Abstract
Clonal genotypes resistant to fungal diseases are an important component of the cocoa production system in southeastern Bahia state (Brazil), so that technologies for faster production of stronger and healthier plantlets are highly desirable. In this study, the effects of inoculated bacterial endophytes isolated from healthy adult cacao plants on seedlings, and aspects related to inoculation methods, colonization patterns, and photosynthesis were investigated. Sequencing of 16S rRNA, hsp-60, and rpo-B genes placed the wild-type isolates within the species Enterobacter cloacae (isolates 341 and 344) and Bacillus subtilis (isolate 629). Spontaneous rifampicin-resistant (rifR) variants for 344 were also produced and tested. Endophytic application was either by immersion of surface sterilized seeds in bacterial suspensions or direct inoculation into soil, 20 days after planting non-inoculated seeds into pots. Results from in vitro recovery of inoculated isolates showed that the wild-type endophytes and rifR variants systemically colonized the entire cacao seedlings in 15–20 days, regardless of the inoculation method. Some endophytic treatments showed significant increases in seedlings’ height, number of leaves, and dry matter. Inoculation methods affected the combined application of endophytes, which maintained the growth-promotion effects, but not in the same manner as in single applications. Interestingly, the 344-3.2 rifR variant showed improved performance in relation to both the wild type and another related variant. Photosynthetic rates and stomatal conductance increased significantly for some endophytic treatments, being partially associated with effects on growth and affected by the inoculation method. The results suggest that E. cloacae and B. subtilis endophytes from healthy adult plants (not transmitted by seeds) were able to promote vegetative growth on cacao seedlings. The development of products for large-scale use in seedlings/plantlets production systems was discussed.
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Abreu-Tarazi MF, Navarrete AA, Andreote FD, Almeira CV, Tsai SM, Almeida M (2010) Endophytic bacteria in long-term in vitro cultivated “axenic” pineapple microplants revealed by PCR–DGGE. World J Microbiol Biotechnol 26:555–560
Aime MC, Phillips-Mora W (2005) The causal agents of witches’ broom and frosty pod rot of cacao (chocolate, Theobroma cacao) form a new lineage of Marasmiaceae. Mycol 97:1012–1022
Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local alignment search tool. J Mol Biol 215:403–410
Andreote FD, Rocha UN, Araújo WL, Azevedo JL, van Overbeek LS (2010) Effect of bacterial inoculation, plant genotype and developmental stage on root-associated and endophytic bacterial communities in potato (Solanum tuberosum). Anton Leeuw 97:389–399
Araújo FF (2008) Seed inoculation with Bacillus subtilis formulated with oyster meal and growth of corn, soybean and cotton. Ciênc Agrotec 32:456–462 (in Portuguese)
Ayres M, Ayres Jr (2007) BioEstat: Aplicações estatísticas nas áreas das ciências biomédicas 2007 (http://wwwmamirauaorgbr/download) Accessed 5 May 2008
Azevedo JL (1998) Microrganismos endofíticos. In: Melo IS, Azevedo JL (eds) Ecologia microbiana. EMBRAPA Meio Ambiente, Jaguariúna, pp 117–137
Azevedo JL, Maccheroni W Jr, Pereira JO, Araújo WL (2000) Endophytic microorganisms: a review on insect control and recent advances on tropical plants. Electron J Biotechnol 3:40–65
Bacon CW, Hinton DM (2002) Endophytic and biological control potential of Bacillus mojavensis and related species. Biol Control 23:274–284
Bashan Y, De-Bashan LE (2005) Bacteria—plant growth-promoting. In: Hillel D (ed) Encyclopedia of soils in the environment, vol 1, Elsevier. Oxford, UK, pp 103–115
Bloemberg GV, Lugtenberg BJJ (2001) Molecular basis of plant growth promotion and biocontrol by rhizobacteria. Curr Opin Plant Biol 4:343–350
Compant S, Clément C, Sessitsch A (2010) Plant growth-promoting bacteria in rhizo- and endosphere of plants: their role, colonization, mechanisms involved and prospects for utilization. Soil Biol Biochem 42:669–678
Compeau G, Al-Achi BJ, Platsouka E, Levy SB (1988) Survival of rifampicin-resistant mutants of Pseudomonas fluorescens and Pseudomonas putida in soil systems. Appl Environ Microbiol 54:2432–2438
Conn KL, Nowak J, Lazarovits G (1997) A gnotobiotic bioassay for studying interactions between potatoes and plant growth-promoting rhizobacteria. Can J Microbiol 43:801–808
Dahllöf I, Baillie H, Kjelleberg S (2000) rpoB-based microbial community analysis avoids limitations inherent in 16S rRNA gene intraspecies heterogeneity. Appl Environ Microbiol 66:3376–3380
Diancourt L, Passet V, Verhoef J, Grimont PAD, Brisse S (2005) Multilocus sequence typing of Klebsiella pneumoniae nosocomial isolates. J Clin Microbiol 43:4178–4182
Donald PF (2004) Biodiversity impacts of some agricultural commodity production systems. Conserv Biol 18:17–37
Faria D, Paciencia MLB, Dixo M, Laps RR, Baumgarten J (2007) Ferns, frogs, lizards, birds and bats in forest fragments and shade cacao plantations in two contrasting landscapes in the Atlantic forest, Brazil. Biodivers Conserv 16:2335–2357
Frommel MI, Nowak J, Lazarovits G (1991) Growth enhancement and developmental modifications of in vitro growth potato (Solanum tuberosum ssp. tuberosum) as affected by a nonfluorescent Pseudomonas sp. Plant Physiol 96:928–936
Giongo A, Beneduzi A, Ambrosini A, Vargas LK, Stroschein MR, Eltz FL, Bodanese-Zanettini MH, Passaglia LMP (2010) Isolation and characterization of two plant growth-promoting bacteria from the rhizoplane of a legume (Lupinus albescens) in sandy soil. Rev Bras Ciênc Solo 34:361–369 (in Portuguese)
Goh SH, Potter S, Wood JO, Hemmingsen SM, Reynolds RTP, Chow AW (1996) Hsp60 gene sequences as universal targets for microbial species identification: studies with coagulase-negative Staphylococci. J Clin Microbiol 34:818–823
Hall TA (1999) BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp Ser 41:95–98
Hallmann J, Quadt-Hallmann A, Mahaffee WF, Kloepper JW (1997) Bacterial endophytes in agricultural crops. Can J Microbiol 43:895–914
Hanada RE, Pomella AWV, Costa HS, Bezerra JL, Loguercio LL, Pereira JO (2010) Endophytic fungal diversity in Theobroma cacao (cacao) and T. grandiflorum (cupuaçu) trees and their potential for growth promotion and biocontrol of black-pod disease. Fungal Biol 114:901–910
Keel C, Weller DM, Natsch A, Défago G, Cook RJ, Thomashow LS (1996) Conservation of the 2,4-diacetylphloroglucinol biosynthesis locus among fluorescent Pseudomonas strains from diverse geographic locations. Appl Environ Microbiol 62:552–563
Khan AA, Jilani G, Akhtar MS, Navqi SMS, Rasheed M (2009) Phosphorus solubilizing bacteria: occurrence, mechanisms and their role in crop production. J Agric Biol Sci 1:48–58
Kim DS, Weller DM, Cook RJ (1997) Population dynamics of Bacillus sp. L324-92R12 and Pseudomonas fluorescens 2-79RN10 in the rhizosphere of wheat. Biol Control 87:559–564
Kloepper JW, Beauchamp CJ (1992) A review of issues related to measuring colonization of plant roots by bacteria. Can J Microbiol 38:1219–1232
Kloepper JW, Ryu C-M, Zhang S (2004) Induced systemic resistance and promotion of plant growth by Bacillus spp. Phytopathology 94:1259–1266
Kumar S, Tamura K, Nei M (2004) MEGA 3: integrated software for molecular evolutionary genetics analysis and sequence alignment. Brief Bioinform 5:150–163
Kwok AYC, Su SC, Reynolds RP, Bay SJ, Av-Gay Y, Dovichi NJ, Chow AW (1999) Species identification and phylogenetic relationships based on partial HSP60 gene sequences within the genus Staphylococcus. Int J Syst Bacteriol 49:1181–1192
Lacava PT, Andreote FD, Araújo WL, Azevedo JL (2006) Characterization of the endophytic bacterial community from citrus by isolation, specific PCR and DGGE. Pesq Agrop Brasileira 41:637–642 (in Portuguese)
Leite HAC (2008) Plant growth-promoting bacterial endophytes in cacao: taxonomy and seedlings colonization studies. Masters Thesis, State University of Santa Cruz (UESC), Ilhéus-BA, Brazil
Lloyd G, McCown B (1981) Commercially feasible micropropagation of Mountain Laurel, Kalmia latifolia, by use of shoot tip culture. Comb Proc Int Plant Prop Soc 30:421–427
Lodewyckx C, Vangronsveld J, Porteous F, Moore ERB, Taghavi S, Mezgeay M, Van Der Lelie D (2002) Endophytic bacteria and their potential applications. Crit Rev Plant Sci 21:583–606
Loguercio LL, Pomella AWV, de Souza JT, Niella GR, Veloso JL, Costa JCB (2012) Controle Biológico das Doenças do Cacaueiro. In: Valle RRM (ed) Ciência, Tecnologia e Manejo do Cacaueiro. CEPLAC, Itabuna, pp 277–292
Lopes VL, Monteiro WR, Pires JL, da Rocha JB, Pinto LRM (2003) On farm selection for witches’ broom resistance in Bahia, Brazil - a historical retrospective. In: 14th International Cocoa Research Conference - Towards a sustainable cocoa economy: what strategies to this end? Vol. 2. COPAL, Accra, Ghana, pp 1001-1006
Magalhães PC, Durães FO, Rodrigues JAS (2008) Ecofisiologia. In: Rodrigues JAS (ed) Cultivo do sorgo. EMBRAPA Milho e Sorgo, Sete Lagoas-MG, http://sistemasdeproducao.cnptia.embrapa.br/FontesHTML/Sorgo/CultivodoSorgo_4ed/ecofisiologia.htm. Accessed 20 Dec 2011
McInroy JA, Kloepper JW (1995) A survey of indigenous bacterial endophytes from cotton and sweet corn. Plant Soil 173:1–6
Melnick RL, Zidack NK, Bailey BA, Maximova SN, Guiltinan MB, Paul A (2008) Bacterial endophytes: Bacillus spp. from annual crops as potential biological control agents of black pod rot of cacao. Biol Control 46:46–56
Oliveira ALM, Urquiaga S, Baldani JI (2003) Processos e mecanismos envolvidos na influência de microrganismos sobre o crescimento vegetal, Documentos 161. EMBRAPA Agrobiologia, Seropédica
Patten CL, Glick BR (2002) Role of Pseudomonas putida indoleacetic acid in development of the host plant root system. Appl Environ Microbiol 68:3795–3801
Peixoto-Neto PAS, Azevedo JL, Araújo WL (2002) Microorganismos endofíticos: interações com plantas e potencial biotecnológico. Biotecnol Cienc Desenv 29:62–76
Pereira JL, Almeida LCC, Santos SM (1996) Witches’ broom disease of cocoa in Bahia: attempts at eradication and containment. Crop Prot 15:743–752
Polesi NPE (2010) Investigation of ubiquitous endophytic microbiota in “axenic” microplants. Masters Thesis, University of São Paulo (ESALQ), Piracicaba-SP, Brazil
Rosenberg GS, Ziber-Rosenberg E (2009) The hologenome theory of evolution contains Lamarckian aspects within a Darwinian framework. Environ Microbiol 11:2959–2962
Rosenblueth M, Martínez-Romero E (2006) Bacterial endophytes and their interactions with hosts. Mol Plant Microbe Interact 19:827–837
Rubini MR, Silva-Ribeiro RT, Pomella AWV, Maki CS, Araújo WL, Dos Santos DR, Azevedo JL (2005) Diversity of endophytic fungal community of cacao (Theobroma cacao L) and biological control of Crinipellis perniciosa, causal agent of witches’ broom disease. Int J Biol Sci 1:24–33
Shi Y, Lou K, Li C (2010) Growth and photosynthetic efficiency promotion of sugar beet (Beta vulgaris L.) by endophytic bacteria. Photosynth Res 105:5–13
Shishido M, Loeb BM, Chanway CP (1995) External and internal root colonization of lodgepole pine seedlings by two growth-promoting Bacillus strains originated from different root microsites. Can J Microbiol 41:707–713
Shishido M, Breuil C, Chanway CP (1999) Endophytic colonization of spruce by growth-promotion rhizobacteria. FEMS Microbiol Ecol 29:191–196
Silva AB (2010) Effects of endophytic bacteria inoculation in cacao: growth promotion and increase in resistance to the black-pod disease. Masters Thesis, State University of Santa Cruz (UESC), Ilhéus-BA, Brazil
Silva HSA, Bettiol W, Terrasan CRF, Tozzi JPL, De Melo IS, Nunes FV (2006) Microrganismos endofíticos: potencial de uso como agentes de biocontrole da ferrugem do cafeeiro, Boletim de Pesquisa e Desenvolvimento 38. EMBRAPA Meio Ambiente, Jaguariúna
Silveira EB, Gomes AMA, Mariano RLR, Silva Neto EB (2004) Bacterization of seeds and development of cucumber seedlings. Hortic Bras 22:217–221 (in Portuguese)
Siqueira JF Jr, Rôças IN (2003) A 16S rDNA-based nested PCR protocol to detect Campylobacter gracilis in oral infections. Pesq Odontol Bras 17:142–146 (in Portuguese)
Steenhoudt O, Vanderleyden J (2000) Azospirillum, a free-living nitrogenfixing bacterium closely associated with grasses: genetic, biochemical and ecological aspects. FEMS Microbiol Rev 24:487–506
Sturz AV, Nowak J (2000) Endophytic communities of rhizobacteria and the strategies required to create yield enhancing associations with crops. Appl Soil Ecol 15:183–190
Sturz AV, Christie BR, Matheson BG, Nowak J (1997) Biodiversity of endophytic bacteria which colonize red clover nodules, roots, stems and foliage and their influence on host growth. Biol Fertil Soils 25:13–19
Tian F, Yanqin D, Hui Z, Liangtong Y, Binghai D (2009) Genetic diversity of siderophore-producing bacteria of tobacco rhizosphere. Braz J Microbiol 40:276–284
Van Loon LC, Bakker PAHM, Pieterse CM (1998) Systemic resistance induced by rhizosphere bacteria. Annu Rev Phytopathol 36:453–483
Wang H, Wen K, Zhao X, Wang X, Li A, Hong H (2009) The inhibitory activity of endophytic Bacillus sp. strain CHM1 against plant pathogenic fungi and its plant growth-promoting effect. Crop Prot 28:634–639
Zilber-Rosenberg I, Rosenberg E (2008) Role of microorganisms in the evolution of animals and plants: the hologenome theory of evolution. FEMS Microbiol Rev 32:723–735
Zinniel DK, Lambrecht P, Harris NB, Feng Z, Kuczmarski D, Higley P, Ishimaru CA, Arunakumari A, Barletta RG, Vidaver AK (2002) Isolation and characterization of endophytic colonizing bacteria from agronomic crops and prairie plants. Appl Environ Microbiol 68:2198–2208
Acknowledgments
The authors are grateful to Dr. George Andrade Sodré for great technical assistance and suggestions with greenhouse experiments and soil analysis. This work was supported by grants from the International Foundation of Science (IFS), CEPLAC, FAPESB, and UESC. H.A.C. Leite and A.B. Silva were supported by Masters Fellowships from FAPESB and CNPq, respectively.
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Hianna Almeida Câmara Leite and Anderson Barbosa Silva contributed equally to this work.
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Leite, H.A.C., Silva, A.B., Gomes, F.P. et al. Bacillus subtilis and Enterobacter cloacae endophytes from healthy Theobroma cacao L. trees can systemically colonize seedlings and promote growth. Appl Microbiol Biotechnol 97, 2639–2651 (2013). https://doi.org/10.1007/s00253-012-4574-2
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DOI: https://doi.org/10.1007/s00253-012-4574-2