AFLP Analysis of Metarhizium Anisopliae Isolates
E. K. K. Fernandes, M. P. Miller, J. Orwin, D. E. N. Rangel, D. W. Roberts
Department of Biology, Utah State University, Logan, UT 84322-5305, USA.
Departamento de Parasitologia Animal, Universidade Federal Rural do Rio de Janeiro, RJ, Brasil
DNA extraction
The fungal cells were ground in liquid nitrogen with a
mortar and pestle, suspended in 1 ml sterile TE buffer and stored
at –20 ºC until use. After defrosting, ca. 70 mg of fungal cells of
each isolate was placed into 2 ml tubes and the genomic DNA
was extracted using the DNeasy® Plant Mini Kit (Qiagen),
following the manufacturer’s protocol.
The DNA extracts were electrophoresed on 0.7% agarose
gels at 120V in Tris-Acetate-EDTA buffer to estimate DNA
quantity and quality. A 100 bp DNA ladder was used as a size
marker. The gels were stained with ethidium bromide and
visualized under ultraviolet light.
AFLP
DNAs were double digested for 1 h at 37 ºC with 5 units
EcoRI and 5 units MseI, 5 l 10 RL buffer, and sterile double
Results and Discussions
There were 123 polymorphic bands produced by all three
primers pairs. A combined dendrogram for all three banding
patterns revealed two clusters of isolates. The first group with
90% of similarity and the other one with 65% of similarity.
Interesting is the fact that the isolates of M. anisopliae var.
PDAY media
ARSEF 324
ARSEF 2575
ARSEF 3341
ARSEF 3609
ARSEF 5749
0.0300%
0.0250%
0.0200%
0.0150%
ATCC 10074
0.0100%
Fungal cultures
The isolates were cultured in flasks containing 100 ml of
Potato Dextrose Broth (DIFCO). Broth cultures were shaken at
130 rpm at 25 ºC for 4 d. The fungal cells (mycelium) were
collected by vacuum filtration and stored at –20 ºC.
Figure 4. Comparison of Abbott-corrected and mycosis-confirmed (sporulation
observed) mortality of C. nenuphar larvae (southern population) infected with
different isolates of M. anisopliae 8 days after treatment (Alston et al. 2005).
0.0080%
Latitude
19 ºS
19 ºS
19 ºS
11 ºN
15 ºN
36 ºC
34 ºN
61.2 ºN
48 ºN
54.4 ºS
34 ºN
0.0060%
Origin
Australia
Australia
Australia
Niger
Thailand
USA
USA
Finland
Austria
Australia
USA
0.0040%
F985
Green Guard
ARSEF 324
ARSEF 3341
ARSEF 3609
ARSEF 23
ARSEF 2575
ARSEF 5626
F52
ARSEF 4343
DWR200
Host / Substract
Autracis guttulosa [Orthoptera: Acrididae]
Autracis guttulosa [Orthoptera: Acrididae]
Autracis guttulosa [Orthoptera: Acrididae]
Ornithacris cavroisi [Orthoptera: Acrididae]
Patanga succinta [Orthoptera]
Conoderus sp. [Coleoptera: Elateridae]
Curculio caryae [Coleoptera: Curculionidae]
Tenebrio molitor [Coleoptera: Tenebrionidae]
Carpocapsa pomonella [Lepidoptera: Olethreutidae]
Soil
Soil
0.0020%
Isolate*
0.0010%
#
1
2
3
4
5
6
7
8
9
10
11
0.0005%
Metarhizium anisopliae isolates
0.0004%
Table 1. Metarhizium anisopliae isolates. Their host or substrate, origin
and latitude.
0.0003%
Metarhizium anisopliae isolates
The different hosts and geographic origins of the eleven
isolates of Metarhizium anisopliae used in this study are listed
in Table 1.
The isolates F985, Green Guard and ARSEF 324,
supposely are the same isolate, and have different names due to
different culture histories. The AFLP results suggest that they
are indeed the same isolate.
The isolate ARSEF 3609 clustered with other isolates of
variety acridum, even though but this isolate was considered
as variety anisopliae by Driver et al. (2000) when the
mitochondrial ITS1 and ITS2 region was analyzed.
Nevertheless, some physiological evidences corroborate that
ARSEF 3609 is an M. anisopliae var. acridum.
Rangel et al. (2005) exposed conidia of 17 isolates of M.
anisopliae to heat. After 8 and 12 h exposure at 45 ºC, only the
isolates ARSEF 324 and 3609 had viability greater than 40%
(Figure 2). The isolate ARSEF 324 (a known M. anisopliae
var. acridum) and ARSEF 3609 demonstrated similar
thermotolerance.
Figure 3. Mean relative percentage culturability of Metarhizium strains after
exposure for 4 h to irradiances of 920 and 1200 mW m-2 (Braga et al. 2001).
0.0002%
Material and Methods
Figure 1. AFLP dendrogram generated by Simple Matching Coefficient for all Metarhizium
anisopliae isolates used in this study.
Another study (not published) showed that ARSEF 3609
and isolates of M. anisopliae var. acridum (ARSEF 324 and
3341) had similar tolerance to a fungicide Dodine (Figure 5).
0.0001%
Metarhizium anisopliae is an important
entomopathogenic fungus that occurs worldwide, and some
isolates are used for biological control of arthropods
population.
In a recent study on characterization of 100 Metarhizium
cultures the isolate ARSEF 3609 was identified as M.
anisopliae var. anisopliae (Driver et al. 2000). This isolate,
however, has physiologic features (e.g. host hange, culture
aspect, UV-B tolerance) more compatible with those of M.
anisopliae var. acridum (Braga et al. 2001, Rangel et al. 2005,
Alston et al. 2005).
This study was conducted to determine if there is
genotypic similarity among ARSEF 3609 and other isolates of
M. anisopliae varieties anisopliae and acridum.
acridum are clustered in the first group and the isolates of M.
anisopliae var. anisopliae in a different group (Figure 1).
Control
Introduction
distilled water up to 50 l. After digestion, we added 10 l of a
solution containing: 0.5 l EcoRI-adapters (10 mM) and 0.5 l
MseI-adapters (100 mM), 1 unit T4 DNA-ligase, 1.2 mM ATP, 1
l 10 RL buffer, and double distilled water up to 10 l. The
incubation was continued for 3 h at 37 ºC. After ligation, the
reaction mixture was stored at –20 ºC.
A PCR pre-amplification was carried out containing 6 l
ligated DNA, 0.5 l EcoA (10 mM), 0.5 l MseA (10 mM), 2 l
dNTP (5 mM), 3 l MgCl2 (25 mM), 1 unit Taq polymerase, 5 l
10 Taq polymerase buffer, and sterile double distilled water up
to 50 l. The PCR pre-amplification cycle included a 2-min 72 ºC
step, 29 cycles of 94 ºC for 30 sec, 98 ºC for 30 sec and 72 ºC for
1 min. Ten l of pre-amplification products and 2 l of a lightcolored loading dye were electrophoresed at 120 V on 1% agarose
gel, using Tris-acetate EDTA buffer. A 100 bp DNA ladder was
run on the same gel as size marker. After electrophoresis, the gels
were stained with ethidium bromide and visualized under
ultraviolet light.
Multilocus AFLP profiles were obtained from three
additional PCR amplifications. Primers used were: A for MseI and
AGC for EcoRI primer extension; A for MseI and AGG for EcoRI
primer extension; and A for MseI and ACC for EcoRI primer
extension. For each primer combination, a PCR amplification was
carried out containing 2.5 l PCR pre-amplification product
diluted 1:10 in water, 0.05 l Eco primer (10 mM), 0.2 l Mse
primer (10 mM), 0.4 l dNTP (5 mM), 0.6 l MgCl2 (25 mM),
0.5 unit Taq polymerase, 1 l 10 Taq polymerase buffer, and
sterile double distilled water up to 10 l. The PCR amplification
was run under the following thermalcycler parameters: 94 ºC for
2 min, then start a ‘touchdown’ PCR procedure using a 30 sec
denature step at 94 ºC, a 30 sec annealing step at 65 ºC, and a 1
min extension step at 72 ºC. This procedure was repeated 9 times,
each time reducing the annealing temperature by 1 ºC. The
annealing temperature of the final ‘touchdown’ step was 57 ºC,
followed by 30 cycles of the following conventional
thermalcycler program: 94ºC for 30 sec, 56 ºC for 30 sec, 72 ºC
for 30 sec.
An appropriate quantity of ABIs ROX 400 size standard
was added and loaded in the ABI 3100. AFLP markers between
50-400 bp were analyzed with Genographer software (freelydistributed at http://hordeum.oscs.montana.edu/genographer/).
Cluster analysis was performed using NTSYS-pc vers2.1
(Exeter Software, Setauket, New York) and a combined
dendrogram from all three primer pairs was derived from the
similarity matrix calculated using simple match coefficient.
Cluster analysis was performed using the unweighted pair-group
method with arithmetic mean (UPGMA). Analyses were
performed on all 11 Metarhizium anisopliae isolates.
14 day old cultures
Figure 2. Mean relative percentage of germination of M. anisopliae isolates after exposure to
temperatures of 40 °C (open bars) and 45 °C (closed bars) for 2, 4, 8, and 12 h (Rangel et al. 2005).
The effects of exposure to UV-B irradiances of 920 and
1200 mW m-2 on conidial relative percentage culturability
of the 30 Metarhizium isolates was evaluated by Braga et al.
(2001). After 4 h exposure, ARSEF 324 and ARSEF 3609
exhibited the highest relative culturability at both irradiance
levels (Figure 3).
Alston et al. (2005) observed that ARSEF 3609 was not
pathogenic to plum curculio (Figure 4). M. anisopliae var.
acridum, at the recommended dose for control of Orthoptera,
under field conditions, was not pathogenic to other insect groups
(Peveling and Demba, 1997; Goettel and Jaronski, 1997 and
Milner et al. 2002).
Figure 5. ARSEF (Metarhizium anisopliae) and ATCC 10074 (Aspergillus) isolates
cultured on PDAY with different concentration of Dodine for 14 d. Control plates do
not contain Dodine (Dettemmeir et al. in preparation).
References
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Acknowledgments
This research was supported by grants from the Utah Department of Agriculture and Food, the Community/University
Research Initiative of Utah State University and PhD scholarships from National Council for Scientific and technological Development
(CNPq) of Brazil for Everton K. K. Fernandes and Drauzio E. N. Rangel.