Dilution curve
The bacterial and fungal dilution curves for the three replicates of samples ZX3 and ZXCK are shown in Fig. 1. As the dilution factor increased, the curve became flatter, which indicated that further sequencing would result in only a small number of new species (OTUs). As the number of bacterial and fungal sequences in the rhizospheric soil samples of A. lancea increased, the dilution curve gradually revealed a flat trends, as the sequence results essentially covered all species information in the sample.
Diversity Analysis
OTUs classification
The statistics on bacterial and fungi OTUs revealed that the bacteria detected in the two groups of samples covered 37 phyla, 187 families, and 395 genera. The number of OTUs detected in ZX3 was 3,696, whereas the number of OTUs detected in ZXCK was 3,849. The fungi detected in the two groups of samples covered 15 phyla, 190 families, and 284 genera. The number of OTUs detected in ZX3 was 2,429, and the number of OTUs detected in ZXCK was 2,354 (Table 2).
Table 1
Species statistics of different classification levels of soil bacterial and fungal microbial communities. ZX3 and ZXCK represent the rhizospheres from the consecutive three-years cultured soils, and those that had never been planted with A. lancea respectively.
Microbe | Sample name | Kingdom | Phylum | Class | Order | Family | Genus | Species | OUTs |
Bacteria | ZX3 | 3 | 37 | 48 | 105 | 186 | 394 | 259 | 3696 |
| ZXCK | 2 | 37 | 49 | 105 | 187 | 395 | 246 | 3849 |
Fungi | ZX3 | 2 | 15 | 44 | 98 | 190 | 284 | 305 | 2429 |
| ZXCK | 2 | 15 | 41 | 85 | 170 | 284 | 286 | 2354 |
Species annotations were made on bacterial and fungal OTUs, and it was found that species could be identified at different taxonomic levels such as phylum, class, order, family, genus, and species. It can be seen from the table that in the two groups of samples, the OUTs detected by bacteria were 3,696 and 3,849, whereas the OUTs detected by fungi were 2,429 and 2,354 respectively. Compared to the ZXCK sample, the OUTs of the bacteria in ZX3 decreased, whereas the OUTs of the fungi increased.
A Venn diagram can intuitively reflect the difference and overlap of the composition of the soil bacterial communities OUTs between the two groups of samples.
As can be seen from Fig. 2a, the number of unique OTUs of ZX3 and ZXCK was 765 and 950, whereas the number of OTUs shared by ZX3 and ZXCK samples was 2,528, and the shared OUTs occupied 59.58% of the total OUTs. It can also be seen from Fig. 2b that the number of OTUs shared by the ZX3 and ZXCK samples was 1,185, and the shared OUTs occupied 37.65% of the total OUTs. The number of unique ZX3 OTUs was 1,050, unique ZXCK OTUs was 912.
Table 2
Alpha diversity index table for bacteria and fungi. The Chao1 index and Ace index represent the community richness, whereas the diversity is represented by the Shannon index and the Simpson index. ZX3 and ZXCK represent the rhizosphere soils from the consecutive three-years cultures and those that had never been planted with A. lancea, respectively.
Microbe | Sample name | Observed species | Shannon | Simpson | Chao1 | Ace |
Bacteria | ZX3 | 1969.33 | 8.82 | 0.993479 | 2103.02 | 2135.13 |
ZXCK | 2160 | 9.12 | 0.994796 | 2312.6 | 2323.52 |
Fungi | ZX3 | 1175 | 6.25 | 0.944552 | 1272.3 | 1237.16 |
ZXCK | 1158.33 | 6.16 | 0.934756 | 1281.4 | 1237.32 |
Alpha Diversity Analysis
It can be seen from the bacteria Alpha diversity index table (Table 2), that the Chao1 index of the experimental group ZX3 was 2,103.02 and the Ace index was 2,135.13, which were both lower than ZXCK. Simultaneously, the Shannon index and Simpson index of ZX3 were lower than ZXCK.
The fungi diversity index revealed that the Chao1 index and Ace index of the experimental ZX3 group were lower than those of the control group ZXCK, while the Shannon and Simpson indices of the experimental group ZX3 were higher than that of the ZXCK control group.
Relative Abundance Analysis
The bacterial species abundance was analyzed at the Phylum classification level (Fig. 3), which showed the top ten species with a relative abundance of bacterial species, which included Proteobacteria, Actinobacteria, Acidobacteria Gemmatimonadetes, Chloroflexi, Bacteroidetes, Verrucobacterium, Firmicutes, Thaumarchaeota and Latescibacteri. The relative abundance of these 10 species in all samples was ≥ 0.7%. The relative abundance ratios of Proteobacteria in samples ZX3 and ZXCK were 38.139% and 38.715%, whereas the relative abundance ratios of Actinobacteria in the ZX3 and ZXCK samples were 21.085% and 18.020%. The relative abundance ratios of Acidobacteria in the ZX3 and ZXCK samples were 20.196% and 22.334%. The relative abundance of the three strains in both samples was > 18%; however, compared to ZX3 and ZXCK, the abundance of the two strains of Proteobacteria and Acidobacteria declined, while the relative abundance of Actinomycota increased.
The species abundance of fungi was analyzed at the level of phylum classification, as shown in Fig. 3, which revealed the relative abundance of bacterial species. They were Ascomycota, Mortierellomycota, Basidiomycota, Mucoromycota, Chytridiomycota, Zoopagomycota, Rhizopusmycota, Glomeromycota, Aphelidiomycota and Neocallimastigomycota. The relative abundance ratios of Ascomycota in the ZX3 and ZXCK samples were 29.602% and 41.530%, whereas the relative abundance ratios of Mortierellomycota in the ZX3 and ZXCK samples were 37.170% and 21.669%. The relative abundance of Basidiomycota in the ZX3 and ZXCK samples were 2.704% and 18.893%. Compared with ZXCK, the relative abundance of Ascomycota and Basidiomycota decreased, and the relative abundance of Mortierellomycota increased. The relative abundances of the three were significantly different in ZX3 and ZXCK.
The bacterial composition of cultivated and uncultivated A. lancea soils was significantly different (Fig. 4). The PCoA ranking showed the changes in the bacterial community in the soil prior to and following the planting of A. lancea (Fig. 4a). The first principal component (41.6% contribution) and second principal component axis (22.46% contribution) distinguished the bacterial communities in the two groups of samples. Moreover, the distribution of bacterial communities varied for different samples. In particular, the difference between the soil samples planted with A. lancea and those without was more obvious (Fig. 4b).
Fungal compositions also differed prior to and following the cultivation of A. lancea, which showed variations based on PCoA ordination analyses (Fig. 5a). The first principal component (41.11% contribution) and second principal component axes (28.39% contribution) differentiated the fungal composition of soils planted with A. lancea and never planted with A. lancea. Furthermore, the distribution of fungal communities varied prior to and following the cultivation of A. lancea (Fig. 5b).
Differential Species Analysis
The differences in bacteria were shown in Fig. 6, where we can see that Bradyrhizobium and Arthrobacter had the most significant species difference between the two groups of samples, where in the ZX3 sample, the Bradyrhizobium had a higher population average. The differences between Cyanobacteria, Sporichthya, Candidatus Koribacter, Jatrophihabitans and Pseudonocardia were obvious in the two groups of samples.
An analysis of fungal differential species revealed that Dactylonectria was significantly different between the two groups of samples. Aspergillus, Humicola and Striaticonidium exhibited significant differences between the two groups of samples.
The ZX3 and ZXCK samples were selected for analysis in the top ten bacterial subordinate levels. The total bacterial abundance of these 10 genera accounted for 73.2% and 76.2% of all detected bacterial levels of the genus level, covering a large portion of the species. The genus name on the abscissa was plotted, as well as the percentage of bacterial species abundance as the ordinate (Fig. 7a). As can be seen from the figure, the richness of Sphingomonas, Solibacter, Rhodanobacter, Bryobacter and Haliangium increased in the ZXCK sample, whereas Burkholderiaceae, Arthrobacter, Gammaproteobacteria, Bradyrhizobium, and Streptomyces declined in abundance. Among these, the proportion of Sphingomonas in the two groups of samples was 4.6% and 5.7%, Burkholderiaceae was 6.3% and 1.0%, Rhodanobacter was 3.0% and 2.1%, and the genus of Arthrobacter was 4.5% and 1.7%. The four genera had obvious differences between the two groups of samples.
According to the cluster heat map of the dominant bacteria genera (Fig. 7b), there were some differences in the species of different groups in the same sample; however, the differences between the ZX3 and ZXCK samples were more obvious. Among these, the abundance of Gammaproteobacteria and Streptomyces were higher in the ZX3 sample, while the abundance of Rhodanobacter was higher in the ZXCK sample.
The top ten fungal subordinate levels in the ZX3 and ZXCK samples were selected for analysis (Fig. 8a). The total abundance of the fungal in the ten genera accounted for 67.1% and 50.4% of the fungi species in all detected genera. It can be seen from the figure that the abundance of Mortierella and Pseudogymnoascus in the ZXCK sample decreased, whereas the abundance of Scleroderma, Fusarium, Penicillium, Metarhizium, Aspergillus, Trichocladium and Epicoccum increased. Among these, the proportions of Scleroderma in the ZX3 and ZXCK samples were 0.001% and 14.2%, Mortierella was 14.2% and 4.0%, and Penicillium was 1.9% and 6.7%, respectively. The fungi of the three genera differed significantly between the two groups of samples.
According to the cluster heat map of the dominant fungi genus (Fig. 8b), Pseudogymnoascus and Mortierella were more abundant in the ZX3 sample, whereas the abundance of Epicoccum and Scleroderma were higher in the ZXCK sample.
Analysis And Discussion
Continuous cropping is a major issue for cultivation of A. lancea., which seriously affects the quality of A. lancea. It is generally acknowledged that the problems associated with A. lancea continuous cropping are intimately associated with rhizosphereic microorganisms. In terms of changes in soil microbial community structures, plant allelopathy and autotoxicity can alter the structures of microorganisms in the soil, which affects the growth of plants [19]. Studies have shown that the root exudates of C. citratus, A. conyzoides and B. Pilosa can decrease the germination rate, root length of seedlings, and seedling heights of radish, rice, and cucumber [20]. The allelopathic substance lycopene in tomato has inhibitory effect on other plants, fungi and insects. Researchers have also discovered that there is an allelopathy phenomenon in A. lancea, which often promotes the increase or decrease in the number of one or more types of microorganisms, and can changes microbial structures [21]. Soil microorganisms play a critical role in the prevention of plant diseases. Once a plant is attacked by root pathogens, it can employ microbial aggregates in the soil to prevent infection [22]. An increasing number of studies have shown that diseases involved in different plant growth processes are closely related to rhizosphere microorganisms [23–25]. Following continuous long-term cultivation of plants such as cotton, the soil microbial communities that support many plants will change, thus affecting their growth [26]. Therefore, changes in the populations of rhizospheric microorganisms and their community structures are the most likely reasons for the continuous problems with A. lancea.
Following an analysis of the dilution curves of bacteria and fungi, we found that sequencing results can essentially cover all species data in the sample; thus, it was feasible to employ this method to treat samples to reflect the diversity of microorganisms. The six curves of the bacterial dilution curve and the fungal dilution curve did not overlap, which indicated that there were certain differences in the microbial species between different soil samples. The analysis of OUTs and Alpha diversity revealed that following the planting of A. lancea, the soil microbial communities changed, the bacterial flora richness was greater than the fungal richness, and the bacteria in the rhizosphere microbial community of the two samples were dominant. Further, the species similarity of bacteria was greater than that of fungi, and the fungi exhibited more significant changes following continuous cropping. After continuous cropping, the diversity and richness of the bacterial communities were reduced; however the richness of the fungi communities was reduced, and the diversity of fungi was increased. Microbiological research by Lanping on soil for planting A. lancea indicated that the microbial community structures of bacteria and fungi in the soil rhizosphere for two consecutive years of A. lancea were lower than that of the annual samples, which was similar to the results of our study [27]. This also occurred in plants such as ginseng and peanuts, which both showed a trend of increasing fungi diversity [28–29]; thus, it was speculated that changes in soil resident fungi are one of the main reasons behind the continuous cropping issues.
Through abundance analysis, it was found that the relative abundances of Ascomycota, Mortierellomycota and Basidiomycota were significantly different between the two groups of samples. The relative abundance of Basidiomycota decreased, whereas the relative abundance of Mortierellomycota increased. Studies have shown that Ascomycota and Basidiomycota are important soil resident decomposers, where most Ascomycota are saprophytic bacteria that can decompose many types of recalcitrant organic matter, which play an important role in nutrient cycling [30]. Following continuous cropping, the abundance of Ascomycota and Basidiomycota decreased, and the nutrient cycling in the rhizosphere was affected, which resulted in a decline in the quality of A. lancea medicinal materials. Mortierellomycota is mostly saprophytic in soil, a few of which are the mycorrhizal fungi of forest trees. Their abundance was observed to increase, which was presumed to be related to the continuous cropping of A. lancea. The differences in bacterial were analyzed, and it was found that Bradyrhizobium and Arthrobacter were significantly different between the two groups of samples. Bradyrhizobium is a rhizobium of the class Proteobacteria [31], which can form a symbiotic relationship with host plants and fix the free nitrogen in the ambient atmosphere into forms that host organisms can use, such as ammonia (NH3) or ammonium (NH4 +), which are intimately related to plant growth [32]. The analysis of different species of fungi revealed that Dactylonectria was significantly different between the two groups of samples. Researchers found that Dactylonectria fungi are primarily related to plant diseases [33–35]. The change of Dactylonectria fungi may be related to the occurrence of pests and diseases of A. lancea.
Further analysis of the species in the two groups of samples at the subordinate level revealed that Sphingomonas, Burkholderiaceae, Rhodanobacter, Arthrobacter, Scleroderma, Mortierella and Penicillium were significantly different between the two groups of samples. It was inferred that the continuous cropping issues of A. lancea radix were related to changes in the bacterial and fungal community structures described above. Research has revealed that Sphingomonas has the capacity to degrade cellulose, and the inhibition of glucose on cellulose is eliminated through its absorption and use [36], and the species of Penicillium are closely related to fruit decay. Simultaneously, studies also have shown that the complex repair of Penicillium and biochar can reduce the content of effective arsenic, while improving the microbial environment in arsenic-contaminated soil, showing good remediation performance for arsenic-contaminated soils [37–38].
This study revealed that soil microorganisms changed following the planting A. lancea, which signified that the continuous cropping issues of A. lancea had an intimate relationship with soil microorganisms. Future experiments should further validate the roles of bacteria and fungi with significant differences in the two groups of continuously cropped A. lancea, and the relationship between the rhizosphere of A. lancea rhizomes, important specific species of bacteria and fungi, and the kinetics of microbial change. Studies have confirmed that inoculation with AV fungi has a positive impact on the growth of functionally symbiotic plants [39]. Further, endophytic actinomycetes and arbuscular mycorrhizal fungi, AMF, and chitosan assist with changing the community structure of rhizospheric microorganisms of A. lancea, and affect the physical and chemical properties of soil [40–41]. In further studies regarding the continuous cropping of A. lancea, AV fungi, endogenous actinomycetes and arbuscular mycorrhizal fungi, AMF, and chitosan might be employed to address continuous cropping obstacles.