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. 2024 Jan 2;14(1):27. doi: 10.1007/s13205-023-03875-7

Diversity of potential nitrogen-fixing bacteria from rhizosphere of the Coffea arabica L. and Coffea canephora L.

Vilian Borchardt Bullergahn 1, Karen Mirella Souza Menezes 1, Tomás Gomes Reis Veloso 1, José Maria Rodrigues da Luz 1, Lucas Ferreira Castanheira 1, Lucas Louzada Pereira 2, Marliane de Cássia Soares da Silva 1,✉
PMCID: PMC10758376  PMID: 38173824

Abstract

Coffea arabica L. and Coffea canephora L. are coffee species most consumed and marketed in the world. The coffee crop requires a large amount of nitrogen, which shows the importance of knowledge of the population of nitrogen-fixing bacteria (NFB) from the rhizosphere of these crops. These microorganisms may help the reduction of nitrogen fertilizing. However, there is no production of NFB inoculum in the coffee. Therefore, our objective was to evaluate the diversity of potential nitrogen-fixing bacteria (PNFB) in the rhizosphere of C. arabica and C. canephora. The microbial DNA of the soil was extracted, amplified through PCR, and sequenced at the Illumina Miseq. platform. The PNFB prediction was performed using the program PICRUSt2. Three hundred and thirty-seven amplicon sequence variants (ASVs) were identified as PNFB in two coffee species. Xanthobacteraceae, Rhizobium multhospitiium, Rhizobium mesosinicum, and Bradyrhizobium sp. were detected in all samples and main components of the core microbiota of the coffee plant rhizosphere. Some ASVs are exclusive from one of the coffee farms, showing that the coffee specie cultivated may influence the PNFB communities. However, edaphoclimatic factors and soil chemical attributes can also influence the distribution of ASVs in coffee soil. In the C. canephora, the PNFB diversity was influenced by the altitude and the soil chemical attributes, while the altitude and the phosphorus content influenced the PNFB population in C. arabica. Our results are important to the understanding of the PNFB dynamic in coffee soil and for the agricultural inputs bioprospecting to coffee.

Keywords: Diazotroph bacteria, Amplicon sequence variants, Soil, Coffee, Microbiome

Introduction

Brazilian agribusiness is one of the country’s most important economic sectors, representing 27.4% of the gross national product in 2021 (CEPEA-ESALQ/USP 2022). Coffee growing is extremely important in this economic sector. Brazil is the world’s greatest cherry coffee producer, with 58.2 million bags of 60 kg in the 2019/2020 harvest of Coffea arabica L. and/or Coffea canephora (International coffee organization 2022).

The C. arabica called arabica coffee has ideal edaphoclimatic conditions for its growing involving mild temperatures (18–23 °C), the reason why in Brazil that specie is cultivated in areas with altitudes above 600 m (Pereira and Moreira 2021). On the other side, C. canephora called robusta coffee, conilon coffee, or canephora coffee has ideal cultivation conditions in higher temperatures and lower altitudes (below 600 m) than arabica coffee (Taques and Dadalto 2007).

To achieve these expressive production numbers in dystrophic/acid tropical soils, the coffee growing in Brazil has to use a great number of inputs such as chemical fertilizers. The growth of both coffee species demands a high amount of nitrogen. Therefore, most fertilizers used in coffee growing are nitrogenous, which represents a considerable part of the production cost (Reis Junior et al. 2011).

A viable alternative to reduce the demand for chemical nitrogenous fertilizers in crops is to make use of the soil’s biological properties for example the use of nitrogen-fixing bacteria (NFB). These bacteria reduce the atmosphere’s nitrogen to ammonia (Rascio and La Rocca 2013). According to these authors, the NFB makes nitrogen assimilable to the plants. Furthermore, the capability of fixing atmosphere nitrogen is exclusive to the prokaryotes, such as diazotrophs that may be symbiotics, free-living or associative (Baldani and Baldani 2005; Rascio and La Rocca 2013).

The exploration of the symbiotic NFB in legume plants is already a reality among crops such as soy and beans. However, studies regarding free-living NFB and their agricultural potential are still incipient, with some studies presenting isolated species described in crop soils of forage, sugarcane, cassava, potato, and sweet potato, which possess the significant potential of N increment to the soil (Adachi et al. 2002; Döbereiner 1997; Videira et al. 2012; Naqqash et al. 2020; Zhang et al. 2022).

There are not inoculants of NFB for coffee crops. Therefore, maintaining and favoring the NFB of the rhizosphere is important to increase nitrogen availability in the soil. The favoring of the native microbiota may be done by planting crops associated with the NFB (green manure) in the coffee lines, use of soil cover management, temperature reduction and greater water retention in the soil; and conscious use of pesticides.

The genetic characterization of NFB populations is the first step to evaluating the potential use of these microorganisms in adding N to the soil. However, there are few studies on the characterization, isolation, and determination of the rate of N fixation by microorganisms from the rhizospheres of the coffee species cultivated in Brazil. For C. canephora, studies about that theme are even scarcer. Recently, Tran (2022) observed a dominance of Burkholderiales in 119 orders of bacteria found in the microbiome of C. canephora. While studying the microbiome of five coffee species, De Sousa et al. (2022) found a predominance of the genera Streptomyces, Bradyrhizobium, and Mycobacterium which are called plant-growing promoters.

Therefore, the objective of this work was to evaluate the diversity of potential nitrogen-fixing bacteria in Coffea arabica and Coffea canephora farms from Espírito Santo, Brazil using next-generation sequencing (NGS).

Materials and methods

Experimental conditions

The soil samples were collected in the state of Espírito Santo, considered the second largest coffee-producing region in Brazil of the species Coffea arabica and Coffea canephora var. conilon (Incaper 2023). The coffee production in this region is done at different levels of altitude, which has influenced the chemical and physical attributes of coffee (Cassamo et al. 2022) and the composition of the soil microbial community (Veloso et al. 2020).

In ten areas with different altitudes and cultivations of C. canephora (C, D, H, J, and AH) and C. arabica (B, F, AG, BB, and X), the soil samples were obtained (Fig. 1). These letters are the initials of the names of the owner of the coffee farm. From each property, three composite soil samples were randomly collected at 10 cm deep, within the projection of the coffee treetop. The soil samples collected were packed in plastic bags and stored under refrigeration at − 20 °C. The crops were grown under a conventional cultivation system and the sample collection occurred during the period of low rainfall between May and September 2020.

Fig. 1.

Fig. 1

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Location map of coffee (Coffea arabica and Coffea canephora) farms in the state of Espírito Santo—Brazil

Analysis of the soil chemical properties

The Mehlich-1 extractor was used for the analysis of P, K, and Na. The P content was determined by the vitamin C method in UV–visible spectrophotometry (Defelipo and Ribeiro 1997). The contents of K and Na were determined in the flame photometer. Ca, Mg, and Al were extracted in KCl solution (1.0 mol/L) and determined by atomic absorption spectrum.

The soil pH was determined in a soil and water solution (1:2.5 m/v), while the potential acidity (H + Al) was determined using calcium acetate (pH 7.0).

The sum of bases (SB), base saturation (V), soil organic matter (SOM), and potential cation-exchange capacity (CEC) were determined according to Defelipo and Ribeiro (1997).

DNA extraction, PCR, and sequencing

The soil’s DNA was extracted from 250 mg of soil using the Nucleo Spin Soil kit (Macherey-Nagel, GmbH & Co. KG, Germany), following the manufacturer’s protocol. To confirm the presence and quality of the extracted DNA, the electrophoresis technique was applied in 0.8% agarose gel stained with ethidium bromide under UV light. A digital camera was used to evaluate the bands.

The DNA libraries of the hypervariable subregions V3-V4 of the 16S rDNA gene were constructed through amplification by the Polymerase Chain Reactions (PCR) technique using the primers pairs 341F (5′ CCTAYGGGRBGCASCAG 3′) and 806R (5′ GGACTACNNGGGTATCTAAT 3′). All libraries were normalized so that all samples had equimolar amounts in the library (2 nM per library) before pooling the samples for the sequencer. The sequencing was performed using the Illumina NovaSeq 6000 platform in the form of paired reads with 250 bp for each read.

Bioinformatic analyses

Demultiplexed raw reads from sequencing had primers, barcodes, and adapters removed using the “Cutadapt” plugin (Martin 2011). The remaining reads were subjected to a filter for removing low-quality sequences using the DADA2 plugin (Callahan et al. 2016) available on Qiime2 (Bolyen et al. 2019). All reads with a maximum expected error above one, DNA chimeras, and singletons were removed by the action of the noise filter (DADA2). The DADA2 algorithm was also used to determine Amplicon Sequence Variants (ASVs) (Callahan et al. 2016). All ASVs were taxonomically classified using a pre-trained algorithm (classify-sklearn) with the SILVA 138 database. All sequences related to chloroplasts and mitochondria were removed using Qiime2 software.

The prediction of potential nitrogen-fixing bacteria (PNFB) was performed by the Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt2) tool (Douglas et al. 2020) using the ASVs sequences. The sequences were placed in a phylogenetic tree with strains of known genomes. For further analyses, only sequences with the Nearest Sequence Taxon Index (NSTI) below 0.15 were maintained to ensure a high-accuracy prediction.

From the predicted metagenome, ASVs with genes related to nitrogen fixation, based on the MetaCyc database (EC:1.18.6.1 and EC:1.19.6.1), were used for the analyses.

Statistical analysis

Data on the absence and presence of PNFB associated with the two coffee species were performed in the PICRUSt2. The non-metric multidimensional Scaling (NMDS) analysis was constructed with Bray–Curtis dissimilarity. The diversity data (Shannon Index), uniformity (Pielou equivalence), and richness were determined in the Microbiome package (Lahti and Shetty 2017) using the R studio software.

The alpha diversity metrics comparison was performed using the one-way ANOVA and Scott–Knott post-hoc test at 5% significance.

Results

In this study, 377 ASVs (1,919,817 sequences) were detected as potential nitrogen-fixing bacteria (NPB) in the rhizosphere of C. arabica (125 ASVs), C. canephora (180 ASVs), and C. arabica/C. canephora (72 ASVs) (Fig. 2A). These ASVs were distributed in 8 phyla, 13 classes, 24 orders, and 39 families. Four ASVs from the bacterial groups Xanthobacteraceae, Bradyrhizobium, Rhizobium multhospitiium, and Rhizobium mesosinicum were identified in all soil samples in both coffee species (Fig. 2B). The bacterial genus Skermanella was found in more than 80% of the C. canephora and C. arabica samples. On the other hand, some PNFB showed exclusivity regarding the coffee species (Fig. 2A, B).

Fig. 2.

Fig. 2

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Venn diagram A and B amplicon sequence variants (ASVs) of potential nitrogen-fixing bacteria (PNFB) associated with each sample of Coffea arabica and Coffea canephora. The letter at the end of the taxon identification represents the level of identification that has been achieved for each ASV. S = species; G = genera; F = family; O = order; C = class, P = phylum. Five coffee cultivation sites (three samples per area) of each species were evaluated, totaling 30 samples. The Venn diagram shows the number of ASVs as PNFB found exclusively or shared in each coffee species

The Xanthobacteraceae, Nitrosomonodaceae, and Beijerinckiaceae families, the Burkholderia, Afipia, Allorhizobium, Ideonella, Mesorhizobium, and Pseudoaminobacter genera, and the Clostridium magnum species were identified exclusively in the ASVs of the C. arabica soils. However, in the soil of C. canephora, it was found the Rhizobiaceae, Xanthobacteraceae, Lachnospiraceae, Peptostreptococcaceae, and Beijerinckiaceae families, the Pseudoaminobacter, Anaeromyxobacter, Allorhizobium, Skermanella, Pseudoaminobacter, and Roseiarcus genera, and the Eubacterium pyruvativorans, Clostridium bornimense, Skermanella aerolata, and Paenibacillus borealis species.

The ordering of samples by non-metric multidimensional scaling (NMDS), based on the Bray–Curtis distance matrix, clearly showed a divergence in the structure of PNFB associated with each coffee species (Fig. 3). The PNFB communities of C. arabica and C. canephora are on opposite sides of the quadrants, which reveals that the PNFB communities associated with these species differ. It is also observed that the PNFB communities in C. canephora were significantly related to the soil chemical characteristics such as soil organic matter, magnesium level, potassium contents, base saturation, and potential acidity, while in C. arabica the PNFB is strongly associated with altitude, phosphorus content, and remaining phosphorus (Fig. 3).

Fig. 3.

Fig. 3

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Beta-diversity of potential nitrogen-fixing bacteria (PNFB) detected in five sites of Coffea canephora cultivation and five sites of Coffea arabica cultivation. Each area is represented by three samples (shaded areas) and a letter (see Fig. 1 for more information). Ordination analysis was based on non-metric multidimensional scaling (NMDS). Edaphic variables were fitted to the ordering using the envfit function available in the vegan package

The microbial diversity, evenness, and richness of each species varied between the areas and altitudes of coffee cultivation (Fig. 4). There were no significant differences in PNFB richness in C. arabica, although differences in diversity (Shannon index) were found. The C. arabica samples from areas B, AG, and BB had higher diversity values than diversity from other areas. For C. canephora, PNFB diversity was greater in high-altitude samples (J and AH) than in low-altitude samples. However, no significant difference in richness was observed in these samples (Figs. 1, 4).

Fig. 4.

Fig. 4

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Metrics of alpha diversity of potential nitrogen-fixing bacteria (PNFB) detected in five sites of Coffea canephora cultivation and five sites of Coffea arabica cultivation. All metrics were calculated using the “Microbiome” package

Discussion

We observed a wide diversity of potential nitrogen-fixing bacteria in the rhizosphere in C. arabica and C. canephora (Fig. 2A). Other studies have also shown this high PNFB in coffee plantations (Grossman et al. 2005; Suharjono and Yulianti 2022). However, our study makes an unprecedented comparative approach of PNFB in C. arabica and C. canephora using samples from different planting areas and altitudes. In addition, Da Silva et al. (2020) evaluated the PNFB diversity in the soil and fruit of C. arabica in Brazil. These authors found 115 ASVs in the soil related to PNFB, while we found 377 ASVs for the two coffee species studied with a percentage of around 20% sharing of ASVs between C. arabica and C. canephora and a difference between the microbial taxa was observed in these analyses.

The Xanthobacteraceae, Bradyrhizobium, Rhizobium multhospitiium, and Rhizobium mesosinicum were the most abundant microbial taxa in arabica coffee and conilon coffee soils (Fig. 2B). The Rhizobium genus stands out as the largest group of nodule-forming symbionts and has the largest number of described diazotrophic species (Lindström and Mousavi 2019). In addition, Rhizobium, Bradyrhizobium, and the Xanthobacteraceae family are important symbiotic NFB members that form nodules and establish a mutualistic relationship with leguminous plants (Kour et al. 2020). These bacterial groups are predominantly found in coffee soils (Grossman et al. 2005; Caldwell et al. 2015; Cabresa-Rodríguez et al. 2020). According to Da Silva et al. (2020), the Bradyrhizobium genre is part of the core microbiota of C. arabica Thus, our study confirms that these members from the Rhizobiales order are part of the core microbiota of arabica coffee and canephora coffee. Besides fixing nitrogen, these microorganisms’ presence in the rhizosphere can bring other benefits to plants by acting as plant growth promoters. The Bradyrhizobium elkanii isolated from the rhizosphere of C. arabica and C. canephora was recommended as a natural fertilizer because of its ability to promote plant growth by solubilizing phosphate and releasing indoleacetic acid into the soil (Suharjono and Yuliatin 2022).

The Skermanella genus was observed in more than 80% of both coffee species samples (Fig. 2). Skermanella has been commonly reported as one of the most abundant bacteria in the soil of some crops of agricultural interest, such as cucumber and vineyard plantations (Gao et al. 2021; Xue et al. 2022) and as a key genus in the structure of bacterial communities (Tran et al. 2020). However, although this genus is phylogenetically close to such genera of diazotrophic bacteria as Azospirillum, Bradyrhizobium, and Mesorhizobium (Körberl et al. 2016), Skermanella spp., have not yet had their nitrogen fixation capacity proven, since they do not have the anfG gene which codes the delta subunit of nitrogenase (Zhu et al. 2015).

Root exudates can select some microbial groups for the rhizosphere that contribute to plant growth and pathogen protection (Fox et al. 2020; Upadhyay et al. 2022). These exudates are released as needed by the plant promoting the formation of a specific environment to attract specific groups of bacteria plant growth promoters (Kamilova et al. 2006). In this study, we found the specificity of certain PNFB groups in both coffee species. For example, a species of the Beijerinckiaceae family was found exclusively in C. arabica (Fig. 2). The presence of this microorganism is interesting for future studies, as it demonstrates that different groups of BFN can be found in the rhizosphere of coffee plants.

We observed an ASV from the Nitrosomonadaceae family specifically in C. arabica. However, this family has been found in the rhizosphere of C. canephora (Tran 2022). Nitrosomonadaceae family members act in the nitrification process in terrestrial, marine, and freshwater environments (Prosser et al. 2014).

In the rhizosphere of C. canephora, we found a greater number of PNFB’s ASVs unique to this coffee species (Fig. 2A). These results show that the PNFB communities of the two coffee species studied are distinct. In the exclusive ASVs, we found the Rhizobiaceae, Xanthobacteraceae, and Beijerinckiaceae families, which are widely known for their nitrogen-fixing capacity (Caldwell et al. 2015; Lindström and Mousavi 2019). They are also present in the soil of C. canephora plants (Tran 2022; Suharjono and Yuliatin 2022). The P. borealis species was found only in C. canephora samples. This bacterium was isolated from the soil of humid forests in Finland and showed the ability to fix nitrogen (Elo et al. 2001). We found no reports in the literature regarding the presence of P. borealis in coffee plantations. This is the first study to demonstrate the presence of this bacterium in the soil of arabica and conilon coffee. Furthermore, this bacterium also can produce phytohormones and inhibit the presence of phytopathogens (Liu et al. 2019).

The Roseiarcus genus was found only in the soil of C. canephora. The presence of this bacterium in soils is correlated with the increase of nitrogen in the leaves of Vaccinium angustifolium (Morvan et al. 2020). However, the abundance of Roseiarcus in the soil depends on the management of nitrogen fertilizers, since high amounts of nitrogen tend to decrease its number (Chaudhari et al. 2020). Other PNFB species present only in the C. canephora were the Skermanella aerolata and Skermanella sp. Although species of this genus are considered diazotrophic, their ability to fix nitrogen is unknown (Zhu et al. 2015), but it may be a plant growth-promoting microorganism. In addition, the phenolic compound released in the root exudate of C. canephora may have been selected for the presence of these bacteria in its rhizosphere. In Arabidopsis plants, the presence of acid γ- aminobutyric in its rhizosphere increased the abundance of Skermanella spp. (Badri et al. 2013).

In this study, we observed that the soil PNFB of coffee species is different (Fig. 3). By studying the microbiome of five coffee species, De Sousa et al. (2022) observed how the rhizosphere microbiomes of C. arabica and C. canephora are similar. The Venn diagram and NMDS show that there are differences in the profile of this PNFB community (Figs. 2A, 3). However, the PNFB population of coffee species located nearby geographically, such as AH and B sites are more similar. Thus, the effect of the host mixes with the effects caused by environmental conditions (altitude, proximity, and chemical properties of the soil). According to Veloso et al. (2020), altitude influences the profile of the soil microbial community in C. arabica. The altitude also contributes to the productivity and quality of coffee beans (Cassamo et al. 2022). Hence, although it is expected differences in the microbial composition between the soils of each coffee species, the populations tend to be similar when cultivated in nearby regions and altitudes.

We noted that in C. arabica soil, the PNFB population was also influenced by the phosphorus content of the soil (Fig. 3). Thus, a large part of the PNFB community may also be capable of promoting phosphate solubilization. This ability is interesting for coffee crops that have a high P demand to grow. The Burkholderia, a genus found in this study, can solubilize phosphorus and other important plant-growing compounds (Curi et al. 2019; Urgiles-Gómez et al. 2021; Suharjono and Yuliatin 2022).

The alpha diversity metrics of each coffee species varied between cultivation areas (Fig. 4). The PNFB diversity was directly proportional to the altitude. This diversity has been driven by greater equity and wealth in C. canephora. This finding is consistent with other results where there is an increase in the bacterial community as a function of high altitudes (Siles and Margesin 2016), reflecting a greater microbial activity (Siles et al. 2016). However, we did not detect changes in diversity in C. arabica depending on the altitude. Changes in the PNFB alpha diversity of C. arabica were observed among crops in geographically close regions, such as AG and BB sites. Nonetheless, in C. arabica, no differences were observed between the richness and evenness of the bacterial population as a function of planting altitude (Veloso et al. 2020).

Conclusions

The cultivation soils of C. arabica and C. canephora have a rich diversity of potential nitrogen-fixing bacteria. Only 19% of this diversity is shared among coffee species. Rhizobium multhospitiium, Rhizobium mesosinicum, Xanthobacteraceae, and Bradyrhizobium represent part of the core microbiota of these soils. These results can direct studies to validate and quantify the PNFB in arabica coffee and conilon coffee plantations. The coffee species and environmental factors, such as altitude, influence the PNFB community. Thus, the bioprospecting of PNFB in soil with C. arabica and C. canephora plantations is promising for the development of inoculants that promote nitrogen addition in the soil.

Acknowledgements

The authors would like to thank the Sul Serrana of Espírito Santo Free Admission Credit Cooperative—Sicoob (23186000886201801), the Coordination of Superior Level Staff Improvement—CAPES, the National Council for Scientific and Technological Development (CNPq), the IFES for supporting the research (PRPPG n°. 12/2021), Incaper, Embrapa, Fapemig, and Fapes.

Authors’ contribution

Conceptualization, validation, investigation, writing, formal analysis: VBB, KMSM, TGRV, JMRL, LLP, and MCSS. Data processing and analysis: KMSM, TGRV, JMRL, LLP, and MCSS. The investigation, writing—review and editing: VBB, KMSM, TGRV, JMRL, LFC, LLP, and MCSS.

Data availability

The authors declare the data availability from the article for interested persons. All authors authorized the submission and publication of the paper.

Declarations

Conflict of interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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