Fungal tyrosine betaine, a novel secondary metabolite from conidia of entomopathogenic Metarhizium spp. fungi

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Fungal tyrosine betaine, a novel secondary metabolite fromconidia of entomopathogenic Metarhizium spp. fungi

Carlos A. CAROLLOa, Ana Luiza A. CALILb, Letıcia A. SCHIAVEb, Thais GUARATINIc,Donald W. ROBERTSd, Norberto P. LOPESa, Gilberto U. L. BRAGAb,*aDepartamento de Fısica e Quımica, Faculdade de Ciencias Farmaceuticas de Ribeirao Preto, Universidade de Sao Paulo-USP,

Ribeirao Preto, SP 14040-903, BrazilbDepartamento de Analises Clınicas, Toxicologicas e Bromatologicas, Faculdade de Ciencias Farmaceuticas de Ribeirao Preto,

Universidade de Sao Paulo-USP, Ribeirao Preto, SP 14040-903, BrazilcDepartamento de Bioquımica, Instituto de Quımica, Universidade de Sao Paulo-USP, Sao Paulo, 05508-900 SP, BrazildDepartment of Biology, Utah State University, Logan, UT 84322-5305, USA

a r t i c l e i n f o

Article history:

Received 16 September 2009

Received in revised form

5 February 2010

Accepted 19 March 2010

Available online 27 March 2010

Corresponding Editor: Judith K. Pell

Keywords:

Conidial metabolites

Fungal secondary metabolite

Fungal tyrosine betaine

Mannitol

Metarhizium anisopliae

Tyrosine betaine

* Corresponding author. Tel.: þ55 16 3602 442E-mail address: [email protected]

1878-6146/$ – see front matter ª 2010 The Bdoi:10.1016/j.funbio.2010.03.009

a b s t r a c t

Fungi, including the entomopathogenic deuteromycete Metarhizium anisopliae, produce a wide

diversity of secondary metabolites that either can be secreted or stored in specific develop-

mental structures, e.g., conidia. Some secondary metabolites, such as pigments, polyols

and mycosporines, are associated with pathogenicity and/or fungal tolerance to several

stress-inducing environmental factors, including temperature and solar radiation extremes.

Extracts of M. anisopliae var. anisopliae (strain ESALQ-1037) conidia were purified by chromato-

graphic procedures and the isolated compounds analyzed by 1H and 13C nuclear magnetic res-

onance spectroscopy and high-resolution mass spectrometry. LC–MS analyses were carried

out to search for mycosporines (the initial targets), but no compounds of this class were

detected. A molecule whose natural occurrence was previously undescribed was identified.

It consists of betaine conjugated with tyrosine, and the structure was identified as

2-{[1-carboxy-2-(4-hydroxyphenyl)ethyl]amino}-N,N,N-trimethyl-2-oxoethanammonium.

Mannitol was the predominant compound in the alcoholic conidial extract, but no amino

acids other than tyrosine were found to be conjugated with betaine in conidia. The fungal

tyrosine betaine was detected also in conidial extracts of three other M. anisopliae var. aniso-

pliae (ARSEF 1095, 5626 and 5749) and three M. anisopliae var. acridum isolates (ARSEF 324, 3391

and 7486), but it was not detected in Aspergillus nidulans conidial extract (ATCC 10074).

ª 2010 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.

Introduction involved in the rhizosphere competence of M. anisopliae or in

Metarhizium is a genus of entomopathogenic fungi used world-

wide as alternatives to chemical insecticides in agricultural

pest and disease-vector control programs (Roberts & St. Leger

2004). In addition to being an entomopathogen, M. anisopliae

also can colonize plant roots and is a common component of

the rhizosphere (root–soil interface). The mechanisms

5.

ritish Mycological Society

its fungal–plant interactions are only partially understood

(Hu & St. Leger 2002; Wang & St. Leger 2007; St. Leger 2008).

The wide utilization of M. anisopliae as bioinsecticide has in-

creased interest in its basic biology, including its fungal second-

ary metabolites. Secondary metabolite production is a complex

process that often is coupled with morphological development

in filamentous fungi. Signaling cascades link sporulation

. Published by Elsevier Ltd. All rights reserved.

Table 1 – Nuclear magnetic resonance data of tyrosinebetaine.

d-1H NMR(500 MHz, D2O)

d-13C NMR(100 MHz, DMSO-d6)

1 – 128.9

2 7.09, d, J¼ 8.2 Hz 129.9

3 6.76, d, J¼ 8.2 Hz 114.6

4 – 155.4

5 6.76, d, J¼ 8.2 Hz 114.6

6 7.09, d, J¼ 8.2 Hz 129.9

7a 3.16, dd, J¼ 4.5 and 14.1 Hz 36.7

7b 2.71, dd, J¼ 10.3 and 14.1 Hz

474 C. A. Carollo et al.

processes with metabolite synthesis (Calvo et al. 2002; Yu &

Keller 2005). Secondary metabolites are predicted to improve

fungal adaptability in their natural habitats (Shwab & Keller

2008); e.g., they are involved in niche establishment, serve as sig-

naling molecules in fungal–plant or fungal–insect interactions,

or act as stress protectors (Moon et al.2008). Inmost cases, the ex-

act function of secondary metabolites for the producing fungi,

including Metarhizium spp., is unknown (Krasnoff et al. 2007).

Metarhizium spp. produce a variety of secondary metabolites in

several chemical classes, including cytochalasins C and D

(Aldridge & Turner 1969), myroridins (Kondo et al. 1980), destrux-

ins A, B and E (Roberts 1969; Paıs et al. 1981), viridoxin (Gupta et al.

1993), swainsonine (Patrick et al. 1993), helvonic acid (Espada &

Dreyfuss 1997), 12-hydroxyovalicin (Kuboki et al. 1999), hydroxy-

fungerin, 7-desmethyl analogues of fusarin C and (8Z)-fusarin C

(Krasnoff et al. 2006), serinocyclins A and B (Krasnoff et al. 2007)

and aurovertins (Azumi et al. 2008). M. anisopliae metabolites

are toxic to a broad range of animals and microbes, including in-

sects (Gupta et al. 1989; Roberts et al. 1992; Vilcinskas et al. 1997;

Roberts & St. Leger 2004; Pal et al. 2007), fungi, bacteria and vi-

ruses (Kondo et al. 1980; Krasnoff et al. 2006). Most of these me-

tabolites were isolated from mycelia or from fermentation

extracts. There is much less information regarding secondary

metabolites exclusively or differentially present in Metarhizium

conidia (Krasnoff et al. 2007; Moon et al. 2008).

M. anisopliae produces green rod-shaped conidia, both dur-

ing its saprophytic existence and on the surface of the insect

cadavers at the end of its pathogenic cycle. Conidia are

asexual specialized structures of filamentous fungi that are

frequently involved in reproduction, dispersal and environ-

mental persistence of these microorganisms. In pathogenic

species, such as M. anisopliae, conidia are also involved in

host recognition and infection (Wang & St. Leger 2007). The co-

nidium is the fungal stage normally used as the field inoculum

in biological control programs (Roberts & St. Leger 2004).

Natural fungal populations are controlled by environmen-

tal factors, such as solar radiation, temperature, and humid-

ity. These factors limit the survival and dispersal of

pathogenic species and represent serious obstacles in the

use of fungi like M. anisopliae as bioinsecticides. Several me-

tabolites present in fungal conidia, such as pigments, sugars,

and polyols have been associated with stress tolerance

(Hallsworth & Magan 1995; Ruijter et al. 2003). The discovery

of new secondary metabolites present in M. anisopliae conidia

will contribute both to (1) the better understanding of the biol-

ogy of this specialized structure, including molecular aspects

of its environmental persistence, germination and entomopa-

thogenicity and (2) the development of entomopathogenic

fungal strains with increased stress tolerance for use in bio-

logical control of insect pests. The aim of the current study

was to isolate and identify secondary metabolites in Meta-

rhizium spp. conidia.

8 4.46, dd, J¼ 4.5 and 10.3 Hz 55.9

9 – 173.1

11 – 170.4

12a 3.95, d, J¼ 14.6 Hz 64.7

12b 3.82, d, J¼ 14.6 Hz

14 3.01, s 53.2

15 3.01, s 53.2

16 3.01, s 53.2

Materials and methods

Fungal strains

Conidia (1 kg) of the M. anisopliae var. anisopliae strain ESALQ-

1037 were purchased from Itaforte-Bioprodutos (Sao Paulo,

Brazil). Other Metarhizium isolates were obtained from the

USDA ARS Collection of Entomopathogenic Fungal Cultures

(Ithaca, NY), viz., three M. anisopliae var. anisopliae (ARSEF

1095, ARSEF 5626 and ARSEF 5749) and three M. anisopliae

var. acridum (ARSEF 324, ARSEF 3391 and ARSEF 7486). Aspergil-

lus nidulans (ATCC 10074) was obtained from the American

Type Culture Collection (Manassas, VA).

Note: A very recent taxonomic revision (Bischoff et al. 2009)

assigns M. anisopliae var. acridum as a new species, viz., M. acri-

dum; and they apply this name to ARSEF 324, 3391 and 7486.

Humber, a coauthor of Bischoff et al. (2009), lists ARSEF 1095

and 5626 as M. brunneum; and ARSEF 5749 as M. anisopliae sensu

lato (Humber et al. 2009). ESALQ-1037 is not mentioned in

either document.

Production of conidia

The ESALQ-1037 conidia were produced by Itaforte-

Bioprodutos by culturing at 28 �C for 12 d on cooked rice

with a daily cycle of 12 h of light and 12 h dark using fluores-

cent light bulbs (TLD 32 W/64 RS, Philips, Brazil). Visible light

irradiance (400–700 nm) was 6 W m�2, as measured with an

USB4000 spectroradiometer (Ocean Optics, Denedin, FL). After

growth, conidia were carefully collected by suction from the

rice substrate. To examine the effect of culture media on fun-

gal tyrosine betaine production, ESALQ-1037 was also grown

(in our laboratory) on 23 mL potato dextrose agar (Difco Labo-

ratories, Detroit, MI) (PDA) and PDA supplemented with 1 g L�1

yeast extract (Technical, Difco) media (PDAY) in petri dishes in

the dark at 28 �C for 14 d. All other isolates were grown on

PDAY in petri dishes in the dark at 28 �C for 14 (Metarhizium)

or 5 d (A. nidulans).

Polarimetry

Optical rotations were measured at 25 �C in MeOH on a Jasco

DIP-370 digital polarimeter using the sodium lamp (589 nm)

with a 50 mm cell.

Fungal tyrosine betaine, a novel secondary metabolite 475

High-performance liquid chromatography

HPLC retention times were determined on a Shimadzu chro-

matograph equipped with two solvent pumps (LC-20A) and

a ShimPack ODS 5-mm Shimadzu, 250� 4.6 mm column using

MeOH/H2O linear elution gradient starting from 3 % MeOH

during 5 min and increased up to 70 % MeOH in 30 min at

a flow rate of 1.0 mL min�1, with detection by UV absorption

from 190 to 600 nm using a diode array detector (SPD-M20A).

Purification was executed by semip-reparative HPLC

(Shimadzu) using a ShimPack ODS 5-mm Shimadzu,

250� 20.0 mm column, MeOH/H2O linear elution gradient

starting from 3 % MeOH during 10 min and increased up to

70 % MeOH in 60 min at a flow rate of 9.6 mL min�1, with de-

tection by UV absorption at 270 nm using a UV–VIS spectro-

photometric detector (Shimadzu SPD-6AV).

Mass spectrometry

Mass measurements were acquired by infusion of methanolic

solutions at 150 mL h�1 using a syringe pump (Hamilton Co,

Fig 1 – Chromatograms obtained by HPLC–DAD (270 nm) with p

tyrosine betaine. (A) Tyrosine betaine standard, (B) supernatant

and (C) uninoculated rice extract.

Reno, Nevada) into a Bruker Daltonics spectrometer (ultrO-

TOFQ). Positive ion mode spectra were recorded at

1000 scans s�1; spectrum interval, 2 s; drying gas flow,

7.0 L min�1; drying gas temperature, 180 �C; nebulizer gas

pressure, 4.

Nuclear magnetic resonance spectroscopy

The 1H NMR spectra were acquired at 500 MHz on a Bruker Ad-

vanced DRX-500 spectrometer and the 13C NMR spectra were

acquired at 100 MHz on a Bruker Advanced DRX-400

spectrometer.

Isolation and characterization of conidial extract constituents

In order to isolate secondary metabolites (the initial target was

mycosporines), 1 kg of conidia (ESALQ-1037) was extracted

with 15 L methanol/water (MeOH/H2O) (9:1) (Fisherchemical,

HPLC grade). The conidial suspension was sonicated 1 h d�1

for 10 d (Ultrasonic Cleaner 1440D, Odontobras, Brazil). The

methanolic extract was filtered (FITEC 19090) and solvent

eaks at approximately 11.8 min corresponding to

of M. anisopliae crude extract (strain ESALQ-1037),

476 C. A. Carollo et al.

removed in vacuo to afford 232 g of crude extract, which was

suspended in 200 mL of MeOH. The suspension was sonicated

and centrifugated. A white solid precipitate (34 g) was sepa-

rated from the extract and identified as mannitol by 1H and13C NMR and high-resolution electron spray ionization mass

spectra (HRESIMS). The supernatant was dried, dissolved at

1 mg mL�1 in MeOH, and then directly analyzed through

high-performance liquid chromatography–diode array detec-

tion/electrospray ionization–mass spectrometry (HPLC–DAD/

ESI–MS) (ShimPack ODS 5-mm Shimadzu, 250� 4.6 mm col-

umn; MeOH/H2O linear elution gradient starting from 3 %

MeOH during 5 min and increased up to 70 % MeOH in

30 min; flow 1.0 mL min�1). The analytical chromatogram

showed one major compound with UV maximum at 270 nm.

For its purification, 2 g of the dried supernatant were dissolved

in MeOH and the major compound was isolated using the

same HPLC methodology previously described, except at

a semi-preparative scale. The procedure yielded 90 mg of

a pure brown amorphous powder. The compound was sub-

jected to polarimetry, 1H and 13C NMR, MS and UV spectrum

analyses for identification and structure elucidation, and it

was identified as tyrosine betaine: [a]Dþ 8.1, absorbance

275 nm, ESI–MS (þ) 281.1492 (observed), molecular formula

C14H21N2O4 (281.1496), ESI–MS–MS (þ) 235 [M–CO2H2]þ, 176

[M–CO2H2–N–Me3]þ. For 1H and 13C NMR data see Table 1.

To verify that fungal tyrosine betaine was not present in rice

used as substrate for the fungal growth, the same extraction pro-

tocol described for conidia was conducted on uninoculated rice.

Results

Preliminary screening

Analysis of the supernatant by HPLC–DAD (190–600 nm) did

not detect any compound with absorption near to 310 nm.

The ESI–MS/MS analysis did not show any characteristic frag-

ments in positive and negative modes (Cardozo et al. 2008,

2009). Since absorbance at 310 nm is characteristic of myco-

sporines, our extracts of conidia of M. anisopliae var. anisopliae

(ESALQ-1037) did not contain detectable quantities of

compounds similar to fungal mycosporines (Leach 1965;

Favre-Bonvin et al. 1976; Young & Patterson 1982; Bernillon

et al. 1984). Conidial extracts were also analyzed for caroten-

oids and xanthophylls with LC–UV and MS as previously

described (Guaratini et al. 2007, 2009), but compounds of these

two groups were not detected.

Isolation and identification of the major conidial polyol

The white precipitate was identified as the sugar mannitol.

Further analysis of the precipitate and the crude extract by

HPLC–MS indicated mannitol as the only major polyol.

Fig 2 – (A) Molecular structure of tyrosine betaine

(2-{[1-carboxy-2-(4-hydroxyphenyl)ethyl]amino}-N,N,

N-trimethyl-2-oxoethanammonium) isolated from

M. anisopliae, and (B) molecular structure of tyrosine with

an N-trimethyl group isolated from non-M. anisopliae

sources.

Structure elucidation of the tyrosine betaine

In the chromatogram of the supernatant, a major compound

was eluted at 11.8 min (Fig 1B ) that was not observed in the

chromatogram of the rice extract, verifying that the

compound was not present in the rice used as substrate for

fungal growth (Fig 1C).

The 1H and 13C NMR spectra of the isolated compound

showed signals similar to those found for the amino acid

tyrosine and the N-trimethylated amino acid derivative be-

taine. Mass-spectrum analysis showed a signal at m/z

281.1492, which suggests that the molecule is a dimer of

these two compounds. This information was confirmed

by the correlation in the Heteronuclear Multiple Bond Cor-

relation (HMBC) contour map between H-8 (d 4.46) and C-11

(d 170.4). Furthermore, the ESI–MS–MS (þ) showed

several fragments (as described in materials and

methods), which are in agreement with the proposed struc-

ture (Fig 2A ).

In a search for other amino acids conjugated with betaine,

HPLC–MS examination of the crude extract was conducted;

and the results carefully monitored for molecular weights of

possible dimers. We did not detect any other conjugate, which

suggests that M. anisopliae conidia specifically accumulate

tyrosine betaine.

Fungal tyrosine betaine also was detected in conidial ex-

tracts of strain ESALQ-1037 grown on PDA and PDAY media

rather than rice (Fig 3) and in conidial extracts of three other

M. anisopliae var. anisopliae (ARSEF 1095, ARSEF 5626 and ARSEF

5749) strains and three M. anisopliae var. acridum (ARSEF 324,

ARSEF 3391 and ARSEF 7486) strains (Fig 4). The metabolite

was not detected in conidial extracts of A. nidulans (ATCC

10074) (Fig 4).

Discussion

Literature shows the occurrence of a molecule named tyro-

sine betaine (Fig 2B) (Bernard et al. 1981; Shrestha & Bisset

Fig 3 – Chromatograms obtained by HPLC–DAD (270 nm) with peaks at approximately 11.9 min corresponding to tyrosine

betaine. (A) Tyrosine betaine standard, (B) supernatant of conidial crude extract of M. anisopliae (strain ESALQ-1037) grown on

rice, (C) grown on PDA, and (D) grown on PDAY.

Fungal tyrosine betaine, a novel secondary metabolite 477

1991; Eggenberger & Rowell-Rahier 1993). It is remarkable

that instead of being betaine conjugated with tyrosine,

this molecule is actually a tyrosine with an N-trimethyl

group. Therefore, tyrosine betaine isolated from M. aniso-

pliae is a novel (i.e. never previously reported) class of

secondary metabolite. The same mistake occurs in

naming other amino acids conjugated with N-trimethyl

groups. Amino acids conjugated with N-trimethyl groups

such as ‘‘glycine betaine’’ (glycine N-trimethyl group)

‘‘proline betaine’’ (proline N-trimethyl group) and ‘‘trypto-

phan betaine’’ (tryptophan N-trimethyl group) were isolated

from plants and microorganisms and act as stress protec-

tors or in microbial-host signaling. ‘‘Glycine betaine’’ and

‘‘proline betaine’’ act as osmo and cryoprotectants in bacte-

ria (Yancey et al. 1982). Hypaphorine, a tryptophan N-tri-

methyl group, was detected in mushrooms and in

ectomycorrhizal fungi and plays an important role

in fungal–plant interaction (Beguiristain & Lapeyrie

478 C. A. Carollo et al.

1997; Nehls et al. 1998; Ditengou & Lapeyrie 2000; Ditengou

et al. 2003).

Mannitol was the predominant compound in the alco-

holic extracts of M. anisopliae var. anisopliae strain ESALQ-

1037 conidia. Mannitol is an acyclic hexitol that is found

in most higher fungi. It is usually the most abundant of

all soluble carbohydrates within the mycelium and conidia

of several fungal species including the entomopathogens

Beauveria bassiana, Paecilomyces farinosus and M. anisopliae

(Lewis & Smith 1967; Hallsworth & Magan 1994;

Witteveen & Visser 1995; Hallsworth & Magan 1995;

Rangel et al. 2008). The precise role(s) of mannitol seems

to differ depending on the fungus (Solomon et al. 2007). In

A. nidulans, mannitol makes up 10–15 % of the conidial

dry weight. The inactivation of the gene mpdA, which en-

codes mannitol 1-phosphate dehydrogenase (the first en-

zyme in the mannitol biosynthesis pathway) reduced the

A. nidulans conidial sensitivity to a variety of stress condi-

tions, including high temperature and oxidative stress

(Ruijter et al. 2003). Mannitol is required for sexual sporula-

tion of the wheat pathogen Stagonospora notorum (Solomon

et al. 2006).

Fungal tyrosine betaine was detected in conidia

obtained from mycelia grown on different culture media

such as rice, PDA and PDY and with the fungus growing

in the presence or absence of visible light. The fungal tyro-

sine betaine was detected in conidia of all seven Meta-

rhizium ssp. Isolates we analyzed (four strains of M.

anisopliae var. anisopliae and three strains of M. anisopliae

var. acridum) suggests that the compound is conserved

and may be important for the biology of the group. Since

all analyses were qualitative, it was not possible to deter-

mine the amounts of fungal tyrosine betaine accumulated

in conidia of the different strains [or species, if the taxo-

nomic revision proposed by Bischoff et al. (2009) is consid-

ered]. Additional experiments are needed to assess the

effect of physical and chemical factors during fungal

growth on the accumulation of the metabolite in conidia

and to assess the variability of fungal tyrosine betaine

accumulation among strains.

We did not detect any other amino acid betaine conjugate,

which suggests that M. anisopliae conidia accumulate fungal

tyrosine betaine specifically. The presence of fungal tyrosine

betaine and other conjugates in fungal mycelium is still

undetermined.

Hopefully, the discovery of fungal tyrosine betaine’s role in

M. anisopliae biology, as well as its possible biotechnological

applications, will provide rewarding intellectual and practical

challenges.

Fig 4 – Chromatograms obtained by HPLC–DAD (270 nm)

with peaks at approximately 11.9 min corresponding to

tyrosine betaine. (A) Tyrosine betaine standard. (B–D)

Supernatant of conidial crude extract of M. anisopliae var.

anisopliae strains ARSEF 1095, 5626, and 5749, respectively.

(E–G) Supernatant of M. anisopliae var. acridum strains

ARSEF 324, 3391, and 7486, respectively. (H) Supernatant

of A. nidulans strain ATCC 10074.

Fungal tyrosine betaine, a novel secondary metabolite 479

Acknowledgements

We thank Ludmilla Tonani for technical assistance. This work

was supported by grants # 03/07702-9 from The State of Sao

Paulo Research Foundation (FAPESP) and # 47.6990/2004-1

from The Brazilian National Council for Scientific and Techno-

logical Development (CNPq).

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