Introduction

Asparagus (Asparagus officinalis L.) is an herbaceous perennial vegetable belonging to the Asparagaceae family. It is known as the “king of vegetables” owing to its tender and unique flavour, rich nutritional value, and important bioactive compounds such as rutin and protodioscin, which are beneficial for human health because of their antioxidant activities1,2,3. In 2020, the annual production of asparagus was 7.3 million metric tons (MMTs), with China being the top producer and accounting for 87% of global production4. The asparagus planting area in China has reached 120,000 hectares, accounting for approximately 49% of the world’s total, indicating that China is the world’s largest asparagus producer and exporter5. With the expansion of the asparagus production area, a large amount of asparagus residue is produced every year in China, especially in southern China, given that the asparagus fern is cut twice a year, in summer and in fall. Owing to the shortage in and rising cost of labour, most residues are discarded in the field, which results in a waste of bioresources and environmental pollution. Therefore, straw removal from the field has become a great burden for asparagus producers and is an urgent problem in asparagus production.

Half of agricultural inputs will eventually be converted to crop straw6. The utilization of crop straw is a tool to prevent the waste of farmland, water resources, and agricultural costs7,8. The current utilization of crop straw in China is not optimal. The effective management of crop residues has become a significant issue faced by the government. Burning is the most popular method for straw disposal by farmers. But straw burning in the field can accelerate soil erosion, environmental pollution, and soil degradation and negatively affect ecosystem functions9,10 and is now banned by the government. Comprehensive technologies for crop straw utilization are still immature, with high costs and poor effects10. Crop straw contains a large amount of organic matter and nutrient elements, including nitrogen, phosphorus, potassium, magnesium, calcium, and sulfur, which are essential nutrient elements for crop growth11,12,13. Directing straw return to the field after crop harvest not only prevents the loss of carbon as CO2 during incineration but also improves the organic C level of the soil, which directly or indirectly improves soil physics, microbial vitality, and crop root development14,15,16. Straw return fields provide a rich carbon source for soil microorganisms. This practice changes the living environment of microorganisms and the structure of the soil microbial community17significantly increasing the amounts of soil bacteria, fungi, and actinomycetes.

Moreover, the plant root-soil interface is a dynamic region in which complex interactions between plant roots, bacteria, and fungi occur. Microbial community compositions change greatly because of their strong selectivity in the environment of plant rhizospheres18. Plant-microbe interactions in the rhizosphere also play a significant role in microbial activity19,20. The plant genotype can play a significant role in shaping the rhizosphere microbiome, as different plant species and varieties produce different types and amounts of root exudates21,22,23. Rhizosphere microbial communities could respond to both straw return and plant type in the straw return field regime, as crops are usually planted in the field following straw return directly or after the completion of decomposition processing. However, the influence of differences in crops and straw return in the same habitat on microbial communities has rarely been investigated to clarify the suitable crops chosen as candidates planted in fields with straw return. In our study, we analysed the rhizosphere microbial community composition of three species of popular vegetables under various intensities of asparagus straw return. Bacterial 16 S rRNA and fungal 18 S rRNA gene amplicon sequencing were used to explore microbial richness, abundance, and diversity in rhizosphere soil samples. We sought to (1) determine the differential effects of the different asparagus straw return treatments on the rhizosphere microbial community composition of three vegetables and (2) clarify the relationships among the asparagus straw return treatments, vegetable species, and the soil microbial community.

Results

Effect of asparagus straw return on the morphology of three vegetables

The percentages of the increase in plant height and fresh weight compared with those in the absence of asparagus straw return are shown in Fig. 1. The heights and fresh weights of all the vegetables improved with asparagus straw return. The height of the eggplant plants (QZ2) increased by more than 30% at a straw return rate of 15 g/kg. In contrast, bitter gourds (KG2) at a straw return rate of 25 g/kg presented the greatest increase in plant height (approximately 60%). The fresh weights of eggplants (QZ2 and QZ3) increased at straw return rates of 15 g/kg and 25 g/kg, with the greatest percentage reaching approximately 200%. Overall, eggplant (QZ) height and fresh weight improved the most in response to asparagus straw return.

Fig. 1
figure 1

The percentage increase of three vegetables produced from asparagus straw as a function of plant height (a) and fresh weight (b). HG2 and HG3 indicate cucumbers planted with 15 and 25 g/kg asparagus straw return, respectively. KG2 and KG3 indicate bitter gourds planted with asparagus straw return rates of 15 and 25 g/kg added, respectively. QZ2 and QZ3 indicate eggplants to which 15 and 25 g/kg asparagus straw was added, respectively.

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Distribution of microbial communities

Principal coordinate analysis (PCoA) revealed that the return of asparagus residue resulted in a differential distribution of the microbial community in the rhizospheres of the three vegetables (Fig. 2). According to the distribution of rhizosphere bacterial flora (Fig. 2a), the first two principal components, PC1 and PC2, explained 45.99 and 10.1% of the distribution, respectively. HG1, KG1, and QZ1 were separated from those of the other two treatments. QZ2, QZ3, and HG3 were separated from each other and from the other treatments. KG2, KG3, and HG2 were nearly mixed.

However, according to the distribution of the fungal community in the rhizosphere soil (Fig. 2b), the first two principal components, PC1 and PC2, explained 50.09% and 17.67%, respectively, of the distribution. The HG1, KG1, and QZ1 samples were also separated from those of the other two treatments. With the exception of the QZ3 samples, which were separated from those of the other treatments, the other treatments were largely not separated from each other. Compared with those under the other treatments, the microbial community under the asparagus residue return treatment differed greatly in terms of the different vegetable rhizospheres.

Fig. 2
figure 2

Principal coordinate analysis (PCoA) ordination highlighting differences in the composition of bacterial (a) and fungal (b) communities (OTUs) in all treatments.

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Moreover, different forms of asparagus straw return significantly affected the separation of both communities in our treatments (Table 1), which indicates that the grouping was reasonable and reliable on the basis of the microbial data. The differences in the PCoA results for the bacterial and fungal communities in the rhizosphere soil among the treatments significantly reflected the original conditions of all the treatments. The R values were 0.7565 for the bacterial PCoA and 0.8361 for the fungal PCoA, whereas the p value was 0.001 for both analyses according to an ANOSIM.

Table 1 Statistical confirmation by analysis of similarity (ANOSIM) for reliability in PCoA.
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Soil microbial community composition

The dominant bacterial phyla included Actinobacteria, Firmicutes, Gemmatimonadetes, Bacteroidetes, Chloroflexi, Acidobacteria, and Proteobacteria (Fig. 3). Notably, the relative abundance of the bacterial phylum Actinobacteria was greater in HG1, KG1, and QZ1 and decreased by approximately 20% with asparagus straw return, whereas that of Firmicutes was greater in the two treatments to which asparagus straw was added to all three vegetable soils, which was nearly 20–30 times greater than that in HG1, KG1, and QZ1. The relative abundances of the bacterial phyla Chloroflexi and Acidobacteria decreased by approximately 45% and 33%, respectively, with increasing degree of asparagus straw return. The dominant fungal phyla were Ciliophora, Ascomycota, and Basidiomycota (Fig. 4). The relative abundance of the fungal phylum Ciliophora was greater in both HG2 and KG2 treatments, and the relative abundance of Ascomycota was highest in the treatment without asparagus residue return and decreased by nearly 14–20% with increasing concentration of returned asparagus straw.

Fig. 3
figure 3

Relative abundances of bacterial phyla in all the treatment groups. All phyla representing < 0.01% relative abundance were combined as “Others” (Fibrobacteres, Deinococcus-Thermus, Elusimicrobia, etc.). HG1, HG2 and HG3 indicate cucumbers planted with 0, 15 and 25 g/kg asparagus straw return, respectively. KG1, KG2 and KG3 indicate bitter gourds planted with asparagus straw return of 0, 15 and 25 g/kg, respectively. QZ1, QZ2 and QZ3 indicate eggplants planted with 0, 15 and 25 g/kg asparagus straw return, respectively.

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Fig. 4
figure 4

Relative abundances of fungal phyla in all the treatment groups. All phyla representing < 0.01% relative abundance were combined as “Others” (Labyrinthulomycetes, Blastocladiomycota, Protosteliida, Ochrophyta, Cryptophyceae, Acanthocystidae, Phragmoplastophyta, Cavosteliida, etc.). HG1, HG2 and HG3 indicate cucumbers planted with 0, 15 and 25 g/kg asparagus straw return, respectively. KG1, KG2 and KG3 indicate bitter gourds planted with asparagus straw return of 0, 15 and 25 g/kg, respectively. QZ1, QZ2 and QZ3 indicate eggplants planted with 0, 15 and 25 g/kg asparagus straw return, respectively.

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Bacterial and fungal alpha diversity

Bacterial alpha diversity

Bacterial alpha diversity was evaluated using the Sobs, Shannon, Simpson, Ace and Chao indices (Table 2). The bacterial Sobs index values for HG1 were significantly greater than those for HG2 and HG3, whereas those for QZ3 were significantly lower than those for QZ1 and QZ2. When 0 g/kg asparagus straw was added, the bacterial Sobs index values for HG1 were significantly greater than those for KG1, whereas when 25 g/kg asparagus straw was added, the bacterial Sobs index values for HG3 were significantly greater than those for KG3. The bacterial Shannon index for QZ2 was significantly greater than that for the other treatments. The bacterial Simpson index for QZ1 was significantly greater than that for the other treatments. The bacterial ACE indices for HG, KG and QZ in response to the addition of 25 g/kg asparagus straw were significantly lower than those in response to the addition of 0 g/kg asparagus straw, while there were no significant differences among the species of vegetables. The bacterial Chao index for QZ3 was significantly lower than those for QZ1 and QZ2.

Table 2 Alpha diversity indices of the bacterial communities in different vegetable rhizospheres under the asparagus straw return treatment.
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Fungal alpha diversity

Fungal alpha diversity was evaluated using the Sobs, Shannon, Simpson, ACE and Chao indices (Table 3). The fungal Sobs index values for HG1 were lower than those for the other treatments, while those of the other treatments were at the same level. The fungal Shannon indices for HG2 and HG3 were significantly greater than that for HG1, while for the same asparagus straw addition treatments, the Shannon indices for HG were significantly greater than those for KG and QZ. The fungal Simpson index for HG1 was significantly greater than that for HG2 and HG3, while that of QZ2 was significantly greater than that of QZ1. Under 15 and 25 g/kg of added asparagus straw, the fungal Simpson index for HG was significantly lower than that under 0 g/kg of returned asparagus straw. The fungal ACE index and Chao1 index did not significantly differ among the treatments.

Table 3 Alpha diversity indices of the fungal communities in different vegetable rhizospheres under the asparagus straw return treatments.
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Varied at the microbial genus level

At the genus level, asparagus straw return significantly affected the bacterial and fungal proportions in the rhizosphere of each vegetable species. Asparagus straw significantly increased bacterial and fungal genera in the rhizospheres of both cucumbers and eggplants, which were greater than those in the bitter gourds (Figs. 5, 6 and 7). The proportions of the bacterial genera Bacillus, Microvirga, Brevibacillus, Paenibacillus, and Lysinibacillus were significantly greater in the rhizosphere of HG2 and HG3 (cucumbers), whereas Actinoallomurus, Pseudonocardia, Sphingomonas, and Actinomadura were significantly lower. At the fungal genus level, the abundances of Chaetomium, Colpodca, Eukaryota, Chaetomiaceae, and Pezizaceae significantly increased in the rhizosphere of cucumbers with asparagus straw return, whereas the abundances of Leotiomycetes, Phialemonium, and Leotiomycetes significantly decreased (Fig. 5). Moreover, bacterial and fungal genera and their varied trends were similarly influenced by asparagus straw in the rhizosphere of the other two vegetables (Figs. 6 and 7), but the number of bacteria and fungi were lower in the rhizosphere of KG (bitter gourds) (Fig. 6). For a specific microbial genus, the mean proportion of Bacillus in the rhizosphere of eggplants with asparagus straw return was approximately 20% (Fig. 7), which was much greater than the 12% reported in the rhizosphere of other vegetables (Figs. 5 and 6). Asparagus straw return significantly decreased the mean proportion of Leotiomycetes in the rhizosphere of the three vegetables (Figs. 5, 6 and 7). The mean proportions of Chaetomium in the rhizospheres of cucumbers, bitter gourds, and eggplants with asparagus straw return were approximately 25%, 35% and 45%, respectively.

Fig. 5
figure 5

Relative abundance difference in bacterial genera in the rhizosphere of three vegetables with asparagus straw return. *0.01 < P ≤ 0.05; **0.001 < P ≤ 0.01; and ***P ≤ 0.001.

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Fig. 6
figure 6

Relative abundance difference in bacterial genera in the rhizosphere of three vegetables with asparagus straw return. *0.01 < P ≤ 0.05; **0.001 < P ≤ 0.01; and ***P ≤ 0.001.

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Fig. 7
figure 7

Relative abundance difference in fungal genera in the rhizosphere of three vegetables with asparagus straw return. *0.01 < P ≤ 0.05; **0.001 < P ≤ 0.01; and ***P ≤ 0.001.

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Discussion

Soil microorganisms are highly diverse and are the driving force for the recycling and transformation of soil organic matter and nutrients24. Straw return can significantly increase the number of bacteria to improve the community structure and functional diversity of soil microorganisms25 resulting in the formation of a new microbiota26. This treatment not only provides a more appropriate soil physical and chemical environment for microorganisms, which is conducive to their survival, but also provides abundant carbon and nitrogen sources7,27. Different straw properties and environmental factors can lead to substantial variation in the diversity of soil microbial communities28. In addition to the treatments in which no asparagus straw was added, the distributions of both the soil bacterial and fungal communities differed from those in the treatments to which no asparagus straw was added (Fig. 1), which included all three vegetable straw treatments. The external properties of each vegetable were more vigorous and stronger when asparagus straw was added than when asparagus straw was not added, with results such as greater plant height and fresh weight, especially in the eggplant treatment (Fig. 1). The intensity of straw return has different effects on soil microbial structure and diversity29. In general, medium and high amounts of straw return are more attractive to soil microorganisms15 which may provide many nutrients for feeding these microorganisms. The number of soil bacteria and actinomycetes increased in response to the different amounts of straw returned to the field. The fungal population increased only at a relatively high rate of straw return. A similar phenomenon was found in our study, which revealed that increasing the intensity of asparagus straw treatment significantly increased the bacterial community composition of the three vegetables, whereas the distribution of fungal communities did not significantly differ among the treatments in which asparagus straw was added. Straw usually increases bacterial richness without the emergence of new bacterial species30,31. The relative abundance of Firmicutes significantly increased after asparagus straw return, whereas its relative abundance in the rhizosphere of the three vegetables without asparagus straw was very low (Fig. 3). Proteobacteria were present in all the treatments, and the greatest number of bacteria was detected in the low-intensity asparagus straw return treatment in the HG2, KG2, and QZ2 groups. The most dominant fungal phylum in all the treatments was Ascomycota (Fig. 4). This trend might have occurred because the community is largely restricted by the soil pH16. The relative abundance of Acidobacteria decreased with increasing intensity of asparagus straw return. A negative correlation was detected between Acidobacteria and soil total carbon32. The relative abundance of the phylum Basidiomycota was high in HG3, KG2, KG3, and QZ3 (Fig. 3) because straw residue degradation affects the abundance of this phylum and can cause rapid metabolism of organic substances in roots33.

Microbial activity is vigorous in the rhizosphere of plants but varies with the species22 because root exudate metabolite profiles are distinctive among various crops. Rhizosphere microbial communities also respond significantly to plant types34. Natural variation within maize species can be used as an approach for understanding the role of root exudate in microbiome selection, in which a specific degree of root exudation impacts root-associated microbiomes35. The fungal composition varied among the species of vegetables (Fig. 2b). Asparagus straw return has a strong influence on microbial species in the rhizospheres of three vegetable species (Supplementary Tables 1 and 2). In terms of bacteria, the abundances of Paenibacillus_cineris, Paenibacillus_faecis, Paenibacillus_lactis, and Aneurinibacillus was highly increased in cucumbers (HG) treatment involving asparagus straw return. Some bacteria that have excellent functions in terms of nitrogen fixation, such as Lysinibacillus, Ramlibacter, Shimazuella, and Bacillusaerophilus, strongly increased in abundance in Solanum eggplant (QZ) treatment. In terms of fungi, Mucoromycota (Fig. 3), Cephaliophora_tropica, and Alternaria_alternata (Supplementary Table 2) were enriched in cucumbers (HGs) and bitter gourds (KGs) with both intensities of asparagus straw treatment, which was lower in the Solanum eggplant (QZ) treatment. This finding might be due to the root exudates in cucumbers (HGs) and bitter gourds (KGs) having a positive effect on these plants, whereas Cephaliophora_tropica and Alternaria_alternata commonly include several fungi that can cause plant diseases. Because the experiment was conducted on vegetables at the seedling stage, disease might not have occurred in bitter gourds. Pathogenic populations with potential harm may increase as favourable conditions for growth are provided by the return of crop straw to soils at relatively high rates and consequently to diseases such as root rot in maize36 common rot and sharp eyespot in wheat37.

Plants grown following maize crops were significantly different, with tomatoes showing the greatest differences, which is consistent with the hypothesis that plant species are important in recruiting specific rhizosphere microbial communities38. A greater abundance of fungal pathogens was also observed with corn straw return than with wheat straw return in the wheat-corn rotation system29. The microbial community was compositionally and functionally distinct and colonized the rhizosphere of tomato plants grown following alfalfa. Asparagus straw return increased the bacterial diversity in the rhizosphere of eggplants. Asparagus straw return decreased the bacterial richness in the rhizospheres of the three vegetables (Table 2). Asparagus straw return increased the fungal diversity in the rhizosphere of cucumbers. Asparagus straw return had no influence on the fungal richness in the rhizospheres of the three vegetables, with no significant changes in the Sobs, ACE and Chao indices (Table 3). The bacterial and fungal diversity are strongly influenced by the vegetable species cucumbers and eggplants. Moreover, at the genus level, the differences in bacteria and fungi were still greater in the rhizospheres of HG and QZ than in those of KG (Figs. 5, 6 and 7). Asparagus straw return increased the relative abundance of dominant microbes, such as Bacillus and Acidobacteria, which are related to soil pH and play important roles in soil material circulation and ecological environment construction. Moreover, there was a decrease in Leotiomycetes, which is a common plant pathogen causing leaf spot disease, cucurbit powdery mildew, and anthracnose39,40.

Conclusion

Asparagus straw return was the dominant factor affecting the rhizosphere microbial genera, but vegetable species were also the determining factor for significant change. Our results indicate that there is a specific correlation between vegetable species and the influence of crop straw return on the soil microbial community. The straw return rates that could be applied in the field should be based on the crop species planted following straw return. Vegetable species should be considered when asparagus straw is returned. In combination with the growth mass and soil microbial conditions, eggplants should be a better choice to plant in asparagus straw return field. However, the reasons that some special microbes are significantly affected by a given vegetable species require further research to explore how asparagus straw and vegetable species interact with those functional microbes.

Methods

Experimental setup

The experiment was conducted at the experimental station of the Institute of Vegetables and Flowers, Jiangxi Academy of Agricultural Sciences, Jiangxi Province, China (28° 11′ N, 115° 56′ E). Two densities of asparagus straw return to the soil were designed for incorporation. Eggplants (Solanum melongena L.), bitter gourds (Momordica charantia L.) and cucumbers (Cucumis sativus L.) were employed as candidates for microbial diversity assessment in soil treated with asparagus straw return. Eggplant cultivar: Long purple from Jingyan Yinong Seed Co. Ltd.; bitter gourd cultivar: Gan4 from the Jiangxi Academy of Agricultural Sciences; and cucumber cultivar: Jinlv from the Tianjin Academy of Agricultural Science. Seeds of the eggplants, bitter gourds and cucumbers were sown after straw return. Soil samples were collected from the rhizospheres of three vegetable species for microbiological testing.

Asparagus residues were collected from five-year-old asparagus at an asparagus planting site. Spears measuring 35–40 cm in length were harvested. The bottom portion (5–10 cm) of the long asparagus spears was taken as the residue after 30 cm of the top portion was cut for commercial use. The residues were washed, dried at 70℃ in an oven, and ground into a powder using a mini grinder.

The incorporation treatments were as follows: ① no residue added as a control, ② 15 g of residue, and ③ 25 g of residue. One kilogram of soil was mixed with 15–25 g of residue powder, placed into a 7 cm×15 cm plastic pot covered with plastic film (with 20 needle holes on the film), and incubated at 80% field water capacity at a temperature of 30 ℃ for 30 d. Water supplementation was applied after every five days. After 30 d, the covered plastic films were removed for sowing vegetable seeds.

The physical and chemical properties of the asparagus straw return soil used in the study are shown in Table 4.

Table 4 Basic physical and chemical properties of the soil treated with asparagus straw.
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The vegetables planted after residues were used in the experiment were cucumbers, bitter gourds, and eggplants. The seeds were soaked in water for 24 h and rinsed every 12 h. Then, a 9 cm diameter Petri dish with two layers of moist filter paper was used for seed germination. Uniformly germinated seeds were sown in pots subjected to the three residue treatments. There were three replicates per treatment and ten pots per replicate. The pots were placed in a greenhouse at a temperature of approximately 25:33 °C (dark: light). The plants were all subjected to the same farm practices.

The experiment consisted of three groups and nine treatments (Table 5). The first group included ①cucumbers + no asparagus straw added as a control (HG1), ②cucumbers + 15 g asparagus straw (HG2), and ③cucumbers + 25 g asparagus straw (HG3); the second group included ①bitter gourds + no asparagus straw added as a control (KG1); ②bitter gourds + 15 g asparagus straw (KG2), and ③ bitter gourds + 25 g asparagus straw (KG3); and the third group included ①eggplants + no asparagus straw added as a control (QZ1), ②eggplants + 15 g asparagus straw (QZ2), and ③eggplants + 25 g asparagus straw (QZ3). Each treatment had ten repetitions.

Table 5 Treatment details used in the experiments.
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Morphological measurement

Morphological features, including the plant height and fresh weight, were investigated using tape scale measurements and a balance. Plant heights and fresh weights were assessed by measuring all the plants.

Soil sampling

After 30 d, soil samples were collected from each of the nine treatments. The soil samples were collected from each plot in the rhizosphere of the plants. The plants were slightly uprooted from the pots to remove the soil. The soil closely adhering to the roots was brushed off, and the samples were immediately stored at − 80 °C for microorganism analysis.

Soil DNA extraction and high-throughput sequencing

The 0.5 g soil (fresh weight) samples were used for the extraction of the genomic DNA of the microorganisms using an E.Z.N.A.® soil DNA kit (Omega Bio-tek, Norcross, GA, U.S.) by following the manufacturer’s instructions. A NanoDrop ND-2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA) was used to determine the quality and concentration of the DNA. The soil bacterial 16 S rRNA gene in the hypervariable V3-V4 regions in the rhizosphere of three vegetables was amplified using the primers 338 F (5′-ACTCCTACGGGAGGCAGCAG-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′). The soil fungal 18 S rRNA gene in the hypervariable V5-V7 regions in the rhizosphere of three vegetables was amplified using the primers SSU0817F (5′-TTAGCATGGAATAATRRAATAGGA-3′) and 1196R (5′-TCTGGACCTGGTGAGTTTCC-3′). The conditions for amplification by PCR were one cycle at 95℃ for 3 min, 27 cycles at 95℃ for 30 s, at 55℃ for 30 s, and at 72℃ for 45s, with a final step of 72℃ for 10 min, and 10℃ until the reaction was stopped by the user. We conducted three PCRs with a 20 µL final volume mixture including 5×TransStartFastPfu Buffer (4 µL), 2.5 mM dNTPs (2 µL), 5 µM forward primer (0.8 µL), 5 µM reverse primer (0.8 µL), TransStartFastPfu DNA Polymerase (0.4 µL), and 10 ng of template DNA. We extracted the PCR products of the soil DNA from a 2% agarose gel and further purified them using the AxyPrepDNA Gel Extraction Kit (Axygen Biosciences, USA) and then quantified them via QuantiFluor™-ST (Promega, USA) according to the manufacturer’s instructions. Purified amplicons were pooled in equimolar amounts and paired-end sequenced (2 × 250) on an Illumina MiSeq PE300 and PE250 platform (Illumina, San Diego, USA) for soil bacteria and fungi, respectively, following the standard protocols of Majorbio Bio-Pharm Technology Co. Ltd. (Shanghai, China).

Processing of sequencing data

Sequences from the Illumina MiSeq platform were processed using the QIIME (version 1.70) software package. We employed Trimmomatic for raw fastq files to be demultiplexed and quality-filtered and then FLASH for merging those data using the following standards: (a) truncating the reads at any site receiving an average quality value < 20 over a 50 bp sliding window; (b) keeping the primers exactly matched (nucleotide sequence be allowed having two mismatches) and removing the reads that contained ambiguous bases; (c) merging sequences that overlapped longer than 10 bp on the basis of their overlap sequence; and (d) differentiating samples on the basis of barcodes and primers at the beginning and end of the sequence and adjusting the sequence direction. Zero barcode mismatches were allowed, and 2 primer mismatches were allowed as the maximum. The clustering for operational taxonomic units (OTUs) was conducted with a 97% similarity cut-off by UPARSE (version 7.1 http://drive5.com/uparse/). The significance of the alpha diversity was analysed with DPS 7.5 software with two-way ANOVA.