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Diversity of Culturable Bacteria Associated with Ascocarps of a Chinese White Truffle, Tuber panzhihuanense (Ascomycota)*

中国白块菌——攀枝花块菌子囊果内可培养细菌的多样性研究



全 文 :中国白块菌———攀枝花块菌子囊果内可培养细菌的多样性研究∗
万山平1ꎬ2ꎬ 刘培贵1∗∗
(1 中国科学院昆明植物研究所东亚植物多样性与生物地理学重点实验室ꎬ 云南 昆明  650201ꎻ
2 中国科学院大学ꎬ 北京  100049)
摘要: 对新近发现的块菌属一新种———攀枝花白块菌 (Tuber panzhihuanense) 子囊果中可培养细菌的多样
性进行了研究ꎮ 采用胰蛋白大豆培养基 (TSA) 对菌株进行分离ꎮ 用毛细管电泳 (HPCE) 对所有获得的
菌株的 16S rDNA V3高变区进行筛选获得不同条带大小的菌株ꎬ 对筛选出的菌株的 16S rDNA 进行测序ꎬ
并进行细菌多样性分析和研究ꎮ 结果显示ꎬ 攀枝花块菌子囊果内可培养细菌在数量及种类上都表现出很高
的多样性ꎬ 所有细菌分属于 5个门的 11个属和 20个种ꎮ 在所分离到的变形菌门的细菌中ꎬ 数量最多的菌
株 (49􀆰 68%) 属于 γ ̄Proteobacteriaꎬ 其中假单胞菌属的 Pseudomonas lurida 为优势类群ꎻ 其次为 α ̄Pro ̄
teobacteriaꎬ 占 37􀆰 42%ꎬ 其中以固氮菌 Bradyrhizobium japonicum和 Phyllobacterium spp.为优势类群ꎮ 其余的
菌株属于放线菌门 (Actinobacteria) (3􀆰 22%) 和厚壁菌门 (Firmicutes) (7􀆰 74%)ꎬ 厚壁菌门中以芽孢杆
菌属 (Bacillus) 为代表菌群ꎮ 酸杆菌门中的 Terriglobus roseus (1􀆰 94%) 首次从块菌中分离获得ꎮ
关键词: 攀枝花块菌ꎻ 子囊果ꎻ 细菌多样性ꎻ 16S rDNAꎻ 序列分析
中图分类号: Q 949􀆰 32ꎬ Q 939􀆰 1      文献标识码: A        文章编号: 2095-0845(2014)01-029-08
Diversity of Culturable Bacteria Associated with Ascocarps of a
Chinese White Truffleꎬ Tuber panzhihuanense (Ascomycota)∗
WAN Shan ̄Ping1ꎬ2ꎬ LIU Pei ̄Gui1∗∗
(1 Key Laboratory for Plant Diversity and Biogeography of East Asiaꎬ Kunming Institute of Botanyꎬ Chinese Academy of Sciencesꎬ
Kunming 650201ꎬ Chinaꎻ 2 University of Chinese Academy of Sciencesꎬ Beijing 100049ꎬ China)
Abstract: Culturable bacterial communities inhabiting ascocarps of Tuber panzhihuanense were investigated. Isolates
obtained on tryptone soy agar (TSA) were screened with high performance capillary electrophoresis (HPCE) ac ̄
cording to differences in size of 16S rDNA V3. Target isolates were identified by analysis of the whole length of 16S
rDNA gene. The results revealed that the ascocarps of T. panzhihuanense harbored a great number of culturable bacte ̄
ria which belonging to 20 species and 11 genera in 5 phyla. Most isolates (49􀆰 68%) were affiliated to the γ ̄Pro ̄
teobacteriaꎬ dominated by Pseudomonas lurida. The second major subclass was α ̄Proteobacteria (37􀆰 42%)ꎬ with
Phyllobacterium and a nitrogen ̄fixing bacterium Bradyrhizobium japonicum also occurring as dominant taxa. The re ̄
maining bacterial isolates contained members of Actinobacteria (3􀆰 22%) and Firmicutes (7􀆰 74%) of which Bacillus
was the commonest bacterium. A novel Tuber ̄associated culturable bacterium speciesꎬ Terriglobus roseusꎬ was isola ̄
ted and detected for the first time in Tuber ascocarps.
Key words: Tuber panzhihuanenseꎻ Ascocarpꎻ Bacteria diversityꎻ 16S rDNA
植 物 分 类 与 资 源 学 报  2014ꎬ 36 (1): 29~36
Plant Diversity and Resources                                    DOI: 10.7677 / ynzwyj201413018

∗∗
Funding: The National Natural Science Foundation of China ( 31270075)ꎬ International Cooperation Yunnan Program of Innovation to
Streng then Provinces by Science & Technology (2009AC013)ꎬ the Joint Founds of the National Science Foundation of China and
Yunnan Province Government (U1202262)
Author for correspondenceꎻ E ̄mail: pgliu@ mail. kib. ac. cn
Received date: 2013-02-15ꎬ Accepted date: 2013-06-10
作者简介: 万山平 (1987-) 女ꎬ 硕士研究生ꎬ E ̄mail: wanshanping@ mail. kib. ac. cn
  True truffles are ascomycetous hypogeous fungi
of the genus Tuber that establish symbiotic relation ̄
ships with some plant roots of gymnosperms and an ̄
giosperms (Harley and Smithꎬ 1993). Truffles are
important fungi not only because of playing important
role in maintaining health of natural ecosystemsꎬ but
also due to the high culinary value of some putative
gourmet speciesꎬ such as T. magnatum Pico.ꎬ T.
melanosporum Vitt.ꎬ and T. indicum Cooke & Mas ̄
see. These stimulate researches on taxonomyꎬ biolo ̄
gyꎬ ecology and cultivation of Tuber. One of interest ̄
ing issues about truffles is the bacteria diversity for
its role in the ecological system. It has been reported
that the symbiotic development of mycorrhizal fungi
on plant roots could be influenced by some bacteria
present in the mycorrhizosphere ( Duponnois and
Garbayeꎬ 1992ꎻ Varese et al.ꎬ 1996ꎻ Johansson et
al.ꎬ 2004ꎻ Jones et al.ꎬ 2004ꎻ de Ridder ̄Duine et
al.ꎬ 2005ꎻ Mello et al.ꎬ 2010). Different microbes
have also been isolated from fruiting bodies and the
hyphae of ectomycorrhizal fungi (Li and Castellanoꎬ
1987ꎻ Garbaye et al.ꎬ 1990ꎻ Varese et al.ꎬ 1996ꎻ
Barbieri et al.ꎬ 2005ꎻ Neal Jr et al.ꎬ 1964ꎻ Andrade
et al.ꎬ 1997ꎻ Mansfeld ̄Giese et al.ꎬ 2002ꎻ Sbrana
et al.ꎬ 2002). In terms of diversity of bacteria asso ̄
ciated with Tuber speciesꎬ the microbial communities
in truffle sporocarps were studied and different types of
bacteria have been isolated and identifiedꎬ such as Ac ̄
tinomycetesꎬ Pseudomonas and aerobic spore ̄forming
bacteria (Gazzanelli et al.ꎬ 1999ꎻ Sbrana et al.ꎬ 2002ꎻ
Barbieri et al.ꎬ 2007). Studies on the diversity of bac ̄
teria associated with Tuber fruiting bodies can help to
understand their ecological role in the truffle life cycle
and can be used for development of biotechnological
applications by utilizing beneficial strains.
The aim of this research is to analyze and evalu ̄
ate the diversity of culturable bacteria inside the asco ̄
carps of T. panzhihuanenseꎬ a new white truffle species
found in southwest of China and described by Deng et
al. (2012) and has potential commercial value for its
special fragrance and ecological significance. The infor ̄
mation of the bacterial inhabitants of this truffle species
can help to understand the bacteria ̄truffle interactions
and will be of practical significance in improving my ̄
corrhizal synthesis for truffle cultivation.
1  Materials and methods
1􀆰 1  Bacteria isolation
Six ascocarps of T. panzhihuanense were ob ̄
tained from natural Pinus armandi forests in Qujing
Prefectureꎬ Yunnanꎬ China. After collectingꎬ fresh
fruiting bodies were kept in an ice box and processed
within a few hours for reducing the storage modifying
the autochthonous bacterial community. The samples
were gently washed with sterile distilled water and
surface ̄disinfected with 96% ethyl alcohol (1 min)ꎬ
5% sodium hypochloride (3 min) and 96% ethyl al ̄
cohol (0􀆰 5 min) (Petrini and Müllerꎬ 1986)ꎬ and
then briefly flamed. About 1 g (wet ̄weight) of gleba
tissue was removed from each ascoma and homoge ̄
nized in 1 mL of filter sterilized physiologic solution
(0􀆰 85% NaCl). 100 μL gleba suspension was dilu ̄
ted from 10-2 to 10-6ꎬ 10-4 to 10-6 were chosen to
culture on tryptone soy agar ( TSA) and inoculated
at 28 ℃ (TSAꎬ Difco) for isolating the fraction of
culturable bacteria (Gazzanelli et al.ꎬ 1999ꎻ Sbrana
et al.ꎬ 2002ꎻ Barbieri et al.ꎬ 2005). After two
days’ incubation on TSAꎬ the number of CFU (col ̄
ony forming unit) was calculated and all colonies
growing on the Petri dishes were picked. Voucher
specimens of ascocarps were deposited in Cryptogamic
Herbarium of Kunming Institute of Botanyꎬ Chinese
Academy of Sciences (KUN ̄HKAS) (Table 1).
1􀆰 2  DNA extraction
The single colony of each isolate was picked up
and cultured in tryptone soy broth ( TSB) for two
days. Total DNA of each strain was extracted by TI ̄
ANamp Bactetia DNA Kit following the manufactur ̄
er’s instruction.
1􀆰 3  Isolate screening
1􀆰 3􀆰 1  PCR amplification of bacterial 16S rDNA V3
region and the 16S rDNA
PCR were carried out on a thermocycler ( Bi ̄
ometra ̄Tgradientꎬ Germany) in a final volume of 25
03                                  植 物 分 类 与 资 源 学 报                            第 36卷
μL containing 1 μL of DNAꎬ 1 μL (5 μM) of each
primersꎬ 2 μL of 10 × buffer (Mg2+ )ꎬ 0􀆰 5 μL of
dNTP (10 μM)ꎬ 0􀆰 5 μL BSA (1%)ꎬ 1􀆰 5 μL MgCl2ꎬ
0􀆰 1 U of Taq DNA polymerase (Takara Tagꎬ Takara
Biotechnologyꎬ Dalian Co. Ltd.ꎬ China).
Variable region fragments of 16S rDNA V3 were
amplified by specific and sensitive primersꎬ f341
(5′ ̄CCTACGGGAGGCAGCAG ̄3′) and r518 ( 5′ ̄
ATTACCGCGGCTGCTGG ̄3′)ꎬ and the following
thermocycling program: 95 ℃ for 5 minꎬ followed by
30 cycles at 94℃ for 1 minꎬ 60℃ for 1 min and 72℃
for 1 min. The final reaction was followed by an ex ̄
tension at 72 ℃ for 10 min. 2 μL PCR products were
checked on the 1% agarose gel. Different sizes of
DNA fragments were screened by high performance
capillary electrophoresis (HPCE) according to the
size of electrophoretic bands (Fig􀆰 1).
Universal prokaryotic primersꎬ 63f ( 5′ ̄CAG ̄
GCCTAACACATGCAAGTC ̄3′) and 1495r (5′ ̄CT ̄
ACGGCTACCTTGTTACGA ̄3′) were used to ampli ̄
fy 16S rDNA genes of the target isolates. Each com ̄
ponent content of the amplification system as same as
the PCR for bacterial 16S rDNA V3 regionꎬ and the
cycling parameters are invariant except annealing
temperature which was changed to 56 ℃ for 1 min.
1􀆰 4  16S rDNA sequencing
16S rDNA sequences of pure bacterial cultures
were performed with ABI Prism BigDye terminator
cycle sequencing kit in ABI PRISM 3730 automatic
sequencer. The Sequence similarity was analyzed u ̄
sing the BLAST algorithm (Basic Local Alignment
Search Tools) available on the National Centre for
Biotechnology Information (NCBI) website (http: / /
www.ncbi.nlm.nih.gov / ) .
1􀆰 5  Data analysis
In order to assess the bacterial diversity of the
16S rDNA culture collectionꎬ nearly full ̄length 16S
rDNA (c. 1 450 nt) sequences were analyzed and com ̄
pared with sequences available from DDBJ / EMBL /
GenBank. The similarity values greater than 97% were
applied to define an operational taxonomic unit (OTU).
Sequences were edited with SeqMan ( DNASTAR
Package). Alignments were performed with Clustal 3
version 1􀆰 81 (Thompson et al.ꎬ 1997) and adjusted
manually with BioEdit Version 5􀆰 0􀆰 9 (Hallꎬ 1999).
Phylogenetic analysis were made following the meth ̄
ods of Saitou & Nei et al. (1987) and Tamura (2007).
The mean value of the number of isolates from differ ̄
ent ascocarp was calculated and analyzed with one ̄
factor ANOVA (identity of the bacteria).
2  Results
2􀆰 1  Size of the culturable bacterial community
A total of 155 bacteria isolates were screened
out from six ascocarps of T. panzhihuanense (Table
1). There was no significant difference in the num ̄
ber of CFU of bacteria per 1 g among the six fresh
sporocarps (P = 0􀆰 093). Howeverꎬ the number of
different bacterial groups associated with each asco ̄
carp is very different.
Fig􀆰 1  Partial results of screened different bands of variable region fragments of 16S rDNA V3
of isolates by high performance capillary electrophoresis (HPCE)
131期      WAN and LIU: Diversity of Culturable Bacteria Associated with Ascocarps of a Chinese White Truffle 􀆺     
Table 1  Number of bacteria isolates screened from six ascocarps of T. panzhihuanense
Bacteria
Sample of ascocarp
T1 T2 T3 T4 T5 T6
Herbarium Code HKAS72014 HKAS72069 HKAS72077 HKAS72016 HKAS72024 HKAS72025
CFU g-1 6􀆰 7±0􀆰 25×106 1􀆰 8±0􀆰 23×107 2􀆰 2±0􀆰 37×107 5􀆰 3±0􀆰 55×107 7􀆰 9±0􀆰 67×106 5􀆰 7±0􀆰 25×106
Number of isolates 36 29 35 18 23 14
OTUs 6 4 7 6 4 3
α ̄Proteobacteria 6 5 20 6 11 0
γ ̄Proteobacteria 28 24 8 12 5 10
Firmicutes 0 0 4 0 2 0
Actinobacteria 2 0 3 0 5 0
Acidobacteria 0 0 0 0 0 4
2􀆰 2  Diversity of the culturable bacterial community
The sequences analysis revealed that 155 iso ̄
lates were identified as five phylaꎬ Firmicutesꎬ Acti ̄
nobacteriaꎬ Acidobacteriaꎬ Alphaproteobacteria and
Gammaproteobacteria. They belonged to 11 genera
and 20 species ( Fig􀆰 2). Among themꎬ the Pro ̄
teobacteria was significantly more numerous than
other group of bacteria.
2􀆰 2􀆰 1  Proteobacteria
Among the 155 isolates 49􀆰 68% (77 of 155) of
bacteria associated with T. panzhihuanense showed a
significant presence of γ ̄Proteobacteria. In γ ̄Pro ̄
teobacteriaꎬ 54􀆰 55% ( 42 of 77 ) isolates were
Pseudomonas lurida and only 25􀆰 97% (20 of 77) of
γ ̄Proteobacteria belonged to P. umsongensisꎬ P.
aeruginosa and P. otitidis ( similarity >99%). There
were two isolatesꎬ T4B4 and T4B10 grouped togeth ̄
er with undescribed Pseudomonas species ( Fig􀆰 2).
In contrast with Pseudomonas groupꎬ only a few iso ̄
lates (13 of 77ꎬ 16􀆰 88%) with Stenotrophomonas
maltophilia on a single branch with a similarity level
of 99% on 1 450 nt (Fig􀆰 2).
Another subclass within the Proteobacteria was
α ̄Proteobacteria ( 58 of 155ꎬ 37􀆰 42%). In this
groupꎬ 55􀆰 17% (32 of 58) isolates belonged to ge ̄
nus of Phyllobacteriumꎬ 31􀆰 03% (18 of 58) to Bra ̄
dyrhizobium japonicumꎬ and 13􀆰 79% (8 of 58) to
Boseaꎬ respectively.
2􀆰 2􀆰 2  Firmicutes and Actinobacteria
The low and high GC Gram ̄positive bacteria
were also obtained from the TSA. Most of the isolates
belonged to low GC Gram ̄positive Firmicutes (12 of
155ꎬ 7􀆰 74%)ꎬ among which 83􀆰 33% (10 of 12)
clustered into genus Bacillus with high similarity (>
99%)ꎬ including B. firmusꎬ B. mojavensis and B.
fortisꎬ and the rest of the isolates were closely relat ̄
ed to Brecibacterium frigoritolerans (Fig􀆰 2). There
were only 3􀆰 22% (5 of 155) of isolates in high GC
Gram ̄positive bacteria which represented by Mi ̄
crobacterium testaceum and Kineococcus xinjiangensis
(Fig􀆰 2).
2􀆰 2􀆰 3  Acidobacteria
A very unique group of bacteriaꎬ Terriglobus ro ̄
seus within ascocarps of the T. panzhihuanense was
detected in this study (3 of 155ꎬ 1􀆰 94%)ꎬ which
was not found from the bacterial communities associ ̄
ated with mycorrhizas or the sporocarps of T. melan ̄
opsporumꎬ T. maculatumꎬ T. bochiiꎬ and T. magna ̄
tum (Li and Castellanoꎬ 1987ꎻ Sbrana et al.ꎬ 2002ꎻ
Barbieri et al.ꎬ 2005ꎬ 2007). This is the first time
that strains of Acidobacteria were detected and re ̄
corded from the ascocarps of Tuber.
3  Discussion
Six ascocarps of T. panzhihuanense were used
for this analysis. There was no obvious difference be ̄
tween different ascocarps in the amount of CFU of
bacteria. Five groups of bacteria were identified from
155 isolates. The majority of them were detected in
previous studiesꎬ for exampleꎬ Pseudomonas and
Stenotrophomonas (γ ̄Proteobacteria)ꎻ Boseaꎬ Bra ̄
dyrhizobiumꎬ and Phyllobacterium ( α ̄Proteobacte ̄
23                                  植 物 分 类 与 资 源 学 报                            第 36卷
ria)ꎻ Firmicutesꎻ Actinobacteria and Acidobacteria
(Lee et al.ꎬ 2008ꎻ Sbrana et al.ꎬ 2000ꎻ Barbieri et
al.ꎬ 2005ꎬ 2007ꎬ 2010). β ̄Proteobacteria was de ̄
tected and reported from the ascoma of T. borchii and
T. magnatum in previous studies ( Barbieri et al.ꎬ
2005ꎬ 2007)ꎬ but nor from all ascocarps studied
here. It was probably due to their different habitats
and hosts.
α ̄Proteobacteria was one of the particularly
constant and important bacterial groupsꎬ indicating it
was probably associated with T. panzhihuanense asco ̄
carps. Phyllobacterium mysinacearum and Bradyrhi ̄
zobium japonicum were the main representatives of this
group. P. mysinacearum was the commonest bacterium
Fig􀆰 2  Neighbor ̄joining tree based on 16S rDNA gene partial sequences of 21 OTUs isolated from fruit bodis of T. panzhihuanense.
It shows that the phylogenetic relationships of isolates gene sequences to closely related sequences obtained from the GenBank
database. The numbers at each branch node are the bootstrap numbers from 1000 re ̄samplings. The sequence
of Aquifex pyrophilus (M83548) was used as the out group
331期      WAN and LIU: Diversity of Culturable Bacteria Associated with Ascocarps of a Chinese White Truffle 􀆺     
and was found consistently associated with truffles
studied (Sbrana et al.ꎬ 2000ꎻ Barbieri et al.ꎬ 2005ꎬ
2007). B. japonicumꎬ which is well known for its a ̄
bility to form symbiotic associations with leguminous
host plants and being responsible of the nitrogen ̄fix ̄
ing activities in the nodulesꎬ was also frequently iso ̄
lated from truffles ( Barbieri et al.ꎬ 2005ꎬ 2007ꎬ
2010). Barbieri et al. (2010) amplified the nitroge ̄
nase gene nifH from T. magnatum ascocarps at dif ̄
ferent stages of maturation using degenerate PCR
primersꎬ and putative amino acid sequences revealed
that the nifH genes from bacteria of α ̄ proteobacteria
affiliated with Bradyrhizobium spp. Besidesꎬ the ni ̄
trogen ̄fixing bacteria have also been detected from
the fruiting bodies of other EM ̄fungi and AM ̄fungi
(Larsen et al.ꎬ 1978ꎻ Spano et al.ꎬ 1982ꎻ Li et al.ꎬ
1992ꎻ Bianciotto et al.ꎬ 1996ꎻ Hurek et al.ꎬ 1997ꎻ
Amora ̄Lazcano et al.ꎬ 1998ꎻ Antoun et al.ꎬ 1998ꎻ
Frey ̄Klett et al.ꎬ 2007 ). Nitrogen fixation and
transfer by the organotrophic bacteria might be im ̄
portant for the nutrition mechanism of truffles and
other fungi.
γ ̄Proteobacteria was quantitatively predomina ̄
ted among the isolates and it was heavily populated
by members of Pseudomonas and Stenotrophomonas.
Previous studies found that the genus Pseudomonas
was representative of the culturable bacterial fraction
associated with truffles ( Gazzanelli et al.ꎬ 1999ꎻ
Sbrana et al.ꎬ 2002ꎻ Barbieri et al.ꎬ 2005ꎬ 2007).
It was reported that certain Pseudomonas had the ca ̄
pability in degrading chitinolitic compoundꎬ and
some strains were shown to restrict growth of phyto ̄
pathogenic fungi. These functions were assumed to be
able to stimulate spore germination and mycelium
growth within fruiting bodies of T. borchii and facilita ̄
ted the morphogenesis of ascus (Bedini et al.ꎬ 1999ꎻ
Gazzanelli et al.ꎬ 1999ꎻ Sbrana et al.ꎬ 2000ꎬ 2002).
Potential relationship between the presence of truffle
fruiting bodies and the γ ̄Proteobacterium Moraxella
osloensis in previous study suggested that M. osloensis
seems to be a promising marker of truffle presence or
productivity (Mello et al.ꎬ 2010). Howeverꎬ this spe ̄
cies was not isolated from the T. panzhihuanense.
Spore ̄forming bacteria isolated from T. panzhi ̄
huanense were mostly referred to Bacillus firmusꎬ B.
mojavensisꎬ and B. fortis. Some previous investiga ̄
tions have shown that Bacillus detected from T.
borchii ascocarps also expressed a clear chitinolitic
degradation activity to affect ascospore germination
and facilitate ascus developing and opening (Gazzanelli
et al.ꎬ 1999). In additionꎬ some cultures isolated
from mycorrhizae and mycorrhizosphere of Laccaria
and Rhizopogon were able to enhance fungal growthꎬ
root colonization and subsequent establishment and
functioning of mycorrhizae (Garbaye et al.ꎬ 1990ꎻ
Garbaye and Duponnoisꎬ 1992).
Previous studies on the T. borchii microbial
communities have described the presence of Acti ̄
nobacteria within ectomycorrhizae and ascocarps
(Bedini et al.ꎬ 1999ꎻ Sbrana et al.ꎬ 2000ꎬ 2002ꎻ
Barbieri et al.ꎬ 2005). Similar results were obtained
from previous isolation experiments carried out on T.
magnatum mycorrhizas collected in a truffle orchard
(29% of isolatesꎬ unpublished results) and from
rhizoplane analysis of T. melanosporum mycorrhizal
roots (Mamoun et al.ꎬ 1986). In our studiesꎬ there
were few numbers associated with high GC Gram ̄
positive bacteria represented by Microbacterium
which have been also isolated from ascocarps of T.
borchii and T. magnatum ( Barbieri et al.ꎬ 2005ꎬ
2007). Besidesꎬ another novel group clustered to ̄
gether with Kineococcus was detected in our research.
The occurrence of these strains confirmed that this
bacterial group represented a common fraction of the
bacterial population inhabiting Tuber mycorrhiza or
fruiting bodies.
Interestinglyꎬ a novel Tuber ̄associated cultur ̄
able speciesꎬ Terriglobus roseusꎬ was detected in this
study. Bacteria belonging to the phylum Acidobacte ̄
ria have been observed in a wide variety of environ ̄
ments and representing dominant and metabolically
active bacteria in rhizosphere soil ( Lee et al.ꎬ
2008). However there was no information available
on the specific interaction of this species with ecto ̄
43                                  植 物 分 类 与 资 源 学 报                            第 36卷
mycorrhizal fungi.
Previous methodsꎬ including plate countsꎬ in
situ microscopic observations and culture ̄independ ̄
entꎬ have found bacteria on the surfaces of truffles
and other fungi’ hyphae and sporesꎬ on mycorrhizal
rootsꎬ and inside the fruiting bodies (Katznelson et
al.ꎬ 1962ꎻ Andrade et al.ꎬ 1998ꎻ Timonen et al.ꎬ
1998ꎻ Bedini et al.ꎬ 1999ꎻ Mansfeld ̄Giese et al.ꎬ
2002ꎻ Sbrana et al.ꎬ 2000ꎻ Barbieri et al.ꎬ 2005).
It suggested that there were some potential interac ̄
tion mechanisms between fungi and bacteria. For in ̄
stanceꎬ the exudates of fungi might be a major or ex ̄
clusive source of nutrients for these bacteria (Danell
et al.ꎬ 1993ꎻ Andrade et al.ꎬ 1997ꎻ Barea et al.ꎬ
2002ꎻ Rangel ̄Castro et al.ꎬ 2002ꎻ Rilligꎬ 2004).
And converselyꎬ bacteria could affect ascospore ger ̄
mination of fungi and facilitate ascus developing and
mycelial growth (Gazzanelli et al.ꎬ 1999ꎻ Sbrana et
al.ꎬ 2002). In terms of the interaction mechanisms
between fungi and bacteriaꎬ there were some parti ̄
cular and significant fungus ̄associated bacteriaꎬ so ̄
called MHBs (mycorrhizal helper bacteria)ꎬ posses ̄
sing the potential activity in the biocontrol or the a ̄
bility to sustain the development of mycorrhizal in ̄
fectionꎬ and positive effects on the plants ̄bacteria ̄
fungus interactionꎬ such as the bacteria aboveꎬ Bra ̄
dyrhizobiumꎬ Pseudomonas and Bacillus.
This study is more qualitative than quantitative
in demonstrating the bacterial community in white
truffle. Although the range of bacterial ̄truffle inter ̄
actions that occur in nature has not yet been fully in ̄
vestigatedꎬ the results obtained from our work extend
the taxonomic information available on Tuber ̄associ ̄
ated bacteria. Based previous studiesꎬ it is reasona ̄
ble to expect that this superficial truffle ̄bacteria con ̄
nection has mutual benefits for both sides. Further
analyses are surely required to improve knowledge on
the interactions between truffles and bacteria. And
by screening out the “helper” microbeꎬ the results
could be utilized to improve establishment and main ̄
tenance of a functional symbiosisꎬ and to enhance
the success of truffle cultivation.
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