Disease severity index of TBW. Disease incidence (I), disease index (DI) and control efficacy with five treatments were calculated at 100 d post-transplantation (Table 1). The I and DI of the control (CK) were significantly higher than those of NaHS treatments. As the concentration of NaHS increased from 200 mg/L to 800 mg/L, the I and DI continuously decreased from 44.22–15.21%, and 13.21 to 4.26, respectively. With the increase of NaHS concentration, the control efficacy increased gradually. All the results suggested that the application of NaHS reduce the disease incidence and disease index of TBW, and the control efficacy of TBW is as high as 89.49%.
Table 1
The occurrence of tobacco bacterial wilt in different concentration of NaHS.
Treatments
|
disease incidence (%)
|
disease index
|
Control efficacy (%)
|
CK
|
89.34 ± 3.69 a
|
40.56 ± 2.29 a
|
0 d
|
NaHS200
|
44.22 ± 5.94 b
|
13.21 ± 1.52 b
|
67.12 ± 2.82 c
|
NaHS400
|
29.35 ± 6.99 c
|
8.76 ± 1.10 bc
|
78.41 ± 2.73 b
|
NaHS600
|
20.58 ± 2.34 cd
|
6.93 ± 1.89 c
|
82.98 ± 3.32 ab
|
NaHS800
|
15.21 ± 1.63 d
|
4.26 ± 1.07 c
|
89.49 ± 1.23 a
|
All data are presented as the mean ± SE. The different lowcase letters in the same column indicate significant differences at p < 0.05 based on LSD test among different concentrations of NaHS. |
Effects of NaHS application on soil physicochemical properties. Seven physicochemical properties of the rhizosphere soil were analyzed (Table 2). The value of pH, alkali-hydrolyzed nitrogen (AN), available phosphorous (AP) and organic matter (OM) were increased as the concentration of NaHS increased from 200 mg/L to 800 mg/L. There was no significant difference in available potassium (AK) and exchangeable calcium (Ca) between NaHS treatments and CK. The results showed that the application of NaHS could increase the soil pH, AN, AP and OM. What’s more, pH, AN, AP and OM showed significantly negative (p < 0.01) correlation with the incidence of TBW (Table S1). These results indicated that the application of NaHS may reduce the incidence of TBW by changing soil physicochemical properties.
Table 2
Effects of different concentrations of NaHS on Soil physicochemical properties.
Treatments
|
pH
|
AN (mg/kg)
|
AP (mg/kg)
|
AK (mg/kg)
|
OM (%)
|
Ca (mg/kg)
|
Mg (mg/kg)
|
CK
|
5.22 ± 0.13 d
|
141.58 ± 7.18 d
|
75.30 ± 3.60 c
|
800.10 ± 15.90 a
|
2.40 ± 0.19 d
|
999.04 ± 251.48 a
|
128.47 ± 6.52 a
|
NaHS200
|
5.59 ± 0.15 c
|
164.39 ± 3.80 c
|
84.00 ± 1.24 bc
|
756.87 ± 25.69 a
|
3.18 ± 0.10 c
|
973.42 ± 127.30 a
|
123.03 ± 2.82 a
|
NaHS400
|
6.14 ± 0.07 b
|
170.31 ± 3.44 bc
|
85.92 ± 4.31 bc
|
744.38 ± 36.12 a
|
3.38 ± 0.16 bc
|
998.58 ± 292.25 a
|
120.25 ± 3.40 a
|
NaHS600
|
6.36 ± 0.09 ab
|
184.48 ± 3.59 ab
|
97.52 ± 5.88 ab
|
750.69 ± 41.77 a
|
3.69 ± 0.07 b
|
990.50 ± 193.05 a
|
112.69 ± 18.00 a
|
NaHS800
|
6.49 ± 0.06 a
|
188.55 ± 6.98 a
|
115.75 ± 11.42 a
|
754.51 ± 8.87 a
|
4.19 ± 0.13 a
|
987.96 ± 138.90 a
|
103.64 ± 2.15 b
|
Soil chemical properties in soils are presented as the mean ± SE. The different lowercase letters in the same column indicate significant differences at p < 0.05 based on LSD test among different concentrations of NaHS. |
Effects of NaHS application on bacterial diversity and community. In total 679,451 high-quality raw sequences with the average length of 252 bps for bacteria were obtained from rhizospherial soil samples after quality filtering. The OTUs, Chao1 and Shannon index were used to evaluate and compare the richness and diversity of bacterial community among different treatments (Table S2). The OTUs, Chao1 and Shannon index were lower in the rhizophere soil of NaHS treatments than the CK. With the increase of NaHS concentration from 0 mg/L to 600 mg/L, the OTUs, Chao1 and Shannon index decreased gradually. Comparing with NaHS600 treatment, the OTUs, Chao1 and Shannon index in NaHS800 treatment was slightly higher (Table S2). While, the OTUs, Chao1 and Shannon index in NaHS800 treatment significantly lower than the CK. This result suggested that NaHS treatments could change the richness and diversity of soil bacterial community.
Principal coordinate analysis (PCoA) were carried out using weighted UniFrac distance in different treatments, and PC1 and PC2 explained 55.21% of total bacterial community. Bacterial community from CK and NaHS200 were clustered together, while the bacterial community of the CK, NaHS400, NaHS600 and NaHS800 were respectively separated from each other (Fig. 1A). This result indicated that the bacterial community structure of NaHS400, NaHS600 and NaHS800 were different from CK. A total of 50 bacterial phyla were identified from all soil samples. The top ten abundant bacterial phyla were selected to compare the changes of bacterial community in rhizosphere soil of five treatments, the relative abundance of the top ten predominant phyla totaled up to 94.27 ~ 97.87% (Fig. 1B). Among the top ten bacterial phyla, Proteobacteria included the pathogen R. solanacearum was the most dominant (48.01 ~ 56.91%), and followed by Verrucomicrobia (5.02 ~ 12.25%), Bacteroidetes (3.28 ~ 10.33%), Firmicutes (4.24 ~ 5.92%), Gemmatimonadetes (3.55 ~ 6.94%), Acidobacteria (3.24 ~ 6.13%), Actinobacteria (2.24 ~ 5.28%), Saccharibacteria (2.24 ~ 3.84%), Cyanobacteria (2.74 ~ 4.89%) and Chloroflexi (0.92 ~ 4.23%). The relative abundance of Proteobacteria in NaHS800 treatment was lower than that in other treatments, while the relative abundance of Verrucomicrobia and Bacteroidetes were higher than that in other treatments (Fig. 1B). The Heatmap analysis of the top 40 genera with hierarchical clusters was used to identify the different composition of bacterial community structure. There were distinctions of bacterial community structures among different treatments in the Heatmap. The application of NaHS significantly increased the abundances of Streptomyces, Microvirga, Rhodococcus, Haliangium, Paenibacillus, Chthonomonas, Bacillus, Solirubrobacter, Gaiella, Lysobacter, Pseudolabrys, Pseudomonas, Granulicella, Stenotrophomonas, Flavobacterium and Rhizobium. In contrast, NaHS significantly decreased the abundances of Massilia, Acidibacter and Ralstonia (pathogen of bacterial wilt) (Fig. 1C). These results suggested that NaHS application play impact on the the structure of bacterial community.
Further analyses were carried out at the genus level, and the different distributions of the top forty abundant bacterial genera among the five treatments were illustrated in Fig. 2. Twelve varied among the five treatments were significantly different, including Solirubrobacter, Rhodococcus, Rhizobium, Ralstonia, Pseudomonas, Paenibacillus, Microvirga, Lysobacter, Haliangium, Granulicella, Flavobacterium and Bacillus. The genus Solirubrobacter which was dominant in NaHS treatments, and occupied low percentage in CK (Fig. 2). The trends in change in the genera Rhodococcus, Rhizobium, Pseudomonas, Paenibacillus, Microvirga, Lysobacter, Haliangium, Granulicella, Flavobacterium and Bacillus were the same as that in Solirubrobacter. In contrast, Ralstonia was dominant in CK, and decreased significantly in NaHS treatments (Fig. 2).
Effects of NaHS application on fungal diversity and community. The difference of the OTUs, Chao1 and Shannon index of fungal community among different treatments were also analyzed (Table S2). There was no significant difference in OTUs, Shannon and Chao1 indexes between CK and NaHS 200. With increase of NaHS concentration from 400 mg/L to 800 mg/L, the OTUs, Chao1 and Shannon index reduced significantly. The results showed that NaHS application could impact the diversity and richness of soil fungi.
According to PCoA analysis, PC1 and PC2 explained 23.47% and 13.75% of the total fungal community variations respectively (Fig. 3A). The distribution of fungi among different treatments was relatively discrete, indicating that there were obvious differences in the fungal community structure among different treatments. 6 main known fungal phyla were identified from all soil samples, including Ascomycota (43.56 ~ 73.45%), followed by Basidiomycota (7.98 ~ 28.21%), Chytridiomycota (6.84 ~ 30.15%), Glomeromycota (1.00 ~ 8.16%), Neocallimastigomycota (0.05 ~ 6.19%) and Zygomycota (0.66 ~ 9.31%) (Fig. 3B). The relative abundance of Ascomycota decreased as the concentration of NaHS increased from 200 mg/L to 600 mg/L, and slightly increased when the concentration of NaHS was 800 mg/L. However, the relative abundance of Ascomycota was lower in NaHS800 than in CK. The relative abundance of Zygomycota increased as the concentration of NaHS increased from 200 mg/L to 600 mg/L, and significantly decreased when the concentration of NaHS was 800 mg/L. The relative abundance of Basidiomycota, Glomeromycota, Chytridiomycota and Zygomycota also varies with the concentration of NaHS. These results indicated that NaHS altered the fungal community composition, which was associated with NaHS concentration. (Fig. 3B). In the Heatmap for fungal community, The relative abundance of Xanthoria, Monograpella, Candida, Paludomyces, Microidium and Sakaguchia in CK were significantly higher than in NaHS treatment. NaHS800 significantly enriched the relative abundance of Batrachochytrium, Gorgonomyces, Populocrescentia, Cladosporium, Rhodosporidium, Aspergillus, Tomentella, Lycogalopsis, Trichoderma, Pseudocamarosporium, Russula, Byssochlamys and Paecilomyces (Fig. 3C).
The different distributions of the top forty abundant fungal at genus level among the five treatments were analyzed (Fig. 4). Three were significantly different among the five treatments, including Trichoderma, Fusarium and Aspergillus. The genus Trichoderma and Aspergillus were dominant in NaHS, and occupied low percentage in CK (Fig. 4). In contrast, Fusarium was dominant in CK, and decreased significantly in NaHS treatments (Fig. 4).
The relationship between rhizosphere soil physicochemical properties and microbial community. The relationship between rhizosphere soil physicochemical properties and microbial community structure were analysed by redundancy analysis (RDA). The results showed that 64.27% and 59.47% of bacterial and fungal community variation, respectively (Fig. 5). The bacterial Rhodococcus, Solirubrobacter, Paenibacillus, Haliangium, Bacillus, Lysobacter, Pseudomonas, Flavobacterium and Granulicella were positively correlated with pH, AN, AP and OM. While, Ralstonia presented contrasting behavior that was negatively correlated with pH, AN, AP and OM (Fig. 5A). The fungal Trichoderma and Aspergillus were positively correlated with AN, Ca, AK, AP and pH. While, Fusarium showed negatively correlated with AN, Ca, AK, AP and pH (Fig. 5B). The redundancy analysis revealed that rhizosphere soil AN, Ca, AK, AP and pH had great influence on microbial community.