Antuie vtm Leeuwenhoek 72: 31,1-325. 1997 .

@) 1997 Kluwer Acadernic Publishers. Printed in rhc Netherlands.311

Exserohilum sodomii, a new species isolated from soil near the Dead Sea(Israel)

Pascale Guiraud, Régine Steiman, Francoise Seigle-Murandi & Lucile SageGroupe pour I'Etude du Devenir des Xénobiotiques dans l'Erwironnement (GEDEXE), UFR Pharmacie(Université Joseph Fourier), BP 138, Avenue de Verdun, j8243 Meylan Cedex, France

Received 14 November 1996; accepted 24 Jnly 7997

Key words: Exserohilum sodomii sp. nov., Israeli mycoflora, antimicrobial activity, enzymatic activities

Abstract

Exserohilum sodomii sp. nov., is described. This new species was isolated from a soil sample from the Dead Sea

surroundings. Its main physiological properties, as well as the influence of temperature and salts concentration inthe culture medium on growth and morphology of the fungus were investigated and discussed.

Introduction

As part of a wide investigation of the microfungal pop-

ulation in various Israeli soils, a new species ofExsero-hilum was isolated from a sample from Sodom (near

the Dead Sea). This area is characteized by a drysummer and high salts level in soil. A survey of micro-fungal population in soil around the Dead Sea has been

recently published (Guiraud et al., 1995; Steiman et al.,

lees).The isolates ofthe new species differed from other

species of Exserohilumktown to date. Another newspecies of Exserohilum was recently isolated from a

soil sample from Timna Park (Israel) (Steiman et a1.,

in press).

The new species is described and its physiologicalfeatures investigated.

Materials and methods

Strain isolation

The soil sample was collected in August 1994 alongthe road to Sodom at the south end of the Dead Sea.

Isolation of the strain was accomplished by the soilplate method of Warcup (Parkinson & Waid, 1960):the soil sample was dispatched into 20 Petri dishes

(90 mm diameter, 5 mg soil each) and sterile malt

extract (1-.5%) - agar (l.SVo) (MEA) - chloramphenicol(0.0570) medium was poured over. After solidification,dishes were in cubated at 22 o C, 30 " C, 3J " C, 45 " C and

the strains were isolated as soon as colonies appeared.

Soluble salt level in soil

1 g of dry soil was suspended in 50 ml of 0.1 N HCIby vigourous shaking. After filtration, sodium (Na),potassium (K), chloride (Cl), calcium (Ca), magne-

sium (Mg), phosphorous (P) were determined withan automated analyzer BM/Hitachi 917 (BoehringerMannheim, France) using the methods adapted byBoehringer Mannheim. Results were expressed as

p,mollg of dry soil. Chloride contained in 0.1 N HClsolution was subtracted.

Culture conditions

Exserohilum sodomiiwas grown on MEA, PDA (pota-to dextrose agar), hyperosmotic medium (malt extract57o, saccharose 37o, NaCl 8%, agar 1,.5Vo) (Pelhate,1968), synthetic agar medium (Galzy & Slonimski,1957). ln each case, plates were incubated at 22 "C,30 oC, 37 oC, 45 'C. Growth was evaluated by dailymeasurement of colony diameter.

318

Enzyme assays

The methods used for the detection of lipase, protease

and amylase production have been described by Han-kin & Anagnostakis (1975). Cellulases were detect-

ed according to Smith (L977); the same method was

adapted to the detection of amylolytic activity accord-

ing to Rinderknecht et al. (1967). Results of these

assays were expressed as arbitrary units corresponding

to the ratio:

diameter of the zone of activitY@

Production of extracellular phenoloxidases (POx) was

researched on PDA and hyperosmotic medium as pre-

viously described (Guiraud et a1.,1992). Ten reagents

were used (benzidine, guaiacol, o-anisidine, pyro-

gallol, a-naphtol, p-cresol, tyrosine, gallic acid as

0.1, M solutions in ethanol; syringaldazite0.l.% (wlv)in ethanol with or without 0.03Vo hydrogen peroxide

aqueous solution; R56 reagent: ethanolic solution con-

taining amidopyrine 17.5 Vo, N-N diethylan ilirre 2.0%,

benzoic acid2.5Vo). Following their intensity, the col-

ored reactions were noted from 0 to 5. The global POx

activity of the strain was obtained by adding the values

corresponding to the different reactions.

These enzymatic assays were performed with 8

day-old cultures, grown and incubated at22 " C,30 " C,

37 "C.

Antimicrobial activiÿ

The strain was grown in two liquid media: malt and

synthetic media described above. The fungus (myceli-

um + spores) was inoculated in 250 m\ Erlenmeyerflasks, containing 70 ml of liquid medium and grown

under shaking (5 days) or static (7 days) conditionsat 30 oC. Media were separated from the mycelia by

filtration and extracted by 2 x 35 ml ethyl acetate.

Organic phases were pooled and dried at 40 oC under

reduced pressure. Crude extracts were dissolved in3 ml ethyl acetate. Target pathogenic strains were 3bacteria, 2 Dermatophytes (Table 5), 3 yeasts (Can-

dida albicans, Candida tropicalis resistant to poly-enic structures, Cryptococcus neoformans), 5 phy-

topathogens (Colletotrichum mL$ae, Drechslera spi-

cifera, Fusarium oxysporum, Geotrichum candidum,

Pyricularia oryzae), 4 entomopathogens (Beauveria

bassiana, Beauveria brongnartii, Metanhizium aniso-

Table 1. Dimensions and frequency of conidia ofExserohilumsodomii (fuom the observation of hundred conidia)

Number of Number of Length (g,m) Width (pm)

\cpla conidia

il'7

6

5

4

2

62-67

46-65

47-52

304429-31

t7 22

14-20

1.4-1'7

7t-171.t-1.7

1 1-14

pliae, Verticillium lecanii). Assessment of antimicro-

bial activity of crude extracts was performed using the

disk diffusion method previously described (Okeke et

aL.,1992).

Poly sacchar ide prod uction

Presence of extracellular polysaccharides in culture

medium was researched at 30 oC according to Aouadi

et al. (1992). The strain was grown in the malt and syn-

thetic media under static (7 days) and shaking (5 days)

conditions. Ifpresent, precipitation of the polysaccha-

rides was obtained by adding 2Vo NaCl5 M and 1.5

volume of absolute ethanol to the f,ltered culture medi-

um. Both media were also tested without fungus.

Results and discussion

Exserohilum sodomii Guiraud, Steiman, Seigle-

Murandi & Sage, sp. nov. (Figures 1A-D).Coloniae (in PDA-chloramphenicol) griseae vel

brunneae, floccose, adversae nigrae. Hyphae ramosae,

septatae, brunneae, laeves, 4.88-6.00 pm diam. Coni-

diophora macronemata, mononemata , erecta, flexuosa,

simplicia, cylindrica ad basim et geniculata ad apicem,

septata, brunnea, laevia, usque 1000 pm alta, 4.80-7.32 pm diam., pauci dilatare ad apicem, 1-8 conidia

portare (usque 8 cicatricis). Conidia clavata, leviter

attenuata ad basim et rotunda ad apicem, 41-65 x1.4-20 p,m, sicca, brunnea, laevia, septata brunnea ad

basim et ad apicem,4-5 pseudoseptata; hilo manifesto,

1..5--2.5 pm diam. praedita est. Germinatio unipolaris

vel bipolaris. Chlamydospora rotunda vel ovata.

Holotypus: ex tera separatus, Dead Sea (Sodom

road), Neguev desert, Israel, August 1994, CMPG

1340. In fungi herbarium, Grenoble, France.

10

40

25

30

5

5

319

Figure 1. Exserohilum sodomii (A) Mycelium, conidiophores and conidia, 600x. (B) Bipolar germination of conidia, 1500x. (C) Conidia withsepta, pseudosepta, and hilum, 600x. (D) Conidia with septa, pseudosepta and hilum, 1500x.

c

,,&;;

,.,1

)r,.*.

'.@,, Y:-'**, 'q, ,: tÿ.

.&,' U. ,.,o.lb §ta 's .-b€'"6\-: ?.1

320

90

FBO

>^ 70c360oo50o40§sos)

Eeoi5 to

0

0 3 6 912 15

Days

0 3 6 912 15

Days

FigLtre2.GrowthofErserchilumsotlomiiondifferentsolitlmecliaasafunctionofthetemperaturefC): -f-24oC-a-30oC-^ 37oCA: malt extract medium; -B: potato dextrose agar medium; C: sl,nthetic medirlml D: hlperosn-rotic medium.

90

Êeo()* 70

tooro 50o40

*roL

Ezo6.,u

0

)-§so

5aooo*30o

#rot§

610

TI*i-

_l*.IIit,l

1t'i

LIl

-tI

+l,

L ^^o uu

c )uo

o 4U

o30L0,

9zo.go 10

0

321

Table 2. Morphological comparison bctrveen Exserohihun sodomii, Erserohilum Llrzeli (Steiman et al., in pü:ss),Exsenhilumrostratum and Exserohilum gedarelense (Leonard, 1976; Sivanesan, 1987)

Exserohilumsodomii Exserohilumisraeli ExsenhiLum gedareJen.se Erserohilnn rostralLtm

Hyphae verrucose

Conidiophore

length

width

septate

branchecl

geniculate

color

Conidia

aspect

dimensions

dist()septLrte

color

hilum sizc

no

up to 10tX) pm

4.8-1.32 trLm

upper part enlarged

yes

no

slightly

dark brown

straight and clavate

never rostrate

,+1-65 x 1;l 2t) pm

2 to 8 (mostl.v 5-7)

brown

1.5 2.5 pm

slightll,

up to 610 pm

3.5 pm base

5 6 /rm upper part

yes

sometimes

yes

brown

straight

never roslrate

-51-88 x 12 18 pm

2 to 10 (mostly 7-10)

brorvnr i I i ,,m

no

up to 570 trrm5.0-7.-5 pm

yes

no

yes (above)

brown to micl brown

paler towards the apex

straight and clavatc

broadest near apex

2-5-100 x 13-28 pnr

I to 7 (sometimes all

thick and dark)

n.rid brown

protuberant

no

up to 200 /rnr6 81rm

,ves

no

yes

olivaceous-brown

straight rc slightl-v

curved

15-200x1 2911m

Ito1u

brou n or olivaceous

protuberant

hble 3. Com:position in Na, Ca, Cl. K, Mg and P of the studied soil sample ancl some

natural media (parts per million)

pHMg

Soil san'rple traces

Dcad Sea rvater* 30820

Hcarth crust'r'* 2830t)

Sea water*+ 10561

120E00 6300 3600 1400 5.0

185310 6912 35186 / 6.6

314 2-5900 20900 1 180 I18980 3E0 1272 < 0.1 i

62200

1 8240

36300

100

- Values liom a previous rvork (Steirran et al.. 1995).*- Values lrom CRC Hanclbook of Chemistry and Phl,sics (1996-1997).

Colonies (on PDA-chloramphenicol medium)greyish brown, floccose, reverse black. Accord-ing to the Methuen Handbook (Kornerup & Wan-

scher, 1978) color codes were: mouse grey (5E3)for aerial mycelium, and negro (6F3) for appressed

mycelium. Colonies were more dense on PDA than

on MEA. Hyphae branched, septate, dark brown,smooth-walled, 4.88-6.00 pm wide. Conidiophoresmacronematous, mononematous, upright, flexuous,

unbranched, slightly geniculated above, cylindricalbelow, septate, dark brown, smooth-walled, up to1000 pm tall, 4.80-7.32 p,mdiameter, slightly enlarged

in the upper geniculated part, bearing 1-8 conidia.Conidia clavate, rounded at the apex, and slightly atten-

uated to the base, dry, brown, smooth, 41-65 x 14-20 p,m, with a distinctly protuberant hilum, diameter:

I.5 to2.5 /rm. Two darkbrown septa to the base and the

apex and pseudoseptate,average 5 to 7 (Table 1). Ger-

mination unipolar or bipolar. Chlamydospores present,

globose to oval, intercalary or at the apex ofhyphae.Holotype: from soil, Dead Sea (road of Sodom),

Neguev desert, Israel, August1994, CMPG 1340. Sub-

cultures of CMPG 1340 have been deposited with the

American Type Culture Collection (ATCC).Exserohilum are graminicolous species and this

genus was segregated from Helminthosporium byLeonard & Suggs (1974) because of the conidial ger-

mination from one or both polar cells and the protu-berant hilum present, the later feature constituting the

main difference with the genus Blpolarls (Shoemaker,

lese).Our isolate was classified into this genus but it

did not correspond to any species described up tonow (Leonard & Suggs, 7974; Leonard, 1976; Llt'

3ZZ

t'il

trell, 1977; Muchovej & Ribeiro-Nesio, 1987; Alcorn,1983, 1985, 1988a, b, 1991; Muchovej et al., 1988;

Sivanesan, 1987, 1992, 1 993 ; Kubatov a, 199 1; U pad-

hyay & Mankau, 1991; Khasatov, 7992; Lacicowa,1993; Castafleda Ruiz et al., 1995; Shahzad & Ghaf-Êar, t995). This species was isolated from a soil sam-

ple taken along the road to Sodom in Israel and was

named Exserohilum sodomii. If compared to Exsero-

hilum israeli, an other recently isolated new species

of Exserohilurz (Steiman et al., in press), it can be

noticed that colours of colonies of E. sodomil weregreyish brown with black reverse versus olivaceous

brown with olivaceous brown reverse for -8. israeli.Moreover, E. sodomii showed longer conidiophores,

never branched, hyphae were never verrucose, conidiawere shorter and clavate and this species was unable

to grow at 45 o C.Table 2 gives the main features of E.

sodomii and the closest related species.

Proteases

Lipases

Figure 3. Extracellular enzvmatic actir,ities in Exserohilum sodontii as a function of the temperature o1'growth.

Soil analysis revealed traces of Na, 62.2 mglgCa (1.68 mmoVg), 120.8 mÿg Cl (3.40 mmoVg),

6.3 mÿg K (0.17 mmol/g), 3.6 mglg Mg (0.1a

mmoVg), 1.4 mÿg P (0.044 mmol/g). The pH of a

soil suspension in distilled water was 5. If compared tovalues conceming some natural media (Tâb1e 3), thissample was rich in Ca and Cl and very poor in Na.

For Ca and R values obtained were close to that foundin heart crust, levels of Cl and K were close to that

found in the Dead Sea, while the Mg level was 3 times

higher than in sea water and 6 to 8 times lower than

in other media. The low pH was in favour of fungaldevelopment.

Four media were used to investigate the influenceof temperature on the growth of E. sodomij: MEA,PDA, hyperosmotic, synthetic (Figures 2A-D). The

best growth rate was observed on MEA and PDA. On

PDA colonies were more densely fructified. The high-

Arbitrary unit

1,8

1,6

1â

1,2

I

0,8

0,6

o,2

0

Amylases

323

Table 4. Extracellular phenoloxidase activity inExserohilum sodomii grownon two media for 8 days

at various temperatures

Medium Potato dextrose agar Hyperosmotic

Specif,city Reagents 220C 30 0C 37 c 22"C 30 0C 37 aC

Phenoloxidases

Mostly laccases

or peroxidases

Laccases

Peroxidases

Tyrosinases

Gallic acid

Pyrogallol

R56

o-Anisidine

Benzidine

Guaiacol

o-Naphtol

Syringaldazine

Syringaldazine +

H:O:p-Cresol

Tvrosine

Total

3+ 5+

3+ 4+

5+ 5+

3++4+ 5+

3+ 5+

4+ 5+

5+ 5+

+l- +

0030.5 36.0

2+ -J+

2+ 5+

00

5+ 5+

5+ -5+

000t)0000

0000

14.0 18.t)

3+

5+

5+

+l-

+

5+

5+

0

0

4+

0

28.5

+

+

+

0

+

0

+l-

+

0

+

5.5

Table 5. Toxicity of crude extracts from 7-day old culture

in liquid synthetic and malt extract media undcr shaken or

static conditions of ExserohiltLm sodomlz tolvards some human

pathogenic dermatophytes and bacteria

Dermatophytes Bacteria

I2

3

4

16

1.4

9

t2

20

15

10

13

0

9

t)

9

t416

15

22

ltlg: Microsporum gypseum; Tm: Trichophyton mentagro'

phytes; Flc: Escherichia coli; Pa: Pseudomonas aeruginosa;

Sa: Staphy lococcus aureus.

1 : Synthetic shaken medium; 2: rnalt exîact shaken medium;

3: synthetic static medium; 4: malt extract static medium.

Results are means of three determinations and are given as

inhibition diameters (mm) of the growth of target strains when

using the disk diffusion method (diameter of the disk: 6 mm).

Inhibition diameters were recorded after 2-3 days of incuba-

tion at 37 o C.

est growth rates were obtained at 22 and 30 oC on

PDA and MEA, while it was lower at37 "C. However,

high temperatures stimulated the growth on hyperos-

motic medium; better results were obtained at 30 oC

aîd37 "C than at24 "C (Figure 2). E. sodomii did not

develop at 45 " C, only germination occurred.

Concerning extracellular e îzyrr;.atic acliYities, phe-

noloxidases, amylases, Iipases and proteases were

detected (Figure 3 and Table 4). The species did not

produce extracellular cellulases. The intensity ofphe-

noloxidases production or activity depended both on

the growth medium used and temperature of growth.

Peroxidases were never detected when using syringal-

dazine and HzOz. On PDA, specific laccase aÇtivity

(evaluated by syringaldazine) increased with tempera-

ture. This was accompanied by a higher reactivity ofgallic acid and benzidine while the responses obtained

with R56 and a-naphtol were maximal at 24 "C, all

being general Pox reagents. Concerning tyrosinases,

a strong positive reaction was obtained at 24 'C withp-cresol, while the second specific reagent, tyrosine,

did not react. It \À/as previously reported that some

fungi reacted with both reagents while others only

with one of them, showing that more than one type

of tyrosinase exist (Rahouti et a1.,1995). The decrease

of tyrosinase activity when increasing the temperature

of growth was only reflected by the response record-

ed with p-cresol and not by the general Pox reagents,

suggesting that these substrates may be more indica-

tive of non-tyrosinase POx. The hyperosmoticmediumwas strongly inhibitory for POx production (no specificresponse for laccase or tyrosinase), however the global

POx activity also increased as a function of the temper-

ature but the optimum was at 30 oC instead of 37 "C.Owing to these observations it may be emphasized that

extracellular Pox activity of this strain may be partly

inhibited in its natural environment, however a good

expression of this activity was recovered when growing

E. sodomii under low osmolar conditions.

EcMg

22

24

24

324

On the whole, an optimal activity was recordedat 37 oC for phenoloxidases, lipases and amylases,although the optimal growth temperature was 30 "C.Protease activity was not influenced by the tempera-ture.

Extracellular polysaccharides were produced bothin malt and synthetic media. The method used gave

only qualitative results, however, the production wasobviously more abundant in malt-extract medium.Controls performed on culture media without funguswere negative. Few data concerning polysaccharidesproduction by species of Exserohilum are available, E.israeli was also a producer of these substances. On thewhole, it is known that species of related genera suchas Drechslera or Bipolaris may be good producers ofpolysaccharides (Aouadi et al., 1992:' Steiman et al.,

tee6).Antimicrobial activity was observed when testing

crude extracts from culture media ofE. sodomii againstpathogenic bacteria and dermatophytes (Table 5). Onthe other hand, these extracts were inactive against theyeasts. The culture conditions did not greatly influ-ence the results. Concerning Entomopathogens and

Phytopathogens, extracts were inactive against Drech-slera spicifera, Fusarium oxysporum and Pyriculariaoryzae, while a slight activity was obtained againstthe other strains only with crude extracts from shaken

culture conditions (MEA and synthetic media).To conclude, a new species of Exserohilum has

been isolated from a desert saline area in the southof Israel. This is the second new taxon in this genuscoming from Israel and isolated in our laboralory. E.sodomii was considered as a thermotolerant species

able to resist (germinate) at 45 "C, and developingwell at 37 oC mostly in a saline medium (hyperosmot-ic). Moreover, some of its physiological potentialities,such as erzymaticactivities increased with the temper-ature of growth.

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