{"paper_id":"41bb5165-6184-442e-b8e4-e14ccfaa8843","body_text":"Different responses of sugarcane and rhizosphere soil microorganisms to single or mixture application of PGPB | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Different responses of sugarcane and rhizosphere soil microorganisms to single or mixture application of PGPB Jiang-Lu Wei, Ying Qin, Qaisar Khan, Wan-Tao Liang, Wan-Ling He, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4643245/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 20 You are reading this latest preprint version Abstract Background: Plant growth-promoting bacteria (PGPB) benefit plant growth and development via different direct and indirect mechanisms. However, our knowledge about rhizosphere soil response at different plant growth stages to diverse PGPB application in sugarcane is limited. In this study, four strains of bacteria genera ( Gluconacetobacter diazotrophicus PAL5, Streptomyces chartreusis WZS021, Bacillus spp . CA1, and Pseudomonas mosselii CN11) were inoculated into two sugarcane varieties (B8, ROC22) as single or mixture in a pot planting experiment. The effects of single or combined application of PGPB on nitrogen metabolism, agronomic traits, rhizosphere soil chemical and biological properties and microbial community were surveyed. Results: It was found that different treatments had different promotion ways for different sugarcane varieties and rhizosphere soils. PAL5 and CA1+CN11 significantly improved the nitrogen fixation efficiency of sugarcane, while WZS021 treatment enhanced phosphorus (available phosphorus and alkaline phosphatase). High-throughput sequencing (HTS) analysis revealed that Proteobacteria, Firmicutes, Chloroflexi, and Actinobacteria were the main microbial community phylum components. Correlation analysis indicates that phyla Proteobacteria and Bacteroidota played a key role in the nitrogen cycle of the soil-microbe-plant interaction system, while phylum Firmicutes had a crucial role in the phosphorus cycle. And we found that, In the varieties with weak bacterial species in the rhizosphere soil, the addition of the composite strain had the best effect, while in the varieties with rich bacterial species, the addition of the composite strain may have the exclusion phenomenon, which was not as good as the addition of the single dominant strain. Conclusions: The PGPB had excellent activities, such as nitrogen fixation, phosphorus and potassium solubilization, which could promote plant growth by decomposing soil nutrients. The inoculated strains can positively enrich the beneficial bacteria in sugarcane. However, there were variations in the quantities of these promoted properties in the treatments with different bacterial strains and sugarcane varieties. It was found that soil-disadvantaged and inoculum-specific bacteria were more favorable to plant development. The considerable variation in soil microbe provides a knowledge base and an experimental system for further mining and utilization of microbial strains. PGPB sugarcane promotion soil High-throughput sequencing (HTS) Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1. Introduction Sugarcane, a significant cash crop for global economy, is facing the problem of excessive fertilizer and pesticide applications. The nutrients required for plant growth in agricultural production are mainly provided by chemical fertilization [ 1 , 2 ]. The heavy use of chemical fertilisers and pesticides have led to the imbalance of nutrient ratios, soil property deterioration, declined agricultural product quality, destruction of environment, and even seriously affected the sustainable development of sugarcane industry [ 3 , 4 ]. In order to avoid the side effects of fertilizers on crops and soil, high attention has been paid to microorganisms as an alternative to chemical fertilizers in agriculture due to their cost-effectiveness and environmental friendliness. One of the ecologically sustainable ways to handle the aforementioned problems is to use the crop-microbe interaction model. In many countries, inoculation of plant growth-promoting bacteria (PGPB) is a promising approach to improve sustainable agricultural production [ 5 ]. PGPB found in the rhizosphere and inside plant, colonize the root cells and subsequently promote plant growth through a variety of mechanisms, such as hormone production, improved nutrient supply for the plant, inhibition of plant pathogens, and modification of the physico-chemical properties of the soil [ 6 , 7 ]. PGPB are essential to the cycling of nutrients in soil [ 8 , 9 ] and has an influence on soil ecosystems, biochemical processes, plant growth and development, and overall health [ 10 ]. The diazotroph which is one of the crucial communities in the PGPB are capable of fixing atmospheric nitrogen. They include the genus Rhizobium, which falls within symbiotic relationships with leguminous plants, and some Pseudomonas sp. When the amount of nitrogen nutrients in the soil decreases, the significance of applying PGPB rises [ 11 ]. Subsequently, the bacteria are capable of efficiently facilitating plant development by supplying the essential limiting factors. Bacillus , Pseudomonas , Agrobacterium , Burkholderia and Streptomyces have been well studied and significantly marketed as biostimulants or biofertilisers [ 12 ]. Although applications of single PGPB have been reported in greenhouse and field environments [ 13 , 14 ], microorganisms have shown poor colonization capacity, high environmental dependence and unstable effects, resulting in that single-strain and single-function fertilisers could not meet the requirements of modern agricultural development. The multi-functionality of composite PGPB will provide a chance to improve its environmental adaptability [ 15 , 16 ]. When a combination of several PGPB strains is applied, complicated chemical signal exchange systems generated by plant-PGPB interactions improve plant growth and defence [ 17 , 18 ]. Microbial functional combination has been shown to be extremely significant in enhancing plant performance, and this strategy may be the first step towards creating bacterial communities that are beneficial to plants [ 19 ]. Therefore, elucidation of the interaction mechanism between mixed PGPB and sugarcane is essential for the selection of bacterial strains that will be used individually or in combination as biofertilizers. Traditional laboratory approaches are tough and time-consuming owing to the majority of unculturable PGPB [ 20 ]. Technological developments in molecular biology have enhanced our comprehension of the rhizosphere microbiota, especially with the introduction of affordable high-throughput sequencing (HTS) and associated data processing methods [ 6 ]. Sequencing provides a practical and comprehensive system for identifying the rhizosphere microbial species regardless of microbial abundance. HTS was also employed to explore the soil microbial dynamics of sugarcane [ 21 , 22 ]. Therefore, evaluation of microbial dynamics in sugarcane and rhizosphere soil by high-throughput sequencing technology will be a potential strategy in revealing the complexity and diversity of microbial communities. Application of a single strain promoted sugarcane growth, increased soil nutrients and enzyme activities [ 23 , 24 ]. Four strains of PGPB( Bacillus spp.CA1, Pseudomonas mosselii CN11, klebsiella sp.DX120E, Streptomyces chartreusis WZS021)have been isolated by our research group[ 25 – 27 ]. And they have been confirmed on sugarcane growth has a promoting effect. In the previous experiment, we have selected the above four PGPB strains for single and combination tests. By using the biological characteristics of PGPB strains, the optimal combination was obtained through factor analysis, and then pot planting test was conducted. Three optimal strain combinations ( Streptomyces chartreusis WZS021, Bacillus spp.CA1 + Pseudomonas mosselii CN11, Pseudomonas mosselii CN11 + Streptomyces chartreusis WZS021) and Gluconacetobacter diazotrophicus PAL5, were selected to apply in two sugarcane varieties (B8, ROC22).The antagonism test against the above combined strains has been carried out, and it has been proved that there is no antagonism.The current study thus aims to comprehend the nutrient uptake by plants from rhizosphere soils and the growth-promoting properties of single PGPB ( Gluconacetobacter diazotrophicus PAL5, Streptomyces chartreusis WZS021) and mixed PGPB ( Bacillus spp.CA1 + Pseudomonas mosselii CN11, Pseudomonas mosselii CN11 + Streptomyces chartreusis WZS021) in sugarcane varieties ROC22 and B8. The 16S rRNA HTS molecular technique was used for analyses of the microbial communities in rhizosphere and sugarcane responsive to PGPB. The purpose of the work was thatdemonstrated how PGPB promote sugarcane nitrogen uptake ability, nitrogen metabolism, fast soil nutrient release, and plant growth and development. This study also revealed the diversity of rhizosphere soil bacterial communities in two sugarcane varieties after PGPB inoculation. These results provided a knowledge of the PGPB influence on plant growth, soil nutrient utilisation, microbial community composition and their interrelationship. 2. Materials and methods 2.1. Bacterial solution preparation Bacterial strains, species, culture media and isolation sources used in this study were shown in supplementary Table 1. Strains Gluconacetobacter diazotrophicus PAL5, Bacillus spp. CA1, Pseudomonas mosselii CN11, and Streptomyces chartreusis WZS021 were taken out from a -80℃ freezer and activated on a suitable solid medium. Single colonies were picked and cultured in a liquid medium overnight until the OD 600 was 1.0. Bacteria were collected by centrifugation (4000 rpm, 4℃, 10 min) and suspended in sterile phosphate buffer solution (PBS, pH 7.4) to get a bacterial concentration of 10 8 CFU/ml. 2.2. Pot experiment Two sugarcane varieties, Xintaitang 22 (ROC22) and RB86-7515(B8), were selected for pot experiment. ROC22 is the main cultivated sugarcane variety, and B8 is the Brazilian nitrogen-fixing sugarcane variety. The experiment was carried out in College of Agriculture, Guangxi University, Nanning (22° 51’ N 108° 17′ E), China. The physico-chemical properties of the soil were as follows: pH 6.7, potassium 7.10 g/kg, phosphorus 0.90 g/kg, nitrogen 0.82 g/kg, available phosphorus (AP) 22.0 mg/kg, hydrolysed nitrogen 47.7 mg/kg, and available potassium (AK) 47.0 mg/kg. The size of the pots used was 24 cm in upper calibre and 30 cm in height. Each pot contained about 17 kg of soil, and 10 mg of ( 15 NH₄)₂SO₄ (product of Shanghai Research Institute of Chemical Industry CO., LTD, China, with 10.12% abundance value of 15 N marker) was added to each kilogram of soil. Sugarcane was planted in a 1:1 substrate ratio of sand and soil. When the plants grew to 3–4 leaves, those with similar growth status were selected, and their roots were washed with water and soaked for 40 min in the strain suspension and then transplanted into pots. The mixed inoculum suspension (1:1) was inoculated on two seedlings per pot with three replicates, with sterile water as the control. No fertiliser was applied during growth, and regular watering and weeding practices were followed. Agronomic traits of the pot-planted sugarcane were investigated and sampled on the 30th, 60th, 90th, and 120th days after inoculation (DAI). The biomass was measured on the 240th DAI. The treatments and codes were shown in Table 1 . Table 1 The treatments and codes Sugarcane variety Inoculation treatment Code Sterile water BCK PAL5 BT0 B8 WZS021 BT1 CA1 + CN11 BT2 CN11 + WZS021 BT3 Sterile water RCK PAL5 RT0 ROC22 WZS021 RT1 CA1 + CN11 RT2 CN11 + WZS021 RT3 2.3. Agronomic traits and enzyme activity determination Plant height [from stem base to leaf ring position of the first fully expanded (+ 1) leaf], chlorophyll content and leaf enzyme activities such as nitrate reductase (NR) [ 28 ], glutamine synthetase (GS) [ 29 ], glutamic pyruvate transaminase (GPT) and glutamate oxaloacetate transaminase (GOT) [ 30 ] were measured on 30th, 60th, 90th, and 120th days after inoculation. Chlorophyll content was determined using a portable chlorophyll analyser (SPAD 502). Sugarcane aboveground and underground parts were taken and weighed separately for biomass determination at 240th DAI. 2.4. Sugarcane nitrogen utilisation assay Whole plants were collected at 240th DAI and weighed freshly. Roots, stems, and leaves were separated and put into an oven at 105℃ for 30 min, then baked to a constant weight at 75℃, and the dry weight were weighed. Crushed samples were used to detect the total nitrogen content (TN) and 15 N content. 15 N atom was calculated according to the nitrogen fixation efficiency formula: Ndfa% = [1 - (inoculated treatment 15 N atom/non-inoculated control 15 N atom)] × 100% [ 31 ] 2.5. Soil physical and chemical properties Soil available nutrients and enzyme activities were determined on the 120th DAI. The available phosphorous (AP) was determined by the UV-visible spectrophotometer based on the standard method (Soil available P leaching with sodium bicarbonate-molybdenum antimony resistance spectrophotometry, LY/T 1232–2015, China). The available potassium (AK) was extracted with neutral 1 mol/L ammonium acetate solution and determined by flame photometer based on the standard method (Determination of soil available K and slow available K, NY/T 889–2004, China). The available nitrogen (AN) was determined by alkali hydrolysis method (LY/T 1228–2015, China). The catalase (CAT), alkaline phosphatase (AKP), and urease (UE) activities were determined according to the kit instructions (Suzhou Grace Biotechnology Co., Ltd, China) 2.6. High-throughput sequencing analysis The sugarcane rhizosphere soil samples were taken at both seedling and elongation stages. The plants with soil were taken out carefully, the soil attached to the roots was shaken off, and kept in sterile bags in an ice box. These samples were quickly frozen in liquid nitrogen and then stored in a -80℃ freezer for 16S rRNA analysis. DNA was extracted using the DNA extraction kit E.Z.N.A.® Soil DNA Kit (Omega Bio-Tek, USA). The extracted DNA was detected in 1% electrophoresis for DNA integrity and Omega Bio-Tek instrument for purity. The DNA was used as a template to amplify the V3-V4 region of the bacterial 16S rDNA gene using 16S primers 338F (5ʹ-ACTCCTACGGGGAGGCAGCA-3ʹ) and 806R (5ʹ-GGACTACHVGGGTWTCTAAT-3ʹ). The PCR amplification for 16S rRNA was done in a total volume of 20 µL comprising of 5× FastPfu Buffer 4 µL, 2.5 mM dNTPs 2 µL, forward primer (5 µM) 0.8 µL, reverse primer (5 µM) 0.8 µL, FastPfu DNA Polymerase 0.4 µL, BSA 0.2 µL, genomic DNA 10 ng, ddH 2 O to 20 µL. The PCR parameters were set as 95℃ for 5 min, 30 cycles (95℃ for 30 s, 55℃ for 30 s, 72℃ for 45 s) and 72℃ for 10 min. The PCR products were identified and purified using 1% agarose gel and AxyPrep DNA Gel Extraction Kit (Axygen, USA). The purified products were quantified by Quantus™ Fluorometer (Promega, USA). The libraries were constructed using the NEXTFLEX Rapid DNA-Seq Kit (Bio Scientific, USA) and sequenced using Illumina's Miseq PE300 (Illumina, USA). 2.7. Operational Taxonomic Unit (OTU) clustering analysis The raw sequences quality was controlled using fastp software ( https://github.com/OpenGene/fastp , version 0.20.0) [ 32 ] and spliced using FLASH software ( http://www.cbcb.umd.edu/software/flash , version 1.2.7) [ 33 ]. OTUs were clustered based on 97% similarity using UPARSE software ( http://drive5.com/uparse/ , version 7.1) [ 34 , 35 ]. To minimise the impact of sequencing depth on subsequent analysis of Alpha diversity and Beta diversity data, the number of sequences in all samples was drawn flat to 20,000. OTU species taxonomy was annotated using the RDP classifier ( http://rdp.cme.msu.edu/ , version 2.11 [ 35 ]) compared to the Silva 16S rRNA gene database (v138), with a confidence threshold of 70%, and community composition was counted for each sample at different levels of species classification. 16S functional prediction analyses were performed using PICRUSt 2 software (version 2.2.0) [ 36 ]. All the high-throughput data analyses were performed on the Meggie BioCloud platform ( https://cloud.majorbio.com ). Mothur soft ( http://www.mothur.org/wiki/Calculators ) was used to calculate α diversity Chao, and Shannon indexes. The Wilcoxon rank sum test was used for group difference analysis of α diversity. R language (version 3.3.1) tools were used for statistics and graphing. Species Venn diagrams were analysed for Qiime to calculate the beta diversity distance matrix, and then graphical trees were drawn in R language (version 3.3.1) for β diversity analysis. The similarity of microbial community structure among samples was examined using Principal Coordinate Analysis (PCoA) based on the bray-curtis distance algorithm and combined with the PERMANOVA non-parametric test to analyse whether the differences in a microbial community structure among sample groups were significant. Bacterial taxa with significant differences in abundance from phylum to genus level between groups were identified using LEfSe analysis (Linear discriminant analysis Effect Size ( http://huttenhower.sph.harvard.edu/LEfSe ) (LDA > 2, P < 0.05) [ 37 ]. Redundancy analysis based on distance (RDA) was used to investigate the effect of soil physico-chemical indicators on soil bacterial community structure. Species were selected for correlation network graph analysis based on spearman correlation |r| > 0.6 p < 0.05 [ 38 ]. MicroPITA analysis was done using R (version 3.3.1) and Python [ 39 ]. Other experimental data were recorded and statistically analysed using Office Excel 2010 and SPSS software version 23.0 (IBM Corp, Armonk, New York). The resulted graph of the data was produced by Origin (2016) software. The raw data of 16S rRNA (accession no. PRJNA1052972) were submitted to the NCBI Sequence Read Archive ( https://www.ncbi.nlm.nih.gov/gene/?term=PRJNA1052972 ). 3. Results 3.1. Nitrogen metabolism enzyme activities and nitrogen fixation efficiency The GPT, GOT, NR, and GS activities in leaves of the inoculated sugarcane plant were generally significantly higher than those in the control. The changes in GPT (Fig. 1 a, e), GOT (Fig. 1 b, f), and GS (Fig. 1 d, h) activities of both sugarcane varieties showed a trend of increasing and then decreasing. The GPT and NR activities in leaves of both varieties generally reached their maximum values after the 60th DAI. The GPT activity in T2 was significantly higher than that in the control for both B8 (96.39%) and ROC22 (116.17%) varieties. On the 90th DAI, the GOT and GS enzyme activities of all treatments reached the maximum. The GS activity in BT0 was significantly increased by 1.09-fold, and that in BT2 was significantly increased up to 89.35% than BCK. Except for BT0, the NR activity in leaves of variety B8 increased significantly on the 120th DAI compared to BCK. The NR activity in leaves of variety ROC22 was significantly higher in RT2 treatment than RCK by 17.76% on the 120th DAI. Although the NR activity in sugarcane leaves gradually decreased, it was significantly higher than the control treatment most of time (Fig. 1 c, g). The results of the 15 N isotope dilution test (Table S2 )revealed that the both sugarcane varieties B8 and ROC22 showed a significant increase in the total nitrogen percentage in roots under PGPB inoculation treatments compared to the controls but a significant decrease in 15 N atoms compared to the controls. The highest nitrogen fixation efficiency for T1 in B8 and ROC22 roots, respectively. 3.2. Agronomic characters The statistics of sugarcane plant height and biomass showed a positive boost after PGPB inoculation. On the 120th DAI, Both BT3 and BT1 treatments showed significantly higher plant height than the BCK by 26.58% and 30.04%, respectively, in B8 (Fig. 2 a). The plant height in variety ROC22 showed an increase up to 41.76% on the 90th DAI (Fig. 2 b). T0 treatment had the most significant effect on the aboveground fresh weight in B8 (Fig. 2 c) and ROC22 (Fig. 2 d), which was 1.59 and 1.81 times, respectively, higher than the controls. Both T2 and T0 treatments increased the underground dry weight in the two sugarcane varieties significantly. The T2 treatment showed the most significant increase in the underground dry weight in both B8 and ROC22, with a 98.60% increase in BT2 and a 1.31-fold increase in RT2, respectively, than the controls. 3.3. Chlorophyll content Chlorophyll content in sugarcane leaves was increased in all the PGPB inoculation treatments compared to the controls. The highest chlorophyll content in B8 was observed in the BT0 treatment at 60th and 90th DAIs. The chlorophyll content in the PGPB inoculation treatments was 19.89–24.91% significantly higher than that in the control on the 120th DAI (Fig. 2 e). The chlorophyll content in ROC22 was also increased in the PGPB inoculation treatments compared to the control, and that was significantly higher in RT2 treatment than in RCK in most cases especially at the 90th and 120th DAIs, respectively (Fig. 2 f). 3.4. Soil physio-chemical properties and enzyme activities Soil is one of the main factors influencing plant growth, development and survival. It was found the available nutrients in the soil were significantly increased after PGPB inoculation (Fig. 3 a, b, c). Soil nitrogenase activity was significantly increased in all the PGPB inoculation treatments compared with the controls (Fig. 3 d). Soil available nutrients and soil nitrogenase activity of different sugarcane cultivars were improved by different treatments.AN content in the soil of sugarcane variety B8 showed the order BT0 > BT3 > BT1 > BT2 > BCK, and the PGPB inoculation treatments showed at least 57.97% higher AN than the control BCK. The AN content in the soil of sugarcane variety ROC22 was highest in the RT2 treatment, which was 92.97% significantly higher than the control RCK. AP content in the soil of sugarcane variety B8 was the highest in RT0 treatment, 2.03-fold significantly higher than that in BCK. AP content in ROC22 soil was highest in RT1 treatment, 3.34-fold significantly higher than that in RCK. AK content in the soil of sugarcane variety B8 showed the order of BT0 > BT2 > BT3 > BT1 > BCK. AK content in the soil of sugarcane variety ROC22 was the highest in the RT0 treatment, which was 1.66-fold significantly increased compared with the control. The other inoculation treatments showed the order RT3 > RT2 > RT1, which were 1.46-fold, 1.37-fold, and 1.34-fold, respectively, significantly higher compared with the control RCK. Soil catalase activity was significantly increased in all the PGPB inoculation treatments compared with the controls (Fig. 3 d). The BT1 treatment showed the highest soil catalase activity, which was 1.08 times higher than BCK. The other PGPB inoculation treatments for B8 showed 46.72–80.48% higher than BCK. The RT3 treatment exhibited the highest soil catalase activity in the ROC22 variety, which was 62.61% higher than that in RCK. All the soils in B8 had considerably stronger alkaline phosphatase activity than the control (Fig. 3 e). The BT1 treatment had 2.45 times more significant alkaline phosphatase activity than the BCK. The remaining treatments showed the order BT3 > BT0 > BT2. The strongest alkaline phosphatase activity in sugarcane ROC22 variety soil was found in the RT0 treatment, which was 1.55-fold higher than that in RCK. The urease activities in the soil were significantly higher in the PGPB inoculation treatments than the control (Fig. 3 f). The order of urease activity in B8 soil showed BT0 > BT2 > BT1 > BT3, which was 88.59%, 78.14%, 60.40%, and 39.10%, respectively, higher than that in BCK soil. The strongest urease activity in ROC22 soil was in the RT2 treatment, which was 64.77% higher than that in RCK soil. 3.5. Sequencing data and operational taxonomic unit (OTU) A total of 611,024 valid sequences (average length of 419 bp) at the seedling and 744,771 valid sequences (average length of 416 bp) at the elongation stage were obtained from the rhizosphere soil samples from different treatments. Following QIIME's filtering, the raw 16S rRNA readings were sorted, grouped, and examined individually for OTUs (Tables S3, 4). Common and unique OTUs among the samples were analysed based on the 16S rRNA sequences. From the 16S rRNA sequences, it was found there were 3220 OTUs in total from all the seedling rhizosphere soil samples, 699 of which were common to all the samples ( Fig. 1 a). The top three ranked OTUs in the treatment were RT1 > BT3 > BT1. The total number of OTUs identified in the rhizosphere soil at elongation stage was 5,511, of which 1,346 were common to all the treatments (Fig. S1 b). Common and unique OTUs were significantly higher compared with those at seedling stage. 3.6. Principal component analysis (PCA) Based on the OTUs data, PCA was done to examine the impact of growth-promoting bacteria in the bacterial community in the rhizosphere soil of sugarcane. The rhizosphere soil microbial communities at seedling stage were present in close proximity at both B8 and ROC22 in T2 and T3 treatments (Fig. S2 a). The PCA results for the elongated soil samples showed that the soil microbial communities in the rhizosphere soil of sugarcane varieties B8 and ROC22 were closer to each other in different treatments except for BCK (Fig. S2 b). 3.7. Diversity index and microbial composition α diversity is the diversity that exists individually within a given sample and is represented by the types of microorganisms enumerated in the test sample. The diversity analysis was characterized using Shannon, Simpson, and Chao exponential rarity curves. Shannon,Chao and Simpson indexes for the seedling soil samples, showed that T1 treatment was the highest in both sugarcane varieties (Fig. S3 a,b). The relatively high indexes for the sugarcane soil samples at the elongation stage were BT2 and BT3 treatments in B8, and RT0 and RT1 treatments in ROC22. Meanwhile, it was found that the Chao indexes for variety ROC22 at the elongation stage were higher than that for variety B8, indicating that the richness of the microorganisms in the rhizosphere soil of variety ROC22 was higher than that in B8. The phylum composition of the soil microbial community in sugarcane at the both stage was shown in the following order Firmicutes > Proteobacteriaes > Chlorofexi > Actinobacteria, which accounted for more than 70% of the microorganisms in all the PGPB inoculation treatments(Fig. 4 , S4). Genera Nocardioides , Streptomyces , Arthrobacter , Sphingomonas , Microvirga , and Bacillus were dominant at elongation stage (Fig. 5 ). The genera in T1 treatment were similar in composition to those in CK at seedling stage. Microbial community analysis of the soil samples at seedling stage showed that the soil samples in the treatments with CN11 bacteria contained Eschercha -Shigella spp. The number of genera and diversity index in each treatment increased at elongation stage compared with those at seedling stage. The genus Bacillus spp. was found higher in T0 treatment, and Sphingomonas spp. higher in variety ROC22 than in B8, while Arthrobacter spp. higher in B8 than in ROC22. The most significant increase in the abundance of Bacillus spp. in the soil of sugarcane variety B8 was observed in the BT0 and BT3 treatments. Bacillus spp. was increased in all the PGPB inoculation treatments and the highest in T3 treatment in the soil of ROC22. The heat map proved that phyla Proteobacteria, Actinobacteriota, Bacteroidota, and Firmicutes had a positive correlation, while Planctomycetota, Armatimonadota, Nitrospirota, Elusimicrobiota had a negative correlation in each treatment. When the heat maps of soil bacterial community abundance at the two stages were compared, it was found that Bacillus spp. was present at the genus level all the time, and exhibited a substantial positive connection ( Fig. S5 , S6), while Escherichia-Shigella and Kosakonia in RT2, RT3, BT2 and BT3 treatments showed a significant positive correlation, and the other treatments showed a negative correlation at seedling stage. 3.8. β diversity Cluster analyses based on PCoA and UniFrac were performed to further understand the β-diversity of 16S rRNAs in sugarcane rhizosphere soils. PCoA analysis revealed that the T0 treatment was separated from all the other treatments, showing higher β diversity. Hierarchical clustering based on UniFrac cluster analysis showed similar results for 16S rRNA data, containing identical sequences showing 0 (blue) distance. RT2 and RT3 showed a greater distance from the other treatments, indicating the difference. The distribution of dominant genera based on their relative abundance using the Bray-Curtis algorithm revealed that Bacillus was the dominant genus in all the samples (Fig. 6 ). Escherichia-Shigella was the second most dominant genus, while no rank_f_JG30-KF-CM45 was the third most dominant genus in sugarcane rhizosphere soils at seedling stage. No rank_f_JG30-KF-CM45 was the second most dominant genus, and Sphingomonas became the third most dominant genus, while the proportion of Arthobacter and Streptomyces genera increased at elongation stage. Soil microbial diversity was similar between the two sugarcane varieties in the same treatment at seedling stage (CK, T0, and T1). At elongation stage, the rhizosphere soil β diversity changed considerably due to sugarcane growth. BT3 and BT1 had similar diversity, and RT2 and RT0 had similar diversity (Figs. S7, S8). 3.9. Spearman’s rank correlation Spearman’s rank correlation analyses were done based on the 16S rRNA abundance of the top 20 genera, diversity indices, and soil variables (Fig. S9 a, c) to assess the bacterial genera correlation and dominance of 16S rRNAs with the soil physico-chemicals. Heat maps revealed that soil physico-chemical factors significantly influenced the relative abundance of bacterial taxa (phylum and genus). The phylum microorganisms, environmental factors, and OTUs were subjected to RDA analysis (Fig. S9 b) and combined with heat maps (Fig. S9 a) to further analyse the relationship between the soil environmental factors, the treatments, and the bacterial community structure. The selected soil chemical factors, enzyme activities, and soil α-diversity were found to explain 88.61% of the variation in the bacterial community. The vector lengths of soil physicochemical properties and microbial communities in the ordination diagrams showed that the soil physicochemical properties and enzyme activities showed a positive correlation with axis 1. Correlation heatmaps at the microbial genus level (Fig. S9 d) revealed that the soil α-diversity index was influenced more by ROC22 compared with B8, while soil available nutrients and enzyme activities affected the bacterial community in the rhizosphere soil of B8 to a greater extent. It was learned that the soil fertility level can lead to changes in the bacterial community and, conversely, may affect the soil fertility status. 3.10. Functional forecasting and analysis Functional prediction of microbial community based on PICRUSt2 was carried out. A comparison of sequencing data in the KEGG database revealed that rhizosphere soils at seedling and elongation stages were involved in six categories of identifiable metabolic pathways, including metabolism, environmental information processing, genetic information processing, cellular processes, human diseases, and organic processes (Fig. S10 ). Metabolism was the most dominant function, with a minimum abundance of 76.06% and 77.24% in the rhizosphere soil at seedling and elongation stages, respectively. Further analyses were carried out at the secondary functional level of the predicted genes, involving a total of 46 functional categories such as global and overview maps, carbohydrate metabolism, amino acid metabolism, and energy metabolism (Figs. S11, S12). Global and overview maps had the highest percentage in all the groups, with the lowest abundance of 39.01% and 40.03% in the rhizosphere soil at seedling and elongation stages, respectively. Global and overview maps, carbohydrate metabolism, amino acid metabolism, and energy metabolism together accounted for more than 50% in each group. The primary function analysis revealed that the metabolic functions in the soils for the treatments RT3, RT2, and BT1 at seedling stage in both varieties were higher than that in the control, and at elongation stage in ROC22, all higher than that in the control. 3.11. MicroPITA analysis According to the distance algorithm, bray curtis, three sample sizes were selected based on OTU classification levels and diversity indices, resulting in a MicroPITA analysis plot (Fig. 7 ). At seedling stage, for the rhizosphere soil samples in different PGPB treatments, multiple selections were RT1, RT3, and maximum diversity selections were BCK and BT1. The most representative selection based on β diversity were BT0, BT3, and RT2. At elongation stage, BCK, BT1, and RCK were selected as multiple selections, BT0 was the most dissimilar, and RT0 and RT1 had maximum diversity among the PGPB treatments. The above results revealed that T1 treatment was highly selective in sugarcane rhizosphere soil. T2 treatment revealed the most extreme β diversity selection in variety ROC22. T3 showed the most extreme β diversity selection in the rhizosphere soil at seedling stage. 4. Discussion Previous research has indicated the impacts of PGPB on the growth of crops. PGPB treatment to maize [ 40 ] and wheat [ 41 ] seeds were found to deliver more nitrogen and enhanced the average yield compared to the untreated control. A combination of PGPB strains delivered more nitrogen for maize than a single-strain supplement and greatly boosted wheat output [ 42 ]. These results suggested that the applied bacterial strains were interacting with each other, and promote the nitrogen-fixation ability of the nitrogen-fixing bacteria. To understand the impacts of single and mixed PGPB application on sugarcane, the current study was performed. So, single (PAL5, WZS021) and mixed (CA1 + CN11, CN11 + WZS021) strains of bacteria were inoculated into two sugarcane varieties, B8 and ROC22. The results revealed that PGPB effectively increased the activities of nitrogen metabolism-related enzymes (GPT, GOT, NR, and GS) in sugarcane plants. The mixed strain CA1 + CN11 had the strongest effect on nitrogenase activity and chlorophyll activity, and significantly increased the dry weight of the underground part of the plant. It is speculated that the interaction effect of bacteria leads to the enhancement of nitrogen fixation capacity.Moreover, it was found that the single dominant strain PAL5 also had a strong effect on nitrogenase activity and nitrogen fixation of sugarcane, and the influence of above biomass was the most significant, so that the height of sugarcane was significantly higher than that of the control.The above results proved that the nitrogen-fixing ability of the growth-promoting bacteria can further promote plant growth by promoting nitrogen utilization and photosynthesis in sugarcane. Inoculation of PGPB in the soil increased the concentration of mineral nitrogen as well as that of phosphorus and potassium in the soil, improved the nutrient utilisation rate, and promoted plant absorption and utilization of nitrogen, phosphorus and potassium by plants [ 43 ]. Soil nutrient detection in this study revealed that PAL5 had a large boosting influence on AN and AK in the rhizosphere soils of both sugarcane varieties, while the combination of CN11 + WZS021 and CA1 + CN11 was the second most important factor influencing these two elements. This might be connected to PAL5's very potent biological nitrogen fixing capabilities [ 44 ]. Sugarcane variety B8 had the highest AP content in response to PAL5, The 15 N isotope dilution experiment also showed that PAL5 treatment significantly increased total nitrogen content in leaves and roots, thereby promoting the increase of aboveground biomass. Streptomyces chartreusis WZS021, participates in the soil phosphorus cycle, decomposing organic matter into available phosphorus, resulting in the accumulation of available phosphorus and available phosphorus components in soil. ROC22 had the highest AP in the WZS021 treatment. The results of CAT and AKP responses showed a similar pattern, with WZS021 being the most effective in variety B8, followed by the mixtures of strains. The most significant reaction response of CAT and AKP content in variety ROC22 was found still in PAL5 treatment, followed by the treatments with combination of CN11 + WZS021 strains. It is consisted that Actinomycetes spp. promote the release of soil insoluble phosphorus through microbial metabolic activities and participate in the soil phosphorus cycle [ 45 – 47 ]. Moreover, WZS021 and CN11 + WZS021 strains significantly affected plant height, indicating that the growthpromoting bacteria had excellent activities, such as nitrogen fixation, phosphorus and potassium solubilization, which could promote plant growth by decomposing soil nutrients. Soil nutrient discrepancies reflect variations in soil enzyme activity in response to various probiotic bacteria in different sugarcane varieties. Based on 16S rRNA sequences, the microbial community composition in sugarcane rhizosphere soil was also investigated. The Chao index for ROC22 variety was higher than that for B8 variety at both seedling and elongation stages, indicating that the total number of bacteria species in the rhizosphere soil of ROC22 variety was higher than that of B8 and the soil microbial community varies at different growth stages of sugarcane and in different sugarcane varieties. A further investigation of its responses to different exogenous PGPB additions revealed that the bacterial microbial richness in sugarcane rhizosphere soils at seedling stage was greater in all the WZS021 treatments than in the other treatments. At elongation stage the microbial abundance in the rhizosphere soil of B8 in the CA1 + CN11 treatment was higher than in the other treatments. It was found that the WZS021 treatment had higher microbial abundance than the other treatments in ROC22 at the elongation stage. At the same time, there were differences in the response mechanism of different sugarcane varieties to the strain. In the varieties with weak bacterial species in the rhizosphere soil, the addition of the composite strain had the best effect, while in the varieties with rich bacterial species, the addition of the composite strain may have the exclusion phenomenon, which was not as good as the addition of the single dominant strain. Proteobacteria, Firmicutes, Actinobacteria, Bacteroidetes, Chloroflexi, Gemmatimonadetes, Planctomycetes, and Nitrospirae phylum-level distribution were shown to predominate in sugarcane [ 22 , 48 ], This is consistent with the present study. Phyla Actinobacteria, Firmicutes, and Proteobacteria were also members of the leading phyla in the sugarcane rhizosphere soil microbial community, whereas Bacteroidota was less represented. Y Gu, J Wang, W Cai, G Li, Y Mei and S Yang [ 49 ] found that phylum Aspergillus, Actinobacteria phylum and Firmicutes phylum were the dominant endophytic bacteria in sugarcane exposed to different treatments. The Bray-Curtis algorithm revealed that Bacillus was the leading genus in all samples, with a substantial increase in abundance in all rhizosphere soils treated with PGPB, which were characterised after the CA1 + CN11 treatment. Furthermore, following PGPB inoculation treatment, the abundance of Streptomyces , Sphingomonas , Arthrobacter , and Nocardioides increased significantly in the B8 rhizosphere soil. As a result, the soil supports the survival of several microbe species for self-regulation. The disadvantaged microbial community, on the other hand, has a poor capacity to compete for its own nutrients, causes minimal change in the soil environment, and performs outstanding activities such as nitrogen fixation, phosphorus and potassium solubilization, all of which contribute to the soil nutrient environment. However, after elongation, sugarcane soil diversity was similar for BT3 vs BT1 treatments. Chao index showed that the diversity index of complex strains in B8 was higher, and RT2 and RT3 treatments in ROC22 were farther apart from the other treatments. It showed the difference between the complex strains and single strain. Therefore, it is speculated that the inoculated strains can positively enrich the beneficial bacteria in sugarcane. Application of bacteria that were disadvantaged and functionally specialised to the soil environment was more favorable to plant development, so it seemed that the microbiota had a more pronounced influence compared to the dominant population. Variation in the composition and diversity of soil microbial communities is closely related to soil physicochemical properties [ 50 , 51 ]. Inoculation of PGPB increased soil nutrients, enzyme activities and improved soil microbial communities [ 52 ]. S Yang, J Xiao, T Liang, W He and H Tan [ 50 ] found that different fertilizer treatments improved sugarcane soil biological properties and soil bacterial diversity. Spearman's correlation analysis showed that The nitrogen-fixing Bacillus species promote plant growth directly via nitrogen fixation, phosphate solubilization and production of phytohormones and indirectly through the production of antibiotics, hydrolytic enzymes and siderophores [ 53 , 54 ]. Bacillus subtilis fixes nitrogen and promotes the growth and development of crops [ 55 ]. It can not only promote the growth of crop plant height but also significantly increase root activity, net photosynthetic rate and yield per plant. RDA analysis indicated that AP, AK, AKP, UE and CAT were all significantly positively correlated with Firmicutes and BRC1. Consistent with the results, higher levels of AP, AK, AKP, UE and CAT were detected in the T2 treatment. This may be the reason why the addition of WZS021 alone had a weaker effect on biomass, SPAD, and AN in sugarcane than the addition of PAL5 and CN11 + CA1 since WZS021 tends to act on the phosphorus cycle, while PAL5 and CA1 tend to enhance the nitrogen cycle. H Minjie [ 56 ] showed that soil effective phosphorus content and AKP activities were closely related to the microbial abundance in soil. This meant that an interaction exists between the level of soil fertility and the bacterial community [ 51 ]. In addition, the results of functional prediction of bacterial flora showed that the bacteria were involved in four metabolism pathways, including global and overview maps, carbohydrate metabolism, amino acid metabolism and energy metabolism in the rhizosphere soil of sugarcane at seedling and elongation stages. It implied the importance of microbial metabolism in soil. 5. Conclusions The application of single and mixed PGPB microbial inoculants in sugarcane increased the activities of nitrogen metabolic enzymes, nitrogen utilization capacity, chlorophyll content, agronomic features, rhizosphere soil chemical and biological properties, and microbial community. The inoculation of PGPB resulted in changes in microbial communities, which improved soil N and P cycling and increased GOT, GPT, NR, GS, SPAD, plant height and biomass of sugarcane. However, there were variations in the quantities of these promoted properties in the treatments with different bacterial strains and sugarcane varieties. It was found that soil-disadvantaged and inoculum-specific bacteria were more favorable to plant development. The strains Gluconacetobacter diazotrophicus PAL5 and Bacillus spp. CA1 showed a positive effect on enhancing nitrogen cycling while Streptomyces chartreusis WZS021 demonstrated a greater benefit in increasing phosphorus cycling in soil. This research offers a reference for investigating advantageous microorganisms in the bacteria found in the rhizosphere of sugarcane, their production, and their utilization as bacterial fertilizers. Abbreviations PGPB Plant growth-promoting bacteria HTS high-throughput sequencing ROC22 Xintaitang 22 B8 RB86-7515 OTU Operational taxonomic unit NR Nitrate reductase GS Glutamine synthetase GPT Glutamic pyruvate transaminase GOT Glutamate oxaloacetate transaminase TN Total nitrogen AP Available phosphorous AN Available nitrogen AK Available potassium CAT Catalase AKP Alkaline phosphatase UE Urease PCA Principal component analysis PCoA Principal coordinated analysis Declarations Ethics approval and consent to participate Not applicable Consent for publication Not applicable Availability of data and materials The raw data of 16S rRNA were deposited in the NCBI Sequence Read Archive (SRA) database under accession number PRJNA1052972 (https://www.ncbi.nlm.nih.gov/gene/?term=PRJNA1052972). Other data generated or analysed during this study are included in this published article [and its supplementary information files]. Conflict of interest The authors declare that they have no competing interests. Funding This work was funded by the National Natural Science Foundation of China (31971858 and 31560353), Fund for Guangxi Key Laboratory of Sugarcane Genetic Improvement (19-185-24-K-03-01), and National Modern Agricultural Production Technology System Guangxi Sugarcane Innovation Team Project (nycytxgxcxtd-2021-03-01), Guangxi Key R & D Program (GK AA22117009), and Fund of Guangxi Academy of Agricultural Sciences (2021YT011). Author contributions JLW, YQ, WTL, and WLH performed the experiment and data analysis. YRL and YXX supervised the project and designed the study. JLW, YQ, QK, YRL, DFD and YXX wrote and reviewed the manuscript. YXX and YRL finalized the manuscript. All authors have read and agreed to the published version of the manuscript. 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Supplementary Files SupplementaryFig.1.docx SupplementaryFig.2.docx SupplementaryFig.3.docx SupplementaryFig.4.docx SupplementaryFig.5.docx SupplementaryFig.6.docx SupplementaryFig.7.docx SupplementaryFig.8.docx SupplementaryFig.9.docx SupplementaryFig.10.docx SupplementaryFig.11.docx SupplementaryFig.12.docx SupplementaryTable1.docx SupplementaryTable2.docx SupplementaryTable3.docx SupplementaryTable4.docx Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 15 Jul, 2024 Reviews received at journal 12 Jul, 2024 Reviews received at journal 12 Jul, 2024 Reviews received at journal 08 Jul, 2024 Reviewers agreed at journal 06 Jul, 2024 Reviewers agreed at journal 05 Jul, 2024 Reviews received at journal 04 Jul, 2024 Reviewers agreed at journal 02 Jul, 2024 Reviewers agreed at journal 02 Jul, 2024 Reviewers agreed at journal 02 Jul, 2024 Reviewers agreed at journal 02 Jul, 2024 Reviewers agreed at journal 02 Jul, 2024 Reviewers agreed at journal 01 Jul, 2024 Reviewers agreed at journal 01 Jul, 2024 Reviewers agreed at journal 01 Jul, 2024 Reviewers invited by journal 01 Jul, 2024 Editor invited by journal 01 Jul, 2024 Editor assigned by journal 01 Jul, 2024 Submission checks completed at journal 01 Jul, 2024 First submitted to journal 26 Jun, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {\"props\":{\"pageProps\":{\"initialData\":{\"identity\":\"rs-4643245\",\"acceptedTermsAndConditions\":true,\"allowDirectSubmit\":false,\"archivedVersions\":[],\"articleType\":\"Research Article\",\"associatedPublications\":[],\"authors\":[{\"id\":326973868,\"identity\":\"7bdc18fe-bfb4-42f5-881b-4b3672930bb7\",\"order_by\":0,\"name\":\"Jiang-Lu Wei\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"College of Agriculture, Guangxi University\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Jiang-Lu\",\"middleName\":\"\",\"lastName\":\"Wei\",\"suffix\":\"\"},{\"id\":326973869,\"identity\":\"cd051790-6a76-4fd2-a2b4-cdefbff08bba\",\"order_by\":1,\"name\":\"Ying Qin\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"College of Agriculture, Guangxi University\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Ying\",\"middleName\":\"\",\"lastName\":\"Qin\",\"suffix\":\"\"},{\"id\":326973870,\"identity\":\"16f708bb-8a54-46c7-80b3-666c4eebf14a\",\"order_by\":2,\"name\":\"Qaisar Khan\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Ecology College, Lishui University\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Qaisar\",\"middleName\":\"\",\"lastName\":\"Khan\",\"suffix\":\"\"},{\"id\":326973871,\"identity\":\"2b7e3dc7-4fb6-41b4-b223-c3778c6e328a\",\"order_by\":3,\"name\":\"Wan-Tao Liang\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"College of Agriculture, Guangxi University\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Wan-Tao\",\"middleName\":\"\",\"lastName\":\"Liang\",\"suffix\":\"\"},{\"id\":326973872,\"identity\":\"8086d9da-da45-478c-9aee-a990141035c8\",\"order_by\":4,\"name\":\"Wan-Ling He\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"College of Agriculture, Guangxi University\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Wan-Ling\",\"middleName\":\"\",\"lastName\":\"He\",\"suffix\":\"\"},{\"id\":326973873,\"identity\":\"3d64bc1e-61a8-458f-a994-743931c1c08a\",\"order_by\":5,\"name\":\"Deng-Feng Dong\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"College of Agriculture, Guangxi University\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Deng-Feng\",\"middleName\":\"\",\"lastName\":\"Dong\",\"suffix\":\"\"},{\"id\":326973874,\"identity\":\"50e567f7-e58a-4e3f-a5a1-9481f25e40a0\",\"order_by\":6,\"name\":\"Yong-Xiu Xing\",\"email\":\"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAsElEQVRIiWNgGAWjYHACxgcQOoF4LcwGJGthkyBNi8GNHLPKr22HGfjZcwwYfu4gQovkjLS02zJnDjNI9rwxYOw9Q4QWfonkY7clKg6DrDNgZmwjQgubRGJbsYTBYQZ7orWAbGH8ALJFglgtkj3PkqUZzqTzSJx5VnCwlxgtBsdzDD/+bLOW429P3vjgJzFaGAQSGJh5GBh4QOwDxGgAeuYAA+MP4pSOglEwCkbBSAUA6vQyWhOqbq0AAAAASUVORK5CYII=\",\"orcid\":\"\",\"institution\":\"College of Agriculture, Guangxi University\",\"correspondingAuthor\":true,\"prefix\":\"\",\"firstName\":\"Yong-Xiu\",\"middleName\":\"\",\"lastName\":\"Xing\",\"suffix\":\"\"},{\"id\":326973875,\"identity\":\"4854a3e0-d7a4-4728-b83e-5c3d8e8f663a\",\"order_by\":7,\"name\":\"Yang-Rui Li\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"College of Agriculture, Guangxi University\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Yang-Rui\",\"middleName\":\"\",\"lastName\":\"Li\",\"suffix\":\"\"}],\"badges\":[],\"createdAt\":\"2024-06-26 14:00:38\",\"currentVersionCode\":1,\"declarations\":\"\",\"doi\":\"10.21203/rs.3.rs-4643245/v1\",\"doiUrl\":\"https://doi.org/10.21203/rs.3.rs-4643245/v1\",\"draftVersion\":[],\"editorialEvents\":[],\"editorialNote\":\"\",\"failedWorkflow\":false,\"files\":[{\"id\":60986351,\"identity\":\"427ae4e7-dc6e-49f8-9ee9-74622e85e967\",\"added_by\":\"auto\",\"created_at\":\"2024-07-24 10:10:26\",\"extension\":\"png\",\"order_by\":1,\"title\":\"Figure 1\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":82210,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eEffects of PGPB inoculation on the activities of nitrogen metabolism enzymes in leaves of sugarcane varieties ROC22 and B8. The same lowercase letters above the bars indicate no significant difference (Duncan’s multiple range test, p \\u0026gt;0.05) between treatments. (a) Glutamic pyruvate transaminase (GPT) activity in B8; (b) Glutamate oxaloacetate transaminase (GOT) activity in B8; (c) Nitrate reductase (NR) activity in B8; (d) Glutamine synthetase (GS) activity in B8; (e) GPT activity in ROC22; (f) GOT activity in ROC22; (g) NR activity in ROC22; (h) GS activity in ROC22. CK, control (sterile water). T0, \\u003cem\\u003eGluconacetobacter diazotrophicus\\u003c/em\\u003e PAL5; T1, \\u003cem\\u003eStreptomyces chartreusis\\u003c/em\\u003e WZS021; T2, \\u003cem\\u003eBacillus \\u003c/em\\u003espp. CA1 + \\u003cem\\u003ePseudomonas mosselii\\u003c/em\\u003e CN11; T3, \\u003cem\\u003ePseudomonas mosselii\\u003c/em\\u003e CN11 + \\u003cem\\u003eStreptomyces chartreusis\\u003c/em\\u003e WZS021.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"OnlineFig.1.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4643245/v1/a0b0164c723eec9b1624217b.png\"},{\"id\":60986354,\"identity\":\"950cf230-797f-4aa4-b9b6-db827346e8b4\",\"added_by\":\"auto\",\"created_at\":\"2024-07-24 10:10:26\",\"extension\":\"png\",\"order_by\":2,\"title\":\"Figure 2\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":126112,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eEffects of PGPB inoculation on plant growth in sugarcane varieties ROC22 and B8. The same lowercase letter above the bars indicate no significant difference (Duncan’s multiple range test, p \\u0026gt;0.05) between treatments. (a) plant height in B8; (b) plant height in ROC22; (c) aboveground fresh weight; (d) underground dry weight; (e) chlorophyll content in B8; (f) chlorophyll content in ROC22. CK, control (sterile water); T0, \\u003cem\\u003eGluconacetobacter diazotrophicus\\u003c/em\\u003e PAL5; T1, \\u003cem\\u003eStreptomyces chartreusis\\u003c/em\\u003e WZS021; T2, \\u003cem\\u003eBacillus \\u003c/em\\u003espp. CA1 + \\u003cem\\u003ePseudomonas mosselii\\u003c/em\\u003e CN11; T3, \\u003cem\\u003ePseudomonas mosselii\\u003c/em\\u003e CN11 + \\u003cem\\u003eStreptomyces chartreusis\\u003c/em\\u003e WZS021.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"OnlineFig.2.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4643245/v1/54cdae555cb6bcf585f00385.png\"},{\"id\":60986356,\"identity\":\"a08a855b-45ce-44ed-8916-52d95e596336\",\"added_by\":\"auto\",\"created_at\":\"2024-07-24 10:10:26\",\"extension\":\"png\",\"order_by\":3,\"title\":\"Figure 3\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":91513,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eSugarcane soil physiochemical properties. The same lowercase letters above the bars indicate no significant difference (Duncan’s multiple range test, \\u003cem\\u003ep\\u003c/em\\u003e \\u0026gt;0.05) between treatments. (a) soil available nitrogen (AN) content; (b) soil available phosphorus (AP) content; (c) soil available potassium (AK) content; (d) soil catalase (CAT) activity; (e) soil alkaline phosphatase (AKP) activity; (f) soil urease (UE) activity. CK, control (sterile water); T0, \\u003cem\\u003eGluconacetobacter diazotrophicus\\u003c/em\\u003e PAL5; T1, \\u003cem\\u003eStreptomyces chartreusis\\u003c/em\\u003e WZS021; T2, \\u003cem\\u003eBacillus \\u003c/em\\u003espp. CA1 + \\u003cem\\u003ePseudomonas mosselii\\u003c/em\\u003e CN11; T3, \\u003cem\\u003ePseudomonas mosselii\\u003c/em\\u003e CN11 + \\u003cem\\u003eStreptomyces chartreusis\\u003c/em\\u003e WZS021.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"OnlineFig.3.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4643245/v1/c13bfd97167ea80631741054.png\"},{\"id\":60987030,\"identity\":\"181d5280-8da6-446d-9bd0-0e3bf8028a1e\",\"added_by\":\"auto\",\"created_at\":\"2024-07-24 10:18:27\",\"extension\":\"png\",\"order_by\":4,\"title\":\"Figure 4\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":162949,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eCircos diagram of PGPB treatment-rhizosphere soil microorganism species relationship on phylum level. Values within the inner circle indicate the number of reads of a phylum within the normalised dataset. (a) sugarcane rhizosphere soil at seedling stage; (b) sugarcane rhizosphere soil at elongation period. BCK, control (sterile water) for sugarcane variety B8; BT0, \\u003cem\\u003eGluconacetobacter diazotrophicus\\u003c/em\\u003e PAL5 for sugarcane variety B8; BT1, \\u003cem\\u003eStreptomyces chartreusis\\u003c/em\\u003e WZS021 for sugarcane variety B8; BT2, \\u003cem\\u003eBacillus \\u003c/em\\u003espp. CA1 + \\u003cem\\u003ePseudomonas mosselii\\u003c/em\\u003e CN11 for sugarcane variety B8; BT3, \\u003cem\\u003ePseudomonas mosselii\\u003c/em\\u003e CN11 + \\u003cem\\u003eStreptomyces chartreusis\\u003c/em\\u003e WZS021 for sugarcane variety B8. RCK, control (sterile water) for sugarcane variety ROC22; RT0, \\u003cem\\u003eGluconacetobacter diazotrophicus\\u003c/em\\u003e PAL5 for sugarcane variety ROC22; RT1, \\u003cem\\u003eStreptomyces chartreusis\\u003c/em\\u003e WZS021 for sugarcane variety ROC22; RT2, \\u003cem\\u003eBacillus \\u003c/em\\u003espp. CA1 + \\u003cem\\u003ePseudomonas mosselii\\u003c/em\\u003e CN11 for sugarcane variety ROC22; RT3, \\u003cem\\u003ePseudomonas mosselii\\u003c/em\\u003e CN11 + \\u003cem\\u003eStreptomyces chartreusis\\u003c/em\\u003e WZS021 for sugarcane variety ROC22.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"OnlineFig.4.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4643245/v1/e5cba4dfb4ed85b0c91a9308.png\"},{\"id\":60987610,\"identity\":\"0442eded-2f80-431e-8493-075b2d3e98ed\",\"added_by\":\"auto\",\"created_at\":\"2024-07-24 10:26:27\",\"extension\":\"png\",\"order_by\":5,\"title\":\"Figure 5\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":96811,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eThe Relative abundance of bacterial groups in rhizosphere soil of sugarcane with different treatments at the genus levels. (a) rhizosphere soil at the seedling stage; (b) rhizosphere soil during the elongation period. BCK, control (sterile water) for sugarcane variety B8; BT0, \\u003cem\\u003eGluconacetobacter diazotrophicus\\u003c/em\\u003e PAL5 for sugarcane variety B8; BT1, \\u003cem\\u003eStreptomyces chartreusis\\u003c/em\\u003e WZS021 for sugarcane variety B8; BT2, \\u003cem\\u003eBacillus \\u003c/em\\u003espp. CA1 + \\u003cem\\u003ePseudomonas mosselii\\u003c/em\\u003e CN11 for sugarcane variety B8; BT3, \\u003cem\\u003ePseudomonas mosselii\\u003c/em\\u003e CN11 + \\u003cem\\u003eStreptomyces chartreusis\\u003c/em\\u003e WZS021 for sugarcane variety B8. RCK, control (sterile water) for sugarcane variety ROC22; RT0, \\u003cem\\u003eGluconacetobacter diazotrophicus\\u003c/em\\u003e PAL5 for sugarcane variety ROC22; RT1, \\u003cem\\u003eStreptomyces chartreusis\\u003c/em\\u003e WZS021 for sugarcane variety ROC22; RT2, \\u003cem\\u003eBacillus \\u003c/em\\u003espp. CA1 + \\u003cem\\u003ePseudomonas mosselii\\u003c/em\\u003e CN11 for sugarcane variety ROC22; RT3, \\u003cem\\u003ePseudomonas mosselii\\u003c/em\\u003e CN11 + \\u003cem\\u003eStreptomyces chartreusis\\u003c/em\\u003e WZS021 for sugarcane variety ROC22.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"OnlineFig.5.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4643245/v1/1beec6a58b6ba6886bc6b923.png\"},{\"id\":60986358,\"identity\":\"8a2a45f3-da13-49e4-af29-ac273a9d34a7\",\"added_by\":\"auto\",\"created_at\":\"2024-07-24 10:10:26\",\"extension\":\"png\",\"order_by\":6,\"title\":\"Figure 6\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":102838,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eβ diversity analysis. Principal coordinated analysis (PCoA) derived from dissimilarity matrix of weighted UniFrac distance, multi-sample differential matrix heat map weighted UniFrac based cluster analysis of bacterial community composition among different samples. (a) rhizosphere soil at seedling stage; (b) rhizosphere soil at elongation stage. BCK, control (sterile water) for sugarcane variety B8; BT0, \\u003cem\\u003eGluconacetobacter diazotrophicus\\u003c/em\\u003e PAL5 for sugarcane variety B8; BT1, \\u003cem\\u003eStreptomyces chartreusis\\u003c/em\\u003e WZS021 for sugarcane variety B8; BT2, \\u003cem\\u003eBacillus \\u003c/em\\u003espp. CA1 + \\u003cem\\u003ePseudomonas mosselii\\u003c/em\\u003e CN11 for sugarcane variety B8; BT3, \\u003cem\\u003ePseudomonas mosselii\\u003c/em\\u003e CN11 + \\u003cem\\u003eStreptomyces chartreusis\\u003c/em\\u003e WZS021 for sugarcane variety B8. RCK, control (sterile water) for sugarcane variety ROC22; RT0, \\u003cem\\u003eGluconacetobacter diazotrophicus\\u003c/em\\u003e PAL5 for sugarcane variety ROC22; RT1, \\u003cem\\u003eStreptomyces chartreusis\\u003c/em\\u003e WZS021 for sugarcane variety ROC22; RT2, \\u003cem\\u003eBacillus \\u003c/em\\u003espp. CA1 + \\u003cem\\u003ePseudomonas mosselii\\u003c/em\\u003e CN11 for sugarcane variety ROC22; RT3, \\u003cem\\u003ePseudomonas mosselii\\u003c/em\\u003e CN11 + \\u003cem\\u003eStreptomyces chartreusis\\u003c/em\\u003e WZS021 for sugarcane variety ROC22.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"OnlineFig.6.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4643245/v1/7effb3438fed7cb9196a30f9.png\"},{\"id\":60986372,\"identity\":\"1e728bab-72f9-445c-87b6-33fc07f4b4b9\",\"added_by\":\"auto\",\"created_at\":\"2024-07-24 10:10:28\",\"extension\":\"png\",\"order_by\":7,\"title\":\"Figure 7\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":69951,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eMicroPITA analysis of microbial community abundance at OTU level. (a) rhizosphere soil at seedling stage; (b) rhizosphere soil at elongation stage. BCK, control (sterile water) for sugarcane variety B8; BT0, \\u003cem\\u003eGluconacetobacter diazotrophicus\\u003c/em\\u003e PAL5 for sugarcane variety B8; BT1, \\u003cem\\u003eStreptomyces chartreusis\\u003c/em\\u003e WZS021 for sugarcane variety B8; BT2, \\u003cem\\u003eBacillus \\u003c/em\\u003espp. CA1 + \\u003cem\\u003ePseudomonas mosselii\\u003c/em\\u003e CN11 for sugarcane variety B8; BT3, \\u003cem\\u003ePseudomonas mosselii\\u003c/em\\u003e CN11 + \\u003cem\\u003eStreptomyces chartreusis\\u003c/em\\u003e WZS021 for sugarcane variety B8. RCK, control (sterile water) for sugarcane variety ROC22; RT0, \\u003cem\\u003eGluconacetobacter diazotrophicus\\u003c/em\\u003e PAL5 for sugarcane variety ROC22; RT1, \\u003cem\\u003eStreptomyces chartreusis\\u003c/em\\u003e WZS021 for sugarcane variety ROC22; RT2, \\u003cem\\u003eBacillus \\u003c/em\\u003espp. CA1 + \\u003cem\\u003ePseudomonas mosselii\\u003c/em\\u003e CN11 for sugarcane variety ROC22; RT3, \\u003cem\\u003ePseudomonas mosselii\\u003c/em\\u003e CN11 + \\u003cem\\u003eStreptomyces chartreusis\\u003c/em\\u003e WZS021 for sugarcane variety ROC22.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"OnlineFig.7.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4643245/v1/d2a52638fa80a50908b75097.png\"},{\"id\":60988103,\"identity\":\"fb580fb4-f83b-4c95-b9b0-82cdf0059f2a\",\"added_by\":\"auto\",\"created_at\":\"2024-07-24 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10:10:27\",\"extension\":\"docx\",\"order_by\":10,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":311925,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"SupplementaryFig.10.docx\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4643245/v1/0e79e1b7a4cbbb18b2c5f450.docx\"},{\"id\":60986368,\"identity\":\"24365162-2579-410a-b894-c162c9d4c482\",\"added_by\":\"auto\",\"created_at\":\"2024-07-24 10:10:27\",\"extension\":\"docx\",\"order_by\":11,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":165258,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"SupplementaryFig.11.docx\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4643245/v1/ed7cbea7c6f2e92c789dea98.docx\"},{\"id\":60987612,\"identity\":\"19d112b1-b080-448d-9ed2-eae49bed8c61\",\"added_by\":\"auto\",\"created_at\":\"2024-07-24 10:26:28\",\"extension\":\"docx\",\"order_by\":12,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":168543,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"SupplementaryFig.12.docx\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4643245/v1/369f8c351aee6d61f178a0b6.docx\"},{\"id\":60986363,\"identity\":\"75036f02-ff27-4844-8317-2a1d31975b91\",\"added_by\":\"auto\",\"created_at\":\"2024-07-24 10:10:26\",\"extension\":\"docx\",\"order_by\":13,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":24396,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"SupplementaryTable1.docx\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4643245/v1/25de2b5699e8a89b65dfeef3.docx\"},{\"id\":60987033,\"identity\":\"b1a97c94-9d08-472c-bf46-d7c7a4f988a9\",\"added_by\":\"auto\",\"created_at\":\"2024-07-24 10:18:28\",\"extension\":\"docx\",\"order_by\":14,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":21846,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"SupplementaryTable2.docx\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4643245/v1/e7dbe86f14fb8e369a9843a5.docx\"},{\"id\":60986366,\"identity\":\"dbffbd57-0148-4e0b-b537-1ef8d6ef3440\",\"added_by\":\"auto\",\"created_at\":\"2024-07-24 10:10:27\",\"extension\":\"docx\",\"order_by\":15,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":21192,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"SupplementaryTable3.docx\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4643245/v1/b287150e9418b3b6690f2037.docx\"},{\"id\":60986375,\"identity\":\"6e6e336e-e41d-4738-8f37-ce724c217ccc\",\"added_by\":\"auto\",\"created_at\":\"2024-07-24 10:10:28\",\"extension\":\"docx\",\"order_by\":16,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":20023,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"SupplementaryTable4.docx\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4643245/v1/14dba15109e802a748e0ee92.docx\"}],\"financialInterests\":\"No competing interests reported.\",\"formattedTitle\":\"Different responses of sugarcane and rhizosphere soil microorganisms to single or mixture application of PGPB\",\"fulltext\":[{\"header\":\"1. Introduction\",\"content\":\"\\u003cp\\u003eSugarcane, a significant cash crop for global economy, is facing the problem of excessive fertilizer and pesticide applications. The nutrients required for plant growth in agricultural production are mainly provided by chemical fertilization [\\u003cspan citationid=\\\"CR1\\\" class=\\\"CitationRef\\\"\\u003e1\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR2\\\" class=\\\"CitationRef\\\"\\u003e2\\u003c/span\\u003e]. The heavy use of chemical fertilisers and pesticides have led to the imbalance of nutrient ratios, soil property deterioration, declined agricultural product quality, destruction of environment, and even seriously affected the sustainable development of sugarcane industry [\\u003cspan citationid=\\\"CR3\\\" class=\\\"CitationRef\\\"\\u003e3\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR4\\\" class=\\\"CitationRef\\\"\\u003e4\\u003c/span\\u003e]. In order to avoid the side effects of fertilizers on crops and soil, high attention has been paid to microorganisms as an alternative to chemical fertilizers in agriculture due to their cost-effectiveness and environmental friendliness.\\u003c/p\\u003e \\u003cp\\u003eOne of the ecologically sustainable ways to handle the aforementioned problems is to use the crop-microbe interaction model. In many countries, inoculation of plant growth-promoting bacteria (PGPB) is a promising approach to improve sustainable agricultural production [\\u003cspan citationid=\\\"CR5\\\" class=\\\"CitationRef\\\"\\u003e5\\u003c/span\\u003e]. PGPB found in the rhizosphere and inside plant, colonize the root cells and subsequently promote plant growth through a variety of mechanisms, such as hormone production, improved nutrient supply for the plant, inhibition of plant pathogens, and modification of the physico-chemical properties of the soil [\\u003cspan citationid=\\\"CR6\\\" class=\\\"CitationRef\\\"\\u003e6\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR7\\\" class=\\\"CitationRef\\\"\\u003e7\\u003c/span\\u003e]. PGPB are essential to the cycling of nutrients in soil [\\u003cspan citationid=\\\"CR8\\\" class=\\\"CitationRef\\\"\\u003e8\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR9\\\" class=\\\"CitationRef\\\"\\u003e9\\u003c/span\\u003e] and has an influence on soil ecosystems, biochemical processes, plant growth and development, and overall health [\\u003cspan citationid=\\\"CR10\\\" class=\\\"CitationRef\\\"\\u003e10\\u003c/span\\u003e]. The diazotroph which is one of the crucial communities in the PGPB are capable of fixing atmospheric nitrogen. They include the genus Rhizobium, which falls within symbiotic relationships with leguminous plants, and some \\u003cem\\u003ePseudomonas\\u003c/em\\u003e sp. When the amount of nitrogen nutrients in the soil decreases, the significance of applying PGPB rises [\\u003cspan citationid=\\\"CR11\\\" class=\\\"CitationRef\\\"\\u003e11\\u003c/span\\u003e]. Subsequently, the bacteria are capable of efficiently facilitating plant development by supplying the essential limiting factors. \\u003cem\\u003eBacillus\\u003c/em\\u003e, \\u003cem\\u003ePseudomonas\\u003c/em\\u003e, \\u003cem\\u003eAgrobacterium\\u003c/em\\u003e, \\u003cem\\u003eBurkholderia\\u003c/em\\u003e and \\u003cem\\u003eStreptomyces\\u003c/em\\u003e have been well studied and significantly marketed as biostimulants or biofertilisers [\\u003cspan citationid=\\\"CR12\\\" class=\\\"CitationRef\\\"\\u003e12\\u003c/span\\u003e].\\u003c/p\\u003e \\u003cp\\u003eAlthough applications of single PGPB have been reported in greenhouse and field environments [\\u003cspan citationid=\\\"CR13\\\" class=\\\"CitationRef\\\"\\u003e13\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR14\\\" class=\\\"CitationRef\\\"\\u003e14\\u003c/span\\u003e], microorganisms have shown poor colonization capacity, high environmental dependence and unstable effects, resulting in that single-strain and single-function fertilisers could not meet the requirements of modern agricultural development. The multi-functionality of composite PGPB will provide a chance to improve its environmental adaptability [\\u003cspan citationid=\\\"CR15\\\" class=\\\"CitationRef\\\"\\u003e15\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR16\\\" class=\\\"CitationRef\\\"\\u003e16\\u003c/span\\u003e]. When a combination of several PGPB strains is applied, complicated chemical signal exchange systems generated by plant-PGPB interactions improve plant growth and defence [\\u003cspan citationid=\\\"CR17\\\" class=\\\"CitationRef\\\"\\u003e17\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR18\\\" class=\\\"CitationRef\\\"\\u003e18\\u003c/span\\u003e]. Microbial functional combination has been shown to be extremely significant in enhancing plant performance, and this strategy may be the first step towards creating bacterial communities that are beneficial to plants [\\u003cspan citationid=\\\"CR19\\\" class=\\\"CitationRef\\\"\\u003e19\\u003c/span\\u003e]. Therefore, elucidation of the interaction mechanism between mixed PGPB and sugarcane is essential for the selection of bacterial strains that will be used individually or in combination as biofertilizers.\\u003c/p\\u003e \\u003cp\\u003eTraditional laboratory approaches are tough and time-consuming owing to the majority of unculturable PGPB [\\u003cspan citationid=\\\"CR20\\\" class=\\\"CitationRef\\\"\\u003e20\\u003c/span\\u003e]. Technological developments in molecular biology have enhanced our comprehension of the rhizosphere microbiota, especially with the introduction of affordable high-throughput sequencing (HTS) and associated data processing methods [\\u003cspan citationid=\\\"CR6\\\" class=\\\"CitationRef\\\"\\u003e6\\u003c/span\\u003e]. Sequencing provides a practical and comprehensive system for identifying the rhizosphere microbial species regardless of microbial abundance. HTS was also employed to explore the soil microbial dynamics of sugarcane [\\u003cspan citationid=\\\"CR21\\\" class=\\\"CitationRef\\\"\\u003e21\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR22\\\" class=\\\"CitationRef\\\"\\u003e22\\u003c/span\\u003e]. Therefore, evaluation of microbial dynamics in sugarcane and rhizosphere soil by high-throughput sequencing technology will be a potential strategy in revealing the complexity and diversity of microbial communities.\\u003c/p\\u003e \\u003cp\\u003eApplication of a single strain promoted sugarcane growth, increased soil nutrients and enzyme activities [\\u003cspan citationid=\\\"CR23\\\" class=\\\"CitationRef\\\"\\u003e23\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR24\\\" class=\\\"CitationRef\\\"\\u003e24\\u003c/span\\u003e]. Four strains of PGPB(\\u003cem\\u003eBacillus\\u003c/em\\u003e spp.CA1, \\u003cem\\u003ePseudomonas mosselii\\u003c/em\\u003e CN11, \\u003cem\\u003eklebsiella\\u003c/em\\u003e sp.DX120E, \\u003cem\\u003eStreptomyces chartreusis\\u003c/em\\u003e WZS021)have been isolated by our research group[\\u003cspan additionalcitationids=\\\"CR26\\\" citationid=\\\"CR25\\\" class=\\\"CitationRef\\\"\\u003e25\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR27\\\" class=\\\"CitationRef\\\"\\u003e27\\u003c/span\\u003e]. And they have been confirmed on sugarcane growth has a promoting effect. In the previous experiment, we have selected the above four PGPB strains for single and combination tests. By using the biological characteristics of PGPB strains, the optimal combination was obtained through factor analysis, and then pot planting test was conducted. Three optimal strain combinations (\\u003cem\\u003eStreptomyces chartreusis\\u003c/em\\u003e WZS021, \\u003cem\\u003eBacillus\\u003c/em\\u003e spp.CA1\\u0026thinsp;+\\u0026thinsp;\\u003cem\\u003ePseudomonas mosselii\\u003c/em\\u003e CN11, \\u003cem\\u003ePseudomonas mosselii\\u003c/em\\u003e CN11\\u0026thinsp;+\\u0026thinsp;\\u003cem\\u003eStreptomyces chartreusis\\u003c/em\\u003e WZS021) and \\u003cem\\u003eGluconacetobacter diazotrophicus\\u003c/em\\u003e PAL5, were selected to apply in two sugarcane varieties (B8, ROC22).The antagonism test against the above combined strains has been carried out, and it has been proved that there is no antagonism.The current study thus aims to comprehend the nutrient uptake by plants from rhizosphere soils and the growth-promoting properties of single PGPB (\\u003cem\\u003eGluconacetobacter diazotrophicus\\u003c/em\\u003e PAL5, \\u003cem\\u003eStreptomyces chartreusis\\u003c/em\\u003e WZS021) and mixed PGPB (\\u003cem\\u003eBacillus\\u003c/em\\u003e spp.CA1\\u0026thinsp;+\\u0026thinsp;\\u003cem\\u003ePseudomonas mosselii\\u003c/em\\u003e CN11, \\u003cem\\u003ePseudomonas mosselii\\u003c/em\\u003e CN11\\u0026thinsp;+\\u0026thinsp;\\u003cem\\u003eStreptomyces chartreusis\\u003c/em\\u003e WZS021) in sugarcane varieties ROC22 and B8. The 16S rRNA HTS molecular technique was used for analyses of the microbial communities in rhizosphere and sugarcane responsive to PGPB. The purpose of the work was thatdemonstrated how PGPB promote sugarcane nitrogen uptake ability, nitrogen metabolism, fast soil nutrient release, and plant growth and development. This study also revealed the diversity of rhizosphere soil bacterial communities in two sugarcane varieties after PGPB inoculation. These results provided a knowledge of the PGPB influence on plant growth, soil nutrient utilisation, microbial community composition and their interrelationship.\\u003c/p\\u003e\"},{\"header\":\"2. Materials and methods\",\"content\":\"\\u003cdiv id=\\\"Sec3\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e2.1. Bacterial solution preparation\\u003c/h2\\u003e \\u003cp\\u003eBacterial strains, species, culture media and isolation sources used in this study were shown in supplementary Table\\u0026nbsp;1. Strains \\u003cem\\u003eGluconacetobacter diazotrophicus\\u003c/em\\u003e PAL5, \\u003cem\\u003eBacillus\\u003c/em\\u003e spp. CA1, \\u003cem\\u003ePseudomonas mosselii\\u003c/em\\u003e CN11, and \\u003cem\\u003eStreptomyces chartreusis\\u003c/em\\u003e WZS021 were taken out from a -80℃ freezer and activated on a suitable solid medium. Single colonies were picked and cultured in a liquid medium overnight until the OD\\u003csub\\u003e600\\u003c/sub\\u003e was 1.0. Bacteria were collected by centrifugation (4000 rpm, 4℃, 10 min) and suspended in sterile phosphate buffer solution (PBS, pH 7.4) to get a bacterial concentration of 10\\u003csup\\u003e8\\u003c/sup\\u003e CFU/ml.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec4\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e2.2. Pot experiment\\u003c/h2\\u003e \\u003cp\\u003eTwo sugarcane varieties, Xintaitang 22 (ROC22) and RB86-7515(B8), were selected for pot experiment. ROC22 is the main cultivated sugarcane variety, and B8 is the Brazilian nitrogen-fixing sugarcane variety. The experiment was carried out in College of Agriculture, Guangxi University, Nanning (22\\u0026deg; 51\\u0026rsquo; N 108\\u0026deg; 17\\u0026prime; E), China. The physico-chemical properties of the soil were as follows: pH 6.7, potassium 7.10 g/kg, phosphorus 0.90 g/kg, nitrogen 0.82 g/kg, available phosphorus (AP) 22.0 mg/kg, hydrolysed nitrogen 47.7 mg/kg, and available potassium (AK) 47.0 mg/kg. The size of the pots used was 24 cm in upper calibre and 30 cm in height. Each pot contained about 17 kg of soil, and 10 mg of (\\u003csup\\u003e15\\u003c/sup\\u003eNH₄)₂SO₄ (product of Shanghai Research Institute of Chemical Industry CO., LTD, China, with 10.12% abundance value of \\u003csup\\u003e15\\u003c/sup\\u003eN marker) was added to each kilogram of soil.\\u003c/p\\u003e \\u003cp\\u003eSugarcane was planted in a 1:1 substrate ratio of sand and soil. When the plants grew to 3\\u0026ndash;4 leaves, those with similar growth status were selected, and their roots were washed with water and soaked for 40 min in the strain suspension and then transplanted into pots. The mixed inoculum suspension (1:1) was inoculated on two seedlings per pot with three replicates, with sterile water as the control. No fertiliser was applied during growth, and regular watering and weeding practices were followed. Agronomic traits of the pot-planted sugarcane were investigated and sampled on the 30th, 60th, 90th, and 120th days after inoculation (DAI). The biomass was measured on the 240th DAI. The treatments and codes were shown in Table\\u0026nbsp;\\u003cspan refid=\\\"Tab1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e.\\u003c/p\\u003e \\u003cp\\u003e \\u003cdiv class=\\\"gridtable\\\"\\u003e\\u003ctable float=\\\"Yes\\\" id=\\\"Tab1\\\" border=\\\"1\\\"\\u003e \\u003ccaption language=\\\"En\\\"\\u003e \\u003cdiv class=\\\"CaptionNumber\\\"\\u003eTable 1\\u003c/div\\u003e \\u003cdiv class=\\\"CaptionContent\\\"\\u003e \\u003cp\\u003eThe treatments and codes\\u003c/p\\u003e \\u003c/div\\u003e \\u003c/caption\\u003e \\u003ccolgroup cols=\\\"3\\\"\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c1\\\" colnum=\\\"1\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c2\\\" colnum=\\\"2\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c3\\\" colnum=\\\"3\\\"\\u003e\\u003c/div\\u003e \\u003cthead\\u003e \\u003ctr\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eSugarcane variety\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eInoculation treatment\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eCode\\u003c/p\\u003e \\u003c/th\\u003e \\u003c/tr\\u003e \\u003c/thead\\u003e \\u003ctbody\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eSterile water\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eBCK\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003ePAL5\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eBT0\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eB8\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eWZS021\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eBT1\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eCA1\\u0026thinsp;+\\u0026thinsp;CN11\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eBT2\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eCN11\\u0026thinsp;+\\u0026thinsp;WZS021\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eBT3\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eSterile water\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eRCK\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003ePAL5\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eRT0\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eROC22\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eWZS021\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eRT1\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eCA1\\u0026thinsp;+\\u0026thinsp;CN11\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eRT2\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eCN11\\u0026thinsp;+\\u0026thinsp;WZS021\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eRT3\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003c/tbody\\u003e \\u003c/colgroup\\u003e \\u003c/table\\u003e\\u003c/div\\u003e \\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec5\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e2.3. Agronomic traits and enzyme activity determination\\u003c/h2\\u003e \\u003cp\\u003ePlant height [from stem base to leaf ring position of the first fully expanded (+\\u0026thinsp;1) leaf], chlorophyll content and leaf enzyme activities such as nitrate reductase (NR) [\\u003cspan citationid=\\\"CR28\\\" class=\\\"CitationRef\\\"\\u003e28\\u003c/span\\u003e], glutamine synthetase (GS) [\\u003cspan citationid=\\\"CR29\\\" class=\\\"CitationRef\\\"\\u003e29\\u003c/span\\u003e], glutamic pyruvate transaminase (GPT) and glutamate oxaloacetate transaminase (GOT) [\\u003cspan citationid=\\\"CR30\\\" class=\\\"CitationRef\\\"\\u003e30\\u003c/span\\u003e] were measured on 30th, 60th, 90th, and 120th days after inoculation. Chlorophyll content was determined using a portable chlorophyll analyser (SPAD 502). Sugarcane aboveground and underground parts were taken and weighed separately for biomass determination at 240th DAI.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec6\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e2.4. Sugarcane nitrogen utilisation assay\\u003c/h2\\u003e \\u003cp\\u003eWhole plants were collected at 240th DAI and weighed freshly. Roots, stems, and leaves were separated and put into an oven at 105℃ for 30 min, then baked to a constant weight at 75℃, and the dry weight were weighed. Crushed samples were used to detect the total nitrogen content (TN) and \\u003csup\\u003e15\\u003c/sup\\u003eN content. \\u003csup\\u003e15\\u003c/sup\\u003eN atom was calculated according to the nitrogen fixation efficiency formula: Ndfa% = [1 - (inoculated treatment \\u003csup\\u003e15\\u003c/sup\\u003eN atom/non-inoculated control \\u003csup\\u003e15\\u003c/sup\\u003eN atom)] \\u0026times; 100% [\\u003cspan citationid=\\\"CR31\\\" class=\\\"CitationRef\\\"\\u003e31\\u003c/span\\u003e]\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec7\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e2.5. Soil physical and chemical properties\\u003c/h2\\u003e \\u003cp\\u003eSoil available nutrients and enzyme activities were determined on the 120th DAI. The available phosphorous (AP) was determined by the UV-visible spectrophotometer based on the standard method (Soil available P leaching with sodium bicarbonate-molybdenum antimony resistance spectrophotometry, LY/T 1232\\u0026ndash;2015, China). The available potassium (AK) was extracted with neutral 1 mol/L ammonium acetate solution and determined by flame photometer based on the standard method (Determination of soil available K and slow available K, NY/T 889\\u0026ndash;2004, China). The available nitrogen (AN) was determined by alkali hydrolysis method (LY/T 1228\\u0026ndash;2015, China). The catalase (CAT), alkaline phosphatase (AKP), and urease (UE) activities were determined according to the kit instructions (Suzhou Grace Biotechnology Co., Ltd, China)\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec8\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e2.6. High-throughput sequencing analysis\\u003c/h2\\u003e \\u003cp\\u003eThe sugarcane rhizosphere soil samples were taken at both seedling and elongation stages. The plants with soil were taken out carefully, the soil attached to the roots was shaken off, and kept in sterile bags in an ice box. These samples were quickly frozen in liquid nitrogen and then stored in a -80℃ freezer for 16S rRNA analysis.\\u003c/p\\u003e \\u003cp\\u003eDNA was extracted using the DNA extraction kit E.Z.N.A.\\u0026reg; Soil DNA Kit (Omega Bio-Tek, USA). The extracted DNA was detected in 1% electrophoresis for DNA integrity and Omega Bio-Tek instrument for purity. The DNA was used as a template to amplify the V3-V4 region of the bacterial 16S rDNA gene using 16S primers 338F (5ʹ-ACTCCTACGGGGAGGCAGCA-3ʹ) and 806R (5ʹ-GGACTACHVGGGTWTCTAAT-3ʹ). The PCR amplification for 16S rRNA was done in a total volume of 20 \\u0026micro;L comprising of 5\\u0026times; FastPfu Buffer 4 \\u0026micro;L, 2.5 mM dNTPs 2 \\u0026micro;L, forward primer (5 \\u0026micro;M) 0.8 \\u0026micro;L, reverse primer (5 \\u0026micro;M) 0.8 \\u0026micro;L, FastPfu DNA Polymerase 0.4 \\u0026micro;L, BSA 0.2 \\u0026micro;L, genomic DNA 10 ng, ddH\\u003csub\\u003e2\\u003c/sub\\u003eO to 20 \\u0026micro;L. The PCR parameters were set as 95℃ for 5 min, 30 cycles (95℃ for 30 s, 55℃ for 30 s, 72℃ for 45 s) and 72℃ for 10 min. The PCR products were identified and purified using 1% agarose gel and AxyPrep DNA Gel Extraction Kit (Axygen, USA). The purified products were quantified by Quantus\\u0026trade; Fluorometer (Promega, USA). The libraries were constructed using the NEXTFLEX Rapid DNA-Seq Kit (Bio Scientific, USA) and sequenced using Illumina's Miseq PE300 (Illumina, USA).\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec9\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e2.7. Operational Taxonomic Unit (OTU) clustering analysis\\u003c/h2\\u003e \\u003cp\\u003eThe raw sequences quality was controlled using fastp software (\\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttps://github.com/OpenGene/fastp\\u003c/span\\u003e\\u003cspan address=\\\"https://github.com/OpenGene/fastp\\\" targettype=\\\"URL\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e, version 0.20.0) [\\u003cspan citationid=\\\"CR32\\\" class=\\\"CitationRef\\\"\\u003e32\\u003c/span\\u003e] and spliced using FLASH software (\\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttp://www.cbcb.umd.edu/software/flash\\u003c/span\\u003e\\u003cspan address=\\\"http://www.cbcb.umd.edu/software/flash\\\" targettype=\\\"URL\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e, version 1.2.7) [\\u003cspan citationid=\\\"CR33\\\" class=\\\"CitationRef\\\"\\u003e33\\u003c/span\\u003e]. OTUs were clustered based on 97% similarity using UPARSE software (\\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttp://drive5.com/uparse/\\u003c/span\\u003e\\u003cspan address=\\\"http://drive5.com/uparse/\\\" targettype=\\\"URL\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e, version 7.1) [\\u003cspan citationid=\\\"CR34\\\" class=\\\"CitationRef\\\"\\u003e34\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR35\\\" class=\\\"CitationRef\\\"\\u003e35\\u003c/span\\u003e]. To minimise the impact of sequencing depth on subsequent analysis of Alpha diversity and Beta diversity data, the number of sequences in all samples was drawn flat to 20,000. OTU species taxonomy was annotated using the RDP classifier (\\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttp://rdp.cme.msu.edu/\\u003c/span\\u003e\\u003cspan address=\\\"http://rdp.cme.msu.edu/\\\" targettype=\\\"URL\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e, version 2.11 [\\u003cspan citationid=\\\"CR35\\\" class=\\\"CitationRef\\\"\\u003e35\\u003c/span\\u003e]) compared to the Silva 16S rRNA gene database (v138), with a confidence threshold of 70%, and community composition was counted for each sample at different levels of species classification. 16S functional prediction analyses were performed using PICRUSt 2 software (version 2.2.0) [\\u003cspan citationid=\\\"CR36\\\" class=\\\"CitationRef\\\"\\u003e36\\u003c/span\\u003e].\\u003c/p\\u003e \\u003cp\\u003eAll the high-throughput data analyses were performed on the Meggie BioCloud platform (\\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttps://cloud.majorbio.com\\u003c/span\\u003e\\u003cspan address=\\\"https://cloud.majorbio.com\\\" targettype=\\\"URL\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e). Mothur soft (\\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttp://www.mothur.org/wiki/Calculators\\u003c/span\\u003e\\u003cspan address=\\\"http://www.mothur.org/wiki/Calculators\\\" targettype=\\\"URL\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e) was used to calculate α diversity Chao, and Shannon indexes. The Wilcoxon rank sum test was used for group difference analysis of α diversity. R language (version 3.3.1) tools were used for statistics and graphing. Species Venn diagrams were analysed for Qiime to calculate the beta diversity distance matrix, and then graphical trees were drawn in R language (version 3.3.1) for β diversity analysis. The similarity of microbial community structure among samples was examined using Principal Coordinate Analysis (PCoA) based on the bray-curtis distance algorithm and combined with the PERMANOVA non-parametric test to analyse whether the differences in a microbial community structure among sample groups were significant. Bacterial taxa with significant differences in abundance from phylum to genus level between groups were identified using LEfSe analysis (Linear discriminant analysis Effect Size (\\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttp://huttenhower.sph.harvard.edu/LEfSe\\u003c/span\\u003e\\u003cspan address=\\\"http://huttenhower.sph.harvard.edu/LEfSe\\\" targettype=\\\"URL\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e) (LDA\\u0026thinsp;\\u0026gt;\\u0026thinsp;2, P\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05) [\\u003cspan citationid=\\\"CR37\\\" class=\\\"CitationRef\\\"\\u003e37\\u003c/span\\u003e]. Redundancy analysis based on distance (RDA) was used to investigate the effect of soil physico-chemical indicators on soil bacterial community structure. Species were selected for correlation network graph analysis based on spearman correlation |r| \\u0026gt; 0.6 p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05 [\\u003cspan citationid=\\\"CR38\\\" class=\\\"CitationRef\\\"\\u003e38\\u003c/span\\u003e]. MicroPITA analysis was done using R (version 3.3.1) and Python [\\u003cspan citationid=\\\"CR39\\\" class=\\\"CitationRef\\\"\\u003e39\\u003c/span\\u003e]. Other experimental data were recorded and statistically analysed using Office Excel 2010 and SPSS software version 23.0 (IBM Corp, Armonk, New York). The resulted graph of the data was produced by Origin (2016) software. The raw data of 16S rRNA (accession no. PRJNA1052972) were submitted to the NCBI Sequence Read Archive (\\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttps://www.ncbi.nlm.nih.gov/gene/?term=PRJNA1052972\\u003c/span\\u003e\\u003cspan address=\\\"https://www.ncbi.nlm.nih.gov/gene/?term=PRJNA1052972\\\" targettype=\\\"URL\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e).\\u003c/p\\u003e \\u003c/div\\u003e\"},{\"header\":\"3. Results\",\"content\":\"\\u003cdiv id=\\\"Sec11\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e3.1. Nitrogen metabolism enzyme activities and nitrogen fixation efficiency\\u003c/h2\\u003e \\u003cp\\u003eThe GPT, GOT, NR, and GS activities in leaves of the inoculated sugarcane plant were generally significantly higher than those in the control. The changes in GPT (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003ea, e), GOT (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eb, f), and GS (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003ed, h) activities of both sugarcane varieties showed a trend of increasing and then decreasing. The GPT and NR activities in leaves of both varieties generally reached their maximum values after the 60th DAI. The GPT activity in T2 was significantly higher than that in the control for both B8 (96.39%) and ROC22 (116.17%) varieties. On the 90th DAI, the GOT and GS enzyme activities of all treatments reached the maximum. The GS activity in BT0 was significantly increased by 1.09-fold, and that in BT2 was significantly increased up to 89.35% than BCK. Except for BT0, the NR activity in leaves of variety B8 increased significantly on the 120th DAI compared to BCK. The NR activity in leaves of variety ROC22 was significantly higher in RT2 treatment than RCK by 17.76% on the 120th DAI. Although the NR activity in sugarcane leaves gradually decreased, it was significantly higher than the control treatment most of time (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003ec, g).\\u003c/p\\u003e \\u003cp\\u003eThe results of the \\u003csup\\u003e15\\u003c/sup\\u003eN isotope dilution test (Table \\u003cspan refid=\\\"MOESM2\\\" class=\\\"InternalRef\\\"\\u003eS2\\u003c/span\\u003e)revealed that the both sugarcane varieties B8 and ROC22 showed a significant increase in the total nitrogen percentage in roots under PGPB inoculation treatments compared to the controls but a significant decrease in \\u003csup\\u003e15\\u003c/sup\\u003eN atoms compared to the controls. The highest nitrogen fixation efficiency for T1 in B8 and ROC22 roots, respectively.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec12\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e3.2. Agronomic characters\\u003c/h2\\u003e \\u003cp\\u003eThe statistics of sugarcane plant height and biomass showed a positive boost after PGPB inoculation. On the 120th DAI, Both BT3 and BT1 treatments showed significantly higher plant height than the BCK by 26.58% and 30.04%, respectively, in B8 (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003ea). The plant height in variety ROC22 showed an increase up to 41.76% on the 90th DAI (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eb). T0 treatment had the most significant effect on the aboveground fresh weight in B8 (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003ec) and ROC22 (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003ed), which was 1.59 and 1.81 times, respectively, higher than the controls. Both T2 and T0 treatments increased the underground dry weight in the two sugarcane varieties significantly. The T2 treatment showed the most significant increase in the underground dry weight in both B8 and ROC22, with a 98.60% increase in BT2 and a 1.31-fold increase in RT2, respectively, than the controls.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec13\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e3.3. Chlorophyll content\\u003c/h2\\u003e \\u003cp\\u003eChlorophyll content in sugarcane leaves was increased in all the PGPB inoculation treatments compared to the controls. The highest chlorophyll content in B8 was observed in the BT0 treatment at 60th and 90th DAIs. The chlorophyll content in the PGPB inoculation treatments was 19.89\\u0026ndash;24.91% significantly higher than that in the control on the 120th DAI (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003ee). The chlorophyll content in ROC22 was also increased in the PGPB inoculation treatments compared to the control, and that was significantly higher in RT2 treatment than in RCK in most cases especially at the 90th and 120th DAIs, respectively (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003ef).\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec14\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e3.4. Soil physio-chemical properties and enzyme activities\\u003c/h2\\u003e \\u003cp\\u003eSoil is one of the main factors influencing plant growth, development and survival. It was found the available nutrients in the soil were significantly increased after PGPB inoculation (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003ea, b, c). Soil nitrogenase activity was significantly increased in all the PGPB inoculation treatments compared with the controls (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003ed). Soil available nutrients and soil nitrogenase activity of different sugarcane cultivars were improved by different treatments.AN content in the soil of sugarcane variety B8 showed the order BT0\\u0026thinsp;\\u0026gt;\\u0026thinsp;BT3\\u0026thinsp;\\u0026gt;\\u0026thinsp;BT1\\u0026thinsp;\\u0026gt;\\u0026thinsp;BT2\\u0026thinsp;\\u0026gt;\\u0026thinsp;BCK, and the PGPB inoculation treatments showed at least 57.97% higher AN than the control BCK. The AN content in the soil of sugarcane variety ROC22 was highest in the RT2 treatment, which was 92.97% significantly higher than the control RCK. AP content in the soil of sugarcane variety B8 was the highest in RT0 treatment, 2.03-fold significantly higher than that in BCK. AP content in ROC22 soil was highest in RT1 treatment, 3.34-fold significantly higher than that in RCK. AK content in the soil of sugarcane variety B8 showed the order of BT0\\u0026thinsp;\\u0026gt;\\u0026thinsp;BT2\\u0026thinsp;\\u0026gt;\\u0026thinsp;BT3\\u0026thinsp;\\u0026gt;\\u0026thinsp;BT1\\u0026thinsp;\\u0026gt;\\u0026thinsp;BCK. AK content in the soil of sugarcane variety ROC22 was the highest in the RT0 treatment, which was 1.66-fold significantly increased compared with the control. The other inoculation treatments showed the order RT3\\u0026thinsp;\\u0026gt;\\u0026thinsp;RT2\\u0026thinsp;\\u0026gt;\\u0026thinsp;RT1, which were 1.46-fold, 1.37-fold, and 1.34-fold, respectively, significantly higher compared with the control RCK.\\u003c/p\\u003e \\u003cp\\u003eSoil catalase activity was significantly increased in all the PGPB inoculation treatments compared with the controls (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003ed). The BT1 treatment showed the highest soil catalase activity, which was 1.08 times higher than BCK. The other PGPB inoculation treatments for B8 showed 46.72\\u0026ndash;80.48% higher than BCK. The RT3 treatment exhibited the highest soil catalase activity in the ROC22 variety, which was 62.61% higher than that in RCK. All the soils in B8 had considerably stronger alkaline phosphatase activity than the control (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003ee). The BT1 treatment had 2.45 times more significant alkaline phosphatase activity than the BCK. The remaining treatments showed the order BT3\\u0026thinsp;\\u0026gt;\\u0026thinsp;BT0\\u0026thinsp;\\u0026gt;\\u0026thinsp;BT2. The strongest alkaline phosphatase activity in sugarcane ROC22 variety soil was found in the RT0 treatment, which was 1.55-fold higher than that in RCK. The urease activities in the soil were significantly higher in the PGPB inoculation treatments than the control (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003ef). The order of urease activity in B8 soil showed BT0\\u0026thinsp;\\u0026gt;\\u0026thinsp;BT2\\u0026thinsp;\\u0026gt;\\u0026thinsp;BT1\\u0026thinsp;\\u0026gt;\\u0026thinsp;BT3, which was 88.59%, 78.14%, 60.40%, and 39.10%, respectively, higher than that in BCK soil. The strongest urease activity in ROC22 soil was in the RT2 treatment, which was 64.77% higher than that in RCK soil.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec15\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e3.5. Sequencing data and operational taxonomic unit (OTU)\\u003c/h2\\u003e \\u003cp\\u003eA total of 611,024 valid sequences (average length of 419 bp) at the seedling and 744,771 valid sequences (average length of 416 bp) at the elongation stage were obtained from the rhizosphere soil samples from different treatments. Following QIIME's filtering, the raw 16S rRNA readings were sorted, grouped, and examined individually for OTUs (Tables S3, 4). Common and unique OTUs among the samples were analysed based on the 16S rRNA sequences. From the 16S rRNA sequences, it was found there were 3220 OTUs in total from all the seedling rhizosphere soil samples, 699 of which were common to all the samples ( Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003ea). The top three ranked OTUs in the treatment were RT1\\u0026thinsp;\\u0026gt;\\u0026thinsp;BT3\\u0026thinsp;\\u0026gt;\\u0026thinsp;BT1. The total number of OTUs identified in the rhizosphere soil at elongation stage was 5,511, of which 1,346 were common to all the treatments (Fig. \\u003cspan refid=\\\"MOESM1\\\" class=\\\"InternalRef\\\"\\u003eS1\\u003c/span\\u003eb). Common and unique OTUs were significantly higher compared with those at seedling stage.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec16\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e3.6. Principal component analysis (PCA)\\u003c/h2\\u003e \\u003cp\\u003eBased on the OTUs data, PCA was done to examine the impact of growth-promoting bacteria in the bacterial community in the rhizosphere soil of sugarcane. The rhizosphere soil microbial communities at seedling stage were present in close proximity at both B8 and ROC22 in T2 and T3 treatments (Fig. \\u003cspan refid=\\\"MOESM2\\\" class=\\\"InternalRef\\\"\\u003eS2\\u003c/span\\u003ea). The PCA results for the elongated soil samples showed that the soil microbial communities in the rhizosphere soil of sugarcane varieties B8 and ROC22 were closer to each other in different treatments except for BCK (Fig. \\u003cspan refid=\\\"MOESM2\\\" class=\\\"InternalRef\\\"\\u003eS2\\u003c/span\\u003eb).\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec17\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e3.7. Diversity index and microbial composition\\u003c/h2\\u003e \\u003cp\\u003eα diversity is the diversity that exists individually within a given sample and is represented by the types of microorganisms enumerated in the test sample. The diversity analysis was characterized using Shannon, Simpson, and Chao exponential rarity curves. Shannon,Chao and Simpson indexes for the seedling soil samples, showed that T1 treatment was the highest in both sugarcane varieties (Fig. \\u003cspan refid=\\\"MOESM3\\\" class=\\\"InternalRef\\\"\\u003eS3\\u003c/span\\u003ea,b). The relatively high indexes for the sugarcane soil samples at the elongation stage were BT2 and BT3 treatments in B8, and RT0 and RT1 treatments in ROC22. Meanwhile, it was found that the Chao indexes for variety ROC22 at the elongation stage were higher than that for variety B8, indicating that the richness of the microorganisms in the rhizosphere soil of variety ROC22 was higher than that in B8.\\u003c/p\\u003e \\u003cp\\u003eThe phylum composition of the soil microbial community in sugarcane at the both stage was shown in the following order Firmicutes\\u0026thinsp;\\u0026gt;\\u0026thinsp;Proteobacteriaes\\u0026thinsp;\\u0026gt;\\u0026thinsp;Chlorofexi\\u0026thinsp;\\u0026gt;\\u0026thinsp;Actinobacteria, which accounted for more than 70% of the microorganisms in all the PGPB inoculation treatments(Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003e, S4). Genera \\u003cem\\u003eNocardioides\\u003c/em\\u003e, \\u003cem\\u003eStreptomyces\\u003c/em\\u003e, \\u003cem\\u003eArthrobacter\\u003c/em\\u003e, \\u003cem\\u003eSphingomonas\\u003c/em\\u003e, \\u003cem\\u003eMicrovirga\\u003c/em\\u003e, and \\u003cem\\u003eBacillus\\u003c/em\\u003e were dominant at elongation stage (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003e). The genera in T1 treatment were similar in composition to those in CK at seedling stage. Microbial community analysis of the soil samples at seedling stage showed that the soil samples in the treatments with CN11 bacteria contained Eschercha\\u003cem\\u003e-Shigella\\u003c/em\\u003e spp. The number of genera and diversity index in each treatment increased at elongation stage compared with those at seedling stage. The genus \\u003cem\\u003eBacillus\\u003c/em\\u003e spp. was found higher in T0 treatment, and \\u003cem\\u003eSphingomonas\\u003c/em\\u003e spp. higher in variety ROC22 than in B8, while \\u003cem\\u003eArthrobacter\\u003c/em\\u003e spp. higher in B8 than in ROC22. The most significant increase in the abundance of \\u003cem\\u003eBacillus\\u003c/em\\u003e spp. in the soil of sugarcane variety B8 was observed in the BT0 and BT3 treatments. \\u003cem\\u003eBacillus\\u003c/em\\u003e spp. was increased in all the PGPB inoculation treatments and the highest in T3 treatment in the soil of ROC22.\\u003c/p\\u003e \\u003cp\\u003eThe heat map proved that phyla Proteobacteria, Actinobacteriota, Bacteroidota, and Firmicutes had a positive correlation, while Planctomycetota, Armatimonadota, Nitrospirota, Elusimicrobiota had a negative correlation in each treatment. When the heat maps of soil bacterial community abundance at the two stages were compared, it was found that \\u003cem\\u003eBacillus\\u003c/em\\u003e spp. was present at the genus level all the time, and exhibited a substantial positive connection ( Fig. \\u003cspan refid=\\\"MOESM5\\\" class=\\\"InternalRef\\\"\\u003eS5\\u003c/span\\u003e, S6), while \\u003cem\\u003eEscherichia-Shigella\\u003c/em\\u003e and \\u003cem\\u003eKosakonia\\u003c/em\\u003e in RT2, RT3, BT2 and BT3 treatments showed a significant positive correlation, and the other treatments showed a negative correlation at seedling stage.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec18\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e3.8. β diversity\\u003c/h2\\u003e \\u003cp\\u003eCluster analyses based on PCoA and UniFrac were performed to further understand the β-diversity of 16S rRNAs in sugarcane rhizosphere soils. PCoA analysis revealed that the T0 treatment was separated from all the other treatments, showing higher β diversity. Hierarchical clustering based on UniFrac cluster analysis showed similar results for 16S rRNA data, containing identical sequences showing 0 (blue) distance. RT2 and RT3 showed a greater distance from the other treatments, indicating the difference. The distribution of dominant genera based on their relative abundance using the Bray-Curtis algorithm revealed that \\u003cem\\u003eBacillus\\u003c/em\\u003e was the dominant genus in all the samples (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003e). \\u003cem\\u003eEscherichia-Shigella\\u003c/em\\u003e was the second most dominant genus, while no rank_f_JG30-KF-CM45 was the third most dominant genus in sugarcane rhizosphere soils at seedling stage. No rank_f_JG30-KF-CM45 was the second most dominant genus, and \\u003cem\\u003eSphingomonas\\u003c/em\\u003e became the third most dominant genus, while the proportion of \\u003cem\\u003eArthobacter\\u003c/em\\u003e and \\u003cem\\u003eStreptomyces\\u003c/em\\u003e genera increased at elongation stage. Soil microbial diversity was similar between the two sugarcane varieties in the same treatment at seedling stage (CK, T0, and T1). At elongation stage, the rhizosphere soil β diversity changed considerably due to sugarcane growth. BT3 and BT1 had similar diversity, and RT2 and RT0 had similar diversity (Figs. S7, S8).\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec19\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e3.9. Spearman\\u0026rsquo;s rank correlation\\u003c/h2\\u003e \\u003cp\\u003eSpearman\\u0026rsquo;s rank correlation analyses were done based on the 16S rRNA abundance of the top 20 genera, diversity indices, and soil variables (Fig.\\u003cspan refid=\\\"MOESM9\\\" class=\\\"InternalRef\\\"\\u003eS9\\u003c/span\\u003ea, c) to assess the bacterial genera correlation and dominance of 16S rRNAs with the soil physico-chemicals. Heat maps revealed that soil physico-chemical factors significantly influenced the relative abundance of bacterial taxa (phylum and genus).\\u003c/p\\u003e \\u003cp\\u003eThe phylum microorganisms, environmental factors, and OTUs were subjected to RDA analysis (Fig. \\u003cspan refid=\\\"MOESM9\\\" class=\\\"InternalRef\\\"\\u003eS9\\u003c/span\\u003eb) and combined with heat maps (Fig. \\u003cspan refid=\\\"MOESM9\\\" class=\\\"InternalRef\\\"\\u003eS9\\u003c/span\\u003ea) to further analyse the relationship between the soil environmental factors, the treatments, and the bacterial community structure. The selected soil chemical factors, enzyme activities, and soil α-diversity were found to explain 88.61% of the variation in the bacterial community. The vector lengths of soil physicochemical properties and microbial communities in the ordination diagrams showed that the soil physicochemical properties and enzyme activities showed a positive correlation with axis 1.\\u003c/p\\u003e \\u003cp\\u003eCorrelation heatmaps at the microbial genus level (Fig. \\u003cspan refid=\\\"MOESM9\\\" class=\\\"InternalRef\\\"\\u003eS9\\u003c/span\\u003ed) revealed that the soil α-diversity index was influenced more by ROC22 compared with B8, while soil available nutrients and enzyme activities affected the bacterial community in the rhizosphere soil of B8 to a greater extent. It was learned that the soil fertility level can lead to changes in the bacterial community and, conversely, may affect the soil fertility status.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec20\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e3.10. Functional forecasting and analysis\\u003c/h2\\u003e \\u003cp\\u003eFunctional prediction of microbial community based on PICRUSt2 was carried out. A comparison of sequencing data in the KEGG database revealed that rhizosphere soils at seedling and elongation stages were involved in six categories of identifiable metabolic pathways, including metabolism, environmental information processing, genetic information processing, cellular processes, human diseases, and organic processes (Fig. \\u003cspan refid=\\\"MOESM10\\\" class=\\\"InternalRef\\\"\\u003eS10\\u003c/span\\u003e). Metabolism was the most dominant function, with a minimum abundance of 76.06% and 77.24% in the rhizosphere soil at seedling and elongation stages, respectively. Further analyses were carried out at the secondary functional level of the predicted genes, involving a total of 46 functional categories such as global and overview maps, carbohydrate metabolism, amino acid metabolism, and energy metabolism (Figs. S11, S12). Global and overview maps had the highest percentage in all the groups, with the lowest abundance of 39.01% and 40.03% in the rhizosphere soil at seedling and elongation stages, respectively. Global and overview maps, carbohydrate metabolism, amino acid metabolism, and energy metabolism together accounted for more than 50% in each group. The primary function analysis revealed that the metabolic functions in the soils for the treatments RT3, RT2, and BT1 at seedling stage in both varieties were higher than that in the control, and at elongation stage in ROC22, all higher than that in the control.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec21\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e3.11. MicroPITA analysis\\u003c/h2\\u003e \\u003cp\\u003eAccording to the distance algorithm, bray curtis, three sample sizes were selected based on OTU classification levels and diversity indices, resulting in a MicroPITA analysis plot (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig7\\\" class=\\\"InternalRef\\\"\\u003e7\\u003c/span\\u003e). At seedling stage, for the rhizosphere soil samples in different PGPB treatments, multiple selections were RT1, RT3, and maximum diversity selections were BCK and BT1. The most representative selection based on β diversity were BT0, BT3, and RT2. At elongation stage, BCK, BT1, and RCK were selected as multiple selections, BT0 was the most dissimilar, and RT0 and RT1 had maximum diversity among the PGPB treatments. The above results revealed that T1 treatment was highly selective in sugarcane rhizosphere soil. T2 treatment revealed the most extreme β diversity selection in variety ROC22. T3 showed the most extreme β diversity selection in the rhizosphere soil at seedling stage.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003c/div\\u003e\"},{\"header\":\"4. Discussion\",\"content\":\"\\u003cp\\u003ePrevious research has indicated the impacts of PGPB on the growth of crops. PGPB treatment to maize [\\u003cspan citationid=\\\"CR40\\\" class=\\\"CitationRef\\\"\\u003e40\\u003c/span\\u003e] and wheat [\\u003cspan citationid=\\\"CR41\\\" class=\\\"CitationRef\\\"\\u003e41\\u003c/span\\u003e] seeds were found to deliver more nitrogen and enhanced the average yield compared to the untreated control. A combination of PGPB strains delivered more nitrogen for maize than a single-strain supplement and greatly boosted wheat output [\\u003cspan citationid=\\\"CR42\\\" class=\\\"CitationRef\\\"\\u003e42\\u003c/span\\u003e]. These results suggested that the applied bacterial strains were interacting with each other, and promote the nitrogen-fixation ability of the nitrogen-fixing bacteria. To understand the impacts of single and mixed PGPB application on sugarcane, the current study was performed. So, single (PAL5, WZS021) and mixed (CA1\\u0026thinsp;+\\u0026thinsp;CN11, CN11\\u0026thinsp;+\\u0026thinsp;WZS021) strains of bacteria were inoculated into two sugarcane varieties, B8 and ROC22. The results revealed that PGPB effectively increased the activities of nitrogen metabolism-related enzymes (GPT, GOT, NR, and GS) in sugarcane plants. The mixed strain CA1\\u0026thinsp;+\\u0026thinsp;CN11 had the strongest effect on nitrogenase activity and chlorophyll activity, and significantly increased the dry weight of the underground part of the plant. It is speculated that the interaction effect of bacteria leads to the enhancement of nitrogen fixation capacity.Moreover, it was found that the single dominant strain PAL5 also had a strong effect on nitrogenase activity and nitrogen fixation of sugarcane, and the influence of above biomass was the most significant, so that the height of sugarcane was significantly higher than that of the control.The above results proved that the nitrogen-fixing ability of the growth-promoting bacteria can further promote plant growth by promoting nitrogen utilization and photosynthesis in sugarcane.\\u003c/p\\u003e \\u003cp\\u003eInoculation of PGPB in the soil increased the concentration of mineral nitrogen as well as that of phosphorus and potassium in the soil, improved the nutrient utilisation rate, and promoted plant absorption and utilization of nitrogen, phosphorus and potassium by plants [\\u003cspan citationid=\\\"CR43\\\" class=\\\"CitationRef\\\"\\u003e43\\u003c/span\\u003e]. Soil nutrient detection in this study revealed that PAL5 had a large boosting influence on AN and AK in the rhizosphere soils of both sugarcane varieties, while the combination of CN11\\u0026thinsp;+\\u0026thinsp;WZS021 and CA1\\u0026thinsp;+\\u0026thinsp;CN11 was the second most important factor influencing these two elements. This might be connected to PAL5's very potent biological nitrogen fixing capabilities [\\u003cspan citationid=\\\"CR44\\\" class=\\\"CitationRef\\\"\\u003e44\\u003c/span\\u003e]. Sugarcane variety B8 had the highest AP content in response to PAL5, The \\u003csup\\u003e15\\u003c/sup\\u003eN isotope dilution experiment also showed that PAL5 treatment significantly increased total nitrogen content in leaves and roots, thereby promoting the increase of aboveground biomass. \\u003cem\\u003eStreptomyces chartreusis\\u003c/em\\u003e WZS021, participates in the soil phosphorus cycle, decomposing organic matter into available phosphorus, resulting in the accumulation of available phosphorus and available phosphorus components in soil. ROC22 had the highest AP in the WZS021 treatment. The results of CAT and AKP responses showed a similar pattern, with WZS021 being the most effective in variety B8, followed by the mixtures of strains. The most significant reaction response of CAT and AKP content in variety ROC22 was found still in PAL5 treatment, followed by the treatments with combination of CN11\\u0026thinsp;+\\u0026thinsp;WZS021 strains. It is consisted that \\u003cem\\u003eActinomycetes\\u003c/em\\u003e spp. promote the release of soil insoluble phosphorus through microbial metabolic activities and participate in the soil phosphorus cycle [\\u003cspan additionalcitationids=\\\"CR46\\\" citationid=\\\"CR45\\\" class=\\\"CitationRef\\\"\\u003e45\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR47\\\" class=\\\"CitationRef\\\"\\u003e47\\u003c/span\\u003e]. Moreover, WZS021 and CN11\\u0026thinsp;+\\u0026thinsp;WZS021 strains significantly affected plant height, indicating that the growthpromoting bacteria had excellent activities, such as nitrogen fixation, phosphorus and potassium solubilization, which could promote plant growth by decomposing soil nutrients. Soil nutrient discrepancies reflect variations in soil enzyme activity in response to various probiotic bacteria in different sugarcane varieties.\\u003c/p\\u003e \\u003cp\\u003eBased on 16S rRNA sequences, the microbial community composition in sugarcane rhizosphere soil was also investigated. The Chao index for ROC22 variety was higher than that for B8 variety at both seedling and elongation stages, indicating that the total number of bacteria species in the rhizosphere soil of ROC22 variety was higher than that of B8 and the soil microbial community varies at different growth stages of sugarcane and in different sugarcane varieties. A further investigation of its responses to different exogenous PGPB additions revealed that the bacterial microbial richness in sugarcane rhizosphere soils at seedling stage was greater in all the WZS021 treatments than in the other treatments. At elongation stage the microbial abundance in the rhizosphere soil of B8 in the CA1\\u0026thinsp;+\\u0026thinsp;CN11 treatment was higher than in the other treatments. It was found that the WZS021 treatment had higher microbial abundance than the other treatments in ROC22 at the elongation stage. At the same time, there were differences in the response mechanism of different sugarcane varieties to the strain. In the varieties with weak bacterial species in the rhizosphere soil, the addition of the composite strain had the best effect, while in the varieties with rich bacterial species, the addition of the composite strain may have the exclusion phenomenon, which was not as good as the addition of the single dominant strain.\\u003c/p\\u003e \\u003cp\\u003eProteobacteria, Firmicutes, Actinobacteria, Bacteroidetes, Chloroflexi, Gemmatimonadetes, Planctomycetes, and Nitrospirae phylum-level distribution were shown to predominate in sugarcane [\\u003cspan citationid=\\\"CR22\\\" class=\\\"CitationRef\\\"\\u003e22\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR48\\\" class=\\\"CitationRef\\\"\\u003e48\\u003c/span\\u003e], This is consistent with the present study. Phyla Actinobacteria, Firmicutes, and Proteobacteria were also members of the leading phyla in the sugarcane rhizosphere soil microbial community, whereas Bacteroidota was less represented. Y Gu, J Wang, W Cai, G Li, Y Mei and S Yang [\\u003cspan citationid=\\\"CR49\\\" class=\\\"CitationRef\\\"\\u003e49\\u003c/span\\u003e] found that phylum Aspergillus, Actinobacteria phylum and Firmicutes phylum were the dominant endophytic bacteria in sugarcane exposed to different treatments. The Bray-Curtis algorithm revealed that \\u003cem\\u003eBacillus\\u003c/em\\u003e was the leading genus in all samples, with a substantial increase in abundance in all rhizosphere soils treated with PGPB, which were characterised after the CA1\\u0026thinsp;+\\u0026thinsp;CN11 treatment. Furthermore, following PGPB inoculation treatment, the abundance of \\u003cem\\u003eStreptomyces\\u003c/em\\u003e, \\u003cem\\u003eSphingomonas\\u003c/em\\u003e, \\u003cem\\u003eArthrobacter\\u003c/em\\u003e, and \\u003cem\\u003eNocardioides\\u003c/em\\u003e increased significantly in the B8 rhizosphere soil. As a result, the soil supports the survival of several microbe species for self-regulation. The disadvantaged microbial community, on the other hand, has a poor capacity to compete for its own nutrients, causes minimal change in the soil environment, and performs outstanding activities such as nitrogen fixation, phosphorus and potassium solubilization, all of which contribute to the soil nutrient environment.\\u003c/p\\u003e \\u003cp\\u003eHowever, after elongation, sugarcane soil diversity was similar for BT3 vs BT1 treatments. Chao index showed that the diversity index of complex strains in B8 was higher, and RT2 and RT3 treatments in ROC22 were farther apart from the other treatments. It showed the difference between the complex strains and single strain. Therefore, it is speculated that the inoculated strains can positively enrich the beneficial bacteria in sugarcane. Application of bacteria that were disadvantaged and functionally specialised to the soil environment was more favorable to plant development, so it seemed that the microbiota had a more pronounced influence compared to the dominant population.\\u003c/p\\u003e \\u003cp\\u003eVariation in the composition and diversity of soil microbial communities is closely related to soil physicochemical properties [\\u003cspan citationid=\\\"CR50\\\" class=\\\"CitationRef\\\"\\u003e50\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR51\\\" class=\\\"CitationRef\\\"\\u003e51\\u003c/span\\u003e]. Inoculation of PGPB increased soil nutrients, enzyme activities and improved soil microbial communities [\\u003cspan citationid=\\\"CR52\\\" class=\\\"CitationRef\\\"\\u003e52\\u003c/span\\u003e]. S Yang, J Xiao, T Liang, W He and H Tan [\\u003cspan citationid=\\\"CR50\\\" class=\\\"CitationRef\\\"\\u003e50\\u003c/span\\u003e] found that different fertilizer treatments improved sugarcane soil biological properties and soil bacterial diversity. Spearman's correlation analysis showed that The nitrogen-fixing \\u003cem\\u003eBacillus\\u003c/em\\u003e species promote plant growth directly via nitrogen fixation, phosphate solubilization and production of phytohormones and indirectly through the production of antibiotics, hydrolytic enzymes and siderophores [\\u003cspan citationid=\\\"CR53\\\" class=\\\"CitationRef\\\"\\u003e53\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR54\\\" class=\\\"CitationRef\\\"\\u003e54\\u003c/span\\u003e]. \\u003cem\\u003eBacillus subtilis\\u003c/em\\u003e fixes nitrogen and promotes the growth and development of crops [\\u003cspan citationid=\\\"CR55\\\" class=\\\"CitationRef\\\"\\u003e55\\u003c/span\\u003e]. It can not only promote the growth of crop plant height but also significantly increase root activity, net photosynthetic rate and yield per plant. RDA analysis indicated that AP, AK, AKP, UE and CAT were all significantly positively correlated with Firmicutes and BRC1. Consistent with the results, higher levels of AP, AK, AKP, UE and CAT were detected in the T2 treatment. This may be the reason why the addition of WZS021 alone had a weaker effect on biomass, SPAD, and AN in sugarcane than the addition of PAL5 and CN11\\u0026thinsp;+\\u0026thinsp;CA1 since WZS021 tends to act on the phosphorus cycle, while PAL5 and CA1 tend to enhance the nitrogen cycle. H Minjie [\\u003cspan citationid=\\\"CR56\\\" class=\\\"CitationRef\\\"\\u003e56\\u003c/span\\u003e] showed that soil effective phosphorus content and AKP activities were closely related to the microbial abundance in soil. This meant that an interaction exists between the level of soil fertility and the bacterial community [\\u003cspan citationid=\\\"CR51\\\" class=\\\"CitationRef\\\"\\u003e51\\u003c/span\\u003e]. In addition, the results of functional prediction of bacterial flora showed that the bacteria were involved in four metabolism pathways, including global and overview maps, carbohydrate metabolism, amino acid metabolism and energy metabolism in the rhizosphere soil of sugarcane at seedling and elongation stages. It implied the importance of microbial metabolism in soil.\\u003c/p\\u003e\"},{\"header\":\"5. Conclusions\",\"content\":\"\\u003cp\\u003eThe application of single and mixed PGPB microbial inoculants in sugarcane increased the activities of nitrogen metabolic enzymes, nitrogen utilization capacity, chlorophyll content, agronomic features, rhizosphere soil chemical and biological properties, and microbial community. The inoculation of PGPB resulted in changes in microbial communities, which improved soil N and P cycling and increased GOT, GPT, NR, GS, SPAD, plant height and biomass of sugarcane. However, there were variations in the quantities of these promoted properties in the treatments with different bacterial strains and sugarcane varieties. It was found that soil-disadvantaged and inoculum-specific bacteria were more favorable to plant development. The strains \\u003cem\\u003eGluconacetobacter diazotrophicus\\u003c/em\\u003e PAL5 and \\u003cem\\u003eBacillus\\u003c/em\\u003e spp. CA1 showed a positive effect on enhancing nitrogen cycling while \\u003cem\\u003eStreptomyces chartreusis\\u003c/em\\u003e WZS021 demonstrated a greater benefit in increasing phosphorus cycling in soil. This research offers a reference for investigating advantageous microorganisms in the bacteria found in the rhizosphere of sugarcane, their production, and their utilization as bacterial fertilizers.\\u003c/p\\u003e\"},{\"header\":\"Abbreviations\",\"content\":\"\\u003ctable border=\\\"1\\\" cellspacing=\\\"0\\\" cellpadding=\\\"0\\\" width=\\\"619\\\"\\u003e\\n \\u003ctbody\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd width=\\\"37.64135702746365%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003ePGPB\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"62.35864297253635%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003ePlant growth-promoting bacteria\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd width=\\\"37.64135702746365%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eHTS\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"62.35864297253635%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003ehigh-throughput sequencing\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd width=\\\"37.64135702746365%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eROC22\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"62.35864297253635%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eXintaitang 22\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd width=\\\"37.64135702746365%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eB8\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"62.35864297253635%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eRB86-7515\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd width=\\\"37.64135702746365%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eOTU\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"62.35864297253635%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eOperational taxonomic unit\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd width=\\\"37.64135702746365%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eNR\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"62.35864297253635%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eNitrate reductase\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd width=\\\"37.64135702746365%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eGS\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"62.35864297253635%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eGlutamine synthetase\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd width=\\\"37.64135702746365%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eGPT\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"62.35864297253635%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eGlutamic pyruvate transaminase\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd width=\\\"37.64135702746365%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eGOT\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"62.35864297253635%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eGlutamate oxaloacetate transaminase\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd width=\\\"37.64135702746365%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eTN\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"62.35864297253635%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eTotal nitrogen\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd width=\\\"37.64135702746365%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eAP\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"62.35864297253635%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eAvailable phosphorous\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd width=\\\"37.64135702746365%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eAN\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"62.35864297253635%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eAvailable nitrogen\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd width=\\\"37.64135702746365%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eAK\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"62.35864297253635%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eAvailable potassium\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd width=\\\"37.64135702746365%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eCAT\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"62.35864297253635%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eCatalase\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd width=\\\"37.64135702746365%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eAKP\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"62.35864297253635%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eAlkaline phosphatase\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd width=\\\"37.64135702746365%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eUE\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"62.35864297253635%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eUrease\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd width=\\\"37.64135702746365%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003ePCA\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"62.35864297253635%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003ePrincipal component analysis\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd width=\\\"37.64135702746365%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003ePCoA\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd width=\\\"62.35864297253635%\\\" valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003ePrincipal coordinated analysis\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003c/tbody\\u003e\\n\\u003c/table\\u003e\"},{\"header\":\"Declarations\",\"content\":\"\\u003cp\\u003eEthics approval and consent to participate\\u003c/p\\u003e\\n\\u003cp\\u003eNot applicable\\u003c/p\\u003e\\n\\u003cp\\u003eConsent for publication\\u003c/p\\u003e\\n\\u003cp\\u003eNot applicable\\u003c/p\\u003e\\n\\u003cp\\u003eAvailability of data and materials\\u003c/p\\u003e\\n\\u003cp\\u003eThe raw data of 16S rRNA were deposited in the NCBI Sequence Read Archive (SRA) database under accession number\\u0026nbsp;PRJNA1052972\\u0026nbsp;(https://www.ncbi.nlm.nih.gov/gene/?term=PRJNA1052972).\\u003c/p\\u003e\\n\\u003cp\\u003eOther data generated or analysed during this study are included in this published article [and its supplementary information files].\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eConflict of interest\\u0026nbsp;\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe authors declare that they have no competing interests.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eFunding\\u003c/strong\\u003e\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003eThis work was funded by the National Natural Science Foundation of China (31971858 and 31560353), Fund for Guangxi Key Laboratory of Sugarcane Genetic Improvement (19-185-24-K-03-01),\\u0026nbsp;and National Modern Agricultural Production Technology System Guangxi Sugarcane Innovation Team Project (nycytxgxcxtd-2021-03-01), Guangxi Key R \\u0026amp; D Program (GK AA22117009), and Fund of Guangxi Academy of Agricultural Sciences (2021YT011).\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eAuthor contributions\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eJLW, YQ, WTL, and WLH performed the experiment and data analysis. YRL and YXX supervised the project and designed the study. JLW, YQ, QK, YRL, DFD and YXX wrote and reviewed the manuscript.\\u0026nbsp;YXX and YRL finalized the manuscript.\\u0026nbsp;All authors have read and agreed to the published version of the manuscript.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eAcknowledgements\\u0026nbsp;\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe authors are thankful to Shanghai Majorbio Bio-pharm Technology Co., Ltd. https://cloud.majorbio.com/ (Shanghai, China) for the microbial diversity analysis.\\u003c/p\\u003e\"},{\"header\":\"References\",\"content\":\"\\u003col\\u003e\\u003cli\\u003e\\u003cspan\\u003eSingh M, Maharjan KL. Organic Farming from Perspective of Three Pillars of Sustainability. Sustainability of Organic Farming in Nepal. Singapore: Springer Singapore; 2017. pp. 179\\u0026ndash;92.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eAmorim F, Santos D, Ospina-Patino M. 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GENOME BIOL. 2011;12(6):R60\\u0026ndash;60.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eBarberan A, Bates ST, Casamayor EO, Fierer N. Using network analysis to explore co-occurrence patterns in soil microbial communities. ISME J. 2014;8(4):952\\u0026ndash;952.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eTickle TL, Segata N, Waldron L, Weingart U, Huttenhower C. Two-stage microbial community experimental design. ISME J. 2013;7(12):2330\\u0026ndash;9.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eG\\u0026oacute;mez-God\\u0026iacute;nez LJ, Fernandez-Valverde SL, Martinez Romero JC, Mart\\u0026iacute;nez-Romero E. Metatranscriptomics and nitrogen fixation from the rhizoplane of maize plantlets inoculated with a group of PGPRs. Syst Appl Microbiol. 2019;42(4):517\\u0026ndash;25.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eSantos MS, Nogueira MA, Hungria M. 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Response of soil microbial communities to contrasted histories of phosphorus fertilisation in pastures. Appl Soil Ecol. 2012;61:40\\u0026ndash;8.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eKruasuwan W, Thamchaipenet A. Diversity of culturable plant growth-promoting bacterial endophytes associated with sugarcane roots and their effect of growth by co-inoculation of diazotrophs and dctinomycetes. J Plant Growth Regul. 2016;35(4):1074\\u0026ndash;87.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eGu Y, Wang J, Cai W, Li G, Mei Y, Yang S. Different amounts of nitrogen fertilizer applications alter the bacterial diversity and community structure in the rhizosphere soil of sugarcane. Front Microbiol 2021, 12.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eYang S, Xiao J, Liang T, He W, Tan H. Response of soil biological properties and bacterial diversity to different levels of nitrogen application in sugarcane fields. AMB Expr. 2021;11(1):172\\u0026ndash;172.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eBender SF, Schlaeppi K, Held A, Van der Heijden MGA. Establishment success and crop growth effects of an arbuscular mycorrhizal fungus inoculated into Swiss corn fields. Agric Ecosyst Environ. 2019;273:13\\u0026ndash;24.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eHuang Z, Ruan S, Sun Y, Cheng X, Dai J, Gui P, Yu M, Zhong Z, Wu J. Bacterial inoculants improved the growth and nitrogen use efficiency of Pyrus betulifolia under nitrogen-limited conditions by affecting the native soil bacterial communities. Appl Soil Ecol. 2022;170:104285.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eAmna, Xia Y, Farooq MA, Javed MT, Kamran MA, Mukhtar T, Ali J, Tabassum T, Rehman Su, Hussain Munis MF, et al. Multi-stress tolerant PGPR Bacillus xiamenensis PM14 activating sugarcane (Saccharum officinarum L.) red rot disease resistance. Plant Physiol Biochem. 2020;151:640\\u0026ndash;9.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eSharma A, Singh RK, Singh P, Vaishnav A, Guo D-J, Verma KK, Li D-P, Song X-P, Malviya MK, Khan N, et al. Insights into the bacterial and nitric oxide-induced salt tolerance in sugarcane and their growth-promoting abilities. Microorganisms. 2021;9(11):2203.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eChandra P, Tripathi P, Chandra A. Isolation and molecular characterization of plant growth-promoting Bacillus spp. and their impact on sugarcane (Saccharum spp. hybrids) growth and tolerance towards drought stress. Acta Physiol Plant. 2018;40(11):1\\u0026ndash;15.\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eMinjie H. Dynamics of phosphorus speciation and the phoD phosphatase gene community in the rhizosphere and bulk soil along an estuarine freshwater-oligohaline gradient. Geoderma. 2020;365:114236\\u0026ndash;v112020114365.\\u003c/span\\u003e\\u003c/li\\u003e\\u003c/ol\\u003e\"}],\"fulltextSource\":\"\",\"fullText\":\"\",\"funders\":[],\"hasAdminPriorityOnWorkflow\":false,\"hasManuscriptDocX\":true,\"hasOptedInToPreprint\":true,\"hasPassedJournalQc\":\"\",\"hasAnyPriority\":false,\"hideJournal\":false,\"highlight\":\"\",\"institution\":\"\",\"isAcceptedByJournal\":true,\"isAuthorSuppliedPdf\":false,\"isDeskRejected\":\"\",\"isHiddenFromSearch\":false,\"isInQc\":false,\"isInWorkflow\":false,\"isPdf\":false,\"isPdfUpToDate\":true,\"isWithdrawnOrRetracted\":false,\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"bmc-plant-biology\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":false,\"externalIdentity\":\"pbio\",\"sideBox\":\"Learn more about [BMC Plant Biology](http://bmcplantbiol.biomedcentral.com/)\",\"snPcode\":\"\",\"submissionUrl\":\"https://www.editorialmanager.com/pbio/default.aspx\",\"title\":\"BMC Plant Biology\",\"twitterHandle\":\"BMC_series\",\"acdcEnabled\":true,\"dfaEnabled\":false,\"editorialSystem\":\"em\",\"reportingPortfolio\":\"BMC Series\",\"inReviewEnabled\":true,\"inReviewRevisionsEnabled\":true},\"keywords\":\"PGPB, sugarcane, promotion, soil, High-throughput sequencing (HTS)\",\"lastPublishedDoi\":\"10.21203/rs.3.rs-4643245/v1\",\"lastPublishedDoiUrl\":\"https://doi.org/10.21203/rs.3.rs-4643245/v1\",\"license\":{\"name\":\"CC BY 4.0\",\"url\":\"https://creativecommons.org/licenses/by/4.0/\"},\"manuscriptAbstract\":\"\\u003cp\\u003e\\u003cstrong\\u003eBackground: \\u003c/strong\\u003ePlant growth-promoting bacteria (PGPB) benefit plant growth and development via different direct and indirect mechanisms. However, our knowledge about rhizosphere soil response at different plant growth stages to diverse PGPB application in sugarcane is limited. In this study, four strains of bacteria genera (\\u003cem\\u003eGluconacetobacter diazotrophicus\\u003c/em\\u003e PAL5, \\u003cem\\u003eStreptomyces chartreusis \\u003c/em\\u003eWZS021, \\u003cem\\u003eBacillus \\u003c/em\\u003espp\\u003cem\\u003e. \\u003c/em\\u003eCA1, and \\u003cem\\u003ePseudomonas mosselii\\u003c/em\\u003e CN11) were inoculated into two sugarcane varieties (B8, ROC22) as single or mixture in a pot planting experiment. The effects of single or combined application of PGPB on nitrogen metabolism, agronomic traits, rhizosphere soil chemical and biological properties and microbial community were surveyed.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eResults: \\u003c/strong\\u003eIt was found that different treatments had different promotion ways for different sugarcane varieties and rhizosphere soils. PAL5 and CA1+CN11 significantly improved the nitrogen fixation efficiency of sugarcane, while WZS021 treatment enhanced phosphorus (available phosphorus and alkaline phosphatase). High-throughput sequencing (HTS) analysis revealed that Proteobacteria, Firmicutes, Chloroflexi, and Actinobacteria were the main microbial community phylum components. Correlation analysis indicates that phyla Proteobacteria and Bacteroidota played a key role in the nitrogen cycle of the soil-microbe-plant interaction system, while phylum Firmicutes had a crucial role in the phosphorus cycle. And we found that, In the varieties with weak bacterial species in the rhizosphere soil, the addition of the composite strain had the best effect, while in the varieties with rich bacterial species, the addition of the composite strain may have the exclusion phenomenon, which was not as good as the addition of the single dominant strain.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eConclusions:\\u003c/strong\\u003eThe PGPB had excellent activities, such as nitrogen fixation, phosphorus and potassium solubilization, which could promote plant growth by decomposing soil nutrients. The inoculated strains can positively enrich the beneficial bacteria in sugarcane. However, there were variations in the quantities of these promoted properties in the treatments with different bacterial strains and sugarcane varieties. It was found that soil-disadvantaged and inoculum-specific bacteria were more favorable to plant development. The considerable variation in soil microbe provides a knowledge base and an experimental system for further mining and utilization of microbial strains.\\u003c/p\\u003e\",\"manuscriptTitle\":\"Different responses of sugarcane and rhizosphere soil microorganisms to single or mixture application of PGPB\",\"msid\":\"\",\"msnumber\":\"\",\"nonDraftVersions\":[{\"code\":1,\"date\":\"2024-07-24 10:10:20\",\"doi\":\"10.21203/rs.3.rs-4643245/v1\",\"editorialEvents\":[{\"type\":\"communityComments\",\"content\":0},{\"type\":\"decision\",\"content\":\"Revision requested\",\"date\":\"2024-07-15T07:24:55+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"editorInvitedReview\",\"content\":\"\",\"date\":\"2024-07-12T09:55:24+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"editorInvitedReview\",\"content\":\"\",\"date\":\"2024-07-12T04:02:52+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"editorInvitedReview\",\"content\":\"\",\"date\":\"2024-07-09T03:53:34+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewerAgreed\",\"content\":\"231829418221729681997366273149054703501\",\"date\":\"2024-07-06T04:54:32+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewerAgreed\",\"content\":\"265689198931331434978284599470974480714\",\"date\":\"2024-07-05T13:00:01+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"editorInvitedReview\",\"content\":\"\",\"date\":\"2024-07-04T10:28:09+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewerAgreed\",\"content\":\"73724835876216623730065348470743316645\",\"date\":\"2024-07-03T03:58:18+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewerAgreed\",\"content\":\"334100847121988193883951870637291633465\",\"date\":\"2024-07-02T17:56:27+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewerAgreed\",\"content\":\"64186726022417066919730831083481563440\",\"date\":\"2024-07-02T10:36:11+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewerAgreed\",\"content\":\"57507315384497421793142557555811764277\",\"date\":\"2024-07-02T06:17:16+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewerAgreed\",\"content\":\"1892803731079133309374490544451570073\",\"date\":\"2024-07-02T04:52:56+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewerAgreed\",\"content\":\"216933456147902126765124471855727123735\",\"date\":\"2024-07-02T03:33:15+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewerAgreed\",\"content\":\"120596322026027081135496088777930470163\",\"date\":\"2024-07-02T02:37:59+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewerAgreed\",\"content\":\"132140639568473789523682279887492517566\",\"date\":\"2024-07-02T01:42:21+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewersInvited\",\"content\":\"\",\"date\":\"2024-07-02T00:57:53+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"editorInvited\",\"content\":\"\",\"date\":\"2024-07-01T14:47:12+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"editorAssigned\",\"content\":\"\",\"date\":\"2024-07-01T14:45:15+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"checksComplete\",\"content\":\"\",\"date\":\"2024-07-01T14:44:02+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"submitted\",\"content\":\"BMC Plant Biology\",\"date\":\"2024-06-26T13:59:24+00:00\",\"index\":\"\",\"fulltext\":\"\"}],\"status\":\"published\",\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"bmc-plant-biology\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":false,\"externalIdentity\":\"pbio\",\"sideBox\":\"Learn more about [BMC Plant Biology](http://bmcplantbiol.biomedcentral.com/)\",\"snPcode\":\"\",\"submissionUrl\":\"https://www.editorialmanager.com/pbio/default.aspx\",\"title\":\"BMC Plant Biology\",\"twitterHandle\":\"BMC_series\",\"acdcEnabled\":true,\"dfaEnabled\":false,\"editorialSystem\":\"em\",\"reportingPortfolio\":\"BMC Series\",\"inReviewEnabled\":true,\"inReviewRevisionsEnabled\":true}}],\"origin\":\"\",\"ownerIdentity\":\"9e14d47d-37b1-4712-b10f-05fff8781d68\",\"owner\":[],\"postedDate\":\"July 24th, 2024\",\"published\":true,\"recentEditorialEvents\":[],\"rejectedJournal\":[],\"revision\":\"\",\"amendment\":\"\",\"status\":\"under-review\",\"subjectAreas\":[],\"tags\":[],\"updatedAt\":\"2026-05-05T18:54:01+00:00\",\"versionOfRecord\":[],\"versionCreatedAt\":\"2024-07-24 10:10:20\",\"video\":\"\",\"vorDoi\":\"\",\"vorDoiUrl\":\"\",\"workflowStages\":[]},\"version\":\"v1\",\"identity\":\"rs-4643245\",\"journalConfig\":\"researchsquare\"},\"__N_SSP\":true},\"page\":\"/article/[identity]/[[...version]]\",\"query\":{\"redirect\":\"/article/rs-4643245\",\"identity\":\"rs-4643245\",\"version\":[\"v1\"]},\"buildId\":\"8U1c8b4HqxoKbykW_rLl7\",\"isFallback\":false,\"isExperimentalCompile\":false,\"dynamicIds\":[84888],\"gssp\":true,\"scriptLoader\":[]}","source_license":"CC-BY-4.0","license_restricted":false}