Effects of Different Proportions of Organic Fertilizer in Place of Chemical Fertilizer on Microbial Diversity and Community Structure of Pineapple Rhizosphere Soil

: With the development of sustainable agriculture, the application of organic fertilizers to crops instead of chemical fertilizers has become an inevitable trend. However, little is known about the proportion of organic fertilizers replacing chemical fertilizers and how it affects the underlying microbial mechanisms of continuous pineapple soil. In this study, we used the Illumina Miseq high-throughput sequencing platform to study the rhizosphere soil of continuously cropped pineapples to study the diversity and community structure of pineapple rhizosphere microorganisms. The re-sults showed that, with 97% similarity, the number of OTUs of all samples obtained using hierarchical clustering analysis is 3645. Both conventional fertilization (CF) and optimal fertilization (YF) increased the relative abundance of Proteobacteria, Actinomycetes, and Sclericutes, and decreased the relative abundance of Acidobacteria, Bacteroidetes, Blastomonas, and Verrucobacteria. The replacement of organic fertilizers increased the relative abundance of Bacteroidetes, among which the relative abundance of chlorocurvus treated with the replacement of organic fertilizers of 80% was the highest. Different fertilization methods also changed the diversity and abundance of bacteria in the soil of the pineapple rhizosphere; the diversity of the species was E > D > C > CK > B > YF > CF. Based on the analysis of the PCoA and NMDS of soil bacterial communities, treatment E was similar to treatment D, treatment CK was similar to treatment YF, and treatment C and treatment D had little structural difference. On the basis of an analysis of the composition and function of the flora, it can be found that different fertilization methods have significant differences in the bacterial groups of the rhizosphere bacterial community of pineapple soil. The relative abundance of beneficial bacteria was increased. When organic fertilizer replaces chemical fertilizer, it promotes the role of bacteria related to the carbon cycle in the rhizosphere.


Introduction
Pineapple (Ananas comosus (Linnaeus) Merrill) is a perennial herb in the Bromeliaceae family and is the third-largest tropical fruit in the world [1,2].It is cultivated in more than 94 countries and regions around the world, and there are at least 100 edible species in the world [3].The nutritional value of the soil and plants could affect the yield and quality of pineapple.In recent years, excessive and unscientific long-term fertilization methods resulted in a series of ecological problems, including soil acidification, soil compaction, and changed soil microbial community structure, and lead to an imbalance in soil nutrients [4][5][6][7][8][9][10].Domestic and foreign experts have conducted research on these issues, but they have mainly focused on the effects of different amounts of fertilizer on pineapple yield and quality [11].Also, with proposals for green agriculture and sustainable agriculture, the application of organic fertilizer and reduced fertilizer has received widespread attention, and an adjusted fertilization pattern has become an inevitable trend [12,13].
Soil microorganisms are an important indicator of soil fertility and health [14].Increased soil bacteria and Actinomycetes can promote plant growth and improve the soil environment.In contrast, an increase in fungi and decrease in bacteria can cause diseases and affect healthy plant growth [15].The species diversity and the community structure of soil microorganisms determines the ability of soil to resist pathogens, which is very important for the sustainable development of soil ecosystems, environmental regulation, and the use of sustainable resources [16,17].At present, there are few studies on the effect of the soil microorganisms of the pineapple rhizosphere, mostly discussing the effects of crop rotation and the combined application of bioorganic fertilizer on the structure of the microbials in the soil of the pineapple rhizosphere.Jing et al. [18] studied how the rotation of pineapple trees in Xuwen and Leizou in summer could induce the abundance and diversity of fungal communities in the rhizosphere; pineapple rotation cultivation has better soil growth and higher yield than continuous cultivation.Beibei Wang et al. [19] studied how pineapple-banana rotation, combined with bio-organic fertilizer application, effectively reduced the abundance of Fusarium.spp.and Fusarium wilt in bananas.Jinming Yang et al. [20] studied how a rotational pineapple-banana crop has the capacity to suppress bananas' Fusarium wilt.Yinghong Hu et al.'s [21] research showed that the application of bio-organic fertilizer increased the richness and diversity of the fungal community, and changed the structure of the fungal community in the soil.The bacterial diversity and community structure in the rhizosphere soil after 2 years of continuous cultivation of pineapple with different fertilization methods have not been reported.
This research uses golden pineapple as the research object.Sequencing analysis was performed using the Illumina Miseq high-throughput sequencing platform.The aim was to investigate the diversity and richness of rhizosphere microorganisms and changes in the composition, function, and difference of the microbial community in pineapple with reduced fertilization and different proportions of organic fertilizer instead of chemical fertilizer.Specifically, we aim to (1) reveal the effects of reduced fertilization and different proportions of organic fertilizer instead of chemical fertilizer on soil microorganisms in the field of pineapple rhizosphere soil; (2) find the optimal ratio of organic fertilizer to chemical fertilizer; and (3) provide reference guidance for subsequent pineapple planting.

Sample Collection and Storage
The soil samples collected on this occasion came from the pineapple test base of Huihai Planting Company, Fushan Town, Chengmai County, Hainan Province (109°04′ E, 19°24′ N).The soil type is brick-red soil developed from basalt parent material.After harvesting, two pineapple fruits were collected from each plot, and the rhizosphere soil of the pineapples was collected using the soil-shaking method and then frozen in liquid nitrogen and stored in a refrigerator −80 °C for the subsequent experiments.

Experimental Design
The field trial was conducted from September 2020 to December 2021 at the pineapple garden experimental base of Huihai Planting Company, Fushan Town, Chengmai County, Hainan Province.The company is a company specializing in tropical fruit cultivation.The field soil pH was 5.00, the organic matter content was 4.78%, total nitrogen was 1.27 mg/kg, available phosphorus was 3.22 mg/kg, and available potassium was 336.96 mg/kg.This region has a tropical monsoon climate with abundant rainfall and sunshine, with 1786-2500 mm of annual rainfall and an average annual temperature of 23.8-24.1 °C.The organic fertilizers and chemical fertilizers used in this experiment were purchased from Hainan Yikang Ecological Agriculture Development Co., Ltd (Hainan, China).The commercial organic fertilizer was fermented and composted with sheep manure as the main raw material.The nutrient index was NP2O5-K2O = 1.2: 1.1: 2, and the organic matter content was ≥40%.Chemical fertilizers included urea (N 46%), superphosphate (P2O5 16%), potassium sulfate (K2O5 2%), compound fertilizer 1 (NP2O5-K2O: 16-16-16), compound fertilizer 2 (NP2O5-K2O: 15-5-25).This study was conducted in a field where pineapples had been grown continuously for 2 years.The field area was 2 acres, close to water sources, and far away from surrounding roads.The experiment included seven treatments; no fertilization (CK); conventional fertilization (CF); optimized fertilization (YF); organic fertilizer replacement YF20%(B); organic fertilizer replacement YF40%(C); organic fertilizer replacement YF60%(D); organic fertilizer replacement YF80%(E).Conventional fertilization was 900 kg•hm −2 (N), 750 kg•hm −2 (P2O5), 1200 kg•hm −2 (K2O).The optimized fertilization was 450 kg•hm −2 (N), 135 kg•hm −2 (P2O5), 1125 kg•hm −2 (K2O).The treatment for replacing chemical fertilizers with organic fertilizers was to substitute organic fertilizers in different proportions on the basis of optimized fertilization.The experiment for each treatment was repeated three times, with a total of twentyone plots, a plant spacing of 35 m, row spacing of 40 m.A total of 190 plants were planted, and the test area of each plot was 63.5 m 2 .

DNA Extraction, PCR Amplification
We used the Power Soil DNA isolation kit (MOBIO company, San Diego, CA, USA) to extract DNA from 500 mg of soil samples.The V4 hypervariable region of 16SrRNA was chosen as the PCR amplified region.The amplification was performed using the forward primer 515F (5′-GTGCCAGCMGCCGCGGTAA-3′) and the reverse primer 907R (5′-CCGTCAATTCMTTTRAGTTT-3′).Preliminary sample purification, quality screening, and paired library preparation were completed by Guangdong Meige Gene Technology Co., Ltd., for high-throughput sequencing.

16SrRNA Gene Sequencing
We used QIIME software to splice and filter the original data [22,23].Standardized analysis of valid data for all samples was performed using the USEARCH program.Sequences with a similarity of 97% were clustered into operational taxonomic units (OTUs).Species classification was performed in the SILVA database (Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures GmbH, Bremen, Germany).

Bioinformatics Analysis
To compare the relative levels of OTU diversity in all soil samples, the bacterial diversity index (Simpson, Shannon, InvSimpson) and richness (ACE, Chao1) were calculated based on Mothur software Version 1.45.3 [24,25].To compare the bacterial community structure of soil samples, R (version 3.2.0)software was used for principal coordinates (PCoAs) based on Bray-Curtis distance [26] and non-metric multidimensional scaling (NMDS) analysis.The authors evaluated the effect of replacing chemical fertilizers with organic fertilizers on the community structure characteristics of pineapple rhizosphere microorganisms.In addition, to exclude the influence of low-abundance species and retain OTUs with relative abundance >0.01%.MEGA7.0 was used to construct a neighborjoining (NJ) phylogenetic tree with 1000 bootstrap replications.

Statistical Analysis
Mothur software was used to calculate the α-diversity index and beta diversity of the bacterial community.Principal coordinate analysis (PCoA) and nonmetric multidimensional scaling (NMDS) analysis were performed with R software (version 2.15.3).Origin software was used for visualization.Linear discriminant analysis effect size (LEfSe) analysis (https://www.bioincloud.tech,accessed on 16 March 2023) was used to calculate significance based on linear discriminant analysis (LDA) effect size ≥ 4 and p < 0.05.The FAPROTAX database was used to predict the function of soil microbes in the rhizosphere.Finally, the phylogenetic tree was constructed using MAGA7.0software and visualized in Adobe Illustrator 2023 software.

Sequencing Results of Different Fertilization Methods
According to the original data obtained using Illumina Miseq sequencing, a total of 2284758 high-quality sequences were obtained from seven samples.Based on the 97% similarity, a total of 3645 OTUs were obtained for the bacterial 16SrRNA genes in all samples.The good coverage value for each group was greater than 0.99, indicating that the results of this sequencing reflect the actual situation of microorganisms in the sample.

Alpha Diversity of Bacteria in Different Fertilization Methods of Rhizosphere Soil
The authors analyzed the α-diversity of bacteria in the soil of pineapples using organic fertilizer instead of chemical fertilizer (from Table 1, without fertilization (CK), conventional fertilization (CF), optimized fertilization (YF), replacement of organic fertilizer YF20% (B), replacement of organic fertilizer YF40% (C), replacement of organic fertilizer YF60% (D), and replacement of organic fertilizer YF80% (E); the seven treatments differed in bacterial diversity and richness).Compared to other treatments, the Chao1 and Shannon indices were the smallest in conventional fertilization (CF) and fertilization (CF), showing the lowest richness and diversity of bacteria.The Chao 1 index and the ACE index were the largest in E, indicating that the bacterial community of the soil rhizosphere had the largest variety.Combined with the replacement of organic fertilizer YF80% (E), it showed that the richness of bacteria followed a descending order from E, C, CK, D, B, YF, to CF.The diversity index (Shannon, InvSimpson index) of organic fertilizer substitution (40%, 60%, 80%) was higher than that of no fertilization (CK), conventional fertilization (CF), and optimized fertilization (YF), showing that the diversity of bacteria followed a descending order from E, D, C, CK, B, YF, to CF.We found that the replacement of organic fertilizer YF40% (C) and the replacement of organic fertilizer YF80% (E) significantly increased soil bacterial diversity and richness compared to CK.It showed that the replacement ratio of these two organic fertilizers has a promoting effect on the abundance and diversity of soil bacteria in the pineapple rhizosphere.However, the bacterial diversity and abundance of conventional fertilization (CF) and optimized fertilization (YF) were significantly reduced, indicating that bacterial diversity and abundance in soil after fertilization were significantly higher than those in the unfertilized area, and the soil microbial diversity and abundance of conventional fertilization (CF) were the lowest in abundance.In summary, different fertilization methods altered the composition of the microbial community of the soil and the index of microbial diversity by varying soil properties.The diversity of species from high to low was E > D > C > CK > B > YF > CF.In addition, the Sobs index of replacing 40% (C) with organic fertilizer was the largest; this suggests that the more uneven community distribution leads to the dominant bacterial [22].

Bydiversity of Bacteria in Different Rhizosphere Soil Fertilization Methods
Principal coordinate analysis (PCoA) based on the Bray-Curtis distance was used to further clarify the impact on the structure of the bacterial community of the soil in samples where organic fertilizers replaced chemical fertilizers.It can be seen in Figure 1a that the contribution of the first principal component axis (PCo1) was 49.35%, and that of the second principal component axis (PCo2) was 17.44%.In the coordinate graph, the closer the distance between the two samples, the more similar the species composition and structure of the two samples.The distance between treatments E and D was very close, indicating that the diversity of species composition of treatments E and D was relatively similar.The distance between treatments YF and CK was very close, indicating that the species composition diversity of treatments E and D was relatively similar.Treatment B and treatment CF were far apart, indicating that there was a large difference in the diversity of microbial communities between them.And there was also a big difference in species diversity between treatments E and D, and treatment YF and treatment CK.Nonmetric multidimensional scaling analysis (NMDS) can reflect the relationship between microbial communities of different samples; the lower stress (<0.1) indicates that NMDS can accurately reflect the degree of difference between samples.The results showed that treatment E, treatment YF, treatment CF, treatment CK, and treatment B were far apart, indicating that there were obvious differences in the rhizosphere bacterial communities of these five treatments.Treatments C and D were close to each other, with two data points next to each other, indicating that there was little difference in the structure of the bacterial community of the soil rhizosphere between 40% replacement of organic fertilizer and 60% replacement of organic fertilizer.The difference from the principal coordinate analysis (PCoA) was that the samples in treatment E were different from those in treatments D and C, indicating that different proportions of organic fertilizer replacement have significant differences in the structure of the pineapple soil bacterial community rhizosphere (Figure 1b).

Composition of the Bacterial Community in the Soil of the Pineapple Rhizosphere under Different Fertilization Methods
To obtain taxonomic information on the species corresponding to each OTU, the SILVA database was used to perform a taxonomic comparison on the representative sequences of OTUs with a level of similarity of 97%.In total, we found evidence for 18 different bacterial genera from 48 different phyla.These dominant phyla represented more than 95% of the bacterial sequences from each soil sample.They included Proteobacteria, Acidobacteria, Bacteroidetes, Chloroflexi, Actinobacteria, Firmicutes, Planctomycetes, Gemmatimonadetes, and Verrucomicrobia (average relative abundance > 0.01%) (Figure 2a).CF and YF increased the relative abundance of Proteobacteria, Actinobacteria, and Firmicutes, while they decreased the relative abundance of Acidobacteria, Bacteroidetes, Gemmatimonadetes, and Verrucomicrobia.This shows that, compared to conventional fertilization, the reduction of chemical fertilizer has no impact on the composition of the microbial communities of the soil rhizosphere.The replacement of chemical fertilizers with organic fertilizers increases the relative abundance of Bacteroidetes.The relative abundance of Chloroflexi was the highest in the treatment where the medium organic fertilizer replaced 80% of chemical fertilizer (Figure 2c).
The 10 most abundant bacterial genera were Chujaibacter, Gp2, Ktedonobacter, Gp3, Gp1, Gp13, Occallatibacter, Paludibaculum, Acidibacter, and Nitrososphaera (average relative abundance >0.01%) (Figure 2b).CK increased the relative abundance of Gp2 and Nitrososphaera compared to treatment with each other.CF and YF decreased the relative abundance of Nitrososphaera, Micropepsis, and Rhodoplanes, while they increased the relative abundance of Chujaibacter.Replacing chemical fertilizers with organic fertilizers increased the relative abundance of Bradyrhizobium.Among these, a 20% replacement of organic fertilizer increased the relative abundance of Gp3, Nitrososphaera, Micropepsis, Gaiella, and Rhodoplanes.Substituting 40% of organic fertilizers increased the relative abundance of Gp1, Gp2, and Occalatibacter, and replacing 80% of organic fertilizers increased the relative abundance of Ktedonobacter (Figure 2d).
analysis was performed to detect bacterial taxa with significantly different abundance between different groups with p value < 0.05 and LDA score > 2 (Figure 3, Supplementary Materials).At the level of bacterial class, Gammaproteobacteria and Clostridia were significantly enriched in YF; Bacilli and Actinobacteria were significantly enriched in CF; (α-, β-, δ-, Proteobacteria), Gemmatimonadetes and Cytophagia were significantly enriched in B; Chitinophagia was significantly enriched in C; and Ktedonobacteria, Burkholderiaceae, Chitinophagaceae, and Ktedonobacteraceae were significantly enriched in C, D, and E. At the level of bacterial genus, Chryseolinea, Rhodoplanes, Gp6, Occallatibacter, Gp1, and Gp3 Ktedonobacter were significantly enriched in B, C, and E. It can be seen from the above that different fertilization methods have significant differences in the bacterial taxa of the bacterial community of the rhizosphere of pineapple soil.Chitinophagia was significantly enriched in C, possibly because manure contains more bacterial taxa from Bacteroidetes, as the proportion of organic fertilizers replacing chemical fertilizers increased.Bacteroidetes became the dominant microorganism and were highly enriched.

Function of Soil Bacteria in the Pineapple Rhizosphere under Different Fertilization Methods
There are also clear differences in the functional types of CK, CF, YF, B, C, D, and E. According to the results of the comparison of the OTU sequences uploaded to the FAP-ROTAX database, 54 functional groups were obtained.The first coordinate PcoA1 (67.56%) and the second coordinate PcoA2 (18.62%) jointly explained 86.18% of the overall change in the function of the soil microbial community (Figure 4a).There were no significant differences between treatments, except that conventional fertilization (CF) and optimized fertilization (YF) could be distinguished.It can be seen from Figure 4b that YF and B, C, and D were close and there is no significant difference.

Functional Differences of TOP 10 Bacteria in Pineapple Rhizosphere Soil under Different Fertilization Methods
There are differences in the functions of soil rhizosphere bacteria under different fertilization methods.The top 10 functional populations with relatively high relative abundance were chemoheterotrophy, aerobic chemoheterotrophy, nitrification, aerobic ammonia oxidation, nitrate reduction, phototrophy, anoxygenic photoautotrophy, S-oxidizing of anoxygenic photoautotrophy, photoautotrophy, and photoheterotrophy.
The abundance values of the chemoheterotrophy and aerobic chemoheterotrophy populations were above 20%.The abundance values of nitrification, aerobic ammonia oxidation, nitrate reduction and phototrophy were above 3%.The lowest were the anaerobic photosynthesis, anaerobic photosynthesis, S-oxidation, photosynthesis, and photoheterotrophy populations.YF had the strongest chemoheterotrophic and oxidative heterotrophic functions.The relative abundance of the anoxygenic photoautotrophy, anoxygenic photoautotrophy S-oxidization, and photoautotrophy functions of organic fertilizer replacing chemical fertilizer were lower than that of CK and CF, and higher than that of YF (Figure 5).

Bioinformatics Analysis
Sequence alignment was performed on some of the seven processed OTUs and Bacillus glycinifermentans MGMM1 related sequences from NCBI using MAGE7.0software.Using the neighbor-joining method (neighbor-joining method, NJ), a phylogenetic tree was constructed, and an exhibit values (on Bootstrap) test was set to repeat 1000 times [27].As can be seen from the figure, it can be divided into three groups.Among them, Bacillus glycinifermentans MGMM1 and thaumarchaeota have a higher homology (Figure 6).

Discussion
The diversity and richness of a bacterial community can be expressed by diversity index (Simpson, Shannon, InvSimpson) and richness index (ACE, Chao1).The diversity index and richness index are important indicators to measure community diversity.The larger the index, the higher the richness and diversity of the microbial community.In this study, the diversity of soil rhizosphere microorganisms under conventional fertilization (CF) and optimized fertilization (YF) was lower compared with the replacement of chemical fertilizers with organic fertilizers, among which the species diversity was highest when organic fertilizers replaced 80% (E).Soil microbial diversity is closely related to microbial stability, soil quality, and nutrient cycles and is susceptible to the input of exogenous organic matter [28][29][30].The application of organic fertilizers improves soil fertility, supplements soil organic matter content, and provides sufficient nutrients for soil microorganisms [21].Therefore, as the proportion of organic fertilizer increases, the proportion of chemical fertilizer decreases, and the diversity of microorganisms increases.In addition, some organic fertilizers replace chemical fertilizers.The combined application of organic fertilizers and chemical fertilizers has a certain complementary effect and can improve the physical and chemical properties of soil [31].Research by Venter et al. [32] showed that high proportions of chemical fertilizers will reduce the diversity and richness of soil bacteria in rapeseed and rice.Chunying's [33] research also showed that soils with higher organic carbon content have higher microbial diversity.It can be seen from the treatments of conventional fertilization (CF) and optimized fertilization (YF) that reducing chemical fertilizer application does not affect the diversity and richness of microorganisms.This shows that reducing the application of chemical fertilizers within a certain range can not only ensure consistency with conventional fertilization, but also effectively improve soil conditions [34][35][36][37][38][39][40].
The dominant bacterial phyla in each treatment in this study were mainly Proteobacteria, Acidobacteria, Bacteroidetes, Chloroflexi, Actinobacteria, Firmicutes, Planctomycetes, Gemtomonads, and Verrucomicrobia.Proteobacteria accounted for the largest proportion [41].This is similar to Han Yafei's [42] study of the dominant bacterial community in poplar rhizosphere soil, and it is also consistent with Roesch Luia Fw et al.'s [26] study of soil microorganisms.But they are not the same in relative abundance.This shows that different treatments have differences in the relative abundance of dominant bacteria in rhizosphere soil.This also shows that the diversity, abundance, and microbial community structure of rhizosphere bacteria are affected by vegetation varieties and soil types [15].Proteobacteria belong to the eutrophic group and generally multiply rapidly in nutrientrich soil.This shows that, after organic fertilizer treatment, soil fertility and the number of microorganisms are increased, which is suitable for the growth of nutrient-rich microorganisms [43,44].Most Acidobacteria are acidophilic bacteria, and their abundance is significantly negatively correlated with soil pH.For continuous-cropping pineapple orchards, pH shows a downward trend [45].
Principal coordinate analysis (PCoA) and nonmetric multidimensional scaling (NMDS) analysis finds that the distances between organic fertilizer substitution (60%, 80%), optimized fertilization (CF), and no fertilization (CK) are very close, and the similarity is high.NMDS is generally consistent with PCoA, and other treatments are further apart.This shows that different fertilization methods can change the soil rhizosphere microbial community structure.With the addition of organic fertilizer, organic fertilizer replacement (60%, 80%) was similar in community structure and composition.In the study by Cai Jie et al., the community structure of no fertilization (R1) and conventional fertilization (R2) was similar.Conventional fertilization only changed the abundance of microorganisms and did not change the community structure of cassava soil microorganisms, but optimized fertilization changed the microbial community structure.In this study, the community structure between optimized fertilization and no fertilization was relatively similar, which was different from Cai Jie's [15] study.The diversity and community structure of soil microbial communities are affected by plant type [46], soil type [47], planting method [43], and fertilization method [48].This study is different from CAI Jie's study in both methods and crop varieties, resulting in differences.
Through LefSe analysis, there are differences in the microbial flora enriched in different treatments, and the bacterial flora enriched in different fertilization methods are different [23].Organic fertilizer replacement 20%(B) significantly enriched Proteobacteria (α-, β-, δ-, Proteobacteria), Gemmatimonadetes (Gemmatimonadetes), and Cytophagia.Organic fertilizer replacement 60%(C) mainly enriched Chitinophagia (Bacteroides), and organic fertilizers replaced 80% enriched nematobacteria (Ktedonobacteria).Studies have shown that nutrient-rich soil contains more α-Proteobacteria that can participate in the nitrogen cycle [26].Chloroflexi bacteria can break down the cellulose contained in the soil, thereby increasing plant biomass [49][50][51].In this study, there are also differences in the functions of pineapple rhizosphere soil bacteria under different fertilization methods.The application of organic fertilizer instead of chemical fertilizer will increase the abundance of chemoheterotrophic functional populations and allow more bacteria to participate in the carbon cycle.Therefore, the function of chemoheterotrophy is crucial for pineapple rhizosphere soil bacteria [51,52].

Conclusions
This study showed that different fertilization methods changed the diversity and abundance of soil bacteria in the pineapple rhizosphere, and the diversity of the species was E > D > C > CK > B > YF > CF.Both conventional fertilization (CF) and optimized fertilization (YF) increased the relative abundance of Proteobacteria, Actinobacteria, and Firmicutes, and decreased the abundance of Acidobacteria, Bacteroidetes, Gemtomonads, and Verrucomicrobia.Replacing chemical fertilizers with organic fertilizers increased the relative abundance of Bacteroidetes, with the highest relative abundance of Chloroflexi in treatments where organic fertilizers replaced 60%(D) and 80%(E).The replacement of chemical fertilizers with organic fertilizers promotes the function of bacteria related to carbon cycling in the rhizosphere.Our next step is to isolate and screen these beneficial microorganisms and further explore the impact of continuous-cropping pineapple rhizosphere exudates on rhizosphere microorganisms when using organic fertilizer instead of chemical fertilizer.In summary, our research provides as much reference material as possible for agricultural production practices to influence soil health and crop growth in an efficient and sustainable way.

Figure 1 .
Figure 1.Structure of the bacterial of the pineapple rhizosphere soil under different fertilization methods.(a) Bacterial community structure of pineapple rhizosphere soil based on principal coordinate analysis; (b) Bacterial community structure of pineapple rhizosphere soil based on NMDS analysis.

Figure 2 .
Figure 2. General distribution of the main bacterial phylum (a) and genus (b), and the average relative abundance of the main phylum (c) and genus (d) in different treatments.

Figure 4 .
Figure 4. Ecological function of the soil bacterial community under different fertilizing treatments.Principal coordinate analysis (PCoA) and nonmetric multidimensional scaling (NMDS) ordination plot based on the Bay-Curtis distance of various samples.(a) Ecological functional diversity of bacteria in different treatments based on principal coordinate analysis; (b) Ecological functional diversity of bacteria in different treatments based on NMDS analysis.

Figure 5 .
Figure 5.The top 10 ecological functions of the soil bacterial community by FAPROTAXtool.

Table 1 .
Diversity and abundance index of rhizosphere soil bacteria in pineapple under different fertilization methods.
Note: CK: no fertilization, CF: conventional fertilization, YF: optimized fertilization, B: organic fertilizer replacement YF20%, C: organic fertilizer replacement YF40%, D: organic fertilizer replacement YF60%, E: organic fertilizer replacement YF80%; the same row of parenthetical data represents the left (minimum value) and the right (maximum value).