fungi (Vey, Hoagland &
Butt 2001). A pending survey of southern Alberta soils
and insects necessitated analysis of these genera with
the M. anisopliae var. acridum-specific primers to
determine specificity of the PCR assay. Further, demonstration of successful amplification of M. anisopliae
var. acridum DNA in the presence of other DNA,
particularly from soil, was essential and has been
demonstrated in this work.
Extraction of PCR-amplifiable DNA from insects
is often difficult due to the number of PCR inhibitors
in the form of tannic acids, quinones, polyphenols,
chelators, etc. coisolated from the insect cuticle
(Hackman 1974). We experienced some weak amplification products using the var. acridum-specific primers
in a single amplification from infected grasshopper
DNA but found that these products could be strongly
amplified with a nested PCR assay using the TW81/
AB28 primers for the first amplification and the
PCR detection of Metarhizium anisopliae var. acridum
1310
Mac-ITS-spF/Mac-ITS-spR primers for the second
amplification. Inhibitory compounds were diluted to a
negligible amount when 1 ml of the first amplification
reaction was used as template for the second amplification. Molting did not interfere with the ability of
the assay to detect M. anisopliae var. acridum DNA
in infected grasshoppers that underwent ecdysis. This
supports a previous observation by Milner & Prior
(1994) that ecdysis did not interfere in the infection
of the Australian plague locust with M. anisopliae var.
acridum.
Studies have demonstrated that, depending on the
dose, the majority of laboratory bioassay mortality in
acridids infected with M. anisopliae var. acridum occurs
between 4–6 d post-infection (Delgado et al. 1997b,
Magalhães et al. 1997, Milner 1997, Lomer, Prior &
Kooyman 1997). In this study, the M. anisopliae var.
acridum-specific PCR assay amplified sequences from
DNA extracted from treated nymphs that died 1–3 d
post-inoculation. Presumably, the majority of nymphs
at this stage died from complications due to contact
with the sunflower oil component of the inoculum
rather than from active fungal infection. Our diagnostic
PCR assay is qualitative and not designed to determine
activity levels of the target organism. However, confirmation of M. anisopliae var. acridum colony growth
for 93.1 % of the treated grasshoppers indicates
that the presence of viable spores can be detected
early post-infection. Moreover, the intensity of amplification products increased with DNA from cadavers
from the later days of the experiment, thus suggesting
a progressive increase in fungal mass in the infected
host.
Surveys for natural incidence of M. anisopliae var.
acridum have indicated that these levels are generally
very low. In northern Benin, Shah et al. (1998) found
levels of 0.3–1.7% and 1.2–3.2 % at different sites, respectively. Also in Benin, Douro-Kpindou et al. (1995)
detected fungal incidence at 15 % in field trial plots
before application of a formulation of M. anisopliae
var. acridum for biocontrol of Zonocerus variegatus.
The ability of our assay to detect levels of M. anisopliae
var. acridum DNA as low as 0.001 % of total DNA
present demonstrates its suitability for detection of this
fungus at low incidence.
Laboratory and field tests indicate differential
impacts of weather affect the operation and efficacy
of entomopathogens (Inglis et al. 1997). Further,
spring temperature, overwintering conditions, and
moisture strongly affect the target insect. Insect body
temperature can be calculated (Lactin & Johnson 1998)
and is largely a result of immediate weather factors ;
however, the probable impact of weather on the
effectiveness of M. anisopliae var. acridum is largely
unknown. Improved knowledge of the biology and
ecology of this fungus in a natural setting is a prerequisite for the development of an effective longlasting pest management strategy for the biological
control of acridids. Our study offers a reliable, specific,
and sensitive diagnostic PCR assay that can be performed on a number of templates including those
with non-target DNA. We plan to use this molecular
method to investigate the geographical extent of
Metarhizium spp. in soils and native insects, to compare this distribution to possible future distributions
under changing weather and climate, and to assess
the opportunities for including Metarhizium spp. in
integrated grasshopper management plans.
ACKNOWLEDGMENTS
Metarhizium anisopliae var. acridum (IMI 330189, Green Muscle1 )
was originally provided by Chris Lomer (IITA). We are also grateful
to Richard A. Humber (USDA-ARS), Lynne Sigler (UAMH),
Jay Yanke (LRC) and Mark Goettel (LRC) for providing isolates.
We thank Craig Andrews for assistance with grasshopper identifications and inoculations, James Lynn for technical assistance, and
Sheila Torgunrud for assistance with figures. We also thank Richard
Humber and two anonymous reviewers for their helpful comments
on the manuscript. S.E. is supported by the University of Lethbridge
and the Climate Change Action Fund.
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Corresponding Editor: R. A. Humber
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