Pakistan J. Zool., vol. 43 (3), pp. 417-425, 2011.
Phylogenetics of Entomopathogenic Fungi Isolated From the Soils of
Different Ecosystems
Shoaib Freed, Feng-Liang Jin and Shun-Xiang Ren*
Engineering Research Center of Biocontrol, Ministry of Education, South China Agricultural University,
Guangzhou, 510642, P.R.China
Abstract.- The entomopathogenic fungi were isolated from the soils of diverse sources i.e., forest, agricultural
and urban lands belonging to different geographical locations. The phylogenetic analysis of the ITS1, ITS2 and 5.8SrDNA sequences of entomopathogenic Paecilomyces spp. from different countries support the polyphyletic character.
In Hypocreales, four key ITS subgroups were found, i.e., Isaria fumosorosea, Paecilomyces marquandii,
Paecilomyces lilacinus and Paecilomyces sp. All of the four foremost subgroups of the Hypocreales showed clear
identification and division. One of the main subgroup showed distinctive characteristics as it showed the majority of
P. marquandii, P. lilacinus and Paecilomyces sp. distant relatedness. In addition the genetic variability among the
isolates of I. fumosorosea was assessed and compared with the strains from other countries; overall genetic variability
among the isolates of different countries was observed that confirms the polymorphic nature of I. fumosorosea. The
majority of the isolates of I. fumosorosea belonged to one of the closely related genotypes. The utmost genetic
distance of 0.169% was observed between the strains of France and USA. This information provides an enhanced
perceptive concerning the isolates of diverse countries with soil source.
Key words: Molecular phylogeny, Paecilomyces spp, ITS-rDNA, Isaria fumosorosea, biocontrol, mycoinsecticide.
INTRODUCTION
F
ungi play an imperative physiological,
meditative and ecological role in the ecosystem
(Miller, 1995). Soil is a multifaceted ecosystem in
which micro-organisms occur in heterogeneous
communities; however, the behaviour of individual
species is often unknown due to the lack of
appropriate detection and identification techniques
(Akkermans et al., 1994). The hypomycete genus
Paecilomyces contains members which are often
thermophilic; the section Isarioidea contains
mesophiles, including numerous entomopathogenic
species such as Isaria farinosus, Isaria
fumosorosea,
Paecilomyces
amoeneroseus,
Paecilomyces lilacinus, Paecilomyces javanicus and
Paecilomyces tenuipes. For the control of insect
pests especially Bemisia tabaci (Gennadius),
considered as a major pest in field and green house
crops, I. fumosorosea is regarded as the most
common pathogen (Lacey et al., 1996). The release
of I. fumosorosea in the field for the effective
____________________________
*
Corresponding author rensxcn@yahoo.com.cn or
shbfkh@gmail.com
0030-9923/2011/0003-0417 $ 8.00/0
Copyright 2011 Zoological Society of Pakistan.
control of insect pests needs the selection of best
genotypes and for this study of population genetics
becomes the prerequisite (Clouet et al., 2005). The
limitations of traditional identification techniques
indicate that non conventional methods need to be
developed for the identification of these fungi
(Inglis and Tigano, 2006).
Comparative RNA (rDNA) gene sequence
information contain a tandem repeat including
codon regions which are conserved at varying
degrees and are also highly divergent due to this
reason these give an enhanced knowledge about the
evolutionary interaction over a broad range (Pace et
al., 1986; Driver et al., 2000). Luangsa-ard et al.
(2004, 2005) examined the nuclear-encoded SSR
DNA sequences of Paecilomyces strains from
Thailand and some of others held in CBS. The
phylogenetic analysis based on the 18S-rDNA
demonstrated that Paecilomyces is polyphyletic
across two subclasses; subsequently the β-tubulin
gene and ITS-rDNA sequences analysis confirmed
the phylogenetic relationship of Paecilomyces sect.
Isarioidea and it showed that this section is also
polyphyletic within the Hypocreales (Luangsa-ard et
al., 2005). In case for the Paecilomyces the analysis
of large and small subunit rRNA gene, ITS-rDNA
and especially for I. fumosorosea has already been
performed. (Obornik et al., 2001; Fargues et al.,
418
S. FREED ET AL.
2002). The genetic variability of I. fumosorosea was
characterized by several methods, 28S-rDNA genes,
RAPD-PCR and tRNA-PCR showed polymorphism
in the isolates of I. fumosorosea (Tigano-Milani et
al., 1995a; Fegan et al., 1993; Leal et al., 1994;
Bidochka et al., 1994; Piatti et al., 1998).
In this study we isolated and selected various
Paecilomyces spp. from the soil samples of different
geological origins and sources to clarify the
taxonomic relationships among them and to uncover
the genetic variability among the isolates of I.
fumosorosea from different countries with diverse
sources of isolation.
MATERIALS AND METHODS
Sampling locations
The soil samples from a variety of habitats
including urban, agricultural and forest ecosystems
were collected from Islamic Republic of Pakistan,
People’s Republic of China, South Korea, Indonesia
and The Netherlands. Soil samples were taken 10
cm deep under the earth from different provinces of
the above described countries. After the collection,
samples were preserved at 4°C untill used for
isolation of Isaria and Paecilomyces spp.
Isolation of fungi
For soil samples collected from different
locations, Isaria and Paecilomyces spp. were
isolated on semi-selective medium described by
(Veen and Ferron, 1966). The medium consisted of
glucose (10 g), peptone (10 g), bile (15 g) (Sigma),
rose Bengal (1:15,000), and agar (30 g) in 1 L of
deionized water. The medium was amended with
0.002
gL-1
dodine
(N-dodecylguanidine
monoacetate; American Cyanamid), 0.12 g L-1
cycloheximide
(Sigma)
and
0.25
gL-1
chloramphenicol (Sigma). Sub-sample of soil (10g)
was placed into 90 ml of sterile water, and the
sample was homogenized using a surface-sanitized
waring blender. The homogenate was diluted three
times in a 10-fold dilution series in sterile phosphate
buffer (100 mM, pH7.0), 100 µl from each dilution
was spread onto the semi-selective medium, and
cultures were maintained at 25°C in the dark.
Cultures were examined after 4 days and daily
thereafter for 3 additional days. Conidia
characteristics of Isaria and Paecilomyces spp. were
transferred onto PDA, the cultures were purified,
and examined microscopically for characteristic
conidiogenesis.
All isolates were subsequently propagated
from a single conidium (Table I). 100 µl of a
suspension containing a low density of conidia in
phosphate buffer was spread onto PDA. After 24 h
growth at 25°C, a single germinated conidium
separated from other conidia was identified by
dissecting microscope using 20 times magnification,
a piece of the medium encompassing only the target
conidium was aseptically removed and transferred
to PDA, and the culture was maintained at room
temperature. After 5 days, morphological
characteristics of Isaria and Paecilomyces spp. were
confirmed microscopically; conidia were collected
and stored in sterile 30 % glycerol at -80°C until
required.
Genomic DNA isolation
The genomic DNA was extracted by the
method described by Liu et al. (2000) with some
modifications. Briefly, the method included the
following steps (i) To a 1.5-ml Eppendorf tube
containing 500 µl of lysis buffer (400mM Tris-HCl
[pH 8.0], 60 mM EDTA [pH 8.0], 150 mM NaCl,
1% sodium dodecyl sulfate), a small lump of
mycelia was added by using a sterile toothpick, with
which the lump of mycelia was disrupted. The tube
was then left at room temperature for 45 min. (ii)
After adding 150 µl of potassium acetate (pH 4.8;
made of 60 ml of 5 M potassium acetate, 11.5 ml of
glacial acetic acid, and 28.5 ml of distilled water),
the tube was then vortexed briefly and spun at
>10,000 x g for 2 min. (iii) The supernatant was
transferred to another 1.5 ml Eppendorf tube and
centrifuged again as described above. After
transferring the supernatant to a new 1.5 ml
Eppendorf tube, an equal volume of isopropyl
alcohol was added. The tube was mixed by
inversion briefly. (iv) The tube was spun at 10,000 x
g for 2 min, while the supernatant was discarded.
The resultant DNA pellet was washed twice with
300 µl of 70% ethanol. After the pellet was spun at
>10,000 rpm for 1 min, the supernatant was
discarded. The DNA pellet was air dried and
dissolved in 70 µl of 1 x Tris-EDTA. Extracted
PHYLOGENETICS OF PAECILOMYCES SPP.
Table I.-
419
Entomopathogenic fungal isolates from different geographical origin and source
S.No.
Isolates
Fungal species
Origin
Province/City
Source
Accession
numbers
1.
2.
3.
4.
5.
6.
7.
8.
9.
CNIM
CNZG
CNZH
CNHG
CNBJ
CNXN
NLHG-1
NLHG-2
NLWG
Isaria fumosorosea
Isaria fumosorosea
Isaria fumosorosea
Isaria fumosorosea
Isaria fumosorosea
Isaria fumosorosea
Isaria fumosorosea
Isaria fumosorosea
Isaria fumosorosea
P.R.China
P.R.China
P.R.China
P.R.China
P.R.China
P.R.China
Netherlands
Netherlands
Netherlands
Riverside soil
Farm land soil
Mountain soil
Farm land soil
Urban land soil
Forest soil
Vegetable field soil
Corn field soil
Grass field soil
FJ765006
FJ765007
FJ765008
FJ765009
FJ765010
FJ765011
FJ765012
FJ765013
FJ765014
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
NLUC
NLAG
SKCH-1
SKCH-2
CNXM
SKCH-4
PAK-1
PAK-2
INTR-1
INTR-2
CNSH
SKCH-5
CNLH
Isaria fumosorosea
Isaria fumosorosea
Isaria fumosorosea
Paecilomyces sp.
Paecilomyces. lilacinus
Paecilomyces. lilacinus
Paecilomyces. lilacinus
Paecilomyces. lilacinus
Paecilomyces. lilacinus
Paecilomyces. lilacinus
Paecilomyces marquandii
Paecilomyces marquandii
Penicilium citrinum
Netherlands
Netherlands
South Korea
South Korea
P.R.China
South Korea
I.R.Pakistan
I.R.Pakistan
Indonesia
Indonesia
P.R.China
South Korea
P.R.China
Inner Mongolia
Guangdong，Zhanjiang
Zhejiang Ningbo
Gansu Huixian
Beijing
Xinjiang
Gelderland, Heteren
Gelderland, Heteren
Gelderland,
Wageningen
Utrecht
Gelderland, Arnhem
Cheju island
Cheju island
Xizang, Motuo
Cheju island
Rawlakot, Kashmir
Muzaffarabad, Kashmir
Tretes
Tretes
Hubei, Shiyan
Cheju Island
Hubei, Liuyang
Urban land soil
Potato field soil
Urban land soil
Seaside soil
Forest soil
Urban land soil
Mountain soil
Mountain soil
Mountain soil
Volcano soil
Mountain soil
Volcano soil
Mountain soil
FJ765015
FJ765016
FJ765017
FJ765018
FJ765019
FJ765020
FJ765021
FJ765022
FJ765023
FJ765024
FJ765025
FJ765026
FJ765031
genomic DNA was identified by 1% TBE–agarose
gel
(Invitrogen
Corp.,
Burlington,
ON)
electrophoresis, stained with ethidium bromide and
visualized under UV light. The DNA was purified
by using the E.Z.N.A. Gel Extraction Kit (OMEGA
bio-tek, USA). The purified DNA was used for the
PCR amplification.
PCR amplification for ITS-rDNA regions
DNA was amplified by PCR using the
complementary
primers
ITS1F
(5'CTTGGTCATTTAGAGGAAGTAA-3') and ITS4A
(5'-GCCGTTACTGGGGCAATCCCTG-3') Larena
et al. (1999). The PCR mixture consisted of a total
volume of 25µl containing 1 x reaction buffer,
0.2mM dNTPs, 2mM MgCl2, 0.5µM of each primer
(Invitrogen, USA), 1U Taq polymerase (TAKARA
Japan) and 2.0µl (20-25 ng) of DNA template. The
amplification conditions consisted of one cycle at
94°C for 5 min, followed by 35 cycles at 94°C for
30s, 58°C for 1 min and 72°C for 50s, finishing up
with 1 cycle at 72°C for 5 min.
DNA visualization, quantification and purification
The PCR products were identified by agarose
gel electrophoresis in a 1.5% TAE-agarose gel.
Quantification of the PCR products was done by
using a 100bp DNA ladder. Products with clear
band were purified by using the E.Z.N.A. Gel
Extraction Kit (OMEGA bio-tek, USA).
DNA sequencing and data analysis
The purified PCR amplified products were
used for nucleotide sequencing. DNA sequencing
was done by using the same primers with the
concentrations as described above. Nucleotide
sequences for the isolates were searched for their
similarity index by using BLAST software
(http://blast.ncbi.nlm.nih.gov/Blast.cgi).
Manual
improvement was also done to the sequences
lacking a few amino acids on their two ends. This
was realized by retrieving the whole subject
sequence from GenBank and translating it with
EditSeq program (version 5.01) of the DNAStar
420
S. FREED ET AL.
package to obtain the absent amino acid residues.
All the sequences were aligned using Muscle online
(www.ebi.ac.uk/Tools/muscle/index.html).
Alignment gaps were treated as missing data and no
characters in the alignment were excluded.
Phylogenetic analyses were conducted on the basis
of 658 aligned positions alignable in all sequences.
Divergence of each pair of sequence was calculated
by DAMBE Xia and Xie (2001).
Table II.-
Isaria and Paecilomyces spp. isolates (ITSrDNA) sequences downloaded from GenBank
used in the phylogenetic tree
S.No.
Accession
No’s
Species
Location
1.
2.
3.
4.
5.
AB265146
AF461747
AF461748
AJ345091
AJ345092
Isaria fumosorosea
Isaria fumosorosea
Isaria fumosorosea
Isaria fumosorosea
Isaria fumosorosea
6.
AJ608982
Isaria fumosorosea
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
AY624182
AY755506
DQ069285
DQ449655
EU886747
EU886748
AM412779
AY213668
EU306174
EU828665
Isaria fumosorosea
Isaria fumosorosea
Isaria fumosorosea
Isaria fumosorosea
Isaria fumosorosea
Isaria fumosorosea
Paecilomyces lilacinus
Paecilomyces lilacinus
Paecilomyces lilacinus
Paecilomyces lilacinus
17.
AB369489
Paecilomyces lilacinus
18.
FJ461773
Paecilomyces lilacinus
19.
20.
AY624193
AB244776
Paecilomyces marquandii
Paecilomyces marquandii
Japan
France
France
Hungary
Hungary
United
Kingdom
Thailand
Germany
Indonesia
Spain
USA
USA
Germany
USA
Taiwan
Thailand
Gansu, P.R.
China
Hunan,
P.R.China
Thailand
Japan
Phylogenetic analyses were conducted using
PAUP 4.0 Beta Win (Swofford, 1998). The aligned
sequences were used to construct the maximum
parsimony tree. Sequences were first aligned in
Muscle and then copied into PAUP window to
prepare nexus files. Maximum parsimony trees were
bootstrapped with 1,000 replicates to provide
information about their statistical reliability.
Maximum parsimony trees were constructed using
PAUP 4.0 Beta Win by executing command
“bootstrap nreps = 100 search = heuristic/addseq =
random”. Other parameters were set to default
values. Relative support for resulting trees was
obtained from bootstrap analyses (Felsenstein,
1985) using 100 heuristic searches with groups
occurring at 70% or greater frequencies being
retained in the consensus trees. Maximum
likelihood trees were constructed using MrBayes 3
(Ronquist and Huelsenbeck, 2003). The trees were
displayed using the TreeView program (version
1.6.6)
(http://taxonomy.zoology.gla.ac.uk/rod/
treeview.html). The ITS regions for the isolates
were sequenced and subsequently submitted to
GenBank under the accession no’s. FJ765006FJ765026 and FJ65031.
RESULTS
Taxonomic relationships of Isaria and
Paecilomyces spp. isolated from different
agricultural, forest and urban land soil of Pakistan,
China, South Korea, Indonesia and The Netherlands
was assessed. The ITS-1, ITS-2 and 5.8S-rRNA
genes were amplified and nucleotide sequencing
was performed. The representative nucleotide
sequences for the Isaria and Paecilomyces spp.
strains were downloaded from GenBank and were
clustered with the laboratory isolated Isaria and
Paecilomyces spp. The maximum parsimony and
maximum likelihood methods generated somewhat
similar
dendrograms,
therefore
only
the
dendrograms of maximum likelihood have been
included in the results. The dendogram obtained by
maximum likelihood (ML) method using MrBayes
3.0, phylogenetic software clustered all of the
isolates into 3 main groups with certain sub groups
(Fig. 1). The group A clustered I. fumosorosea
isolates from different countries. The clade 1-A
clustered the isolates of laboratory isolated I.
fumosorosea, while the clade 1-B grouped together
the isolates of I. fumosorosea from different
countries viz., USA, Thailand, Germany etc. The
genetic variability among all the isolates of I.
fumosorosea used in the study was found to be
minimal, even with different geographical origins.
The Group B clustered P. lilacinus and
Paecilomyces
sp.
isolates
from
different
geographical origins. The grouping of P. lilacinus
from Asia, Europe and America, demonstrated the
results somewhat similar to that of group-A, as
it illustrated same kind of diversification i.e., the
PHYLOGENETICS OF PAECILOMYCES SPP.
85
1-A
87
A
98
1-B
100
95
100
97
79
Hypocreales
76
B
95
99
C
421
CNIM I. fumosorosea
CNHG I. fumosorosea
NLHG1 I. fumosorosea
NLGHW I. fumosorosea
CNZG I. fumosorosea
NLAG I. fumosorosea
SKCHI I. fumosorosea
CNBJ I. fumosorosea
NLWG I. fumosorosea
CNZH I. fumosorosea
NLUC I. fumosorosea
CNXJ I. fumosorosea
AF461747 I. fumosorose FR*
DQ069285 I. fumosorose IND*
AY755506 I. fumosorosea GER*
AY624182 I. fumosorosea TL*
AJ345091 I. fumosorosea HUN*
AB265146 I. fumosorosea JP*
AJ608982 I. fumosorosea UK*
EU886747 I. fumosorosea FR*
DQ449655 I. fumosorosea SP*
EU886748 I. fumosorosea USA*
AJ345092 I. fumosorosea HUN*
INTR1 P. lilacinus
INTR2 P. lilacinus
SKCH2 Paecilomyces sp.
AB369489 P. lilacinus CN*
AY213668 P. lilacinus USA*
EU306174 P. lilacinus TW*
PAK2 P. lilacinus
CNXM P. lilacinus
PAK1 P. lilacinus
SKCH4 P. lilacinus
AM412779 P. lilacinus GER*
FJ461773 P. lilacinus CN*
EU828665 P. lilacinus
AY624193 P. marquandii TL*
SKCH5 P. marquandii
CNSH P. marquandii
AB244776 P. marquandii JP*
CNLH Penicilium citrinum
Fig. 1. Dendogram based on Maximum Likelihood (ML) method by analysing the internal transcribed spacer
(ITS) 1, 2 and the 5.8S ribosomal gene of Paecilomyces spp. from different geographical origins. Penicillium citrinum
was used as out group. The numbers at the nodes indicate for the internal branches within the tree obtained by
bootstrap analysis (% of 1000 bootstraps). (CN = China, IM = Inner Mongolia, HG = Huixian, BJ = Beijing, ZH =
Zhejiang, XJ = Xinjiang, ZG= Zhanjiang, SH = Shiyan, SC = Sichuan, XM = Xizang (Tibet) and LH = Liuyang),
(PAK = Pakistan) (SK = South Korea, CH = Cheju), (NL= Netherlands, HG = Heteren, WG = Wageningen, UC =
Utrecht and AG = Arnhem), (IN = Indonesia, TR = Tretes). (TL = Thailand, JP = Japan, Hun = Hungary, UK = United
Kingdom, SP = Spain, FR = France). The Strains marked with asterisk* are GenBank accessions not sequenced in the
study.
clustering of isolates from Asia, Europe and USA.
In contrast to this P. marquandii from different
geographical origins (Asian countries i.e., China,
South Korea, Thailand and Japan) was grouped in
clade C. The clustering of all the isolates of P.
marquandii shows the grouping and its difference in
422
S. FREED ET AL.
genetic make up from that of other Paecilomyces
spp. The dendogram obtained by ML method clearly
indicates the presence of four main sub groups of
Paecilomyces i.e., I. fumosorosea, P. marquandii, P.
lilacinus and Paecilomyces sp. of the order
Hypocreales of Ascomycota, while outgroup
consisted of Eurotiales (Fig. 1). The isolates of P.
lilacinus and Paecilomyces sp. showed a basal clade
and the presence of these species together in one
group shows that some characters are being shared
by both species.
Genetic variability of I. fumosorosea
The overall genetic variability among the
laboratory isolated I. fumosorosea isolates was
determined (Fig. 2). The dendogram obtained by
ML analysis clustered the isolates into two main
groups. The group-A clustered mainly isolates from
Europe, UK and USA while other lab isolates viz.,
CNIM, CNHG, CNZH and CNXJ didn’t cluster in
this group. Group-B assembled mainly the
laboratory isolated samples and in addition it also
grouped together few isolates from France,
Germany and Indonesia. The genetic variability
among the isolates seemed to be minimal, the reason
for which can be the sharing of the same host.
Genetic distances of I. fumosorosea isolates
according to locations
The genetic distances among the isolates I.
fumosorosea from different ecosystems were
calculated by Kimura 2-parameter model. The
results showed different genetic distances for the
isolates of I. fumosorosea. The genetic distances
among the isolates range from 0.000 to 0.169%. The
maximum genetic distance of 0.169% was observed
between the isolates of France and USA.
DISCUSSION
Phylogenetic analysis based on the ITSrDNA regions revealed a high level of
polymorphism within the isolates of I. fumosorosea
and other related Paecilomyces species. Four
distinct groups of Paecilomyces spp. were observed
from the ITS-rDNA sequence data. All I.
fumosorosea isolates were clustered in group-A.
The second group i.e. B, clustered P. lilacinus while
the third group C, contained P. marquandii from
different countries (Table I) (Fig. 1). The molecular
analysis of the Paecilomyces spp. demonstrates that
the insect associated Paecilomyces are polyphyletic
within the order Hypocreales (Fig. 1). The order
Hypocreales was clearly divided into different sub
groups
as
the
group-A
contained
the
entomopathogenic fungi I. fumosorosea, while the
presence of the nematophagous fungi P. lilacinus in
group-B proved the findings of Luangsa-ard et al.
(2005) that the section Isarioidea of the order
Hypocreales is not monophyletic. P. lilacinus
isolates were grouped together with another related
species i.e., Paecilomyces sp. that confirms the
relationship of the order Hypocreales with its
subgroups. In contrast to this P. marquandii was
clustered group-C that shows the division of
Paecilomyces groups.
The ITS-rDNA sequence data showed a high
level of variability that divided the three species of
Paecilomyces into four groups i.e., I. fumosorosea,
P. marquandii, P. lilacinus and Paecilomyces sp.
Our results are in confirmation with the results of
Inglis and Tigano (2006), that a high level of
variability was observed among the Paecilomyces
spp. isolates from different countries and hosts. The
same results were obtained by using different DNA
markers (Tigano-Milani et al., 1995a,b). The
isolates of Paecilomyces spp. were clustered into
three distinct groups while the genetic variability
among the isolates of the same Paecilomyces spp.
seemed to be minimal, even the isolates were
isolated from different geographical locations and
hosts (Fig. 1). The group A confers comprehensive
detail regarding the relationship of I. fumosorosea,
as it clustered together the isolates from three
different continents viz., Asia, Europe and America.
The maximum genetic distance of 0.169% was
observed between the strains of I. fumosorosea from
France and USA. It can be assumed that the
entomopathogenic fungi possess little genetic
variability as in case of Metarhizium anisopliae var.
anisopliae (Freed et al., 2010; Inglis et al., 2008).
The ITS region was found to be useful for resolving
the difficulties in the taxonomy of Paecilomyces
spp. The nucleotide sequences of different
Paecilomyces spp. were downloaded from GenBank
for their relationship with sequence data from our
PHYLOGENETICS OF PAECILOMYCES SPP.
423
EU886747 I. fumosorosea USA*
DQ449655 I. fumosorosea SP*
AB265146 I. fumosorosea JP*
AJ345092 I. fumosorosea HUN*
100
AY624182 I. fumosorosea TL*
AJ608982 I. fumosorosea UK*
AJ345091 I. fumosorosea HUN*
EU886748 I. fumosorosea USA*
CNIM I. fumosorosea
CNHG I. fumosorosea
A
CNZH I. fumosorosea
CNXJ I. fumosorosea
AY755506 I. fumosorosea GER*
CNZG I. fumosorosea
SKCHI I. fumosorosea
NLAG I. fumosorosea
87
NLWG I. fumosorosea
NLGH1 I. fumosorosea
NLHG2 I. fumosorosea
95
B
AF461747 I. fumosorosea FR*
DQ069285 I. fumosorosea IND*
NLUC I. fumosorosea
CNBJ I. fumosorosea
CNLH Penicillium citrinium
Fig. 2. Dendogram based on Maximum Likelihood (ML) method by analysing the internal transcribed spacer
(ITS) 1, 2 and the 5.8S ribosomal gene of isolates of Isaria fumosoroseus from different geographical origins.
Pencillium citrinum was used as an out group. The numbers at the nodes indicate for the internal branches within the
tree obtained by bootstrap analysis (% of 1000 bootstraps), CN = China, IM = Inner Mongolia, HG = Huixian, BJ =
Beijing, ZH = Zhejiang, XJ = Xinjiang, ZG= Zhanjiang, SH = Shiyan, SC = Sichuan, XM = Xizang(Tibet) and LH =
Liuyang), (PAK = Pakistan) (SK = South Korea, CH = Cheju), (NL= Netherlands, HG = Heteren, WG = Wageningen,
UC = Utrecht and AG = Arnhem) and (IN = Indonesia, TR = Tretes). The Strains marked with asterisk* are GenBank
accessions not sequenced in the study.
study. In this case furthermore same results were
obtained. All the nucleotide sequences together with
laboratory isolates were clustered into 3 main
groups with sub groups
This suggests that
Paecilomyces spp. are closely related to each other
and also with other entomopathogenic fungi as
reported by Obornik et al. (2001).
Previous phylogenetic studies on I.
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S. FREED ET AL.
fumosorosea showed that there are at least three
monophyletic groups within this complex and that
host selection pressure may be significant in the
selection of genotypes (Fargues et al., 2002), as it is
significant mycoinsecticide for the control of B.
tabaci (Lacey et al., 1996; 2001). Our studies
showed that I. fumosorosea isolates were only
clustered with the genetically identical I.
fumosorosea isolates, none of the other
Paecilomyces spp. isolates were clustered with these
isolates (Fig. 2). This gives strong evidence that
these isolates are genetically different from the other
Paecilomyces spp. isolates and less genetic
variability among these isolates proposes that it may
possibly be due to the same source of isolation i.e.,
soil sources, while the other isolates used in
previous studies were isolated from the insect hosts
i.e., B. tabaci (Tignao-Milani et al., 1995a; Cantone
and Vandenberg, 1998).
Our studies confirm the results as that of
Luangsa-ard et al. (2004), studying the large and
small subunits rRNA gene sequences, where
Paecilomyces probably represents a genus only. The
molecular results based on the nucleotide sequences
of ITS-rDNA region support the previous studies of
Paecilomyces spp. and will facilitate further studies
related to the phylogenetics of Paecilomyces spp.
ACKNOWLEDGMENTS
This research was funded by a grant from the
National Natural Science Foundation of China
(31071685), the Special Scientific Research Fund
for Commonwealth Trade of China (200803005).
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(Received 10 January 2010, revised 2 November 2010)
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