Variability of the Mitochondrial SSU rDNA of NomuraeaSpecies and Other Entomopathogenic Fungi fromHypocreales

Daniel R. Sosa-Gomez Æ Richard A. Humber ÆKathie T. Hodge Æ Eliseu Binneck ÆKarina Lucas da Silva-Brandao

Received: 4 June 2008 / Accepted: 10 September 2008

� Springer Science+Business Media B.V. 2008

Abstract Hypocrealean arthropod pathogenic fungi

have profound impact on the regulation of agricul-

tural and medical pests. However, until now the

genetic and phylogenetic relationships among species

have not been clarified, such studies could clarify

host specificity relationships and define species

boundaries. Our purpose was to compare the

sequences of the mitochondrial SSU rDNA fragments

from several mitosporic entomopathogenic Hypocre-

ales to infer relationships among them and to evaluate

the possibility to use these sequences as species

diagnostic tool in addition to the more commonly

studied sequences of nuclear SSU rDNA. The SSU

mt-rDNA proved to be useful to help in differenti-

ation of species inside several genera. Clusters

obtained with Parsimony, Bayesian, and Maximum

Likelihood analyses were congruent with a new

classification of the Clavicipitaceae (Sung et al. Stud

Mycol. 2007;57:5–59) in which the anamorphic

genera Nomuraea and Metarhizium species remain

in the Clavicipitaceae and Isaria species sequenced

here are assigned to the family Cordycipitaceae.

Mitochondrial genomic information indicates the

same general pattern of relationships demonstrated

by nuclear gene sequences.

Keywords Phylogeny � Mitochondrial DNA �Ribosomal DNA � Insect fungi

Introduction

Hypocrealean arthropod pathogenic fungi have pro-

found impact on the regulation of agricultural and

medical pests such as insects of the orders Lepidop-

tera, Hemiptera, Coleoptera, and Diptera, and species

from the order Acarina and Araneae [1–4]. Studies

focusing on their use as control agents to induce

epizootics on arthropod pests have been performed

for more than 100 years [2]. Until now, genetic and

D. R. Sosa-Gomez (&) � E. Binneck

Embrapa - National Soybean Research Center, Embrapa

Soja, Londrina, Cx. P. 231, 86001-970, PR, Brazil

e-mail: drsg@cnpso.embrapa.br; drsgxx@gmail.com

E. Binneck

e-mail: binneck@cnpso.embrapa.br

R. A. Humber

Biological Integrated Pest Management, Robert W.

Holley Center for Agriculture and Health, Tower Road,

Ithaca, NY 14853-2901, USA

e-mail: Richard.Humber@ars.usda.gov

K. T. Hodge

Department of Plant Pathology, Cornell University,

Ithaca, NY 14853, USA

e-mail: kh11@cornell.edu

K. L. d. Silva-Brandao

Departamento de Zoologia, Instituto de Biologia,

Universidade Estadual de Campinas, Cx. P. 6109

Campinas 13083-970, SP, Brazil

e-mail: klucas@unicamp.br

123

Mycopathologia

DOI 10.1007/s11046-008-9157-5

phylogenetic relationships among these fungi species

have not been clarified, nevertheless such studies

should enlighten host specificity relationships and

define species boundaries. For most of these conidial

species, the sexual stages remain unknown, and many

of these conidial fungi remain difficult to distinguish

morphologically. Due to this, morphological charac-

ters have limited power to distinguish members of

different genera or varieties within species. Also the

relative importance of phenotypic characters for

discriminating species within phylogenetically reclas-

sified genera remains to be demonstrated.

The use of molecular approaches to define bound-

aries among taxa has been applied to clarify the

taxonomic position of entomopathogenic fungi or to

infer the phylogenetic relationships among them.

Comparative DNA sequence studies can bring new

insights on the linkage between entomopathogenic

mitosporic fungi (anamorphs) and their teleomorphic

stages and help to clarify phylogenetic relationships

among them. However, limited molecular genetic and

phylogenic information is available within this group.

To the best of our knowledge, most of the studies

concerning the interrelationships among entomopath-

ogenic fungi of the family Clavicipitaceae using

DNA sequences have been done on specific nuclear

genes such as b-tubulin [5, 6], the ITS1–5.8S-ITS2

regions of the nuclear ribosomal DNA [5, 7–12], and

introns from the same region [9, 13]. Phylogenetic

analysis on entomopathogenic Clavicipitaceae with

mitochondrial SSU rDNA sequences has been

explored by Nikoh and Fukatsu [7] in their study on

phylogenetic relationship among Cordyceps species,

but that study, however, included no species from the

genera Nomuraea, Tolypocladium, and Lecanicillium.

This study sought to determine whether the

mitochondrial gene for the ribosomal small subunit

(SSU mt-rDNA) might reveal a level of genetic

variability in Nomuraea rileyi (Farlow) Samson to

indicate whether this fungal biocontrol agent is a

single globally distributed species or a species

complex. This locus was chosen because the

mtSSU-rDNA data has been used in fungal system-

atics [14, 15]. Another aim of this study was to test

whether this gene might be useful addition to the

nuclear and mitochondrial genes informative for

phylogenetic studies or for diagnoses of individual

species of entomopathogenic fungi from the Hypo-

creales. This study emphasized N. rileyi, one of the

major global pathogens of agriculturally important

species of the Noctuidae (Lepidoptera), but included

comparisons of sequences of the SSU mt-rDNA

fragments from several other related fungi: Beauve-

ria bassiana (Bals.) Vuill., B. brongniartii (Sacc.)

Petch, Isaria amoenorosea P. Henn., I. fumosorosea

Wize, I. javanica (Friederichs & Bally) Samson &

Hywel-Jones, Metarhizium anisopliae (Metsch.)

Sorok., M. cylindrosporae Q.T. Chen & H.L. Guo,

M. flavoviride W. Gams & Rozsypal, M. viridulus

S.S. Tzean, L.S. Hsieh, J.L. Chien & W.J. Wu,

Nomuraea anemonoides Hocking, N. rileyi, Tolypoc-

ladium cylindrosporum W. Gams, and Lecanicillium

lecanii (Zimmerm.) Zare & W. Gams. Sequences

from these fungi and others available from GenBank

provide a basis to evaluate the usefulness of the SSU

mt-rDNA sequences but also address some specific

taxonomic questions about some of the classification

of clavicipitoid conidial pathogens affecting insects.

Material and Methods

DNA Extraction

Entomopathogenic fungi were stored either on silica

gel at the Embrapa Soybean Research Center [16] or

under low-temperature (-196�C) at the USDA-ARS

Collection of Entomopathogenic Fungi (ARSEF, Ith-

aca, NY) [17]. Cultures were plated on Sabouraud

maltose plus 1% yeast extract (SMY) agar plates and

grown at 26�C for 5–7 days. The colonies were used to

inoculate flasks containing 50 ml of Sabouraud dex-

trose plus 1% yeast extract (SDY) broth. Broth cultures

were shaken at 250 rpm at 26�C for 8–10 days. The

fungal mycelia were collected by filtration, washed

with sterile distilled water, and the DNA extracted.

Fungal samples placed in pre-cooled mortar were

frozen and crushed with a pestle in liquid nitrogen.

The DNA was extracted using a modified CTAB

protocol [18]. A total of 150 mg of harvested

mycelial preparations were homogenized in liquid

nitrogen and transferred to DNA extraction buffer

(100 mM Tris–HCl pH 8, 20 mM EDTA pH 8, 2%

CTAB, 1.4 M NaCl, 1% b-mercaptoethanol).

Homogenates were mixed and incubated at 65�C

for 1 h. After centrifugation, the upper phase was

collected and added with 1 volume of chloroform:iso-

amyl alcohol (24:1). After centrifugation, the aqueous

Mycopathologia

123

layer was precipitated with isopropanol. Nucleic

acids were pelleted by centrifugation and washed

with 70% ethanol. The dried pellet was resuspended

at room temperature in TE buffer pH 8.0 containing

RNase. After a 30 min digestion at 37�C, the DNA

was again purified by adding 1/10 volume of 5 M

NaCl and 2 volumes of 95% ethanol and collected by

centrifugation. The resulting pellets were dried at

37�C and resuspended in TE. DNA was quantified in

comparison with known standards in 1.3% agarose

gel electrophoresis.

PCR Amplification and Sequencing

PCR was used to amplify the region of the mito-

chondrial SSU rDNA using primers MSA0-s (50-CTT

GACACATGCTAATCGAACG-30, modified from

Nikoh and Fukatsu [7]), MS597R (50-AACACTA

GTCTCTTACGTATTACC-30 designed by K.T.

Hodge), MS531 (50-TTTGTTTATATATCGATAAT-

GACG-30, designed by K.T. Hodge), and MS2 (50-GCGGATTATCGAATTAAATAAC-30, from White

et al. [19]. The reaction mix was prepared with Taq

DNA polymerase (Promega, USA) under the tem-

perature program of 95�C for 3 min, followed by 10

cycles of 94�C for 1.5 min, 53�C for 1 min, 74�C for

1.5 min, followed by 25 cycles of 94�C for 1 min,

53�C for 1 min, and 74�C for 1.5 min, and a final

extension of 74�C for 5 min. The amplified products

were purified with centrifugal filter devices Amicon

Microcon-PCR (Millipore, Billerica, MA, USA).

The PCR products were analyzed with the ABI

3700 DNA sequencer using the referred primers with

BigDye terminators DNA sequencing kit (Applied

Biosystems, Foster City, CA, USA), at the BioRe-

source Center, Cornell University.

Molecular Analyses

A unique sequence was generated with two fragments

generated with two pairs of primers. Multiple align-

ments were performed using a ClustalW package

[20]. The final alignment was adjusted manually.

These sequences were aligned with the most similar

published mitochondrial SSU rDNA data: B. bassi-

ana (AB027360), Cordyceps militaris (Linn.: Fr.) Fr.

(AB027357), Isaria tenuipes Peck (AB027358), I. fu-

mosorosea (AY755523), Torrubiella confragosa

Mains (AY556052), Lecanicillium muscarium (Petch)

Zare & W. Gams (AF487277), C. kanzashiana

Kobayasi & Shimizu (AB027347), C. prolifica

Kobayashi (AB027346), Elaphocordyceps inegoensis

(Kobayasi) G.H. Sung, J.M. Sung & Spatafora,

(AB027344), E. konnoana (Kobayasi & Shimizu)

G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora

(AB031194), E. paradoxa Kobayashi (Kobayasi)

G.H. Sung, J.M. Sung & Spatafora (AB027345),

Ophiocordyceps sobolifera (Hill ex Watson)

G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora

(AB027350), O. cochlidiicola (Kobayasi & Shimizu)

G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora,

(AB027355), O. coccidiicola (Kobayasi) G.H. Sung,

J.M. Sung, Hywel-Jones & Spatafora, (AB031197),

and Metarhizium anisopliae (NC008068), isolate

ME1)(NCBI GenBank database; http://www.ncbi.

nlm.nih.gov/) were included in the analysis. The

Overall Mean Distance computed in MEGA v. 4.0

[21] indicated that the global alignment score (1 - d)

is 0.847, considered well within the acceptable

range of identity to obtain a reliable phylogenetic tree

[22, 23].

Phylogenetic Analyses

After alignment, the phylogenetic analyses were

performed with PAUP* v. 4.0 b10 [24], using

Maximum Parsimony. Bayesian analysis was carried

out with MrBayes 3.08 v [25], and Maximum

Likelihood analysis was performed with PHYML

[26]. The purpose of doing Bayesian and Maximum

Likelihood analyses was to investigate the effect of

more restrictive assumptions of substitutions models

on the results. For all analyses, Aspergillus nidulans

was used as outgroup to root the tree.

Maximum Parsimony analysis (MP) was per-

formed on the entire data set and with ambiguously

aligned sites excluded, using heuristic search with

1,000 random taxon addition replicates, TBR branch-

swapping, gaps scored as missing data, and all

characters equally weighted. A strict consensus tree

was computed whenever multiple equally parsimoni-

ous trees were obtained. The consistency index (CI)

and the retention index (RI) were calculated by the

PAUP ‘‘tree scores’’ option. The robustness of each

branch was determined using the non-parametric

bootstrap test [27], with 1,000 replicates and 10

random taxon additions.

Mycopathologia

123

The program Modeltest v. 3.06 [28] was used to

determine the available substitution model with the

best fit to the data set. The best fit model was found to

be the GTR ? G [General Time-Reversible model

[29], with gamma distribution (G)], and both Bayes-

ian and Maximum Likelihood analyses were carried

out for the combined data set under this model. For

Bayesian analysis, four simultaneous chains were

conducted for 5.0 9 106 generations, sampling trees

every 500 cycles for a total of 10,000 sampled trees.

The first 2,500 trees were discarded as ‘‘burn in.’’

Maximum Likelihood analysis was performed

using the parameters estimated with Modeltest-

frequencies [28] of A = 0.3724; C = 0.1261,

G = 0.1959, and T = 0.3055, and a = 0.4058. The

reliability of each branch was estimated using the

approximate Likelihood Ratio Test (aLRT), followed

by Shimodaira–Hasegawa (SH) procedure to assess

significance of the statistic [23].

Results

We obtained partial sequences from the mitochon-

drial SSU rDNA gene of 10 species of

entomopathogenic fungi: T. cylindrosporum (isolate

ARSEF 963), N. rileyi (isolates CNPSo 188, CNPSo

220, CNPSo 238, CNPSo 250, CNPSo 286, CNPSo

289, CNPSo 359, CNPSo 370, CNPSo 374, CNPSo

381, CNPSo 393, CNPSo 416, ARSEF 558, ARSEF

1760, ARSEF 1761, ARSEF 2202, ARSEF 2395,

ARSEF 3940, ARSEF 6877, ARSEF 6879, ARSEF

6880, ARSEF 6881, ARSEF 6882), N. anemonoides

(ARSEF 2467), M. flavoviridae (ARSEF 1184),

M. viridulus (ARSEF 6927), M. cylindrosporae (AR-

SEF 6926), M. anisopliae var. anisopliae (ARSEF

5161), B. brongniartii (ARSEF 1830), I. amoenoro-

sea (ARSEF 744) and I. javanica (ARSEF 322). The

sizes of the amplified products were approximately of

500 pb and 550 bp, respectively (data not shown)

after the overlap of the two sequences, trimming and

alignment the whole sequence ranged from 854 bp

for I. javanica to 918 bp for M. cylindrosporae and

M. viridulus. No variation was observed among the

nucleotide sequences (907 bp) from among the 24

N. rileyi isolates.

The amplified region of the 10 taxa of this study

corresponds to a similar sequence located between

position 20,140 and 21,097 when compared to the

complete fungal mt-DNA of Lecanicillium muscari-

um (GenBank accession AF 487277) [30].

Of the 1,047 characters, 291 (27.8%) were vari-

able, 421 (40.2%) were constant, and 335 (31.9%)

were parsimony informative. The strict consensus

tree of two most parsimonious trees with 1,344 steps

(CI = 68.5; RI = 74.1) is presented in Fig. 1. The

taxon N. rileyi is closely related to the clade

composed by all representatives of the genus Meta-

rhizium, with high value of Bootstrap (Fig. 1). The

Metarhizium clade comprising by M. anisopliae var.

anisopliae, M. cylindrosporae, M. viridulus, M. flav-

oviride, and N. rileyi is supported by a 100%

bootstrap value in the mitochondrial phylogeny.

The close relationship between N. rileyi and the

representatives of the genus Metarhizium was also

recovered by both Bayesian and Maximum Likeli-

hood analyses, with high values of posterior

probability and aLRT, respectively.

Fig. 1 Strict consensus tree of two most parsimonious trees

based on the analysis of partial mitochondrial SSU rDNA

sequences of entomopathogenic fungi. The sequences related

to scientific names (bold) were obtained in this study.

Alignment consisted of 1,025 nucleotides positions. Numbers

on the branches indicate bootstrap values (where it exceeds

50%). Values obtained with 1,000 replicates

Mycopathologia

123

The cluster including B. bassiana, Cordy-

ceps brongniartii, and N. anemonoides showed

100% bootstrap consistency (Fig. 1). This cluster

was distantly related to Metarhizium group, but close

related to members of the genus Isaria.

The topologies obtained with the Bayesian and

Maximum Likelihood analyses were mostly similar

to that obtained by Maximum Parsimony (Figs. 2, 3),

and both analyses suggested that the most divergent

taxon is Cordyceps kanzashiana. Elaphocordyceps

species grouped with T. cylindrosporum with mod-

erate to weak support (Bootstrap value = 53) when

Maximum Likelihood analysis was applied. How-

ever, this value was higher when the analysis was

performed using Parsimony (Bootstrap value = 100).

Discussion

The new sequences of I. amoenorosea, I. javanica,

and N. anemonoides clustered in the same group with

B. brongniartii and B. bassiana. Distinct divergences

between the genetic sequences of N. anemonoides

ARSEF 2467, and N. rileyi have been observed after

RAPD, AFLP, ISSR, and ITS1-5.8-ITS2 sequence

analyses [31–33]. The present results lead us to

Fig. 2 Tree obtained by Bayesian analysis of mitochondrial

SSU rDNA sequences under the substitution model GTR ? G.

Values on the branches indicate posterior probability values

([50%). The Cordyceps kanzashiana branch is truncated for

better visualization

Fig. 3 Maximum Likelihood tree obtained based on the

analysis of mitochondrial SSU rDNA sequences under the

substitution model GTR ? G (Ln = - 7057.90622). Values

on the branches indicate bootstrap values, obtained with 200

replicates, and aLRT values, respectively. The bars indicate

familial classifications of species as proposed by Sung et al.

[35] based on sequences of one mitochondrial (atp6) and six

nuclear genes. The C. kanzashiana branch is truncated for

better visualization

Mycopathologia

123

assume that N. anemonoides should be excluded from

the genus Nomuraea unless it were established that

all sequence information derived from the ex-type

isolate of N. anemonoides are from a contaminant

fungus differing genetically from the holotype spec-

imen. Unfortunately, other than the widely distributed

ex-type isolate of this species, no other culture of

N. anemonoides appears to be available from any

source to compare with the ex-type isolate or

holotype specimen of this species.

Tolypocladium cylindrosporum (ARSEF 963), a

fungus usually found on dipterans, grouped with

E. inegoensis and E. paradoxa, both of which known

only from cicadas (Figs. 1, 2 and 3), both belong to

the family Ophiocordicipitaceae [34]. The sequences

analyzed provided additional support for the findings

of Rakotonirainy et al. [35], and confirmed the

divergence observed between T. cylindrosporum

and B. bassiana, who compared sequences of the

D1 and D2 domains located at the 50 end of the 28-S

subunit of the rRNA gene.

Earlier studies of a fragment of the b-tubulin gene

of several N. rileyi isolates revealed no significant

intraspecific variability in that nuclear gene [32]. In a

similar manner, no significant intraspecific variation

was detected among SSU mt-rDNA sequences of

N. rileyi isolates from the perspective of either

biogeographical origins (the isolates sequenced here

came from the Philippines, Indonesia, United States,

Argentina, and Brazil; see Table 1) or any obvious

host specificity. N. rileyi is known almost exclusively

from hosts in the Noctuidae (Lepidoptera) but

without any clear preference for genera or species

within that family [33]. In a seemingly anomalous

exception, however, the putative source of N. rileyi

ARSEF 558 is from an Indonesian specimen of

white-backed plant-hopper, Sogatella fucifera (Horv-

ath) (Hemiptera: Delphacidae). Unfortunately, this

unprecedented host cannot be confirmed since the

source specimen was not preserved. Such an unex-

pected host might be recorded if the plant-hopper was

contaminated by spores from an infected lepidopteran

or if some later contamination in the laboratory by

N. rileyi replaced a fungus more characteristically

pathogenic for such Hemipterans; no experimentation

has confirmed whether ARSEF 558 is able to infect

any delphacid or other related Hemipteran.

The results of our phylogenetic molecular analysis

suggest that N. rileyi is closely related to

M. anisopliae var. anisopliae and other Metarhizium

species, as has been noted by other authors for either

the ITS1-5.8s-ITS2 region [5, 31] or an even more

comprehensive sampling of the genome [35]. Our

analysis of the mitochondrial rDNA also appears to

demonstrate a relatively close relationship between

N. rileyi and Metarhizium species, since both taxa

grouped in a well-supported cluster with a bootstrap

value of 100% in our analysis. The genetic divergence

between N. rileyi and M. cylindrosporae and M. vir-

idulus was also observed phenotypically, since neither

of these Metarhizium species has the dimorphic

(yeast) phase observed for N. rileyi on Sabouraud

maltose agar medium plus 1% yeast extract. Also,

mycelia of M. viridulus and M. cylindrosporae grow

rapidly and sporulate easily, even after repeated

subcultures on potato dextrose agar; this is not

observed for N. rileyi isolates. Pathogenicity bioas-

says with high dosages of M. cylindrosporae and

M. viridulus applied to the neotropical brown stink

bug, Euschistus heros (Fabr.), and to the velvetbean

caterpillar, A. gemmatalis, a host susceptible to

N. rileyi infections, caused no infections of either

species (Sosa-Gomez, unpub.). Comparisons of the

mt-rDNA sequences show that M. cylindrosporae is

more closely related to M. anisopliae than M. virid-

ulus (Fig. 2), and these studies also validate the

correct placement of M. cylindrosporae and M. vir-

idulus in the genus Metarhizium as proposed by

Huang et al. [36] rather than in Nomuraea; the generic

placements of these two species has been problematic.

In addition, studies on partial sequences of the

b-tubulin gene and the ITS1-5.8-ITS2 rDNA placed

M. cylindrosporae and M. flavoviridae in the same

clade [5]. The sequence of 885 bp from M. anisopliae

ARSEF 5161 shared 99% of the identity with the

strain AB027361 and AY884128.1 determined by

Nikoh and Fukatsu [7] and Ghikas et al. [37].

Neither Sung et al. [35] nor we were able to assign

C. kanzashiana and C. prolifica to a clade (or family)

in the new phylogenetic reclassification of the

Clavicipitaceae in the broad sense. The rDNA data

show divergence of both species from the remaining

species studied here (Fig. 3).

The SSU mt-rDNA proved to be useful in

differentiating species inside several genera, and the

previously defined phylogenetic relationships among

different families were confirmed. Nuclear and mito-

chondrial genomic information seem to indicate the

Mycopathologia

123

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Mycopathologia

123

same general pattern of relationships among the fungi

of the order Hypocreales tested here, and the

introduction of additional mitochondrial gene

sequences information might help in the overall

refinement of the taxonomy of many of the problem-

atic genera of entomopathogens in the this major

fungal order as has been demonstrated recently for

the genus Lecanicillium [38]. Clusterings obtained

through the differing Parsimony, Bayesian and Max-

imum likelihood analytic techniques (Figs. 1–3) were

congruent with the new familial taxonomy proposed

by Sung et al. [35] in which the anamorphic genera

Nomuraea and Metarhizium are assigned to the

family Clavicipitaceae sensu stricto while the Beau-

veria and Isaria species sequenced here are assigned

to family Cordycipitaceae.

Several conclusions about Nomuraea species, as

the primary fungal focus of this study, were validated

by the results: N. anemonoides belongs in the Cord-

ycipitaceae rather than in the Clavicipitaceae (with

N. rileyi, the type species of the genus), and will

eventually have to be placed in some other genus.

Further, the genetic evidence presented studied here

suggests that N. rileyi is a globally distributed species

comprising surprisingly little genetic variability (at

least in the SSU mt-rDNA gene) rather than a species

complex as is now being shown for the major species

of Beauveria and Metarhizium. The results presented

here do not clarify whether Nomuraea should con-

tinue to be treated as a distinct genus closely related to

Metarhizium or, because of some strong genomic

similarities [31], be synonymized with Metarhizium.

Acknowledgments The study was supported by the

Conselho Nacional de Desenvolvimento Cientıfico

e Tecnologico (CNPq) from Brazil and USDA Agricultural

Research Service (Ithaca, New York). We wish to

acknowledge John Vandenberg for allowing to use his

facilities, Drion G. Boucias for the critical review of the

manuscript and providing some N. rileyi isolates, Karen

Hansen and Louela Castrillo for helping with laboratory

work. DRSG thanks CNPq (Project 490348/2004-1 and

fellowship 303997/2004–4) and KLSB thanks CNPq

(fellowship 151004/2005–6) and Fundacao de Amparo a

Pesquisa––FAPESP (grants #06/60127–0 and #07/53919–0).

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