全 文 ：Research on Liquid Culture of Mycobiont and
Photobiont Isolated from Cladonia pyxidata
SUMin1
, WEI Jiang-chun1 ,2
(1.College of LifeScience , Hebei University , Baoding 071002 , China;2.Key Laboratory of Systematic
Mycology &Lichenology , Institute of Microbiology , Chinese Academy of Sciences , Beijing 100101 ,
China)
Abstract:This paper deals with the cell growth of both the mycobiont and photobiont of Cladonia pyxi-
data collected from China in liquid culture.The experiments results showed that the better conditions of
mycobiontic growth were cultured by using liquid Lilly-Barnett medium containing 40 g/L inositol as a
carbon source , 2 g/L L-glutamine as a nitrogen source , adjusting pH to 7.0 before autoclaving , and in-
cubating cultures at 20℃.The better conditions of photobiontic growth were cultured by using liquid
Bold s basal medium containing 160 g/L glucose as a carbon source , 1.75 g/L NaNO3 as a nitrogen
source , adjusting pH to 5.0 before autoclaving , and incubating cultures at 20℃.
Key words:lichen;Cladonia pyxidata;mycobiont;photobiont;liquid culture
CLC number:Q932　　　Document code:A　　　Article ID:1672-3538(2008)01-0057-06
喇叭石蕊共生菌藻液体培养的研究
苏 　敏1 , 魏江春1 , 2＊＊
(1.河北大学生命科学学院 ,保定 071002;2.中国科学院微生物研究所真菌地衣系统学重点
实验室 ,北京 100101)
摘　要:对喇叭石蕊共生菌 、藻液体培养条件进行了研究。结果表明:共生菌生长在以 40 g/L 肌醇为碳源 、
2 g/ L L-谷氨酰胺为氮源 、起始 pH值为 7.0 的 LB 液体培养基中 ,培养温度为 20℃时表现最佳。其共生藻的生
长在以 160 g/L葡萄糖为碳源 、1.75 g/ L NaNO3 为氮源 、起始 pH 值为 5.0 的 BBM 液体培养基中 ,培养温度为
20℃时表现最佳。
关键词:地衣;喇叭石蕊;共生菌;共生藻;液体培养
中图分类号:Q932　　　文献标识码:A　　　文章编号:1672-3538(2008)01-0057-06
　　Biological soil crusts result from an intimate asso-
ciation between soil particles and cyanobacteria , algae ,
microfungi , lichens , and bryophytes (in different pro-
portions)which live within , or immediately on top , the
uppermost millimeters of soil.Soil particles are aggre-
gated through the presence and activity of these biota ,
and the resultant living crust covers the surface of the
ground as a coherent layer[ 1].Such structure of the the
biological soil(microbiotic crusts)enable them to with-
stand the erosive forces of wind and water.The micro-
biotic crusts are distributed in the arid and semiarid
desert zones of NW China.The project on the culture
conditions of mycobionts and photobionts isolated from
Cladonia pyxidata collected in microbiotic crusts from
Shaanxi is one of the feasibility studies for artificial in-
oculation in desert.

Corresponding author:WEI Jiang-chun, E-mail:weijc2004@126.com
Biography:SU Min(1976-), female , master degree candidate , studying on microbiology.
Received date:2007-06-18
　第 6卷第 1 期 菌　物　研　究 Vol.6 No.1　
　2008 年 3月 Journal　of　Fungal　Research Mar.2008　
1　Materials and Methods
1.1　Materials
Cladonia pyxidata (L.)Hoffm.(Fig.1-2)was
collected by Su M.on the microbiotic crusts , Mt.
Duntan shan , Ansai , Shaanxi on 2006-11-11(HMAS-
L-109927).The mycobiont and photobiont examined
were isolated from C.pyxidata using Yamamoto s cul-
ture method[ 2].
Fig.1.The habit of Cladonia pyxidata examined 　　　　　　Fig.2.A scyphus of Cladonia pyxidata examined
1.2　Methods
1.2.1　DNA extraction　The total DNA of C.pyxi-
data was extracted from both the lichen thallus and the
axenic cultures of its mycobiont and photobiont using
Lee &Taylor′s method[ 3] modified by us as follows:
Transfer water-phase extract to SGM (silica-gel-mem-
brane)mini-column inserted a collection tube , add
binding-solution , washing-solution and eluting-solution
to extract DNA.
1.2.2 　PCR amplification and sequencing 　The
primers using to amplify the nrDNA ITS region(ITS1 ,
5.8S , and ITS2)of the mycobiont from C.pyxidata:
E9 is -TTGTACACACCGCCCGT-, and SL4R is -TTC-
GATCACTCTACTTGTGC-.The primers using to am-
plify the nrDNA ITS region (ITS1 ,5.8S , and ITS2)of
the photobiont from C.pyxidata[ 4] :nr-SSU-1780-5′
is -CTGCGGAAGGATCATTGATTC-, and nr-LSU-
0012-3′is -AGTTCAGCGGGTGGTCTTG- Amplifica-
tions were performed in 50 μL reactions [ H2O
34.7μL;MgCl2 4 μL;Buffer 5 μL;dNTP 3.2 μL;
each primer 0.8 μL;Taq enzyme(5 U)0.5 μL;DNA
template 1μL] .After initial denaturation at 95℃ for
3 min , amplification was run for 38 cycles(30 s de-
naturation at 94℃, 30 s annealing at 52℃,90 s exten-
sion at 72℃)with Taq Plus Polymerase(SABC), fol-
lowing by 1 cycle of 10min extension at 72℃.The ob-
tained products were verified by electrophoresis on
8 g/L agarose gels.Purified PCR products were se-
quenced by Beijing Tiangen Biotech Co.,Ltd.
1.2.3 　Culture conditions and media 　Cultured
colonies of the mycobiont were transferred to 100 mL of
liquid Lilly-Barnett(LB)medium in a 300 mL Erlen-
meyer flask and incubated at 20℃on a rotary shaker at
140 r/min in a light-dark cycle of 12 h light and 12 h
dark.To examine the effect of the carbon source , the
mycelia were cultured for 3 weeks at 20℃ in LB solu-
tion(100 mL)containing glucose(G), fructose(F),
sucrose(S), inositol(I), or mannitol(M)at 40 g/L.
To examine the effect of amino acids , the mycelia were
cultured for 3 weeks at 20℃ in LB solution(100 mL)
containing L-alanine(Ala), L-asparagine(Asn), L-
glutamine(Gln), Glycine(Gly), or L-proline (Pro)at
2 g/L.To examine the effect of initial medium pH , the
pH of the LB solution (100 mL)was adjusted with
NaOH or HCl to 3.0 , 4.0 , 5.0 , 6.0 , 7.0 before au-
toclaving , and the mycelia were cultured in liquid me-
dia with various pH levels for 3 weeks at 20℃.All 2
replicate cultures were incubated in 300 mL Erlenmeyer
flasks on a rotary shaker at 140 r/min in a light-dark
cycle of 12 h light and 12 h dark.
Cultured colonies of the photobiont were trans-
ferred to 200 mL of Bold′s basal medium (BBM)in a
500 mL Erlenmeyer flask and subcultures were estab-
lished after 3 weeks of culture.The cell suspension of
58 菌　物　研　究 2008 年
the photobiont was transferred to 100 mL of liquid BBM
in a 300 mL Erlenmeyer flask and incubated at 20℃
on a rotary shaker at 140 r/min in a light-dark cycle of
12 h light and 12 h dark.To examine the effect of the
carbon source , the algal cells were cultured for 3 weeks
at 20℃ in BBM solution(100 mL)containing glucose
at 20 , 40 , 80 , 160 , or 200 g/L.To examine the ef-
fect of the nitrogen source , the algal cells were cultured
for 3 weeks at 20℃ in BBM solution (100 mL)con-
taining NaNO3 at 0.25 , 0.75 , 1.25 , 1.75 , or
2.25 g/L.To examine the effect of initial medium pH ,
the pH of the BBM solution (100 mL)was adjusted
with NaOH orHCl to 3.0 , 4.0 , 5.0 , 6.0 , 7.0 before
autoclaving , and the algal cells were cultured in liquid
media with various pH levels for 3 weeks at 20℃.All
three replicate cultures were incubated in 300 mL Er-
lenmeyer flasks on a rotary shaker at 140 r/min in a
light-dark cycle of 12 h light and 12 h dark.
2　Results and discussions
2.1　Solid culture of the mycobiont and photo-
biont of C.pyxidata
Two weeks after inoculation , mycobiont and pho-
tobiont grew out of the thallus fragments. About
1 month small mycobiont and photobiont colonies ap-
peared on the surface of the agar medium.After
3 months , mycobiont (Fig.3 -1) and photobiont
(Fig.3-2 , 3) growing colonies were transferred to
fresh medium.The photobiont is identified as Treboux-
ia sp.
1.Colony of mycobiont;2.Colony of photobiont;3.Algal cells of photobiont under microscope
Fig.3.Cladonia pyridate
2.2 　 The isolations checked by analysis of
molecular systematics
About 1 100 bp length fungal ITS rDNA product
and 1 024 bp length algal ITS rDNA product were ob-
tained respectively.Meg 3.1 software was used for the
analysis of phylogenetic tree.The fungal ITS rDNA se-
quence of C.pyxidata as well as the other 19 se-
quences of the Cladoniaceae downloaded from the Gen-
Bank was used for the analyses.Cladia aggregata was
used as outgroup.
The algal ITS rDNA sequence of C.pyxidata as
well as the other 20 sequences of the algal genus Tre-
bouxia downloaded from the GenBank was used for the
analyses.Chlorella saccharophila was used as out-
group.The result of the analyses based on molecular
data gave us much more important information for sys-
tematics.It is well known that the photobiont of Clado-
nia belongs to the algal genus Trebouxia.So the phylo-
genetic trees(Fig.4 ,Fig.5)showed that both the my-
cobiont and photobiont examined are the symbionts of
C.pyxidata.
59　第 6卷　第 1 期 苏　敏等:喇叭石蕊共生菌藻液体培养的研究
Fig.4.The phylogenetic tree of C.pyxidata mycobiont generated from ITS1 , 5.8S and ITS2 rDNA sequences
Fig.5.The phylogenetic tree of C.pyxidata photobiont generated from ITS1 , 5.8S and ITS2 rDNA sequences
2.3　The liquid culture of mycobiont
Inositol , mannitol and glucose accelerated growth
more than fructose and sucrose (Fig.6).Growth was
best by using liquid Lilly-Barnett medium containing
40 g/L inositol as a carbon source.Growth was
strongest in the liquid Lilly-Barnett medium containing
60 菌　物　研　究 2008 年
2 g/L L-glutamine as a nitrogen source among five
amino acids including L-alanine , L-asparagine , L-glu-
tamine , Glycine and L-proline(Fig.7).Growth of the
C.pyxidata mycobiont was best adjusting pH to 7.0
before autoclaving(Fig.8).These results would ref lect
the nature of their habitats of these lichens.
Fig.6. Effect of carbon source on growth of the
Cladonia pyxidata mycobiont
Fig.7.Effect of amino acids on growth of the Cladonia
pyxidata mycobiont
Fig.8.Effect of initial medium pH levels on growth of
the Cladonia pyxidata mycobiont
2.4　The liquid culture of photobiont
After a short lag phase , the photobiont grew
rapidly.Optimal concentration of glucose in BBM for
growth of C. pyxidata photobiont was 160 g/L
(Fig.9).These results showed a higher concentration
of sugar than in the original BBM promoted photobiont
growth.Photobiont growth was promoted by the addi-
tion of high concentration of NaNO3 , and the optimal
concentration of NaNO3 was 1.75 g/L(Fig.10).How-
ever , the cell growth was not remakably affected by
NaNO3 , the photobiont grew very slowly in the liquid
inorganic BBM.Growth of the C.pyxidata photobiont
was best adjusting pH to 7.0 before autoclaving.
(Fig.11).
Fig.9.Effect of glucose concentration on growth of the
Cladonia pyxidata photobiont
Fig.10.Effect of initial medium pH levels on growth of
the Cladonia pyxidata photobiont
61　第 6卷　第 1 期 苏　敏等:喇叭石蕊共生菌藻液体培养的研究
Fig.11. Effect of NaNO3 concentration on growth of
the Cladonia pyxidata photobiont
Reference:
[ 1] 　Belnap J , Bǜdel B, Lange O L.Biological soil crusts:charcteristi cs
and distribution in Jayne Belnap and Otto L.Lange [M] .Berlin:
Springer-Verlag , 2001.
[ 2] 　Yamamoto Y , Mizuguchi R , Yamada Y.Tissue cultures of Usnea
rubescens and Ramalina yasudae and production of usnic acid in
their cultures[ J] .Agric Biol Chem , 1985 , 49:3347-3348.
[ 3] 　Innis M A , Gelfand D H , Sninsky J J , et al.PCR Protocols-a
guide to methods and applications[ M ] . San Diego:Academic
press , 1990:282-306.
[ 4] 　Michele D , Piercey Normore , Paula T.Depriest algal switching a-
mong lichen symbioses[ J] .American Journal of Botany , 2001 , 88
(8):1490-1498.
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