J Hattori Bot. Lab. No. 90: 307-314 (July 2001)

SCREENING OF HALOPHILIC OR SALT TOLERANT LICHEN MYCOBIONTS CULTURED ON SODIUM CHLORIDE

ENRICHED MEDIA

YOSHIKAZU YAMAMOT0 1*, TOSHIKAZU TAKAHAGI2, FUMIHIKO SAT02,

YASUHIRO KTNOSHITA3, HIROYUKI NAKASHIMA4

,

and ISAO YOSHIMURA5

ABSTRACT. Halophilic or salt tolerant lichen mycobionts were screened for 77 species on an NaCl enriched medium. Only the Niebla homalea mycobiont collected from the North American Pacific seashore showed evident halophilic growth: 145% growth on malt-yeast extract agar-medium supple­mented with 0.9 M NaCl and 129% growth at 1.2 M NaCl in comparison with that of the zero NaCl medium. In addition, mycobionts of Arthonia cinnabarina, Chaenotheca brunneola, Caloplaca scop­ularis, Ramalina subbreviuscula and Vermilacinia combeoides harvested from seashore showed rela­tively better growth on NaCl enriched medium; however, some mycobionts are sensitive to salt. These selected mycobionts were also tolerant to KC! and glycerol added.

INTRODUCTION

Lichens grow in extreme environments such as polar regions, hot arid deserts, seashores, and high mountains. Many investigations have studied influences of extreme en­vironments on lichens in nature (see Kappen, 1973), but we have limited information of stress mechanisms in lichens. Generally, many of environmental stresses are caused by an excess of water. Salinity stress is a good model system to evaluate water stress in lichens. Ramkrer (1978) and Takahagi et al. (2000) reported effects of salinity on spore germination and consequent hyphal penetration. Watanabe et al. (1997) showed that photobionts isolat­ed from maritime lichens are salt tolerant. However, the growth of lichen mycobiont under salinity stress conditions has not been investigated in vitro.

The mechanism of salt tolerance in lichens is unclear. Two defensive mechanisms to high salt are reported for yeasts, namely, osmotic equilibration (Adler et al. 1985) and reg­ulation of ion distribution (Rodriguez-Navarro et al. 1994, Watanabe et al. 1991). Lichens are symbiotic associations of fungi and algae; therefore, it is thought that they have unique

1 Department of Biological Production, Faculty of Bioresource Sciences, Akita Prefectural Uni­versity, Akita 0I0--0195, Japan.

2 Department of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Sakyo, Kyoto 606-8502, Japan.

3 Basic Research Department, Nippon Paint Co., Ltd., 4-1- 15, Minamishinagawa, Shinagawa, Tokyo 140-8675, Japan.

4 Department of Biotechnology, Kurume Technological College, Komorinocho 1232, Kurume, Fukuoka 830-8555, Japan.

5 Kochi Gakuen College, Asahitenjincho 292, Kochi 780-0955, Japan. * The author to whom correspondence should be sent.

308 J. Hattori Bot. Lab. No. 90 2 0 0 I

tolerance mechanisms for extreme environments. To elucidate this, we investigated the ef­fect of the addition of NaCl on the growth of mycobionts, and characterized the tolerance of isolated mycobionts using other salts and glycerol to distinguish the tolerance to ion dis­turbance and osmotic potential induced by NaCL Here we report the isolation of halophilic and salt tolerant mycobionts of lichens which can grow on NaCl enriched media and their characteristics.

MATERIALS

Seventy-seven species of lichen mycobionts used to evaluate halophilicity and salt tol­erance (Table l) were induced from spores or thalli of lichens collected worldwide by the modified Ahmadjian's method (Ahmadjian 1973, Yamamoto et al. 1998) and the Yamamo­to's method (Yamamoto et al. 1985). Twenty-one specimens marked with an asterisk (Table l) were collected from maritime areas. Air-dried specimens were stored in Akita Prefectur­al University. Spore-derived mycobionts as well as thallus-derived cultures have also been maintained in the culture collection of Akita Prefectural University.

METHODS

Five pieces (total fresh weight, 30±5 mg per dish) from growing mycobiont cell-ag­gregates were aseptically inoculated onto 5 ml of the malt-yeast extract (MY) medium (Ah­madjian 1961) supplemented with various concentrations of NaCl, KCI, Li Cl or glycerol in a Petri dish (60 mm in diam.). NaCl at concentrations of 0, 0.6 and 1.2 M were used for the fist evaluation and several chloride salts of 0, 0.3, 0.6, 0.9 and 1.2 M and glycerol of 0, 0.6, 1.2, 1.8 and 2.4 M were used for the characterization of tolerant mycobionts. Three dishes each with five inocula were incubated at l 5°C in the dark for 3 months, growing myco­bionts were harvested, and their total weights were measured. The growth ratio (total weight of harvest/total weight of inoculum) and the relative growth value [(the growth ratio of culture on the medium with treatment - l) X l 00 I (the growth ratio without treat­ment - l)] were calculated. Mycobionts with more than ten or one hundred relative growth values on 0.6 or 1.2 M NaCl medium were judged to be salt tolerant or halophilic, respec­tively.

RESULTS

Seventy-seven species of lichen mycobionts were examined for their salt tolerance (Table 1). Among 21 maritime lichens tested, 16 species (76%) on the MY medium en­riched with 0.6 M NaCl and six species (29%) on the medium enriched with 1.2 M NaCl showed salt tolerance for mycobiont growth (Table 1). Thirty-six species on the medium enriched with 0.6 M NaCl and six maritime species (Arthonia cinnabarina, Chaenotheca brunneo/a, Calop/aca scopularis, Niebla homalea, Ramalina subbreviuscula and Vermi­/acinia combeoides) on the medium enriched with 1.2 M NaCl showed more than ten for the relative growth value (Fig. 1 ). Only the mycobiont of N homa/ea showed a superior growth (relative growth= 145% on the medium supplemented with 0.9 M NaCl and relative growth = 129% on the medium supplemented with 1.2 M NaCl), thus we concluded that N homalea mycobiont is halophilic (Fig. 2).

Y. YAMAMOTO et al.: Screening of Halophilic or Salt Tolerant Lichen Mycobionts 309

Table I. Growth of mycobionts of maritime(*) and non-maritime lichens cultured at l 5°C for 3 months on MY agar-medium containing 0, 0.6 or 1.2 M NaCl.

Growth Ratio (GR) Relative GR Species Locality

OM 0.6M 1.2 M OM 0.6M 1.2 M

Acarospora chlorophana Tome Lappmark, Sweden 7.84 3.49 1.18 JOO 36 3 Alectoria ochroleuca Tome Lappmark, Sweden 3.52 I.OJ I.OJ JOO 0 0 Arthonia cinnabarina \Vakayama,Japan* 13.18 5.73 3.01 100 39 17 Asahinae kurodakensis Fukushima, Japan 8.84 1.06 0.98 100 I 0 Baeomyces fungoides Fukushima, Japan 4.03 0.95 0.95 100 0 0 Bryoria smithii Hawaii, USA* 2.65 1.07 1.10 100 4 6 Buellia sp. Ishikawa, Japan* 6.64 3.24 0.99 100 40 0 Ca/icium japonicum Okayama, Japan 8.22 4.08 1.01 JOO 43 0 Caloplaca scopularis Hokkaido, Japan* 9.08 5.65 2.43 JOO 58 18 Caloplaca sp. \Vakayama, Japan* 3.00 1.06 0.87 100 3 0 Chaenotheca brunneola Hawaii, USA 8.14 6.88 1.99 100 82 14 Cladia aggregata Kyoto, Japan 5.49 0.84 0.96 100 0 0 Cladonia bacilioformis Oulun, Finland 9.46 1.09 0.99 100 I 0 Cladonia cristatella North Carolina, USA 3.02 1.03 1.07 100 2 4 Cladonia subpityrea Kochi, Japan 8.07 5.18 1.00 100 59 0 Cladonia vulcani Aomori, Japan 6.89 3.50 1.01 100 42 0 Dermatocarpon British Columbia, Canada 7.29 2.29 0.87 JOO 21 0

miniatum Dermatocarpon British Columbia, Canada 5.76 3.12 1.00 100 45 0

reticulatum Dibaeis absoluta Kyoto, Japan 5.46 0.93 0.90 JOO 0 0 Evernia esorediosa British Columbia, Canada 4.77 1.08 1.02 100 2 I Flavocetraria nivalis British Columbia, Canada 9.04 2.31 0.97 100 16 0 Graphis cervina Kyoto, Japan 12.48 8.97 1.03 JOO 69 0 Graphis cognata \Vakayama,Japan 7.71 6.22 1.00 100 78 0 Graphis proserpens Okayama, Japan 9.56 0.93 0.96 100 0 0 Graphis tenella Kyoto, Japan 11.47 7.70 0.99 100 64 0 Hypogymnia vittata \Vakayama,Japan 6.62 1.12 0.89 JOO 2 0 Icmadophila ericetorum Oulun, Finland 3.33 0.79 0.94 JOO 0 0 Lasallia papulosa Tennessee, USA 9.81 0.98 0.90 100 0 0 Lasa/lia pennsylvanica Aomori, Japan J0.66 3.75 0.98 100 28 0 Lecanora argopholi Kuusamo, Finland 9.73 1.02 I.OJ JOO 0 0 Lecanora expectans South Pole 6.04 1.38 1.13 100 7 3 Lecanora muralis Hokkaido, Japan* 3.26 2.78 1.01 100 79 0 Letharia columbiana California, USA 3.53 1.09 1.13 JOO 3 5 Menegazzia terebrata \Vakayama, Japan 5.42 1.02 0.88 JOO 0 Myelochroa crassata Kochi, Japan 3.26 1.32 0.85 JOO 14 0 Nephromopsis ornata Tochigi, Japan J0.16 1.28 0.97 JOO 3 0 Niebla homalea California, USA* 4.33 6.54 5.59 JOO 166 138 Ochrolechia trochophora Yamanashi, Japan 2.53 1.05 0.93 100 3 0 Parmelia \Vakayama,Japan 7.05 1.42 0.86 JOO 7 0

pseudoshinanoana Parmelia saccatiloba Tokyo, Japan• 7.43 1.63 1.06 JOO JO

310 J. Hattori Bot. Lab. No. 90 2 0 0 I

Table 1. (Continued.)

Growth Ratio (GR) Relative GR Species Locality

OM 0.6M 1.2 M OM 0.6M 1.2M

Foraminella ambigua Uusimaa, Finland 6. J3 1.02 0.98 JOO 0 0 Parmotrema Hiroshima, Japan 3.30 1.07 1.00 JOO 3 0

austrosinense Parmotrema Tokyo, Japan* 5.62 4.30 0.99 JOO 72 0

parahypotropa Parmotrema Tokyo, Japan* 7.35 2.03 1.02 JOO J6 0

praesorediosa Phaeographis exaltata Kyoto, Japan 7.16 1.24 1.04 JOO 4 J Platismatia glauca Oulun, Finland 4.16 1.09 0.97 100 3 0 Porpidia Kyoto, Japan 11.47 7.22 0.94 100 59 0

albocaerulescens Pseudephebe pubescens British Columbia, Canada 6.45 1.15 0.87 100 3 0 Rama/ina boninensis Tokyo, Japan* 3.44 1.74 1.20 100 30 8 Ramalina crassa \¥akayama,Japan* 3.49 2.69 1. 10 100 68 4 Ramalina exi/is Tokyo, Japan• 4.19 1.28 0.92 100 9 0 Ramalina peruviana Kyoto, Japan 4.92 3.32 0.95 100 59 0 Ramalina litoralis \¥akayama,Japan* 2.91 2.65 1.00 JOO 86 0 Ramalina roesleri Hokkaido, Japan* 5.29 2.26 1.02 JOO 46 1 Ramalina subbreviuscula Aomori, Japan* 5.56 3.94 1.48 100 65 11 Sphaerophorus fragilis Nagano, Japan 10.47 6.96 0.83 100 40 0 Sphaerophorus globosus British Columbia, Canada 7.18 1.21 1.12 100 3 2 Sphaerophorus \¥akayailla,Japan 5.79 1.07 1.00 100 0

melanocarpus Stereocaulon alpinum Oulun, Finland 5.8J 0.94 0.96 100 0 0 Stereocaulon Oulun, Finland 4.6J 0.89 0.87 100 0 0

subcoralloides Stereocaulon vesuvianum Oulun, Finland 5.84 1.15 1.05 JOO 3 1 Teloschistes jf.avicans Hawaii, USA* 4.66 1.59 1.02 100 16 I Thamnolia vermicularis Nagano, Japan 4.64 0.96 0.88 100 0 0 Tremolecia atrata British Columbia, Canada 3.54 0.99 1.0 1 100 0 0 Tuckermannopsis Oulun, Finland 6.41 2.07 0.88 100 20 0

sepincola Umbilicaria caroliniana Aomori, Japan 10.18 3.74 0.99 100 30 0 Umbilicaria cylindrica British Columbia, Canada 11.01 2.90 1.36 100 J9 4 Usnea australis Hawaii, USA* 6.67 1.00 1.01 100 0 0 Usnea bismolliuscula Nagano, Japan 6.67 1.07 l.10 100 J 2 Usnea fiexilis Saba, Malaysia 9.7J 1.16 0.94 100 2 0 Usnea rubescens Kyoto, Japan 4.34 1.07 1.05 100 2 2 Usnea rubicunda \¥akayama, Japan 4.36 1.02 0.95 100 1 0 Vermilacinia combeoides California, USA* 5.14 3.45 2.11 100 59 27 Verrucaria sp. \¥akayama, Japan* 2.86 1.83 l.10 100 45 5 Vulpicida juniperinus Hokkaido, Japan 3.43 1.06 1.07 100 3 3 Xanthoria canadensis British Columbia, Canada 9.05 1.22 1.04 100 3 0 Xanthoria mandschurica Hokkaido, Japan* 4.00 2.51 0.94 100 50 0

Y. YAMAMOTO et al.: Screening of Halophilic or Salt Tolerant Lichen Mycobionts 311

Furtheremore, cultured mycobionts of four lichen species, Arthonia cinnabarina, Chaenotheca brunneola, Niebla homalea and Vermilacinia combeoides, all of which showed salt tolerance in the first evaluation, were studied for the effects on their growth of various concentrations of KC!, LiCI and glycerol (Fig. 2). The effect of KC! is similar as that of NaCl and addition ofLiCI to the medium completely damaged the growth. Addition of relatively low concentrations of glycerol promoted growth of A. cinnabarina, C. brun­neola and V. combeoides; however, high concentrations inhibited their growth. The growth of the N. homalea mycobiont was accelerated by the addition of glycerol in all tested cases.

DISCUSSION

Screening of Salt Tolerance of Cultured Lichen Mycobiont From the first step in screening salt tolerance for 77 cultured lichen mycobionts, only

Niebla homalea, a halophilic mycobiont, and five mycobionts (Arthonia cinnabarina, Chaenotheca brunneola, Caloplaca scopularis, Ramalina subbreviuscu/a and Vermilacinia combeoides) showing the salt tolerant property were collected at seashore. Ramkrer (1978) also found maritime Caloplaca marina as a halophilic species based on the observations of spore germination on an enriched salinity medium. Among 25 maritime lichens tested, 76% at 0.6 M NaCl and 29% at 1.2 M showed salt tolerance (Table 1 ). Takahagi et al. (2000) obtained a similar result, the spores of half of the tested maritime lichens germinating at 0.6 M NaCl. These results indicate that not all mycobionts of maritime lichens have a salt tolerant property. On the other hand, Watanabe et al. (1997) observed good or excellent growth at ea. 0.5 M NaCl of all photobionts derived from tested maritime lichens. It is as-

200

..c: 150 ~ 0 ~

~ Q)100 > ·~ a:; c:: 50

0

·· O· ·Nthonio~

-c.i.i-.,,.,,,,,.,;.

-~-··O··--~ RMIMlilM •ubbr.viuaoula -- '6 -- V~~.

0

... 13 . . . ...... •.

0.6 NaCl Cone. (M)

1.2

Fig. I. Effects of NaCl concentration on the growth of six salt tolerant lichen myco­bionts cultured on MY medium at I 5°C in the dark for 3 months.

312 J. Hattori Bot. Lab. No. 90 2 0 0 I

sumed that maritime lichens whose mycobionts show a salt sensitive property overcome saline environments by structural tolerance mechanisms based on their symbiotic associa­tion with algae. Comparison of Tolerance Properties between Salt Tolerant Mycobionts

All of the four lichen species, Arthonia cinnabarina, Chaenotheca brunneola, Niebla homalea and Vermilacinia combeoides, which showed remarkable salt tolerance in the first evaluation, showed similar effects to KCl and glycerol on their mycobiont growth as those of NaCL Additional LiCl (0.3 M) perfectly inhibited the growth of A. cinnabarina and N.

homalea mycobionts. On the other hand, growth promotion of A. cinnabarina, C. brunneo­la and V combeoides mycobionts by the addition of low concentration of glycerol (0.6 M) and a N. homalea mycobiont in all cases of the additional glycerol may indicate that myco­bionts metabolize glycerol or use it as osmoprotectants (Fig. 2). This probably shows that

(A) Arthonia cinnabarina ~NaCl - KCI _._ LiCI - Glycerol

150

..c

1 100 .... CJ

~ ·~ 50 a; c:::

0 0 2 3

Concentration

(C) Vermilacinia combeioides ~NaCl - KCI

150 - Glycerol

..c

1100 ....

" CD > ~ 50 a; c:::

0 0 2 3

Concentration

4

4

(B) Chaenotheca brunneo/a

150

..c

1100 .... CJ

CD >

·~ 50 a; c:::

0

~NaCl - KCI

- Glycerol

0 2 3 Concentration

(D) Niebla homa/ea

~NaCl - KCI

4

-.-uc1 -Glycerol 150

..c

1100 ....

" CD > ·~ 50 a; c:::

0 0 2 3 4

Concentrat ion

Fig. 2. Effects of various concentrations of NaCl, KCI, LiCI and glycerol on the growth of four salt tolerant lichen mycobionts cultured on MY medium at I 5°C in the dark for 3 months. Concentrations were 0 (0), 0.3 (1), 0.6 (2), 0.9 (3) and 1.2 (4) M for NaCl , KC! and LiCI, and 0 (0), 0.6 (1), 1.2 (2), 1.8 (3) and 2.4 (4) M for glycerol.

Y. YAMAMOTO et al.: Screening of Halophilic or Salt Tolerant Lichen Mycobionts 313

such mycobionts can survive in a high osmotic condition. It is well known that natural lichens accumulate lichen substances such as sugar alcohols [arabitol, mannitol and ribitol] (Huneck & Yoshimura 1996) and amino acid betaines [glycine betaine (Chapman et al. 1994), sticticin (Bernard et al. 1980) and solorinine (Matsubara et al. 1994)]. However, it is not clear whether these substances really act as compatible solutes in lichens or not.

Many lichen species adapt themselves to extreme environments and the mechanisms of the tolerance to environmental stresses are poorly known. It is important to clarify stress-related receptors and enzymes, as well as their genes, in halophilic lichen myco­bionts such as Niebla homalea. Further research on halophilic and salt tolerant lichen my­cobionts would reveal the mechanisms involved, which might be quite different to those in salt tolerant yeasts and fungi.

ACKNOWLEDGMENTS

We thank Mr Stephen Sharnoff and the late Mrs Sylvia Sharnoff for their kind help in supplying Niebla homa/ea and Vermilacinia combeoides at our request, and Prof. M. Nakanishi of Okayama Science University, Dr H. Miyawaki of Saga University and Dr G. Thor of the Swedish University of Agricultural Sciences for their identifications of speci­mens.

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