全 文 :攀枝花块菌-华山松菌根根际土壤可培养细菌的多样性研究∗
万山平1ꎬ2ꎬ3ꎬ 郑 毅1ꎬ 汤 利1ꎬ 刘培贵2ꎬ 王 冉2ꎬ 于富强2∗∗
(1 云南农业大学 资源与环境学院ꎬ 昆明 650100ꎻ 2 中国科学院昆明植物研究所 资源植物与生物技术所级
重点实验室ꎬ 昆明 650201ꎻ 3 云南农业大学 植物保护学院ꎬ 昆明 650100)
摘要: 块菌作为可食用的地下外生菌根真菌ꎬ 有着重要的经济价值和生态学意义ꎮ 中国白块菌资源虽然被
不断的描述和报道ꎬ 但形成机制尚未为人所知ꎮ 前人研究表明ꎬ 块菌的菌根根际土壤微生物群落对块菌的
形成有着重要的影响ꎮ 因此ꎬ 本研究以攀枝花块菌 (Tuber panzhihuanense) ̄华山松 (Pinus armandii) 菌根根
际土壤为研究对象ꎬ 用可培养的方法揭示了其根际土壤的细菌多样性ꎮ 结果显示ꎬ 在所分离到的细菌中ꎬ
β ̄Proteobacteria占了最大的比例 (30 98%)ꎬ 以 Burkholderia 为优势类群ꎬ 其次是以 Pseudomonas 为代表类
群的 γ ̄Proteobacteria (28 8%)ꎬ 另外ꎬ α ̄Proteobacteria (14 67%) 的主要代表类群为 Phyllobacterium 和根
瘤菌 Rhizobiumꎻ 此外ꎬ 还分离到了分别以 Arthrobacter和 Bacillus为优势菌群代表的 Actinobacteria (12 5%)
和 Firmicutes (7 6%)ꎻ Bacteroidetes中只有唯一的代表菌株 Chryseobacterium ureilyticumꎮ 另外ꎬ 就目前对块
菌属子实体及其根际土壤内的可培养细菌多样性研究进行了比较和探讨ꎮ
关键词: 攀枝花块菌ꎻ 根际土壤ꎻ 细菌多样性ꎻ 16S rDNA
中图分类号: Q 938 1ꎬ Q 16 文献标志码: A 文章编号: 2095-0845(2015)06-861-10
Diversity of Culturable Bacteria Associated with
Tuber panzhihuanense ̄Pinus armandii
Ectomycorrhizosphere Soil
WAN Shan ̄ping1ꎬ2ꎬ3ꎬ ZHENG Yi1ꎬ TANG Li1ꎬ WANG Ran2ꎬ LIU Pei ̄gui2ꎬ YU Fu ̄qiang2∗∗
(1 College of Resource and Environmentꎬ Yunnan Agricultural Universityꎬ Kunming 650100ꎬ Chinaꎻ 2 Key Laboratory of Economic
Plants and Biotechnologyꎬ Kunming Institute of Botanyꎬ Chinese Academy of Sciencesꎬ Kunming 650201ꎬ Chinaꎻ
3 College of Plant Protectionꎬ Yunnan Agricultural Universityꎬ Kunming 650100ꎬ China)
Abstract: Truffles are edible hypogeous ectomycorrhizal fungi which have great economic importance for their orga ̄
noleptic properties and have significant ecological interests for forestry. Although some new precious Chinese white
truffle have been described constantlyꎬ the molecular mechanisms that control truffle body formation are largely un ̄
known. It has been hypothesized that ectomycorrhizosphere soil communities may have influences on truffle produc ̄
tion. Thusꎬ isolation and molecular characterisation of culturable bacteria were carried out to investigate the bacteria
diversity in mycorrhizosphere soil of Tuber panzhihuanense ̄Pinus armandii in this work. Sequencing results showed a
significant presence mostly affiliated with Burkholderia was β ̄Proteobacteria (30 98%). The second culturable frac ̄
tion which dominated by Pseudomonas was γ ̄Proteobacteria (28 8%) other isolates were mostly Phyllobacterium
and Rhizobiumꎬ members of α ̄Proteobacteria (14 67%)ꎬ actinobacteria (12 5%) and Firmicutes (7 6%) repre ̄
sented by Arthrobacter and Bacillusꎬ respectively. Chryseobacterium ureilyticum was the only bacterial strain belong ̄
ing to Bacteroidetes. Similarities and differences of culturable bacterial community of ascocarps and ectomycorrhizos ̄
植 物 分 类 与 资 源 学 报 2015ꎬ 37 (6): 861~870
Plant Diversity and Resources DOI: 10.7677 / ynzwyj201515032
∗
∗∗
Funding: National Natural Science Foundation of China (31460551ꎬ 31370070)ꎬ the Joint Founds of the National Science Foundation of
China and Yunnan Province Government (U1202262)
Author for correspondenceꎻ E ̄mail: fqyu@mail kib ac cn
Received date: 2015-03-04ꎬ Accepted date: 2015-06-23
作者简介: 万山平 (1987-)ꎬ 女ꎬ 在读博士生ꎬ 主要从事菌物研究ꎮ E ̄mail: wsp871117@163 com
phere soil associated with Tuber were discussed.
Key words: Tuber panzhihuanenseꎻ Ectomycorrhizosphere soilꎻ Bacteria diversityꎻ 16S rDNA
The term ‘rhizosphere’ was introduced by Hilt ̄
ner in 1904 to denote the region of the soil that is
subjected to the influence of plant roots ( Hiltnerꎬ
1904). Besidesꎬ The roots of most terrestrial plants
are colonised by mycorrhiza ̄forming symbiotic fungi
(Molina et al.ꎬ 1992)ꎬ the fungi also grow on the
root surface and extend into the surrounding soilꎬ
significantly expanding the functional space and ca ̄
pabilities of the rhizosphere (Allenꎬ 1992). Henceꎬ
the rhizosphere concept has been widened to associ ̄
ate this fungal effectꎬ resulting in the introduction of
terms “mycorrhizosphere” and “hyphosphere” (Ram ̄
belliꎬ 1973ꎻ Lindermanꎬ 1988). The microbial com ̄
munities of the soil surrounding mycorrhizal roots
and extrametrical mycelium are different from those
of the rhizosphere of non mycorrhizal plants and the
bulk soilꎬ due to higher availability of water and nu ̄
trients (Katznelson et al.ꎬ 1962ꎻ Garbaye and Bow ̄
enꎬ 1987ꎬ 1989ꎻ Garbayeꎬ 1991). Microorganisms
that inhabit the mycorrhizosphere serve as an inter ̄
mediary between the plantsꎬ which require soluble
inorganic nutrientsꎬ the mycorrhiza fungiꎬ which
transfer energy and materialꎬ and the soilꎬ which
contains the necessary nutrients but mostly in com ̄
plex and inaccessible forms. Rhizosphere microbes
thus provide a critical link between plantꎬ mycorrhi ̄
za and soil environments.
Based on decades of researchesꎬ the fungal
symbionts forming ectomycorrhizas (ECMs)ꎬ form a
significant component of forest ecosystems in the bo ̄
realꎬ temperate and Mediterranean climate zones
(Courty et al.ꎬ 2010). The ECMsꎬ as well as their
associated bacteriaꎬ benefit the growth and health of
forest trees and also the formation and development
of ectomycorrhiza in a number of ways although the
most important role is enhancing soil nutrient mobili ̄
zation and uptake. Bacterial species and strains most
commonly isolated in the inner and outer ectomycor ̄
rhizosphere or vicinity productive niches were Acti ̄
nomycetesꎬ Azotobacterꎬ Pseudomonasꎬ Burkholderiaꎬ
Acetobacterꎬ Herbaspirillumꎬ Bacillus and Agrobacte ̄
rium etc. (Oswald and Ferchauꎬ 1968ꎻ Malajczuk
and McCombꎬ 1979ꎻ Garbaye and Bowenꎬ 1989ꎻ
Varese et al.ꎬ 1996ꎻ Frey et al.ꎬ 1997bꎻ Timonen et
al.ꎬ 1998ꎻ Glickꎬ 1995ꎻ Probanza et al.ꎬ 1996ꎻ
Poole et al.ꎬ 2001ꎻ Aspray et al.ꎬ 2006ꎻ Founoune
et al.ꎬ 2001ꎬ 2002ꎻ Mello et al.ꎬ 2010). Those
strains enhancing mycorrhiza formation have been
termed mycorrhization helper bacteria ( MHB )
(Garbayeꎬ 1994). Pioneering work in this field had
been carried out on ectomycorrhizal fungiꎬ Suillusꎬ
Paxillusꎬ Rhizopogon and Laccaria synthesed with
Pinusꎬ Piceaꎬ Pseudotsuga and Quercus ( Garbaye
and Duponnoisꎬ 1992ꎻ Garbayeꎬ 1994ꎻ Schelkle
and Petersonꎬ 1996ꎻ Frey et al.ꎬ 1997aꎻ Dunstan et
al.ꎬ 1998ꎻ Pedersen et al.ꎬ 1999ꎻ Poole et al.ꎬ
2001ꎻ van Tichelen et al.ꎬ 2001). Howeverꎬ few
data are available about microbial populations thri ̄
ving in the precious ECMF ̄truffle ectomycorrhizaꎬ
and their potential role in the truffle lifecycle
(Mamoum et al.ꎬ 1986ꎻ Sbrana et al.ꎬ 2000ꎬ 2002ꎻ
Zacchi et al.ꎬ 2003). In Chinaꎬ where possesses
tremendous truffle resources containing multitudinous
species and potential biomassꎬ only a handful of in ̄
vestigations were carried out on truffle ̄relevant re ̄
searchesꎬ especially in the studies of Chinese white
truffle. In this paperꎬ we focus on the culturable mi ̄
crobes associated with ectomycorrhizosphere soil of a
significant China white truffle Tuber panzhihuanense ̄
Pinus armandiiꎬ to investigate the microorganism di ̄
versity in the ectomycorrhizosphere soil of this spe ̄
ciesꎬ to collect and screen MHB samples which will
likely be utilized for promoting and controlling the
mycelia growthꎬ the formation and development of
Tuber ascocarpsꎬ the ecological restorationꎬ and also
the artificial cultivation of Chinese white truffleꎬ
which have not been reported in previous resear ̄
ches.
268 植 物 分 类 与 资 源 学 报 第 37卷
1 Materials and methods
1 1 Bacteria isolation
Ectomycorrhizas of T panzhihuanense ̄P armandii
and six rhizosphere soil samples ( each sample had
two replications) were collected in October in three
towns of Qujing Prefectureꎬ Yunnanꎬ China (Table
1). We found the fruiting bodies of T panzhihuanense
in nature pure Pinus armandii woodlandꎬ and then
collected the ectomycorrhizas and rhizosphere soil
( the identification work of fruiting bodies and ecto ̄
mycorrhizas were carried out in the lab). Each sam ̄
ple was composed of freshꎬ identified and ungalled
mycorrhizal roots and their adhering soil. Rhizo ̄
sphere soil was collected from the roots after remo ̄
ving loosely attached soil particlesꎬ and stored in ice
for few hours until processing ( Fujiiꎬ 2004). One
gram of soil from each fresh sample was aseptically
weighed and was homogenized in 9 mL of filter steri ̄
lized physiologic solution (0 85% NaCl). Bacteria
were isolated by plating serial dilutions (10-3 throu ̄
gh 10-6 in triplicate) of the soil sample prepared in
sterile saline on tryptone soy agar (TSA) plates and
inoculated at 28 ℃ (TSAꎬ Difco). After 48 hꎬ the
number of CFU (colony forming unit) on the plates
was calculated and all colonies were picked and in ̄
oculated into 1 5 mL of tryptic soy broth (TSB) for
24 h at 28 ℃ .
1 2 Isolate screening and characterisation
1 2 1 PCR amplification of 16S rDNA V3 region and
the HPCE PCR were carried out on a thermocycler
(Biometra ̄Tgradientꎬ Germany) in a final volume of
25 μL containing 1 μL of DNAꎬ 1μL (5 μmolL-1) of
each primersꎬ 2 μL of 10 × buffer (Mg2+)ꎬ 0 5 μL of
Dntp (10 μmolL-1)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′-ATT ̄
ACCGCGGCTGCTGG-3′) were used. The polymerase
chain reaction (PCR) protocol was as follows: 1 cy ̄
cle of 5 min at 95 ℃ꎬ followed by 30 cycles of 1 min
at 94 ℃ꎬ 1 min at 60 ℃ and 72 ℃ for 1 minꎬ fol ̄
lowed by 1 cycle of 10 min at 72 ℃ . 2 μL PCR
products were checked on the 1% agarose gel. Dif ̄
ferent sizes of DNA fragments were screened by high
performance capillary electrophoresis ( HPCE) ac ̄
cording to the size of electrophoretic bands (Fig 1).
Table 1 Number of bacterial isolates screened from 6 rhizosphere soil samples
Sample ̄No Samples location Altitude / m Isolates No. OTUs CFU g-1
SB1 Laochanꎬ Huizeꎬ Qujing 2026 31 21 5 8 ± 0 60 × 108
SB2 Laochan Huizeꎬ Qujing 2130 37 11 2 7 ± 0 22 × 109
SB3 Yiche Huizeꎬ Qujing 1957 24 12 4 4 ± 0 33 × 108
SB4 Yiche Huizeꎬ Qujing 1950 27 6 1 6 ± 0 62 × 109
SB5 Jinzhong Huizeꎬ Qujing 2390 26 12 8 2 ± 0 46 × 107
SB6 Jinzhong Huizeꎬ Qujing 2307 29 8 3 4 ± 0 21 × 108
Fig 1 Partial results of screened different bands of variable region fragments of 16S rDNA V3 of isolates by
high performance capillary electrophoresis (HPCE)
3686期 WAN Shan ̄ping et al.: Diversity of Culturable Bacteria Associated with Tuber panzhihuanense ̄Pinus
1 2 2 16S rDNA sequencing In order to assess the
richness and relative abundance of bacteria associat ̄
ed with the rhizosphere soilꎬ isolates were further i ̄
dentified by partial 16S rDNA gene sequence analy ̄
sis. Universal prokaryotic primersꎬ 63f (5′-CAGG ̄
CCTAACACATGCAAGTC-3′) and 1495r (5′-CTA ̄
CGGCTACCTTGTTACGA-3′) were used to amplify
16SrDNA genes of the target isolates. Each compo ̄
nent 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.
The PCR products were purified and sequenced at
Sango Biotech Corporation in Shanghaiꎬ China. The
Sequence similarity was analyzed using the BLAST
algorithm (Basic Local Alignment Search Tools) a ̄
vailable on the National Centre for Biotechnology In ̄
formation (NCBI) website ( http: / / www. ncbi. nlm.
nih.gov / ) by using the program FASTA.
1 3 Data analysis
The similarity values greater than 97% were ap ̄
plied to define an operational taxonomic unit (OTU).
Phylogenetic relationships were inferred using the
neighbor ̄joining methodꎬ molecular evolutionary ge ̄
netics analysis (MEGA) software version 4 0 (Saitou
and Neiꎬ 1987ꎻ Tamuraꎬ 2007). The mean value of
the number of isolates from different soil was statisti ̄
cally analyzed with one ̄factor ANOVA ( identity of
the bacteria) .
2 Results
2 1 Size of the culturable bacterial community
In totalꎬ 184 T panzhihuanense ̄P armandii rhi ̄
zobacterial strains were isolated. The samples loca ̄
tionꎬ altitude and number of isolates were shown in
Table 1. There was no significant relationship between
bacterial abundance and soil type and altitude differ ̄
ence. No obvious difference in the number of CFU of
bacteria per 1 g among the six soil samples (P =
0 087).
2 2 The composition of the culturable bacterial
community
Sequences analysis identified six major groups
of the bacterial domainꎬ which included members of
Proteobacteria comprising the αꎬ β and γ subdivi ̄
sionsꎻ the Firmicutesꎻ Actinobacteria and the Bacte ̄
roidetes phyla. All 184 strains belonged to 55 spe ̄
cies of 25 genera. According to the analysesꎬ mem ̄
bers (137 of 184ꎬ 74 46%) belonging to Proteobac ̄
teria represented the prevalent bacterial groupꎬ and
they were significantly more abundant than the iso ̄
lates belonging to the other bacterial groups.
2 2 1 Members of the Proteobacteria division In the
bacterial collectionꎬ 14 67% of the bacterial isolates
were affiliated with the α ̄Proteobacteria (27 of 184)
(Fig 2). The majority of them clustered with Phyl ̄
lobacterium myrsinacearum ( 44 44%ꎬ 12 of 27)ꎬ
and 26% (7 of 27) were related to Rhizobiumꎬ the
rest of the isolates were closely related to Agrobacterium
Fig 2 Neighbor ̄joining tree of α ̄Proteobacteria based on 16S rDNA gene. The numbers at each branch node are the bootstrap
numbers from 1 000 re ̄samplings. Aquifex pyrophilus (M83548) was used as the out group
468 植 物 分 类 与 资 源 学 报 第 37卷
rhizogenes ( 18 5%ꎬ 5 of 27) and Brevundimonas
nasdae (11 11%ꎬ 3 of 27).
Among the isolatesꎬ 30 98% (57 of 184) of
them showed a most significant presence of β ̄Pro ̄
teobacteria ( Fig 3 ). They mostly represented by
Burkholderia (50 88%ꎬ 29 of 57) and the majority
had a significant similarity with B sediminicolaꎬ the
remainder belonged to B hospitaꎬ B sordidicolaꎬ
B xenovoransꎬ B graminisꎬ B phytofirmans and an
undescribed Burkholderia sp.
In contrast with Burkholderiaꎬ only a few iso ̄
lates grouped together with seven genera: Duganella
zoogloeoideꎬ Achromobacter spaniusꎬ Comamonas tes ̄
tosteroneꎬ Cupriavidus necatorꎬ Herbaspirillum hiltne ̄
riꎬ Variovorax paradoxusꎬ Collimonas arenae and
C pratensis.
The second numerous subclass within the Pro ̄
teobacteria was γ ̄Proteobacteria (28 8%ꎬ 53 of 184)
(Fig 4)ꎬ comprising the communityꎬ were assigned to
six genera: Pseudomonas (60 38%ꎬ 32 of 53)ꎬ Sten ̄
otrophomonas rhizophila (9%ꎬ 5 of 53)ꎬ Cedecea ne ̄
teri (9%ꎬ 5 of 53)ꎬ Raoultella terrigena (7 5%ꎬ 4 of
53)ꎬ Erwinia rhapontici (7 5%ꎬ 4 of 53) and Enter ̄
obacter aerogenes (3 78%ꎬ 2 of 53). The genus Pse ̄
udomonas was the most diverseꎬ and it was composed
of nine different species: P aspleniiꎬ P frederiksberg ̄
ensisꎬ P kilonensisꎬ P luridaꎬ P palleronianaꎬ P sp.ꎬ
P umsongensisꎬ P vancouverensis and P koreensis.
Fig 3 Neighbor ̄joining tree of β ̄Proteobacteria based on 16S rDNA gene. The numbers at each branch node are the
bootstrap numbers from 1 000 re ̄samplings. Aquifex pyrophilus (M83548) was used as the out group
5686期 WAN Shan ̄ping et al.: Diversity of Culturable Bacteria Associated with Tuber panzhihuanense ̄Pinus
Fig 4 Neighbor ̄joining tree of γ ̄Proteobacteria based on 16S rDNA gene. The numbers at each branch node are the
bootstrap numbers from 1 000 re ̄samplings. Aquifex pyrophilus (M83548) was used as the out group
2 2 2 Members of the Actinobacteria division A sig ̄
nificant group of OTUs occurring within the ectomy ̄
corrhizosphere soil of T panzhihuanense ̄P armandii
also represented by members of the Actinobacteria
phylum (12 5%ꎬ 23 of 184) (Fig 5). The Arthrob ̄
acter (39 13%ꎬ 9 of 23) group comprised many dif ̄
ferent speciesꎬ with the most abundant being A alka ̄
liphilusꎬ A defluviiꎬ A humicolaꎬ A methylotrophusꎬ
A nitroguajacolicus and A ramosus. The second major
group within Actinobacteria was Streptomyces (30 43%ꎬ
7 of 23)ꎬ including S atratusꎬ S atroaurantiacusꎬ
S aureusꎬ S mirabilis and S sioyaensis. Few strains
clustered within Acinetobacter calcoaceticus (13 04%ꎬ
3 of 23)ꎬ Rhodococcus (17 39%ꎬ 4 of 23)ꎬ R bai ̄
konurensis and R qingshengii.
2 2 3 Members of the Firmicutes and Bacteroidetes
division Most of the members of the Firmicutes (7 6%ꎬ
14 of 184) (Fig 5) represented by Bacillus (64 29%ꎬ
9 of 14 ) and belonged to B weihenstephanensis、
B circulans、 B acidiceler and B luciferensis. Among
the other bacteriaꎬ 21 43% Staphylococcus epidermi ̄
dis (3 of 14) and 14 29% Brevibacterium frigoritol ̄
erans (2 of 14) were identified. In additionꎬ only
one isolate T6B34 of Bacteroidetes (Fig 5) was de ̄
tected on a single branch related to Chryseobacterium
ureilyticum with a similarity level of 99%.
668 植 物 分 类 与 资 源 学 报 第 37卷
Fig 5 Neighbor ̄joining tree of Actinobacteriaꎬ Firmicutesꎬ Bacteroidetes based on 16S rDNA gene. The numbers at each branch
node are the bootstrap numbers from 1 000 re ̄samplings. Aquifex pyrophilus (M83548) was used as the out group
3 Discussion
According to the analysisꎬ there were no obvi ̄
ous differences between six different soil samples in
the amount of CFU of bacteriaꎬ this mostly coincided
with the results of the previous studies in the com ̄
munity of truffle associated ectomycorrhizosphere or
vicinity productive niches baceria ( Sbrana et al.ꎬ
2002ꎻ Mamoum et al.ꎬ 1986). Howeverꎬ in contrast
with the microorganism which have been consistently
isolated within the ascocarps of T borchiiꎬ T magn ̄
atumꎬ T maculatum and T panzhihuanenseꎬ the di ̄
versity of ectomycorrhizosphere soil bacteria is more
plentiful both in the abundance and quantity (Citte ̄
rio et al.ꎬ 1995ꎻ Gazzanelli et al.ꎬ 1999ꎻ Barbieri et
al.ꎬ 2005ꎬ 2007ꎻ Wan and Liuꎬ 2014).
In this studyꎬ the culturable approach was used
to analysis the structural compositon of a natural
bacterial community of a Chinese white truffle ecto ̄
mycorrhizosphere soil. The results of 16S rDNA se ̄
quences generated a high variety from six phyloge ̄
7686期 WAN Shan ̄ping et al.: Diversity of Culturable Bacteria Associated with Tuber panzhihuanense ̄Pinus
netic groups: αꎬ βꎬ γ ̄Proteobacteriaꎻ Firmicutesꎻ
Actinobacteria and Bacteroidetes. The largest portion
of the operational taxonomic units ( OTUs) within
the isolates were identified as β ̄Proteobacteria and
mostly with close relatives among the Burkholderia.
Burkholderia was also detected from the ascocarps of
T magnatum with a tiny number and only one species
(Barbieri et al.ꎬ 2007). Howeverꎬ this genus had not
been observed from the ascocarps of T borchii and
T panzhihuanense (Barbieri et al.ꎬ 2005ꎻ Wan and
Liuꎬ 2014). Such comparisons would provide addi ̄
tional information as to the specificity of the associa ̄
tion between Tuber and their related bacterial strains
and aid researchers in focusing future studies to ̄
wards fungus ̄specific bacteria interactions. Besidesꎬ
Variovorax paradoxus of β ̄Proteobacteria had also
been detected in the prior researches and had been
characterized as a plant ̄growth ̄promoting rhizobac ̄
terium by containing 1 ̄aminocyclopropane ̄1 ̄carbox ̄
ylate deaminase (Belimov et al.ꎬ 2001ꎻ Barbieri et
al.ꎬ 2005ꎬ 2007). Howeverꎬ no reports described a ̄
bout the rest six genera affiliated with β ̄Proteobacte ̄
ria in Tuber spp. associated microbes. γ ̄Proteobacte ̄
riaꎬ as the second major community dominated by
Pseudomonas and contained various specieses. The
relations between this bacterial group and truffle as ̄
sociate bacteria were described in previous investiga ̄
tions (Citterio et al.ꎬ 1995ꎻ Citterio et al.ꎬ 2001ꎻ
Gazzanelli et al.ꎬ 1999ꎻ Sbrana et al.ꎬ 2002ꎻ Bar ̄
bieri et al.ꎬ 2005ꎬ 2007ꎻ Mello et al.ꎬ 2010ꎻ Wan
and Liuꎬ 2014 ). Previous studies indicated that
Pseudomonas isolated from fruitbodies of T borchii
were capable to adhere to the hyphal matrix and
have an antifungal activity for producing biological
control or phytostimulatory compounds to restrict
growth of pathogen and influence mycelial growth
and morphogenesis (Bedini et al.ꎬ 1999ꎻ Sbrana et
al.ꎬ 2000ꎻ Barbieri et al.ꎬ 2001ꎻ Citterio et al.ꎬ
2001). In additionꎬ Pseudomonas isolated from ecto ̄
mycorrhizas and ectomycorrhizosphere of Laccaria
and Rhizopogonꎬ Lactariusꎬ were able to enhance
fungal growthꎬ root colonisation and subsequent es ̄
tablishment and functioning of mycorrhizas (Garbaye
and Bowenꎬ 1989ꎻ Garbaye et al.ꎬ 1990ꎻ Garbaye
et al.ꎬ 1994ꎻ Poole et al.ꎬ 2001). α ̄Proteobacteria
was a particularly constant and important bacterial
group which mainly represented by Phyllobacterium
and Rhizobium. P mysinacearum was found consist ̄
ently associated with truffles studied (Sbrana et al.ꎬ
2000ꎻ Barbieri et al.ꎬ 2005ꎬ 2007ꎻ Wan and Liuꎬ
2014). Rhizobiumꎬ which has the ability of nitrogen ̄
fixing activities in the nodules have also been de ̄
scribed in the ascocarps of other ectomycorrhizal fungi
(Spano et al.ꎬ 1982ꎬ Barbieri et al.ꎬ 2007). It sug ̄
gests that these associated microrganisms could play a
role in the fungal growth or nutrition during ascocarps
development.
Actinobacteria were dominanted by Arthrobacter
and Streptomyces in our study. This clade was con ̄
firmed as a considerable fraction of the total bacterial
population inhabiting ectomycorrhiza and ascocarps
of truffle (Mamoun et al.ꎬ 1986ꎻ Bedini et al.ꎬ 1999ꎻ
Sbrana et al.ꎬ 2002ꎻ Barbieri et al.ꎬ 2005ꎻ Wan
and Liuꎬ 2014)ꎬ and it was considered as a common
presence representative within Tuber spp.. Another
essential group obtained from the ectomycorrhizos ̄
phere soil of T panzhihuanense ̄P armandii was Fir ̄
micutes and referred to Bacillus. The framework
demonstrated that Bacillus spp. were able to increase
the mycelial growth of T borchii and other ectomy ̄
corrhizal fungi ( Garbaye and Bowenꎬ 1989ꎻ Gar ̄
baye et al.ꎬ 1990ꎻ Garbaye et al.ꎬ 1994ꎻ Sbrana et
al.ꎬ 2002). Moreoverꎬ Bacillus spp. as a N ̄fixing
bacterial species present in the mycorrhizosphere of
forest trees played an indispensable role in energy
exploitation and substance recycling of the environ ̄
ment (Li et al.ꎬ 1992ꎻ Senꎬ 2000).
In conclusionꎬ our results obtained in this study
greatly extended the taxonomic information available
on Tuber ̄associated bacteriaꎬ but none prticular in ̄
teraction between the truffle and their bacterial floras
were identified. For the first timeꎬ our work de ̄
scribed the soil microbial communities inhabiting a
natural Chinese white truffle ̄ground. We opening
868 植 物 分 类 与 资 源 学 报 第 37卷
new perspectives on diversity of interactions occur ̄
ring ectomycorrhizosphere of T panzhihuanense and
for a better understanding of this plant ̄fungal ̄bacte ̄
rial interaction. Through comparing with the previous
workꎬ five genera: Pseudomonasꎬ Bacillusꎬ Stenotr ̄
ophomonasꎬ Brevibacterium and Phyllobacteriumꎬ were
isolated in the ascocarps of T panzhihuanense (Wan
and Liuꎬ 2014)ꎬ and also detected from ectomycor ̄
rhizosphere soil of T panzhihuanense in this study. It
indicates that these strains perhaps as the represent ̄
atives of the culturable bacterial fraction associated
with T panzhihuanenseꎬ and could be play an indis ̄
pensable character in the mycorrhiza colonization
and ascocarp formation of this truffle. Some certain
strainsꎬ such as Pseudomonads and Bacilliꎬ which
enabled N ̄fixing activity and express chitinolitic ac ̄
tivityꎬ were charactered as MHBs and were assumed
to be able to affect ascospore germination within fruit
bodies of Tuber and to facilitate ascus opening and
mycelium growth (Gazzanelli et al.ꎬ 1999ꎻ Sbrana et
al.ꎬ 2002). Generallyꎬ the dominant and prevalent
bacterial strains investigated from Tuber may be use ̄
ful for further studies on the mechanisms of the MH ̄
Bs and will be efficiently utilized for the artificial ec ̄
tomycorrhiza synthesis and cultivation of Chinese
white truffle.
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