Mycol. Res. 106 (11): 1307–1314 (November 2002).

f The British Mycological Society 1307

DOI: 10.1017/S0953756202006676 Printed in the United Kingdom.

Genetic variability of Beauveria bassiana associated with the

Coffee Berry Borer Hypothenemus hampei and other insects

Alvaro GAITAN*, Ana M. VALDERRAMA, Gabriel SALDARRIAGA, Patricia VELEZ

and Alex BUSTILLO
National Coffee Research Center, CENICAFE, Plan Alto, Chinchiná, Caldas, Colombia.
E-mail : alvaro.gaitan@cafedecolombia.com

Received 19 February 2002; accepted 25 July 2002.

Genetic variability of 95 isolates of Beauveria bassiana from different geographical regions and hosts was assessed using
RAPDs and ITS-RFLP molecular markers in order to characterize its genetic variability. Clusterings were obtained
using UPGMA and Principal Coordinates Analysis. Four groups were identified with RAPDs. A 930 bp fragment was
amplified with primers ITS1 and PN16, and polymorphisms were observed with AluI and MspI, generating two main clusters.
Results indicate low genetic variability present in the Colombian B. bassiana population, no association to host type or
geographic locality, and a population structure that is not clonal. This characterization sets criteria for determination of
bio-insecticide potentiality, introduction of foreign strains and improvement of entomopathogens within an IPM program.

INTRODUCTION analyze 138 B. bassiana isolates obtained from a wide

range of geographical regions and hosts, and a clonal
The coffee berry borer, Hypothenemus hampei (Col-
population structure was suggested as a result. Maurer
eoptera: Scolytidae) is a major pest in Colombia and in
et al. (1997) used RAPDs and RFLPs in 38 isolates from
the coffee zones of the other countries where it has been
around the world and supported the observation of
introduced. It perforates the berry and reproduces in the
clonal populations when most of the isolates presented
endosperm, causing total bean loss, alteration in cup
identical banding patterns. On the other hand, Castrillo
quality and premature fruit fall (Damon 2000). In
& Brooks (1998) and Castrillo, Wiegmann & Brooks
Colombia, the hyphomycete Beauveria bassiana has
(1999), using RAPDs on 81 isolates from North Caro-
been found naturally infecting the coffee berry borer
lina and West Virginia, found no evidence of clonal
throughout the country. The fungus has also been iso-
propagation among isolates associated with the dark-
lated from more than 30 insect species, most of them
ling beetle Alphitobius diaperinus.
coleopterans and lepidopterans (Bustillo et al. 1998),
Differences among reports can be explained in part
exhibiting the generalized mycosis ‘White Muscardine ’.
because the detection of genetic variability depends not
This suggests an important role for B. bassiana as a
only on the intrinsic polymorphism of the population
biocontrol agent in the tropics.
under study, but also on the number of samples, the
A coffee berry borer control programme that includes
markers, parts of the genome that are being studied, and
the use of entomopathogens has been under develop-
the collection strategies (Castrillo, Wiegmann & Brooks
ment for ten years at the Colombian National Coffee
1999), making it difficult to compare host–pathogen sys-
Research Center (Cenicafé). The research uses a collec-
tems. Nonetheless, for a biocontrol programme, some
tion of 106 B. bassiana isolates obtained from different
understanding of the dynamics of the populations in
localities and ecological conditions. Phenotypic data
the particular ecosystems where they interact is necess-
such as spore dimensions, pathogenicity or host speci-
ary to provide guidelines for the selection, introduction,
ficity are not sufficient to characterize and select strains
or modification of biocontrol agents in a way that causes
to be used experimentally. A study of genetic variation
minimal alterations to the environment.
through molecular markers was therefore required.
We report on the genetic variability of 68 B. bassiana
Isozymes were used initially by St Leger et al. (1992) to
isolates collected in Colombia from H. hampei and other
hosts, and compare it with 27 isolates from around the
* Corresponding author. world ; RAPDs and ITS-RFLP molecular markers were
Genetic variability of Beauveria bassiana 1308

used to determine the population structure of this ento- CCGAGCTG) ; OPF01 (5k-ACGGATCCTG) ; OPC20
mopathogen and provide criteria to improve the cur- (5k-ACTTCGCCAC); and OPX07 (5k-GAGCGAG-
rent strategies for use in an IPM programme. GCT). PCR reactions were carried out in 500 ml
microcentrifuge tubes with a total volume of 25 ml
containing 0.2 mM of each dNTP, 0.8 mM of the primer,
MATERIALS AND METHODS
0.625 U Taq DNA polymerase (Gibco BRL, Rockville,
Fungal isolates MD), 50 ng of template DNA in 1rPCR buffer (20 mM
of Tris HCl pH 8.4, 50 mM KCl) and 2.0 mM of MgCl2 in
The isolates studied consisted of 95 Beauveria bassiana
ultrafiltered water. Microtubes were placed in a Ther-
from parasitized insects found in different regions in
mocycler (MJ Research PTC 200) with the following
Colombia and around the world, as well as one isolate
program : 94 x for 5 min followed by 40 cycles with a
of B. brongniartii included as an outgroup (Table 1).
denaturation at 94 x for 1 min, annealing at 36 x for
For each isolate, five polypropilene vials containing
1 min and extension at 72 x for 2 min, and a final exten-
108 spores in 1.8 ml of 10 % glycerol were preserved at
sion at 72 x for 5 min. A negative control was included
x22 xC in the Mycological Collection of Cenicafé.
in every amplification reaction. All the reactions were
made at least twice in order to observe consistent band
Mycelium collection
patterns.
Mycelia and conidia from each isolate were plated on For ITS reactions, primers ITS 1 (5k-TCCGTAGG-
Sabouraud agar and inoculated into liquid GYM (glu- TGAACCTGCGG) and PN16 (5k-TCCCTTTCAA-
cose–yeast extract medium), incubated with orbital CAATTTCACG) that amplify the internal transcrip-
agitation (150 r.p.m.) at 25 x for 6 d. Mycelium was re- tion spacers (ITS1 and ITS2), the complete 5.8S rRNA
covered by vacuum filtration through a Whatman No. 1 gene and the 5k end of the 28S rRNA gene were evaluated
filter paper, washed twice with ultrafiltrated water, (White, Bruns & Taylor 1990). PCR reactions were set
lyophilized, and stored at x20 x until extraction. up in a 500 ml microcentrifuge tube with a total volume
of 100 ml containing 0.2 mM of each primer, 2.5 U of
DNA extraction Taq DNA polymerase (Gibco BRL), 100 ng of template
DNA, in 1rPCR buffer (20 mM of Tris HCl pH 8.4,
The CTAB extraction procedure (Zolan & Pukkila
50 mM KCl) and 1.5 mM of MgCl2 in ultrafiltrated
1996) was used with few modifications. About 50–
water. Amplification reactions consisted of an initial
100 mg of lyophilized mycelium was macerated in a
denaturation at 94 x for 5 min followed by 40 cycles with
prefrozen mortar, adding liquid nitrogen, until a pow-
denaturation at 94 x for 1.5 min, annealing at 55 x for
dered mycelium was obtained. The powder was then
1 min, extension at 72 x for 2 min, and a final extension
transferred into a polypropylene test tube containing
at 72 x for 5 min. A set of negative controls was included.
5 ml extraction buffer (700 mM NaCl, 50 mM Tris HCl
Amplification products were monitored by electro-
pH 8.0, 10 mM EDTA. 2 % CTAB, and 1 % mercapto-
phoresis in 1.5 % agarose gels, running 10 ml of ampli-
ethanol). Samples were incubated for 1 h at 60 x,
fication reaction using TBE Buffer 1r (89 mM tris
followed by two consecutive extractions with 5 ml
borate – 2 mM EDTA pH 8.0) at 4 V cmx1 during 2 h.
chloroform–isoamylalcohol (24 :1). The emulsions were
The gels were stained with ethidium bromide and visu-
centrifuged at 3500 r.p.m. for 15 min and the aqueous
alized under UV light.
phase recovered and taken to another tube. DNA was
precipitated by adding 0.54 v/v of cold isopropanol and
chilled at x20 x for at least 30 min. RFLP’s
DNA was collected by centrifugation at 10 000 r.p.m.
PCR products were precipitated with 0.2 M NaCl and
at 4 x for 15 min. The supernatant was discarded and
two volumes of ethanol, and resuspended in water.
the pellet dried at room temperature. DNA was dis-
Eight restriction enzymes were evaluated: AluI, EcoRI,
solved in 500 ml of TE buffer (10 mM Tris HCl pH 8.0,
MboI, HincII, SstI, MspI, ClaI and HaeIII (Promega,
1 mM EDTA pH 8.0) and treated with 5 ml of RNAse
Madison, WI) following the conditions recommended
A (10 mg mlx1) at 37 x during 1 h. After RNA diges-
by the supplier. Digestion products were analyzed in
tion, samples were extracted with 500 ml chloroform–
agarose gels at 2 % and in polyacrilamide gels at 12%,
isoamylalcohol (24 :1), centrifuged at 10 000 r.p.m. and
stained with ethidium bromide, and visualized with UV
DNA was precipitated from the supernatant at x20 x
light. Digestions were repeated at least twice to avoid
after addition of 2 v/v of absolute ethanol and 0.3 M
the possibility of incomplete digestions of the fragments
sodium acetate. DNA was finally collected by centri-
that could alter the final results.
fugation at 3500 r.p.m. and dissolved in 100 ml of TE
buffer.
Data analysis
Amplification
Only reproducible bands were taken into account to
For RAPD analysis, five random sequence primers develop a presence-absence binary data matrix. With
were used : C3 (5k-CGGCTTGGGT); OPA02 (5k-TG- this, Jaccard similarity coefficients were calculated
A. Gaitan and others 1309

Table 1. Genotype information for the 95 Beauveria bassiana (Bb) and one Beauveria brongniartii (Bbr) isolates compared.

Isolate Host Family Origin

Bb 9001 Hypothenemus hampei Coleoptera: Scolytidae Colombia (Nariño)

Bb 9002 H. hampei Coleoptera: Scolytidae Colombia (Nariño)
Bb 9003 H. hampei Coleoptera: Scolytidae Colombia (Nariño)
Bb 9004 Premnotripes vorax Coleoptera: Curculionidae Colombia (Nariño)
Bb 9005 – – Colombia (Nariño)
Bb 9006 Metamasius hemipterus Coleoptera: Curculionidae Colombia
Bb 9007 – Coleoptera: Scarabaeidae Colombia (Antioquia)
Bb 9008 Leptopharsa gibbicarina Hemiptera: Tingidae Colombia (Magdalena)
Bb 9009 Spodoptera frugiperda Lepidoptera: Noctuidae Colombia (Antioquia)
Bb 9010 H. hampei Coleoptera: Scolytidae Colombia
Bb 9011 Monalonium dissimulatum Hemiptera: Miridae Colombia (Huila)
Bb 9012 H. hampei Coleoptera: Scolytidae Colombia (Antioquia)
Bb 9013 – – Philippines
Bb 9014 H. hampei Coleoptera: Scolytidae Brazil
Bb 9015 Nilaparvata lugens Homoptera: Delphacidae China
Bb 9016 – – Thailand
Bb 9017 – – Thailand
Bb 9018 Cossus cossus Lepidoptera: Cossidae Italy
Bb 9019 Ostrinia furnacalis Lepidoptera: Pyralidae China
Bb 9020 Nilaparvata lugens Homoptera: Delphacidae Philippines
Bb 9021 H. hampei Coleoptera: Scolytidae Ecuador
Bb 9022 Nephotettix cincticeps Homoptera: Cicadellidae China
Bb 9023 Leptocorisa sp. Hemiptera: Coreidae Philippines
Bb 9024 Drycoetes confusus Coleoptera: Scolytidae Canadá
Bb 9025 Ips sp. Coleoptera: Scolytidae Canadá
Bb 9026 – – Philippines
Bb 9027 Nilaparvata lugens Homoptera: Delphacidae China
Bb 9028 Cosmopolites sordidus Coleoptera: Curculionidae Colombia
Bb 9029 Ancognatha scarabaeoides Coleoptera: Scarabaeidae Colombia (Nariño)
Bb 9101 H. hampei Coleoptera: Scolytidae Colombia (Nariño)
Bb 9102 H. hampei Coleoptera: Scolytidae Colombia (Antioquia)
Bb 9103 H. hampei Coleoptera: Scolytidae Colombia (Antioquia)
Bb 9106 Leucoptera coffeellum Lepidoptera: Geometridae Colombia (Quindı́o)
Bb 9107 Bombyx mori Lepidoptera: Bombicidae Colombia (Caldas)
Bb 9108 H. hampei Coleoptera: Scolytidae Colombia (Antioquia)
Bb 9112 Cargolia arana Lepidoptera: Geometridae Colombia (Caldas)
Bb 9114 H. hampei Coleoptera: Scolytidae Colombia (Huila)
Bb 9115 H. hampei Coleoptera: Scolytidae Colombia (Antioquia)
Bb 9116 H. hampei Coleoptera: Scolytidae Colombia (Risaralda)
Bb 9117 H. hampei Coleoptera: Scolytidae Colombia (Valle)
Bb 9118 H. hampei Coleoptera: Scolytidae Colombia (Quindı́o)
Bb 9119 H. hampei Coleoptera: Scolytidae Colombia (Risaralda)
Bb 9120 H. hampei Coleoptera: Scolytidae Colombia (Risaralda)
Bb 9201 H. hampei Coleoptera: Scolytidae Colombia (Caldas)
Bb 9202 H. hampei Coleoptera: Scolytidae Colombia (Quindı́o)
Bb 9203 H. hampei Coleoptera: Scolytidae Colombia (Putumayo)
Bb 9204 Antaeotricha sp. Lepidoptera: Stenomidae Colombia (Santander)
Bb 9205 Diatraea saccharalis Lepidoptera: Pyralidae Colombia (Valle)
Bb 9206 H. hampei Coleoptera: Scolytidae Colombia (Valle)
Bb 9207 H. hampei Coleoptera: Scolytidae Colombia (Risaralda)
Bb 9208 H. hampei Coleoptera: Scolytidae Colombia (Risaralda)
Bb 9209 Cyclocephala sp. Coleoptera: Scarabaeidae Colombia (Antioquia)
Bb 9210 – – –
Bb 9212 H. hampei Coleoptera: Scolytidae Colombia (Risaralda)
Bb 9213 H. hampei Coleoptera: Scolytidae Colombia (Valle)
Bb 9215 H. hampei Coleoptera: Scolytidae Colombia (Valle)
Bb 9216 Metamasius hemipterus Coleoptera: Curculionidae Colombia (Antioquia)
Bb 9217 – – –
Bb 9218 Cosmopolites sordidus Coleoptera: Curculionidae Colombia
Bb 9219 C. sordidus Coleoptera: Curculionidae USA (Florida)
Bb 9301 Rhynchophorus palmarum Coleoptera: Curculionidae Colombia (Caldas)
Bb 9302 R. palmarum Coleoptera: Curculionidae Colombia (Caldas)
Bb 9305 H. hampei Coleoptera: Scolytidae Colombia (Huila)
Bb 9307 H. hampei Coleoptera: Scolytidae Colombia (Antioquia)
Bb 9308 Bombus sp. Hymenoptera: Apidae Colombia (Caldas)
Bb 9309 Oiketicus sp. Lepidoptera: Psichidae Colombia (Caldas)
Bb 9312 Leptopharsa gibbicarina Hemiptera: Tingidae Colombia (Santander)
Genetic variability of Beauveria bassiana 1310

Table 1. (cont.)

Isolate Host Family Origin

Bb 9313 Anthonomus grandis Coleoptera: Curculionidae Colombia (Caldas)

Bb 9315 Periplaneta sp. Orthoptera: Blattidae Colombia (Risaralda)
Bb 9316 – Lepidoptera: Saturniidae Colombia (Caldas)
Bb 9401 – – –
Bb 9402 Cryptophlebia sp. Lepidoptera: Tortricidae Colombia (Quindı́o)
Bb 9403 H. hampei Coleoptera: Scolytidae Brazil
Bb 9404 Cryptophlebia sp. Lepidoptera: Tortricidae –
Bb 9407 H. hampei Coleoptera: Scolytidae Colombia (Risaralda)
Bb 9408 Metamasius hemipterus Coleoptera: Curculionidae Colombia (Santander)
Bb 9409 – – –
Bb 9416 H. hampei Coleoptera: Scolytidae Colombia (Risaralda)
Bb 9417 Hypothenemus sp. Coleoptera: Scolytidae Colombia (Santander)
Bb 9418 – – –
Bb 9501 Cephalonomia stephanoderis Hymenoptera: Bethylidae Colombia (Caldas)
Bb 9502 H. hampei Coleoptera: Scolytidae Guatemala
Bb 9503 H. hampei Coleoptera: Scolytidae Guatemala
Bb 9504 H. hampei Coleoptera: Scolytidae Guatemala
Bb 9505 H. hampei Coleoptera: Scolytidae Guatemala
Bb 9506 H. hampei Coleoptera: Scolytidae Guatemala
Bb 9508 H. hampei Coleoptera: Scolytidae Colombia (Santander)
Bb 9509 Araecerus fasciculatus Coleoptera: Antribidae Colombia (Caldas)
Bb 9510 Megalopyge orsilochus Lepidoptera: Megalopygidae Colombia (Caldas)
Bb 9511 H. hampei Coleoptera: Scolytidae Colombia (Valle)
Bb 9512 – Lepidoptera Colombia (Caldas)
Bb 9601 – Lepidoptera: Arctiidea Colombia (Valle)
Bb 9602 Corinus sp. Coleoptera: Coccinelidae Colombia (Valle)
Bb 9603 Periplaneta americana Orthoptera: Blattidae Colombia (Valle)
Bb 9604 Paracompsus sp. Coleoptera: Curculionidae Colombia (Valle)
Bbr 9301B.brong Antaeotricha sp. Lepidoptera: Stenomidae Colombia (Santander)

(a/(a+b+c)), and a dendrogram generated using hier-
archical methods by the UPGMA clustering algorithm
(Sneath & Sokal 1973). Additionally, an ordering
analysis was made using a Principal Coordinates
Analysis (Sneath & Sokal 1973). Similarity coefficient
calculations, dendrograms and Principal Coordinates
Analysis were carried out with the NTSYSpc 2.02I
program (Exeter Software, Setauket, NY).
RESULTS
RAPD’s
The five primers produced 60 bands, the number gen-
erated by each primer varying between 7 (for OPX 07)
and 15 (for primer C3). As shown in Fig. 1, 25 bands
occurred as rare alleles, present in less than 10 % of the
isolates tested, while 10 were fixed alleles, present in
more than 90 % of the isolates, leaving most variation in
the remaining 25 bands. This initial bandmap shows
relatively low variability in the isolates studied, even the
ones from around the world included. Also, three iso-
lates (Bb9001, Bb9023 and Bb9024), were distinct from
the rest, and finally, the B. brongniartii isolate was in-
cluded amongst those of B. bassiana.
Pairwise comparisons (data not shown) produced an
average similarity value of 0.7063. Isolates exhibiting
exactly the same patterns with the five primers were not
found. The highest values were between Bb9118 and
Bb9309, with a similarity index of 0.9655 ; The isolates
were from two different Colombian coffee regions, on
coffee pests of the orders Coleoptera (H. hampei) and
Lepidoptera (Perileucoptera coffeella). The lowest simi-
larity value was found for Bb9023 and Bb9504, with a
similarity index of 0.1944 ; isolates from the Philippines
and Guatemala, on insects of the order Hemiptera and
Coleoptera (Leptocorisa sp. and H. hampei, respect-
ively).
The dendrogram generated with the similarity data
(Fig. 2a) separated the 96 isolates into four groups.
Group one, the three isolates mentioned above (Bb9001,
Bb9023 and Bb9024), from Colombia, Philippines and
Canada, respectively. Group two, of ten isolates, nine
from Colombia and one from China (Bb9027), and
mostly obtained from H. hampei. Group three, the
largest one, comprising 75 isolates. And the fourth
group, consisting of eight isolates from different coun-
tries (Thailand, China, Colombia, Brazil and the Phi-
lippines), and associated to various insect orders. This
last group had the lowest similarity index (0.4836), and
the isolate of B. brongniartii (Bbr9301) was included in it.
In general, no direct relationship was observed with
respect to host or geographical origin, although small
groups comprised isolates from geographically close
areas. This was the case for Bb9003, Bb9004 and
Bb9005, with an average similarity index of 0.9355,
from the southern Colombian region of Nariño ; Bb9118
and Bb9309, which came from Caldas and Quindı́o,
A. Gaitan and others
Fig. 1. Bandmap based on RAPD markers (rows) for all the Beauveria bassiana isolates (columns). Marker presence is denoted
by a black square. The white triangle indicates B. brongniartii. Black triangles indicate genotypes Bb9001, Bb9023 and
Bb9024. Column on the right : white, rare alleles (less than 0.1 frequency) ; grey, allele frequency between 0.1 and 0.9 ; black,
very frequent alleles (higher than 0.9).
located in the Colombian central coffee zone, with a
similarity index of 0.9655, and obtained from H. hampei
and P. coffeella ; Bb9117, Bb9206 and Bb9215 from
Valle del Cauca, southwestern Colombia, with an aver-
age similarity of 0.8571 ; Bb9102, Bb9106 and Bb9107
proceeding from Antioquia, Caldas and Risaralda, all in
the main Colombian coffee zone, and from H. hampei,
P. coffeella and Bombix mori, with an average similarity
of 0.9524. Finally, Bb9007, Bb9315 and Bb9316 isolated
from Antioquia, Caldas and Risaralda on insects of the
families Scarabeidae, Saturniidea and Blattidae, with an
average similarity of 0.8751.
Principal Coordinates Analysis (Fig. 3) of the binary
matrix data clearly separated individuals belonging to
Group One, whereas a densely populated cloud con-
tained 80 isolates, and left out 14 that belonged indis-
tinctively to the groups two (Bb9114), three (Bb9006,
Bb9026, Bb9112, Bb9206, Bb9213, Bb9510, and
Bb9604) and four (Bb9016, Bb9022 and B. brongniartii)
of the dendrogram.
ITS-RFLP’s
Primers ITS1 and PN16 amplified a 930 bp fragment
for all the 95 isolates of Beauveria bassiana and the
isolate of B. brongniartii. From the eight enzymes used,
polymorphisms were observed with AluI and MpsI
(Table 2), while MboI, HaeIII, HincII and ClaI pro-
duced monomorphic band patterns, and no restriction
fragments were obtained with EcoRI and SstI. The
dendrogram generated (Fig. 4) clustered the B. bassiana
isolates into three well-differentiated groups, while the
B. brongniartii sample was clearly separated from B.
bassiana. A second group was formed, once again, by
Bb9011, Bb9023 and Bb9024. In the third group, four
different RFLP patterns were found : two were unique
for isolates Bb9011 and Bb9016. The other two were
associated with a subgroup of 32 isolates (Bb : 9005,
1311
9012, 9002, 9003, 9004, 9006, 9007, 9008, 9009, 9010,
9013, 9014, 9015, 9017, 9018, 9019, 9020, 9021, 9022,
9025, 9028, 9029, 9102, 9106, 9120, 9203, 9204, 9205,
9215, 9218, 9512, 9601) and a subgroup of the 59 ad-
ditional isolates. Again, none of the groups could be
related to either host or geographic location.
DISCUSSION
In order to determine the genetic variability of Beauveria
bassiana in Colombia, a broad range of samples were
studied using markers unaffected by environmental
factors and targeting the whole genome at random
(RAPD’s), and also markers relevant to species-level
taxonomy (ITS). Using five random sequence RAPD
primers on the 95 B. bassiana and one B. brongniartii
isolates, no identical banding patterns were found.
However, a 0.7063 average similarity index indicates low
genetic variation especially considering that isolates
from different continents and hosts were included, and
that a molecular marker technique that covers the
whole genome (RAPD’s) was used, which allowed to
find differences amongst genetically close individuals.
RAPD data, therefore, does not indicate that the B.
bassiana population in Colombia comprises clonal sub-
populations, due to the variability observed, and the
lack of association among the 25 (41.6%) highly poly-
morphic bands obtained (Fig. 1).
The 930 bp ITS fragment amplified in this experiment
matches the reports by Neuvéglise et al. (1994), that
permitted the differentiation of Paecilomyces from
Beauveria, but not between Beauveria species. In that
study, ITS markers were useful in evaluating the
variability of B. brongniartii isolates from different
geographical origins and hosts. However, very few
B. bassiana isolates were studied in that work or else-
where to date, and they display a conserved ITS
Genetic variability of Beauveria bassiana 1312

Fig. 2. Genetic similarity dendrogram based on 60 RAPD markers in Beauveria bassiana. Black squares indicate individuals
belonging to the first large subgroup obtained by ITS-RFLP. Individuals with unique ITS-RFLPs are labelled with an
asterisk. Origins : P, Philippines ; C, Canada; Ch, China ; E, Ecuador; G, Guatemala ; I, Italy ; T, Thailand ; U, USA.
br9301, B. brongniartii.
A. Gaitan and others 1313

Fig. 3. Principal Coordinates Analysis output for Beauveria bassiana. br9301, B. brongniartii.

Table 2. Band patterns (in bp) for polymorphic ITS-RFLP in No concordance was observed between the grouping
Beauveria bassiana. obtained with RAPD’s and that from ITS-RFLP
Enzyme Pattern 1 Pattern 2 Pattern 3 Pattern 4 Pattern 5 analysis (Fig. 2b). This indicates that a mechanism that
may result in diminished gene exchange and isolation of
AluI 470 600 670 genetic types, such as heterokaryon incompatibility, is
165 140 175
140 110 140
not at work. On the contrary, the presence of the two
110 60 110 main ITS types scattered among most of the RAPD’s
60 groups, together with the common absence of the teleo-
MspI 460 460 460 460 460 morph (Cordyceps staphylinidaecola), suggests that an
280 280 280 280 300 open gene transfer system could generate the observed
130 100 100 100 100 genetic diversity of the population. Under laboratory
100 80 80 60 90
conditions, both heterokaryon incompatibility and ana-
80 60 60 60
60 30 stomosis have been reported in B. bassiana (Paccola-
Meirelles & Azevedo 1991). However, further research
is required to establish the presence and significance of
sequence length. In fungi, ITS length polymorphisms parasexual recombination in nature for B. bassiana.
are not frequent within the same species; in most of the In addition to gene exchange, two other factors can
cases, variation in the ITS size does not exceed a few be related to genetic diversity in populations : host
nucleotides among species of the genus, even closely range and ecological conditions. Maurer et al. (1997)
related ones (Nazar et al. 1991). Our results, with a used RAPD markers and observed clusterings based
larger number of isolates, confirm these observations on the host insect, independently from the geographic
since the 95 isolates evaluated were monomorphic in origin. Similarly, Castrillo et al. (1999) observed a
length, and exhibited only six different variations in ITS common presence of a B. bassiana genotype associated
restriction patterns; four of these patterns were rep- with Alphitobius diaperinus, as well as genetic variability
resented by only one (Bb9001, Bb9011, Bb9016) or in the population, represented by the presence of 77
two (Bb9023 and Bb9024) isolates. Contrary to RAPDs, genotype-specific bands among 78 isolates evaluated.
ITS-RFLP data only indicates two very conserved and This indicates that a predominant factor that affects
widely dispersed genotypes of B. bassiana, which can- the population structure of B. bassiana is the host range,
not sufficiently differentiate the isolates in this study. In exerting a selection pressure favoring one or few geno-
addition, the recovery of conserved genotypes from types. In contrast, the population under study revealed
different continents indicates that the two ITS genotypes a clustering of isolates from H. hampei (40 isolates)
of B. bassiana may be distributed worldwide. together with ones from other coleopterans, and with
Genetic variability of Beauveria bassiana
Fig. 4. Genetic similarity dendrogram based on ITS-RFLP markers for Beauveria bassiana. To facilitate reading, only the
first member of the large subgroups mentioned in the text is indicated.
isolates obtained from other orders (42 isolates from
30 different hosts, and 14 from unknown hosts). This
suggests that host range is not a determining selec-
tion factor, at least in Colombia, where B. bassiana
has been isolated from more than 27 identified species
mostly belonging to Lepidoptera and Coleoptera
(Bustillo et al. 1998), and where H. hampei is ubiquitous
in the Colombian coffee zone.
With respect to ecological conditions, Glare & In-
wood (1998) found evidence of association between
certain B. bassiana genotypes and specific geographic
regions in New Zealand. Although, small clusters at a
regional level were found, as reported by Castrillo et al.
(1999), our data indicate that isolates from different
regions in Colombia (68 isolates) were clustered mixed
together with isolates from other countries (22 isolates
plus five of unknown origin), rejecting, in consequence,
a grouping based on geographic origin (Fig. 2c).
Our results confirm the low level of genetic variability
in B. bassiana, pointing out the similarity between
B. bassiana isolates from Colombia and ones from
other parts of the world. This implies that, for such a
widely dispersed species, the introduction of foreign
strains will not alter significantly the diversity already
present. However, because of the possibility of active
recombination, new traits, either endogenous (such as
mutations) or exogenous (viral RNA or genetic trans-
formation), could also be easily shared in the genetic
pool. Finally, a question remains as to the importance
of the genetic variability of entomopathogens in in-
sect epizootic effectiveness. These aspects must be con-
sidered in further refinements of the implementation of
biocontrol measures of the coffee berry borer using
B. bassiana.
ACKNOWLEDGEMENTS
This research was funded by the National Federation of Coffee
Growers of Colombia and the Colombian Institute for the Develop-
ment of Science and Technology (COLCIENCIAS), to we express
gratefulness.
1314
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Corresponding Editor: N. Magan