Characterization of bacterial endophytes from the roots of native and cultivated Brazil nut trees (Bertholletia excelsa)

Caracterização de bactérias endofíticas de raízes de castanha-do-Brasil (Bertholletia excelsa) em habitats nativos e cultivados

ABSTRACT

Brazil nut is a very important nontimber forest product in the Amazon region. Propagation of this tree still represents a challenge due to slow and uneven seed germination. In this context, plant growth-promoting bacteria can facilitate the process of propagation. The aims of this study were to isolate and characterize endophytic bacteria from the roots of Brazil nut trees in native terra firme forest and cultivation areas in northern Brazil, and to identify mechanisms by which bacteria act in plant growth promotion. Overall, 90 bacterial isolates were obtained from the roots of Brazil nut trees in monoculture, agroforestry and native forest areas by using different semisolid media. The isolates were characterized by sequencing the 16S rRNA gene. Plant growth-promoting characteristics were evaluated by the presence of the nifH gene, aluminum phosphate solubilization and the production of indole compounds. The isolates were affiliated with 18 genera belonging to 5 different classes (α-Proteobacteria, β-Proteobacteria, γ-Proteobacteria, Bacilli and Actinobacteria). The genus Bacillus was predominant in the forest and monoculture areas. Fourteen isolates presented the nifH gene. Most of the bacteria were able to solubilize aluminum phosphate and synthetize indole compounds. The results indicated high diversity of endophytic bacteria present among the roots of Brazil nut trees, mainly in the agroforestry area, which could be related to soil attributes. Among the 90 isolates, the 22 that presented the best results regarding plant growth promotion traits were good candidates for testing in seedling production of Brazil nut trees.

Keywords:
16S rRNA; phosphate solubilization; indole compounds; biological nitrogen fixation; plant growth-promotion bacteria; Amazon

RESUMO

A castanha-do-brasil é um produto florestal não madeireiro muito importante na região amazônica. A propagação desta árvore ainda representa um desafio, devido ao lento e irregular processo de germinação das sementes. Neste contexto, bactérias promotoras do crescimento vegetal podem facilitar o processo de propagação. O objetivo deste estudo foi isolar e caracterizar bactérias endofíticas em raízes de castanha-do-Brasil em floresta de terra firme e em áreas cultivadas no norte do Brasil, e identificar alguns mecanismos de promoção do crescimento vegetal executados por essas bactérias. No total, 90 isolados bacterianos foram obtidos de raízes de castanha-do-Brasil em monocultura, agrofloresta e floresta nativa, usando diferentes meios de cultivo semi-sólidos. Os isolados foram caracterizados pelo sequenciamento do gene 16S rRNA. As características de promoção do crescimento vegetal foram avaliadas através da presença do gene nifH, solubilização de fosfato de alumínio e produção de compostos indólicos. Os isolados foram afiliados a 18 gêneros, pertencentes a cinco diferentes classes (α-Proteobacteria, β-Proteobacteria, γ-Proteobacteria, Bacilli e Actinobacteria). O gênero Bacillus foi predominante, principalmente nas áreas de floresta e monocultura. Quatorze isolados apresentaram o gene nifH. A maioria dos isolados foi capaz de solubilizar fosfato de alumínio e sintetizar compostos indólicos. Os resultados indicam uma elevada diversidade de bactérias endofíticas presente em raízes de castanha-do-Brasil, principalmente em área de agrofloresta, que pode estar relacionado aos atributos do solo. Entre os 90 isolados, 22 apresentaram os melhores resultados relacionados às características de promoção do crescimento vegetal, e são bons candidatos para testes em produção de mudas de castanha-do-Brasil.

Palavras-chave:
16S rRNA; solubilização de fosfatos; compostos indólicos; fixação biológica de nitrogênio; bactéria promotora do crescimento vegetal; Amazônia

INTRODUCTION

Brazil nut (Bertholletia excelsa H.B.K.) is native to Brazil, Bolivia, Venezuela, Colombia, Peru and the Guianas (Mori 1992; Lorenzi 2002) and represents the most important commercial nontimber forest product in the Amazon region. Brazil nut trees are long-living organisms, and large trees have been carbon dated with ages of more than 300 years (Vieira et al. 2005). As the nuts are solely harvested in the wild, the sustainable exploitation of Brazil nut contributes to promote Amazonian forest conservation and has enjoyed widespread and longstanding economic success in the international market (Wadt et al. 2008). However, the species also suffers from increasing habitat loss due to deforestation for illegal timber trade, opening of pasture areas, and forest fires. Studies on the introduction of cultivated Brazil nut trees are needed (Camargo et al. 2010), as habitat loss puts the species at risk due to loss of pollinators and seed-dispersing agents, and increased pollination distances (Ortiz 2002). A problem with Brazil nut cultivation is the time required for seed germination (20-60 days) and the uneven time germination of its seeds (Müller et al. 1995).

An alternative for improving the production of Brazil nut seedlings could be the use of plant growth-promoting bacteria (PGPB). These bacteria are able to improve the growth of plants and protect them from disease and abiotic stress (Glick 2012; Souza et al. 2015). PGPB can be found in the rhizosphere, on the root surfaces, or in the internal tissues of plants. PGPB that colonize internal tissues are known as endophytes and are found in nearly every plant worldwide (Santoyo et al. 2016) and do not harm the plant (Hallman et al. 1997). Endophytic bacteria can accelerate seedling emergence and increase plant growth (Chanway 1997). The mechanisms involved in plant growth promotion by these bacteria are similar to those observed among rhizobacteria, such as nitrogen fixation, phytohormone production, phosphate solubilization, and the production of antifungal compounds, induction of systemic resistance and production of siderophores (Vessey 2003; Compant et al. 2010; Glick 2012; Chanway 1997), which are bioactive compounds produced by bacteria that are readily available to the plant (Afzal et al. 2019). To date, there is no information about the isolation or characterization of endophytic PGPB from Brazil nut trees. The isolation and characterization of endophytic plant growth-promoting bacteria from Brazil nut roots would be important for the identification of new biological inoculants which may provide an alternative approach for the improvement of seed germination and seedling development.

Bacterial diversity in Amazon soils is higher in association with more intensive land uses than in undisturbed primary forest (Carvalho et al. 2016). Land use and changes in soil fertility, especially pH, alter the bacterial community composition (Jesus et al. 2009, Carvalho et al. 2016). Therefore, the main hypothesis of our study was that cultivated Brazil nut trees may harbor a greater diversity of endophytic bacteria than those in native forest areas. An additional hypothesis was that endophytic bacteria associated with Brazil nut roots exhibit mechanisms for promoting plant growth, such as the ability to solubilize phosphates and produce phytohormones. To test these hypotheses, we isolated and characterized endophytic bacteria from Brazil nut trees in an area of native terra firme forest and two experimental cultivation areas in the state of Roraima (northern Brazilian Amazon region), one containing only a monoculture of Brazil nut trees, and an agroforestry system containing Brazil nut trees in combination with other agricultural activities. The isolates were then tested to identify the mechanisms of plant growth promotion.

MATERIAL AND METHODS

Origin of bacteria

Root samples were collected between April and October 2013 in three areas in Roraima State, Brazil (Figure 1): (a) from 11 trees in a monoculture of Brazil nut trees planted in 2007 in the Serra da Prata experimental field of Embrapa Roraima in the city of Mucajaí (02º22’28.2”N, 60º59’46.8”W) [the Brazil nut seedlings received 100 g of triple superphosphate at the tips at planting, and, after two years, 30 kg ha^-1 of N (ammonium sulfate) and 30 kg ha^-1 of K₂O (potassium chloride) in the crown projection area]; (b) from 10 trees in an agroforestry system planted in 1995 in the Confiança experimental field of Embrapa Roraima, in the city of Cantá (02º15’00”N, 60º39’54”W) [the area was plowed and received 2000 kg ha^-1 liming (PRNT 100%), 40 kg ha^-1 P₂O₅ and 50 kg ha^-1 of FTE BR12 Nutriplant (7.1% Ca, 5.7% S, 1.8% B, 0.8% Cu, 2.0% Mn, 0.1% Mo, and 9.0% Zn) as a source of micronutrients]. Apart from Brazil nut, other species planted were cupiúba (Goupia glabra Aubl.), peach palm (Bactris gasipaes Kunth.), cupuassu (Theobroma grandiflorum Schum.), coffee (Coffea canephora Pierre.), rain tree (Samanea saman (Jacq.) Merr.) and andiroba (Carapa guianensis Aubl.)]; and (c) from seven trees in a dense ombrophilous forest in a private estate located in São João da Baliza (0º57’024”N, 59º54’41”W).

Figure 1
Location of the three sampling areas for bacterial isolates from roots of Brazil nut (Bertholletia excelsa) trees in the state of Roraima, in the northern Brazilian Amazon. This figure is in color in the electronic version.

The climate in the region of the monoculture and agroforestry areas is Ami (Köppen system), with mean annual temperatures ranging from 26 to 29 °C, and rainfall from around 1800 to 2400 (Oliveira Junior et al. 2003), and the soil type is a red-yellow ultisol, representing an area of savanna-forest transition on a flat relief. In the forest area the relief is flat to wavy, with climate of type Awi (Köppen system), with mean annual temperature of 27 ^oC and rainfall range from 1700 to 2000 mm; the soils were not classified, but yellow ultisols and oxisols are predominant in the region (Femact 1993; Seplan 2010). Soil samples from the three sampling areas were chemically and physically characterized by Embrapa (1997) (parameters are presented in Table 1).

Table 1
Chemical and physical attributes of the soil in areas where roots of Brazil nut (Bertholletia excelsa) trees were sampled in Roraima state (northern Brazil). Data from Embrapa (1997).

Areas	Measure unit	Agroforestry	Monoculture	Native terra firme Forest
Chemical attributes
pH (water)		5.9	4.8	4.5
Ca2+	cmolc dm-3	3.5	1.2	0.33
Mg2+	cmolc dm-3	0.7	0.4	0.19
K+	cmolc dm-3	0.2	0.04	0.19
Al3+	cmolc dm-3	0.1	1	0.82
H+Al	cmolc dm-3	5.12	2.3	5.77
P	mg dm-3	19.33	3	1.95
V	%	44.9	41	11
O.M.	g dm-3	41.4	7	23
Physical attributes
Argila	%	23	28	28
Silte	%	8	15	15
Areia	%	69	57	57

• V (base saturation), O. M (organic matter)

Bacteria were isolated from nonwoody roots in different semisolid culture media that included NFb, LGI (both of which favor the growth of Azospirillum spp.) (Döbereiner et al. 1995), JMV (Burkholderia spp.) (Estrada De Los Santos et al. 2001) and DYG’s (nonselective) (Rodrigues Neto et al. 1986). The roots were washed with water and cut into fragments of approximately 1 cm in length. then subjected to surface disinfection with sodium hypochlorite (2%) for 2 min and then hydrogen peroxide (5%) for 1 min, after which they were washed six times with sterilized water. The media with roots (5 replicates) were incubated for 10 days in a growth chamber at 28 °C. The isolation was performed from all the cultures that presented growth, i.e., the formation of a pellicle in the medium. After confirmation of bacterial growth, a loopful of the pellicle was streaked onto the corresponding solid medium to isolate the bacterium based on the phenotypic characteristics of the colonies (Döbereiner et al. 1995). After isolation, the following colony characteristics were evaluated: growth time, average diameter, shape, margin, surface, elevation, color and gum production. Ninety bacteria were selected from 303 obtained isolates (41 isolates from the agroforestry system, 30 from the monoculture system and, 19 from the native terra firme forest). All isolates obtained were stored at -80 °C in 20% glycerol.

16S rRNA partial sequencing

Bacteria were grown in DYG’s liquid medium (Rodrigues Neto et al. 1986) for 24 h, after which their DNA was extracted from 1 ml of cell suspension with an RBC extraction kit (cat. YGB300, Taiwan), following the instructions provided by the manufacturer. Amplification of the 16S rRNA gene was performed using the 27F (5’-AGAGTTTGATCCTGGCTCAG-3’) and 1492R (5’-GGTTACCTTGTTACGACTT-3’) primers (Lane 1991), and partial sequencing was performed using the 27F primer. Sequencing was performed using a 3730xl DNA sequencer (Applied Biosystems, Foster City, CA, USA). The sequences were aligned using the ClustalW program (Thompson et al. 1994) in MEGA 5.01 software (Tamura et al. 2011). A phylogenetic tree was constructed using the “neighbor-joining” method and Kimura’s 2-parameter model (Kimura 1980) with MEGA 5.01 software, performing 1000 repetitions. The 16S rRNA gene sequences were deposited in the GenBank database under accession numbers MF442264-MF442353.

nif H gene amplification

The nifH gene was amplified using the primers 19F (5’-CCI WTYTAYGGIAARGGIGG-3’) and 407R (5’-AAICCRCCRCAIACIACRTC-3’) (Ueda et al. 1995).

Aluminum phosphate solubilization assays

An initial qualitative estimation of the P-solubilizing activity of the bacteria was carried out on modified National Botanical Research Institute Phosphorus (NBRIP) agar (Nautiyal 1999). Tricalcium phosphate was substituted with aluminum phosphate (AlPO₄, 0.236%), and the pH of the medium was adjusted to 4.5. All isolates were cultured in DYG’S liquid medium for 24 h at 28 °C until the cell concentration measured via the optical density at 630 nm (OD₆₃₀) reached 0.5−0.7. Ten-microliter samples of the bacterial cultures were inoculated into NBPRIP medium at three equidistant points on a plate, which was subsequently incubated for 18 days at 28 °C.

A quantitative assay was also performed using NBPRIP liquid medium. One milligram of cells from the isolates cultured in DYG’S solid medium was inoculated into 30 mL of NBPRIP with AlPO₄ at a concentration of 12 mg P l^-1. These cultures were incubated at 28 °C for 4 days under 150 rpm agitation. At the end of this period, the pH was determined; each sample was centrifuged (10 000 rpm for 5 min), and the levels of soluble P in the supernatant were quantified using the phosphomolybdate method (Murphy and Riley 1962). The concentration of P was estimated using a standard curve previously prepared with 0, 0.1, 0.5, 0.75, 1, 2, 3, 4, 4.5, 5 and 6 mg l^-1 P in the form of KH₂PO₄.

In all tests, BR 11001^T (Azospirillum brasiliense), BR 11340 (Burkholderia sp.), BR11175^T (Herbaspirillum seropedicae), BR 11790^T (Herbaspirillum frisigense), and ERR 532 (Bacillus sp. isolated from the roots of Brachiaria sp.) were included for comparison. The experiments were completely randomized with three replications. The data were statistically analyzed using the SISVAR program, version 4.3 (Ferreira 2011), with the effects from the treatments evaluated by the Scott-Knott test (Scott and Knott 1974) at a 5% level of significance.

Production of indole compounds

The isolates and the type and reference strains, as well as Azospirillum brasilense BR 11001^T (Radwan et al. 2002), Herbaspirillum seropedicae BR11175^T (Baldani et al. 1986), Burkholderia sp. BR11340, Herbaspirillum frisigense BR11790^T, and ERR 532 (Bacillus sp. isolate from roots of Brachiaria sp.), were cultured in DYG’S liquid medium (Rodrigues Neto et al. 1986) for 24 h. After growth, the cultures were adjusted to an OD₆₃₀ ranging from 0.6−0.8. Then, 500 µl aliquots of the bacterial cultures were inoculated into 6 ml of DYG’s medium (without _L-tryptophan or supplemented with 100 mg L^-1 _L-tryptophan), followed by incubation for 48 h at 28 °C under constant stirring at 150 rpm. The cultures were recovered after centrifugation at 10.000 rpm g for 5 min, and 3 ml of the recovered supernatant and 2 ml of Salkowski reagent were then mixed together (Sarwar and Kremer 1995). This mixture was incubated in the dark for 20 min, until a pink color developed that is indicative of indole production. The color intensity was measured in a spectrophotometer at 535 nm. The concentration of indoles was estimated using a standard curve prepared with 0, 10, 25, 50, 75 and 100 μg AIA ml^-1 (Sigma-Aldrich, cod. I3750). The experiments were completely randomized with three replications. The data were then statistically analyzed as described for the phosphate solubilization assays.

Selection of bacteria

For the selection of bacteria exhibiting growth-promoting characteristics, histograms were constructed to visualize the data and group the isolates (data not shown). Bacteria associated with relatively higher phosphate solubilization and indole compound production were selected.

RESULTS

16S rRNA partial sequencing

The closest matches for the 90 bacterial isolates within the NCBI database are presented in Table 2. The 90 endophytic bacteria were affiliated with 18 genera belonging to 5 different classes: α-Proteobacteria, β-Proteobacteria, γ-Proteobacteria, Bacilli and Actinobacteria. Members of the classes Bacilli (43.3%) and γ-Proteobacteria (32.2%) were predominant followed by β-Proteobacteria (12.2%), α-Proteobacteria (6.7%) and Actinobacteria (5.6%). In the class Bacilli, the genus Bacillus was predominant, with 30 isolates. Stenotrophomonas (10 isolates) was predominant in the class γ-Proteobacteria, and Burkholderia and Achromobacter, both with four isolates, in the class β-Proteobacteria. Members of the predominantly occurring genus Bacillus were obtained mainly from the native−forest and monoculture samples. The greatest diversity of bacterial genera was isolated from the agroforestry samples.

Table 2
Identification of 90 bacterial isolates sampled from roots of Brazil nut (Bertholletia excelsa) trees in native and cultivated habitats in Roraima (northern Brazil). The closest match in the NCBI database of the partial sequencing of the 16S rRNA gene is indicated, as well as the origin and medium of isolation of the isolates.

Isolates	Closest match	Origin	Medium
Class α -Proteobacteria
ERR 739	Ochrobactrum	Agroforestry	DYG’S
ERR 745	Ochrobactrum	Agroforestry	DYG’S
ERR 792	Ochrobactrum	Forest	DYG’S
ERR 858	Agrobacterium/ Rhizobium	Agroforestry	NFb
ERR 865	Agrobacterium/Rhizobium	Agroforestry	NFb
ERR 870	Agrobacterium/Rhizobium	Agroforestry	NFb
Class β - Proteobacteria
ERR 584	Burkholderia	Agroforestry	JMV
ERR 587	Burkholderia	Forest	JMV
ERR 589	Burkholderia	Monoculture	JMV
ERR 594	Burkholderia	Monoculture	JMV
ERR 689	Achromobacter/ Uncultured	Monoculture	DYG’S
ERR 737	Achromobacter/ Uncultured	Agroforestry	DYG’S
ERR 738	Achromobacter	Agroforestry	DYG’S
ERR 744	Uncultured	Agroforestry	DYG’S
ERR 763	Achromobacter/ Uncultured	Agroforestry	DYG’S
ERR 772	Delftia	Agroforestry	DYG’S
ERR 762	Delftia	Agroforestry	DYG’S
Class γ - Proteobacteria
ERR 596	Pantoea	Monoculture	LGI
ERR 602	Klebsiella	Agroforestry	LGI
ERR 604	Enterobacter	Agroforestry	LGI
ERR 626	Klebsiella	Agroforestry	LGI
ERR 640	Stenotrophomonas	Agroforestry	LGI
ERR 652	Enterobacter	Monoculture	DYG’S
ERR 692	Enterobacter	Monoculture	DYG’S
ERR 710	Pseudomonas	Agroforestry	DYG’S
ERR 711	Pseudomonas	Agroforestry	DYG’S
ERR 719	Serratia	Agroforestry	DYG’S
ERR 720	Pseudomonas	Agroforestry	DYG’S
ERR 721	Pseudomonas	Agroforestry	DYG’S
ERR 723	Pseudomonas	Agroforestry	DYG’S
ERR 727	Enterobacter	Agroforestry	DYG’S
ERR 750	Uncultured	Agroforestry	DYG’S
ERR 752	Pseudomonas	Agroforestry	DYG’S
ERR 761	Uncultured/ Stenotrophomonas	Agroforestry	DYG’S
ERR 765	Pseudomonas	Agroforestry	DYG’S
ERR 770	Stenotrophomonas/Uncultured	Agroforestry	DYG’S
ERR 771	Stenotrophomonas	Agroforestry	DYG’S
ERR 779	Pseudomonas	Forest	DYG’S
ERR 782	Uncultured	Forest	DYG’S
ERR 828	Stenotrophomonas	Agroforestry	NFb
ERR 833	Enterobacter	Forest	NFb
ERR 838	Uncultured	Agroforestry	NFb
ERR 843	Stenotrophomonas/ Uncultured	Agroforestry	NFb
ERR 849	Uncultured	Agroforestry	NFb
ERR 859	Stenotrophomonas	Agroforestry	NFb
ERR 873	Uncultured	Agroforestry	NFb
Class Bacilli
ERR 575	Paenibacillus	Agroforestry	JMV
ERR 651	Bacillus	Monoculture	DYG’S
ERR 654	Bacillus	Monocuture	DYG’S
ERR 656	Lysinibacillus	Monoculture	DYG’S
ERR 657	Bacillus	Monoculture	DYG’S
ERR 660	Bacillus	Monoculture	DYG’S
ERR 665	Bacillus	Monoculture	DYG’S
ERR 667	Bacillus	Monoculture	DYG’S
ERR 673	Bacillus	Monoculture	DYG’S
ERR 676	Bacillus	Monoculture	DYG’S
ERR 677	Paenibacillus	Monoculture	DYG’S
ERR 678	Paenibacillus	Monoculture	DYG’S
ERR 679	Bacillus	Monoculture	DYG’S
ERR 680	Bacillus	Monoculture	DYG’S
ERR 684	Paenibacillus	Monoculture	DYG’S
ERR 691	Bacillus	Monoculture	DYG’S
ERR 694	Bacillus	Monoculture	DYG’S
ERR 701	Bacillus	Monoculture	DYG’S
ERR 703	Lysimibacillus	Monoculture	DYG’S
ERR 705	Bacillus	Monoculture	DYG’S
ERR 706	Bacillus	Monoculture	DYG’S
ERR 708	Bacillus	Monoculture	DYG’S
ERR 709	Bacillus	Monoculture	DYG’S
ERR 717	Lysinibacillus	Agroforestry	DYG’S
ERR 741	Bacillus	Agroforestry	DYG’S
ERR 785	Bacillus	Forest	DYG’S
ERR 794	Lysinibacillus	Forest	DYG’S
ERR 795	Bacillus	Forest	DYG’S
ERR 797	Bacillus	Forest	DYG’S
ERR 799	Bacillus	Forest	DYG’S
ERR 800	Paenibacillus	Forest	DYG’S
ERR 803	Bacillus cereus	Forest	DYG’S
ERR 805	Bacillus	Forest	DYG’S
ERR 807	Bacillus	Forest	DYG’S
ERR 809	Bacillus	Forest	DYG’S
ERR 813	Bacillus	Forest	DYG’S
ERR 819	Bacillus	Forest	DYG’S
ERR 821	Bacillus	Forest	DYG’S
ERR 830	Bacillus	Forest	NFb
Class Actinobacteria
ERR 644	Curtobacterium	Forest	LGI
ERR 733	Microbacterium	Agroforestry	DYG’S
ERR 753	Microbacterium	Agroforestry	DYG’S
ERR 773	Microbacterium	Agroforestry	DYG’S
ERR 822	Curtobacterium	Forest	DYG’S

nif H gene amplification

Fourteen bacterial isolates were positive for the amplification of DNA with primers 19F and 407R, corresponding to a region of the nifH gene (approximately 400 bp). These bacteria were identified as belonging to Bacillus (ERR 667, ERR 679, ERR 785, ERR 819, ERR 821 and ERR 830), Paenibacillus (ERR 684), Enterobacter (ERR 604 and ERR 838), Klebsiella (ERR 602), Stenotrophomonas (ERR 640), Pseudomonas (ERR 711), Delftia (ERR 762) and Microbacterium (ERR 773) (Table 2). Seven of these isolates were from the agroforestry system; four from the forest, and three from the monoculture. Among the 14 isolates, five were obtained from media selective for Azospirillum spp. (NFb and LGI) (ERR 602, ERR 604, ERR 640, ERR 830, ERR 838) and nine from nonselective medium (DYG’s medium) (ERR 667, ERR 679, ERR 684, ERR 711, ERR 762, ERR 773, ERR 785, ERR 819, ERR 821).

Aluminum phosphate solubilization assays

None of the bacteria or type strains showed a halo of solubilization on solid medium. In liquid medium, however, 88.9% of the isolates were able to solubilize aluminum phosphate (Table 3). Some isolates, such as ERR 587, ERR 589, ERR 733, ERR 719 and ERR 584, had significantly higher values and made available up to 14% of the P added to the medium (Table 3). Type or reference strains such as BR 11001^T (A. brasilense), BR 11340 (Burkholderia sp.), BR 11790^T (H. frisigense), BR 11175^T (H. seropedicae) and ERR 532 (Bacillus sp.) could also solubilize aluminum phosphate but showed lower solubilization compared to ERR 587, ERR 589, ERR 733 and ERR 719. These isolates were identified as Burkholderia sp. (ERR 587 and ERR 589), Microbacterium sp. (ERR 733) and Serratia sp. (ERR 719). A reduction in the pH of the media was observed with increasing solubilization for the majority of the bacteria (Table 3).

Table 3
Soluble phosphate after solubilization by bacterial isolates sampled from roots of Brazil nut (Bertholletia excelsa) trees in native and cultivated habitats in Roraima (northern Brazil), and reference strains in liquid media. Different letters in columns indicate statistical differences (p < 0.05) according to the Skott-Knott test. The coefficient of variation was 35.9%. Isolates in bold were used as controls.

Isolate	Soluble P (mg l-1)	pH	Soluble P (%)		Isolate	Soluble P (mg l-1)	pH	Soluble P (%)
ERR 587	1.69 a	3.7	14.1		ERR 805	0.30 e	3.7	2.5
ERR 589	1.54 a	3.0	12.8		ERR 677	0.25 e	3.9	2.1
ERR 733	1.38 a	3.0	11.5		ERR 828	0.24 e	4.4	2.0
ERR 719	1.23 b	3.1	10.2		ERR 640	0.24 e	3.8	2.0
ERR 584	1.14 b	3.3	9.5		ERR 779	0.23 e	4.0	1.9
ERR 723	0.99 c	3.3	8.3		ERR 799	0.23 e	4.4	1.9
ERR 833	0.98 c	3.4	8.1		ERR 809	0.22 e	4.7	1.9
ERR 727	0.96 c	3.4	8.0		ERR 813	0.22 e	4.4	1.9
ERR 773	0.93 c	4.1	7.8		ERR 738	0.22 e	4.7	1.8
ERR 753	0.84 c	4.2	7.0		ERR 800	0.20 f	4.1	1.7
ERR 765	0.83 c	4.1	6.9		ERR 706	0.20 f	4.3	1.6
ERR 721	0.78 c	4.1	6.5		ERR 772	0.20 f	4.2	1.6
ERR 694	0.78 c	3.9	6.5		ERR 858	0.20 f	4.3	1.6
ERR 752	0.76 c	4.0	6.3		BR 11001	0.19 f	3.8	1.6
ERR 678	0.72 d	4.0	6.0		ERR 657	0.19 f	3.5	1.6
ERR 594	0.72 d	3.4	6.0		ERR 744	0.19 f	4.0	1.6
ERR 602	0.69 d	3.3	5.8		ERR 692	0.15 f	3.4	1.3
ERR 821	0.69 d	3.7	5.8		ERR 830	0.15 f	4.2	1.3
ERR 708	0.68 d	3.6	5.7		ERR 575	0.14 f	3.7	1.2
BR 11340	0.63 d	3.6	5.3		ERR 673	0.14 f	3.0	1.2
ERR 604	0.62 d	3.9	5.2		ERR 782	0.14 f	4.5	1.1
ERR 596	0.61 d	3.4	5.1		ERR 849	0.14 f	4.4	1.1
ERR 626	0.61 d	3.3	5.1		ERR 803	0.13 f	4.5	1.1
ERR 652	0.60 d	3.4	5.0		ERR 865	0.13 f	4.4	1.1
ERR 711	0.58 d	3.9	4.8		ERR 651	0.12 f	4.2	1.0
ERR 720	0.58 d	4.1	4.8		ERR 870	0.12 f	4.6	1.0
ERR 838	0.58 d	3.5	4.8		ERR 795	0.11 f	4.4	0.9
ERR 739	057 d	3.8	4.8		ERR 761	0.10 f	4.6	0.9
ERR 771	0.57 d	3.7	4.7		ERR 656	0.10 f	4.3	0.8
ERR 785	0.55 d	4.1	4.6		ERR 750	0.09 f	4.3	0.8
ERR 684	0.53 d	3.8	4.4		ERR 807	0.09 f	4.5	0.8
ERR 667	0.52 d	3.6	4.3		ERR 859	0.09 f	4.3	0.8
ERR 680	0.48 e	3.7	4.0		ERR 770	0.09 f	4.5	0.7
ERR 745	0.45 e	3.9	3.7		ERR 665	0.08 f	4.2	0.7
ERR 763	0.45 e	3.7	3.7		ERR 737	0.08 f	4.4	0.7
BR 11790	0.44 e	4.1	3.6		ERR 822	0.08 f	3.9	0.7
ERR 532	0.42 e	3.6	3.5		ERR 843	0.08 f	4.2	0.7
ERR 741	0.40 e	3.9	3.3		ERR 703	0.07 f	4.0	0.6
ERR 717	0.39 e	4.4	3.2		ERR 644	0.07 f	3.9	0.6
ERR 710	0.37 e	4.0	3.1		ERR 660	0.06 f	4.2	0.5
ERR 762	0.37 e	4.1	3.1		ERR 679	0.06 f	4.3	0.5
BR 11175	0.36 e	4.2	3.0		ERR 654	0.05 f	4.4	0.4
ERR 819	0.35 e	4.3	2.9		ERR 691	0.01 f	4.4	0.1
ERR 792	0.35 e	3.9	2.9		ERR 701	0.01 f	4.4	0.1
ERR 794	0.34 e	4.2	2.8		ERR 705	0.01 f	4.3	0.1
ERR 797	0.34 e	4.5	2.8		ERR 873	0.01 f	4.4	0.1
ERR 689	0.30 e	3.9	2.5		ERR 676	0.01 f	4.4	0.1

Production of indole compounds

Fifty-eight bacteria were able to synthetize indole compounds in media supplemented with tryptophan (Figure 2a). The production of isolates ERR 596 (184.47 µg ml^-1), ERR 626 (104.61 µg ml^-1), and ERR 723 (92.43 µg ml^-1), as well as the type strain H. seropedicae BR 11175^T (104.67 µg ml^-1), was significantly greater than that of the other bacteria and the type strain A. brasilense BR 11001^T. These bacteria were identified as Pantoea sp. (ERR 596), Klebsiella sp. (ERR 626) and Pseudomonas (ERR 723). However, in the absence of tryptophan, the production decreased from ERR 744 (Achromobacter sp.) with 33.77 µg mL^-1, to ERR 779 (Pseudomonas sp.) (31.61 µg mL^-1), ERR 710 (Pseudomonas sp.) (30.96 µg mL^-1), ERR 656 (Lysinibacillus sp.) (30.81 µg mL^-1) and ERR 626 (Klebsiella sp.) (30.24 µg mL^-1) (Figure 2b).

Figure 2
Production of indole compounds by endophytic bacterial isolates obtained from the roots of Brazil nut (Bertholletia excelsa) trees in native and cultivated habitats in media supplemented with (A) and without (B) tryptophan.

Selection of bacteria

After all analyses, 22 bacterial isolates (Table 4) were selected for best results in growth promotion, and as potential candidates for inoculation during seed germination and seedling production of Brazil nut trees. They belonged to 12 genera (Burkholderia, Pantoea, Enterobacter, Klebsiella, Stenotrophomonas, Lysinibacillus, Bacillus, Pseudomonas, Serratia, Microbacterium, Achromobacter and Delftia) and were obtained from the agroforestry system (10), monoculture (6) and terra firme forest (4).

Table 4
Genus, origin and growth promotion characteristics of selected isolates among 90 bacterial isolates sampled from roots of Brazil−nut (Bertholletia excelsa) trees in native and cultivated habitats in Roraima (northern Brazil). *PS = phosphate solubilization; IC⁺ = indole compounds production with tryptophan; IC = indole compounds production without tryptophan; NF = nitrogen fixation.

Isolate	Genus	Origin	Growth promotion*
ERR 584	Burkholderia	Agroforestry	PS
ERR 587	Burkholderia	Native Forest	PS
ERR 589	Burkholderia	Monoculture	PS
ERR 596	Pantoea	Monoculture	IC+
ERR 604	Enterobacter	Agroforestry	IC+/ NF
ERR 626	Klebsiella	Agroforestry	IC
ERR 640	Stenotrophomonas	Agroforestry	IC+/NF
ERR 656	Lysinibacillus	Monoculture	IC-
ERR 680	Bacillus	Monoculture	IC+
ERR 692	Enterobacter	Monoculture	IC
ERR 703	Lysinibacillus	Monoculture	IC
ERR 710	Pseudomonas	Agroforestry	IC
ERR 719	Serratia	Agroforestry	PS/IC+
ERR 723	Pseudomonas	Agroforestry	PS/IC+
ERR 733	Microbacterium	Agroforestry	PS
ERR 744	Achromobacter	Agroforestry	IC-
ERR 753	Microbacterium	Agroforestry	PS
ERR 773	Microbacterium	Agroforestry	OS/NF
ERR 779	Pseudomonas	Native Forest	IC-
ERR 833	Enterobacter	Native Forest	PS
ERR 762	Delftia	Agroforestry	IC/NF
ERR 779	Pseudomonas	Native Forest	IC

DISCUSSION

We report the first isolation and characterization of endophytic bacteria from the roots of Brazil nut trees. The bacterial classes identified are generally dominated by endophytes in diversity analyses (Santoyo et al. 2016). However, most studies using plant growth-promoting bacteria have been performed with grasses (Moreira et al. 2010), whereas few such works have involved trees. In Brazil, only one study involved endophytic plant growth-promoting bacteria isolated from a native tree species [Araucaria angustifolia (Bertol.) Kuntze.] in southern Brazil (Neroni and Cardoso 2007), which identified diazotrophic bacteria in soil and root samples as belonging to the genus Burkholderia, while the PGPB belonged mainly to Bacillus and Enterobacter. In our Brazil−nut tree root samples, Bacillus was the predominant genus, followed by Stenotrophomonas and Pseudomonas.

The highest diversity of bacterial isolates in our samples was found in the agroforestry system (15 genera), relative to Brazil nut trees in native forest and monoculture (eight and seven genera, respectively). The cultivation of agroforestry systems represents a sustainable practice for carbon sequestration in soil (Abbas et al. 2017). Agroforestry can enrich soil organic carbon, improve soil nutrient availability and fertility due to the presence of trees in the system, litter decomposition and mineralization, and the increase of soil microbial dynamics (Dollinger and Jose 2018). Our agroforestry area also received limestone and fertilizer application, which could be related to an increase in the quantity and quality of litter inputs and, consequently, more soil organic matter accumulation in the soil. In Amazon soils, bacterial community structure and composition are related to land use, likely through the effects of soil attributes, particularly those related to soil acidity (Jesus et al. 2009). These characteristics may explain the higher bacterial diversity associated with Brazil nut roots agroforestry system in our samples, as the soil in this area had lower acidity and higher nutrient content (see Table 1).

The observed diversity was also influenced by the medium utilized for isolation, as the highest diversity was found in DYG’S medium (12 genera), that is rich and nonselective (Rodrigues Neto et al. 1986). For example, the JMV medium is selective for Burkholderia (Estrada De Los Santos et al. 2001), and most isolates obtained from this medium belonged to Burkholderia. We did not detect the presence of Azospirillum spp. using NFb or LGI media, which are known to favor these species (Silva et al. 2011a).

Endophytic bacteria can increase host fitness via many different functions, such as N₂ fixation, P solubilization, and phytohormone production (Compant et al. 2010). Nitrogen fixation has been the focus of many studies seeking alternatives to reduce the use of nitrogen fertilizers (Moreira et al. 2010; Glick 2012). Fourteen of the 90 isolates showed positive amplification of the nifH gene, which qualifies them as potential N₂-fixing bacteria that could presumably be considered diazotrophs. The majority were obtained using LGI, NFb and DYG’s media and belonged to Klebsiella, Enterobacter, Burkholderia, Stenotrophomonas, Bacillus, Paenibacillus, Pseudomonas, Delftia and Microbacterium.

In acidic soils in tropical regions, most P is precipitated with iron and aluminum, as is the case in soils from Roraima, which have low natural fertility and contain aluminum (Vale Junior and Leitão Sousa 2005). There are fewer studies involving the solubilization of aluminum phosphate than that of calcium phosphate, but some have reported a halo of solubilization on solid media (Marra et al. 2011, 2012; Oliveira-Longatti et al. 2014), while others have indicated the absence of a halo of solubilization on media with aluminum phosphate (Pérez et al. 2007; Silva et al. 2011a). For our isolates, as well as the type or reference strains tested, no halo of solubilization was observed on the utilized media. Phosphate solubilization tests (Ca, Al and Fe) have been widely used for the selection of plant growth-promoting bacteria (Hara and Oliveira 2004, 2005; Marra et al. 2011, 2012; Nautiyal, 1999; Oliveira-Longatti et al. 2014; Silva Filho and Vidor 2000; Silva Filho et al. 2002; Silva et al. 2011b; Silva et al. 2012). However, solubilization tests in solid media show relatively poor effectiveness for universal selection to isolate solubilizing bacteria in vitro (Bashan et al. 2012). Bacteria that have performed solubilization many times could grow on solid media without a visible halo and may present other mechanisms of solubilization that do not result in a visible halo (Bashan et al. 2012; Fankem et al. 2008; Illmer et al. 1995). In liquid media, most of our bacterial isolates could solubilize aluminum phosphate, therefore, a visible halo on solid media should not be used as a unique test for solubilizing bacteria, and quantitative tests should also be used (Bashan et al. 2012). As happened in our samples, Illmer et al. (1995) also reported a pH reduction in cultures of bacteria and fungi that are able to solubilize aluminum phosphate and attributed the decrease to the production of organic acids and/or H⁺ excretion accompanying NH₄ assimilation. Among the five best phosphorus-solubilizing bacteria in our work, three belong to the Burkholderia genus.

Indole compound production was variable among the isolates, but most of them could produce indoles with and without tryptophan addition. Tryptophan has been identified as a main precursor for IAA biosynthesis pathways in bacteria (Spaepen et al. 2007), which could explain our results regarding greater numbers of bacterial isolates able to produce indolic compounds with tryptophan. The IAA produced by bacteria acts in seed germination and in the radicular system, increasing the size and number of adventitious roots and deep ramifications, allowing a greater volume of soil to be explored by the roots (Glick 2012). This mechanism is an important tool for use in seed germination and seedlings production of Brazil nut.

CONCLUSIONS

Our results indicated that there is a great diversity of endophytic bacteria in the roots of Brazil nut trees in Roraima state, in the northern Brazilian Amazon. The diversity of genera was higher in isolates obtained from an agroforestry area than in those from a Brazil nut monoculture and a native terra firme forest area. The obtained endophytic bacteria presented plant growth−promotion characteristics, such as nitrogen fixation, phosphate solubilization and indole compound production. Among the 90 isolates obtained, at least 22 that presented the best results for plant-growth promotion traits are promising candidates for use in the seed germination and seedling production of Brazil nut.

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• CITE AS:

  Chalita, P.B.; Farias, E. do N.C.; Costa, I.B. da; Sousa, B.F.; Santos, M.A.O. dos; Albuquerque, T.C.S. de; Vital, M.J.S.; Silva, K. da. 2019. Characterization of bacterial endophytes from the roots of native and cultivated Brazil nut trees (Bertholletia excelsa). Acta Amazonica 49: 257-267

Edited by

ASSOCIATE EDITOR:

Oscar Valverde

Publication Dates

• Publication in this collection
  04 Nov 2019
• Date of issue
  Oct-Dec 2019

History

• Received
  26 Dec 2018
• Accepted
  21 Aug 2019