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Nodule microbiome from cowpea and lima bean grown in composted tannery sludge-treated soil
Applied Soil Ecology 151 (2020) 103542
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Nodule microbiome from cowpea and lima bean grown in composted tannery sludge-treated soil
T
Sandra Mara Barbosa Rochaa, Lucas William Mendesb, Louise Melo de Souza Oliveiraa, Vania Maria Maciel Meloc, Jadson Emanuel Lopes Antunesa, Fábio Fernando Araujod, ⁎ Mariangela Hungriae, Ademir Sergio Ferreira Araujoa, a
Soil Quality Lab., Agricultural Science Center, Federal University of Piauí, Teresina, PI, Brazil Center for Nuclear Energy in Agriculture, University of Sao Paulo, Piracicaba, SP, Brazil c Laboratório de Ecologia Microbiana e Biotecnologia, Federal University of Ceara, Fortaleza, CE, Brazil d Universidade do Oeste Paulista, Presidente Prudente, SP, Brazil e Embrapa Soja, Cx. Postal 231, Londrina, PR, Brazil b
A R T I C LE I N FO
A B S T R A C T
Keywords: BNF Legumes Nodulation Rhizobia
Root nodules can present a diverse bacterial community that contributes to plant growth. However, this bacterial community may vary among different plant species and their response to soil contamination, such as the application of composted tannery sludge (CTS) and Cr contamination. In this study, we assessed the bacterial community in nodules of cowpea and lima bean grown in soils with low and high rates of Cr-rich CTS. Bulk soil samples and nodules from cowpea and lima bean were collected for assessing the bacterial community by highthroughput sequencing. The bacterial diversity and the proportion of specialist bacterial species in the nodules of cowpea were higher than the lima bean. However, the bacterial diversity of the nodule was not influenced by CTS and Cr rate. The microbiome of the nodules was dominated by Proteobacteria (97.6%), followed by Actinobacteria (1.1%), and Firmicutes (0.4%). The dominant bacterial group in the nodules was Bradyrhizobium, accounting for > 90% of the sequences. The functional prediction showed 26 groups, with the core functions represented by chemoheterotrophy (32.3%), followed by aerobic chemoheterotrophy (32%), nitrification (12.7%), and ammonia oxidation (11.2%). This study revealed specific differences in the nodule microbiome between the two plants species and, although suggested that the nodule microbiome was not affected by the CTS application, the functional prediction data showed that the treatments with Cr-rich CTS increased the abundance of sequences affiliated to aerobic ammonia oxidation and nitrification in the nodules.
1. Introduction Biological N fixation (BNF) is one of the most important biological processes influencing the environment and agriculture, being responsible for 60% of the fixed N on Earth, while N fertilizers account for about 25% (Olivares et al., 2013). BNF allows plants, mainly legumes species, to supply their needs in N by the symbiosis with diazotrophic bacteria. These bacteria can be found in nodules that are produced by the plants in response to bacterial infection. When an effective symbiosis is established, there are typically increases in plant health, yield and resilience under stress (Dashti et al., 1998). Rhizobia, as common term, comprise the majority bacterial groups found in legume nodules, belonging to α-rhizobia (for example, Rhizobium and Bradyrhizobium) and β-rhizobia (for example, Cupriavidus and Burkholderia)
⁎
(Gyaneshwar et al., 2011). Although there are several bacterial groups, the process of nodulation is usually specific where there is a close recognition between the bacteria and the host plant. The majority of the studies performed so far on microsymbionts of nodules have focused on rhizobia and the host specificity; however, little is known about the microbial community variability inside the nodules. Martínez-Hidalgo and Hirsch (2017) have proposed that the rhizobia do not live alone in nodules and that there would be a microbiome, i.e. a diverse microbial community inhabiting the nodule. Recently, culture-dependent sampling and microbiome sequencing have also described the occurrence of other bacterial taxa inhabiting the nodules (Xiao et al., 2017; Sharaf et al., 2019). These other bacterial groups could act in facilitating plant growth and health. For example, the nodule microbiome of soybean is not only dominated by
Corresponding author at: Agricultural Science Center, Federal University of Piauí, Teresina, PI, Brazil. E-mail address: [email protected] (A.S.F. Araujo).
https://doi.org/10.1016/j.apsoil.2020.103542 Received 2 October 2019; Received in revised form 16 January 2020; Accepted 2 February 2020 0929-1393/ © 2020 Elsevier B.V. All rights reserved.
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Bradyrhizobium, but there is a high variability of other bacteria, including groups of Pseudomonaceae and Enterobacteriaceae (Sharaf et al., 2019). In this sense, new studies must be conducted to disentangle the composition of the nodules microbiome, trying to reveal how these microbial communities vary among different plant species, their functional roles, and how they respond to abiotic stresses. Soil abiotic stresses, such as those caused by the application of metals-rich industrial wastes, can affect nodule communities and their functions on nitrogen fixation and plant growth. Specifically, the longterm application of composted tannery sludge (CTS) increases soil pH, salinity and chromium (Cr) accumulation (Araújo et al., 2018). This condition, mainly the high accumulation of Cr in soil, promotes negative changes in soil microbial biomass and activity and bacterial community (Sousa et al., 2017; Miranda et al., 2018). Previous studies have shown that the process of nodulation and BNF in cowpea (Vigna unguiculata L.) and lima bean (Phaseolus lunatus L.) were negatively affected by the increase in Cr accumulation in the soil after the application of CTS (Miranda et al., 2014; Rocha et al., 2019). Although the nodulation decreases in soil contaminated with Cr (Miranda et al., 2014; Rocha et al., 2019), the impact on nodule microbiome of cowpea and lima bean are not known. Also, there is a lack of knowledge about the natural bacterial community found in the nodule of these two different plant species. Describing and understanding the variability of the nodule microbiome is important to deciphering mechanisms of the symbiosis that lead to an increase in plant production under stress conditions. In this work, we hypothesize that (1) the nodule microbiomes are different in cowpea and lima bean, and (2) the CTS application and Cr accumulation change nodule microbiome in both species. Thus, the aim of this study was to assess bacterial community in nodules of cowpea (Vigna unguiculata) and lima bean (Phaseolus lunatus) grown in soil with low and high rates of CTS and Cr concentration.
Table 2 Chemical properties of the soil after permanent applications of composted tannery sludge (CTS). CTS Mg ha−1 0 2.5 20
Soil samples were collected at 0–20 cm depth from an experimental field with the application of composted tannery sludge (CTS) during eight years. The experimental field is located at the Agricultural Science Centre, Teresina, Piauí (05°05 S; 42°48′ W, 75 m). The regional climate is dry tropical (Köppen) and is characterised by two distinct seasons: rainy summer and dry winter, with annual average temperatures of 30 °C and rainfall of 1200 mm. The rainy season extends from January to April, when 90% of the total annual rainfall occurs. The soil is classified as a Fluvisol (IUSS Working Group WRB, 2007) with the following composition at 0–20-cm depth: 100 g kg−1 clay, 280 g kg−1 silt, and 620 g kg−1 sand. The tannery sludge, a solid waste produced by tannery industries, was composted by mixing with sugarcane straw and cattle manure for 90 days before application in the soil. The chemical characteristics of CTS are shown in Table 1. The experiment started in 2009 by application of CTS at five rates: 0 (control), 2.5, 5, 10, and 20 Mg ha−1. The experiment had a completely block design with four replicates. The CTS was applied in experimental plots (20 m2 each one) along eight years (one application per year from 2009 to 2017). Annually, CTS was amended on the soil surface and
% 68
g kg 7.5
201
N
P
K
Ca
Mg
Na
−1
4.9
2.9
121
7.2
49.1
Ca
g kg−1
mg kg−1
mmolc kg−1
5.5 7.0 8.5
6.0 6.8 12.5
2.2 2.4 2.1
13.3 20.0 27.3
Mg
6.5 6.8 7.8
Na
4.6 5.0 5.6
EC
Cr
dS m−1
mg kg−1
0.71 0.83 0.94
4.4 29.3 150.1
Total DNA from the nodules (0.5 g) and soil (0.5 g) were extracted using the PowerLyzer PowerSoil DNA Isolation Kit (MoBIO Laboratories, Carlsbad, CA, USA), according to the manufacturer's instructions. The DNA extraction was performed in triplicate for each sample. The quality and concentration of the extracted DNA were determined using NanoDrop 2000 spectrophotometer (Thermo Scientific, Waltham, USA). The V4 region of the 16S rRNA gene was amplified with regionspecific primers (515F/806R) (Caporaso et al., 2011). Each 25 μL PCR reaction contained the following: 12.25 μL of nuclease-free water (Certified Nuclease-free, Promega, Madison, WI, USA), 5.0 μL of buffer solution 5× (MgCl2 2 Mm), 0,75 μL of solution of dNTP's (10 mM), 0,75 μL of each primer (515 YF 40 μM e 806 R 10 μM), 1.0 unit of Platinum Taq polymerase High Fidelity in concentration of 0,5 μL (Invitrogen, Carlsbad, CA, USA), and 2.0 μL of template DNA. Moreover, a control reaction was performed by adding water instead of DNA. The conditions for the PCR reaction were as follows: 95 °C for 3 min, 35 cycles at 98 °C for 20 s, 55 °C for 20 s, and 72 °C for 30 s, and a final extension of 3 min at 72 °C. After indexing, the PCR products were cleaned up using Agencourt AMPure XP – PCR purification beads (Beckman Coulter, Brea, CA, USA), according to the manufacturer's manual, and quantified using the dsDNA BR assay kit (Invitrogen, Carlsbad, CA, USA) on a Qubit 2.0
Cr mg kg−1
15
K
2.2. Bulk soil and nodules DNA extraction, PCR and sequencing
Table 1 Composition of composted tannery sludge. TOC
P
incorporated into the 20-cm layer. More detailed information about this experiment with CTS can be found in Miranda et al. (2018). In this study, we used soil samples from the following treatments with CTS: 0 Mg ha−1 (control without CTS); 2.5 Mg ha−1 (lowest CTS rates) and 20 Mg ha−1 (highest CTS rates). Soil samples were taken, sieved, and sent to the laboratory for chemical analyses. The main chemical properties of the soil are shown in Table 2. To assess the nodules microbiomes, two legume species were used: cowpea (Vigna unguiculata) and lima bean (Phaseolus lunatus). These plant species were chosen because they are the most important legume plants cultivated in Piauí state and there is no information about their nodule microbiome until now. The surface-sterilized seeds of these plants were sown in polyvinyl chloride (PVC) pots (diameter 180 mm, length 160 mm) containing 5 kg of soil from each treatment and the experiment was laid out in a completely randomized design with three replicates. Five days after germination, plants were thinned, leaving one individual plant per pot. Pots were irrigated daily with sterilized water to maintain soil moisture at 80% of field capacity. Nodules from cowpea and lima bean were collected at 35 and 45 days, respectively, which corresponded to the flowering period of each plant species. Plants were excised at the cotyledonal node to separate shoots from roots. Roots and adhering soil of each plant were placed onto a 1 mm mesh sieve and washed thoroughly with a gentle stream of tap water to remove the soil. Ten nodules per plant were randomly selected for DNA extraction. Intact and undamaged nodules were immersed in 70% (v/v) ethanol (C2H6O) for 1 min and then left to soak in 6% (v/v) sodium hypochlorite (NaOCl) for 3 min before being carefully rinsed off six times in sterile water.
2.1. Soil sampling and rhizobia isolation
pH
6.5 6.7 7.8
TOC
pH - CaCl2; TOC - Total organic carbon.
2. Materials and methods
Moisture
pH
1943
pH - CaCl2; TOC - Total organic carbon. 2
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fluorometer (Invitrogen, Carlsbad, CA, USA). Once quantified, equimolar concentrations of each library were pooled into a single tube. After quantification, the molarity of the pool was determined and diluted to 2 nM, denatured, and then diluted to a final concentration of 8.0 pM with a 20% PhiX (Illumina, San Diego, CA, USA) spike for loading into the Illumina MiSeq sequencing machine (Illumina, San Diego, CA, USA).
not clear. To further investigate the community's dynamics, we tested the niche occupancy in bulk soil and nodules, and the proportion of specialists in the nodules of cowpea was higher than in those of lima bean (Fig. 2). 3.2. Bacterial community composition The 16S rRNA gene sequencing generated approximately 2,100,000 sequences. After quality trimming and rarefaction, the sequences were clustered into approximately 4000 OTUs. The microbiome composition was very distinct between bulk soil and the nodules. In the bulk soil, the most abundant bacterial groups were Unclassified Bacteria (24%), followed by Proteobacteria (20%), Actinobacteria (18%), and Firmicutes (14%). The microbiome of the nodules was dominated by Proteobacteria (97.6%), followed by Actinobacteria (1.1%), and Firmicutes (0.4%). Further analysis on a deeper taxonomical level showed that bulk soil presented a high proportion of the family Bacillales, Acidobacteria group 6, Bacillus, and unclassified member of Actinobacteria (Fig. 3A). As expected, the dominant bacterial group in the nodules was Bradyrhizobium (Alphaproteobacteria class), representing > 90% of the sequences in the nodules, in contrast with < 0.5% in bulk soil. Also, we observed that the lima bean presented a higher abundance of Bradyrhizobium than cowpea (Fig. 3B). On the other hand, cowpea nodules presented a higher abundance of other specific groups such as Microbacterium (average proportion = 0.10%, P = 0.024), Chitinophagales (0.06%, P = 0.28), Rhizobiaceae (0.06%, P = 0.037), and Acetobacteraceae (0.039%, P = 0.04) compared to lima bean (Fig. 3C). At a deeper taxonomic level we observed that CTS application in bulk soil affected 35 OTUs, while in the nodules of cowpea and lima bean affected four and three OTUs, respectively (Supplementary Table 1).
2.3. Data processing Sequence data were processed using QIIME 2 version 2017.11. First, the sequences were demultiplexed and quality control was carried out using DADA2 (Callahan et al., 2016), using the consensus method to remove any remaining chimeric and low-quality sequences. Afterward, samples were rarefied to 21,000 sequences, following the number of the lowest sample, and singletons, doubletons, chloroplast, and mitochondria sequences were removed from further analysis. The taxonomic affiliation was performed at 97% similarity using the Silva database v. 132 (Quast et al., 2013), and the generated matrix was further used for statistical analyses. The sequences are deposited in the NCBI database under the accession number ‘PRJNA575533’. 2.4. Data analysis To compare the bacterial community structure between bulk soil and the nodules of both plant species we used Principal Component Analysis (PCA) with Canoco 4.5 (Biometrics, Wageningen, The Netherlands). For this, the data were first evaluated using Detrended Correspondence Analysis (DCA), which indicated linearly distributed data (length of gradient < 3), revealing that the best-fit mathematical model for the data was PCA. Shannon diversity index was calculated based on the OTU table using the software Past v.3 (Hammer et al., 2001) and compared using Tukey's HSD test. To understand the community dynamics we tested the niche occupancy by classifying the OTUs into specialists and generalists. The niche occupancy was verified by the multinomial species classification method ‘clamtest’ available in ‘vegan’ package in R. This method compares the microbial abundance between two habitats and classifies the group of species that are similarly distributed across both habitats as generalists, and classifies as specialists the species more abundant in one habitat compared to the other (Chazdon et al., 2011). For CLAM analysis, it was considered a significant level for individual test of alpha 0.005 and a specialization threshold of 0.66. In order to compare the differential abundance of bacteria between bulk soil and the nodules of the two plant species, the OTU table was used as input in the software STAMP (Parks and Beiko, 2010). P-values were calculated based on two-sided Welch's t-test and correction using Benjamini-Hochberg FDR. To further predict the relevant potential functions related to the N-cycle, we performed a functional annotation using FAPROTAX database (Louca et al., 2016). For this, a table of frequency of taxa at the genus level was used as input and converted into a putative functional table and we selected only the functions related to the N-cycle.
3.3. Functional prediction of microbial community related to N-cycle
3. Results
We then used the FAPROTAX database to predict the potential functions of the bulk soil and nodules microbial communities with a focus on the N-cycle. The general functional profile clustered the samples according to the niche, i.e. bulk soil and nodules (Fig. 4A). However, there were no differences in functional diversity between bulk soil and nodules (Fig. 4B). The sequences were classified into 26 functional groups, with the core functions represented by chemoheterotrophy (32.3%), followed by aerobic chemoheterotrophy (32%), nitrification (12.7%), and ammonia oxidation (11.2%) (Fig. 5). The functional profile revealed some shifts in the functionality of the bacterial community from bulk soil to nodules. In general, bulk soil presented a higher abundance of sequences associated with nitrification, and ammonia and nitrite oxidation, while the nodules presented a higher abundance of chemoheterotrophy and symbionts (Figs. 5 and 6). Regarding the functions related to N-cycle, bulk soil presented a higher abundance of sequences related to aerobic ammonia oxidation, aerobic nitrite oxidation, and nitrification (P < 0.05), while nitrate reduction, nitrate respiration, and nitrogen respiration were detected at similar levels in all samples (Fig. 6). Interestingly, in the treatments with CTS and Cr contamination, there was an increase in the aerobic ammonia oxidation and nitrification in the nodules (Fig. 5; P < 0.05).
3.1. Bacterial community structure and diversity
4. Discussion
The principal component analysis (PCA) explained 98% of the variation in the first two axes of the plot, clustering the samples according to the niche, i.e. bulk soil and nodules of cowpea and lima bean (Fig. 1A; PERMANOVA F = 159.8, P = 0.0001). The Shannon index showed a decrease in bacterial diversity from bulk soil to nodules of cowpea and lima bean (Fig. 1B, P < 0.05), but the bacterial diversity in the nodules of cowpea was higher than the nodules of lima bean (P < 0.05). Therefore, the effect of CTS and Cr on nodule diversity was
In this study, we used a high-throughput sequencing method to assess the nodule microbiome of two important legume species for tropical regions that were exposed to low and high rates of CTS and Cr concentration. In bulk soil, unclassified bacteria were the dominant group. In addition, Proteobacteria, Actinobacteria, and Firmicutes were the most abundant identified phyla in both bulk soil and nodules showing a relationship between soils and nodule microbiome, at least in a phylum level. Nevertheless, the abundance of Actinobacteria and 3
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Fig. 1. (A) Principal component analysis (PCA) based on bacterial 16S rRNA from bulk soil and nodules of cowpea and lima bean grown in soils treated with composted tannery sludge. The dashed lines in the graph indicate significant clusters based on PERMANOVA (P < 0.05). (B) Diversity measurement based on bacterial 16S rRNA at OTU level at 97% similarity. Lower case letters refer to significant differences between the treatments, based on Tukey's test (P < 0.05). T1 = 0 Mg ha−1; T2 = 2.5 Mg ha−1; T3 = 20 Mg ha−1.
Fig. 2. Multinomial species classification method (CLAM) for the niche occupancy test. The pairwise treatments evaluated were as follow: (A) bulk and cowpea nodules, (B) bulk and lima bean nodules, and (C) cowpea and lima bean nodules. The proportion of generalists, specialists, and rare is displayed in the graphs.
As hypothesized, the nodule microbiome of cowpea showed differences in microbial groups in comparison to lima bean, although Bradyrhizobium was the most abundant genus in both species. Several studies have reported the genus Bradyrhizobium as the main symbiont of cowpea (Leite et al., 2017; Marinho et al., 2017; Ndungu et al., 2018) and lima bean (Ormeño-Orrillo et al., 2017). However, the idea that the nodules host a diverse microbiome began to be cleared with recent studies in cowpea (Leite et al., 2017) and soybean (Sharaf et al., 2019). Interestingly, lima bean nodules hosted Bradyrhizobium more abundantly than cowpea. In addition, the analysis of 16S rRNA and gyrB gene sequencing identified co-habitant bacteria in nodules of lima bean (Silva, 2017). The bacterial groups Microbacterium, Chitinophagales, Rhizobiaceae, and Acetobacteraceae were significantly more abundant in the cowpea nodules, suggesting that this plant species selects distinct co-habitant bacteria. Interestingly, we observed a different microbiome composition at a deeper taxonomical level, revealing that plant genotype exerts an effect on the assembly of the nodule microbiome, a result similar to Leite et al. (2017) who assessed the effect of soil type and plant genotype on nodule microbiome of cowpea. On the contrary to our second hypothesis, the application of CTS
Firmicutes was higher in bulk soil than nodules, where Proteobacteria prevailed. A previous study in cowpea also found Proteobacteria as the most abundant group (Leite et al., 2017) altogether indicating that it is possibly representing rhizobia. Comparing the bacterial diversity in bulk soil and nodules, the results showed a decrease in the bacterial diversity in nodules and it is in agreement with previous studies that reported lower bacterial diversity in nodules compared to rhizosphere or bulk soils (Lu et al., 2017; Sharaf et al., 2019). Indeed, soil microbiome is composed of a complex bacterial community (native or introduced) that is driven by the soil environment (Nandasena et al., 2007), while in the nodules, plants regulate the composition of bacterial community through the capabilities of bacteria to establish and interact with the plants (Peix et al., 2015). Interestingly, our functional prediction showed a high abundance of sequences affiliated to symbionts in the nodules, which may explain the selection of a specific microbiome in this environment. The results also showed that nodules of cowpea presented higher bacterial diversity than nodules of lima bean. This observation was confirmed by the niche occupancy analysis, which found more specialists in the nodules of cowpea than lima bean, indicating a stronger selective effect of lima bean on the bacterial assembly in the nodules. 4
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Fig. 3. (A) Distribution of the most abundant bacterial phyla (based on 16S rRNA gene) in bulk soil and nodules of cowpea and lima bean grown in soils treated with composted tannery sludge. (B) Proportion of sequences affiliated to the genus Bradyrhizobium. Lower case letters refer to significant differences between the treatments, based on Tukey's test (P < 0.05). (C) Scatter-plot showing the differential abundance of bacterial groups in the nodules of cowpea and lima bean. Pvalues were calculated based on two-sided Welch's t-test corrected using Benjamini-Hochberg FDR.
ecosystems. The functional prediction based on the DNA amplicon approach from the bacterial community can provide some correlations between taxonomy and function, and these tools have been used in recent studies (Hariharan et al., 2017; Dube et al., 2019). In our study, the dominant functional group was chemoheterotrophy, indicating that the most proportion of the microbes, in both bulk soil and nodules, cannot fix carbon and have to obtain carbon and energy from the oxidation of organic compounds (Zhang et al., 2018). In the bulk soil samples, the proportion of chemoheterotrophic bacteria can be explained by the high rate of organic matter, while in the nodules, the sugars are one of the carbon sources incorporated in large amounts inside the nodule cells (Hernández et al., 2013). Considering that BNF is the most important ecological service in the nodules, we explored soil bacterial functional groups related to N-cycle. The prediction of functions related to N-cycle is important since the bacterial community plays essential roles in ammonification, nitrogen fixation, denitrification, and nitrification (Yoon et al., 2015). Our analysis revealed that the general functional profile was distinct between bulk soil and the nodules, with specific functions presenting differential abundance
and the Cr concentration in the soil did not influence the bacterial communities, especially in nodules. Although our PCA analysis did not separate the bulk soil samples according to the CTS treatment, we found that 35 OTUs were affected by CTS in the bulk soil. This observation is supported by a previous study that has reported that less abundant bacterial groups in bulk soil were affected by the CTS application (Miranda et al., 2018). In the nodules, the application of CTS and Cr concentration did not influence the bacterial diversity, where only four and three OTUs were affected in cowpea and lima bean, respectively. However, previous studies have shown that CTS application and the accumulation of Cr affected nodulation and BNF in cowpea (Miranda et al., 2014) and lima bean (Rocha et al., 2019). Thus, although the nodulation decreases, the plant, through root nodules, could protect the bacteria against environmental stress and, therefore, avoiding that the bacterial community inside nodules being affected. Monitoring the soil bacterial community function in the soil and nodules can improve our knowledge about the response of the nodule community to the CTS application and Cr concentration, providing a theoretical basis for the restoration of degraded and/or contaminated
Fig. 4. (A) Principal component analysis (PCA) based on the functional profile of the microbial communities from bulk soil and nodules of cowpea and lima bean grown in soils treated with composted tannery sludge. The functional prediction was based on 16S rRNA genes using FAPROTAX database. The dashed lines in the graph indicate significant clusters based on PERMANOVA (P < 0.05). (B) Diversity measurement based on predicted functional profile. T1 = 0 Mg ha−1; T2 = 2.5 Mg ha−1; T3 = 20 Mg ha−1.
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Lima Bean
S.M.B. Rocha, et al.
FT3 FT2 FT1 CT3 CT2 CT1
Bulk
BT3 BT2 BT1 0%
10%
20%
30%
40%
50%
Proporons of Sequences
60%
70%
aerobic ammonia oxidaon
cyanobacteria
methanol oxidaon
oxygenic photoautotrophy
sulfate respiraon
aerobic chemoheterotrophy
dark hydrogen oxidaon
methylotrophy
photoautotrophy
ureolysis
aerobic nitrite oxidaon
fermentaon
nitrate reducon
phototrophy
animal parasites or symbionts
human pathogens all
nitrate respiraon
predatory or exoparasic
aromac compound degradaon
intracellular parasites
nitrificaon
reducve acetogenesis
chemoheterotrophy
manganese oxidaon
nitrogen respiraon
respiraon of sulfur compounds
80%
90%
100%
Fig. 5. Distribution of the functional categories from the bacterial communities of bulk soil and nodules of cowpea and lima bean grown in soils treated with composted tannery sludge. The functional prediction was based on 16S rRNA genes using FAPROTAX database.
step in nitrification and is carried out by distinct groups of microorganisms. Interestingly, our data showed that the treatments with CTS and Cr contamination increased the abundance of sequences affiliated to aerobic ammonia oxidation and nitrification in the nodules. This pattern could be related to the disturbed condition of the soil, since several studies have shown an increased abundance of nitrification genes in disturbed soils (Merloti et al., 2019; Pedrinho et al., 2019). Also, using the functional prediction approach we did not detect sequences affiliated to nitrogen fixation, which can be a limitation of the method. However, our results highlight the fact that important functions, other than BNF, are probably occurring in the nodule environment, making the nitrogen available to the plant in different forms. For
between them. In the bulk soil, functions related to nitrogen transformations in soil were highlighted, such as nitrification, and ammonia and nitrite oxidation, which it is expected since the bulk soil samples presented high organic matter and N content together with higher microbial diversity. On the other hand, the nodule microbiome presented a high abundance of functions related to ‘animal symbionts’, which could be explained by the high abundance of Bradyrhizobium (> 90% of the sequences). Among the N-related functions, the most abundant in both niches were nitrification and ammonia oxidation. Nitrification is mediated by the nitrifying microorganisms and is responsible for regulating the availability of N in soils (Merloti et al., 2019) and probably in the nodules. The ammonia oxidation is the first
Fig. 6. Proportion of sequences related to nitrogen cycle from the bacterial communities of bulk soil and nodules of cowpea and lima bean grown in soils treated with composted tannery sludge. The functional prediction was based on 16S rRNA genes using FAPROTAX database. 6
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example, we found sequences related to nitrification in the nodules although in lower abundance compared to the bulk soil. As previous mentioned, the nitrification is a process that converts ammonium to nitrate. We also found sequences related to aerobic ammonia oxidation in the nodules, which is the first step in the nitrification process, performed by aerobic ammonia-oxidizing bacteria and archaea (Mohamed et al., 2010). Despite the amount of nitrogen available, a factor that can affect plant growth is the nitrate:ammonium ratio (Barker and Mills, 1980), because nitrate is more mobile than ammonium. Then, the nitrification of ammonium to nitrate occurring inside the nodules makes nitrogen more available for plant uptake.
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Lu, J., Yang, F., Wang, S., Ma, H., Liang, J., Chen, Y., 2017. Co-existence of rhizobia and diverse non-rhizobial bacteria in the rhizosphere and nodules of Dalbergia odorifera seedlings inoculated with Bradyrhizobium elkanii, Rhizobium multihospitium-like and Burkholderia pyrrocinia-like strains. Front. Microbiol. 8, 2255. Marinho, R.C.N., Ferreira, L.V.M., Silva, A.F., Martins, L.M.V., Nóbrega, R.S.A., FernandesJúnior, P.I., 2017. Symbiotic and agronomic efficiency of new cowpea rhizobia from Brazilian Semi-Arid. Bragantia 76, 273–281. Martínez-Hidalgo, P., Hirsch, A.M., 2017. The nodule microbiome: N2-fixing rhizobia do not live alone. Phytobiomes Journal 1, 70–82. Merloti, L.F., Mendes, L.W., Pedrinho, A., Souza, L.F., Ferrari, M.B., Tsai, S.M., 2019. Forest-toagriculture conversion in Amazon drives soil microbial communities and N-cycle. Soil Biol. Biochem. 137, 107567. Miranda, A.R.L., Araújo, A.S.F., Nunes, L.A.P.L., Oliveira, M.L.J., Melo, W.J., 2014. 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Forest-to-pasture conversion and recovery based on assessment of microbial communities in Eastern Amazon Rainforest. FEMS Microbiol. Ecol. 95, 236. Peix, A., Ramírez-Bahena, M.H., Velázquez, E., Bedmar, E.J., 2015. Bacterial associations with legumes. Critical Review in Plant Science 34, 17–42. Quast, C., Pruesse, E., Yilmaz, P., Gerken, J., Schweer, T., Yarza, P., Peplies, J., Glöckner, F.O., 2013. The SILVA ribosomal RNA gene database project: improved data processing and web-based tools. Nucleic Acids Res. 4, 590–596. Rocha, S.M.B., Antunes, J.E.L., Oliveira, L.M.S., Aquino, J.P.A., Figueiredo, M.V.B., Melo, W.J., Araújo, A.S.F., 2019. Nodulation, nitrogen uptake and growth of lima bean in a composted tannery sludge-treated soil. Ciencia Rural 49, e20190301. Sharaf, H., Rodrigues, R.R., Moon, J., Zhang, B., Mills, K., Williams, M.A., 2019. Unprecedented bacterial community richness in soybean nodules vary with cultivar and water status. Microbiome 7, 63. Silva, C.P., 2017. Polyphasic characterization of nitrogen-fixing and co-resident bacteria in nodules of Phaseolus lunatus inoculated with soils from Piauí State, northeast Brazil. Brazilian Meeting of Soil Science 1, 1 Belem:SBCS. Sousa, R.S., Santos, V.M., Melo, W.J., Nunes, L.A.P.L., van den Brink, P.J., Araújo, A.S.F., 2017. Time-dependent effect of composted tannery sludge on the chemical and microbial properties of soil. Ecotoxicology 26, 1366–1377. Xiao, X., Chen, W., Zong, L., Yang, J., Jiao, S., Lin, Y., et al., 2017. Two cultivated legume plants reveal the enrichment process of the microbiome in the rhizocompartments. Mol. Ecol. 26, 1641–1651. Yoon, S., Cruz-García, C., Sanford, R., Ritalahti, K.M., Löffler, F.E., 2015. Denitrification versus respiratory ammonification: environmental controls of two competing dissimilatory NO3(−)/NO2(−) reduction pathways in Shewanella loihica strain PV-4. ISME J. 9, 1093–1104. Zhang, X., Hu, B.X., Ren, H., Zhang, J., 2018. Composition and functional diversity of microbial community across a mangrove-inhabited mudflat as revealed by 16S rDNA gene sequences. Sci. Total Environ. 633, 518–528.
5. Conclusion In summary, our study provides new information on the diverse nodule microbiome of two leguminous species cultivated in soils treated or not with composted tannery sludge. Our data revealed that the nodule microbiome was dominated by rhizobia but also by a diverse bacterial community inhabiting this niche. We showed specific differences in the nodule microbiome between the two plant species. Regarding the functional profile, we showed that important functions related to the nitrogen cycle are present in the nodules. Overall, these results reveal a previously undescribed non-rhizobia community in the nodules that could play important role. Considering that the nodule microbiome was not affected by the CTS application, we suggest that this community is helping the plant to thrive under stress condition. Further studies should explore the beneficial potential of nodule microbiome to improve plant growth and health. Supplementary data to this article can be found online at https:// doi.org/10.1016/j.apsoil.2020.103542. Acknowledgments This study was funded by Conselho Nacional de Desenvolvimento Científico e Tecnológico – CNPq (grant 305069/2018-1) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES. The authors thank to the Centro de Genetica e Bioinformatica (CeGenBio) from the Unit of Research (NPDM/UFC). Sandra Mara Barbosa Rocha thanks Fundacao de Amparo a Pesquisa do Estado do Piaui (FAPEPI) for her scholarship. Jadson Emanuel Lopes Antunes thanks CAPES for his fellowship. Ademir Sergio Ferreira Araujo, Vania Maria Maciel Melo, and Mariangela Hungria thank CNPq for their fellowship of research. Ethical approval This article does not contain any studies with human participants or animals performed by any of the authors. Declaration of competing interest The authors declare that they have no conflict of interest. References Araújo, A.S.F., Santos, V.M., Miranda, A.R.L., Nunes, L.A.P.L., Dias, C.T.S., Melo, W.J., 2018. Chemical variables influencing microbial properties in composted tannery sludge-treated soil. Int. J. Environ. Sci. Technol. 15, 1793–1800. Barker, A.V., Mills, H.A., 1980. Ammonium and nitrate nutrition of horticultural crops. Horticultural Review 2, 395–423. Callahan, B.J., McMurdie, P.J., Rosen, M.J., Han, A.W., Johnson, A.J.A., Holmes, S.P., 2016. DADA2: high-resolution sample inference from Illumina amplicon data. Nat. Methods 13, 581–583. Caporaso, J.G., et al., 2011. Global patterns of 16S rRNA diversity at a depth of millions of sequences per sample. Proceedings of National Academy of Science 108, 4516–4522. Chazdon, R.L., Chao, A., Colwell, R.K., Lin, S.-Y., Norden, N., Letcher, S.G., et al., 2011. A novel statistical method for classifying habitat generalists and specialists. Ecology 92, 1332–1343. Dashti, N., Zhang, F., Hynes, R., Smith, D.L., 1998. Plant growth promoting rhizobacteria
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Applied Soil Ecology journal homepage: www.elsevier.com/locate/apsoil
Nodule microbiome from cowpea and lima bean grown in composted tannery sludge-treated soil
T
Sandra Mara Barbosa Rochaa, Lucas William Mendesb, Louise Melo de Souza Oliveiraa, Vania Maria Maciel Meloc, Jadson Emanuel Lopes Antunesa, Fábio Fernando Araujod, ⁎ Mariangela Hungriae, Ademir Sergio Ferreira Araujoa, a
Soil Quality Lab., Agricultural Science Center, Federal University of Piauí, Teresina, PI, Brazil Center for Nuclear Energy in Agriculture, University of Sao Paulo, Piracicaba, SP, Brazil c Laboratório de Ecologia Microbiana e Biotecnologia, Federal University of Ceara, Fortaleza, CE, Brazil d Universidade do Oeste Paulista, Presidente Prudente, SP, Brazil e Embrapa Soja, Cx. Postal 231, Londrina, PR, Brazil b
A R T I C LE I N FO
A B S T R A C T
Keywords: BNF Legumes Nodulation Rhizobia
Root nodules can present a diverse bacterial community that contributes to plant growth. However, this bacterial community may vary among different plant species and their response to soil contamination, such as the application of composted tannery sludge (CTS) and Cr contamination. In this study, we assessed the bacterial community in nodules of cowpea and lima bean grown in soils with low and high rates of Cr-rich CTS. Bulk soil samples and nodules from cowpea and lima bean were collected for assessing the bacterial community by highthroughput sequencing. The bacterial diversity and the proportion of specialist bacterial species in the nodules of cowpea were higher than the lima bean. However, the bacterial diversity of the nodule was not influenced by CTS and Cr rate. The microbiome of the nodules was dominated by Proteobacteria (97.6%), followed by Actinobacteria (1.1%), and Firmicutes (0.4%). The dominant bacterial group in the nodules was Bradyrhizobium, accounting for > 90% of the sequences. The functional prediction showed 26 groups, with the core functions represented by chemoheterotrophy (32.3%), followed by aerobic chemoheterotrophy (32%), nitrification (12.7%), and ammonia oxidation (11.2%). This study revealed specific differences in the nodule microbiome between the two plants species and, although suggested that the nodule microbiome was not affected by the CTS application, the functional prediction data showed that the treatments with Cr-rich CTS increased the abundance of sequences affiliated to aerobic ammonia oxidation and nitrification in the nodules.
1. Introduction Biological N fixation (BNF) is one of the most important biological processes influencing the environment and agriculture, being responsible for 60% of the fixed N on Earth, while N fertilizers account for about 25% (Olivares et al., 2013). BNF allows plants, mainly legumes species, to supply their needs in N by the symbiosis with diazotrophic bacteria. These bacteria can be found in nodules that are produced by the plants in response to bacterial infection. When an effective symbiosis is established, there are typically increases in plant health, yield and resilience under stress (Dashti et al., 1998). Rhizobia, as common term, comprise the majority bacterial groups found in legume nodules, belonging to α-rhizobia (for example, Rhizobium and Bradyrhizobium) and β-rhizobia (for example, Cupriavidus and Burkholderia)
⁎
(Gyaneshwar et al., 2011). Although there are several bacterial groups, the process of nodulation is usually specific where there is a close recognition between the bacteria and the host plant. The majority of the studies performed so far on microsymbionts of nodules have focused on rhizobia and the host specificity; however, little is known about the microbial community variability inside the nodules. Martínez-Hidalgo and Hirsch (2017) have proposed that the rhizobia do not live alone in nodules and that there would be a microbiome, i.e. a diverse microbial community inhabiting the nodule. Recently, culture-dependent sampling and microbiome sequencing have also described the occurrence of other bacterial taxa inhabiting the nodules (Xiao et al., 2017; Sharaf et al., 2019). These other bacterial groups could act in facilitating plant growth and health. For example, the nodule microbiome of soybean is not only dominated by
Corresponding author at: Agricultural Science Center, Federal University of Piauí, Teresina, PI, Brazil. E-mail address: [email protected] (A.S.F. Araujo).
https://doi.org/10.1016/j.apsoil.2020.103542 Received 2 October 2019; Received in revised form 16 January 2020; Accepted 2 February 2020 0929-1393/ © 2020 Elsevier B.V. All rights reserved.
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Bradyrhizobium, but there is a high variability of other bacteria, including groups of Pseudomonaceae and Enterobacteriaceae (Sharaf et al., 2019). In this sense, new studies must be conducted to disentangle the composition of the nodules microbiome, trying to reveal how these microbial communities vary among different plant species, their functional roles, and how they respond to abiotic stresses. Soil abiotic stresses, such as those caused by the application of metals-rich industrial wastes, can affect nodule communities and their functions on nitrogen fixation and plant growth. Specifically, the longterm application of composted tannery sludge (CTS) increases soil pH, salinity and chromium (Cr) accumulation (Araújo et al., 2018). This condition, mainly the high accumulation of Cr in soil, promotes negative changes in soil microbial biomass and activity and bacterial community (Sousa et al., 2017; Miranda et al., 2018). Previous studies have shown that the process of nodulation and BNF in cowpea (Vigna unguiculata L.) and lima bean (Phaseolus lunatus L.) were negatively affected by the increase in Cr accumulation in the soil after the application of CTS (Miranda et al., 2014; Rocha et al., 2019). Although the nodulation decreases in soil contaminated with Cr (Miranda et al., 2014; Rocha et al., 2019), the impact on nodule microbiome of cowpea and lima bean are not known. Also, there is a lack of knowledge about the natural bacterial community found in the nodule of these two different plant species. Describing and understanding the variability of the nodule microbiome is important to deciphering mechanisms of the symbiosis that lead to an increase in plant production under stress conditions. In this work, we hypothesize that (1) the nodule microbiomes are different in cowpea and lima bean, and (2) the CTS application and Cr accumulation change nodule microbiome in both species. Thus, the aim of this study was to assess bacterial community in nodules of cowpea (Vigna unguiculata) and lima bean (Phaseolus lunatus) grown in soil with low and high rates of CTS and Cr concentration.
Table 2 Chemical properties of the soil after permanent applications of composted tannery sludge (CTS). CTS Mg ha−1 0 2.5 20
Soil samples were collected at 0–20 cm depth from an experimental field with the application of composted tannery sludge (CTS) during eight years. The experimental field is located at the Agricultural Science Centre, Teresina, Piauí (05°05 S; 42°48′ W, 75 m). The regional climate is dry tropical (Köppen) and is characterised by two distinct seasons: rainy summer and dry winter, with annual average temperatures of 30 °C and rainfall of 1200 mm. The rainy season extends from January to April, when 90% of the total annual rainfall occurs. The soil is classified as a Fluvisol (IUSS Working Group WRB, 2007) with the following composition at 0–20-cm depth: 100 g kg−1 clay, 280 g kg−1 silt, and 620 g kg−1 sand. The tannery sludge, a solid waste produced by tannery industries, was composted by mixing with sugarcane straw and cattle manure for 90 days before application in the soil. The chemical characteristics of CTS are shown in Table 1. The experiment started in 2009 by application of CTS at five rates: 0 (control), 2.5, 5, 10, and 20 Mg ha−1. The experiment had a completely block design with four replicates. The CTS was applied in experimental plots (20 m2 each one) along eight years (one application per year from 2009 to 2017). Annually, CTS was amended on the soil surface and
% 68
g kg 7.5
201
N
P
K
Ca
Mg
Na
−1
4.9
2.9
121
7.2
49.1
Ca
g kg−1
mg kg−1
mmolc kg−1
5.5 7.0 8.5
6.0 6.8 12.5
2.2 2.4 2.1
13.3 20.0 27.3
Mg
6.5 6.8 7.8
Na
4.6 5.0 5.6
EC
Cr
dS m−1
mg kg−1
0.71 0.83 0.94
4.4 29.3 150.1
Total DNA from the nodules (0.5 g) and soil (0.5 g) were extracted using the PowerLyzer PowerSoil DNA Isolation Kit (MoBIO Laboratories, Carlsbad, CA, USA), according to the manufacturer's instructions. The DNA extraction was performed in triplicate for each sample. The quality and concentration of the extracted DNA were determined using NanoDrop 2000 spectrophotometer (Thermo Scientific, Waltham, USA). The V4 region of the 16S rRNA gene was amplified with regionspecific primers (515F/806R) (Caporaso et al., 2011). Each 25 μL PCR reaction contained the following: 12.25 μL of nuclease-free water (Certified Nuclease-free, Promega, Madison, WI, USA), 5.0 μL of buffer solution 5× (MgCl2 2 Mm), 0,75 μL of solution of dNTP's (10 mM), 0,75 μL of each primer (515 YF 40 μM e 806 R 10 μM), 1.0 unit of Platinum Taq polymerase High Fidelity in concentration of 0,5 μL (Invitrogen, Carlsbad, CA, USA), and 2.0 μL of template DNA. Moreover, a control reaction was performed by adding water instead of DNA. The conditions for the PCR reaction were as follows: 95 °C for 3 min, 35 cycles at 98 °C for 20 s, 55 °C for 20 s, and 72 °C for 30 s, and a final extension of 3 min at 72 °C. After indexing, the PCR products were cleaned up using Agencourt AMPure XP – PCR purification beads (Beckman Coulter, Brea, CA, USA), according to the manufacturer's manual, and quantified using the dsDNA BR assay kit (Invitrogen, Carlsbad, CA, USA) on a Qubit 2.0
Cr mg kg−1
15
K
2.2. Bulk soil and nodules DNA extraction, PCR and sequencing
Table 1 Composition of composted tannery sludge. TOC
P
incorporated into the 20-cm layer. More detailed information about this experiment with CTS can be found in Miranda et al. (2018). In this study, we used soil samples from the following treatments with CTS: 0 Mg ha−1 (control without CTS); 2.5 Mg ha−1 (lowest CTS rates) and 20 Mg ha−1 (highest CTS rates). Soil samples were taken, sieved, and sent to the laboratory for chemical analyses. The main chemical properties of the soil are shown in Table 2. To assess the nodules microbiomes, two legume species were used: cowpea (Vigna unguiculata) and lima bean (Phaseolus lunatus). These plant species were chosen because they are the most important legume plants cultivated in Piauí state and there is no information about their nodule microbiome until now. The surface-sterilized seeds of these plants were sown in polyvinyl chloride (PVC) pots (diameter 180 mm, length 160 mm) containing 5 kg of soil from each treatment and the experiment was laid out in a completely randomized design with three replicates. Five days after germination, plants were thinned, leaving one individual plant per pot. Pots were irrigated daily with sterilized water to maintain soil moisture at 80% of field capacity. Nodules from cowpea and lima bean were collected at 35 and 45 days, respectively, which corresponded to the flowering period of each plant species. Plants were excised at the cotyledonal node to separate shoots from roots. Roots and adhering soil of each plant were placed onto a 1 mm mesh sieve and washed thoroughly with a gentle stream of tap water to remove the soil. Ten nodules per plant were randomly selected for DNA extraction. Intact and undamaged nodules were immersed in 70% (v/v) ethanol (C2H6O) for 1 min and then left to soak in 6% (v/v) sodium hypochlorite (NaOCl) for 3 min before being carefully rinsed off six times in sterile water.
2.1. Soil sampling and rhizobia isolation
pH
6.5 6.7 7.8
TOC
pH - CaCl2; TOC - Total organic carbon.
2. Materials and methods
Moisture
pH
1943
pH - CaCl2; TOC - Total organic carbon. 2
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fluorometer (Invitrogen, Carlsbad, CA, USA). Once quantified, equimolar concentrations of each library were pooled into a single tube. After quantification, the molarity of the pool was determined and diluted to 2 nM, denatured, and then diluted to a final concentration of 8.0 pM with a 20% PhiX (Illumina, San Diego, CA, USA) spike for loading into the Illumina MiSeq sequencing machine (Illumina, San Diego, CA, USA).
not clear. To further investigate the community's dynamics, we tested the niche occupancy in bulk soil and nodules, and the proportion of specialists in the nodules of cowpea was higher than in those of lima bean (Fig. 2). 3.2. Bacterial community composition The 16S rRNA gene sequencing generated approximately 2,100,000 sequences. After quality trimming and rarefaction, the sequences were clustered into approximately 4000 OTUs. The microbiome composition was very distinct between bulk soil and the nodules. In the bulk soil, the most abundant bacterial groups were Unclassified Bacteria (24%), followed by Proteobacteria (20%), Actinobacteria (18%), and Firmicutes (14%). The microbiome of the nodules was dominated by Proteobacteria (97.6%), followed by Actinobacteria (1.1%), and Firmicutes (0.4%). Further analysis on a deeper taxonomical level showed that bulk soil presented a high proportion of the family Bacillales, Acidobacteria group 6, Bacillus, and unclassified member of Actinobacteria (Fig. 3A). As expected, the dominant bacterial group in the nodules was Bradyrhizobium (Alphaproteobacteria class), representing > 90% of the sequences in the nodules, in contrast with < 0.5% in bulk soil. Also, we observed that the lima bean presented a higher abundance of Bradyrhizobium than cowpea (Fig. 3B). On the other hand, cowpea nodules presented a higher abundance of other specific groups such as Microbacterium (average proportion = 0.10%, P = 0.024), Chitinophagales (0.06%, P = 0.28), Rhizobiaceae (0.06%, P = 0.037), and Acetobacteraceae (0.039%, P = 0.04) compared to lima bean (Fig. 3C). At a deeper taxonomic level we observed that CTS application in bulk soil affected 35 OTUs, while in the nodules of cowpea and lima bean affected four and three OTUs, respectively (Supplementary Table 1).
2.3. Data processing Sequence data were processed using QIIME 2 version 2017.11. First, the sequences were demultiplexed and quality control was carried out using DADA2 (Callahan et al., 2016), using the consensus method to remove any remaining chimeric and low-quality sequences. Afterward, samples were rarefied to 21,000 sequences, following the number of the lowest sample, and singletons, doubletons, chloroplast, and mitochondria sequences were removed from further analysis. The taxonomic affiliation was performed at 97% similarity using the Silva database v. 132 (Quast et al., 2013), and the generated matrix was further used for statistical analyses. The sequences are deposited in the NCBI database under the accession number ‘PRJNA575533’. 2.4. Data analysis To compare the bacterial community structure between bulk soil and the nodules of both plant species we used Principal Component Analysis (PCA) with Canoco 4.5 (Biometrics, Wageningen, The Netherlands). For this, the data were first evaluated using Detrended Correspondence Analysis (DCA), which indicated linearly distributed data (length of gradient < 3), revealing that the best-fit mathematical model for the data was PCA. Shannon diversity index was calculated based on the OTU table using the software Past v.3 (Hammer et al., 2001) and compared using Tukey's HSD test. To understand the community dynamics we tested the niche occupancy by classifying the OTUs into specialists and generalists. The niche occupancy was verified by the multinomial species classification method ‘clamtest’ available in ‘vegan’ package in R. This method compares the microbial abundance between two habitats and classifies the group of species that are similarly distributed across both habitats as generalists, and classifies as specialists the species more abundant in one habitat compared to the other (Chazdon et al., 2011). For CLAM analysis, it was considered a significant level for individual test of alpha 0.005 and a specialization threshold of 0.66. In order to compare the differential abundance of bacteria between bulk soil and the nodules of the two plant species, the OTU table was used as input in the software STAMP (Parks and Beiko, 2010). P-values were calculated based on two-sided Welch's t-test and correction using Benjamini-Hochberg FDR. To further predict the relevant potential functions related to the N-cycle, we performed a functional annotation using FAPROTAX database (Louca et al., 2016). For this, a table of frequency of taxa at the genus level was used as input and converted into a putative functional table and we selected only the functions related to the N-cycle.
3.3. Functional prediction of microbial community related to N-cycle
3. Results
We then used the FAPROTAX database to predict the potential functions of the bulk soil and nodules microbial communities with a focus on the N-cycle. The general functional profile clustered the samples according to the niche, i.e. bulk soil and nodules (Fig. 4A). However, there were no differences in functional diversity between bulk soil and nodules (Fig. 4B). The sequences were classified into 26 functional groups, with the core functions represented by chemoheterotrophy (32.3%), followed by aerobic chemoheterotrophy (32%), nitrification (12.7%), and ammonia oxidation (11.2%) (Fig. 5). The functional profile revealed some shifts in the functionality of the bacterial community from bulk soil to nodules. In general, bulk soil presented a higher abundance of sequences associated with nitrification, and ammonia and nitrite oxidation, while the nodules presented a higher abundance of chemoheterotrophy and symbionts (Figs. 5 and 6). Regarding the functions related to N-cycle, bulk soil presented a higher abundance of sequences related to aerobic ammonia oxidation, aerobic nitrite oxidation, and nitrification (P < 0.05), while nitrate reduction, nitrate respiration, and nitrogen respiration were detected at similar levels in all samples (Fig. 6). Interestingly, in the treatments with CTS and Cr contamination, there was an increase in the aerobic ammonia oxidation and nitrification in the nodules (Fig. 5; P < 0.05).
3.1. Bacterial community structure and diversity
4. Discussion
The principal component analysis (PCA) explained 98% of the variation in the first two axes of the plot, clustering the samples according to the niche, i.e. bulk soil and nodules of cowpea and lima bean (Fig. 1A; PERMANOVA F = 159.8, P = 0.0001). The Shannon index showed a decrease in bacterial diversity from bulk soil to nodules of cowpea and lima bean (Fig. 1B, P < 0.05), but the bacterial diversity in the nodules of cowpea was higher than the nodules of lima bean (P < 0.05). Therefore, the effect of CTS and Cr on nodule diversity was
In this study, we used a high-throughput sequencing method to assess the nodule microbiome of two important legume species for tropical regions that were exposed to low and high rates of CTS and Cr concentration. In bulk soil, unclassified bacteria were the dominant group. In addition, Proteobacteria, Actinobacteria, and Firmicutes were the most abundant identified phyla in both bulk soil and nodules showing a relationship between soils and nodule microbiome, at least in a phylum level. Nevertheless, the abundance of Actinobacteria and 3
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Fig. 1. (A) Principal component analysis (PCA) based on bacterial 16S rRNA from bulk soil and nodules of cowpea and lima bean grown in soils treated with composted tannery sludge. The dashed lines in the graph indicate significant clusters based on PERMANOVA (P < 0.05). (B) Diversity measurement based on bacterial 16S rRNA at OTU level at 97% similarity. Lower case letters refer to significant differences between the treatments, based on Tukey's test (P < 0.05). T1 = 0 Mg ha−1; T2 = 2.5 Mg ha−1; T3 = 20 Mg ha−1.
Fig. 2. Multinomial species classification method (CLAM) for the niche occupancy test. The pairwise treatments evaluated were as follow: (A) bulk and cowpea nodules, (B) bulk and lima bean nodules, and (C) cowpea and lima bean nodules. The proportion of generalists, specialists, and rare is displayed in the graphs.
As hypothesized, the nodule microbiome of cowpea showed differences in microbial groups in comparison to lima bean, although Bradyrhizobium was the most abundant genus in both species. Several studies have reported the genus Bradyrhizobium as the main symbiont of cowpea (Leite et al., 2017; Marinho et al., 2017; Ndungu et al., 2018) and lima bean (Ormeño-Orrillo et al., 2017). However, the idea that the nodules host a diverse microbiome began to be cleared with recent studies in cowpea (Leite et al., 2017) and soybean (Sharaf et al., 2019). Interestingly, lima bean nodules hosted Bradyrhizobium more abundantly than cowpea. In addition, the analysis of 16S rRNA and gyrB gene sequencing identified co-habitant bacteria in nodules of lima bean (Silva, 2017). The bacterial groups Microbacterium, Chitinophagales, Rhizobiaceae, and Acetobacteraceae were significantly more abundant in the cowpea nodules, suggesting that this plant species selects distinct co-habitant bacteria. Interestingly, we observed a different microbiome composition at a deeper taxonomical level, revealing that plant genotype exerts an effect on the assembly of the nodule microbiome, a result similar to Leite et al. (2017) who assessed the effect of soil type and plant genotype on nodule microbiome of cowpea. On the contrary to our second hypothesis, the application of CTS
Firmicutes was higher in bulk soil than nodules, where Proteobacteria prevailed. A previous study in cowpea also found Proteobacteria as the most abundant group (Leite et al., 2017) altogether indicating that it is possibly representing rhizobia. Comparing the bacterial diversity in bulk soil and nodules, the results showed a decrease in the bacterial diversity in nodules and it is in agreement with previous studies that reported lower bacterial diversity in nodules compared to rhizosphere or bulk soils (Lu et al., 2017; Sharaf et al., 2019). Indeed, soil microbiome is composed of a complex bacterial community (native or introduced) that is driven by the soil environment (Nandasena et al., 2007), while in the nodules, plants regulate the composition of bacterial community through the capabilities of bacteria to establish and interact with the plants (Peix et al., 2015). Interestingly, our functional prediction showed a high abundance of sequences affiliated to symbionts in the nodules, which may explain the selection of a specific microbiome in this environment. The results also showed that nodules of cowpea presented higher bacterial diversity than nodules of lima bean. This observation was confirmed by the niche occupancy analysis, which found more specialists in the nodules of cowpea than lima bean, indicating a stronger selective effect of lima bean on the bacterial assembly in the nodules. 4
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Fig. 3. (A) Distribution of the most abundant bacterial phyla (based on 16S rRNA gene) in bulk soil and nodules of cowpea and lima bean grown in soils treated with composted tannery sludge. (B) Proportion of sequences affiliated to the genus Bradyrhizobium. Lower case letters refer to significant differences between the treatments, based on Tukey's test (P < 0.05). (C) Scatter-plot showing the differential abundance of bacterial groups in the nodules of cowpea and lima bean. Pvalues were calculated based on two-sided Welch's t-test corrected using Benjamini-Hochberg FDR.
ecosystems. The functional prediction based on the DNA amplicon approach from the bacterial community can provide some correlations between taxonomy and function, and these tools have been used in recent studies (Hariharan et al., 2017; Dube et al., 2019). In our study, the dominant functional group was chemoheterotrophy, indicating that the most proportion of the microbes, in both bulk soil and nodules, cannot fix carbon and have to obtain carbon and energy from the oxidation of organic compounds (Zhang et al., 2018). In the bulk soil samples, the proportion of chemoheterotrophic bacteria can be explained by the high rate of organic matter, while in the nodules, the sugars are one of the carbon sources incorporated in large amounts inside the nodule cells (Hernández et al., 2013). Considering that BNF is the most important ecological service in the nodules, we explored soil bacterial functional groups related to N-cycle. The prediction of functions related to N-cycle is important since the bacterial community plays essential roles in ammonification, nitrogen fixation, denitrification, and nitrification (Yoon et al., 2015). Our analysis revealed that the general functional profile was distinct between bulk soil and the nodules, with specific functions presenting differential abundance
and the Cr concentration in the soil did not influence the bacterial communities, especially in nodules. Although our PCA analysis did not separate the bulk soil samples according to the CTS treatment, we found that 35 OTUs were affected by CTS in the bulk soil. This observation is supported by a previous study that has reported that less abundant bacterial groups in bulk soil were affected by the CTS application (Miranda et al., 2018). In the nodules, the application of CTS and Cr concentration did not influence the bacterial diversity, where only four and three OTUs were affected in cowpea and lima bean, respectively. However, previous studies have shown that CTS application and the accumulation of Cr affected nodulation and BNF in cowpea (Miranda et al., 2014) and lima bean (Rocha et al., 2019). Thus, although the nodulation decreases, the plant, through root nodules, could protect the bacteria against environmental stress and, therefore, avoiding that the bacterial community inside nodules being affected. Monitoring the soil bacterial community function in the soil and nodules can improve our knowledge about the response of the nodule community to the CTS application and Cr concentration, providing a theoretical basis for the restoration of degraded and/or contaminated
Fig. 4. (A) Principal component analysis (PCA) based on the functional profile of the microbial communities from bulk soil and nodules of cowpea and lima bean grown in soils treated with composted tannery sludge. The functional prediction was based on 16S rRNA genes using FAPROTAX database. The dashed lines in the graph indicate significant clusters based on PERMANOVA (P < 0.05). (B) Diversity measurement based on predicted functional profile. T1 = 0 Mg ha−1; T2 = 2.5 Mg ha−1; T3 = 20 Mg ha−1.
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Fig. 5. Distribution of the functional categories from the bacterial communities of bulk soil and nodules of cowpea and lima bean grown in soils treated with composted tannery sludge. The functional prediction was based on 16S rRNA genes using FAPROTAX database.
step in nitrification and is carried out by distinct groups of microorganisms. Interestingly, our data showed that the treatments with CTS and Cr contamination increased the abundance of sequences affiliated to aerobic ammonia oxidation and nitrification in the nodules. This pattern could be related to the disturbed condition of the soil, since several studies have shown an increased abundance of nitrification genes in disturbed soils (Merloti et al., 2019; Pedrinho et al., 2019). Also, using the functional prediction approach we did not detect sequences affiliated to nitrogen fixation, which can be a limitation of the method. However, our results highlight the fact that important functions, other than BNF, are probably occurring in the nodule environment, making the nitrogen available to the plant in different forms. For
between them. In the bulk soil, functions related to nitrogen transformations in soil were highlighted, such as nitrification, and ammonia and nitrite oxidation, which it is expected since the bulk soil samples presented high organic matter and N content together with higher microbial diversity. On the other hand, the nodule microbiome presented a high abundance of functions related to ‘animal symbionts’, which could be explained by the high abundance of Bradyrhizobium (> 90% of the sequences). Among the N-related functions, the most abundant in both niches were nitrification and ammonia oxidation. Nitrification is mediated by the nitrifying microorganisms and is responsible for regulating the availability of N in soils (Merloti et al., 2019) and probably in the nodules. The ammonia oxidation is the first
Fig. 6. Proportion of sequences related to nitrogen cycle from the bacterial communities of bulk soil and nodules of cowpea and lima bean grown in soils treated with composted tannery sludge. The functional prediction was based on 16S rRNA genes using FAPROTAX database. 6
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example, we found sequences related to nitrification in the nodules although in lower abundance compared to the bulk soil. As previous mentioned, the nitrification is a process that converts ammonium to nitrate. We also found sequences related to aerobic ammonia oxidation in the nodules, which is the first step in the nitrification process, performed by aerobic ammonia-oxidizing bacteria and archaea (Mohamed et al., 2010). Despite the amount of nitrogen available, a factor that can affect plant growth is the nitrate:ammonium ratio (Barker and Mills, 1980), because nitrate is more mobile than ammonium. Then, the nitrification of ammonium to nitrate occurring inside the nodules makes nitrogen more available for plant uptake.
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5. Conclusion In summary, our study provides new information on the diverse nodule microbiome of two leguminous species cultivated in soils treated or not with composted tannery sludge. Our data revealed that the nodule microbiome was dominated by rhizobia but also by a diverse bacterial community inhabiting this niche. We showed specific differences in the nodule microbiome between the two plant species. Regarding the functional profile, we showed that important functions related to the nitrogen cycle are present in the nodules. Overall, these results reveal a previously undescribed non-rhizobia community in the nodules that could play important role. Considering that the nodule microbiome was not affected by the CTS application, we suggest that this community is helping the plant to thrive under stress condition. Further studies should explore the beneficial potential of nodule microbiome to improve plant growth and health. Supplementary data to this article can be found online at https:// doi.org/10.1016/j.apsoil.2020.103542. Acknowledgments This study was funded by Conselho Nacional de Desenvolvimento Científico e Tecnológico – CNPq (grant 305069/2018-1) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES. The authors thank to the Centro de Genetica e Bioinformatica (CeGenBio) from the Unit of Research (NPDM/UFC). Sandra Mara Barbosa Rocha thanks Fundacao de Amparo a Pesquisa do Estado do Piaui (FAPEPI) for her scholarship. Jadson Emanuel Lopes Antunes thanks CAPES for his fellowship. Ademir Sergio Ferreira Araujo, Vania Maria Maciel Melo, and Mariangela Hungria thank CNPq for their fellowship of research. Ethical approval This article does not contain any studies with human participants or animals performed by any of the authors. Declaration of competing interest The authors declare that they have no conflict of interest. References Araújo, A.S.F., Santos, V.M., Miranda, A.R.L., Nunes, L.A.P.L., Dias, C.T.S., Melo, W.J., 2018. Chemical variables influencing microbial properties in composted tannery sludge-treated soil. Int. J. Environ. Sci. Technol. 15, 1793–1800. Barker, A.V., Mills, H.A., 1980. Ammonium and nitrate nutrition of horticultural crops. Horticultural Review 2, 395–423. Callahan, B.J., McMurdie, P.J., Rosen, M.J., Han, A.W., Johnson, A.J.A., Holmes, S.P., 2016. DADA2: high-resolution sample inference from Illumina amplicon data. Nat. Methods 13, 581–583. Caporaso, J.G., et al., 2011. Global patterns of 16S rRNA diversity at a depth of millions of sequences per sample. Proceedings of National Academy of Science 108, 4516–4522. Chazdon, R.L., Chao, A., Colwell, R.K., Lin, S.-Y., Norden, N., Letcher, S.G., et al., 2011. A novel statistical method for classifying habitat generalists and specialists. Ecology 92, 1332–1343. Dashti, N., Zhang, F., Hynes, R., Smith, D.L., 1998. Plant growth promoting rhizobacteria
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