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Effect of Chicken Manure-Based Fertiliser on Bacterial Communities and Diversity of Tomato Endosphere Microbiota

Effect of Chicken Manure-Based Fertiliser on Bacterial Communities and Diversity of Tomato... Agriculture (Poľnohospodárstvo), 67, 2021 (3): 144 − 154 DOI: 10.2478/agri-2021-0013 Original paper EFFECT OF CHICKEN MANURE-BASED FERTILISER ON BACTERIAL COMMUNITIES AND DIVERSITY OF TOMATO ENDOSPHERE MICROBIOTA HAFEEZ UL HAQ, YE LI, LINGYUE JIN, TING ZHANG, LINJIE CHENG, ZHE LI, BAOYU TIAN* College of Life Sciences, Fujian Normal University, Fuzhou, China Haq, H.U., Li, Y., Jin, L., Zhang, T., Cheng, L., Li, Z., and Tian, B. 2021: Effect of chicken manure-based fertiliser on bacterial communities and diversity of tomato endosphere microbiota. Agriculture (Poľnohospodárstvo), 67(3), 144 – 154. The frequent use of chemical fertiliser produces harmful effects on the soil ecosystem. Therefore, biocompatible methods are used to improve plant health and production through natural fertiliser or plant beneficial microorganisms. This study aims to investigate the effect of amended chicken manure-based natural fertiliser on bacterial communities and plant beneficial bacteria of tomato endosphere microbiome using the high throughput 16S rRNA gene amplicon sequencing. The results showed Proteobacteria (89.4 ± 4.7% to 86.7 ± 3.9%), Actinobacteria (6.03 ± 2.9% to 3.56 ± 2.1%), and Firmicutes (3.34 ± 1.3% to 0.59 ± 0.3%) as the dominant bacterial phyla of tomato endophytic microbiome. Pseudomonas and Bacillus were the most abundant identifiable genera in the chicken manure root (CMR) (amended manure treatments) sample. There was no significant difference in alpha bacterial diversity (Shannon index: p = 0.48) and species richness (Chao 1: p = 0.43) between control original root (OR) and CMR. However, the distribution of the dominant phyla was mainly affected by manure fertilisation. The non-metric multidimensional scaling (NMDS) and PCoA of beta diversity analysis suggested a significant separation in bacterial communities of tomato endophytes between CMR and OR. The most differently abundant bacterial taxa belong to Bacteroidetes in the OR group, whereas the beneficial bacterial communities of Actinobacteria and Firmicutes were more abundant in the CMR group. Therefore, the chicken manure application can significantly affect bacterial communities of tomatoes´ root endophytic microbiome and effectively improve the abundance of the beneficial microbes as biofertilisers. Key words: manure addition, endosphere microbiome, 16S rRNA gene amplicon sequencing, tomato With an increase in the world population and be alternative tools to chemical fertiliser (Hallmann the need for food, agricultural systems have be- et al. 1997; Xu et al. 2019; Sturz et al. 2000). It come more productive and sustainable (Hartmann is widely accepted that as ubiquitous colonizers of et al. 2015). The frequent use of chemical fertiliser plants, endophytes improve plant health and pro- can produce harmful effects on the soil ecosystem ductivity (Eo & Park 2016). Endophytes may have (Bebber & Richards 2020). Organic fertiliser, espe- beneficial effects on host plants by protecting the cially animal manure or plant residue-based natural host from pathogen or pests, providing the nutrients, fertiliser can be considered an important measure increasing competition for space, producing phyto- for improving soil quality and efficient ecological hormones to promote plant growth, etc. (Bulgarelli agricultural production (Li et al. 2014). Besides nat- et al. 2013; Turner et al. 2013; Tian et al. 2017). ural fertilisers, endophytes-based biofertiliser can Extensive research has been carried out to as- Hafeez Ul Haq, Ye Li, Lingyue Jin, Ting Zhang, Linjie Cheng, Zhe Li, Baoyu Tian (*Corresponding author), College of Life Sciences, Fujian Normal University, No.8, Shangsan Road, Cangshan District, Fuzhou, 350108, Fujian, China. E-mail: tianby@fjnu.edu.cn © 2021 Authors. This is an open access article licensed under the Creative Commons Attribution-NonComercial-NoDerivs License (http://creativecommons.org/licenses/by-nc-nd/4.0/). 144 Agriculture (Poľnohospodárstvo), 67, 2021 (3): 144 − 154 sess the potential of endophytes as biological con- has been done to evaluate the contribution of biofer- trol agents and biofertilisers (Tian et al. 2017; Sturz tiliser on plant beneficial bacteria of plant root endo- et al. 2000). Recently, high throughput sequenc- phytic microbiota. In this paper, the influence of ma- ing-based metagenomic analysis has revealed that nure addition was investigated by using 16S rRNA Actinobacteria and Proteobacteria were present in gene amplicon sequencing. Community structure the endosphere of Arabidopsis, rice, and tomatoes and composition, bacterial diversity of tomato root (Bulgarelli et al. 2013). Endophytic bacteria from endosphere microbiome, and plant beneficial bac- genera of Streptomyces, Pseudomonas, and Bacil- teria between control (OR) and manure treatments lus have been demonstrated as beneficial microbes (CMR) were compared in detail. Therefore, based with remarkable activities in different studies (Eo on the results, we are convinced that our study will & Park 2016). Several Pseudomonas have shown open a new way for the development of the environ- antagonism against pathogen bacteria, fungi, and ment-friendly practice in agriculture with fertiliser nematodes. Pseudomonas also played important additions in the future. role in promoting plant growth through the produc- tion of secondary metabolites, such as phenazine derivatives, and hydrogen cyanide (Weller et al. MATERIAL AND METHODS 2002; Loper et al. 2012; Lundberg et al. 2012). Re- cent studies have revealed that Pseudomonas and Experimental setup and sample collection In this experiment, the soil was collected from Bacillus have a significant probiotic effect on the lawns without a known history of organic fertiliser expression of Indole-3-acetic acid (IAA) in toma- on the campus of Fujian Normal University. Chick- toes, weakening the abscisic acid (ABA), the ex- en manure was collected as a nutrient from the pression of ethylene, and also provides protection to chicken farmhouse near campus because of its high plants from phytopathogens through preproduction concentration of micronutrients (Duncan 2005). The clottase, antibiotics, and antibacterial peptides sub- physicochemical properties of the soil and manure stances (Lin et al. 2012; Dombrowski et al. 2017; were examined with an inductively coupled plasma Tian et al. 2017; Xu et al. 2019). atomic emission spectrometer (Thermo Scientific, The diversity of endosphere bacterial commu- Waltham, MA, USA) (Peiffer et al. 2013). The soil nities is critical to the sustainability, stability, and and manure properties were as follows: pH 6.7, total integrity of endosphere ecosystems (Wang et al. carbon (2.9%; 35.0%), total nitrogen (0.35%; 4.7%), 2015). The use of natural organic fertilisers signifi- (16; 3,700 mg/kg) NH , and (255.0; 57 mg/kg) cantly impacts the bacterial structure, diversity of NO , respectively. A pot experiment was performed soil, and plant microbiome, from soil-plant systems in flower pots with 25 cm in height and 15 cm in di- to the ecological environment (Beecher 1998). Bac- ameter. The first treatment contained a total of 1.5 kg terial diversity significantly decreases in the field of soil with chicken manure (1 kg soil + 0.5 kg with continuous input of chemical nitrogen content, chicken manure) as a treated sample (CMR), and which may cause stress conditions and inhibit bacte- the other contained 1.5 kg soil without chicken ma- rial growth (Yang et al. 2017). In contrast, bacterial nure as a control (OR) (Zhang et al. 2020b). Each diversity in the organic fertilised soil is significantly treatment was performed with three replicates. 3 ‒ 5 higher than that of chemically fertilised soils in the tomato seeds were planted in each flower pot under legume and tomato system (Manching et al. 2014). 25°C in daylight (14 h) and 20°C in dark (10 h) (70% Field experiments in tomato gardens to study the ef- humidity) in a climate-controlled glasshouse (Table fects of different fertilisation on the soil microbial S1). After 45 days of planting, tomato plants were community indicate that organic fertiliser treatment pulled from every flower pot (Dong et al. 2019). has a higher diversity of soil bacterial communities The roots were shaken to remove soil particles com- (van Elsas et al. 2012; Xiao et al. 2018). Howev- pletely and followed by washing with water. After er, previous studies only focused on the impact of that, root samples were separately immersed in 30% different organic matter on bacterial diversity of hydrogen peroxide for 30 min, and 70% ethanol for endophytic microbiota in the plant, and little work 145 Agriculture (Poľnohospodárstvo), 67, 2021 (3): 144 − 154 1 min to remove the bacterial community residing for subsequent analysis. A summary of the obtained on the surface of roots, and then washed with ster- data used for processing steps is provided in Table ilized water up to three times. Finally, 0.5 g of the S2. To correct the difference in sequencing depth, lateral root samples were chosen and cut into 0.1 to the number of bacterial reads per sample was sep- 0.5 mm sections using sterile scissors and placed in arately elevated to the minimum number of reads. a 1.5 mL Eppendorf tube for further DNA extraction For individual samples, effective bacterial sequenc- process (Zhang et al. 2019). es were separately subsampled for the subsequent statistical analysis. DNA extraction After subsampling, the data were processed ac- Total genomic DNA was extracted with Pow- cording to a modified standard operating procedure er Soil DNA Isolation Kit (Mo Bio Laboratories, (SOP) pipeline based on software package QIIME Carlsbad, CA, USA), following the manufacturer´s and USEARCH (Caporaso et al. 2010; Edgar 2010). protocols. The extracted DNA was run on 1% aga- Briefly, the effective sequence was clustered to op- rose gels at 90 V for 45 min in 1× TAE (Tris-ac- erational taxonomic units (OTUs) using USEARCH etate-EDTA) buffer to examine the DNA size and at 97% sequence similarity (Edgar 2010). Each integrity. The concentration and purity of the ex- OTU’s representative sequence was aligned to the tracted DNA were assessed using an ND2000c 16S rDNA SILVA database (Release 128) (Yilmaz spectrophotometer (Thermo Scientific, Dreieich, et al. 2014). Taxonomy was subsequently assigned Germany). Extracted DNA was stored at ‒80°C for to each representative sequence with 90% confi- high-throughput sequencing analysis. dence using the ribosomal database project (RDP) classifier. The community richness and diversity in- PCR amplification and high throughput sequencing dex (α-diversity) within the sample were measured The V5-V7 hypervariable regions of bacterial (Chao1 Index, ACE Index, Shannon diversity, and 16S rRNA gene were amplified using primer pair Simpson diversity). The community richness, chao1 799F (5’ AACMGGATTAGATACCCKG-3’) and index, and abundance-based coverage (ACE). ACE 1193R (5’-ACGTCATCCCCACCTTCC-3’) fused index were used to assess the abundance of bacte- with the appropriate Illumina adapters and a 12-bp rial taxa, while the Shannon and Simpson indices index sequence unique to each sample (Cheng et were used to assess alpha diversity. Statistical anal- al. 2019). All the Polymerase chain reaction (PCR) ysis was carried out using an analysis of variance amplification reactions were carried out in 10 ng (ANOVA) and alpha = 0.05 to determine whether metagenomic DNA per 50 μL reaction. DNA ampli- the diversity index or species richness estimator con was subsequently purified, combined in equim - was statistically significant among root samples. To olar ratios, and sent to Sangon Biotech (Shanghai, estimate the beta diversity, the weighted UniFrac China) for producing paired 300-nucleotide reads at distance was used to calculate the similarities of an Illumina Miseq platform. the membership and structures at the OTUs levels Data processing and analysis found in both groups. Principal coordinate analysis The raw sequence data generated from a paired- (PCoA) and non-metric multidimensional scaling end library was first linked using FLASH (version (NMDS) plots using the Vegan package in R (ver- 1.2.3) (Magoč & Salzberg 2011). Cutadapt (version sion 3.3.1) were used to observe bacterial commu- 1.9.1) (Martin 2011) was used to remove adapters, nity differences and composition between individ- barcodes, and primers sequences. Low-quality se- uals or in groups. The significance of the effects of quence, lengths shorter than 200 bp, or sequences manure addition on beta diversity was statistically with ambiguous bases were removed to control se- measured using permutational multivariate analysis quence quality using USEARCH (version 8.1.1861) of variance (PERMANOVA), Adonis, or analysis of (Edgar 2010). Chimeric sequences were identi- similarities (ANOSIM) performed using the Vegan fied and removed with a de novo method using package in R (version 3.3.1). Linear discriminant USEARCH (version 8.1.1861) (Edgar 2010), final- analysis effect size (LEfse) is an algorithm that can ly resulted in high-quality and effective sequences identify the characteristics of microbial communi- 146 Agriculture (Poľnohospodárstvo), 67, 2021 (3): 144 −154 ties in different treatment methods as well as em- to meet the further analysis requirements. phasizes the statistical significance and biological Estimating the bacterial diversity and richness of relevance at the same time (http://huttenhower.sph. the endosphere microbiota harvard.edu/lefse/). The non-parametric factorial To quantify the diversity (Shannon diversity and Kruskal-Wallis sum rank test for LEfse was per- Simpson diversity) and richness (ACE Index and formed to identify significant differences in bacte - Chao1 Index) of the bacterial community of root rial taxa among the groups and then estimate each samples among both groups, we analysed the α-di- feature’s effect on differentially abundant features versity index using the OTU-based analysis meth- through linear discriminant analysis size. Function- od (Figure S2). The results showed that the manure al annotation of protein was searched against the addition did not affect the diversity of bacterial evolutionary genealogy of genes: non-supervised communities. No significant differential OTU rich- Orthologous groups (eggnog, version 3.0) databas- ness (ACE index: P = 0.4252, and Chao1 index: P es using basic local alignment search tool program = 0.4304) and bacterial diversity (Shannon diversi- (BLASTP) (version 2.2.26) (Altschul et al. 1990) ty: P = 0.4843, and Simpson diversity: P = 0.3716) with the E-value of 105 cutoffs. The abundance of were observed between CMR and OR group. a certain eggnog entry in each sample was calculat- ed by the total found genes and weighted by their Community structures and compositions of the en- coverage. The eggnog database provides multiple dosphere microbiota sequence alignments and broad term functional an- The sequences of both group samples were clas- notation. The raw sequencing data were deposited in sified at the phylum, class, order, family, and ge- the National Center for Biotechnology Information nus levels. 27 phylum, 52 classes, 110 orders, 189 (NCBI) sequence read archive (SRA) under acces- families, and 448 genera were identified in the root sion number PRJNA593402. samples. Proteobacteria, Actinobacteria, Firmic- utes, Bacteroidetes were the dominant phyla while the remaining phyla were in low abundance in to- RESULTS mato root samples (Figure 1A). Only the two most abundant phyla Proteobacteria and Firmicutes were Data processing significantly abundant in CMR compared to that of A total of 255,551 original sequences were ob- the OR sample (Figure 2). The relative abundances tained from both groups through high-throughput of Proteobacteria were 89.4 ± 4.7% in CMR sam- sequencing. After quality control filtration (removal ples. In comparison, 86.7 ± 3.9% to the OR samples of primer sequences, low-quality fraction sequences, their abundances were significantly lower than those and chimeric sequences, etc.), 234,813 high-quality, in the CMR sample. Top fifty genera with sequence effective sequences were obtained, with an average abundances > 1% were identified, including other, of 39,135 sequences per sample (Table S2). The re- and unclassified (Figure 1B). The most abundant sultant filtered sequences were classified as 5,963 genera in both groups were Pseudomonas, Bacillus, operational taxonomic units (OTUs) at a similarity Herminiimonas, Propionibacterium, Staphylococ- level of 97%. A certain number of sequences were cus, Corynebacterium, Sphingomonas, Flavobacte- randomly selected from the sequence as horizontal rium, Gaiella, and Variovorax Ramlibacter. The un- coordinates. The number of OTUs represented by classified genera were the second most abundant in these sequences is shown in the OTUs accumulation the CMR and OR. Among identifiable genera, Pseu- curve (Figure S1). It showed that as the sample size domonas (81.61 ± 7.36%), Bacillus (1.71 ± 1.15%), increased, the OTUs accumulation curve tended to Herminiimonas (0.76 ± 0.14%), Propionibacterium flatten, indicating that most of the OTUs in the sam- (0.71 ± 0.48%), and Staphylococcus (0.55 ± 0.45%) ples were detected. The average coverage of 6 sam- were the most abundant in the CMR sample (Figure ples was 0.98 (min-0.96, max-0.99), which support- 1B). While the most abundant identifiable genera in ed the above observation that sequencing had cov- the OR sample were Pseudomonas (66.6 ± 12.4%), ered the vast majority of the species in each sample 147 Agriculture (Poľnohospodárstvo), 67, 2021 (3): 144 −154 Sphingomonas (0.65 ± 1.1%), Herminiimonas (0.61 OR group (p = 0.03) (Figure 3). In addition, Micro- ± 0.37%), Ramlibacter (0.53 ± 0.28) and Gaiella bacteriaceae, Rhizobiaceae, Bacillus, Bacillacea, (0.50 ± 0.64%). The results indicated that the ma- Bacillales Bacilli, and Actinomycetales were most nure addition had a significant effect on the structure abundant in CMR groups (Figure 3). of the tomato roots’ endogenous bacterial commu- Estimating the dissimilarity and similarities of the nities and with a higher abundance of the beneficial endosphere microbiota using the beta diversity microbes, such as Pseudomonas and Bacillus (Fig- The principal coordinate analysis (PCoA) based ure 1B). Bacterial taxa with significantly differential on the Bray-Curtis algorithm clearly shows that the abundances were detected by using linear discrimi- endosphere microbial community structure differed nant analysis effect size (LEfSe) between the CMR between the CMR and OR groups (Figure 4A). CMR and OR groups (Figure 3). The most differently and OR were separated from each other. In the total abundant bacterial taxa belong to Proteobacteria variance of the data set, the two principal compo- Phylum in the OR group, whereas the phylum Ac- nents explain 59% of the total bacterial community. tinobacteria, Firmicutes, and Proteobacteria were Furthermore, the first principal coordinate (PCoA1) more abundant in the CMR group. Moreover, at the is the most important, which displayed a 40% dif- genus level, 9 genera showed significantly enriched ference in bacterial community’s variation, and between the CMR and OR groups. Specifically, at the principal coordinate (PCoA2) explained 19% the order level, Burkholderiales were enriched in Figure 1. The composition and relative abundance of major bacterial taxa in tomato endosphere sample between CMR and OR while the bar represent the relative abundance of bacterial taxa. Heatmap was drawn using the average value of three replicates of each sample group. A ‒ the composition and relative abundance of major bacterial Phylum; B ‒ the composition and relative abundance of major bacterial genera. 148 Agriculture (Poľnohospodárstvo), 67, 2021 (3): 144 −154 bacterial variation. A non-metric multidimensional based on the eggNOG database were shared by both scale (NMDS) was obtained to compare and analyse group samples (Figure 5). Functional gene annota- the differences between and within groups, direct- tions based on COG classes showed that the relative ly reflecting the differences in bacterial communi - distribution of the 26 basic metabolic categories of ty structure. The NMDS analysis results were in CMR and OR was similar. Additionally, in gener- alignment with these results. However, these results al functional prediction, two groups contain more indicate significant differences in endosphere bac- genes related to energy production and transfor- terial communities between CMR and OR (Figure mation, amino acid transport and metabolism, tran- 4B). The PERMANOVA test (ADONIS) revealed scription, carbohydrate transport and metabolism, that the CMR group accounted for high differences signal transduction mechanisms, transcription, inor- (ADONIS: R = 0.2471, p = 0.0015), while it was ganic ion transport, and metabolism. notable that ANOSIM tests between the two group bacterial communities were more similar (R = 0.530, p = 0.001). DISCUSSION Functional annotation of protein The diversity of the soil microbial population For functional annotations, the read was anno- is critical to the stability, integrity, and survival of tated with the eggNOG database. Most functions Figure 2. Fisher exact test plot of bacterial taxa abundance (means percent) in the CMR and OR samples at the phylum levels. The plot was drawn using the average value of three replicates of each sample group. 149 Agriculture (Poľnohospodárstvo), 67, 2021 (3): 144 −154 the soil environment (Cheng et al. 2019). In agro- rial growth (Lee et al. 2019). In contrast, bacterial ecosystems, different fertilisation applications can diversity was substantially higher in soils with or- affect the soil microenvironment. Chemical fertili- ganic fertilisation than in soils with chemical fertili- sation has been shown to harm soil bacterial diver- sation in wheat and the legume system (Allard et al. sity in a variety of agricultural ecosystems (Turn- 2016). Similarly, a field study in tomato cultivation er et al. 2013). A substantial decrease in bacterial was performed to investigate the impact of various diversity was found at high nitrogen levels, which fertilisation on soil bacterial communities, show- might produce severe situations and inhibit bacte- ing that organic manure application had the most Figure 3. Linear discrimination analysis (LEfse) shows statistically different bacterial communities in CMR and OR. The LEfse and linear discriminant analysis (LDA) were drawn using the average value of three replicates of each sample group. Figure 4. Microbial community differentiation between all endosphere samples. A ‒ principal coordinate analysis (PCoA); B ‒ non-metric multidimensional scaling (NMDS) analysis of bacterial communities based on Bray-Curtis similarity matrices. 150 Agriculture (Poľnohospodárstvo), 67, 2021 (3): 144 −154 diverse soil bacterial population (Lee et al. 2019). on soil microorganism diversity. The composition of However, prior research only examined the effect endosphere communities was susceptible to various of various organic matter on bacterial diversity but fertilisation applications in agriculture practices, didn’t address the effect of endosphere bacterial di- Proteobacteria, Chloroflexi, Acidobacteria, Acti - versity in tomato cultivation. In our recent study, we nobacteria, and Bacteroidetes were the most abun- focused on the complex changes in root bacterial di- dant phyla in agricultural soils (Berg et al. 2005). versity in tomato cultivation that had been fertilised Proteobacteria, Actinobacteria, and Acidobacteria with chicken manure in a pot experiment. The result were the most abundant phyla in soils with various showed that the manure application had no signif- fertilisation levels in tomato cultivations (Green et icant effect on bacterial diversity in term of OTUs al. 2012). Here, in the endosphere community com- richness and alpha-diversity (Figure S2), however, position was examined and the result revealed that led to a significant effect on bacterial community’s Proteobacteria, Actinobacteria, Firmicutes, and diversity (Figure 4) a similar trend presented in the Bacteroidetes were the most prevalent phyla in to- previous study which described that fertilisation mato cultivation (Figure 1). Recognizing variations has a positive impact on beta-diversity on a tomato in endosphere bacteria between fertilised and unfer- plantation (Luo et al. 2019). The complete utiliza- tilised soils can provide more insight to determine tion of nutrients in chicken manure by root bacte- soil fertility and the impact of manure fertilisers. ria may be the possible reason for high diversity in Proteobacteria and Actinobacteria are the two most the tomato endosphere according to prior research common phyla in the bacterial community (Xu et (Ye et al. 2021). Therefore, we strongly believe that al. 2019). A significant proportion of Proteobacte- seasonal changes such as fertilisation would affect ria and Actinobacteria can enhance important nutri- root bacterial diversity in tomato cultivation. Sea- ents cycles, resulting in sustainable utilization and increased soil fertility. Proteobacteria have evolved son and fertilisation can have a synergistic impact Figure 5. Functional protein annotation of CMR and OR samples against eggnog database. 151 Agriculture (Poľnohospodárstvo), 67, 2021 (3): 144 −154 to survive in habitats that are rich in resources (Haas ure 2) suggesting that the soil in tomato cultivation & Défago 2005; Zhen et al. 2014). Actinobacteria without chicken manure was good for the growth were formerly assumed to prefer oligotrophic en- of Bacteroidetes. They may actively participate in vironments which resource-limited. According to a series of soil activities and make important con- previous research, the proportion of Proteobacteria tributions to soil nutrient conversion and material to Actinobacteria can represent soil nutrition status: recycling. Flavobacterium belonged to the Bacte- a higher ratio indicates that more organic matter is roidetes, and its relative abundance was higher in being added to the soil (Zhang et al. 2020a; Ye et al. soils without manure (Figure 1). It was reported to 2021). A previous study identified that a relatively be capable of degrading macromolecular organic higher abundance of Proteobacteria significantly matter such as lipid and protein, as well as poten- correlated with the organic substitution ratio in to- tial denitrification and nitrification ability (Zhang et mato cultivation, but the abundance of Actinobacte- al. 2017). The bacteria in the genus Pseudomonas ria was negatively correlated with this ratio (Schreit- were aerobic denitrifiers and could participate in er et al. 2018). Although, there is no evidence about the action of aerobic denitrification which was the the features of Proteobacteria and Actinobacteria in main process of natural nitrogen cycling (Zhang et tomato cultivation with chicken manure. In the cur- al. 2017). A relatively high abundance of Pseudo- rent research, the relative abundance of Proteobac- monas in soils with manure application suggested teria increased significantly in soils with chicken that organic fertilisation can encourage bacteria to manure application, suggesting that soils containing participate in the nitrogen cycle and enhanced the chicken manure may provide a suitable environment biochemical cycle in tomato cultivation. for soil bacteria that is intermediate between oligo- trophic and copiotrophic conditions. Bacteroidetes and Firmicutes are the two main phyla of bacteria CONCLUSIONS present in soil and manure (Guron et al. 2019). Fir- micutes are known to have a wide range of functions We concluded that as a biofertiliser, chicken ma- and to aid in the degradation of organic materials nure application significantly improved the abun- (Smit et al. 2001; Torsvik & Øvreås 2002). They dance of the beneficial microbes and the effects on were comparatively inactive opponents under high community structure and bacterial diversity of the nutrient environments compared to other microbes, endosphere in tomato cultivation. In addition, our occupying a lower role than Proteobacteria or Ac- study provides a principled approach to the rational tinobacteria. However, they had a competitive ad- use of chicken manure for tomato cultivation, which vantage over other bacteria due to their slow growth is crucial to reducing the consumption of chemical and ability to decompose complex substrates (Ji et fertilizers. al. 2018). The relative abundance of Firmicutes in Acknowledgments. This work was support- chicken manure-treated soils was significantly high- ed by grants from the Department of Science er than in the control soils, suggesting that manure and Technology of Fujian Province, China (Nos. application produced a nutrient-rich condition that 2020J01173; 2017J01625) and the National Natural favoured Firmicutes. These findings were consistent Science Foundation of China (No. Nos. 31670125; with previous research (Kamau et al. 2008). Bacte- 31660544). roidetes are the most common microbes in manure (Zhong et al. 2018). The prior analysis revealed that Conflict of interest. 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Effect of Chicken Manure-Based Fertiliser on Bacterial Communities and Diversity of Tomato Endosphere Microbiota

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Abstract

Agriculture (Poľnohospodárstvo), 67, 2021 (3): 144 − 154 DOI: 10.2478/agri-2021-0013 Original paper EFFECT OF CHICKEN MANURE-BASED FERTILISER ON BACTERIAL COMMUNITIES AND DIVERSITY OF TOMATO ENDOSPHERE MICROBIOTA HAFEEZ UL HAQ, YE LI, LINGYUE JIN, TING ZHANG, LINJIE CHENG, ZHE LI, BAOYU TIAN* College of Life Sciences, Fujian Normal University, Fuzhou, China Haq, H.U., Li, Y., Jin, L., Zhang, T., Cheng, L., Li, Z., and Tian, B. 2021: Effect of chicken manure-based fertiliser on bacterial communities and diversity of tomato endosphere microbiota. Agriculture (Poľnohospodárstvo), 67(3), 144 – 154. The frequent use of chemical fertiliser produces harmful effects on the soil ecosystem. Therefore, biocompatible methods are used to improve plant health and production through natural fertiliser or plant beneficial microorganisms. This study aims to investigate the effect of amended chicken manure-based natural fertiliser on bacterial communities and plant beneficial bacteria of tomato endosphere microbiome using the high throughput 16S rRNA gene amplicon sequencing. The results showed Proteobacteria (89.4 ± 4.7% to 86.7 ± 3.9%), Actinobacteria (6.03 ± 2.9% to 3.56 ± 2.1%), and Firmicutes (3.34 ± 1.3% to 0.59 ± 0.3%) as the dominant bacterial phyla of tomato endophytic microbiome. Pseudomonas and Bacillus were the most abundant identifiable genera in the chicken manure root (CMR) (amended manure treatments) sample. There was no significant difference in alpha bacterial diversity (Shannon index: p = 0.48) and species richness (Chao 1: p = 0.43) between control original root (OR) and CMR. However, the distribution of the dominant phyla was mainly affected by manure fertilisation. The non-metric multidimensional scaling (NMDS) and PCoA of beta diversity analysis suggested a significant separation in bacterial communities of tomato endophytes between CMR and OR. The most differently abundant bacterial taxa belong to Bacteroidetes in the OR group, whereas the beneficial bacterial communities of Actinobacteria and Firmicutes were more abundant in the CMR group. Therefore, the chicken manure application can significantly affect bacterial communities of tomatoes´ root endophytic microbiome and effectively improve the abundance of the beneficial microbes as biofertilisers. Key words: manure addition, endosphere microbiome, 16S rRNA gene amplicon sequencing, tomato With an increase in the world population and be alternative tools to chemical fertiliser (Hallmann the need for food, agricultural systems have be- et al. 1997; Xu et al. 2019; Sturz et al. 2000). It come more productive and sustainable (Hartmann is widely accepted that as ubiquitous colonizers of et al. 2015). The frequent use of chemical fertiliser plants, endophytes improve plant health and pro- can produce harmful effects on the soil ecosystem ductivity (Eo & Park 2016). Endophytes may have (Bebber & Richards 2020). Organic fertiliser, espe- beneficial effects on host plants by protecting the cially animal manure or plant residue-based natural host from pathogen or pests, providing the nutrients, fertiliser can be considered an important measure increasing competition for space, producing phyto- for improving soil quality and efficient ecological hormones to promote plant growth, etc. (Bulgarelli agricultural production (Li et al. 2014). Besides nat- et al. 2013; Turner et al. 2013; Tian et al. 2017). ural fertilisers, endophytes-based biofertiliser can Extensive research has been carried out to as- Hafeez Ul Haq, Ye Li, Lingyue Jin, Ting Zhang, Linjie Cheng, Zhe Li, Baoyu Tian (*Corresponding author), College of Life Sciences, Fujian Normal University, No.8, Shangsan Road, Cangshan District, Fuzhou, 350108, Fujian, China. E-mail: tianby@fjnu.edu.cn © 2021 Authors. This is an open access article licensed under the Creative Commons Attribution-NonComercial-NoDerivs License (http://creativecommons.org/licenses/by-nc-nd/4.0/). 144 Agriculture (Poľnohospodárstvo), 67, 2021 (3): 144 − 154 sess the potential of endophytes as biological con- has been done to evaluate the contribution of biofer- trol agents and biofertilisers (Tian et al. 2017; Sturz tiliser on plant beneficial bacteria of plant root endo- et al. 2000). Recently, high throughput sequenc- phytic microbiota. In this paper, the influence of ma- ing-based metagenomic analysis has revealed that nure addition was investigated by using 16S rRNA Actinobacteria and Proteobacteria were present in gene amplicon sequencing. Community structure the endosphere of Arabidopsis, rice, and tomatoes and composition, bacterial diversity of tomato root (Bulgarelli et al. 2013). Endophytic bacteria from endosphere microbiome, and plant beneficial bac- genera of Streptomyces, Pseudomonas, and Bacil- teria between control (OR) and manure treatments lus have been demonstrated as beneficial microbes (CMR) were compared in detail. Therefore, based with remarkable activities in different studies (Eo on the results, we are convinced that our study will & Park 2016). Several Pseudomonas have shown open a new way for the development of the environ- antagonism against pathogen bacteria, fungi, and ment-friendly practice in agriculture with fertiliser nematodes. Pseudomonas also played important additions in the future. role in promoting plant growth through the produc- tion of secondary metabolites, such as phenazine derivatives, and hydrogen cyanide (Weller et al. MATERIAL AND METHODS 2002; Loper et al. 2012; Lundberg et al. 2012). Re- cent studies have revealed that Pseudomonas and Experimental setup and sample collection In this experiment, the soil was collected from Bacillus have a significant probiotic effect on the lawns without a known history of organic fertiliser expression of Indole-3-acetic acid (IAA) in toma- on the campus of Fujian Normal University. Chick- toes, weakening the abscisic acid (ABA), the ex- en manure was collected as a nutrient from the pression of ethylene, and also provides protection to chicken farmhouse near campus because of its high plants from phytopathogens through preproduction concentration of micronutrients (Duncan 2005). The clottase, antibiotics, and antibacterial peptides sub- physicochemical properties of the soil and manure stances (Lin et al. 2012; Dombrowski et al. 2017; were examined with an inductively coupled plasma Tian et al. 2017; Xu et al. 2019). atomic emission spectrometer (Thermo Scientific, The diversity of endosphere bacterial commu- Waltham, MA, USA) (Peiffer et al. 2013). The soil nities is critical to the sustainability, stability, and and manure properties were as follows: pH 6.7, total integrity of endosphere ecosystems (Wang et al. carbon (2.9%; 35.0%), total nitrogen (0.35%; 4.7%), 2015). The use of natural organic fertilisers signifi- (16; 3,700 mg/kg) NH , and (255.0; 57 mg/kg) cantly impacts the bacterial structure, diversity of NO , respectively. A pot experiment was performed soil, and plant microbiome, from soil-plant systems in flower pots with 25 cm in height and 15 cm in di- to the ecological environment (Beecher 1998). Bac- ameter. The first treatment contained a total of 1.5 kg terial diversity significantly decreases in the field of soil with chicken manure (1 kg soil + 0.5 kg with continuous input of chemical nitrogen content, chicken manure) as a treated sample (CMR), and which may cause stress conditions and inhibit bacte- the other contained 1.5 kg soil without chicken ma- rial growth (Yang et al. 2017). In contrast, bacterial nure as a control (OR) (Zhang et al. 2020b). Each diversity in the organic fertilised soil is significantly treatment was performed with three replicates. 3 ‒ 5 higher than that of chemically fertilised soils in the tomato seeds were planted in each flower pot under legume and tomato system (Manching et al. 2014). 25°C in daylight (14 h) and 20°C in dark (10 h) (70% Field experiments in tomato gardens to study the ef- humidity) in a climate-controlled glasshouse (Table fects of different fertilisation on the soil microbial S1). After 45 days of planting, tomato plants were community indicate that organic fertiliser treatment pulled from every flower pot (Dong et al. 2019). has a higher diversity of soil bacterial communities The roots were shaken to remove soil particles com- (van Elsas et al. 2012; Xiao et al. 2018). Howev- pletely and followed by washing with water. After er, previous studies only focused on the impact of that, root samples were separately immersed in 30% different organic matter on bacterial diversity of hydrogen peroxide for 30 min, and 70% ethanol for endophytic microbiota in the plant, and little work 145 Agriculture (Poľnohospodárstvo), 67, 2021 (3): 144 − 154 1 min to remove the bacterial community residing for subsequent analysis. A summary of the obtained on the surface of roots, and then washed with ster- data used for processing steps is provided in Table ilized water up to three times. Finally, 0.5 g of the S2. To correct the difference in sequencing depth, lateral root samples were chosen and cut into 0.1 to the number of bacterial reads per sample was sep- 0.5 mm sections using sterile scissors and placed in arately elevated to the minimum number of reads. a 1.5 mL Eppendorf tube for further DNA extraction For individual samples, effective bacterial sequenc- process (Zhang et al. 2019). es were separately subsampled for the subsequent statistical analysis. DNA extraction After subsampling, the data were processed ac- Total genomic DNA was extracted with Pow- cording to a modified standard operating procedure er Soil DNA Isolation Kit (Mo Bio Laboratories, (SOP) pipeline based on software package QIIME Carlsbad, CA, USA), following the manufacturer´s and USEARCH (Caporaso et al. 2010; Edgar 2010). protocols. The extracted DNA was run on 1% aga- Briefly, the effective sequence was clustered to op- rose gels at 90 V for 45 min in 1× TAE (Tris-ac- erational taxonomic units (OTUs) using USEARCH etate-EDTA) buffer to examine the DNA size and at 97% sequence similarity (Edgar 2010). Each integrity. The concentration and purity of the ex- OTU’s representative sequence was aligned to the tracted DNA were assessed using an ND2000c 16S rDNA SILVA database (Release 128) (Yilmaz spectrophotometer (Thermo Scientific, Dreieich, et al. 2014). Taxonomy was subsequently assigned Germany). Extracted DNA was stored at ‒80°C for to each representative sequence with 90% confi- high-throughput sequencing analysis. dence using the ribosomal database project (RDP) classifier. The community richness and diversity in- PCR amplification and high throughput sequencing dex (α-diversity) within the sample were measured The V5-V7 hypervariable regions of bacterial (Chao1 Index, ACE Index, Shannon diversity, and 16S rRNA gene were amplified using primer pair Simpson diversity). The community richness, chao1 799F (5’ AACMGGATTAGATACCCKG-3’) and index, and abundance-based coverage (ACE). ACE 1193R (5’-ACGTCATCCCCACCTTCC-3’) fused index were used to assess the abundance of bacte- with the appropriate Illumina adapters and a 12-bp rial taxa, while the Shannon and Simpson indices index sequence unique to each sample (Cheng et were used to assess alpha diversity. Statistical anal- al. 2019). All the Polymerase chain reaction (PCR) ysis was carried out using an analysis of variance amplification reactions were carried out in 10 ng (ANOVA) and alpha = 0.05 to determine whether metagenomic DNA per 50 μL reaction. DNA ampli- the diversity index or species richness estimator con was subsequently purified, combined in equim - was statistically significant among root samples. To olar ratios, and sent to Sangon Biotech (Shanghai, estimate the beta diversity, the weighted UniFrac China) for producing paired 300-nucleotide reads at distance was used to calculate the similarities of an Illumina Miseq platform. the membership and structures at the OTUs levels Data processing and analysis found in both groups. Principal coordinate analysis The raw sequence data generated from a paired- (PCoA) and non-metric multidimensional scaling end library was first linked using FLASH (version (NMDS) plots using the Vegan package in R (ver- 1.2.3) (Magoč & Salzberg 2011). Cutadapt (version sion 3.3.1) were used to observe bacterial commu- 1.9.1) (Martin 2011) was used to remove adapters, nity differences and composition between individ- barcodes, and primers sequences. Low-quality se- uals or in groups. The significance of the effects of quence, lengths shorter than 200 bp, or sequences manure addition on beta diversity was statistically with ambiguous bases were removed to control se- measured using permutational multivariate analysis quence quality using USEARCH (version 8.1.1861) of variance (PERMANOVA), Adonis, or analysis of (Edgar 2010). Chimeric sequences were identi- similarities (ANOSIM) performed using the Vegan fied and removed with a de novo method using package in R (version 3.3.1). Linear discriminant USEARCH (version 8.1.1861) (Edgar 2010), final- analysis effect size (LEfse) is an algorithm that can ly resulted in high-quality and effective sequences identify the characteristics of microbial communi- 146 Agriculture (Poľnohospodárstvo), 67, 2021 (3): 144 −154 ties in different treatment methods as well as em- to meet the further analysis requirements. phasizes the statistical significance and biological Estimating the bacterial diversity and richness of relevance at the same time (http://huttenhower.sph. the endosphere microbiota harvard.edu/lefse/). The non-parametric factorial To quantify the diversity (Shannon diversity and Kruskal-Wallis sum rank test for LEfse was per- Simpson diversity) and richness (ACE Index and formed to identify significant differences in bacte - Chao1 Index) of the bacterial community of root rial taxa among the groups and then estimate each samples among both groups, we analysed the α-di- feature’s effect on differentially abundant features versity index using the OTU-based analysis meth- through linear discriminant analysis size. Function- od (Figure S2). The results showed that the manure al annotation of protein was searched against the addition did not affect the diversity of bacterial evolutionary genealogy of genes: non-supervised communities. No significant differential OTU rich- Orthologous groups (eggnog, version 3.0) databas- ness (ACE index: P = 0.4252, and Chao1 index: P es using basic local alignment search tool program = 0.4304) and bacterial diversity (Shannon diversi- (BLASTP) (version 2.2.26) (Altschul et al. 1990) ty: P = 0.4843, and Simpson diversity: P = 0.3716) with the E-value of 105 cutoffs. The abundance of were observed between CMR and OR group. a certain eggnog entry in each sample was calculat- ed by the total found genes and weighted by their Community structures and compositions of the en- coverage. The eggnog database provides multiple dosphere microbiota sequence alignments and broad term functional an- The sequences of both group samples were clas- notation. The raw sequencing data were deposited in sified at the phylum, class, order, family, and ge- the National Center for Biotechnology Information nus levels. 27 phylum, 52 classes, 110 orders, 189 (NCBI) sequence read archive (SRA) under acces- families, and 448 genera were identified in the root sion number PRJNA593402. samples. Proteobacteria, Actinobacteria, Firmic- utes, Bacteroidetes were the dominant phyla while the remaining phyla were in low abundance in to- RESULTS mato root samples (Figure 1A). Only the two most abundant phyla Proteobacteria and Firmicutes were Data processing significantly abundant in CMR compared to that of A total of 255,551 original sequences were ob- the OR sample (Figure 2). The relative abundances tained from both groups through high-throughput of Proteobacteria were 89.4 ± 4.7% in CMR sam- sequencing. After quality control filtration (removal ples. In comparison, 86.7 ± 3.9% to the OR samples of primer sequences, low-quality fraction sequences, their abundances were significantly lower than those and chimeric sequences, etc.), 234,813 high-quality, in the CMR sample. Top fifty genera with sequence effective sequences were obtained, with an average abundances > 1% were identified, including other, of 39,135 sequences per sample (Table S2). The re- and unclassified (Figure 1B). The most abundant sultant filtered sequences were classified as 5,963 genera in both groups were Pseudomonas, Bacillus, operational taxonomic units (OTUs) at a similarity Herminiimonas, Propionibacterium, Staphylococ- level of 97%. A certain number of sequences were cus, Corynebacterium, Sphingomonas, Flavobacte- randomly selected from the sequence as horizontal rium, Gaiella, and Variovorax Ramlibacter. The un- coordinates. The number of OTUs represented by classified genera were the second most abundant in these sequences is shown in the OTUs accumulation the CMR and OR. Among identifiable genera, Pseu- curve (Figure S1). It showed that as the sample size domonas (81.61 ± 7.36%), Bacillus (1.71 ± 1.15%), increased, the OTUs accumulation curve tended to Herminiimonas (0.76 ± 0.14%), Propionibacterium flatten, indicating that most of the OTUs in the sam- (0.71 ± 0.48%), and Staphylococcus (0.55 ± 0.45%) ples were detected. The average coverage of 6 sam- were the most abundant in the CMR sample (Figure ples was 0.98 (min-0.96, max-0.99), which support- 1B). While the most abundant identifiable genera in ed the above observation that sequencing had cov- the OR sample were Pseudomonas (66.6 ± 12.4%), ered the vast majority of the species in each sample 147 Agriculture (Poľnohospodárstvo), 67, 2021 (3): 144 −154 Sphingomonas (0.65 ± 1.1%), Herminiimonas (0.61 OR group (p = 0.03) (Figure 3). In addition, Micro- ± 0.37%), Ramlibacter (0.53 ± 0.28) and Gaiella bacteriaceae, Rhizobiaceae, Bacillus, Bacillacea, (0.50 ± 0.64%). The results indicated that the ma- Bacillales Bacilli, and Actinomycetales were most nure addition had a significant effect on the structure abundant in CMR groups (Figure 3). of the tomato roots’ endogenous bacterial commu- Estimating the dissimilarity and similarities of the nities and with a higher abundance of the beneficial endosphere microbiota using the beta diversity microbes, such as Pseudomonas and Bacillus (Fig- The principal coordinate analysis (PCoA) based ure 1B). Bacterial taxa with significantly differential on the Bray-Curtis algorithm clearly shows that the abundances were detected by using linear discrimi- endosphere microbial community structure differed nant analysis effect size (LEfSe) between the CMR between the CMR and OR groups (Figure 4A). CMR and OR groups (Figure 3). The most differently and OR were separated from each other. In the total abundant bacterial taxa belong to Proteobacteria variance of the data set, the two principal compo- Phylum in the OR group, whereas the phylum Ac- nents explain 59% of the total bacterial community. tinobacteria, Firmicutes, and Proteobacteria were Furthermore, the first principal coordinate (PCoA1) more abundant in the CMR group. Moreover, at the is the most important, which displayed a 40% dif- genus level, 9 genera showed significantly enriched ference in bacterial community’s variation, and between the CMR and OR groups. Specifically, at the principal coordinate (PCoA2) explained 19% the order level, Burkholderiales were enriched in Figure 1. The composition and relative abundance of major bacterial taxa in tomato endosphere sample between CMR and OR while the bar represent the relative abundance of bacterial taxa. Heatmap was drawn using the average value of three replicates of each sample group. A ‒ the composition and relative abundance of major bacterial Phylum; B ‒ the composition and relative abundance of major bacterial genera. 148 Agriculture (Poľnohospodárstvo), 67, 2021 (3): 144 −154 bacterial variation. A non-metric multidimensional based on the eggNOG database were shared by both scale (NMDS) was obtained to compare and analyse group samples (Figure 5). Functional gene annota- the differences between and within groups, direct- tions based on COG classes showed that the relative ly reflecting the differences in bacterial communi - distribution of the 26 basic metabolic categories of ty structure. The NMDS analysis results were in CMR and OR was similar. Additionally, in gener- alignment with these results. However, these results al functional prediction, two groups contain more indicate significant differences in endosphere bac- genes related to energy production and transfor- terial communities between CMR and OR (Figure mation, amino acid transport and metabolism, tran- 4B). The PERMANOVA test (ADONIS) revealed scription, carbohydrate transport and metabolism, that the CMR group accounted for high differences signal transduction mechanisms, transcription, inor- (ADONIS: R = 0.2471, p = 0.0015), while it was ganic ion transport, and metabolism. notable that ANOSIM tests between the two group bacterial communities were more similar (R = 0.530, p = 0.001). DISCUSSION Functional annotation of protein The diversity of the soil microbial population For functional annotations, the read was anno- is critical to the stability, integrity, and survival of tated with the eggNOG database. Most functions Figure 2. Fisher exact test plot of bacterial taxa abundance (means percent) in the CMR and OR samples at the phylum levels. The plot was drawn using the average value of three replicates of each sample group. 149 Agriculture (Poľnohospodárstvo), 67, 2021 (3): 144 −154 the soil environment (Cheng et al. 2019). In agro- rial growth (Lee et al. 2019). In contrast, bacterial ecosystems, different fertilisation applications can diversity was substantially higher in soils with or- affect the soil microenvironment. Chemical fertili- ganic fertilisation than in soils with chemical fertili- sation has been shown to harm soil bacterial diver- sation in wheat and the legume system (Allard et al. sity in a variety of agricultural ecosystems (Turn- 2016). Similarly, a field study in tomato cultivation er et al. 2013). A substantial decrease in bacterial was performed to investigate the impact of various diversity was found at high nitrogen levels, which fertilisation on soil bacterial communities, show- might produce severe situations and inhibit bacte- ing that organic manure application had the most Figure 3. Linear discrimination analysis (LEfse) shows statistically different bacterial communities in CMR and OR. The LEfse and linear discriminant analysis (LDA) were drawn using the average value of three replicates of each sample group. Figure 4. Microbial community differentiation between all endosphere samples. A ‒ principal coordinate analysis (PCoA); B ‒ non-metric multidimensional scaling (NMDS) analysis of bacterial communities based on Bray-Curtis similarity matrices. 150 Agriculture (Poľnohospodárstvo), 67, 2021 (3): 144 −154 diverse soil bacterial population (Lee et al. 2019). on soil microorganism diversity. The composition of However, prior research only examined the effect endosphere communities was susceptible to various of various organic matter on bacterial diversity but fertilisation applications in agriculture practices, didn’t address the effect of endosphere bacterial di- Proteobacteria, Chloroflexi, Acidobacteria, Acti - versity in tomato cultivation. In our recent study, we nobacteria, and Bacteroidetes were the most abun- focused on the complex changes in root bacterial di- dant phyla in agricultural soils (Berg et al. 2005). versity in tomato cultivation that had been fertilised Proteobacteria, Actinobacteria, and Acidobacteria with chicken manure in a pot experiment. The result were the most abundant phyla in soils with various showed that the manure application had no signif- fertilisation levels in tomato cultivations (Green et icant effect on bacterial diversity in term of OTUs al. 2012). Here, in the endosphere community com- richness and alpha-diversity (Figure S2), however, position was examined and the result revealed that led to a significant effect on bacterial community’s Proteobacteria, Actinobacteria, Firmicutes, and diversity (Figure 4) a similar trend presented in the Bacteroidetes were the most prevalent phyla in to- previous study which described that fertilisation mato cultivation (Figure 1). Recognizing variations has a positive impact on beta-diversity on a tomato in endosphere bacteria between fertilised and unfer- plantation (Luo et al. 2019). The complete utiliza- tilised soils can provide more insight to determine tion of nutrients in chicken manure by root bacte- soil fertility and the impact of manure fertilisers. ria may be the possible reason for high diversity in Proteobacteria and Actinobacteria are the two most the tomato endosphere according to prior research common phyla in the bacterial community (Xu et (Ye et al. 2021). Therefore, we strongly believe that al. 2019). A significant proportion of Proteobacte- seasonal changes such as fertilisation would affect ria and Actinobacteria can enhance important nutri- root bacterial diversity in tomato cultivation. Sea- ents cycles, resulting in sustainable utilization and increased soil fertility. Proteobacteria have evolved son and fertilisation can have a synergistic impact Figure 5. Functional protein annotation of CMR and OR samples against eggnog database. 151 Agriculture (Poľnohospodárstvo), 67, 2021 (3): 144 −154 to survive in habitats that are rich in resources (Haas ure 2) suggesting that the soil in tomato cultivation & Défago 2005; Zhen et al. 2014). Actinobacteria without chicken manure was good for the growth were formerly assumed to prefer oligotrophic en- of Bacteroidetes. They may actively participate in vironments which resource-limited. According to a series of soil activities and make important con- previous research, the proportion of Proteobacteria tributions to soil nutrient conversion and material to Actinobacteria can represent soil nutrition status: recycling. Flavobacterium belonged to the Bacte- a higher ratio indicates that more organic matter is roidetes, and its relative abundance was higher in being added to the soil (Zhang et al. 2020a; Ye et al. soils without manure (Figure 1). It was reported to 2021). A previous study identified that a relatively be capable of degrading macromolecular organic higher abundance of Proteobacteria significantly matter such as lipid and protein, as well as poten- correlated with the organic substitution ratio in to- tial denitrification and nitrification ability (Zhang et mato cultivation, but the abundance of Actinobacte- al. 2017). The bacteria in the genus Pseudomonas ria was negatively correlated with this ratio (Schreit- were aerobic denitrifiers and could participate in er et al. 2018). Although, there is no evidence about the action of aerobic denitrification which was the the features of Proteobacteria and Actinobacteria in main process of natural nitrogen cycling (Zhang et tomato cultivation with chicken manure. In the cur- al. 2017). A relatively high abundance of Pseudo- rent research, the relative abundance of Proteobac- monas in soils with manure application suggested teria increased significantly in soils with chicken that organic fertilisation can encourage bacteria to manure application, suggesting that soils containing participate in the nitrogen cycle and enhanced the chicken manure may provide a suitable environment biochemical cycle in tomato cultivation. for soil bacteria that is intermediate between oligo- trophic and copiotrophic conditions. Bacteroidetes and Firmicutes are the two main phyla of bacteria CONCLUSIONS present in soil and manure (Guron et al. 2019). Fir- micutes are known to have a wide range of functions We concluded that as a biofertiliser, chicken ma- and to aid in the degradation of organic materials nure application significantly improved the abun- (Smit et al. 2001; Torsvik & Øvreås 2002). They dance of the beneficial microbes and the effects on were comparatively inactive opponents under high community structure and bacterial diversity of the nutrient environments compared to other microbes, endosphere in tomato cultivation. In addition, our occupying a lower role than Proteobacteria or Ac- study provides a principled approach to the rational tinobacteria. However, they had a competitive ad- use of chicken manure for tomato cultivation, which vantage over other bacteria due to their slow growth is crucial to reducing the consumption of chemical and ability to decompose complex substrates (Ji et fertilizers. al. 2018). The relative abundance of Firmicutes in Acknowledgments. This work was support- chicken manure-treated soils was significantly high- ed by grants from the Department of Science er than in the control soils, suggesting that manure and Technology of Fujian Province, China (Nos. application produced a nutrient-rich condition that 2020J01173; 2017J01625) and the National Natural favoured Firmicutes. These findings were consistent Science Foundation of China (No. Nos. 31670125; with previous research (Kamau et al. 2008). Bacte- 31660544). roidetes are the most common microbes in manure (Zhong et al. 2018). The prior analysis revealed that Conflict of interest. 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Journal

Agriculturede Gruyter

Published: Oct 1, 2021

Keywords: manure addition; endosphere microbiome; 16S rRNA gene amplicon sequencing; tomato

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