Introduction
Endophytic
bacteria refer to such types of bacteria during a certain stage in their life
cycle or their entire life, wherein they occur in healthy growing plant tissues
or cells. The host plant does not exhibit obvious disease symptoms because of
this class of microbes, but they can play an important part in the
micro-ecosystem of the plant and play an important role during the process of
long-term cooperative coevolution. Furthermore, the plants may form a mutually
beneficial relationship with these bacteria (Santoyo et al. 2015; Donoso et al. 2016).
As an important reproductive organ of plants, seeds carry abundant
microbial populations on the surface and inside (Verma et al. 2017).
Compared with other plant organs, the plant seed endophyte has received
relatively less attention. Because seeds are of significance to plant
reproduction and agricultural development, studies focused on endophytic
bacteria of seeds should receive more attention (Chowdhary and Kaushik 2015; Fouda et al. 2015; Shade et al. 2017).
Therefore, it is necessary to conduct research related to endophytes of plant
seeds, especially that of medicinal and spice plant seed microorganisms for
which their endogenous community structure and diversity in the seed should be
understood.
Alpinia zerumbet (Pers.) Burtt et Smith is an important aromatic plant in China,
which has been widely planted in Southern Sichuan in recent years.
Studies have shown that it is rich in volatile oils and has antibacterial,
antioxidant, analgesic, and anti-inflammatory properties. Furthermore, it
regulates the cardiovascular system and various biological activities, such as
those of the nervous system (Indrayan et al.
2009; Tao et al. 2009; Araujo et al. 2010; Shen et al.
2010). In the southern part of Sichuan, A. zerumbet is widely
distributed and there are special climatic conditions. Growing A. zerumbet
may have potential endophytic species resources that are completely different from those from other areas and in other species.
At present, the research on A. zerumbet has mainly focused on the
function of chemical and active ingredients, and research on endophytes that
may affect its growth health and active ingredients is lacking (Elzaawely et al. 2007; Chompoo
et al. 2012; Chen et
al. 2017). Although researchers
have studied endophytes in more than 100 plants for over 20 years, there are
still a large number of plants in nature with unknown endophytic resources,
especially highly utilized and functional plant species. Thus, research on
endophytes is of great significance. This study was aimed to establish the
relationship between endophytic bacterial communities and their plant
developmental stages. High-throughput sequencing was used to explore the
diversity of the endophytic communities of A. zerumbet seeds at five different
developmental stages. The results presented in this paper provide a scientific
basis for the reasonable implementation of microbiological control and
strengthen the natural fermentation of A. zerumbet products, which is
necessary to ensure the quality of products.
Materials and
Methods
Experimental
materials
The samples of A.
zerumbet seeds were in five different growth periods, consisting of the
fruit formation period, young fruit period, early mature period, middle mature
period, and late mature period. A 5.0 g sample of seeds was collected in Yibin, Sichuan Province (28°53′17″N,
104°43′7″E), and stored at 4°C. After the samples were sterilized,
they were incubated at 28°C for 3 d to test the effect of surface disinfection.
Uncontaminated samples were used in the next experiment.
DNA extraction and
PCR amplification
The OMEGA kit was
used to extract DNA. For PCR amplification, bacteria with a length of
approximately 450 bp were selected as the target amplified fragments for subsequent
high-throughput sequencing. The primer sequences were the forward primer
ACTCCTACGGGAGGCAGCA and the reverse primer GGACTACHVGGGTWTCTAAT. The PCR
reaction system (25 μL)
consisted of 5 μL
5 × reaction buffer, 5 μL
5 × GC buffer, 2 μL
dNTP (100 mmol·L-1), forward primer (10 μmol·L-1) 1 μL, 1 μL reverse primer (10 μmol·L-1), 0.25 μL Q5 high-fidelity DNA
polymerase, 2 μL
DNA template, and 8.75 μL
ddH2O. The PCR reaction conditions were denaturation for 2 min at 98°C,
denaturation for 15 s at 98°C, annealing for 30 s at 55°C, extension for 30 s
at 72°C, and expansion for 25–30 cycles, with a final extension at 72°C for 5
min. After cutting the target fragment, it was recovered by the Axygen gel recovery kit.
Construction of
gene clone library
The purified PCR
products were ligated to the T3 vector and transformed into Escherichia coli
DH5α competent cells (Xiang et al. 2018). The transformation
product was spread on Luria–Bertani (LB) agar plates containing ampicillin (100
mg/L), white clones were randomly selected for streaking, and a cloning library
was constructed.
Sequencing and phylogenetic analysis
Two-hundred clones
from each sample were randomly selected for partial sequencing of the 16S rRNA
gene. BLASTN was used to compare approximately 700 base nucleotide sequences to
the NCBI (https://blast.ncbi.nlm.nih.gov/Blast)
database (Naveed et al. 2014). The sequences were aligned using CLUSTALW
(Hung and Weng 2016), and the neighbor-joining method was used to construct the
tree with the MEGA 4 program package (Sudhir et al. 2018). Finally, the
extent of the cloned library was evaluated by rarefaction analysis.
Results
A 16S rRNA gene
clone library of endophytic bacteria from A.
zerumbet was constructed using the purified PCR products. Two-hundred
clones were randomly selected for sequencing and submitted to GenBank
(accession no. MF508571—MF508602, MF508535—MF508570,
MF541320—MF541368, MF803088—MF803146, and MG346174—MG346221). The
endophytic bacteria were detected during the five different seed growth periods
in A. zerumbet. These were the fruit formation period, young fruit
period, early mature period, middle mature period, and late mature period,
which included 31, 36, 49, 58, and 46 OTUs. The coverage was calculated as
94.70, 93.55, 91.40, 94.45 and 93.75%, respectively.
Among the endophytic bacteria during the five
seed growth periods in A. zerumbet, the Curtobacterium
played a role in four growth periods, which made up the largest fraction of the first four
growth periods. At the fruit formation period, 200 clones were analyzed, of
which 43 clones (21.50%) belonged to α-proteobacteria, 3 clones (1.50%)
belonged to β-proteobacteria, 140 clones (70.00%) belonged to
γ-proteobacteria, 6 clones (3.00%) belonged to Bacteroidetes, and 8 clones
(4.00%) belonged to Actinobacteria, Actinobacteria, Bacteroidetes,
α-proteobacteria, β-proteobacteria, and
γ-proteobacteria were made up of 1, 2, 8, 2 and 18 bacterial OTU,
respectively. In the clone library, Curtobacterium (25.0%), Pantoea (25.0%), and Aureimonas
(10.0%) were the dominant genera (Table 1).
During the young fruit period, among 200 clones analyzed, 31 clones
(15.50%) belonged to α-proteobacteria, 67 clones (33.50%) belonged to
β-proteobacteria, 99 clones (49.50%) belonged to γ-proteobacteria,
and three clones (1.50%) belonged to Bacteroidetes. Bacteroidetes, α-proteobacteria,
β-proteobacteria and γ-proteobacteria were made up of 2, 9, 7 and 18
bacterial OTU, respectively. Pseudomonas (34.00%), Acidovorax
(31.00%), Curtobacterium (7.50%), and Sphingomonas (7.00%) were the dominant genera (Table 2).
Of the 200 clones analyzed during the early mature growth period, 6
clones (3%) belonged to α-proteobacteria, 15 clones (7.50%) belonged to
β-proteobacteria, 158 clones (79%) belonged to γ-proteobacteria, 19
clones (9.50%) belonged to Firmicutes, and 2 clones (1%) belonged to Actinobacteria. α-proteobacteria, β-proteobacteria, γ-proteobacteria,
Firmicutes, and Actinobacteria were made up of
6, 11, 20, 10, and 2 bacterial OTU, respectively. Kosakonia
(37.50%), Curtobacterium (10.50%), Erwinia
(7.50%), Pantoea (7.50%), and Luteimonas (7.00%) were the dominant genera (Table
3).
Table
1: Distribution of 16S rRNA clones detected from endophytes in the fruit formation period of Alpinia zerumbet
|
Group |
OTUs |
clones |
% total clones |
Closest NCBI match |
% identity |
|
α-proteobacteria |
8 |
1 |
0.5 |
Roseomonas aerophila 7515T-07(T) |
98.74 |
|
|
|
3 |
1.5 |
Sphingomonas pseudosanguinis
G1-2(T) |
99.57 |
|
|
|
3 |
1.5 |
S. aeria R1-3(T) |
99.10 |
|
|
|
2 |
1.0 |
S. parapaucimobilis NBRC 15100(T) |
99.72 |
|
|
|
5 |
2.5 |
S. sanguinis NBRC 13937(T) |
100 |
|
|
|
1 |
0.5 |
Methylobacterium gossipiicola Gh-105(T) |
99.72 |
|
|
|
8 |
4.0 |
Neokomagataea tanensis AH13(T) |
98.87 |
|
|
|
20 |
10.0 |
Aureimonas ureilytica NBRC 106430(T) |
99.29 |
|
β-proteobacteria |
2 |
1 |
0.5 |
Comamonas kerstersii LMG 3475(T) |
97.37 |
|
|
|
2 |
1.0 |
Acidovorax wautersii DSM 27981(T) |
100 |
|
γ-proteobacteria |
18 |
1 |
0.5 |
Pantoea conspicua LMG 24534(T) |
99.25 |
|
|
|
2 |
1.0 |
P. vagans LMG 24199(T) |
99.57 |
|
|
|
1 |
0.5 |
P. rodasii LMG 26273(T) |
99.55 |
|
|
|
1 |
0.5 |
P. eucalypti LMG
24198(T) |
99.41 |
|
|
|
4 |
2.0 |
P. rwandensis LMG 26275(T) |
98.96 |
|
|
|
20 |
10.0 |
P. ananatis LMG 2665(T) |
100 |
|
|
|
15 |
7.5 |
P. anthophila LMG 2558(T) |
99.72 |
|
|
|
1 |
0.5 |
Tatumella citrea LMG 22049(T) |
99.10 |
|
|
|
1 |
0.5 |
Pseudomonas seleniipraecipitans CA5(T) |
98.52 |
|
|
|
4 |
2.0 |
P. psychrotolerans DSM 15758(T) |
100 |
|
|
|
1 |
0.5 |
P. oleovorans subsp. lubricantis RS1(T) |
95.94 |
|
|
|
1 |
0.5 |
Rouxiella chamberiensis 130333(T) |
99.44 |
|
|
|
2 |
1.0 |
Erwinia rhapontici ATCC 29283(T) |
99.44 |
|
|
|
1 |
0.5 |
Rosenbergiella nectarea 8N4(T) |
99.86 |
|
|
|
15 |
7.5 |
Acinetobacter nectaris SAP 763.2(T) |
99.86 |
|
|
|
8 |
4.0 |
Flavobacterium acidificum LMG 8364(T) |
99.57 |
|
|
|
50 |
25.0 |
Curtobacterium plantarum CIP 108988(T) |
99.58 |
|
|
|
12 |
6.0 |
Tatumella saanichensis NML 06-3099(T) |
99.30 |
|
Bacteroidetes |
2 |
5 |
2.5 |
Chryseobacterium hagamense RHA2-9(T) |
99.25 |
|
|
|
1 |
0.5 |
C. lineare XC0022(T) |
99.00 |
|
Actinobacteria |
1 |
8 |
4.0 |
Microbacterium testaceum DSM 20166(T) |
99.30 |
Of the 200 clones analyzed during the middle mature period, 72 clones
(36%) belonged to α-proteobacteria, 33 clones (16.50%) belonged to
β-proteobacteria, 66 clones (33%) belonged to γ-proteobacteria, 8
clones (4%) belonged to δ-proteobacteria, 7 clones (3.50%) belonged to
Firmicutes, 7 clones (3.50%) belonged to Actinobacteria, and 7 clones (3.50%)
belonged to Bacteroidetes. Firmicutes, Actinobacteria, Bacteroidetes,
α-proteobacteria, β-proteobacteria, γ-proteobacteria, and
δ-proteobacteria were made up of 6, 3, 3, 26, 9, 10 and 1 bacterial OTU,
respectively. Curtobacterium (14%), Methylobacterium (10.50%), Paraburkholderia
(9%), Rhizobium (8.50%), and Caulobacter (8%) were the dominant
genera (Table 4).
During the late mature period, among 200 clones, 35 clones (17.5%)
belonged to α-proteobacteria, 10 clones (5%) belonged to
β-proteobacteria, 80 clones (40%) belonged to γ-proteobacteria, 4
clones (2%) belonged to δ-proteobacteria, 9 clones (4.50%) belonged to
Firmicutes, 62 clones (31%) belonged to Actinobacteria. α-proteobacteria,
β-proteobacteria, γ-proteobacteria, δ-proteobacteria,
Firmicutes, and Actinobacteria were made up of 17, 6, 10, 2, 4, and 7 bacterial
OTU, respectively. Sphingobacterium (28.50%), Stenotrophomonas
(13.50%), Luteimonas (12.50%), and Methylobacterium
(8.00%) were the dominant genera (Table 5).
A consistent succession of community structures could be observed in endophytic bacteria of A. zerumbet seeds. At the
growth period one, Curtobacterium was the
dominant genera, with 25%, and the sec and third genera were Pantoea and Acinetobacter,
with 22 and 7.5%, respectively. At growth period two, Pseudomonas was
the dominant genera, with 34%, and the sec and third genera were Acidovorax and Curtobacterium,
with 31 and 7.5%, respectively. At growth period three, Kosakonia
was the dominant genera, with 37.5%, and the sec and third genera were Curtobacterium, Erwinia, and Pantoea, with 10.5, 7.5, and 7.5%, respectively. At
growth period four, Curtobacterium was the
dominant genera, with 14%, and the sec and third genera were Methylobacterium and Paraburkholderia,
with 10.5 and 9%, respectively. At growth period five, Sphingobacterium
was the dominant genera, with 28.5%, and the sec and third genera were Stenotrophomonas
and Luteimonas, with 13.5 and 12.5%,
respectively. It is clear that Curtobacterium
appeared during the first four growth periods, and reached 25, 7.5, 10.5 and
14%, respectively. The tendency for the occurrence of Curtobacterium
first decreased and then increased. Similarly, Pantoea
appeared during growth periods one and three, and reached 22 and 7.5%,
respectively, dipping gradually. Noticeably, during growth period three, the
clone of Kosakonia reached its peak at 37.5%.
In addition, both Erwinia and Pantoea reached
their lowest point of 7.5%. In addition, the genera in Acinetobacter during
growth period one, Curtobacterium during
growth period two, Erwinia and Pantoea during
growth period three, all reached their lowest occurrence at 7.5% (Table 6).
Table
2: Distribution of
16S rRNA clones detected from endophytes
in the young fruit period of Alpinia zerumbet
|
Group |
OTUs |
clones |
% total clones |
Closest NCBI match |
% identity |
|
α-proteobacteria |
9 |
2 |
1.0 |
Allorhizobium oryzae Alt505 (T) |
97.18 |
|
|
|
1 |
0.5 |
Sphingomonas yabuuchiae GTC 868 (T) |
99.72 |
|
|
|
1 |
0.5 |
S. pseudosanguinis G1-2 (T) |
99.86 |
|
|
|
10 |
5.0 |
S. aeria R1-3 (T) |
99.40 |
|
|
|
1 |
0.5 |
S. sanguinis NBRC 13937 (T) |
99.86 |
|
|
|
1 |
0.5 |
S. parapaucimobilis NBRC 15100 (T) |
99.72 |
|
|
|
1 |
0.5 |
Rhizobium larrymoorei ATCC 51759 (T) |
99.44 |
|
|
|
10 |
5.0 |
R. qilianshanense CCNWQLS01 (T) |
97.11 |
|
|
|
4 |
2.0 |
Aureimonas ureilytica NBRC 106430 (T) |
99.01 |
|
β-proteobacteria |
7 |
1 |
0.5 |
Delftia lacustris LMG 24775 (T) |
99.01 |
|
|
|
1 |
0.5 |
Comamonas kerstersii LMG 3475 (T) |
97.54 |
|
|
|
1 |
0.5 |
Curvibacter gracilis 7-1 (T) |
98.87 |
|
|
|
1 |
0.5 |
Methylovorus menthalis MM (T) |
99.13 |
|
|
|
2 |
1.0 |
Acidovorax oryzae ATCC 19882 (T) |
93.32 |
|
|
|
60 |
30.0 |
A. wautersii DSM 27981 (T) |
100 |
|
|
|
1 |
0.5 |
Herbaspirillum chlorophenolicum CPW301 (T) |
98.30 |
|
γ-proteobacteria |
18 |
1 |
0.5 |
Pseudomonas caricapapayae ATCC 33615 (T) |
99.86 |
|
|
|
1 |
0.5 |
P. rhodesiae CIP 104664 (T) |
98.88 |
|
|
|
1 |
0.5 |
P. hibiscicola ATCC 19867 (T) |
97.85 |
|
|
|
6 |
3.0 |
P. psychrotolerans DSM 15758 (T) |
99.16 |
|
|
|
1 |
0.5 |
P. libanensis CIP 105460 (T) |
99.86 |
|
|
|
1 |
0.5 |
P. trivialis DSM 14937 (T) |
99.16 |
|
|
|
3 |
1.5 |
P. paralactis WS4992 (T) |
99.55 |
|
|
|
6 |
3.0 |
P. cerasi 58 (T) |
99.72 |
|
|
|
12 |
6.0 |
P. tolaasii ATCC 33618 (T) |
99.44 |
|
|
|
30 |
15.0 |
P. azotoformans DSM 18862 (T) |
99.86 |
|
|
|
6 |
3.0 |
P. simiae OLi (T) |
99.69 |
|
|
|
2 |
1.0 |
Flavobacterium acidificum LMG 8364 (T) |
98.30 |
|
|
|
5 |
2.5 |
Pantoea anthophila LMG 2558 (T) |
99.44 |
|
|
|
6 |
3.0 |
P. ananatis LMG 2665 (T) |
100 |
|
|
|
1 |
0.5 |
Kosakonia oryziphila REICA_142 (T) |
97.79 |
|
|
|
1 |
0.5 |
Xanthomonas codiaei LMG 8678 (T) |
99.58 |
|
|
|
15 |
7.5 |
Curtobacterium plantarum CIP 108988 (T) |
99.86 |
|
|
|
1 |
0.5 |
Acinetobacter lwoffii NCTC 5866 (T) |
99.86 |
|
Bacteroidetes |
2 |
1 |
0.5 |
Pedobacter sandarakinus DS-27 (T) |
98.14 |
|
|
|
2 |
1.0 |
Chryseobacterium hagamense RHA2-9 (T) |
99.25 |
Discussion
Plant seeds are
infected by microorganisms, which are necessary for their normal germination (Stöckel et al. 2014; Mehra
et al. 2017). Zhang et al. (2017) found that many microbial
communities occurred in the seeds and on the surface of the seeds. The
structure of the microbial community was affected by their physiological state,
had a significant effect on the health of plant seeds. However, compared to the
extensive research conducted on plant rhizosphere microorganisms, less research
has been devoted to seed-related endophytes (Stroheker
et al. 2018). Therefore, the research on the endophytic community
structure of A. zerumbet seeds provides new
information regarding the influence of endophytic bacteria. The bacteria
present on the surface of A. zerumbet seeds were
washed and disinfected. These bacteria were ignored in the subsequent analysis
because it is difficult to prevent pollution from the environment and the sources of such bacteria are
highly diverse.
To understand the
correlation between seed endophytic bacteria and community diversity in
different growth periods, A. zerumbet seeds were selected that were in
five critical growth periods (fruit formation period, young fruit period, early
mature period, middle mature period, and late mature period). Some studies have
found that Bacillus, Agrobacterium, Burkholderia,
and Enterobacter groups do not change during different growth stages of
rice, although their numbers gradually increased from the seedling stage to the
booting stage and decreased from the filling stage to the milk ripening stage.
The changes in the number of endophytic bacterial groups tended to be similar
to their overall change (Zhang et al. 2015). Wang et al.
(2015) analyzed the diversity of endophytic bacteria in the seedling stage, tillering stage, flowering stage, and seed setting stage in
rice. Among the Burkholderia, Herbaspirillum, and Flavobacterium
isolated, Burkholderia was dominant. Lin et
al. (2018) studied Pennisetum spp., and
their endophytic bacterial diversity was analyzed. Cyanobacteria
and Proteobacteria were Table 3: Distribution of 16S rRNA
clones detected from endophytes in the early mature
period of Alpinia zerumbet
|
Group |
OTUs |
clones |
% total clones |
Closest NCBI match |
% identity |
|
α-proteobacteria |
6 |
1 |
0.5 |
Sphingomonas sanguinis NBRC 13937 (T) |
99.72 |
|
|
|
1 |
0.5 |
S. aquatilis JSS7 (T) |
99.86 |
|
|
|
1 |
0.5 |
S. hankookensis ODN7 (T) |
99.16 |
|
|
|
1 |
0.5 |
S. herbicidovorans NBRC 16415 (T) |
100.00 |
|
|
|
1 |
0.5 |
Croceicoccus naphthovorans PQ-2 (T) |
97.62 |
|
|
|
1 |
0.5 |
Azospirillum massiliensis URAM1 |
98.45 |
|
β-proteobacteria |
11 |
1 |
0.5 |
Pelomonas saccharophila DSM 654 (T) |
99.86 |
|
|
|
3 |
1.5 |
Duganella ginsengisoli DCY83 (T) |
99.15 |
|
|
|
1 |
0.5 |
Burkholderia thailandensis E264 (T) |
99.29 |
|
|
|
1 |
0.5 |
Paraburkholderia bannensis NBRC 103871 (T) |
99.58 |
|
|
|
1 |
0.5 |
P. susongensis L226 (T) |
98.44 |
|
|
|
1 |
0.5 |
P. oxyphila NBRC 105797 (T) |
99.58 |
|
|
|
1 |
0.5 |
Ralstonia pickettii ATCC 27511 (T) |
99.86 |
|
|
|
2 |
1.0 |
Massilia varians CCUG 35299 (T) |
99.70 |
|
|
|
1 |
0.5 |
Piscinibacter defluvii SH-1 (T) |
99.58 |
|
|
|
2 |
1.0 |
P. aquaticus IMCC1728 (T) |
99.58 |
|
|
|
1 |
0.5 |
Delftia lacustris LMG 24775 (T) |
99.72 |
|
γ-proteobacteria |
20 |
21 |
10.5 |
Curtobacterium plantarum CIP 108988 (T) |
99.86 |
|
|
|
4 |
2.0 |
Pantoea ananatis LMG 2665 (T) |
100.00 |
|
|
|
1 |
0.5 |
P. conspicua LMG 24534 (T) |
99.40 |
|
|
|
8 |
4.0 |
P. anthophila LMG 2558 (T) |
99.72 |
|
|
|
1 |
0.5 |
P. beijingensis LMG 27579 (T) |
99.27 |
|
|
|
1 |
0.5 |
P. brenneri LMG 5343 (T) |
99.84 |
|
|
|
2 |
1.0 |
Klebsiella pneumoniae subspp. pneumoniae DSM 30104 (T) |
99.30 |
|
|
|
2 |
1.0 |
Acinetobacter lwoffii NCTC 5866 (T) |
100.00 |
|
|
|
1 |
0.5 |
A. nectaris SAP 763.2 (T) |
99.86 |
|
|
|
1 |
0.5 |
A. junii CIP 64.5 (T) |
99.86 |
|
|
|
1 |
0.5 |
A. bouvetii DSM 14964 (T) |
99.72 |
|
|
|
1 |
0.5 |
Luteibacter anthropi CCUG 25036 (T) |
100.00 |
|
|
|
2 |
1.0 |
Erwinia persicina NBRC 102418 (T) |
99.86 |
|
|
|
13 |
6.5 |
E. gerundensis EM595 (T) |
99.85 |
|
|
|
7 |
3.5 |
Stenotrophomonas humi DSM 18929 (T) |
100.00 |
|
|
|
1 |
0.5 |
Dyella koreensis BB4 (T) |
99.58 |
|
|
|
1 |
0.5 |
Enhydrobacter aerosaccus LMG 21877 (T) |
99.15 |
|
|
|
1 |
0.5 |
Moraxella osloensis CCUG 350 (T) |
99.86 |
|
|
|
14 |
7.0 |
Luteimonas terrae
THG-MD21 (T) |
99.86 |
|
|
|
75 |
37.5 |
Kosakonia cowanii JCM 10956 (T) |
99.30 |
|
Firmicutes |
10 |
3 |
1.5 |
Bacillus tequilensis KCTC 13622 (T) |
99.58 |
|
|
|
5 |
2.5 |
B. nakamurai NRRL B-41091 (T) |
100.00 |
|
|
|
2 |
1.0 |
B. solani FJAT-18043 (T) |
99.86 |
|
|
|
1 |
0.5 |
Lactobacillus fermentum CECT 562 (T) |
99.58 |
|
|
|
1 |
0.5 |
L. kefiri LMG 9480 (T) |
99.71 |
|
|
|
1 |
0.5 |
Leuconostoc mesenteroides subspp. suionicum DSM 20241 (T) |
99.72 |
|
|
|
1 |
0.5 |
Pediococcus pentosaceus DSM 20336 (T) |
99.58 |
|
|
|
1 |
0.5 |
Thermoactinomyces daqus H-18 (T) |
99.44 |
|
|
|
2 |
1.0 |
Clostridium akagii CK58 (T) |
100.00 |
|
|
|
2 |
1.0 |
C. saccharoperbutylacetonicum N1-4 (HMT)(T) |
99.44 |
|
Actinobacteria |
2 |
1 |
0.5 |
Nocardia ninae OFN 02.72 (T) |
99.02 |
|
|
|
1 |
0.5 |
Cutibacterium acnes
DSM 1897 (T) |
99.44 |
the main bacterial genera. In this study, there were relatively few types
of endophytic bacteria because the seeds were in the process of rapid growth
and required more nutrients during the fruit formation period. From the young
fruit stage to the middle maturity stage, the number and variety of bacteria in
the seeds increased significantly because of the softness of the seeds, which
reached a peak during the middle maturity period, explaining the relative
endophytic bacterial diversity during this period. Because the internal
environment during the early stages of seed development was similar, the same
dominant species can be expected and were found in previous samples.
Zhang et al. (2018) suggested
that the contents and concentrations of water and dry matter in seeds vary
greatly during the maturation process, which affects the types of endophytic
bacteria that can survive in seeds. If endophytic bacteria are more adapted to
the new environment of the seeds, they can accumulate as dominant bacteria
(Shahzad et al. 2017). This conclusion was proven by the results
obtained in this study. For example, Table 4: Distribution of 16S rRNA
clones detected from endophytes in the middle mature
period of Alpinia zerumbet
|
Group |
OTUs |
clones |
% total clones |
Closest NCBI match |
% identity |
|
α-proteobacteria |
26 |
1 |
0.5 |
Sphingomonas kyungheensis THG-B283 (T) |
99.86 |
|
|
|
1 |
0.5 |
S. aquatilis JSS7 (T) |
100.00 |
|
|
|
1 |
0.5 |
S. aeria R1-3 (T) |
98.21 |
|
|
|
1 |
0.5 |
S. abaci C42
(T) |
99.86 |
|
|
|
1 |
0.5 |
S. yunnanensis YIM 003 (T) |
99.58 |
|
|
|
1 |
0.5 |
Sphingobium abikonense NBRC 16140 (T) |
99.15 |
|
|
|
1 |
0.5 |
Rhizobium yantingense H66 (T) |
99.15 |
|
|
|
10 |
5.0 |
R. nepotum 39/7 (T) |
99.86 |
|
|
|
2 |
1.0 |
R. qilianshanense CCNWQLS01 (T) |
99.71 |
|
|
|
4 |
2.0 |
R. larrymoorei ATCC 51759 (T) |
99.58 |
|
|
|
2 |
1..0 |
Devosia subaequoris HST3-14 (T) |
98.59 |
|
|
|
1 |
0.5 |
Mesorhizobium plurifarium LMG 11892 (T) |
100.00 |
|
|
|
2 |
1.0 |
Roseomonas aerophila 7515T-07 (T) |
98.60 |
|
|
|
1 |
0.5 |
Aureimonas ureilytica NBRC 106430 (T) |
99.29 |
|
|
|
1 |
0.5 |
A. frigidaquae
JCM 14755 (T) |
99.86 |
|
|
|
5 |
2.5 |
Shinella yambaruensis MS4 (T) |
99.44 |
|
|
|
15 |
7.5 |
Caulobacter segnis ATCC 21756 (T) |
99.01 |
|
|
|
1 |
0.5 |
C. mirabilis FWC38
(T) |
97.74 |
|
|
|
10 |
5.0 |
Methylobacterium komagatae 002-079 (T) |
97.75 |
|
|
|
3 |
1.5 |
M. brachiatum B0021 (T) |
100.00 |
|
|
|
2 |
1.0 |
M. extorquens IAM 12631 (T) |
100.00 |
|
|
|
1 |
0.5 |
M. aerolatum 5413S-11 (T) |
99.27 |
|
|
|
2 |
1.0 |
M. phyllostachyos BL47 (T) |
99.58 |
|
|
|
1 |
0.5 |
M. rhodesianum DSM 5687 (T) |
99.29 |
|
|
|
1 |
0.5 |
M. phyllosphaerae CBMB27 (T) |
99.86 |
|
|
|
1 |
0.5 |
M. suomiense NCIMB 13778 (T) |
99.44 |
|
β-proteobacteria |
9 |
16 |
8.0 |
Paraburkholderia bannensis NBRC 103871 (T) |
99.43 |
|
|
|
1 |
0.5 |
P. tropica Ppe8 (T) |
99.55 |
|
|
|
1 |
0.5 |
P. phytofirmans PsJN
(T) |
99.86 |
|
|
|
1 |
0.5 |
Herbaspirillum aquaticum IEH 4430 (T) |
100.00 |
|
|
|
8 |
4.0 |
Limnobacter thiooxidans CS-K2 (T) |
99.72 |
|
|
|
1 |
0.5 |
Duganella zoogloeoides IAM 12670 (T) |
99.86 |
|
|
|
2 |
1.0 |
Burkholderia thailandensis E264 (T) |
99.15 |
|
|
|
1 |
0.5 |
Pelomonas saccharophila DSM 654 (T) |
99.86 |
|
|
|
1 |
0.5 |
Aquabacterium commune
B8 (T) |
98.51 |
|
|
|
1 |
0.5 |
Xylophilus ampelinus ATCC 33914 (T) |
99.44 |
|
γ-proteobacteria |
10 |
8 |
4.0 |
Xanthomonas cucurbitae LMG 690 (T) |
99.86 |
|
|
|
12 |
6.0 |
Luteimonas terrae THG-MD21
(T) |
100.00 |
|
|
|
1 |
0.5 |
Pseudomonas tolaasii ATCC 33618 (T) |
99.72 |
|
|
|
1 |
0.5 |
Acinetobacter junii CIP 64.5 (T) |
100.00 |
|
|
|
28 |
14.0 |
Curtobacterium plantarum CIP 108988 (T) |
99.44 |
|
|
|
7 |
3.5 |
Pantoea anthophila LMG 2558 (T) |
99.86 |
|
|
|
5 |
2.5 |
Luteibacter anthropi CCUG 25036 (T) |
100.00 |
|
|
|
1 |
0.5 |
Stenotrophomonas humi DSM 18929 (T) |
99.72 |
|
|
|
1 |
0.5 |
Acinetobacter guillouiae CIP 63.46 (T) |
100.00 |
|
|
|
2 |
1.0 |
Escherichia hermannii GTC 347 (T) |
99.72 |
|
δ-proteobacteria |
1 |
8 |
4.0 |
Cystobacter miniatus DSM 14712 (T) |
99.86 |
|
Firmicutes |
6 |
1 |
0.5 |
Leuconostoc mesenteroides subspp. suionicum |
99.72 |
|
|
|
1 |
0.5 |
Bacillus maritimus KS16-9 (T) |
99.44 |
|
|
|
2 |
1.0 |
Lactococcus lactis subspp. tructae L105 (T) |
100.00 |
|
|
|
1 |
0.5 |
Paenibacillus kyungheensis DCY88 (T) |
99.86 |
|
|
|
1 |
0.5 |
Nocardia jinanensis NBRC 108249 (T) |
99.72 |
|
|
|
1 |
0.5 |
Moraxella osloensis CCUG 350 (T) |
98.87 |
|
Actinobacteria |
3 |
5 |
2.5 |
Micrococcus yunnanensis YIM 65004 (T) |
99.86 |
|
|
|
1 |
0.5 |
Geodermatophilus brasiliensis Tu 6233 (T) |
99.72 |
|
|
|
1 |
0.5 |
Leifsonia soli TG-S248
(T) |
98.94 |
|
Bacteroidetes |
3 |
5 |
2.5 |
Chryseobacterium hagamense RHA2-9 (T) |
99.25 |
|
|
|
1 |
0.5 |
Flavobacterium akiainvivens DSM 25510 (T) |
99.00 |
|
|
|
1 |
0.5 |
Pedobacter ureilyticus THG-T11 (T) |
99.41 |
Kosakonia
became the dominant genus in the early maturity period after accumulating
during the first two periods, whereas Sphingobacterium
became the dominant bacterium in the late maturity period after a long period
of accumulation. Abdallah et al. (2016)
reported that many endophytic bacteria resistant to high osmotic pressure are
present in rice seeds at the late growth stage and the abundance of endophytic
bacteria with amylase activity is significantly increased. However, the results
obtained in this study were different. In Table 5: Distribution of 16S rRNA
clones detected from endophytes in the late mature
period of Alpinia
zerumbet
|
Group |
OTUs |
clones |
% total clones |
Closest NCBI match |
% identity |
|
α-proteobacteria |
17 |
1 |
0.5 |
Brevundimonas viscosa CGMCC 1.10683 (T) |
98.54 |
|
|
|
1 |
0.5 |
B. vesicularis NBRC 12165 (T) |
98.84 |
|
|
|
2 |
1.0 |
Rhizobium rubi NBRC 13261 (T) |
99.58 |
|
|
|
1 |
0.5 |
R. larrymoorei
ATCC 51759 (T) |
99.58 |
|
|
|
2 |
1.0 |
R. nepotum 39/7 (T) |
99.86 |
|
|
|
1 |
0.5 |
Methylobacterium phyllostachyos BL47 (T) |
99.58 |
|
|
|
1 |
0.5 |
M. platani PMB02 (T) |
98.73 |
|
|
|
1 |
0.5 |
M. aerolatum 5413S-11 (T) |
98.83 |
|
|
|
5 |
2.5 |
M. komagatae 002-079 (T) |
97.75 |
|
|
|
5 |
2.5 |
M. brachiatum B0021 (T) |
100 |
|
|
|
1 |
0.5 |
M. goesingense iEII3 (T) |
98.87 |
|
|
|
2 |
1.0 |
M. suomiense NCIMB 13778 (T) |
98.45 |
|
|
|
1 |
0.5 |
Caulobacter mirabilis FWC38 (T) |
97.88 |
|
|
|
5 |
2.5 |
C. fusiformis ATCC 15257 (T) |
97.88 |
|
|
|
1 |
0.5 |
Sphingomonas aquatilis JSS7 (T) |
99.58 |
|
|
|
1 |
0.5 |
S. asaccharolytica NBRC 15499 (T) |
98.74 |
|
|
|
4 |
2.0 |
Aureimonas frigidaquae JCM 14755 (T) |
99.44 |
|
β-proteobacteria |
6 |
1 |
0.5 |
Xenophilus aerolatus 5516S-2 (T) |
99.71 |
|
|
|
2 |
1.0 |
Paraburkholderia bannensis NBRC 103871 (T) |
99.58 |
|
|
|
2 |
1.0 |
P. nodosa R-25485 (T) |
99.57 |
|
|
|
1 |
0.5 |
Herbaspirillum aquaticum IEH 4430 (T) |
99.58 |
|
|
|
2 |
1.0 |
Delftia lacustris LMG 24775 (T) |
99.72 |
|
|
|
2 |
1.0 |
Ideonella sakaiensis 201-F6 (T) |
98.44 |
|
γ-proteobacteria |
10 |
1 |
0.5 |
Xanthomonas cucurbitae LMG 690 (T) |
99.30 |
|
|
|
1 |
0.5 |
Acinetobacter bouvetii DSM 14964 (T) |
99.86 |
|
|
|
1 |
0.5 |
Pantoea anthophila LMG 2558 (T) |
99.72 |
|
|
|
3 |
1.5 |
Luteibacter anthropi CCUG 25036 (T) |
100 |
|
|
|
7 |
3.5 |
Stenotrophomonas humi DSM 18929 (T) |
99.72 |
|
|
|
20 |
10.0 |
S. rhizophila DSM 14405 (T) |
99.44 |
|
|
|
2 |
1.0 |
Escherichia coli NCTC9001 (T) |
99.30 |
|
|
|
12 |
6.0 |
Erwinia persicina NBRC 102418 (T) |
99.86 |
|
|
|
25 |
12.5 |
Luteimonas terrae THG-MD21 (T) |
99.58 |
|
|
|
8 |
4.0 |
Curtobacterium plantarum CIP 108988 (T) |
98.73 |
|
δ-proteobacteria |
2 |
2 |
1.0 |
Melittangium lichenicola ATCC 25946 (T) |
99.71 |
|
|
|
2 |
1.0 |
Cystobacter miniatus DSM 14712 (T) |
99.71 |
|
Firmicutes |
4 |
2 |
1.0 |
Leuconostoc suionicum DSM 20241 (T) |
99.58 |
|
|
|
5 |
2.5 |
Bacillus solani
FJAT-18043 (T) |
99.44 |
|
|
|
1 |
0.5 |
B. maritimus KS16-9 (T) |
99.58 |
|
|
|
1 |
0.5 |
Planomicrobium soli XN13 (T) |
99.44 |
|
Actinobacteria |
7 |
2 |
1.0 |
Microbacterium testaceum DSM 20166 (T) |
99.58 |
|
|
|
1 |
0.5 |
M. proteolyticum RZ36 (T) |
99.30 |
|
|
|
1 |
0.5 |
Sphingobacterium lactis DSM 22361 (T) |
97.99 |
|
|
|
56 |
28.0 |
Sphingobacterium hotanense XH4 (T) |
99.85 |
|
|
|
1 |
0.5 |
Hymenobacter fastidiosus VUG-A124 (T) |
95.44 |
|
|
|
1 |
0.5 |
Pedobacter humi THG S15-2 (T) |
99.56 |
the
fourth and fifth periods, the dominant bacteria in the seeds were almost
non-exclusive, which may have been caused by the different selectivity of the
species in different regions.
Sphingomonas, Methylobacterium, Microbacterium, Pseudomonas
and Rhizobium are common dominant bacteria in plant seeds. (Chaudhry et al. 2016; Antunes et al. 2017; Durand et
al. 2017; Tavares et al. 2018; Verma and White 2018; Zhang et al.
2018). The first dominant genera in the five
growth stages were Curtobacterium, Pseudomonas, Kosakonia,
Curtobacterium, and Sphingobacterium,
respectively. Curtobacterium was the
dominant bacteria in the first four stages of the fruit formation process in A.
zerumbet, indicating that it was closely related
to seed development. Therefore, it is speculated that endophytic bacteria are
able to adapt and survive in new internal environments in the seeds, and
permanently become one of the dominant bacteria. Some researchers have studied
the growth-promoting characteristics of rice endophytic Curtobacterium
citreum and found that it capable of producing
IAA, dissolving phosphorus, and fixing nitrogen (Xu et al. 2014).
Studies have also found that Kosakonia has
good nitrogen-fixing effects; Pseudomonas has a certain bacteriostatic
activity, and Sphingobacterium is an
antagonistic bacterium (Li et al. 2016; Shcherbakov
et al. 2017; Yang et al. 2017). In addition, Pantoea
was considered to be the major dominant genus in rice seeds and common bacteria
that promote plant growth (Campestre et al.
2016; Megías et al. 2017). Pantoea
was the dominant genus in the first and third Table 6: Comparison of dominant genera of samples at
five different growth stages
|
Growth stages |
Genera |
|
P1 (fruit formation period) |
Curtobacterium (25.0%) |
|
|
Pantoea (22.0%) |
|
|
Acinetobacter (7.5%) |
|
P2 (young fruit period) |
Pseudomonas (34.0%) |
|
|
Acidovorax (31.0%) |
|
|
Curtobacterium (7.5%) |
|
P3 (early mature period) |
|
|
|
Curtobacterium (10.5%) |
|
|
Erwinia (7.5%) |
|
|
Pantoea (7.5%) |
|
P4 (middle mature period) |
Curtobacterium (14.0%) |
|
|
Methylobacterium (10.5%) |
|
|
Paraburkholderia (9.0%) |
|
P5 (late mature period) |
|
|
|
Stenotrophomonas (13.5%) |
|
|
Luteimonas (12.5%) |
growth stages in this study. Therefore, it can be inferred that the results
of this study will be of great significance for future microbial regulation of A. zerumbet-related products using the endophytes with special functions.
Conclusion
The first
population of dominant bacteria from A. zerumbet seeds belonged to Curtobacterium (25.0%), Pseudomonas (34.0%), Kosakonia (37.5%), Curtobacterium
(14.0%), and Sphingobacterium (28.5%) during
different growth stages, which may have been caused by the complex
environmental conditions in the area where A. zerumbet plants are
located. Related research from the direction of endophytic bacterial
communities may bring new ideas for the exploitation of plant resources. The
results are of great significance to the development of the theory of plant
microbial ecology.
Acknowledgments
The first author
acknowledges that this work was co-supported by Scientific Research Project of Yibin University (grant no.
2018RC09) and Science and Technology Bureau Project of Yibin
(grant no. 2017ZSF009-6).
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