Izolarea si selectia lui Metarhizium.pdf

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CTC medium: A novel dodine-free selective medium for isolatingentomopathogenic fungi, especially Metarhizium acridum, from soil

Éverton K.K. Fernandes a, Chad A. Keyser a, Drauzio E.N. Rangel a,1, R. Nelson Foster b, Donald W. Roberts a,*

a Department of Biology, Utah State University, Logan, UT 84322-5305, USAb USDA/APHIS/PPQ/CPHST Lab, Phoenix, AZ 85040-2931, USA

a r t i c l e i n f o

Article history:Received 6 January 2010

Accepted 14 May 2010

Available online 12 June 2010

Keywords:Selective medium

Entomopathogenic fungi

Beauveria bassianaBeauveria brongniartiiMetarhizium brunneumMetarhizium acridumChloramphenicol

Thiabendazole

Cycloheximide

a b s t r a c t

The selective media most commonly used for isolating hyphomycetous species of entomopathogenic

fungi from non-sterile substrates rely on N -dodecylguanidine monoacetate (dodine) as the selective fun-

gicide. Although these media are effective for isolating many species of Metarhizium and Beauveria from

soil, they are inefficient media for isolation of an important Metarhizium species, Metarhizium acridum,

from non-sterile soil. Our current study was directed to formulating a dodine-free selective medium that

is efficient for isolating naturally occurring Beauveria spp. and Metarhizium spp., especially M. acridum,

from soil. The selective medium (designated CTC medium) consists of potato dextrose agar plus yeast

extract (PDAY) supplemented with chloramphenicol, thiabendazole and cycloheximide. In comparisons

with selective media previously reported in the literature, the CTC medium afforded colonies that were

larger and had both earlier and more abundant conidiation of entomopathogenic fungi, features which

greatly facilitated identification of the emerging entomopathogenic fungi. In addition to efficient re-iso-

lation of M. acridum, this medium also is an effective tool for selective isolation of Metarhizium brunneum,

Metarhizium robertsii, Beauveria bassiana and Beauveria brongniartii from non-sterile field-collected soil

samples inoculated (spiked) with fresh conidia in the laboratory.

2010 Elsevier Inc. All rights reserved.

1. Introduction

The use of entomopathogenic fungi for biological control of

arthropods dates back to the 1880s (Krassilstschik, 1888), and

there are current examples worldwide of successful fungus-based

insect control programs (Roberts and St. Leger, 2004; Shah and Pell,

2003). Metarhizium anisopliae sensu lato (s.l.) (Metschnikoff) Soro-

kin and Beauveria bassiana (Balsamo) Vuillemin are the fungi most

commonly employed for pest control. Another Metarhizium spe-

cies, Metarhizium acridum (Driver and Milner) Bischoff, Rehner

and Humber was found to be an effective pathogen of locusts

and grasshoppers (Acrididae), and it is quite host-specific to these

and closely related insects. Two M. acridum (one African and one

Australian) have been developed as commercial products for bio-

logical control of this insect group (Lomer et al., 2001).

Fungal species tend to occur in nature as mixtures of strains

with widely diverse physiological traits. Exploration of this rich

diversity by isolating new fungal isolates from the field is expected

to yield strains with attributes superior to those currently available

for biological pest control. The methods for these searches are still

under development. Conidia produced on cadavers of insects killed

in nature by entomopathogenic fungi are routinely deposited on or

in nearby soil. Pathogenic fungi, however, are a distinct minority

amidst a myriad of diverse microorganisms in soil (Tsao, 1970),

and this probably is especially true for entomopathogenic fungi.

The isolation of entomopathogenic fungi from soil is often difficult,

and requires ‘‘finely tuned” methods to maximize success. Two of

the most commonly employed methods are: (1) baiting the envi-

ronment with a susceptible insect host (Zimmermann, 1986) or

(2) using specific selective media containing chemicals that pre-

clude or reduce the growth of contaminants (Beilharz et al.,

1982; Doberski and Tribe, 1980; Joussier and Catroux, 1976; Liu

et al., 1993; Veen and Ferron, 1966).

In 1966, Veen and Ferron developed a selective medium that

contained oxgall, chloramphenicol and cycloheximidine (Actidi-

one) for the isolation of two entomopathogenic fungi, Beauveriabrongniartii (Saccardo) Petch and M. anisopliae s.l. In 1982, the fun-

gicide dodine (N-dodecylguanidine monoacetate) was successfully

incorporated into a culture medium for isolation of M. anisopliae s.l.

from soil (Beilharz et al., 1982). This report led to the development

of a widely utilized dodine-based selective medium for isolation of

B. bassiana and M. anisopliae s.l. (Chase et al., 1986). Later, the addi-

tion of low dodine concentrations (10 and 50lg/ml) to Veen’s

1049-9644/$ - see front matter 2010 Elsevier Inc. All rights reserved.doi:10.1016/j.biocontrol.2010.05.009

* Corresponding author. Fax: +1 435 797 1575.

E-mail address: [email protected] (D.W. Roberts).1 Present address: Instituto de Pesquisa e Desenvolvimento, Universidade do Vale

do Paraíba, São José dos Campos, SP 12244-000, Brazil.

Biological Control 54 (2010) 197–205

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Biological Control

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http://dx.doi.org/10.1016/j.biocontrol.2010.05.009

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medium was found to reduce the number of contaminants by over

90% and to improve the isolation of M. anisopliae s.l. from soil (Liu

et al., 1993).

Recently, potato dextrose agar enriched with yeast extract

(PDAY), when supplemented with dodine and gentamicin, proved

to be a very efficient substrate for selecting several entomopatho-

genic fungi from contaminated environments (Rangel et al., 2010).

Nevertheless, this study also demonstrated thatM. acridum

is more

susceptible to dodine than other Metarhizium spp.; i.e., it grows

and sporulates poorly on PDAY containing very low dodine concen-

trations. M. acridum is an Orthoptera-host-specific fungus (Alston

et al., 2005; Bridge et al., 1997; Goettel and Jaronski, 1997; Milner

et al., 2002; Peveling and Demba, 1997) that has been isolated in

hot climates and deserts in Australia, Thailand, Brazil, Mexico, Sen-

egal, Benin, Tanzania, Madagascar and Niger. In fact, M. acridumisolates from Africa and Australia are currently registered for com-

mercial use in their respective continents. The importation of these

products, however, currently is not permitted by some countries,

e.g., USA, for which M. acridum is an exotic species. The isolation

of native M. acridum should facilitate importation of exotic com-

mercial isolates into the restrictive nations, and/or newly discov-

ered native M. acridum isolates could be developed as novel

domestic products for biological control of orthopteran pests. With

these goals in mind, we are conducting an energetic search for na-

tive USA M. acridum isolates. The project, however, was severely

impeded initially by the absence of an effective medium for selec-

tively isolating M. acridum from non-sterile soil.

Effective selective media for the isolation of a specific fungus

should contain both a nutrient source and anti-microbial agents

(i.e., fungicides and antibiotics), with the latter in appropriate con-

centrations to allow the target-fungus to grow while impeding the

growth of common contaminants (Luz et al., 2007). Although

widely used for isolation of most species of entomopathogenic fun-

gi, selective media containing dodine are not suitable for isolating

M. acridum from non-sterile soil due to this species’ high sensitivity

to dodine (Rangel et al., 2010). Accordingly, a selective medium

with a suitable dodine alternative was needed to efficiently searchfor new isolates of M. acridum in soil samples.

The study reported here investigates the effectiveness of several

selective media with different concentrations of several anti-

microbial compounds for re-isolation of Metarhizium spp. and

Beauveria spp. from non-sterile soil inoculated in the laboratory

with fresh conidia of several species of entomopathogenic fungi.

A novel selective medium is described. It is based on modifications

of previous media (Veen and Ferron, 1966; Luz et al., 2007; Rocha

and Luz, 2009; Rangel et al., 2010) and is designed to maximize

recovery of naturally occurring Beauveria spp. and Metarhiziumspp., especially M. acridum, from soil. The new medium, ‘‘CTC med-

ium”, follows an uncomplicated preparation method and utilizes a

simple nutritive substrate; i.e., potato dextrose agar enriched with

yeast extract (PDAY), supplemented with chloramphenicol, thia-bendazole and cycloheximide. The acronym ‘‘CTC” refers to these

three chemicals.

2. Materials and methods

2.1. Soil samples and fungal isolates

Four soil samples were collected within the state of Utah, USA

(Table 1). The sampling protocol was as follows: (1) the most obvi-

ous surface duff and growing vegetation were cleared away from

an area of approximately 225 cm2; (2) approximately 200 g of soil

was taken at each site from within the top 5 cm of the surface; (3)

the samples were placed in plastic bags and held at room temper-ature (20 ± 2 C) until experimentation began. Soil sample physical

and chemical characterization was performed by the Analytical

Laboratory of Utah State University. Soil physical traits are listed

in Table 2, and concentration of 19 elements in Table S1 (see Sup-

plementary data available online).

Six isolates representing five fungal species (viz., B. bassiana, B.brongniartii, M. acridum, Metarhizium brunneum Petch, and Meta-rhizium robertsii Bischoff, Rehner and Humber) were investigated.

Details of the isolates are given in Table 3.

2.2. Media preparation

Four selective media were prepared as follows: (1) ‘‘Dodine

medium”, consisting of PDAY [potato dextrose agar (Difco Labora-

tories, Sparks, MD, USA) supplemented with 1 g/l yeast extract

(Technical; Difco Laboratories)], +0.05 g/l gentamicin (BioWhittak-

er, Walkersville, MD, USA), and one of four dodine (N -dodecylgua-

nidine monoacetate) concentrations [0.001%, 0.002%, 0.004% or

0.006% active ingredient (A.I.)]. The commercial fungicide ‘‘Syllit”

(65% A.I.) (Chimac-Agriphar S.A., Aceto Agricultural Chemicals

Table 1

Collection sites of soil samples in 2009.

Soil sample Sites (Utah, USA) Longitude Latitude

# 1 Piute 11238010W 3843170N

# 2 Sevier 11222950W 3851260N

# 3 Millard 11204300W 3909540N

# 4 Millard 11205190W 3911510N

Table 2

Physical characterization of soil samples.

Soil

sample

pH EC

(dS/m)*Sand

(%)

Silt

(%)

Clay

(%)

Texture

# 1 7.3 4.18 39 41 20 Loam

# 2 7.2 2.49 48 38 14 Loam

# 3 7.3 4.08 52 37 11 Sandy Loam/Loam# 4 7.6 1.28 36 46 18 Loam

* EC, eletrical conductivity in deci-Siemens/meter.

Table 3

Origins of fungal isolates used to compare several potential selective media forrecovering conidia added to soil.

Fungal

isolate*Origin Host Latitude Collection

year

Metarhizium acridumARSEF 324 Queensland, Australia Orthoptera:

Acrididae

19000S 1979

ARSEF 5628 Shelsela, Ethiopia Orthoptera:Acrididae

12280

S 1996

Metarhizium robertsiiARSEF 2575 South

Carolina, USA

Coleoptera:

Curculionidae

34000N 1988

Metarhizium brunneumARSEF 5626 Pälkäne, Finland Coleoptera:

Tenebrionidae 61200N 1986

Beauveria bassianaARSEF 252 Orono, Maine, USA Coleoptera:

Chrysomelidae 44530N 1978

Beauveria brongniartiiATCC 58798 Czechoslovakia Diptera:

Tipulidae

50050N Unknown

* Culture collection: ARSEF, ARS Collection of Entomopathogenic Fungal Cultures –

ARSEF, USDA, Ithaca, NY, USA; ATCC, American Type Culture Collection, Manassas,VA, USA.

198 É.K.K. Fernandes et al. / Biological Control 54 (2010) 197–205

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Corp., Lake Success, NY, USA) was used as the dodine source. An

aqueous stock solution of dodine (0.1% A.I.) was autoclaved

(121 C for 15 min) separately and then thoroughly mixed with

autoclaved PDAY in appropriate quantities to obtain the designated

concentration. (2) ‘‘CTC medium”, consisting of PDAY supple-

mented with 0.5 g/l chloramphenicol (Sigma Chemical Co., St.

Louis, MO, USA), 0.001 g/l thiabendazole (Sigma) and 0.25 g/l

cycloheximide (A.G. Scientific Inc., San Diego, CA, USA) was pre-

pared and autoclaved. In addition, CTC was prepared with higher

concentrations of thiabendazole, i.e., 0.002 and 0.004 g/l. CCT med-

ia with the three thiabendazole concentrations were labeled ‘‘CTC

1T”, ‘‘CTC 2T” and ‘‘CTC 4T.” ‘‘CTC” without thiabendazole concen-

tration designated is equivalent to ‘‘CTC 1T.” (3) ‘‘Veen’s medium”,

according to Veen and Ferron (1966) and (4) ‘‘Liu’s medium”, con-

sisting of Veen’s medium plus 0.001% dodine (Liu et al., 1993).

The pH of all media was adjusted to 6.9 with 10% NaOH solu-

tion, as needed, before autoclaving at 121 C for 15min, and

23 ml of each medium were individually pipetted into a polysty-

rene Petri plate (95 15 mm, Fisherbrand, Santa Clara, CA,

USA). Plates of media were stored at room temperature in the dark

and used within 4 days of preparation.

2.3. Re-isolation of entomopathogenic fungi from ‘‘spiked” soil

Entomopathogenic fungal isolates were cultured on 23 ml PDAY

in polystyrene Petri plates (95 15 mm, Fisherbrand) in the dark

at 25 C for 15 days. The conidia were harvested with a microbio-

logical loop and immediately suspended in polyoxyethylene sorbi-

tan monooleate (Tween 80, Sigma) solution (0.01% v/v). The

suspension was vigorously agitated for 30 s with a vortex mixer

and filtered through an 8-lm pore size polycarbonate membrane

(Nucleopore, Acton, MA, USA) to remove hyphal and conidial

aggregates. The conidial suspensions were then quantified by

hemacytometer and the concentration adjusted to 1.0 106 coni-

dia ml1. Conidial viability was estimated by placing 20-ll aliquot

of conidial suspension on 4-ml PDAY medium plus 0.002% (w/v)

benomyl (25% active ingredient, Hi-Yield Chemical Company, Bon-ham, TX, USA) in Petri plates (35 10 mm) (Braga et al., 2001; Mil-

ner et al., 1991). Plates were incubated 48h inthe darkat 25 ± 1 C.

One drop of lactophenol methyl blue and a coverslip was then

placed on the medium of each plate, and germination observed

at 400 magnification. A minimum of 300 conidia/plate were eval-

uated and percentage germination was calculated, revealing great-

er than 98% viable conidia for all six fungal isolates.

For the four field-collected soil samples, six 25-g aliquots were

placed into separate plastic zipper storage bags (160 160 mm).

One bag of each of the four soil samples was individually inocu-

lated with 1 ml of a conidial suspension (1.0 106 conidia ml1)

of one of the six fungal isolates tested. Inoculations were done

immediately after conidial suspension preparation. The bags were

held open inside a laminar-flow hood overnight to allow the inoc-ulum to dry. The soil samples were then homogenized by massag-

ing the bags manually.

Aliquots of 0.35 ± 0.05 g of soil were removed from each bag

and placed in a 2-ml microcentrifuge tube. One ml of sterile dou-

ble-distilled water was added to the microcentrifuge tube, agitated

vigorously (vortexed) for approximately 30 s, and 50 ll (with

approximately 700 conidia of entomopathogenic fungi) was pipet-

ted onto individual 95-mm Petri plates of each media type. The

inocula were spread evenly on the selective media with bent glass

rods and the plates incubated at 27 ± 1 C. Colonies were counted

and photographed at days 7, 14 and 21. This procedure was re-

peated three times on separate days 48 h apart, using the same

spiked soil. Soil was stored between repetitions at 5 C.

The CTC medium was compared to PDAY supplemented with0.001, 0.002, 0.004 or 0.006% dodine as to effectiveness in isolating

the entomopathogenic fungi. Also, the fungus re-isolation efficacy

of CTC medium with the concentration of thiabendazole set at

0.001, 0.002 or 0.004 g/l was compared to that of two selective

media: ‘‘Veen’s medium” (Veen and Ferron, 1966) and ‘‘Liu’s med-

ium” (Veen’s medium plus 0.001% dodine) (Liu et al., 1993).

2.4. Statistical analysis

The effect of medium on colony formation was assessed using

an analysis of variance of a two-way factorial in a randomized

block design; the fixed-effects factors included media and repeti-

tion and media repetition, while the random-effects factors are

soil (the replicating factor), media soil, repetition soil and resid-

ual error; no significant interaction was observed. Prior to analysis,

data were square root transformed to better meet the assumptions

of normality and homogeneity of variance. Each of the six isolates

was analyzed separately. Mean comparisons among treatments

were adjusted for family-wise Type I error rate using the Tukey–

Kramer method; P -values less than 0.05 were considered as signif-

icant. Data analyses were generated using the MIXED procedure in

SAS/STAT software, Version 9.1.3 of the SAS System for Windows.

3. Results

The PDAY dodine-based media at concentrations of dodine that

selectively limited the growth of non-entomopathogenic fungi

present in the soil samples allowed growth of M. brunneum (ARSEF

5626), M. robertsii (ARSEF 2575), B. bassiana (ARSEF 252) and B.brongniartii (ATCC 58798) (Fig. 1). Nevertheless, neither of the M.acridum isolates (ARSEF 324 and ARSEF 5628) grew on any of the

PDAY plus dodine concentrations; i.e., no colonies of M. acridumwere recovered on PDAY supplemented with low concentrations

of dodine, 0.002%, 0.004% or 0.006% (see Fig. 1). Even 0.001% dodine

did not allow isolation of M. acridum from soil (data not shown). In

contrast, an average of 200 M. acridum colonies (approximately

30% of the inoculum) was recovered on CTC medium plates. In gen-eral, recovery rates of conidia were approximately 20–40% for all

species of entomopathogenic fungi when analyzed with CTC med-

ium (see Fig. 1).

Metarhizium robertsii and B. brongniartii tended to be more sen-

sitive to dodine than M. brunneum and B. bassiana (see Fig. 1). The

number of colonies of M. robertsii (P < 0.0001, F 3,9 = 39.09) and B.brongniartii (P = 0.0150, F 3,9 = 6.10) recovered on the various media

differed significantly; it appears that the colonies recovered on the

dodine-supplemented media decreased proportionally to the in-

creased concentration of dodine (see Fig. 1). Statistically significant

differences among the number of target-fungus colonies were ob-

served between the CTC and PDAY + dodine media for both M.brunneum (P = 0.0104, F 3,9 = 6.91) and M. robertsii. For both isolates,

the number of colonies recovered on CTC medium was statisticallysimilar to the number of colonies recovered on PDAY + 0.002% A.I.

dodine, but significantly higher than on PDAY + 0.004 or 0.06% A.I.

dodine. The number of colonies of B. brongniartii recovered on CTC

medium was significantly higher than on PDAY + 0.006% dodine,

but similar to those recovered on PDAY + 0.002% or 0.004% A.I. do-

dine. No difference was observed between the number of colonies

recovered on any of the media tested with soil spiked with B. bas-siana (P = 0.0585, F 3,9 = 3.61) (see Fig. 1).

Colonies of M. acridum recovered on CTC 1T or CTC 2T, contain-

ing 0.001 or 0.002 g/l thiabendazole, respectively, did not differ

significantly in numbers compared to Veen’s or Liu’s media 14 days

after inoculation; however, the number of colonies recovered on

CTC 4T (0.004 g/l thiabendazole) was drastically reduced (ARSEF

324: P = 0.0002, F 4,12 = 13.08; ARSEF 5628: P = 0.0063, F 4,12 = 6.14)(Fig. 2). On the other hand, differences in aspect (= appearance)

É.K.K. Fernandes et al. / Biological Control 54 (2010) 197–205 199



of M. acridum colonies were observed with each medium type, and

both M. acridum isolates (ARSEF 324 or ARSEF 5628) were more

easily identifiable among the contaminants on CTC 1T and CTC

2T than on CTC 4T, Veen’s or Liu’s media. This difference was nota-

ble as early as 7 days after inoculation, as well as onday 21. M. acri-dum colonies tended to be larger and with more sporulation on CTC

1T or CTC 2T than on CTC 4T, Veen’s or Liu’s media. On day 7, no M.acridum colonies were recovered on CTC 4T; but some were iso-

lated on days 14 and 21 (see Fig. 2).

The number of colonies of M. brunneum on CTC 4T was signifi-

cantly higher than on Veen’s or Liu’s media, but CTC 1T and CTC

2T were similar to Veen’s or Liu’s media (P = 0.0314, F 4,12 = 3.83).

The number of M. robertsii colonies recovered at 14 days after inoc-

ulation on CTC 4T was higher than on Liu’s, but not Veen’s medium

(P = 0.0203, F 4,12 = 4.40) (Fig. 3). Colonies of both M. brunneum and

M. robertsii tended to be larger, especially M. robertsii (ARSEF 2575),

with more sporulation on CTC 1T or CTC 2T than on CTC 4T, Veen’s

or Liu’s media. This made the fungi more easily identifiable on CTC

1T and CTC 2T, especially 7 days after inoculation (see Fig. 3).

The number of B. bassiana or B. brongniartii colonies recovered

on CTC (1T, 2T or 4T) did not differ significantly from the num-

ber of colonies recovered on Veen’s or Liu’s media 14 days after

inoculation (ARSEF 252: P = 0.0816, F 4,12 = 2.70; ATCC 58798:

P = 0.2037, F 4,12 = 1.75). At 7 days after treatment, however, the

Metarhizium acridum (ARSEF 324) Metarhizium acridum (ARSEF 5628)

n i e s

f c o l o n

m b e r

o f

N u m

Metarhizium brunneum (ARSEF 5626) Metarhizium robertsii (ARSEF 2575)

n i e s

A f c o l o n

AB A

BBB

AB

A

m b e r

o f

B

C N u m

Beauveria bassiana (ARSEF 252) Beauveria brongniartii (ATCC 58798)A

AA e

s A

A

A

c o l o n i e

AA

AB b e r o f c

AB

N u m b

B

Medium (PDAY + % Dodine, or + CTC) Medium (PDAY + % Dodine, or + CTC)

A B

C D

E F

Fig. 1. Isolation of entomopathogenic fungi (EF) from ‘‘spiked” soil (conidia added to non-sterile soil). The 50 ll of a suspension of ‘‘spiked” soil spread onto each plate of

selective medium contained approximately 700 conidia of EF. Number of EF colonies isolated from four different spiked soil samples using two different selective media:

PDAY (potato dextrose agar supplemented withyeast extract) withthree dodine (N -dodecylguanidine monoacetate) concentrations (0.002%, 0.004% or 0.006%); or CTC(PDAY

plus chloramphenicol, thiabendazole and cycloheximide). Colonies were counted at day 7. Bars represent standard errors of three trials with four different soil samples.

Means depicted by the columns with the same letter are not significantly different from each other [ Metarhizium brunneum (ARSEF 5626): P = 0.0104, F = 6.91; M. robertsii(ARSEF 2575): P < 0.0001, F = 39.09; Beauveria bassiana (ARSEF 252): P = 0.0585, F = 3.61; B. brongniartii (ATCC 58798): P = 0.0150, F = 6.10].




B. bassiana colonies tended to be larger on CTC (1T, 2T or 4T)

than on Veen’s or Liu’s media; and after both 14 and 21 days,

the colonies were both larger and had considerably more sporu-lation than on the other two media (Fig. 4). Colonies of B. bron-

gniartii were notably larger with more sporulation on CTC 1T

than on CTC 2T, CTC 4T, Veen’s or Liu’s media at day 21. On

CTC 4T, B. brongniartii colonies had a dark color and weak spor-ulation at day 21 (see Fig. 4).

Fig. 2. Metarhizium acridum (ARSEF 324 or ARSEF 5628) conidia were added (spiked) to non-sterile soil and then re-isolated using five different selective media: CTC [PDAY

(potato dextrose agar supplemented with yeast extract) plus chloramphenicol, thiabendazole and cycloheximide], Veen’s, and Liu’s medium. The CTC was tested at three

different concentrations of thiabendazole: 1 mg/l (CTC 1T), 2 mg/l (CTC 2T) or 4 mg/l (CTC 4T). The 50 ll of a suspension of ‘‘spiked” soil spread onto each plate contained

approximately 700 conidia of EF. (A)Colonies countedat day14; bars representstandarderrors of three trials with four different soil samples. Meansdepictedby thecolumns

with the same letter are not significantly different from each other (ARSEF 324: P = 0.0002, F = 13.08; ARSEF 5628: P = 0.0063, F = 6.14); (B) Plates at day 7 and 21 after

inoculation. Notedifferent appearance (aspect) and number of M. acridum coloniesin each medium type.Arrows indicate at least oneM. acridum colonyper plate; non-similar

colonies are contaminant fungi isolated from soil. For color plates of the artwork, please refer to the online version of the figure.




4. Discussion

Our current study succeeded in developing an effective dodine-free selective medium (CTC medium) for isolation of entomopath-

ogenic fungi, including M. acridum, from soil. The CTC medium uses

PDAY as the nutrient substrate, since our previous studies estab-

lished that PDAY was more effective than Czapeck or Oatmeal agarin dodine-based selective media (Rangel et al., 2010). PDAY med-

Fig. 3. Metarhizium brunneum (ARSEF 5626) or M. robertsii (ARSEF 2575) conidia were added (spiked) to non-sterile soil and then re-isolated using five different selective

media: CTC [PDAY (potato dextrose agar supplemented with yeast extract) plus chloramphenicol, thiabendazole and cycloheximide], Veen’s, and Liu’s medium. The CTC was

testedat three differentconcentrations of thiabendazole: 1 mg/l (CTC 1T), 2 mg/l (CTC 2T) or 4 mg/l (CTC 4T). The 50ll of a suspension of ‘‘spiked” soil spreadontoeach plate

contained approximately 700 conidia of EF. (A) Colonies counted at day 14; bars represent standard errors of three trials with four different soil samples. Means depicted by

thecolumns with thesame letterare not significantlydifferentfrom each other (ARSEF5626: P = 0.0314, F = 3.83; ARSEF 2575: P = 0.0203, F =4.40); (B) Plates atday 7 and 21

after inoculation. Note different appearance (aspect) and number of Metarhizium spp. colonies in each medium type. Arrows indicate at least one Metarhizium spp. colony per

plate; non-similar colonies are contaminant fungi isolated from soil. For color plates of the artwork, please refer to the online version of the figure.




ium is commercially available, easily prepared and relatively trans-

parent, which allows easy visualization of colonies from the re-verse of Petri plates and, when needed, visualizing conidial

germination on the agar surface by compound-microscope obser-

vation. In addition to the primary ingredient (PDAY), CTC mediumis supplemented with several chemicals that have been previously

Fig. 4. Beauveria bassiana (ARSEF 252) or B. brongniartii (ATCC 58798) conidia were added(spiked) to non-sterile soil and thenre-isolated using five different selective media:

CTC[PDAY (potato dextrose agar supplementedwith yeast extract) plus chloramphenicol, thiabendazole and cycloheximide], Veen’s, and Liu’s medium. The CTC was tested at

three different concentrations of thiabendazole: 1 mg/l (CTC 1T), 2 mg/l (CTC 2T) or 4 mg/l (CTC 4T). The 50ll of a suspension of ‘‘spiked” soil spread onto each plate

contained approximately 700 conidia of EF. (A) Colonies counted at day 14; bars represent standard errors of three trials with four different soil samples. Means depicted by

the columns withthe same letter are not significantly different fromeach other (ARSEF 252: P = 0.0816, F = 2.70; ATCC 58798: P = 0.2037, F = 1.75); (B) Plates at day 7 and21

after inoculation.Note different appearance (aspect) and number of Beauveria spp. colonies in each medium type. Arrows indicate at least one Beauveria spp. Colony.For color

plates of the artwork, please refer to the online version of the figure.




described for isolation of entomopathogenic fungi: chlorampheni-

col, thiabendazole and cycloheximide (Luz et al., 2007; Rocha and

Luz, 2009; Veen and Ferron, 1966). Chloramphenicol is a broad-

spectrum antibiotic that is effective against a wide variety of

Gram-positive and Gram-negative bacteria, including most anaer-

obic organisms (Falagas et al., 2008). Thiabendazole is used in agri-

culture as a fungicide or parasiticide. In a recent search for

selective chemicals, thiabendazole was proposed as the most

appropriate of several antifungal compounds for selective isolation

of entomopathogenic fungi (Luz et al., 2007; Rocha and Luz, 2009).

Cycloheximide is an inhibitor of biosynthesis in eukaryotic organ-

isms (Taber and Vincent, 1969). Cycloheximide has a wide anti-

fungal effect, but it allows growth and development of a few

fungi, including many entomopathogenic fungi. Accordingly, it is

an excellent selective agent (Tsao, 1970; Whiffen, 1948).

Thiabendazole generally has been shown to have low acute der-

mal mammalian toxicity; and it is neither irritating to the eyes or

skin nor a dermal sensitizer. Toxic categories, which range from 1

(most toxic) to 4 (least toxic), were mostly 4 for thiabendazole

(United States Environmental Protection Agency, Washington,

DC). Cycloheximide is a specific-hazard-type toxicant, classified

as most severe. The mammalian LC50 for cycloheximide is 2 mg/

kg. The Hazard Evaluation System and Information Service (HESIS)

recommends that cycloheximide be considered potentially harm-

ful to the human reproductive system. Persons working with cyclo-

heximide should avoid hand-to-mouth exposure and wear tight-

fitting, disposable, impermeable gloves to prevent skin contamina-

tion and subsequent skin absorption (HESIS, Richmond, CA). De-

spite its toxicity, cycloheximide is used in treatment of cancer

and in management of graft-versus-host reactions following organ

transplantations. Chloramphenicol has an irritating effect to skin,

eyes, and mucous membranes. Its LD50 is 2500 mg/kg when in-

gested by rats, and 171 mg/kg when administrated intravenously

in rats (Neogen Co., Material Safety Data Sheet, Lansing, MI). Chlor-

amphenicol is possibly carcinogenic to humans. In contrast, dodine

did not cause allergic skin reactions when tested in humans; how-

ever, because it may cause severe eye irritation, dodine is consid-ered a highly toxic material. Dodine is not acutely toxic via

inhalation or ingestion; the oral LD50 for technical dodine in rats

is 1000 mg/kg (Pesticide Information Profile, Extension Toxicology

Network, Cornell University, Ithaca, NY).

For the past three decades, dodine has been an important ingre-

dient in media designed for selective isolation of entomopatho-

genic fungi. In recent years, however, this compound has

experienced considerably reduced use in its primary market, i.e.,

as an USA orchard fungicide. It is now difficult to obtain in many

localities (Luz et al., 2007; Rocha and Luz, 2009), but currently it

is still available in the USA (manufactured by Chimac-Agriphar

S.A., and distributed by Aceto Agricultural Chemicals Corporation,

Lake Success, NY). This lack of ready availability of dodine to many

scientists, combined with the fact that M. acridum is more suscep-tible to dodine than most other entomopathogenic fungi (Rangel

et al., 2010), prompted our search for a suitable alternative. Conid-

ial germination of three isolates of M. acridum was not severely

inhibited by 0.002% or 0.004% A.I. dodine in PDAY medium (Rangel

et al., 2010). In our current study, using conidia mixed with soil, no

colonies of M. acridum were recovered from PDAY supplemented

with low concentrations of dodine (0.002%, 0.004% or 0.006% A.I.)

(see Fig. 1). This disparity suggests fungal inhibitors in the soil,

possibly associated with naturally occurring microbes. Accord-

ingly, in addition to considering the variation among target-fungus

susceptibility, selective media should be designed to tolerate com-

petition between target fungi and the diversity of microorganisms

and microbial metabolites occurring in the soil.

Metarhizium acridum conidia in soil were not re-isolated onPDAY containing even a very low concentration (0.001% A.I.) of do-

dine. It is not known whether this is directly due to dodine inhibi-

tion or because the low concentration of dodine was unable to

inhibit fast-growing non-target fungi which overran the isolation

plates. The other species of entomopathogenic fungi in soil tested

in our study, i.e., M. brunneum, M. robertsii, B. bassiana and B. brongniartii, were re-isolated on PDAY + 0.001% A.I. dodine media de-

spite the high levels of contamination. These results suggest that

contamination alone was not sufficient to impede re-isolation.

Conversely, M. acridum colonies were re-isolated on Liu’s medium

which contains 0.001% A.I. dodine, indicating that this concentra-

tion in this specific medium does not impede M. acridum isolation

from soil. It is likely that the lack of M. acridum growth on PDAY

plates with 0.001% dodine was due to a combination of susceptibil-

ity to the fungicide and competition from other microorganisms.

Liu et al. (1993) reported that dodine was less inhibitory in Veen’s

medium than in ‘germinating medium’ of Milner et al. (1991). Ran-

gel et al. (2010) also noted that the effectiveness of dodine varied

with the media.

Veen’s medium (Veen and Ferron, 1966) is a dodine-free selec-

tive medium recommended for isolation of B. brongniartii and M.anisopliae s.l. The substrate, which is glucose and peptone, is sup-

plemented with chloramphenicol, cycloheximide and oxgall. The

addition of dodine to Veen’s medium afforded a medium with im-

proved effectiveness in isolating Metarhizium spp. from soil and re-

duced the number of contaminants by over 90% (Liu et al., 1993).

Nevertheless, because of differences between isolates of Metarhiz-ium spp. in their susceptibility to dodine, a significantly low con-

centration of 10lg/ml (0.001%) was recommended for selective

Metarhizium spp. isolation from soil (Liu et al., 1993). Despite the

efficiency of Veen’s and Liu’s media in isolating entomopathogenic

fungi, CTC produced colonies that, in general, were larger and had

both earlier and greater sporulation. These features facilitated

identification of the fungi, especially with the CTC 1T medium

(see Figs. 2–4). Early and heavy sporulation of target fungi is

known to aid in their rapid identification (Flowers and Hendrix,

1969; Papavizas, 1967).

Appropriate agar concentration is an important consideration indeveloping selective media, as it determines the rigidity of the

growth substrate (Flowers and Hendrix, 1969). Veen’s and Liu’s

media both contain 35 g of agar per liter, while CTC medium con-

tains only 15 g; this concentration makes the CTC medium less ri-

gid than the others, possibly allowing greater access to nutrients.

Oxgall is a growth-inhibiting chemical (Durbin, 1961) that reduces

the speed of fungal development. In our current study, colonies on

media containing oxgall developed much slower. The thiabenda-

zole in CTC medium helps maintain fungal contaminants at accept-

able levels. While statistical differences in the number of colony re-

isolations were not observed between CTC and Veen’s or Liu’s med-

ia, the efficiency, quality, and confidence in identification of the re-

isolated colonies were greatly enhanced using CTC medium.

The M. acridum isolates tested in our study were severely inhib-ited by the fungicide thiabendazole in the amount (0.004 g/l) sug-

gested by Rocha and Luz (2009). On the other hand, when this

amount was reduced to 0.001 g or 0.002 g/l (CTC 1T or CTC 2T,

respectively), the recovery of M. acridum colonies was drastically

increased (see Fig. 2). These results demonstrate and agree with

previous studies that found different species of the same fungal

genus and even different isolates of the same species often differ

in sensitivity to a toxicant (Eckert and Tsao, 1962; Papavizas,

1964, 1967; Rangel et al., 2010). The low thiabendazole concentra-

tion still avoided the interference of most fungal contaminants, and

the combination of the chemicals in CTC medium completely pre-

cluded bacterial contamination. However, successful isolation of

entomopathogenic fungi in the presence of contaminants will vary

according to the diversity of microorganisms occurring in the soiland to the inoculation method employed.




In conclusion, CTC medium (which is equivalent to CTC 1T in

the current study) proved to be a competent medium for the isola-

tion of M. acridum, as well as other Metarhizium and Beauveria spe-

cies, from soil. Conversely, the dodine-based selective media

(PDAY + dodine) proved to be ineffective or less effective (Liu’s

medium) for the isolation of M. acridum. The efficiency of the

CTC medium may be explained by the low concentration of agar,

the absence of oxgall, and the addition of thiabendazole in CTC

medium. Due to the variability in the response to fungicides among

species and isolates of the same species of entomopathogenic fun-

gi, further studies should be conducted to confirm the effectiveness

of CTC medium as an appropriate selective media for many isolates

of Metarhizium spp. and Beauveria spp., and other entomopatho-

genic fungi not investigated in our present study. In addition, the

use of more than one selective medium is recommended to maxi-

mize the recovery of strains or clones which might be different in

their sensitivity to toxicants (Papavizas, 1964, 1967). Nevertheless,

CTC medium is the most efficient of the several media examined

here, and we expect it to be a powerful tool for isolating entomo-

pathogenic fungi from soil.

Acknowledgments

We are grateful to Susan Durham (Utah State University, Logan,

UT, USA) for the statistical analyses, and to Richard A. Humber

(USDA/ARS, Ithaca, NY, USA) for providing the fungal isolates used

in this study. We very much appreciate helpful comments by our

anonymous reviewers and especially the detailed observations of

our Biological Control Editor (H.K.K.). We also thank our laboratory

technicians: Alessandra Fernandes, Carmina Moore, Fabiana Alva-

rez and Scott Treat for their careful attention to detail in the exper-

iments of this study. This research was supported by grants from

the Utah Department of Agriculture and Food, the United States

Department of Agriculture (USDA, APHIS) and the Community/Uni-

versity Research Initiative of Utah State University. Paper 8173 of

the Utah Agricultural Experiment Station.

This article reports the results of research only. Mention of aproprietary product does not constitute an endorsement or a

recommendation for its use by the United States Department of

Agriculture.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, in

the online version, at doi:10.1016/j.biocontrol.2010.05.009.

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