Bioprospecting and selection of growth-promoting bacteria for Cymbidium sp. orchids

ABSTRACT:

Inoculants containing bacteria which promote growth in plants can increase productivity and both the economic and the environmental cost in plant crop systems. Similarly, in the flower and ornamental plant sector, the use of diazotrophic bacteria is a promising approach for improving orchid propagation from tissue culture to the ex vitro environment. We isolated diazotrophic bacteria from the roots and leaves of Cymbidium sp. The isolates were used to inoculate Cymbidium sp. plantlets during acclimatization in the nursery. After 150 days, plants were collected and their morphological and nutritional characteristics assessed. Eight bacterial strains were isolated containing traits that promote plant growth: Bacillus thuringiensis, Burkholderia cepacia, Burkholderia gladioli, Herbaspirillum frisingense, Pseudomonas stutzeri, Rhizobium cellulosilyticum, Rhizobium radiobacter, and Stenotrophomonas maltophilia. The isolated Herbaspirillum frisingense and Stenotrophomonas maltophilia increased 26 % and 29 % in dry matter in Cymbidium sp. plants, respectively, compared to the control. In addition, H. frisingense led to higher contents of N and P, by 68 % and 28 %, respectively, than those found in the control plants. These isolates, therefore, have potential for application as biostimulants and biofertilizers to promote growth and development of Cymbidium sp. during acclimatization.

Keywords:
orchidaceae; diazotrophic bacteria; phosphate-solubilizing bacteria; endophytic bacteria; acclimatization

Introduction

Sustainable agriculture requires strategies that increase productivity and minimize environmental damage. Among the new sustainable technologies being developed, the formulation and application of inoculants and/or biofertilizers containing bacteria that promote growth in plants have shown promising results in a variety of crops (Hallmann et al., 1997Hallmann, J.; Quadt-Hallmann, A.; Mahaffee, W.F.; Kloepper, J.W. 1997. Bacterial endophytes in agricultural crops. Canadian Journal of Microbiology 43: 895-914.). The promotion of plant growth through bacterial inoculation is mediated in part by biological nitrogen fixation as well as by factors such as the solubilization of rock phosphate, synthesis of phytohormones and siderophores, and the biological control and systemic resistance of host plants (Hallmann et al., 1997Hallmann, J.; Quadt-Hallmann, A.; Mahaffee, W.F.; Kloepper, J.W. 1997. Bacterial endophytes in agricultural crops. Canadian Journal of Microbiology 43: 895-914.; Hardoim et al., 2008Hardoim, P.R.; van Overbeek, L.S.; van Elsas, J.D. 2008. Properties of bacterial endophytes and their proposed role in plant growth. Trends in Microbiology 16: 463-471.; Ryan et al., 2008Ryan, R.P.; Germaine, K.; Franks, A.; Ryan, D.J.; Dowling, D.N. 2008. Bacterial endophytes: recent developments and applications. FEMS Microbiological Letters 278: 1-9.; Singh et al., 2011Singh, J.S.; Pandey, V.C.; Singh, D.P. 2011. Efficient soil microorganisms: a new dimension for sustainable agriculture and environmental development. Agriculture, Ecosystems and Environment 140: 339-353.).

The expansion of the flower and ornamental plant industry, in association with the use of diazotrophic bacteria as growth and plant protection agents, has encouraged research into not only bacteria-orchid interactions but also isolating, characterizing, and re-introducing growth-promoting bacteria into the cultivation environment. In order to cultivate in vitro propagated plants, for example, inoculants containing growth-promoting bacteria might be of value for accelerating the development of plantlets and decreasing the long acclimatization period (Baldotto et al., 2010Baldotto, L.E.B.; Baldotto, M.A.; Olivares, F.L.; Viana, A.P.; Bressan-Smith, R. 2010. Selection of growth-promoting bacteria for pineapple ‘Vitória’ during acclimatization. Revista Brasileira de Ciência do Solo 34: 349-360 (in Portuguese, with abstract in English).; Faria et al., 2013Faria, D.C.; Dias, A.C.F.; Melo, I.S.; Costa, F.E.C. 2013. Endophytic bacteria isolated from orchid and their potential to promote plant growth. World Journal of Microbiology and Biotechnology 29: 217-221).

Among cultivated orchids, Cymbidium sp. hybrids form a large group of epiphytic, terrestrial, and rhizomatous plants that originate in Asia (Choi et al., 2006Choi, S.H.; Kim, M.J.; Lee, J.S.; Kyu, K.H. 2006. Genetic diversity and phylogenetic relationships among and within species of oriental cymbidiums based on RAPD analysis. Scientia Horticulturae 108: 79-85.). Since they are popular ornamental plants, they require propagation methods that ensure quick formation of a large number of vigorous and healthy plantlets. In this respect, in vitro propagation is an appropriate method, as it allows for the production of a large number of plantlets free of pests and diseases, with requirement for little space and the possibility of establishing production and marketing schedules (Chugh et al., 2009Chugh, S.; Guha, S.; Usha, R.I. 2009. Micropropagation of orchids: a review on the potential of different explants. Scientia Horticulturae 122: 507-520.). Strategies that accelerate the growth of Cymbidium sp. plantlets during acclimatization are attractive, and offer the possibility of reestablishing the association between orchids and beneficial microbiota.

This study was undertaken in order to (1) isolate diazotrophic bacteria found in the roots and the leaves of orchids; (2) assess diazotrophic bacterial strains for their ability to solubilize phosphate and zinc oxide, and synthesize indole compounds; (3) identify the bacterial isolates; and (4) evaluate the growth and development of Cymbidium sp. plantlets in response to bacterial inoculation during acclimatization.

Materials and Methods

Plant material for bacterial isolation

The study was carried out in Florestal, in the state of Minas Gerais, in Brazil (19°53′22″ S, 44°25′57″ W, 776 m above sea level). Samples of roots and leaves of Cymbidium sp. orchids were collected from the orchid collection at Florestal. The one mother plant is kept in a greenhouse, and is grown in a ceramic pot containing coconut fiber as a substrate.

Isolation of diazotrophic bacteria

Endophytic diazotrophic bacteria were isolated as previously described by Döbereiner et al. (1995)Döbereiner, J.; Baldani, V.L.D.; Baldani, J.I. 1995. How to isolate and identify diazotrophic bacteria from non-leguminous plants = Como isolar e identificar bactérias diazotróficas de plantas não-leguminosas. Embrapa Agrobiologia, Seropédica, RJ, Brazil (in Portuguese). with a number of alterations. One gram of root sample and 1.0 g of leaf sample were macerated in 9.0 mL of saline solution (NaCl, 8.5 g L−1). Serial dilutions of this suspension were obtained by adding 1.0 mL of the suspension to 9 mL of the saline solution; this series was concluded at 10−6 dilution. Aliquots (100 µL) from the different dilutions were transferred in triplicate to glass bottles containing 5 mL of the semi-solid, semi-selective culture media JMV (mannitol 5.0 g L−1, K2HPO4 0.6 g L−1, KH2PO4 1.8 g L−1, MgSO4.7H2O 0.2 g L−1, NaCl 0.1 g L−1, CaCl2.2H2O 0.02 g L−1, Na2MoO4.2H2O 0.002 g L−1, MnSO4.H2O 0.00235 g L−1, H3BO3 0.0028 g L−1, CuSO4.5H2O 8.0 × 10−5 g L−1, ZnSO4.7H2O 0.002 g L−1, bromothymol blue (0.5 % solution in 0.2 M KOH) 2 mL, iron ethylenediamine tetraacetic acid (FeEDTA) (solution 1.64 %) 4 mL, biotin 1.0 × 10−4, HCl- pyridoxine 2.0 × 10−4 and agar 2.1 g L−1, pH between 4.2 and 4.5), JMVL (mannitol 5.0 g L−1, K2HPO4 0.6 g L−1, KH2PO4 1.8 g L−1, MgSO4.7H2O 0.2 g L−1, NaCl 0.1 g L−1, CaCl2.2H2O 0.02 g L−1, Na2MoO4.2H2O 0.002 g L−1, MnSO4.H2O 0.00235 g L−1, H3BO3 0.0028 g L−1, CuSO4.5H2O 8.0 × 10−5 g L−1, ZnSO4.7H2O 0.002 g L−1, bromothymol blue (0.5 % solution in 0.2 M NaOH) 2 mL, FeEDTA (solution 1.64 %) 4 mL, biotin 1.0 × 10−4, HCl- pyridoxine 2.0 × 10−4, yeast extract 0.02 g L−1 and agar 1.6 g L−1, pH between 5.0 and 5.4), NFb (malic acid 5.0 g L−1, K2HPO4 0.5 g L−1, MgSO4.7H2O 0.2 g L−1, NaCl 0.1 g L−1, CaCl2.2H2O 0.01 g L−1, Na2MoO4.2H2O 0.002 g L−1, MnSO4.H2O 0.00235 g L−1, H3BO3 0.0028 g L−1, CuSO4.5H2O 8.0 × 10−5 g L−1, bromothymol blue (0.5 % solution in 0.2 M KOH) 2 mL, FeEDTA (solution 1.64 %) 4 mL, agar 1.6 g L−1, at pH 6.8), JNFb (malic acid 5.0 g L−1, K2HPO4 0.6 g L−1, KH2PO4 1.8 g L−1, MgSO4.7H2O 0.2 g L−1, NaCl 0.1 g L−1, KOH 4.5 g L−1, CaCl2.2H2O 0.02 g L−1, Na2MoO4.2H2O 0.002 g L−1, MnSO4.H2O 0.00235 g L−1, H3BO3 0.0028 g L−1, CuSO4.5H2O 8.0 × 10−5 g L−1, ZnSO4.7H2O 0.002 g L−1, bromothymol blue (0.5 % solution in 0.2 M KOH) 2 mL, FeEDTA (solution 1.64 %) 4 mL, biotin 1.0 × 10−4 g L−1 and HCl- pyridoxine 2.0 × 10−4 g L−1, agar 1.7 g L−1, at pH 5.8), and LGI (sacarose 5.0 g L−1, K2HPO4 0.2 g L−1, KH2PO4 0.6 g L−1, MgSO4.7H2O 0.2 g L−1, CaCl2.2H2O 0.02 g L−1, Na2MoO4.2H2O 0.002 g L−1, KOH 4.5 g L−1, bromothymol blue (0.5 % solution in 0.2 M KOH) 2 mL, FeEDTA (solution 1.64 %) 4 mL, biotin 1.0 × 10−4 g L−1 and HCl-pyridoxine 2.0 × 10−4 g L−1, agar 1.4 g L−1, pH between 6.0 and 6.2) (Baldani et al., 1986Baldani, J.I.; Baldani, V.L.D.; Seldin, L.; Döbereiner, J. 1986. Characterization of Herbaspirillum seropedicae ge. nov., sp. nov., a root-associated nitrogen-fixing bacterium. International Journal of Systematic Bacteriology 36: 86-93.; Baldani et al., 1996Baldani, V.L.D.; Baldani, J.I.; Döbereiner, J. 1996. Specific culture media for the isolation of endophytic bacteria that fix atmospheric N2 = Meios de cultura específicos para o isolamento de bactérias endofíticas que fixam N2 atmosférico. CNPAB, Seropédica, RJ, Brazil (in Portuguese).; Döbereiner et al., 1995Döbereiner, J.; Baldani, V.L.D.; Baldani, J.I. 1995. How to isolate and identify diazotrophic bacteria from non-leguminous plants = Como isolar e identificar bactérias diazotróficas de plantas não-leguminosas. Embrapa Agrobiologia, Seropédica, RJ, Brazil (in Portuguese).), all without added nitrogen. The formation of a typical aerotaxic film on the surface of the medium was observed 7 days after incubation in a growth chamber at 30 °C and was considered positive growth. Subsequently, bacteria grown at greater dilution factors were transferred to new semisolid media, where they were grown for 7 days, followed by plating on the appropriate solid medium. Individual colonies with different morphological characteristics were transferred to fresh semi-solid media and then to solid DYGS medium to check the purity of the isolates (Döbereiner et al., 1995Döbereiner, J.; Baldani, V.L.D.; Baldani, J.I. 1995. How to isolate and identify diazotrophic bacteria from non-leguminous plants = Como isolar e identificar bactérias diazotróficas de plantas não-leguminosas. Embrapa Agrobiologia, Seropédica, RJ, Brazil (in Portuguese).). Once purified, the colonies were kept in sterile distilled water.

Evaluation of the capacity to synthesize indole compounds

Bacterial isolates were grown in liquid DYGS medium for 24 h at 30 °C and 120 rpm. To evaluate the synthesis of indole compounds, 10 µL of bacterial cultures were transferred to plates containing 1/10 TSA (Trypticase Soy Agar) medium (Brick et al., 1991Brick, J.M.; Bostock, R.M.; Silversone, S.E. 1991. Rapid in situ assay for indole acetic acid production by bacteria immobilized on nitrocellulose membrane. Applied Environmental Microbiology 57: 535-538.). After the transfer, the medium was covered with a nitrocellulose membrane and incubated at 28 °C for 24 h. Subsequently, the membrane was transferred to another plate, saturated with Salkowski solution (Gordon and Weber, 1951Gordon, S.A.; Weber, R.P. 1951. Colorimetric estimation of indoleacetic acid. Plant Physiology 26: 192-195.), and incubated at room temperature for 30 min. Formation of a reddish halo on the membrane indicated the presence of indole synthesized by the bacteria. Three replicates for each bacterial strain were conducted.

Evaluation of the calcium phosphate and zinc oxide solubilization capability

Bacteria were grown in DYGS liquid medium for 24 h at 30 °C and 120 rpm. Bacterial solution samples of 20 µL were placed on Petri plates with solid culture media containing insoluble phosphate (10.0 g L−1 glucose, 5.0 g L−1 of NH4Cl, 1.0 g L−1 MgSO4.7H2O, 4.0 g L−1 of CaHPO4, 15.0 g L−1 of agar, pH 7.2) or zinc oxide (10.0 g L−1 glucose, 1.0 g L−1 (NH4)2SO4, 0.2 g L−1 of KCl, 0.1 g L−1 K2HPO4, 0.2 g L−1 of MgSO4.7H2O, 1.0 g L−1 ZnO, 15.0 g L−1 agar, pH 7.0), and incubated at 30 °C for 72 h (Baldotto et al., 2010Baldotto, L.E.B.; Baldotto, M.A.; Olivares, F.L.; Viana, A.P.; Bressan-Smith, R. 2010. Selection of growth-promoting bacteria for pineapple ‘Vitória’ during acclimatization. Revista Brasileira de Ciência do Solo 34: 349-360 (in Portuguese, with abstract in English).). The solubilization activities for calcium phosphate and zinc oxide were evaluated by examining the plates for the presence of a translucent halo around the solubilizing colonies on each medium. Three replicate experiments were carried out for each bacterial strain.

Identification of bacterial isolates

A number of bacterial isolates were identified by analyzing fatty acid methyl esters (GC-FAME) (Sasser, 2006Sasser, M. 2006. Bacterial Identification by Gas Chromatographic Analysis of Fatty Acids Methyl Esters (GC-FAME). MIDI, Newark, DE, USA. (Technical Note, 101).) at Viçosa, in the state of Minas Gerais. Bacterial isolates were grown on TSA medium for 24 h at 30 °C, and then again in fresh medium for a second incubation. A cell sample of approximately 3 mg was collected to extract fatty acids. Fatty acids were extracted and obtained using the Instant Fame kit (Midi, Newark, DE), following the manufacturer's recommendations. The Sherlock® Microbial Identification (Midi, Newark, DE) system was used to determine the composition of the bacterial fatty acids. Following extraction and quantification, the fatty acid profile was compared with a library to identify the sample.

Further bacterial isolates were identified by sequencing the 16S rRNA gene at Piracicaba, in the state of São Paulo. Genomic DNA was extracted from bacterial isolates using the protocol described by Stirling (2003)Stirling, D. 2003. DNA extraction from fungi, yeast and bacteria. p.53-54. In: Bartlett, J.M.S.; Stirling, D., eds. Methods in molecular biology: PCR Protocols. 2ed. Humana Press, Totowa, NJ, USA.. The 16S rRNA gene was amplified with the following oligonucleotide primers for Eubacteria (5′-AGA GTT TGA TCC TGG CTC AG-3′) and rD1 (5′-AAG GAG GTG ATC CAG CC-3′) (Weisburg et al., 1991Weisburg, W.G.; Barns, S.M.; Pelletier, D.A.; Lane, D.J. 1991. 16S Ribosomal DNA amplification for phylogenetic study. Journal of Bacteriology 173: 697-703.). The 16S rRNA gene amplifications were conducted in a final volume of 25 µL that contained 5 pmol of oligonucleotide primers, 200 µM of each dNTP, 1 × Taq buffer, 1.5 mM MgCl2, 2 U Platinum Taq polymerase DNA and 10 ng DNA. The reaction was initiated with 3 min of denaturation at 94 °C, followed by 30 cycles of denaturation at 94 °C for 1 min, annealing at 55 °C for 30 s, extension at 72 °C for 30 s, and final extension at 72 °C for 32 min. The sequencing polymerase chain reaction (PCR) of the 16S rRNA fragments was conducted in a final volume of 10 µL with 5 pmol of initiator oligonucleotides, 1.0 µL of 2.5 × buffer; 3.0 µL of Big Dye Terminator 30 Cycle Sequencing v.3 (Applied Biosystems, USA). The oligonucleotides used were the same as in the previous amplification (Weisburg et al., 1991Weisburg, W.G.; Barns, S.M.; Pelletier, D.A.; Lane, D.J. 1991. 16S Ribosomal DNA amplification for phylogenetic study. Journal of Bacteriology 173: 697-703.). Amplification conditions were 2 min of denaturation at 96 °C, followed by 30 cycles of denaturation at 95 °C for 20 s, annealing at 55 °C for 15 s, and extension at 60 °C for 1 min. After amplification of the fragments, unincorporated dNTPs were eliminated by precipitation. The reading of the labeled bases was performed on an Automatic Sequencer ABI Prism 3130 Genetic Analyzer, which uses capillary electrophoresis to separate and detect the amplified fragments. The sequences were compared in the BLAST database (NCBI).

Plant material for greenhouse experiments

Cymbidium sp. plantlets of the Angelica variety were grown in glass bottles containing MS medium (Murashigue and Skoog, 1962Murashigue, T.; Skoog, F. 1962. A revised medium for rapid growth and bioassays with tobacco tissue culture. Physiologia Plantarum 15: 473-497.) with no added growth regulators or vitamins. To carry out the subsequent experimental steps, plantlets with approximately 1.0 g of fresh matter were selected.

Bacterial growth and inoculation

The bacteria were grown in DYGS liquid medium in a shaker for 24 h, at 30 °C and 120 rpm, and inoculation was performed by immersing the orchid plantlets in 50 mL of bacterial medium for 2 h, with further application of the same bacteria on the substrate (Baldotto et al., 2010Baldotto, L.E.B.; Baldotto, M.A.; Olivares, F.L.; Viana, A.P.; Bressan-Smith, R. 2010. Selection of growth-promoting bacteria for pineapple ‘Vitória’ during acclimatization. Revista Brasileira de Ciência do Solo 34: 349-360 (in Portuguese, with abstract in English).). The control was immersed in autoclaved liquid DYGS medium. Later, the propagative materials were transferred to 1.0 dm3 plastic pots (two plantlets per pot) containing Bioplant® commercial substrate for acclimatization in a greenhouse for 150 days. Each pot was fertilized every 15 days with 5 mL liquid mineral fertilizer BeG Orquídeas®.

Growth analysis

After acclimatization, the plants were collected for measurement of the following variables: number of leaves; plant height, which was measured by the distance between the lower internode to the leaf apex using a tape-measure: base diameter, measured with a Starrett 727 digital caliper; fresh matter from the root and aerial parts; and dry matter of the root and aerial parts, which were obtained after material had been kiln dried under forced air ventilation at 65 °C for 48 h. Total fresh matter, total dry matter, and the relationship between root and aerial parts were calculated.

Nutritional analysis

After drying, the leaves of the orchids were ground in a Wiley mill coupled to six 50 cm2 mesh sieves. Next, the powder obtained was subjected to sulfuric acid digestion combined with hydrogen peroxide and the total contents of nitrogen (N), phosphorus (P), potassium (K), calcium (Ca) and magnesium (Mg) were determined. For the measurement of N, Nessler's method was used; P content was obtained by molecular absorption spectrophotometry (colorimetry, at a wavelength of 725 nm) after a reaction with vitamin C and ammonium molybdate; K was measured by using flame photometry, and the Ca and Mg contents were obtained by atomic absorption spectrophotometry. The contents of N, P, K, Ca, and Mg were estimated by multiplying the dry matter of the aerial part by the nutrient content.

Statistical analysis

The experiment was conducted in a random block design with six replicates and each experimental unit consisted of a pot containing two plants. The data were analyzed by analysis of variance (ANOVA) and LSD t-tests using the SISVAR software program v. 5.4 at Lavras, Brazil, with significance set at the 5 % probability level.

Results and Discussion

We found that at least eight strains of diazotrophic bacteria were naturally inhabiting leaves and roots of Cymbidium sp. and differed in their ability to promote plant growth (Table 1). Analysis of fatty acid methyl esters (FAME-GC) and sequencing of the 16S rRNA gene identified the isolated bacterial strains as Bacillus thuringiensis, Burkholderia cepacia, Burkholderia gladioli, Herbaspirillum frisingense, Pseudomonas stutzeri, Rhizobium cellulosilyticum, Rhizobium radiobacter, and Stenotrophomonas maltophilia. In addition to the biological fixation of atmospheric nitrogen, most of the isolates showed potential for solubilizing calcium phosphate and zinc oxide and for synthesizing indolic compounds, which are predecessors of the auxin class of phytohormones and reflect the ability of these bacteria to promote plant growth (Table 1).

Table 1
Identification and characteristics of growth-promoting bacteria isolated from Cymbidium sp.

These results are consistent with the work of Wilkinson et al. (1989Wilkinson, K.G.; Dixon, K.W.; Sivasithamparam, K. 1989. Interaction of soil bacteria, mycorrhizal fungi and orchid seed in relation to germination of Australian orchids. New Phytologist 112: 429-435.; 1994) who presented the first descriptions of an association between orchids and plant growth-promoting bacteria; these authors isolated Pseudomonas, Bacillus, Xanthomonas, Arthrobacter, and Kurthia bacteria with the potential to synthesize an auxin class phytohormone. Subsequently, Tsavkelova et al. (2001Tsavkelova, E.A.; Cherdyntseva, T.A.; Lobakova, E.S.; Kolomeitseva, G.; Netrusov, A.I. 2001. Microbiota of the orchid rhizoplane. Mikrobiologiya 70: 567-573.; 2003Tsavkelova, E.A.; Lobakova, E.S.; Kolomeitseva, G.L.; Cherdyntseva, T.A.; Netrusov, A.I. 2003. Localization of associative cyanobacteria on the roots of epiphytic orchids. Mikrobiologiya 72: 99-104.; 2004Tsavkelova, E.A.; Cherdyntseva, T.A.; Netrusov, A.I. 2004. Bacteria associated with the roots of epiphytic orchids. Mikrobiologiya 73: 825-831.) isolated Acinetobacter, Bacillus, Cellulomonas, Flavobacterium, Gluconobacter, Micrococcus, Mycobacterium, Pseudomonas, Rhodococcus, and Streptomyces bacterial genera from Acampe papillosa and Dendrobium moschatum orchids. Recently, Faria et al. (2013)Faria, D.C.; Dias, A.C.F.; Melo, I.S.; Costa, F.E.C. 2013. Endophytic bacteria isolated from orchid and their potential to promote plant growth. World Journal of Microbiology and Biotechnology 29: 217-221, isolated strains belonging to the Paenibacillus genus from Cymbidium eburneum. Notably, bacteria with biotechnological potential inhabit plants belonging to the Orchidaceae family.

Growth data (Table 2) of acclimatized Cymbidium sp. indicated that a number of bacterial strains helped the growth and adaptation of plantlets under ex vitro conditions. The bacteria Stenotrophomonas maltophilia and Herbaspirillum frisingense promoted the greatest increases in growth of Cymbidium sp., indicated by an increase in total dry matter of 29 % and 26 %, respectively, compared to control plants. The acclimatization of Cymbidium plants propagated in vitro is a slow process (Chugh et al., 2009Chugh, S.; Guha, S.; Usha, R.I. 2009. Micropropagation of orchids: a review on the potential of different explants. Scientia Horticulturae 122: 507-520.) which might benefit from applications of both biostimulants (Baldotto et al., 2014Baldotto, L.E.B.; Baldotto, M.A.; Gontijo, J.B.; Oliveira, F.M.; Gonçalves, J. 2014. Acclimatization of orchid (Cymbidium sp.) in response to the application of humic acids. Ciência Rural 44: 830-833 (in Portuguese, with abstract in English).) and, as verified in this work, and plant growth-promoting bacteria.

Table 2
Cymbidium sp. growth characteristics in response to inoculation of plant growth-promoting bacteria.

The bacterial genus Stenotrophomonas comprises gram-negative, rod-like species. Stenotrophomonas maltophilia is found in a variety of environments and geographical regions, and it promotes growth or is a symbiotic agent in several species of plants, such as sugarcane (Beneduzi et al., 2013Beneduzi, A.; Moreira, F.; Costa, P.B.; Vargas, L.K.; Lisboa, B.B.; Favreto, R.; Baldani, J.I.; Passaglia, L.M.P. 2013. Diversity and plant growth promoting evaluation abilities of bacteria isolated from sugarcane cultivated in the south of Brazil. Applied Soil Ecology 63: 94-104.), fruit from the Brazilian Cerrado (Dias et al., 2015Dias, M.; Cruz, M.G.P.M.; Duarte, W.F.; Silva, C.F.; Schwan, R.F. 2015. Epiphytic bacteria biodiversity in Brazilian Cerrado fruit and their cellulolytic activity potential. Annals of Microbiology 65: 851-864.) and agricultural crops in Korea (Park et al., 2005Park, M.; Kim, C.; Yang, J.; Lee, H.; Shin, W.; Kim, S.; Sa, T. 2005. Isolation and characterization of diazotrophic growth promoting bacteria from rhizosphere of agricultural crops of Korea. Microbiological Research 160: 127-133.). According to Jeong et al. (2010)Jeong, J.; Lee, O.; Jeon, Y.; Kim, J.; Lee, N.; Lee, C.; Son, H. 2010. Production of keratinolytic enzyme by a newly isolated featherdegrading Stenotrophomonas maltophilia that produces plant growth-promoting activity. Process Biochemistry 45: 1738-1745., this bacterial species shows a high capacity for producing indole acetic acid (IAA), an observation also verified in several other species of plant growth- promoting bacteria (Spaepen et al., 2007Spaepen, S.; Vanderleyden, J.; Remans, R. 2007. Indole-3-acetic-acid in microbial and microorganism-plant signaling. FEMS Microbiology Reviews 31: 425-448.). As IAA has a beneficial effect on plant growth, our observation here that inoculation of Cymbidium sp. with Stenotrophomonas maltophilia gave the highest resultant relative increase in dry matter may be due to hormone activity.

The bacterial isolate Herbaspirillum frisingense caused the highest increases in the nutritional contents of N and P, of 68 % and 28 %, respectively, compared to control samples (Table 3). Representatives of the genus Herbaspirillum are considered mandatory endophytic bacteria and have a low survival rate in the soil. Herbaspirillum frisingense has biological nitrogen fixation capability, and is a potential candidate for calcium phosphate and zinc oxide solubilization and indolic compound synthesis (Kirchhof et al., 2001Kirchhof, G.; Eckert, B.; Stoffels, M.; Baldani, J.I.; Reis, V.M.; Hartmann, A. 2001. Herbaspirillum frisingense sp. nov., a new nitrogen-fixing bacterial species that occurs in C4-fibre plants. International Journal of Systematic and Evolutionary Microbiology 51: 157-168.; Montañez et al., 2012Montañez, A.; Blanco, A.R.; Barlocco, C.; Beracochea, M.; Sicardi, M. 2012. Characterization of cultivable putative endophytic plant growth promoting bacteria associated with maize cultivars (Zea mays L.) and their inoculation effects in vitro. Applied Soil Ecology 58: 21-28.; Straub et al., 2013Straub, D.; Yang, H.; Liu, Y.; Tsap, T.; Ludewig, U. 2013. Root ethylene signalling is involved in Miscanthus sinensis growth promotion by the bacterial endophyte Herbaspirillum frisingense GSF30T. Journal of Experimental Botany 64: 4603-4615.). Potential for the promotion of growth was reflected in the relative increases in dry matter and nutritional contents of N, P and K obtained in our greenhouse experiment.

Table 3
Nutritional composition of Cymbidium sp. in response to inoculation of plant growth-promoting bacteria.

Bacteria belonging to the genus Bacillus can be easily isolated from the soil and rhizosphere of various plants (Seldin et al., 1998Seldin, L.; Rosado, A.S.; Cruz, D.W.; Nobrega, A.; Van Elsas, J.D.; Paiva, E. 1998. Comparison of Paenibacillus azotofixans strains isolated from rhizoplane, rhizosphere, and non-root-associated soil from maize planted in two different Brazilian soils. Applied Environmental Microbiology 64: 3860-3868.). Many Bacillus species contribute to the health of plants in many ways, such as biological nitrogen fixation and biological phytopathogen control agents (Lacey et al., 2001Lacey, L.A.; Frutos, R.; Kaya, H.K.; Vanderleyden, J. 2001. Insect pathogens as biological control agents: do they have a future? Biological Control 21: 230-248.). Bacillus thuringiensis is known for its use in biological control programs (Sadfi et al., 2001Sadfi, N.S.; Cherif, M.; Fliss, I.; Boudabbous, A.; Antoun, H. 2001. Evaluation of bacterial isolates from salty soils and Bacillus thuringiensis strains for the biocontrol of fusarium dry rot of potato tubers. Journal of Plant Pathology 83: 101-117.); however, there are only a few reports of use of this bacterial species as an endophytic growth promoting microorganism, for example, in the banana tree “prata-anã” (Andrade et al., 2014Andrade, L.F.: Souza, G.L.O.D.; Nietsche, S.; Xavier, A.A.; Costa, M.R.; Cardoso, A.M.S.; Pereira, M.C.T.; Pereira, D.F.G.S. 2014. Analysis of the abilities of endophytic bacteria associated with banana tree roots to promote plant growth. Journal of Microbiology 52: 27-34.) and when isolated from soybean root nodules (Bai et al., 2002Bai, Y.; D'Aoust, F.; Smith, D.; Driscoll, B. 2002. Isolation of plant-growth-promoting Bacillus strains from soybean root nodules. Canadian Journal of Microbiology 48: 230-238.). Andrade et al. (2014)Andrade, L.F.: Souza, G.L.O.D.; Nietsche, S.; Xavier, A.A.; Costa, M.R.; Cardoso, A.M.S.; Pereira, M.C.T.; Pereira, D.F.G.S. 2014. Analysis of the abilities of endophytic bacteria associated with banana tree roots to promote plant growth. Journal of Microbiology 52: 27-34. also identified the calcium phosphate solubilization and IAA production capability of diazotrophic bacteria belonging to the genus Bacillus which was also identified in this study (Table 1).

Two bacterial strains belonging to the genus Burkholderia were isolated from Cymbidium sp. leaves. Bacteria from the genus Burkholderia are gram-negative in the form of motile rods, with three or more flagella. Currently, this genus includes 62 species with great functional diversity. Species within this genus may be plant growth-promoters or human, animal, and vegetable pathogens (Eberl and Vandamme, 2016Eberl, L.; Vandamme, P. 2016. Members of the genus Burkholderia: good and bad guys. F1000Research 1007: 1-10.), as in the case of Burkholderia gladioli, which was isolated in this study and characterized as pathogenic in orchids by Keith et al. (2005)Keith, L.M.; Sewake, K.T.; Zee, F.T. 2005. Isolation and characterization of Burkholderia gladioli from orchids in Hawaii. Plant Disease 89: 1273-1278., a fact that may be related to the negative effect on the relative increase of dry matter after inoculation. In addition, the Burkholderia bacteria identified here had the ability to solubilize calcium phosphate, consistent with the results from Rodríguez and Fraga (1999)Rodríguez, H.; Fraga, R. 1999. Phosphate solubilizing bacteria and their role in plant growth promotion. Biotechnology Advances 17: 319-339..

The genus Pseudomonas is characterized as being composed of gram-negative, aerobic, and mobile bacilli and stands out because of its great nutritional versatility in agricultural production systems and its ability to grow in a wide variety of environmental conditions (Peix et al., 2009Peix, A.; Ramírez-Bahena, M.H.; Velázquez, E. 2009. Historical evolution and current status of the taxonomy of genus Pseudomonas. Infection, Genetics and Evolution 9: 1132-1147.). Pseudomonas stutzeri was isolated from cucumber (Islam et al., 2016Islam, S.; Akanda, A.M.; Prova, A.; Islam, M.T.; Hossain, M.M. 2016. Isolation and identification of plant growth promoting rhizobacteria from cucumber rhizosphere and their effect on plant growth promotion and disease suppression. Frontiers in Microbiology 6: 1-12.), rice (Pham et al., 2017Pham, V.T.K.; Rediers, H.; Gheguire, M.G.K.; Nguyen, H.H.; De Mot, R.; Vanderleyden, J.; Spaepen, S. 2017. The plant growthpromoting effect of the nitrogen-fixing endophyte Pseudomonas stutzeri A15. Archives of Microbiology 199: 513-517.), sunflower (Pandey et al., 2013Pandey, R.; Chavan, N.; Walokar, N.M.; Khetmalas, M. 2013. Pseudomonas stutzeri RP1: a versatile plant growth promoting endorhizospheric bacteria inhabiting sunflower (Helianthus annus). Research Journal of Biotechnology 8: 48-55.), and has significant potential for biological nitrogen fixation. Diazotrophic bacteria from the genus Pseudomonas can solubilize calcium phosphate (Rodríguez and Fraga, 1999Rodríguez, H.; Fraga, R. 1999. Phosphate solubilizing bacteria and their role in plant growth promotion. Biotechnology Advances 17: 319-339.) and zinc oxide, and can synthesize IAA (Islam et al., 2016Islam, S.; Akanda, A.M.; Prova, A.; Islam, M.T.; Hossain, M.M. 2016. Isolation and identification of plant growth promoting rhizobacteria from cucumber rhizosphere and their effect on plant growth promotion and disease suppression. Frontiers in Microbiology 6: 1-12.), which was also observed in this study.

The Rhizobium genus is composed of microorganisms generally identified as rhizobia and gram-negative bacteria, and is usually related to nitrogen-fixing bacteria that form nodules on leguminous plants (Stroschein, 2010Stroschein, M.R.D.; Eltz, F.L.F.; Antoniolli, Z.I.; Lupatini, M.; Vargas, L.K.; Giongo, A.; Pontelli, M.P. 2010. Symbiotic efficiency and genetic characteristics of Bradyrhizobium sp. strain UFSM LA 1.3 isolated from Lupinus albescens (H. et Arn). Scientia Agricola 67: 702-706.). Singh et al. (2015)Singh, N.P.; Singh, R.K.; Meena, V.S.; Meena, R.K. 2015. Can we use maize (Zea mays) rhizobacteria as plant growth promoter? International Journal of Plant Research 28: 86-99. isolated Rhizobium radiobacter from corn and identified its growth-promoting activity. Recently, Diez-Mendez et al. (2015)Diez-Mendez, A.; Menendez, E.; García-Fraile, P.; Celador-Lera, L.; Rivas, R.; Mateos, P.F. 2015. Rhizobium cellulosilyticum as a co-inoculant enhances Phaseolus vulgaris grain yield under greenhouse conditions. Symbiosis 67: 135-141. identified the most efficient approach to biological nitrogen fixation from co-inoculation of Rhizobium cellulosilyticum with beans. Isolated bacterial strains from Cymbidium sp. were identified as belonging to the genus Rhizobium and have the ability to solubilize calcium phosphate, consistent with the findings of Rodríguez and Fraga (1999)Rodríguez, H.; Fraga, R. 1999. Phosphate solubilizing bacteria and their role in plant growth promotion. Biotechnology Advances 17: 319-339..

The above findings indicate that the inoculation of orchid plantlets with diazotrophic bacteria during in vitro propagation can be a viable strategy for accelerating the acclimatization of plants; this approach can, thus, reduce production costs by improving growth and nutritional efficiency. The promotion of plant growth as a result of bacterial metabolic processes, the focus of this study, has the potential to contribute to achieving sustainability in the agribusiness sector. Therefore, inoculating orchid plantlets with selected bacterial strains (Herbaspirillum frisingense and Stenotrophomonas maltophilia) could provide the floriculture and ornamental plant sector with a greater competitive edge in the production and marketing of their agricultural products. The biotechnological applications of bacterial inoculation technology in agriculture might contribute to the reduction in use and consequent impact of fertilizers, leading to significant economic benefits.

  • Edited by: Fernando Dini Andreote

Acknowledgments

This work was supported by CNPq (Brazilian National Council for Scientific and Technological Development - 470567-2011-12), FAPEMIG (Minas Gerais State Foundation for Research Support - 03929-10), and FUNARBE (Arthur Bernardes Foundation - Funarbe Program to Support Research for Young Researchers (FUNARPEQ, 2011)). We are also grateful to the following individuals: Prof. Marcos Tótola (Federal University of Viçosa) and Prof. Siu Tsai (University of São Paulo/CENA), who provided guidance on the identification of bacterial isolates; and Fernanda Oliveira (Federal University of Viçosa) and Joelma Gonçalves (Federal University of Viçosa), who assisted in conducting the greenhouse experiments.

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Publication Dates

  • Publication in this collection
    Sep-Oct 2018

History

  • Received
    02 Apr 2017
  • Accepted
    11 June 2017
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