Incidence rate and incidence index of Verticillium wilt
Before RTL, the incidence rate of Verticillium wilt in cotton fields was as high as 87.50%, while it decreased to 62.50% after RTL. When compared to CK, the incidence index of Verticillium wilt in cotton fields decreased by 42.64% after RTL (Table 1, Fig S2). These results showed that RTL can simultaneously decrease the incidence rate and incidence index of cotton Verticillium wilt.
Table 1
Incidence rate and incidence index of Verticillium wilt after CK and restructuring tilth layers (RTL) treatments
Treatment | Incidence rate | Incidence index |
CK | 87.50 ± 6.45% | 41.93 ± 2.80% |
RTL | 62.50 ± 6.45% | 24.05 ± 2.60% |
Soil Physical Property
The soil physical properties changed significantly after RTL (Table 2). When compared to CK, soil bulk density decreased 4.12% in the 20–40 cm soil layer after RTL. Total soil porosity increased significantly in the 0–20 cm and 20–40 cm soil layer, but capillary porosity did not change significantly. Non-capillary porosity increased 30.50% and 101.47% in the 0–20 cm and 20–40 cm soil layer, respectively (Table 2). These results suggest that the increase in non-capillary porosity contributed to the total soil porosity.
Table 2
Soil physical properties in both soil depths after CK and restructuring tilth layers (RTL) treatments
Soil depth | Soil bulk density(g๒cm− 3) | Total soil porosity (%) | Capillary porosity (%) | Non-capillary porosity (%) |
CK | RTL | CK | RTL | CK | RTL | CK | RTL |
0-20cm | 1.431 ± 0.006a | 1.424 ± 0.007a | 46.050 ± 0.288b | 46.967 ± 0.127a | 34.74 ± 0.68a | 32.20 ± 0.80a | 11.31 ± 0.40b | 14.76 ± 0.85a |
20-40cm | 1.498 ± 0.010a | 1.436 ± 0.005b | 44.516 ± 1.277b | 48.428 ± 0.217a | 39.08 ± 1.12a | 37.26 ± 0.15a | 5.44 ± 0.49b | 10.96 ± 0.44a |
Different letters following the data of the same column mean significant difference at P < 0.05.
Table 3
Soil Average temperature and daily range of both two growth stages and soil depth of bacterial after CK and restructuring tilth layers (RTL) treatments
Date | 6/20 | 8/21 |
Soil depth | Treatment | Average temperature | Daily temperature range | Average temperature | Daily temperature range |
10cm | CK | 23.83 ± 0.84a | 5.67 ± 0.45b | 32.83 ± 0.60a | 5.83 ± 0.60b |
RTL | 23.00 ± 0.30ab | 7.87 ± 0.70a | 32.20 ± 0.36a | 7.40 ± 0.62a |
30cm | CK | 22.67 ± 0.50ab | 2.03 ± 0.31c | 30.77 ± 0.65b | 2.10 ± 0.30c |
RTL | 22.17 ± 0.47b | 2.07 ± 0.42c | 30.70 ± 1.05b | 2.23 ± 0.30c |
Different letters following the data of the same column mean significant difference at p < 0.05
Soil moisture content was measured from April to September (Fig. 1). When compared to CK, the moisture content of the 0–20 cm and 20–40 cm layers increased by 23.40% and 24.00%, 18.10% and 9.30%, and 22.10% and 13.10% after RTL at the bud stage (6/20), full blooming stage (7/20) and boll opening stage (9/21), respectively. The effects of RTL on moisture content in the topsoil (0–20 cm) were greater than in the subsoil (20–40 cm). Overall, soil moisture content was increased after RTL, which may have been related to changes in soil porosity.
The average soil temperature at the same soil depth did not differ significantly between CK and RTL. In August, the average soil temperature at 30 cm was lower than that at 10 cm (Table 3). Additionally, the daily temperature range increased at 10 cm in both June and August.
Taxonomic Characterization Of Soil Microbiota
After quality filtering, a total of 1,705,825 bacterial 16S rRNA reads (ranging from 68,746 to 73,951 per sample) (Table S1, S2) and 1,568,465 fungal ITS reads (ranging from 62,745 to 70,146 per sample) were obtained from the 24 analyzed soil samples (Table S1, S3). All of the sequences clustered into 2257 bacterial OTUs and 1358 fungal OTUs with 97% similarity (Table S4).
Bacterial analysis revealed no significant differences in OTUs between RTL and CK during June, but the number of OTUs increased significantly in the subsoil in August (Fig. 2a). Comparison of the number of OTUs among soil layers revealed that, under CK, the number of OTUs in the subsoil was significantly lower than that in the topsoil in August, but there was no significant difference in the number of OTUs in the two layers of soil under RTL. From June to August, the number of OTUs in topsoil remained stable in CK, but decreased significantly in the subsoil. In RTL, the number of OTUs in topsoil increased significantly, while the number of OTUs in subsoil showed no significant difference.
There was no significant difference in the number of fungal OTUs between CK and RTL in June (Fig. 2b), but the number of OTUs in the 20–40 cm layer was significantly higher than that in the 0–20 cm layer. In August, the number of OTUs in the topsoil of the CK treatment was significantly higher than that in the RTL treatment, whereas the number of OTUs in subsoil did not differ significantly between CK and RTL. We also found that the number of OTUs in topsoil was significantly higher in August than June, and that it tended to be uniform between topsoil and subsoil in August.
Changes In Microorganism Diversity And Composition
We further evaluated the microbial diversity of all soil samples based on the Chao1 index and the Shannon index (Table 4). The Chao1 and Shannon indexes of bacteria in June did not differ significantly between CK and RTL. But these two indexes increased in subsoil after RTL in August, However, these indexes indicated that RTL treatment could significantly enhance bacterial richness and evenness in subsoil relative to CK in August. Moreover, the results suggested that the alpha-diversity of bacteria changed over time.
Table 4
Chao1 index and Shannon index in both two growth stages and soil depth of bacterial after CK and restructuring tilth layers (RTL) treatments
Stage | Treatments | Chao1 index | Shannon index |
June-top | CK | 2064.62ab | 6.65ab |
RTL | 2045.99b | 6.32b |
June-sub | CK | 2059.42ab | 6.54ab |
RTL | 2163.69ab | 6.75a |
August-top | CK | 2099.27ab | 6.70ab |
RTL | 2218.22a | 6.92a |
August-sub | CK | 1858.78c | 6.33b |
RTL | 2165.05ab | 6.87a |
Different letters following the data of the same column mean significant difference at P < 0.05.
As we known, 20-40cm soil layer in RTL was replaced from the 0-20cm soil layer in CK, while 0-20cm soil layer in RTL was replaced from 20-40cm soil layer in CK. When compared with the 20–40 cm soil layer in CK, the Chao1 and Shannon indexes of the bacterial community increased significantly in the 0–20 cm soil layer in the RTL treatment in August; however, there was no significant difference in June. Comparison of samples from different soil depths in the same treatment revealed that the Shannon index of subsoil was higher than that of topsoil after RTL, while there was no significant difference between soil depths in CK in June. The Chao1 and Shannon index became more uniform between topsoil and subsoil after RTL in August.
Evaluation of the fungal community revealed no significant change in the Chao1 and Shannon index after RTL, except for samples collected from the 0–20 cm soil during June (Table 5). However, the RTL treatment decreased the variation in fungal evenness and richness between the topsoil and subsoil. Comparison of topsoil from CK with subsoil from RTL revealed that the Chao1 index increased significantly after RTL in June, while the Shannon index decreased significantly after RTL in August. Comparison of subsoil from CK with topsoil from RTL revealed that the Chao1 and Shannon indexes did not change significantly in June or August. Taken together, these results indicated that the bacterial and fungal diversity respond differently to RTL.
Table 5
Chao1 index and Shannon index in both two growth stages and soil depth of fungal after CK and restructuring tilth layers (RTL) treatments
Stage | Treatments | Chao1 index | Shannon index |
June-top | CK | 786.11c | 5.13ab | |
RTL | 903.86b | 4.71bc | |
June-sub | CK | 918.18ab | 5.10ab | |
RTL | 922.02ab | 5.31a | |
August-top | CK | 978.50a | 5.30a | |
RTL | 910.76ab | 4.75abc | |
August-sub | CK | 900.08b | 4.42c | |
RTL | 926.11ab | 4.56bc | |
Different letters following the data of the same column mean significant difference at P < 0.05.
PCoA based on the Bray–Curtis distance was conducted to evaluate differences in the microbial community composition of soil samples subjected to different tillage practices (Fig. 3). For bacteria, PCoA1 and PcoA2 explained 43.03% and 15.12% of the bacterial community composition variance, respectively (Fig. 3a). Moreover, PCoA analysis showed that samples in the CK and RTL treatments fell into different groups, indicating that RTL could change the microbial community composition. The samples from the 20–40 cm layer in the RTL treatment were clustered with samples from the 0–20 cm layer in CK in June and August. Moreover, the number of OTUs and the Chao1 index did not differ significantly between samples from the two soil layers in August. These findings indicated that the richness and community composition of bacteria did not change over time after moving soil from the 0–20 cm layer to a depth of 20–40 cm. Samples collected from the 0–20 cm layer in the RTL treatment and the 20–40 cm layer in CK in August formed two groups. When combined with alpha diversity results, these findings suggested that the community composition changed significantly after moving soil from 20–40 cm to 0–20 cm.
Among fungi, PCoA1 and PCoA2 contributed 34.68% and 19.49% of the total variation, respectively (Fig. 3b). PCoA analysis showed that the samples were mainly divided into three groups. Samples from the 0–20 cm layer in June in CK and RTL were clustered together, as were samples from the 20–40 cm layers in CK and RTL; however, samples from June and August were divided into two groups. These results suggested that the fungal community composition changed with the original soil layer and was affected by the soil layer depth and cotton growth stage.
Species Distribution
Species distribution
The dominant phyla (top 10) identified from the bacterial 16s rRNA genes were the same across both treatments and soil depths (Proteobacteria, Acidobacteria, Actinobacteria, Gemmatimonadetes, Choroflexi, Rokubacteria, Planctomycetes, Bacteroidetes, Nitrospirae and Verrucomicrobia) (Fig. 4a). However, the relative abundance of the dominant phyla varied among treatments. For example, the relative abundance of Proteobacteria in the RTL treatment was higher than that in CK at 0–20 cm and 20–40 cm. The dominant families (top 15) which can be recognized was 8 in both soil layers, there were Gemmatimonadaceae, Pyrinomonadaceae, Nitrosomonadaceae, Sphingomonadaceae, Nitrospiraceae, bacteriap25 and Soilbacteraceae_subgrop3 and A4b, respectively (Fig. 4c). When compared with CK, the relative abundance of Sphingobacteriaceae and Nitrospiraceae increased significantly, while that of Pyrinomonadaceae decreased significantly in the topsoil after RTL treatment in June. In August, the relative abundance of Nitrosomonadaceae and Pyrinomonadaceae changed significantly after RTL treatment in topsoil, while that of Sphingomonadaceae, Gemmatimonadaceae and Nitrospiraceae changed significantly after RTL treatment in the subsoil. Among these organisms, the abundance of Sphingomonadaceae increased, whereas that of all other species decreased (Table S6).
The dominant phyla (top 10) identified for the fungal ITS2 gene across both treatments and soil depths are shown in Fig. 4b. The relative abundance of Ascomycota, Mortierellomycota and Basidiomycota represented more than half of the total, ranging from 67.6–92.7% in the different treatments. The dominant families (top 15) in both soil layers were Mortierellaceae, Aspergillaceae, Nectriaceae, Cladosporiaceae, Chaetomiaceae, Plectosphaerellaceae, Mycosphaerellaceae, Debaryomycetaceae, Stachybotryaceae, Lasiosphaeriaceae, Hypocreales_fam_Incertae_sedis, Bulleribasidiaceae, Pleosporaceae, Clavicipitaceae and Cordycipitaceae (Fig. 4d). In June, the relative abundance of Pleosporaceae, Plectosphaerellaceae, Clavicipitaceae, Aspergillaceae and Mortierellaceae changed significantly after RTL treatment compared with CK. Specifically, the abundance of Pleosporaceae increased in topsoil, whereas that of Plectosphaerellaceae and Clavicipitaceae decreased. Additionally, the abundance of Aspergillaceae increased in subsoil, while that of Mortierellaceae decreased. The abundance of Chaetomiaceae, Lasiosphaeriaceae and Plectosphaerellaceae also changed significantly after RTL treatment in August, with that of Lasiosphaeriaceae decreasing in the topsoil and that of Chaetomiaceae and Plectosphaerellaceae increasing in the subsoil (Table S7).
Metastats analysis showed that, in June, the relative abundance of V. dahliae decreased significantly after RTL in the 0–20 cm soil layer in June, but increased in the 20–40 cm soil layer. In August, the relative abundance of V. dahliae did not differ significantly between CK and RTL (Table 6). RT-PCR showed that the abundance of V. dahliae decreased after RTL in the 0–20 cm soil layer in both June and August, while there was no significant difference between CK and RTL in the 20–40 cm layer (Fig. 5). The change in V. dahliae in topsoil after RTL may be one of the reasons for the decline in the incidence rate and index of cotton Verticillium wilt that was observed in this study.
Sequencing of the 16S gene revealed the presence of biocontrol bacteria, such as Bacillus, Pseudomonas and Pseudoxanthomonas (Table 7). The relative abundance of Bacillus and Pseudomonas increased by 61.24% and 887.22% in topsoil, respectively, while it decreased by 63.77% and 87.98% in subsoil after RTL treatment. Pseudoxanthomonas can inhibit root knot nematodes, which can cause plant root wounds and promote the occurrence of soil borne diseases[24, 25]. The relative abundance of Pseudoxanthomonas increased more than 6 times and 14 times in June and August after RTL treatment, respectively, in the 0–20 cm soil layer.
Redundancy Analysis (RDA)
Redundancy analysis (RDA) was conducted to investigate the relationship between the relative abundance of V. dahliae, Bacillus, Pseudomonas and Pseudoxanthomonas and soil physical properties in August (Fig. 6). A Monte Carlo test showed that the soil bulk density (SBD), total soil porosity (TSP) and non-capillary porosity (NCP) were significantly correlated with changes in soil pathogens and biocontrol bacteria. Specifically, V. dahliae and Pseudoxanthomonas were correlated with SBD and TSP, while Bacillus and Pseudomonas were correlated with NCP. Additionally, Bacillus were negatively correlated with V. dahliae. The relationships between the relative abundance of V. dahliae, Bacillus, Pseudomonas and Pseudoxanthomonas and soil physical properties of all soil samples (June and August) as well as soil samples only from June were also analyzed (Fig S3a, b). In June and August, the soil moisture content (SMC) and soil average temperature (AT) were significantly correlated (Table S8), indicating that microbial community structure was affected by temperature and humidity in the long-term. The relationship in June showed that environmental factors had no significant correlation (Table S9).
Table 6
The differential species between CK and restructuring tilth layers (RTL) treatments of fungi in species level
Treatment | species | CK | RTL | P-value | Fold Change |
Mean | Std.err | Mean | Std.err |
June-top | Aspergillus insuetus | 0.00004900 | 0.00005000 | 0.001835 | 0.0002938 | 0.002735 | 37.45 |
Thanatephorus cucmeris | 0.003123 | 0.0009283 | 0.0007994 | 0.0002018 | 0.03974 | 0.2559 |
Verticillium dahliae | 0.02074 | 0.002263 | 0.01201 | 0.001480 | 0.01915 | 0.5789 |
June-sub | Acremonium acutatum | 0.00073501 | 0.00008210 | 0.00001692 | 0.00001692 | 0.001441 | 0.02301 |
Acremonium fusidioides | 0 | 0 | 0.0003131 | 0.0001176 | 0.0318 | - |
Acremonium rutilum | 0.00004259 | 0.00002418 | 0.0006387 | 0.0002135 | 0.02963 | 15.00 |
Acremonium tubakii | 0.009270 | 0.001065 | 0.01325 | 0.0007904 | 0.02543 | 1.429 |
Verticillium dahliae | 0.01052 | 0.0005683 | 0.01390 | 0.001225 | 0.03982 | 1.322 |
August-top | Acremonium tubakii | 0.01439 | 0.002710 | 0.006412 | 0.001520 | 0.03666 | 0.4457 |
Aspergillus austroafricanus | 0.001522 | 0.00001326 | 0.0003054 | 0.0002832 | 0.01201 | 0.1997 |
Aspergillus cibarius | 0.0003946 | 0.0001038 | 0.002573 | 0.0005491 | 0.01462 | 6.521 |
Aspergillus insuetus | 0.003081 | 0.0004360 | 0.00003436 | 0.00001759 | 0.003053 | 0.01115 |
Trichothecium roseum | 0.001778 | 0.0001414 | 0.004231 | 0.001020 | 0.04496 | 2.380 |
August-sub | - | - | - | - | - | - | - |
Table 7
The differential species between CK and restructuring tilth layers (RTL) treatments of bacteria in genus level
Treatment | genus | CK | RTL | P value | Fold Change |
Mean | Std.err | Mean | Std.err |
June-top | Pseudoxanthomonas | 0.0005259 | 0.0001049 | 0.00006981 | 0.00001714 | 0.01401 | 7.533 |
Bryobacter | 0.004133 | 0.001525 | 0.01320 | 0.001084 | 0.01127 | 0.3130 |
Gemmatimonas | 0.0018415 | 0.0007707 | 0.006413 | 0.00029501 | 0.007501 | 0.2872 |
Nitrosomonas | 0.001274 | 0.0001560 | 0.0002215 | 0.00005356 | 0.006090 | 5.752 |
Nitrosospira | 0.0006961 | 0.0003158 | 0.001817 | 0.0002327 | 0.04781 | 0.3838 |
Nitrospira | 0.02170 | 0.001468 | 0.01243 | 0.001205 | 0.01056 | 1.745 |
June-sub | Bacillus | 0.004212 | 0.0009566 | 0.01163 | 0.002089 | 0.02786 | 0.3623 |
August-top | Bacillus | 0.004401 | 0.0001293 | 0.002729 | 0.0002211 | 0.01257 | 1.612 |
Pseudomonas | 0.005756 | 0.0008132 | 0.0005831 | 0.0001165 | 0.01351 | 9.872 |
Pseudoxanthomonas | 0.003057 | 0.0004514 | 0.0002136 | 0.00008777 | 0.01397 | 14.31 |
Bryobacter | 0.007399 | 0.0006748 | 0.01496 | 0.001217 | 0.01813 | 0.4947 |
Gemmatimonas | 0.005068 | 0.0005087 | 0.007757 | 0.0005696 | 0.03989 | 0.6534 |
Sphingomonas | 0.01366 | 0.001952 | 0.02426 | 0.001579 | 0.02967 | 0.5632 |
Nitrosomonas | 0.0005085 | 0.00003202 | 0.0001247 | 0.00003068 | 0.007219 | 4.077 |
Nitrosospira | 0.0004482 | 0.00004937 | 0.001832 | 0.00004505 | 0.0009318 | 0.2447 |
August-sub | Pseudomonas | 0.002703 | 0.0008453 | 0.022491 | 0.004967 | 0.02905 | 0.1202 |
Bryobacter | 0.008889 | 0.0009668 | 0.003332 | 0.0002979 | 0.01242 | 2.668 |
Gemmatimonas | 0.005539 | 0.0002797 | 0.0005622 | 0.0001299 | 0.0004310 | 9.853 |
Sphingomonas | 0.01437 | 0.0003230 | 0.004957 | 0.0002656 | 0 | 2.899 |
Nitrospira | 0.01957 | 0.001199 | 0.03095 | 0.0003265 | 0.003673 | 0.6323 |
Table 8
Correlation among biocontrol bacteria, soil borne pathogen and environmental variables in August identified by Monte Carlo permutation tests.
Environment | r2 | P-value |
SBD | 0.6162 | 0.025* |
TSP | 0.7745 | 0.001* |
NCP | 0.7459 | 0.003* |
SMC | 0.0415 | 0.836 |
AT | 0.1312 | 0.529 |
*P < 0.05. |