soil physical and chemical indices and community structure of rhizosphere symbiotic bacteria of ginseng.
Comparison of soil physical and chemical indices in different ecological environments
The findings indicated that the main physicochemical properties differ significantly between forestland and farmland soils and that the same greenhouse-collected soils differ from each other in their physicochemical properties owing to different original soil sources, without significant changes in soil physicochemical properties owing to short-term environmental changes (Table 1).
Table 1
The determination results of soil physical and chemical properties of four ginseng planting sites
Sample
|
Organic matter(g/kg)
|
Available phosphorus
(mg/kg)
|
Available potassium
(mg/kg)
|
Total nitrogen(g/kg)
|
Total phosphorus
(g/kg)
|
pH
|
Water content(%)
|
Altitude(m)
|
Forestland
|
48.07a
|
10.66c
|
331a
|
2.45b
|
0.17a
|
5.71a
|
10.75b
|
792a
|
Farmland
|
23.36c
|
28.04b
|
195c
|
2.34b
|
0.19a
|
5.65ab
|
9.90b
|
482b
|
Greenhose S
|
44.53b
|
65.43a
|
259b
|
2.45b
|
0.20a
|
5.59ab
|
14.50a
|
225c
|
Greenhose Z
|
48.77a
|
3.43d
|
339a
|
2.73a
|
0.24a
|
5.48b
|
7.56c
|
225c
|
Correlation between soil factors and community structure of rhizosphere symbiotic bacteria of ginseng.
There are substantial differences in environmental factors such as organic matter, available phosphorus, and altitude among soils in different ecological environments. The abundance of rhizosphere symbiotic bacteria Proteobacteria, Acidobacteria, Actinobacteria, Firmicutes, Gemmatimonadetes, and Cyanobacteria were significantly affected by soil factors such as soil total phosphorus, pH value, total nitrogen, organic matter, and rapidly available potassium. Among these, soil total phosphorus and pH were the most important factors affecting the level of the ginseng bacterial phylum community structure (Table 2). The abundance of the ginseng rhizosphere bacteria Methylotenera, Chitinophaga, Bacillus, Nocardioides, Inquilinus and Mesorhizobium was significantly affected by soil organic matter content. Rhizobium and Agrobacterium were closely related to soil available phosphorus. Chitinophaga and Streptomyces were significantly affected by soil available K. Luteimonas and Solibacter were significantly affected by soil total nitrogen. Chitinophaga and Streptomyces were significantly affected by soil available K. The genera affected by altitude included Burkholderia and Luteibacter, where soil organic matter was the most important factor affecting the horizontal community structure of the ginseng bacterial genera. Soil organic matter was the most important factor affecting the horizontal community structure of ginseng bacteria (Table 3).
Table 2
Pearson correlation between rhizosphere bacteria (class) and soil parameters in different environments
Phylum
|
Organic matter
|
Available phosphorus
|
Available potassium
|
Total nitrogen
|
Total phosphorus
|
pH
|
Water content
|
Altitude
|
Proteobacteria
|
-0.255
|
0.306
|
-0.325
|
-0.861
|
-0.993**
|
0.972*
|
0.570
|
0.751
|
Acidobacteria
|
0.424
|
-0.522
|
0.556
|
0.952*
|
0.924
|
-0.890
|
-0.689
|
-0.551
|
Actinobacteria
|
-0.988*
|
-0.071
|
-0.863
|
-0.702
|
-0.347
|
0.404
|
-0.096
|
0.216
|
Gemmatimonadetes
|
0.127
|
-0.228
|
0.184
|
0.779
|
0.989*
|
-0.968*
|
-0.537
|
-0.800
|
Verrucomicrobia`
|
0.868
|
0.330
|
0.602
|
0.413
|
0.159
|
-0.255
|
0.490
|
-0.273
|
Bacteroidetes
|
0.365
|
0.802
|
-0.008
|
-0.262
|
-0.314
|
0.197
|
0.942
|
-0.127
|
Chloroflexi
|
0.142
|
-0.246
|
0.204
|
0.791
|
0.990*
|
-0.968*
|
0.549
|
-0.790
|
Planctomycetes
|
0.221
|
-0.263
|
0.279
|
0.836
|
0.996**
|
-0.977*
|
-0.543
|
-0.780
|
Nitrospirae
|
0.365
|
-0.599
|
0.538
|
0.928
|
0.896
|
-0.849
|
-0.764
|
-0.485
|
Firmicutes
|
-0.984*
|
0.162
|
-0.895
|
-0.776
|
-0.422
|
0.468
|
0.015
|
0.237
|
Cyanobacteria
|
0.448
|
0.482
|
-0.996**
|
-0.738
|
-0.259
|
0.266
|
0.250
|
-0.097
|
Armatimonadetes
|
0.356
|
-0.439
|
0.465
|
0.924
|
0.962*
|
-0.933
|
-0.649
|
-0.639
|
Note: “*” and “**” indicate significant difference at 0.05 and 0.01 level. |
Table 3
Pearson correlation between rhizosphere bacteria genus and soil parameters in different environment
Genus
|
Organic matter
|
Available phosphorus
|
Available potassium
|
Total nitrogen
|
Total phosphorus
|
pH
|
Water content
|
Altitude
|
|
Stenotrophomonas
|
0.367
|
0.397
|
0.483
|
0.684
|
0.178
|
0.703
|
0.008
|
0.901
|
Sphingobium
|
0.364
|
0.881
|
0.667
|
0.192
|
0.281
|
0.268
|
0.609
|
0.706
|
Burkholderia
|
0.100
|
0.546
|
0.146
|
0.719
|
0.374
|
0.786
|
0.249
|
-0.983*
|
Pseudomonas
|
0.379
|
0.387
|
0.177
|
0.734
|
0.422
|
0.662
|
0.732
|
0.506
|
Methylotenera
|
-0.984*
|
0.040
|
0.847
|
0.337
|
0.686
|
0.398
|
0.126
|
0.225
|
Janthinobacterium
|
0.754
|
0.137
|
0.574
|
0.723
|
0.789
|
0.787
|
0.072
|
0.708
|
Luteimonas
|
0.633
|
0.342
|
0.662
|
0.903
|
-0.984*
|
0.903
|
0.459
|
0.617
|
Novosphingobium
|
0.330
|
0.414
|
0.458
|
0.696
|
0.199
|
0.719
|
0.013
|
0.918
|
Sphingomonas
|
0.406
|
0.679
|
0.606
|
0.839
|
0.931
|
0.786
|
0.802
|
0.383
|
Pantoea
|
0.372
|
0.401
|
0.489
|
0.679
|
0.171
|
0.698
|
0.004
|
0.898
|
Rhizobium
|
0.085
|
0.951*
|
0.458
|
0.125
|
0.217
|
0.228
|
0.778
|
0.661
|
Kaistobacter
|
0.914
|
0.007
|
0.765
|
0.561
|
0.790
|
0.622
|
0.063
|
0.469
|
Rhodoplanes
|
0.016
|
0.547
|
0.220
|
0.879
|
0.743
|
0.815
|
0.814
|
0.524
|
Phenylobacterium
|
0.306
|
0.772
|
0.577
|
0.401
|
0.085
|
0.468
|
0.451
|
0.838
|
Nocardioides
|
-0.992**
|
0.100
|
0.878
|
0.333
|
0.703
|
0.387
|
0.076
|
0.186
|
Agrobacterium
|
0.160
|
0.999**
|
0.537
|
0.138
|
0.439
|
0.033
|
0.897
|
0.445
|
Dokdonella
|
0.557
|
0.838
|
0.813
|
0.089
|
0.426
|
0.147
|
0.553
|
0.598
|
CandidatusSolibacter
|
0.489
|
0.610
|
0.648
|
0.865
|
0.967*
|
0.825
|
0.726
|
0.440
|
Rhodanobacter
|
0.915
|
0.260
|
0.872
|
0.626
|
0.906
|
0.656
|
0.199
|
0.380
|
Flavobacterium
|
0.233
|
0.914
|
0.170
|
0.046
|
0.175
|
0.075
|
0.902
|
0.455
|
Inquilinus
|
0.986*
|
0.021
|
0.844
|
0.199
|
0.590
|
0.262
|
0.195
|
0.104
|
Mesorhizobium
|
0.996**
|
0.089
|
0.878
|
0.251
|
0.647
|
0.306
|
0.118
|
0.114
|
Lysobacter
|
0.803
|
0.629
|
0.936
|
0.044
|
0.511
|
0.062
|
0.311
|
0.456
|
Streptomyces
|
0.926
|
0.524
|
-0.992**
|
0.396
|
0.831
|
0.397
|
0.346
|
0.012
|
Chitinophaga
|
-0.972*
|
0.382
|
-0.977*
|
0.179
|
0.668
|
0.199
|
0.124
|
0.118
|
Microbacterium
|
0.822
|
0.172
|
0.620
|
0.611
|
0.735
|
0.684
|
0.166
|
0.616
|
Pseudoxanthomonas
|
0.176
|
0.929
|
0.226
|
0.015
|
0.165
|
0.133
|
0.877
|
0.526
|
Luteibacter
|
0.310
|
0.468
|
0.063
|
0.755
|
0.516
|
0.827
|
0.227
|
0.957*
|
Caulobacter
|
0.103
|
0.936
|
0.291
|
0.095
|
0.147
|
0.209
|
0.831
|
0.610
|
Nitrospira
|
0.386
|
0.526
|
0.526
|
0.929
|
0.939
|
0.891
|
0.705
|
0.556
|
Bacillus
|
-0.984*
|
0.007
|
0.835
|
0.267
|
0.629
|
0.331
|
0.182
|
0.176
|
Paenibacillus
|
0.876
|
0.052
|
0.714
|
0.608
|
0.791
|
0.671
|
0.076
|
0.542
|
Bradyrhizobium
|
0.577
|
0.240
|
0.402
|
0.606
|
0.206
|
0.538
|
0.610
|
0.482
|
Methylibium
|
0.076
|
0.092
|
0.087
|
0.980*
|
0.712
|
-0.972*
|
0.427
|
0.872
|
Flavisolibacter
|
0.050
|
0.880
|
0.389
|
0.582
|
0.652
|
0.483
|
0.990*
|
0.052
|
Thermomonas
|
-0.548
|
0.196
|
0.530
|
0.949
|
0.936
|
0.961*
|
0.370
|
0.750
|
Note: “*” and “**” indicate significant difference at 0.05 and 0.01 level. |
Comparison of the structure and function of the symbiotic bacterial community in the rhizosphere of Chinese ginseng varieties in different ecological environments
The common rhizosphere-dominant phyla of ginseng in different environments were Proteobacteria, Acidobacteria, Actinobacteria, and Bacteroidetes; however, the proportions of their respective rhizosphere-dominant phyla were different (Fig. 1a). The variation range of ginseng rhizosphere abundance of different dominant bacteria in different environments varies, including significant differences in rhizosphere bacterial abundance of Actinobacteria and Bacteroidetes in forestland and farmland environments.
There were differences in the dominant bacterial genera of Chinese ginseng rhizosphere bacteria in different environments, and different environments had their own dominant bacterial genera that were different from other environments (Fig. 1b).
The distribution of the dominant bacterial genera Stenotrophomonas, Novosphingobium, Pantoea in HRrh was significantly different from that in other environments; the dominant bacterial genera Burkholderia, Rhizobium, Sphingobium in DCrh, CRSrh, and CRZrh were significantly different from their distribution in HRrh, and the distribution of Sphingomonas in CRZrh was significantly different from that in other environments, indicating that the proportions of dominant phyla and genera in the ginseng rhizosphere of various groups in different environments were significantly different.
The non-rhizosphere bacterial species were the least abundant in the forestland environment and differed significantly compared to the farmland and greenhouse environments, while the difference between the two samples in the greenhouse was not significant; the rhizosphere bacterial species were the least abundant in the forestland and differed significantly compared to the farmland environment (Fig. 2). Rhizosphere and non-rhizosphere bacterial species differed under different ecological conditions.
The proportion of endemic bacteria enriched in DCrh was the largest and that enriched in CRZrh was the smallest. The proportion of endemic bacteria did not differ significantly among DCrh, HRrh, and CRSrh, but differed significantly from CRZrh (Fig. 3).
The bacterial population alpha diversity index includes Shannon, Simpson, Chao1, ACE, etc., and mainly focuses on species richness and uniformity in a local uniform ecological environment. The diversity and richness of HRrh rhizosphere bacteria were significantly different from those of DCrh, CRSrh, and CRZrh, whereas there were no significant differences between DCrh, CRSrh, and CRZrh. There were no significant differences between the diversity and richness of HRrh rhizosphere bacteria in the other environments (Table 4). This indicates that ecological differences have an effect on rhizosphere bacterial diversity and richness, with Chinese ginseng HRrh having the richest rhizosphere bacterial diversity.
Table 4
Alpha diversity index of Chinese ginseng community in different environment
Sample name
|
Shannon
|
Simpson
|
Chao1
|
ACE
|
HRrh
|
8.878a
|
0.925b
|
1393.294a
|
1409.215a
|
DCrh
|
7.246b
|
0.983a
|
1275.899b
|
1311.298b
|
CRSrh
|
7.124b
|
0.976a
|
1275.130b
|
1290.809b
|
CRZrh
|
7.879b
|
0.986a
|
1295.949b
|
1333.086b
|
There were 458 rhizosphere bacterial OTUs coexisting in the ginseng rhizosphere in all four environments, accounting for 12.49% of the total OTUs (Fig. 4a). As shown in Fig. 1b, the total abundance ratios of HRrh, DCrh, and CRSrh were all higher than 65%, whereas that of CRZrh was lower. Therefore, the OTU distribution of Chinese ginseng rhizosphere bacteria in the first three ecological environments was restored (Fig. 4b). A total of 1372 OTUs were obtained in the three ecological environments, and the number of OTUs in each ecological environment ranged from 808 to 956, with an average value of 891; 499 rhizosphere bacterial OTUs coexisted in the rhizosphere in the three ecological environments, accounting for 36.37% of the total OTUs. Thus, information on the Chinese ginseng core rhizosphere bacteria in different ecological environments was preliminarily obtained. The corresponding bacterial genera of these common OTUs included Stenotrophomonas, Burkholderia, Sphingobium, Rhizobium, Pantoea, and Agrobacterium.
Specific bacterial OTUs are present in the rhizosphere of Chinese ginseng in different ecological environments. Compared to farmland, the relative proportions of Bacteroidetes, Actinobacteria, Planctomycetes, and Gemmatimonadetes were higher in forestland ginseng. However, more strains of Proteobacteria, Acidobacteria, Verrucomicrobia, Chloroflexi, and Firmicutes were included in farmland ginseng-specific rhizosphere bacteria, and Nitrospirae and Cyanobacteria only existed in farmland rhizosphere-specific bacteria, but not in forestland. In addition, there was a higher proportion of unclassified phyla in forestland ecosystems.
At the genus level, forestland-endemic bacterial genera included Prosthecobacter, Cellulomonas, Sporocytophaga, Geobacter, Gemmatimonas, Hymenobacter, Bdellovibrio, Fimbriimonas, and Adhaeribacter. Compared with farmland ginseng, the relative abundance of rhizosphere bacteria in forest ginseng was significantly higher, including Stenotrophomonas, Novosphingobium, Pantoea and Pseudomonas. Compared with the forest ginseng rhizosphere bacteria, the relative abundance of farmland ginseng rhizosphere bacteria was significantly higher in Sphingobium, Burkholderia, Rhizobium, Kaistobacter, Phenylobacterium and Rhodanobacter (Fig. 5). This indicated that there were significant differences in the composition of ginseng rhizosphere bacteria in forestland and farmland, and that the corresponding bacterial community was formed in a specific ecological environment.
Comparison of ginseng bacterial gene function prediction under different ecological environment conditions
As a phylogenetic marker gene, the 16S rRNA gene is a key tool for studying microbial communities; however, it cannot directly demonstrate the functional capacity of the community. PICRUSt was used to perform functional prediction based on the KEGG database for 16S sequencing data of root-related bacterial communities in woodland and farmland environments. A total of 5760 predicted functions were found, and the top 30 predicted functions can be seen in the heat map of the third classification level (Fig. 6).
According to the function prediction heat map, there were differences in the gene functions of ginseng root-related bacterial communities between forestland and farmland. Forestland ginseng root-associated bacterial communities had superior peptidases, chromosomes, molecular chaperones, folding catalysts, membrane and intracellular structural molecules, ribosome production, and bacterial motor proteins. In contrast, glycolysis/gluconeogenesis, pyruvate metabolism, glycolytic serine threonine metabolism, fatty acid metabolism, arginine and proline metabolism, propionate metabolism, valine leucine isoleucine degradation, and butyric acid metabolism were inferior to those of farmland ginseng. The above results indicate that the functions of the rhizosphere bacterial community of forest ginseng are more likely to be related to bacterial chemotaxis to root secretions, biofilm formation, and co-evolution with the host, whereas most of the functions of the rhizosphere bacterial community of farmland are related to nutrient metabolism, such as carbohydrates, amino acids, organic acids, and fatty acids.
Comparison of community structure and function of symbiotic bacteria in the rhizosphere of ginseng of different genotypes
The range of variation in the abundance of the dominant phylum in the rhizosphere of ginseng of different genotypes also differed. Actinobacteria and Bacteroidetes showed significant differences in the rhizosphere bacterial abundance of HRrh and HXrh(Fig. 7a), and there were significant differences in the abundance of Acidobacteria and Actinobacteria between DCrh and DKrh(Fig. 7b). The comparison results of LRSrh, TRSrh, and CRSrh showed that Proteobacteria were significantly different between LRSrh and CRSrh, while the abundance of Actinobacteria and Acidobacteria in LRSrh was significantly different from that of other cultivars, and the abundance of Bacteroidetes in TRZrh was significantly different from that of other cultivars (Fig. 7c). There were significant differences in the proportion of dominant genera in the ginseng rhizosphere among all groups in different environments and in the abundance of HRrh and HXrh at the genus level in Stenotrophomonas, Pantoea and Pseudomonas (Fig. 7d). The abundances of DCrh and DKrh in Sphingobium, Burkholderia and Kaistobacter were significantly different (Fig. 7e). The abundance of Sphingobium and Burkholderia in CRSrh was significantly different from that of other rhizosphere bacteria, and the abundance of Burkholderia and Pseudomonas in LRZrh was significantly different from that of other rhizosphere bacteria (Fig. 7f).
These results indicated that there were significant differences in the abundance distribution of rhizosphere bacteria among different genotypes at specific phylum and genus levels, and genotypes had a great influence on the rhizosphere bacterial community structure of ginseng.
As shown in Table 5, differences in ginseng bacterial diversity among different genotypes in each habitat were compared horizontally. Shannon and Simpson indices reflected species diversity, while Chao1 and ACE indices reflected species richness; the results showed that, in the forest environment, the rhizosphere bacteria Shannon, Simpson, Chao1, and ACE indices of Chinese ginseng were significantly higher than those of American ginseng; in the farmland environment, the rhizosphere bacteria Shannon, Simpson, Chao1, and ACE indices of Korean ginseng varieties were significantly higher than those of Chinese ginseng Damaya. In the greenhouse environment, the Chao1 and ACE indices of the rhizosphere of Korean Tianfeng varieties were between those of Korean Lianfeng and Chinese varieties, and there was no significant difference in rhizosphere bacterial diversity between the Korean ginseng varieties Tianfeng, Lianfeng, and Chinese varieties. This indicated that the rhizosphere bacterial diversity of Chinese ginseng species was better than that of western ginseng, the rhizosphere bacterial diversity of Korean ginseng species was better than that of Chinese ginseng damiana, and the rhizosphere bacterial richness of Korean ginseng Tianfeng was between that of Korean Lianfeng and Chinese ginseng species.
Table 5
Alpha diversity index of rhizosphere bacteria in different varieties of ginseng
Sample name
|
Shannon
|
Simpson
|
Chao1
|
ACE
|
HRrh
|
8.878*
|
0.925
|
1393.294*
|
1409.215*
|
HXrh
|
6.795
|
0.828
|
1223.456
|
1212.347
|
DCrh
|
7.246
|
0.983
|
1275.899
|
1311.298
|
DKrh
|
7.803*
|
0.988
|
1392.497*
|
1421.277*
|
LRSrh
|
7.148a
|
0.971a
|
1398.879a
|
1426.541a
|
TRSrh
|
7.533a
|
0.977a
|
1348.606a
|
1386.268ab
|
CRSrh
|
7.124a
|
0.976a
|
1275.130a
|
1290.809b
|
LRZrh
|
7.271a
|
0.968a
|
1135.124a
|
1185.967b
|
TRZrh
|
8.038a
|
0.991a
|
1196.259a
|
1224.556ab
|
CRZrh
|
7.879a
|
0.986a
|
1295.949a
|
1333.086a
|
The results showed that the proportion of unique OTUs to all OTUs was greater in Chinese ginseng than in Western ginseng, with significant differences in composition and structure. The proportion of OTUs common to both Chinese and Korean ginseng species was higher than 63%. The genera corresponding to these shared OTUs included Novosphingobium, Pantoea, Rhizobium, Sphingomonas, Luteimonas, Rhodanobacter, Agrobacterium (Fig. 8). The percentage of specific OTUs differed for each species. This indicates that differences in ginseng genotypes have a large effect on their rhizosphere bacterial species and distribution.
By comparing the differences in the number of bacterial OTUs of different varieties of ginseng in each habitat, the results showed that the number of bacterial OTUs in the rhizosphere of Chinese ginseng was greater than that of American ginseng (Fig. 9a) and Korean ginseng (Fig. 9b), but the difference was not significant; the number of rhizosphere bacteria of Korean ginseng Tianfeng > Korean ginseng Lianfeng > Chinese ginseng (Fig. 9c), and the differences among the three varieties were significant; the number of rhizobacterial OTUs was Chinese ginseng > Korean ginseng Tianfeng > Korean ginseng Lianfeng (Fig. 9d), but there was no significant difference among the three. This indicates that the number of rhizosphere bacterial OTUs between different varieties in the same environment may or may not be different.
The UPGMA clustering analysis based on weighted UniFrac distance (Fig. 10) could reflect the differences in the community structure of ginseng bacteria of different genotypes. Using 0.05 as the threshold can be divided mainly into HXrh and other genotypes, of which all the other genotypes are ginseng. At a threshold of 0.12, LRZrh, TRZrh, and CRZrh clustered together, TRZrh and CRZrh clustered together first and then with LRZrh, and the distance between them was not correlated with genotypes; at a threshold of 0.17, LRSrh, TRSrh, and CRZrh clustered together, where LRSrh and TRSrh clustered together first and with CRSrh clustered together and correlated with the distance between genotypes. The results showed that the distance between ginseng genotypes was correlated with the hierarchical cluster map of rhizosphere bacterial community structure.